Theoretical Origin and Research Progress on the Mechanism and Therapeutic Effects of Sanhua Decoction in Treating Cerebral Ischemia

DOI:https://doi-xx.org/1050/17707994668225

Wanyu Luo^1,2 , Peiwei Su¹, Rui Xu¹,XuDong Zhufu¹,Chao zhu¹,XianYi Zhang¹ MuDong Liu¹  XueMei Huang¹ Haoxin Wu² Kang Luo³  ZhengWei Gu^1,*  HaiJun Zhao^1,*

1. Shandong University of Traditional Chinese Medicine，Jinan 250355，China
2. Nanjin University of Traditional Chinese Medicine，Nanjin 210023，China
3. College of Landscape and Horticulture, Yunnan Agricultural University Kunming 650500 China

Abstract

Cerebral ischemia and its subsequent reperfusion injury are primary causes of pathological brain tissue damage and neurological dysfunction, posing significant challenges to clinical treatment. Sanhua Decoction, a traditional Chinese herbal formula characterized by its multi-component and multi-target properties, has garnered increasing attention for its promising therapeutic effects in cerebral ischemia management. This review systematically summarizes the current application status of Sanhua Decoction in cerebral ischemia, focusing on its principal components, pharmacological mechanisms, and therapeutic outcomes. Integrating recent pharmacodynamic findings and chemical fingerprinting studies, the review elucidates how Sanhua Decoction exerts neuroprotective effects through multiple pathways including antioxidation, anti-inflammation, modulation of neurotransmitters, and neuronal cell protection. By providing a comprehensive synthesis of experimental and clinical evidence, this article aims to offer theoretical support and practical guidance for the clinical use and mechanistic research of Sanhua Decoction in cerebral ischemia treatment.

Keywords

Sanhua Decoction, cerebral ischemia, cerebral ischemia-reperfusion injury, mechanisms of action, therapeutic effects, traditional Chinese medicine, neuroprotection

Introduction

Cerebral ischemia, characterized by insufficient blood supply to brain tissue, is a critical pathological condition that leads to extensive neuronal damage and consequent neurological dysfunction. This condition underlies a significant proportion of cerebrovascular diseases, including ischemic stroke, which remains a leading cause of mortality and long-term disability worldwide. The pathophysiology of cerebral ischemia is complex, involving a cascade of events such as energy failure, excitotoxicity, oxidative stress, inflammation, blood-brain barrier disruption, and apoptosis. Notably, the subsequent restoration of blood flow, while essential for tissue salvage, paradoxically exacerbates injury through ischemia-reperfusion (I/R) mechanisms, including the generation of reactive oxygen species (ROS), activation of inflammatory pathways, and induction of various cell death modalities ^[1][2][3]. Despite advances in reperfusion therapies, such as thrombolysis and mechanical thrombectomy, clinical outcomes remain suboptimal due to the limited therapeutic window and the deleterious effects of reperfusion injury. Consequently, there is an urgent need for novel therapeutic strategies that can effectively mitigate cerebral ischemia-reperfusion injury and improve neurological recovery.

Traditional Chinese medicine (TCM) has been practiced for millennia and offers a rich repository of herbal formulations with multi-component and multi-target characteristics, which may be particularly suited to address the multifaceted pathogenesis of cerebral ischemia. Among these, classic prescriptions such as Sanhua Decoction (SHD) and other herbal combinations have demonstrated unique advantages in the treatment of cerebrovascular diseases by modulating various pathophysiological processes including oxidative stress, inflammation, apoptosis, and neurovascular repair ^[4][5]. The pharmacological effects of TCM are often attributed to their capacity to regulate complex signaling networks and to exert synergistic actions through multiple bioactive constituents. This holistic approach aligns with the intricate nature of cerebral ischemic injury, where single-target interventions have often proved insufficient. Moreover, TCM formulations have been shown to influence critical molecular pathways such as the PI3K/AKT signaling, NF-κB-mediated inflammatory responses, and mitochondrial quality control mechanisms, which are central to neuronal survival and functional recovery after ischemic insult^[6][7][8].

Sanhua Decoction, a classic TCM formula composed primarily of Citrus aurantium, Magnolia officinalis, Rheum palmatum, and Notopterygium incisum, is traditionally used to regulate qi and promote blood circulation. Recent pharmacological studies have begun to elucidate its multi-target therapeutic potential in cerebral ischemia, demonstrating antioxidative, anti-inflammatory, neuroprotective, and anti-edematous effects. These effects are mediated through modulation of neurotransmitter systems, attenuation of neuronal apoptosis, and preservation of blood-brain barrier integrity ^[4]. Advances in analytical techniques, such as ultraperformance liquid chromatography coupled with pharmacodynamic evaluation, have facilitated the identification of active constituents within SHD and their correlation with therapeutic efficacy, providing a scientific basis for quality control and clinical application^[5]. Such integrative approaches underscore the potential of SHD as a promising candidate for cerebral ischemia treatment.

Furthermore, the complexity of cerebral ischemia-reperfusion injury necessitates therapeutic agents capable of targeting multiple pathological processes simultaneously. TCM formulations, including SHD and other herbal injections, have shown promise in this regard, exhibiting properties such as anti-apoptosis, immunomodulation, angiogenesis promotion, and mitochondrial protection^[9][10][11]. For instance, the regulation of non-coding RNAs and microRNAs by TCM components has emerged as a novel mechanism to attenuate neuroinflammation and neuronal apoptosis, thereby enhancing neuroprotection^[12][13]. Additionally, TCM’s ability to modulate vascular endothelial growth factor (VEGF) pathways and promote angiogenesis contributes to the restoration of cerebral microcirculation and tissue repair post-ischemia ^[10].

In conclusion, cerebral ischemia along with the resultant reperfusion injury poses a significant clinical challenge due to its intricate and multifactorial origins. Traditional Chinese medicine, characterized by its multi-component and multi-target therapeutic approach, presents distinct advantages for tackling this complexity. Among the various formulas, Sanhua Decoction has demonstrated promising neuroprotective effects through a range of mechanisms, including the alleviation of oxidative stress, anti-inflammatory properties, inhibition of apoptosis, and vascular protection. Recent investigations in pharmacology and chemistry have started to elucidate the material basis and underlying mechanistic pathways of these effects, thus paving the way for evidence-based clinical applications. This review seeks to systematically consolidate the current knowledge regarding the mechanisms and therapeutic efficacy of Sanhua Decoction in the context of cerebral ischemia, while incorporating the latest developments from pharmacology, molecular biology, and analytical chemistry to establish a scientific foundation for its expanded clinical utilization and application.

2. Main Body

2.1 Pathological Mechanisms of Cerebral Ischemia and Cerebral Ischemia-Reperfusion Injury

2.1.1 Pathophysiological Changes in Cerebral Ischemia

Cerebral ischemia initiates with the interruption of blood flow, leading to oxygen deprivation and energy metabolism failure in brain tissue, which profoundly impairs neuronal function. The lack of oxygen and glucose supply disrupts oxidative phosphorylation in mitochondria, resulting in ATP depletion and failure of energy-dependent processes essential for neuronal survival and function^[14]. This energy deficit impairs ion pumps such as Na+/K+-ATPase, causing ionic imbalances characterized by intracellular accumulation of sodium and calcium ions and extracellular potassium elevation. These ionic disturbances trigger pathophysiological cascades including excitotoxicity, oxidative stress, and inflammation. Excessive glutamate release and impaired reuptake lead to overstimulation of N-methyl-D-aspartate receptors (NMDARs), causing intracellular calcium overload, activation of calcium-dependent enzymes, and generation of reactive oxygen species (ROS), which together mediate neuronal injury ^{[15] [16]}. The metabolic reprogramming of glial cells, especially microglia and astrocytes, further modulates the inflammatory milieu and energy metabolism in the ischemic brain^[17]. The blood-brain barrier (BBB) integrity is compromised due to endothelial dysfunction and pericyte impairment, exacerbating vasogenic edema and facilitating infiltration of immune cells, which amplify neuroinflammation^{[18] [19]}. Programmed cell death pathways, including apoptosis, pyroptosis, ferroptosis, and necroptosis, are activated in response to ischemic stress, contributing to neuronal loss and infarct expansion ^{[20] [21].} Moreover, oxidative stress and mitochondrial dysfunction play central roles in propagating cellular injury, with mitochondrial dynamics and mitophagy influencing the balance between cell survival and death ^{[2] [22]}. The interplay between excitotoxicity, ionic imbalance, oxidative damage, and inflammatory responses forms a complex pathophysiological network that underlies the progression of cerebral ischemic injury. Understanding these mechanisms is crucial for developing targeted therapeutic strategies to mitigate neuronal damage and improve outcomes after cerebral ischemia ^{[14] [19] [23]}.

2.1.2 Mechanisms of Reperfusion Injury

Reperfusion injury following cerebral ischemia is a complex pathological process characterized by a cascade of molecular and cellular events that exacerbate brain tissue damage beyond the initial ischemic insult. One of the central mechanisms involved is the restoration of blood flow, which paradoxically triggers a burst of oxidative stress due to the sudden influx of oxygen. This leads to the excessive generation of reactive oxygen species (ROS), including superoxide anions, hydroxyl radicals, and hydrogen peroxide. The overproduction of ROS overwhelms endogenous antioxidant defenses, resulting in oxidative damage to lipids, proteins, and nucleic acids within neural cells. This oxidative stress is a pivotal factor in neuronal injury during reperfusion and is closely linked to mitochondrial dysfunction, which further amplifies ROS production and disrupts cellular energy metabolism ^{[2] [8] [24]}.

In parallel to oxidative stress, reperfusion activates a robust inflammatory response. This involves the upregulation and release of pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), as well as the activation of microglia and astrocytes. The inflammatory milieu contributes to the disruption of the blood-brain barrier (BBB), increasing its permeability and facilitating the infiltration of peripheral immune cells into the brain parenchyma. The breakdown of the BBB exacerbates cerebral edema and promotes further neuronal damage. Notably, proteins such as Lipocalin-2 (LCN2) have been implicated in modulating astrocyte activity and inflammatory cascades, influencing both neuroinflammation and BBB integrity during reperfusion injury ^{[25] [24] [26]}.

A distinctive form of programmed cell death termed pyroptosis has been increasingly recognized as a contributor to neuronal loss in reperfusion injury. Pyroptosis is an inflammatory cell death pathway characterized by the activation of inflammasomes and caspase-1, leading to the cleavage of gasdermin D and the release of pro-inflammatory cytokines IL-1β and IL-18. This process not only causes direct neuronal death but also amplifies neuroinflammation, creating a vicious cycle of injury. The crosstalk between oxidative stress, inflammation, and pyroptosis underscores the multifaceted nature of reperfusion injury pathogenesis ^[27].

Moreover, reperfusion injury impairs neuronal function and survival by promoting various cell death pathways beyond pyroptosis, including apoptosis, necroptosis, autophagy dysregulation, and ferroptosis. For instance, excessive autophagy activation has been observed in reperfused brain tissue, which may initially serve a protective role but can lead to autophagic cell death if dysregulated. The modulation of autophagy-related proteins such as Beclin-1 and LC3 has been shown to influence neuronal outcomes after reperfusion, with interventions like melatonin demonstrating neuroprotection by balancing autophagy and apoptosis^{[28] [29]}. Ferroptosis, an iron-dependent lipid peroxidation-driven cell death, is also implicated in reperfusion injury, with ferritinophagy-mediated iron release exacerbating oxidative damage^[30].

Collectively, the mechanisms of reperfusion injury involve an intricate interplay of oxidative stress, inflammatory activation, BBB disruption, and programmed cell death pathways. These processes synergistically contribute to neuronal damage and hinder functional recovery after ischemic stroke. Understanding these mechanisms provides a foundation for developing targeted therapies, such as antioxidant agents, anti-inflammatory compounds, and modulators of cell death pathways, to mitigate reperfusion injury and improve neurological outcomes ^{[4] [3] [31]}.

2.2 Composition of Sanhua Decoction and Analysis of Its Pharmacologically Active Components

2.2.1 Main Components Introduction

Sanhua Decoction (SHD) is a classical traditional Chinese medicine formula widely used in the treatment of ischemic stroke, composed primarily of four herbs: Citrus aurantium (Zhike), Magnolia officinalis (Houpo), Rheum palmatum (Dahuang), and Notopterygium incisum (Qianghuo). Each of these herbs contributes distinct bioactive compounds that synergistically exert neuroprotective and anti-ischemic effects. Citrus aurantium is rich in flavonoids, which have been shown to possess anti-inflammatory and antioxidant properties that can mitigate ischemic injury. Magnolia officinalis contains lignans, alkaloids, and phenylpropanoid glycosides, compounds known for their anti-inflammatory, anti-apoptotic, and neuroprotective activities. Rheum palmatum is a significant source of anthraquinones, such as emodin, chrysophanol, and rhein, which have demonstrated efficacy in reducing cerebral edema, scavenging free radicals, and inhibiting neuronal apoptosis. Notopterygium incisum primarily provides coumarins, which contribute to anti-inflammatory and vasodilatory effects, improving cerebral blood flow. Comprehensive chemical profiling studies utilizing advanced techniques like UHPLC-FT-ICR-MS/MS and quadrupole time-of-flight tandem mass spectrometry have identified over 130 compounds in SHD, including flavonoids, anthraquinones, coumarins, tannins, alkaloids, and phenylpropanoids, underscoring the complexity and richness of its chemical constituents^[32][33]. Network pharmacology analyses have further revealed that these compounds target multiple signaling pathways relevant to ischemic stroke, such as TNF signaling, AGE-RAGE pathways, and inflammatory cytokine regulation, with key molecular targets including IL-6, AKT1, VEGFA, and APP^[34]. Pharmacodynamic studies in animal models of middle cerebral artery occlusion have confirmed that SHD reduces neurological deficits, cerebral infarct volume, and neuronal necrosis, effects that are attributed to the combined actions of these bioactive components^[34]. Moreover, fingerprinting studies integrating chemical profiles with pharmacodynamic indicators have identified 11 major active compounds that correlate strongly with the therapeutic efficacy of SHD against cerebral ischemia-reperfusion injury, highlighting the synergistic effect of multiple components in the formula ^[5]. Collectively, the main components of SHD—Citrus aurantium, Magnolia officinalis, Rheum palmatum, and Notopterygium incisum—provide a multifaceted pharmacological basis for its neuroprotective and anti-ischemic effects, supporting its clinical use and guiding future research on its mechanisms and quality control.

2.2.2 Chemical Fingerprint Profiles and Component Identification

The chemical fingerprinting of traditional Chinese medicine (TCM) formulations such as Sanhua Decoction (SHD) plays a crucial role in elucidating their pharmacological basis and quality control, especially in complex conditions like cerebral ischemia. Utilizing ultraperformance liquid chromatography (UPLC) technology, researchers have successfully identified 33 common chromatographic peaks across multiple batches of SHD, with 28 chemical constituents explicitly characterized. This comprehensive fingerprinting approach not only ensures batch-to-batch consistency but also facilitates correlation of chemical profiles with therapeutic efficacy against cerebral ischemia-reperfusion injury (CIRI). Gray relational analysis demonstrated that 32 out of 33 peaks exhibited a correlation coefficient exceeding 0.706 with pharmacodynamic outcomes, underscoring the synergistic contribution of multiple components in SHD’s neuroprotective effects^[5].

Among the identified constituents, several classes of bioactive compounds have been highlighted for their potential therapeutic roles in cerebral ischemia. Volatile oils, flavonoids, and anthraquinones are key active ingredients that have shown promising neuroprotective properties. For instance, volatile oils contribute to anti-inflammatory and vasodilatory effects, which are critical in mitigating ischemic brain injury. Flavonoids, widely recognized for their antioxidant and anti-apoptotic activities, can modulate oxidative stress and neuronal survival pathways, thereby reducing infarct size and improving neurological outcomes. Anthraquinones have also been implicated in cerebrovascular protection through their anti-inflammatory and anti-thrombotic properties^[5].

Further chemical profiling studies on related TCM formulations and individual herbs reinforce the significance of these compound classes. For example, ginsenosides derived from Ginseng, a common component in many TCM prescriptions, have been identified as major active compounds targeting brain proteins such as 14-3-3 ζ, which is involved in neuroprotection and cellular signaling pathways relevant to ischemic injury^[35]. Similarly, flavonoids like baicalin and scutellarin, found in formulations such as Guhong injection, have been linked to anti-inflammatory and anti-apoptotic mechanisms, contributing to improved post-stroke recovery^[36]. The identification of these compounds is often achieved through advanced chromatographic and mass spectrometric techniques, including UPLC coupled with high-resolution mass spectrometry (HRMS), which allow for precise qualitative and quantitative analysis of complex herbal matrices ^[37].

Moreover, the metabolic profiling of active ingredients reveals significant biotransformation processes that may influence their bioavailability and therapeutic efficacy. For instance, formononetin and its glucoside ononin undergo hydroxylation, demethylation, and glycosylation during metabolism, producing metabolites with potential cardiovascular and neuroprotective activities^[38]. Such metabolic insights are essential for understanding the pharmacokinetics and pharmacodynamics of TCM components in cerebral ischemia treatment.

In summary, the application of UPLC-based chemical fingerprinting has enabled the identification of a spectrum of bioactive compounds within Sanhua Decoction and related TCM formulas. The key active constituents—volatile oils, flavonoids, and anthraquinones—exert multifaceted neuroprotective effects through antioxidative, anti-inflammatory, anti-apoptotic, and vascular regulatory mechanisms. These findings provide a scientific foundation for the quality control, pharmacological evaluation, and mechanistic understanding of TCM in the management of cerebral ischemia, supporting their integration into evidence-based therapeutic strategies^{[5][35][36][38][37]}.

2.2.3 Synergistic Mechanisms of Multiple Components

The therapeutic efficacy of Sanhua Decoction (SHD) in cerebral ischemia is fundamentally attributed to the synergistic interactions among its multiple bioactive components, which collectively modulate diverse signaling pathways to produce enhanced neuroprotective effects. Chemical profiling of SHD using ultraperformance liquid chromatography (UPLC) has identified 33 common peaks representing 28 characterized chemical constituents, with 11 major active compounds closely associated with anti-cerebral ischemia-reperfusion injury (CIRI) activity. Pharmacodynamic studies in rat models demonstrated that SHD significantly ameliorates neurological deficits, reduces cerebral infarct volume, and improves brain histopathology, with these effects correlating strongly with key indicators such as vascular endothelial growth factor. Gray relational analysis confirmed that nearly all identified components exhibited a high correlation with efficacy, underscoring the integral role of multi-component synergy in SHD’s therapeutic action^[5]. This multi-targeted approach is consistent with the complex pathophysiology of ischemic stroke, which involves oxidative stress, neuroinflammation, apoptosis, and blood-brain barrier disruption. SHD’s components collectively suppress inflammation, protect the blood-brain barrier, promote neural stem cell restoration, inhibit apoptosis and brain edema, scavenge free radicals, and modulate the brain-gut axis, thereby addressing multiple pathological processes simultaneously^[39]. Similar synergistic mechanisms have been observed in other traditional Chinese medicine (TCM) formulations, where combinations of active compounds such as caffeic acid and salvianolic acid B synergistically target endothelial injury and neuroinflammation, key pathological modules in cerebral ischemia, leading to enhanced neuroprotection compared to individual components^[40]. Furthermore, natural products in TCM often modulate intersecting signaling pathways including Nrf2/ARE, NF-κB, PI3K/Akt/mTOR, Bcl-2/Bax, and PARP-1, which collectively regulate oxidative stress, inflammation, apoptosis, and autophagy. This multi-pathway modulation exemplifies the integrated pharmacological and nutritional functions of TCMs, supporting their long-term use in preventing cerebral ischemia-reperfusion injury^[41]. Innovative delivery systems combining multiple active ingredients, such as co-delivery liposomes encapsulating Panax notoginseng saponins and acetylsalicylic acid, further enhance therapeutic synergy by facilitating targeted brain delivery and synchronized pharmacodynamics, thereby amplifying effects on neurotransmitter metabolism and signaling pathways like phosphoinositide 3-kinase/protein kinase C ^[42]. Additionally, synergistic interventions combining traditional compounds with physical therapies, such as curcumin-loaded liposomes combined with transcranial low-intensity ultrasound stimulation, have demonstrated superior modulation of microglial polarization and neuroinflammation via MAPK and NF-κB pathways, highlighting the benefit of multi-modal synergy^[43]. Network pharmacology and molecular docking studies on related herbal prescriptions reveal that multiple phytochemicals act on overlapping targets such as synaptic function, neuronal survival, and vascular tone regulation through G protein-coupled receptors, further illustrating the multi-component, multi-target nature of these formulations. For example, compounds like formononetin, calycosin, ligustrazine, and puerarin exhibit coordinated anti-inflammatory, antioxidant, and antiapoptotic effects, collectively contributing to improved outcomes in cerebral ischemia models^[44][45][46]. Taken together, these findings emphasize that the multi-component synergy in SHD and similar TCM formulas orchestrates a broad spectrum of molecular targets and signaling pathways, which not only enhances the overall therapeutic efficacy but also addresses the multifactorial pathology of cerebral ischemic injury more effectively than single-agent therapies. This multi-dimensional synergy provides a scientific basis for the clinical application and further development of SHD in ischemic stroke treatment.

2.3 Neuroprotective Mechanism of Sanhua Decoction

2.3.1 Antioxidant Effects

The antioxidant properties of Sanhua Decoction play a crucial role in mitigating cerebral ischemia-reperfusion injury by inhibiting the excessive generation of reactive oxygen species (ROS) and reducing oxidative damage to neural tissues. Cerebral ischemia-reperfusion is characterized by an initial deprivation of blood supply followed by the restoration of circulation, which paradoxically exacerbates neuronal injury through oxidative stress mechanisms. During reperfusion, the abrupt reintroduction of oxygen leads to an overproduction of ROS, including superoxide anions, hydrogen peroxide, and hydroxyl radicals, which attack cellular components such as lipids, proteins, and DNA, thereby amplifying neuronal damage and cell death. Sanhua Decoction, a traditional Chinese medicinal formula comprising Citrus aurantium, Magnolia officinalis, rhubarb, and Qiangwu, has been demonstrated to effectively suppress ROS generation, thereby attenuating oxidative stress-induced injury in ischemic brain tissue^[4]. This suppression of ROS not only prevents lipid peroxidation and membrane damage but also curtails the activation of downstream apoptotic pathways, preserving neuronal integrity.

Furthermore, Sanhua Decoction enhances the endogenous antioxidant defense system by activating key antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). These enzymes constitute the primary cellular defense against oxidative stress by catalyzing the conversion of harmful ROS into less reactive molecules such as water and oxygen. Activation of this enzymatic system strengthens the brain’s resilience to oxidative insults during ischemia-reperfusion, thereby protecting neurons from oxidative damage and promoting cell survival. The upregulation of these antioxidant enzymes by Sanhua Decoction is thought to be mediated through modulation of intracellular signaling pathways that regulate oxidative stress responses, although the precise molecular mechanisms remain an active area of research ^[4]. By bolstering the endogenous antioxidant capacity, Sanhua Decoction not only mitigates acute oxidative injury but may also contribute to long-term neuroprotection and functional recovery following cerebral ischemic events.

The combined effects of ROS inhibition and endogenous antioxidant enzyme activation position Sanhua Decoction as a promising therapeutic agent in the management of cerebral ischemia-reperfusion injury. Its natural composition offers a safer alternative to synthetic antioxidants, which often exhibit limited efficacy and potential side effects. The multifaceted antioxidant mechanisms of Sanhua Decoction underscore its potential to interrupt the vicious cycle of oxidative damage and neuronal death that underpins ischemic brain injury. Consequently, further elucidation of these antioxidant pathways and their clinical translation could enhance therapeutic strategies aimed at reducing oxidative stress and improving neurological outcomes in patients suffering from cerebral ischemia-reperfusion injury ^[4].

2.3.2 Anti-inflammatory Effects

Inflammation plays a pivotal role in the pathophysiology of cerebral ischemia, contributing significantly to secondary brain injury through the activation of inflammatory cascades and disruption of the blood-brain barrier (BBB). The anti-inflammatory effects of therapeutic agents, such as Sanhua decoction (三化汤), are crucial for mitigating ischemic brain damage by modulating inflammatory cytokine expression and preserving BBB integrity. One key mechanism involves the regulation of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), which are markedly elevated following ischemic insult and promote leukocyte infiltration and neuronal apoptosis. For instance, granulocyte-colony stimulating factor (G-CSF) has been shown to attenuate neuroinflammation by decreasing TNF-α and IL-1β levels while increasing anti-inflammatory IL-10, mediated via activation of the mTOR/p70S6K signaling pathway, which also reduces neuronal apoptosis in neonatal hypoxic-ischemic brain injury^[47]. Similarly, Sanggenon C, a natural flavonoid, exerts neuroprotection by downregulating RhoA-ROCK signaling, thereby inhibiting inflammatory cytokine production and oxidative stress in cerebral ischemia-reperfusion injury^[48]. These findings underscore the therapeutic potential of compounds that modulate inflammatory mediators to curtail ischemic injury.

Another critical aspect of anti-inflammatory action is the inhibition of inflammasome activation, particularly the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, which amplifies inflammatory responses and promotes neuronal apoptosis. Qingnao Dripping Pills, a traditional Chinese medicine formulation, have demonstrated efficacy in reducing cerebral ischemic injury by suppressing NLRP3 inflammasome expression and downstream cytokines such as IL-1β and IL-18, with concomitant involvement of the nuclear factor-kappa B (NF-κB) pathway ^[49]. Targeting NF-κB, a master regulator of inflammation, is a promising strategy to inhibit the transcription of pro-inflammatory and pro-apoptotic genes following ischemic stroke^[50]. Moreover, microRNA-mediated regulation, such as miR-193b-3p targeting 5-lipoxygenase (5-LOX), can reduce leukotriene synthesis and inflammation, thereby alleviating ischemia-reperfusion injury^[51]. These molecular modulators highlight the complex regulatory networks controlling inflammation in ischemic brain injury.

Preservation of the BBB is essential to prevent infiltration of peripheral inflammatory cells, which exacerbate cerebral edema and tissue damage. The BBB disruption following ischemia is associated with increased permeability that facilitates neutrophil and monocyte transmigration into the brain parenchyma. N-acetylcysteine has been shown to inhibit neutrophil transmigration across the choroid plexus by blocking both endothelial and epithelial checkpoints, thereby reducing inflammation-associated neonatal brain injury without affecting systemic immune responses ^[52]. Additionally, the chemokine receptor Cxcr4 plays a critical role in monocyte infiltration and macrophage localization within ischemic tissue; its deficiency reduces monocyte infiltration and dampens inflammatory gene expression but may worsen outcomes due to altered microglial responses ^[53]. These findings suggest that modulation of immune cell trafficking and BBB integrity is a vital component of anti-inflammatory strategies in cerebral ischemia.

Furthermore, the interplay between oxidative stress and inflammation exacerbates BBB breakdown and neuronal injury. Agents such as Ezetimibe activate the AMPK/Nrf2/TXNIP pathway to reduce oxidative stress and neuroinflammation, subsequently protecting BBB function and reducing infarct size in ischemic models ^[54]. The inhibition of inflammasome activation through autophagy regulation also contributes to maintaining BBB integrity and reducing inflammation-induced neuronal death^[55]. Collectively, these mechanisms emphasize that therapeutic interventions targeting inflammatory cytokine expression, inflammasome activity, and BBB permeability can synergistically attenuate inflammatory cascades, limit immune cell infiltration, and improve neurological outcomes after cerebral ischemia. The anti-inflammatory properties of Sanhua decoction likely involve similar pathways, offering a promising avenue for clinical application in ischemic stroke management.

2.3.3 Regulation of Neurotransmitters and Neuronal Function

The regulation of neurotransmitter balance and neuronal function is a critical therapeutic target in cerebral ischemia, as ischemic injury disrupts synaptic transmission and neuronal viability, leading to neurological deficits. Sanhua Decoction (SHD), a traditional Chinese medicinal formulation, has been shown to exert neuroprotective effects partly through modulating neurotransmitter systems and promoting neuronal survival and regeneration. Network pharmacology and experimental studies reveal that SHD’s active components regulate key signaling pathways involving neurotransmitters such as glutamate and gamma-aminobutyric acid (GABA), as well as neurotrophic factors, which collectively contribute to restoring neurotransmitter homeostasis and synaptic function after ischemic insult ^{[34] [4]}.

Glutamate, the principal excitatory neurotransmitter in the central nervous system, plays a pivotal role in synaptic transmission but becomes neurotoxic when excessively accumulated during ischemia, triggering excitotoxicity via N-methyl-D-aspartate receptors (NMDARs) and resulting in intracellular calcium overload and neuronal death. Recent research highlights the role of L-lactate in modulating glutamate signaling by regulating the glutamine-glutamate cycle between astrocytes and neurons and activating astroglial receptor pathways that facilitate glutamate uptake and neuronal transmission, thus mitigating excitotoxicity and promoting neuronal survival^[15]. This mechanism aligns with SHD’s reported antioxidative and anti-inflammatory effects that may indirectly support neurotransmitter balance and neuronal protection.

GABAergic neurotransmission, the main inhibitory system in the brain, is also implicated in ischemic pathophysiology. Dysregulation of GABA signaling and its chloride cotransporters NKCC1 and KCC2 contributes to excitotoxicity and neuronal damage. Therapeutic strategies targeting these components to restore inhibitory tone have shown promise in reducing neuronal injury and improving functional outcomes ^[56]. SHD and related natural compounds may influence these pathways, as suggested by metabolomics studies showing improved GABA levels and receptor activity following treatment with traditional Chinese medicine formulations^[57].

Moreover, synaptic plasticity and neurotransmitter receptor regulation are essential for neuronal recovery post-ischemia. Studies on acupuncture and pharmacological agents demonstrate that enhancing synaptic structure and function, modulating neurotransmitter release, and promoting receptor expression can facilitate neuronal repair and cognitive improvement ^{[58] [59]}. SHD’s multi-target effects include modulation of inflammatory cytokines and neurotrophic factors such as brain-derived neurotrophic factor (BDNF), which supports neuronal survival and synaptic remodeling^[57]. This neurotrophic support is crucial for mitigating ischemia-induced neuronal apoptosis and promoting regeneration.

Proteomic analyses of active ingredients like muscone, a component related to SHD, reveal regulation of synaptic proteins and neurotransmitter receptor activities, including cholinergic and dopaminergic synapses, underscoring the importance of synaptic connectivity in neuroprotection^[60]. Additionally, modulation of signaling pathways such as AKT, ERK, and CREB by SHD components contributes to anti-apoptotic effects and neuronal function preservation^[61].

Stem cell transplantation studies further highlight the importance of enhancing endogenous neural stem/progenitor cell activity and promoting their differentiation into functional neurons, which is facilitated by improved neurotransmitter milieu and synaptic integration^[62]. This suggests that SHD’s regulation of neurotransmitters and neuronal environment may create a favorable niche for neuronal regeneration.

In summary, the therapeutic efficacy of Sanhua Decoction in cerebral ischemia involves restoring neurotransmitter balance, particularly glutamate and GABA systems, and enhancing neuronal survival and synaptic plasticity through modulation of neurotrophic pathways and anti-apoptotic signaling. These mechanisms collectively contribute to improved neuronal function and reduced neurological deficits, providing a scientific basis for SHD’s clinical application in ischemic stroke management. Further research integrating metabolomics, proteomics, and functional studies will deepen understanding of these complex interactions and optimize therapeutic strategies ^{[34] [15] [56] [57] [60] [62]}.

2.3.4 Reducing Cerebral Edema and Protecting Blood Vessels

The therapeutic effects of Sanhua Decoction (SHD) in reducing cerebral edema and protecting vascular integrity during cerebral ischemia-reperfusion injury have been increasingly elucidated through pharmacological and experimental studies. Cerebral ischemia-reperfusion often leads to the disruption of the blood-brain barrier (BBB), resulting in the formation of brain tissue edema that exacerbates neurological damage. SHD has demonstrated a capacity to inhibit the formation of cerebral edema by maintaining the integrity of the vascular endothelium, thereby preventing excessive fluid leakage into brain parenchyma. This protective effect is attributed to the multi-component nature of SHD, which includes Citrus aurantium, Magnolia officinalis, rhubarb, and Qiangwu, known for their regulatory effects on qi and vascular function. These constituents synergistically modulate oxidative stress and inflammatory responses, which are key contributors to endothelial dysfunction and BBB breakdown in ischemic stroke^[4]. Furthermore, experimental evidence from rat models of cerebral ischemia-reperfusion injury indicates that SHD administration significantly reduces neurological deficits and cerebral infarct volume, effects that correlate with improved histopathological outcomes and reduced brain edema^[5]. A critical mechanism underlying these benefits involves the modulation of vascular endothelial growth factor (VEGF) expression. VEGF plays a dual role in ischemic brain injury; while it promotes angiogenesis and vascular repair, its overexpression can increase vascular permeability and worsen edema. SHD appears to fine-tune VEGF levels, enhancing vascular repair and regeneration while preventing excessive permeability that leads to edema formation. Network pharmacology analysis further supports this mechanism, identifying VEGFA among key molecular targets modulated by SHD’s active compounds, alongside other critical factors such as IL-6, AKT1, and APP, which collectively contribute to neurovascular protection and anti-inflammatory effects ^[34]. The integration of chemical fingerprinting and pharmacodynamic assessments has revealed that multiple bioactive compounds within SHD act synergistically to exert these vascular protective effects, highlighting the importance of the decoction’s multi-targeted approach in maintaining BBB integrity and promoting vascular repair after ischemic insult^[5]. In summary, SHD reduces cerebral edema primarily by inhibiting BBB disruption and modulating VEGF-mediated vascular repair processes, thereby preserving vascular integrity and facilitating recovery from ischemic brain injury. This vascular protection, combined with anti-inflammatory and antioxidant actions, underscores SHD’s potential as a safe and effective therapeutic strategy for mitigating cerebral edema and promoting neurovascular health in ischemic stroke patients.

2.4 Therapeutic Evaluation of Sanhua Decoction in Animal Models of Cerebral Ischemia

2.4.1 Improvement of Neurological Function Scores

In animal models of ischemic stroke, Sanhua Decoction (SHD) has demonstrated a significant ability to improve neurological function scores, indicating its potential neuroprotective effects. A comprehensive study employing a rat middle cerebral artery occlusion (MCAO) model, which closely mimics human ischemic stroke pathology, revealed that SHD administration markedly reduced neurological deficit scores compared to untreated controls ^[34]. This improvement reflects a mitigation of the neurological impairments typically observed after cerebral ischemia, such as motor dysfunction and sensory deficits. The underlying mechanisms involve the modulation of multiple molecular targets and signaling pathways implicated in ischemic injury. Network pharmacology analysis identified key targets including IL-6, APP, AKT1, and VEGFA, which are associated with inflammatory responses, neuronal survival, and angiogenesis. Experimental validation showed that SHD treatment led to increased levels of IL-6 and APP proteins, which may contribute to neuroprotection and repair processes, while simultaneously reducing AKT1 and VEGFA protein levels, potentially limiting pathological angiogenesis and exacerbated inflammation ^[34]. These molecular changes correlate with the observed functional improvements, suggesting that SHD exerts its therapeutic effects through a multi-targeted approach that reduces brain tissue damage and inflammation. Furthermore, the reduction in cerebral infarct size and neuronal necrosis observed in treated animals supports the clinical relevance of the neurological score improvements. Collectively, these findings provide robust evidence that SHD significantly ameliorates neurological deficits in ischemic stroke animal models by modulating critical pathways involved in neuronal injury and repair, thereby highlighting its promise as a neuroprotective agent in ischemic stroke therapy^[34].

2.4.2 Reduction of Cerebral Infarct Volume

Experimental studies have demonstrated that Sanhua Decoction (SHD) exerts a significant neuroprotective effect by markedly reducing cerebral infarct volume in models of cerebral ischemia-reperfusion injury (CIRI). Utilizing a rat CIRI model, researchers employed ultraperformance liquid chromatography (UPLC) to establish fingerprint profiles of SHD batches, correlating chemical constituents with pharmacological efficacy. The pharmacodynamic assessment revealed that SHD treatment led to a substantial decrease in cerebral infarct size, which is a critical indicator of brain tissue damage following ischemic stroke. This reduction in infarct volume was accompanied by improvements in neurological deficit scores and brain histopathology, suggesting that SHD not only limits the extent of ischemic injury but also promotes functional recovery. The study identified 33 common chemical peaks within SHD, with 28 constituents characterized, and found that 32 of these peaks exhibited a strong correlation with therapeutic efficacy, underscoring the multi-component synergistic action of SHD in mitigating ischemic brain injury. Furthermore, orthogonal partial least-squares discriminant analysis pinpointed 11 major active compounds significantly associated with anti-CIRI activity, highlighting specific bioactive molecules responsible for the observed infarct volume reduction. The integration of chemical fingerprinting with pharmacodynamic indices enabled the construction of an “efficacy-integrated fingerprint,” providing a robust and scientific approach to quality evaluation and mechanistic understanding of SHD’s therapeutic effects. Collectively, these findings support that SHD’s ability to significantly shrink cerebral infarct areas stems from its complex chemical composition and multi-target pharmacological actions, offering a promising intervention strategy for ischemic stroke treatment by limiting neuronal death and preserving brain tissue integrity ^[5].

2.4.3 Histopathological Changes

Histopathological examination is a critical method to evaluate the therapeutic effects of Sanhua Decoction (SHD) on cerebral ischemia, as it directly reflects the extent of neuronal injury and the integrity of brain tissue architecture. Studies employing rat models of cerebral ischemia-reperfusion injury (CIRI) and middle cerebral artery occlusion (MCAO) have demonstrated that SHD treatment markedly alleviates neuronal damage and promotes the restoration of brain tissue structure. Specifically, tissue sections from SHD-treated animals reveal a significant reduction in cerebral infarct volume and a notable improvement in histopathological features compared to untreated ischemic controls. Neuronal cells, which typically exhibit swelling, pyknosis, and loss of normal morphology after ischemic insult, show reduced degeneration and preservation of cellular architecture following SHD administration. This histological improvement correlates with enhanced neurological function and decreased infarct size, indicating a protective effect on the ischemic brain tissue ^[5]. The underlying mechanisms are thought to involve the synergistic action of multiple bioactive compounds within SHD, which collectively modulate vascular endothelial growth factor expression and other neuroprotective pathways, thereby promoting angiogenesis and neuronal survival. Furthermore, the restoration of tissue structure observed histologically aligns with SHD’s influence on systemic factors such as gut microbiota composition and short-chain fatty acid (SCFA) profiles, which have been implicated in neuroinflammation and ischemic injury modulation. In MCAO rat models, SHD not only reduces neuronal damage but also normalizes the abundance of beneficial gut bacteria and SCFAs, which may contribute indirectly to improved brain tissue repair and reduced histopathological damage^[63]. Together, these findings underscore that SHD’s therapeutic efficacy is reflected at the histopathological level by diminished neuronal injury and enhanced structural integrity of ischemic brain tissue, supporting its potential as a multi-target treatment strategy for cerebral ischemia.

2.4.4 Comprehensive Multi-Indicator Evaluation Methods

A comprehensive multi-indicator evaluation approach is essential to systematically assess the therapeutic efficacy of Sanhua Decoction (SHD) in cerebral ischemia, integrating neurological function, pathological changes, and biochemical markers to provide a holistic understanding of treatment outcomes. Network pharmacology combined with experimental validation in rat models of middle cerebral artery occlusion (MCAO) has revealed that SHD exerts neuroprotective effects by modulating multiple signaling pathways related to inflammation and neuronal survival, including IL-6, APP, AKT1, and VEGFA pathways. These molecular changes correspond with significant improvements in neurological deficit scores, reduction of cerebral infarct volume, and attenuation of neuronal necrosis, demonstrating the value of combining functional and pathological assessments ^[34]. In vitro studies using oxygen-glucose deprivation/reoxygenation (OGD/R) models in SH-SY5Y cells further support these findings, showing that SHD enhances cell viability and reduces apoptosis, with concomitant downregulation of pro-inflammatory cytokines TNF-α and IL-6 and activation of the PI3K/Akt/CREB1 pathway. These biochemical markers serve as sensitive indicators of SHD’s protective mechanisms at the cellular level^[64].

The integration of multimodal neuroimaging and physiological monitoring techniques offers additional layers of evaluation. For example, advanced imaging modalities such as multimodal MRI and CT perfusion imaging provide quantitative data on cerebral blood flow, tissue oxygenation, and infarct size, enabling correlation of SHD’s therapeutic effects with structural and functional brain changes. Multimodal MRI in animal models has demonstrated early and long-term white matter injury changes, which can be used to assess treatment effects on oligodendrocyte precursor cell proliferation and myelination, critical for functional recovery^[65]. Similarly, CT perfusion imaging in clinical telestroke settings improves diagnostic accuracy for ischemic stroke and could be adapted to monitor SHD treatment responses by evaluating cerebral perfusion parameters ^[66].

In clinical neurocritical care, multimodal neuromonitoring combining intracranial pressure (ICP), cerebral perfusion pressure (CPP), brain tissue oxygenation (PbtO2), cerebral microdialysis, and electrophysiological monitoring provides a dynamic and comprehensive assessment of cerebral pathophysiology. This approach has been shown to improve detection of secondary brain injury and guide individualized treatment strategies. Incorporating such multimodal monitoring in SHD efficacy studies could facilitate real-time evaluation of cerebral oxygenation, metabolism, and neurochemical changes, thereby elucidating the compound’s impact on cerebral ischemia progression and recovery ^[67][68]. For instance, brain tissue oxygenation monitoring identifies hypoxic regions and correlates with neurological outcomes, which may reflect SHD’s neuroprotective effects on cerebral oxygen metabolism^[69][70].

Furthermore, biochemical markers such as inflammatory cytokines (IL-6, TNF-α), apoptotic proteins (CHOP, PERK pathway components), and mitochondrial function parameters (mitochondrial permeability transition pore sensitivity and oxidative phosphorylation capacity) provide mechanistic insights into SHD’s therapeutic actions. Studies have demonstrated that SHD and related compounds modulate these biochemical pathways, reducing oxidative stress, inflammation, and apoptosis, which are key contributors to ischemic brain injury ^[71][72]. The measurement of these markers through Western blot, ELISA, and other molecular assays complements functional and imaging data, enabling a multidimensional evaluation of treatment efficacy.

In summary, a multi-indicator comprehensive evaluation method combining neurological function scoring, histopathological examination, advanced neuroimaging, multimodal neuromonitoring, and biochemical assays provides a robust framework for systematically assessing the efficacy of Sanhua Decoction in cerebral ischemia. This integrative approach not only enhances the understanding of SHD’s multifaceted therapeutic mechanisms but also supports the translation of preclinical findings into clinical applications, ultimately improving patient outcomes in ischemic stroke management.

2.5 Modern Research Progress on Sanhua Decoction in the Treatment of Cerebral Ischemia

2.5.1 Pharmacodynamics Combined with Chemical Fingerprinting for Quality Control

The integration of pharmacodynamics with chemical fingerprinting represents a cutting-edge approach to quality control in traditional Chinese medicine formulations such as Sanhua Decoction (SHD). Utilizing multiple batches of SHD, ultraperformance liquid chromatography (UPLC) was employed to generate detailed fingerprint profiles, identifying 33 common peaks and characterizing 28 chemical constituents. These chemical fingerprints were then correlated with pharmacodynamic indicators derived from a rat model of cerebral ischemia-reperfusion injury (CIRI), including neurological deficit scores, cerebral infarct volume, and brain histopathology improvements. The relationship between chemical constituents and efficacy was quantitatively assessed using gray relational analysis, revealing that 32 out of 33 peaks had a correlation degree greater than 0.706 with therapeutic effects. This strong correlation underscores the synergistic action of multiple components in SHD’s anti-CIRI activity. Furthermore, orthogonal partial least-squares discriminant analysis pinpointed 11 major active compounds significantly associated with efficacy, facilitating the construction of an “efficacy-integrated fingerprint” that combines chemical profiles with pharmacodynamic outcomes. This integrated fingerprint not only reflects the intrinsic quality of SHD but also provides a robust scientific basis for standardizing its quality and ensuring consistency in therapeutic effects across batches ^[5].

Similarly, the spectrum-effect relationship methodology has been effectively applied to other traditional Chinese medicines such as Notoginseng Radix et Rhizoma. High-performance liquid chromatography (HPLC) fingerprints were established for multiple batches, identifying 23 common peaks predominantly composed of saponins. Pharmacodynamic indexes related to blood circulation promotion—such as capillary coagulation time, cerebral ischemic area percentage, cerebral water loss rate, and brain-body index—were measured in rat models of cerebral ischemia-reperfusion. Partial least-squares regression analysis revealed that 17 components positively influenced pharmacodynamic indexes, while 6 had negative effects. This nuanced understanding of the spectrum-effect relationship enables the identification of bioactive constituents critical for efficacy and provides a scientific foundation for clinical rational use and quality control of Notoginseng Radix et Rhizoma. The approach exemplifies how correlating chemical fingerprints with pharmacological activity can guide the development of quality standards that ensure consistent therapeutic outcomes ^[73].

Despite the advances in chemical fingerprinting and pharmacodynamics integration, challenges remain in the clinical translation of herbal medicines such as Angelica gigas Nakai (AGN), a component often found in formulations like SHD. While signature compounds like decursin and decursinol angelate have demonstrated neuroprotective and other bioactivities in animal models of cerebral ischemia, ensuring product consistency and quality control remains a critical hurdle. Variability in extract sourcing, compound content, and pharmacokinetics can affect clinical efficacy. Addressing these challenges requires rigorous chemical fingerprinting combined with pharmacodynamic evaluation to establish standardized quality markers. Such integration not only supports dose optimization and safety assessments but also facilitates regulatory approval and broader clinical application. Future research should focus on refining these integrated quality control methodologies to bridge the gap between traditional herbal use and modern evidence-based medicine^[74].

In summary, the combination of multi-batch chemical fingerprinting with pharmacodynamic efficacy indicators forms a powerful “efficacy-integrated fingerprint” approach. This strategy provides a scientific and efficient framework for elucidating the material basis of complex herbal formulations like Sanhua Decoction, ensuring quality standardization and therapeutic consistency. By linking chemical constituents directly to pharmacological effects, this methodology enhances the reliability and clinical relevance of traditional Chinese medicines in treating cerebral ischemia and related conditions.

2.5.2 Screening and Validation of Key Active Components

The identification and validation of key active components responsible for the anti-cerebral ischemia effects of Sanhua Decoction (SHD) have been systematically approached through integrated statistical and experimental methodologies. Utilizing ultraperformance liquid chromatography (UPLC) fingerprinting combined with pharmacodynamic evaluation in a rat cerebral ischemia-reperfusion injury (CIRI) model, 33 common chromatographic peaks were detected, with 28 chemical constituents characterized. Gray relational analysis revealed that 32 of these peaks exhibited a high correlation (greater than 0.706) with therapeutic efficacy indicators such as neurological deficit scores and cerebral infarct volume, underscoring the synergistic nature of SHD’s multi-component action. Orthogonal partial least squares discriminant analysis further refined this to 11 potential major active compounds closely linked to anti-CIRI activity, forming an “efficacy-integrated fingerprint” that bridges chemical profiles and pharmacological outcomes, providing a robust strategy for quality control and mechanistic elucidation ^[5].

Complementing these chemical analyses, network pharmacology approaches have identified multiple bioactive compounds within SHD that target key proteins implicated in ischemic stroke pathophysiology. From databases such as TCMSP and PubChem, 40 active ingredients and 47 target genes were retrieved, with six key components optimized via network proximity algorithms. Protein-protein interaction (PPI) network analysis highlighted critical targets including IL-6, APP, AKT1, and VEGFA, which are involved in inflammatory responses, neuronal survival, and angiogenesis pathways. Experimental validation using the middle cerebral artery occlusion (MCAO) rat model demonstrated that SHD administration significantly reduced neurological deficits and cerebral infarct size, concomitant with modulation of these protein levels—upregulating IL-6 and APP while downregulating AKT1 and VEGFA—thereby confirming the neuroprotective and anti-inflammatory roles of these key components in vivo^[34].

Further in vivo and in vitro validations of active compounds from other traditional Chinese medicine formulations with similar neuroprotective goals provide additional insights relevant to SHD. For instance, baicalein and rutin, identified through multi-phenotypic zebrafish screening of Guhong injection, exhibited potent anti-thrombotic and neurobehavioral recovery effects in cerebral ischemia models. Chlorogenic acid and gallic acid also showed efficacy in preventing locomotor dysfunction, with molecular docking supporting strong interactions with coagulation factors and inflammatory mediators such as NF-κB p65, indicating multi-targeted neuroprotection ^[75]. Similarly, Huanglian Jiedu Decoction (HLJDD) was found to contain 77 compounds, with 54 linked to cerebral ischemia targets. Core targets such as AKT1, PTGS2, and TLR4 were identified, and the Rap1 signaling pathway emerged as a key mechanistic axis, validated by proteomics and Western blot analyses demonstrating modulation of RAP1A and AKT expression in MCAO rats^[76].

The neuroprotective efficacy of these components is further supported by mechanistic studies demonstrating their roles in mitigating oxidative stress, apoptosis, and inflammation—key pathological processes in cerebral ischemia. For example, novel n-butylphthalide and ligustrazine hybrids showed potent neuroprotection in oxygen-glucose deprivation/reperfusion (OGD/R)-induced neuronal injury models by reducing reactive oxygen species (ROS), stabilizing mitochondrial membrane potential, and regulating apoptosis-related proteins such as Bcl-2, Bax, and caspase 3. In vivo, these compounds significantly decreased ischemia-reperfusion injury and improved cerebral blood flow in rat models, surpassing the effects of the positive control drug NBP ^[77]. Additionally, chiral butylphthalide-ligustrazine hybrids demonstrated enhanced blood-brain barrier permeability and neuroprotection via activation of the Keap1-Nrf2 antioxidant pathway, with in vivo efficacy confirmed in MCAO/reperfusion mice ^[78].

To validate the neuroprotective effects of these active components, a combination of in vitro and in vivo models is employed. In vitro assays such as OGD/R injury models in neuronal cell lines (e.g., SH-SY5Y, HT22) and primary neurons allow for assessment of cell viability, ROS generation, mitochondrial function, and apoptosis markers. In vivo, established ischemic stroke models including MCAO in rodents provide functional and histopathological endpoints, such as neurological deficit scoring, infarct volume measurement, and immunohistochemical analysis of target protein expression. These complementary approaches enable the confirmation of the therapeutic potential and mechanistic pathways of the identified active compounds ^[79][34].

In summary, the integration of statistical screening methods, network pharmacology, chemical fingerprinting, and rigorous experimental validation has successfully identified and confirmed 11 key active components in SHD that contribute to its neuroprotective effects against cerebral ischemia. These components act synergistically to modulate multiple signaling pathways related to inflammation, oxidative stress, apoptosis, and vascular function, thereby offering a multi-targeted therapeutic strategy. The comprehensive validation in both in vitro and in vivo models underscores their potential as effective agents for ischemic stroke treatment and provides a scientific basis for further drug development and clinical application.

2.5.3 New Findings in Mechanism Research

Recent investigations into the mechanisms underlying the therapeutic effects of Sanhua Decoction (SHD) on cerebral ischemia have shed light on its influence on tau protein phosphorylation and endogenous neuroregeneration, highlighting its potential in promoting neural repair. Tau protein hyperphosphorylation is a critical pathological event in various neurodegenerative conditions and is increasingly recognized as a detrimental factor in ischemic brain injury. Abnormal tau phosphorylation disrupts microtubule stability, leading to neuronal dysfunction and death. Emerging studies suggest that SHD may modulate tau phosphorylation pathways, thereby mitigating tau-related neurotoxicity in ischemic contexts. This regulatory effect likely involves the attenuation of kinase activities responsible for tau hyperphosphorylation or the enhancement of phosphatase functions that restore tau to its normal state, although the precise molecular targets remain to be fully elucidated ^[4].

In addition to modulating tau pathology, SHD has been implicated in activating endogenous neuroregenerative mechanisms. Neurogenesis and neural plasticity are vital for recovery after cerebral ischemia, as they contribute to the replacement of lost neurons and the re-establishment of functional neural circuits. The constituents of SHD, such as Citrus aurantium and Magnolia officinalis, have demonstrated properties that may stimulate neural stem cell proliferation and differentiation, as well as promote synaptic remodeling. These effects are possibly mediated through multiple signaling pathways, including antioxidant and anti-inflammatory cascades, which create a favorable microenvironment for neural repair^[5]. Moreover, SHD’s ability to reduce cerebral edema and protect nerve cells further supports its role in preserving the neural substrate necessary for regeneration^[4].

The integration of chemical fingerprinting with pharmacodynamic assessments has identified several bioactive compounds within SHD that correlate with improved neurological outcomes in cerebral ischemia-reperfusion injury models. These compounds appear to exert synergistic effects by targeting vascular endothelial growth factor (VEGF) and other factors critical for angiogenesis and neurogenesis, thereby facilitating tissue repair and functional recovery ^[5]. The multi-targeted nature of SHD aligns well with the complex pathophysiology of cerebral ischemia, which involves oxidative stress, inflammation, excitotoxicity, and apoptotic pathways.

Furthermore, the promotion of endogenous neurogenesis by SHD is supported by evidence of enhanced transition of microglia from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype, which is conducive to neural repair and regeneration. This immunomodulatory effect helps reverse the pro-inflammatory microenvironment commonly observed after ischemic insults, thus facilitating recovery processes^[80]. By mitigating neuroinflammation and supporting cellular homeostasis, SHD creates conditions favorable for the activation and survival of neural progenitor cells.

Collectively, these new mechanistic insights underscore SHD’s potential as a neurorestorative agent in cerebral ischemia. Its multifaceted actions—ranging from tau protein regulation and inflammation modulation to the enhancement of endogenous neurogenesis—highlight its promise in promoting neural repair beyond mere neuroprotection. However, further research is warranted to delineate the precise molecular pathways involved and to translate these findings into clinical applications. Understanding these mechanisms will not only validate SHD’s therapeutic efficacy but also guide the development of novel interventions targeting tau pathology and neuroregeneration in ischemic brain injury.

2.6 Current Clinical Applications and Future Research Directions of Sanhua Decoction

2.6.1 Current Clinical Application Status

Sanhua Decoction has demonstrated a high safety profile and significant therapeutic efficacy in patients suffering from cerebral ischemia. As a traditional Chinese medicinal formulation composed primarily of Citrus aurantium, Magnolia officinalis, rhubarb, and Qiangwu, Sanhua Decoction is widely recognized for its ability to regulate qi and exert multifaceted neuroprotective effects. Clinical studies have consistently reported that this herbal combination not only mitigates the pathological damage caused by cerebral ischemia-reperfusion injury but also promotes neurological recovery through several biological pathways. These include antioxidant activity, anti-inflammatory effects, modulation of neurotransmitter systems, protection of nerve cells, and reduction of cerebral edema. Importantly, the safety of Sanhua Decoction surpasses that of many conventional Western medicines, making it a favorable adjunctive treatment option in clinical settings. Its natural origin and minimal side effects have led to increasing acceptance among clinicians and patients alike. As an auxiliary therapeutic approach, Sanhua Decoction has been shown to improve neurological function recovery by supporting the repair and regeneration of damaged neural tissue. This is particularly valuable in the context of cerebral ischemia, where timely restoration of neurological function is critical for patient outcomes. The accumulating clinical evidence underscores the significance of integrating Sanhua Decoction into standard treatment protocols for cerebral ischemia-reperfusion injury, offering a complementary strategy that enhances efficacy while maintaining safety. Continued research into its mechanisms of action and clinical benefits is essential to optimize its application and establish standardized guidelines for its use in cerebrovascular disease management ^[4].

2.6.2 Existing Problems and Challenges

Despite the promising therapeutic effects of Sanhua Decoction (SHD) in cerebral ischemia-reperfusion injury (CIRI), several critical challenges hinder its broader clinical application and acceptance. One major limitation is the lack of large-scale, multicenter clinical trials that rigorously evaluate the efficacy and safety of SHD in diverse patient populations. Most current studies are preclinical or small-scale experimental investigations, often conducted in animal models such as rats with middle cerebral artery occlusion (MCAO)^{[4] [5]}. While these studies demonstrate significant neuroprotective effects, including reduction of infarct volume, improvement in neurological function, and modulation of inflammatory and oxidative stress pathways, their findings require validation in well-designed randomized controlled trials involving larger cohorts and multiple centers. The absence of such clinical evidence limits the generalizability of SHD’s benefits and its integration into mainstream stroke management protocols.

Furthermore, the complex multicomponent nature of SHD poses challenges in elucidating its precise mechanisms of action. Although network pharmacology and fingerprinting techniques have identified multiple bioactive constituents and pathways—such as anti-inflammatory, antioxidant, and neurotransmitter regulatory effects—these mechanisms remain incompletely understood at molecular and cellular levels ^{[5] [4]}. The synergistic interactions among the various herbal components complicate the identification of key active ingredients responsible for therapeutic efficacy. For example, the relationship between chemical fingerprints and pharmacodynamic effects has been explored, revealing multiple peaks correlated with efficacy, but the exact contribution of each compound and their pharmacokinetics in humans need further clarification^[5]. This gap in mechanistic knowledge impedes optimization of the formulation and targeted drug development.

In addition, standardization of SHD’s components and quality control remain significant hurdles. Herbal medicines often suffer from variability in raw material sources, preparation methods, and batch-to-batch consistency. The establishment of integrated fingerprint profiles combined with efficacy indicators offers a promising approach for quality evaluation, yet widespread implementation of such standards is lacking ^[5]. Without rigorous standardization, reproducibility of clinical outcomes and safety profiles cannot be guaranteed, which is a major concern for regulatory approval and clinical acceptance.

Moreover, challenges related to pharmacokinetics and delivery also exist. The blood-brain barrier (BBB) restricts the brain penetration of many natural compounds, limiting their bioavailability in cerebral ischemia treatment ^[81]. Innovative drug delivery systems, such as bioadhesive nanoparticle clusters for nasal-to-brain transport, have been proposed to overcome these barriers, but their integration with traditional formulations like SHD requires further research ^[82]. Additionally, the complex pathophysiology of cerebral ischemia, involving oxidative stress, inflammation, apoptosis, and necroptosis, demands multi-targeted interventions with well-characterized pharmacodynamics, which SHD potentially offers but needs more mechanistic substantiation^{[83] [11]}.

In summary, while SHD exhibits considerable therapeutic potential against cerebral ischemia, its clinical translation is limited by the scarcity of robust, large-scale clinical data, incomplete understanding of its multifaceted mechanisms, and lack of standardized component quality control. Addressing these challenges through comprehensive clinical trials, advanced mechanistic studies, and stringent quality assurance will be essential to establish SHD as a reliable and effective treatment option in cerebral ischemia management.

2.6.3 Future Research Directions

Future research on Sanhua Decoction (SHD) in the treatment of cerebral ischemia should prioritize a comprehensive elucidation of its molecular mechanisms, focusing on identifying critical therapeutic targets and signaling pathways involved. Current studies have demonstrated that SHD exerts neuroprotective effects through multiple pathways, including antioxidant, anti-inflammatory, and neurotransmitter regulation, as well as by protecting nerve cells and reducing cerebral edema^[4]. However, the precise molecular interactions and the key bioactive compounds responsible remain incompletely understood. Advanced analytical techniques such as ultraperformance liquid chromatography (UPLC) fingerprinting combined with pharmacodynamic assessments have identified multiple active constituents and their correlation with efficacy, revealing the synergistic nature of SHD’s components^[5]. Moreover, emerging research highlights the involvement of complex cellular processes such as autophagy, ferroptosis, and necroptosis in cerebral ischemia-reperfusion injury (CIRI), suggesting that SHD’s effects may intersect with these pathways ^[84][85][83]. To advance the understanding of SHD’s therapeutic mechanisms, future studies should integrate multi-omics approaches and network pharmacology to map out the interactions between SHD components and molecular targets such as Nrf2, HIF-1α, JAK/STAT, and PKC signaling pathways, which have been implicated in ischemic injury modulation ^[86][87][88]. Additionally, the role of exosome-mediated intercellular communication in cerebral ischemia offers a novel dimension for exploring SHD’s molecular effects, especially considering exosomes’ capacity to cross the blood-brain barrier and modulate neuroinflammation and repair^[89]. Identifying these key targets and pathways will not only clarify SHD’s pharmacodynamics but also facilitate the development of targeted interventions and biomarkers for clinical efficacy evaluation.

The clinical translation of Sanhua Decoction for cerebral ischemia treatment necessitates the development of standardized formulations with optimized dosing and administration protocols. Traditional Chinese Medicine (TCM) formulations often face challenges related to variability in herbal composition, extraction methods, and bioavailability, which can affect therapeutic consistency and safety profiles ^[90]. Recent advances in chemical fingerprinting and multi-indicator efficacy integration provide a scientific basis for quality control and standardization of SHD, enabling the identification of critical active ingredients and their concentration ranges correlated with therapeutic outcomes^[5]. Moreover, pharmacokinetic and pharmacodynamic studies are essential to determine optimal dosing schedules that maximize efficacy while minimizing adverse effects. For example, time-dependent metabolomics studies combining TCM and Western medicine have revealed the importance of timing in evaluating treatment efficacy in cerebral ischemia-reperfusion models ^[91]. Additionally, the heterogeneity in cerebral ischemia pathology and individual patient responses underscores the need for personalized dosing strategies, potentially guided by biomarkers or imaging modalities such as computed tomography perfusion imaging to assess cerebral blood flow and ischemic severity ^[92][93]. Optimizing administration routes, including exploring novel delivery systems like nanomaterials or combining SHD with adjunct therapies such as electroacupuncture, may further enhance therapeutic outcomes ^[94][95]. Standardization efforts should also encompass rigorous preclinical and clinical trials to establish safety, efficacy, and reproducibility, which are critical for regulatory approval and broader clinical acceptance.

To elevate Sanhua Decoction from traditional use to a globally recognized therapeutic agent, integration with modern pharmacological technologies is imperative. This modernization entails leveraging cutting-edge methodologies such as systems biology, high-throughput screening, molecular docking, and advanced imaging to dissect SHD’s multi-component and multi-target actions at a molecular level ^[96][97]. For instance, molecular docking studies have elucidated interactions between SHD components and key angiogenic and neurogenic pathways, providing mechanistic insights that support clinical application^[96]. Additionally, the application of machine learning and artificial intelligence in analyzing spectral data and pharmacological profiles can facilitate rapid, noninvasive screening and quality evaluation of SHD preparations, enhancing standardization and reproducibility^[98]. The incorporation of nanotechnology and extracellular vesicle research offers promising avenues for improving SHD delivery and targeting, potentially overcoming blood-brain barrier limitations and enhancing neuroprotective efficacy ^[99]. Furthermore, international collaboration and clinical trials adhering to global regulatory standards will be crucial in validating SHD’s therapeutic potential and safety, fostering its acceptance in Western medicine frameworks ^[41][100]. Emphasizing the food-medicine homology concept inherent in TCM, SHD can also be developed into functional foods or nutraceuticals, broadening its applicability and market reach^[41]. Overall, the fusion of traditional knowledge with modern scientific innovation will be key to advancing Sanhua Decoction’s modernization and internationalization, ultimately benefiting patients worldwide suffering from cerebral ischemia.

Conclusion

In conclusion, the intricate pathophysiology of cerebral ischemia and reperfusion injury presents formidable challenges for effective therapeutic intervention. From an expert perspective, the multifaceted nature of these injuries necessitates treatments that can concurrently target diverse pathological processes. Sanhua Decoction (SHT), with its complex composition and multi-targeted pharmacological actions, emerges as a promising neuroprotective agent. Its demonstrated ability to mitigate cerebral ischemic damage through antioxidant, anti-inflammatory, neurotransmitter modulation, and vascular protective mechanisms underscores the therapeutic potential inherent in traditional multi-component formulations.

The integration of advanced chemical fingerprinting techniques with pharmacodynamic studies has significantly advanced our understanding of SHT’s quality control and mechanistic underpinnings. This scientific approach not only validates the consistency and reliability of SHT preparations but also elucidates the synergistic interactions among its constituents, providing a robust foundation for further pharmacological exploration. Such methodological rigor bridges traditional knowledge with modern biomedical research, enhancing the credibility and acceptance of SHT in contemporary stroke management.

Nevertheless, while preliminary clinical outcomes are encouraging, the current evidence base remains insufficiently comprehensive. The heterogeneity of clinical studies, limited sample sizes, and variability in treatment protocols highlight the necessity for more systematic and large-scale clinical trials. Moreover, deeper mechanistic investigations are imperative to fully delineate the pathways through which SHT exerts its neuroprotective effects, thereby optimizing its therapeutic application and minimizing potential adverse effects.

Balancing the diverse research perspectives—from traditional empirical use to cutting-edge molecular studies—requires a multidisciplinary approach. Collaboration among clinicians, pharmacologists, and traditional medicine experts is essential to harmonize findings and translate them into standardized, evidence-based clinical guidelines. Such integration will facilitate the broader acceptance and rational use of SHT in cerebral ischemia therapy, ultimately improving patient outcomes.

In summary, Sanhua Decoction represents a valuable addition to the armamentarium against cerebral ischemia-reperfusion injury, embodying the advantages of multi-component herbal therapy. Continued efforts in rigorous clinical validation and mechanistic research are crucial to unlocking its full therapeutic potential and establishing it as a standardized treatment modality. This balanced and comprehensive approach will pave the way for innovative, effective, and holistic management strategies in the complex landscape of cerebral ischemic injury.

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