Mechanism Study of Guishen Decoction in Treating Ischemic Stroke Based on Network Pharmacology and Molecular Docking

Stroke, which also known as cerebrovascular accident or brain vascular accident, is regarded as an acute cerebrovascular disease evoked by the rhexis or blockage of blood vessels in the brain, resulting in damage to brain tissue. It is the primary cause of disability among adults and the second leading cause of death in humans. Stroke includes two capital categories: ischemic stroke and hemorrhagic stroke, with ischemic stroke being the most prevalent (Wang et al., 2022). Ischemic stroke is individualized by high disability, mortality, and recurrence rates. In accordancce with survey statistics, the onset age of strokes is decreasing year by year (Merel S. Ekker et al., 2023), and it has now become one of the material agents of death among habitants in China (Wen-Jun Tu et al., 2023).

The temporary or permanent occlusion of cerebral blood vessels can lead to cerebral tissue ischemia, causing local brain tissue and functional damage. The extent of contaminate is associated to the duration of ischemia and reliquus blood flow: short-term incomplete ischemia only induces reversible impairment, while long-term complete or severe ischemia can reflect on cerebral ischemic infarction. Ischemic stroke causes neuronal damage through multiple pathways and targets, including apoptosis, transformation of astrocytes and neurons mediated by neural precursor cells (NPCs), and loss of neurotrophic factors (NT). In the ischemic core, neuronal cell bodies and axons disappear, and glial cells exhibit cytoplasmic swelling and nucleolar loss; in the ischemic border zone, there is disintegration of endoplasmic reticulum ribosomes and mitochondria. At this time, microglia are activated, exhibiting amoeboid-like swelling, concomitted with the generation of inflammatory substances such as cytokines, chemokines, and oxygen free radicals. Additionally, blood-brain barrier (BBB) permeability increases, allowing immune cells, like leukocytes, monocytes, and macrophages, to infiltrate the ischemic lesion, causing neurotoxicity and damaging NT (Chuan Qin et al., 2022). Cerebral tissue suffers from hypoxia and glucose deprivation due to interrupted blood flow, resulting in inadequate ATP synthesis, energy imbalance, and disturbances in ion and acid-base balance. Upon reperfusion of cerebral blood vessels, the brain tissue undergoes repeated injury, presenting pathological changes such as mitochondrial swelling, cristae rupture, endoplasmic reticulum swelling, and increased intercellular gaps of vascular endothelial cells, leading to irreversible damage (Gao et al., 2022). The concrete pathological mechanisms occurred in the cerebral ischemia reperfusion injury (CIRI), for example, the release of oxygen free radicals, calcium ion overload (Han et al., 2022), excitatory amino acids, inflammatory responses (Jurcau & Simion, 2021; Zhang et al., 2019), apoptosis (Shi et al., 2022), and depletion of high-energy phosphates (X. Li et al., 2021). These pathological changes cause cerebral tissue necrosis, structural damage, neuronal death, and local neurological dysfunction (Y. Zhao et al., 2022).

The existence of a time window for CIRI poses a dominating challenge in the diagnosis and cure process of ischemic stroke. With prolonged cerebral ischemia time, the difficulty of using drugs or surgical methods to restore cerebral blood flow increases, and the severity of CIRI worsens. Currently, the only evidence-based treatment drug accepted for treating acute ischemic stroke is recombinant tissue-type plasminogen activator (r-tPA) (Powers et al., 2019). However, this drug cannot alleviate reperfusion injury after cerebral ischemia. Ischemic stroke is among the primary element conditions threatening human life and causing disability and cognitive impairment (Benjamin et al., 2017).

TCM has build up abundant experience in the obviattion and iateria of ischemic stroke. In “Synopsis Golden Chamber”, there is a method of “pounding the juice of sedge and pouring it into the ears, blowing the powder of honey locust into the nose” to treat blindness due to stroke. Since the Jin and Yuan Dynasties, with the gradual rise of Mingmen theory, the theory of “kidney brain correlation” has also become one of the main guiding ideas for the prevention and treatment of ischemic stroke. “Huangdi Neijing” states that the kidneys and brain are connected through the brain marrow. “Huayang Zangxiang Yuanbian” agrees with this statement and believes that the brain marrow is the main material basis for the brain to exert its physiological functions: “Within the human body, spanning from the waist to the spine, governing the limbs, connecting the joints, lies the pivotal point of the soul and the essence of life - the brain and spinal cord.” Zhang Xichun's “Yixue Zhongxi Canxi Lu” describes in detail the relationship between the brain and the kidneys under the theory of TCM: the kidneys contain innate essence, and are related to The acquired essence and Qi in the kidneys are combined into kidney essence. The kidney essence goes up the spine and perfuses into the brain. The clear part is transformed into the marrow. It is called the spinal cord in the spine. It flows into the bones and is called the bone marrow. When it enters the brain, it is called the cerebral marrow. The kidneys and brain communicate with each other through the transformation of the essence, and are connected by the bladder meridian, governor meridian, and kidney meridian, forming a “kidney-brain mutual support” relationship that is interconnected in terms of material relationships, meridians, and organ functions. Therefore, during the onset of ischemic stroke, the physiological activities occuring in the brain are severely damaged, and the method of nourishing the kidneys and replenishing essence can be used to indirectly replenish the brain marrow, abundant the prognosis methods of ischemic stroke patients and improve the prognosis of ischemic stroke patients. In modern medical research, cerebral blood flow is interrupted during cerebral ischemia injury, glucose and oxygen in the brain are depleted, ATP synthesis is interrupted, and energy is insufficient (Aleksandra Steliga et al., 2020), ion homeostasis is damaged, acid-base balance is disordered, causing brain neuroinflammation, nerve cell death, and mitochondrial dysfunction; in the core area of ischemia, brain neuron cell bodies and axons disappear (Derek Barthels & Hiranmoy Das, 2020), cytoplasmic swelling of neurons and glial cells, disappearance of nucleoli, disintegration of endoplasmic ribosomes and Nissl bodies in the ischemic penumbra area, morphology of microglia and astrocytes Changes in (Chuan Qin et al., 2022), damage to brain endothelial cells (Lena Bourhy et al., 2022), and reduction in the number of neurons and neural stem cells (Ji Ma et al., 2017), reducing the content of brain-derived neurotrophic factor (Karantali et al., 2021), corresponding to the insufficient permeability of Qi and blood in the brain, malnutrition of brain marrow, and ineffectiveness of Yuanshen in the theory of TCM, and the hypothalamus-pituitary-adrenal axis can regulate Ca²⁺ concentration and Ca²⁺ channel activity (Maria Calvo-Rodriguez et al., 2020), inhibit neuroinflammatory response (Ji Ma et al., 2017; Lena Bourhy et al., 2022; S.K. Kritas et al., 2014), reduces oxygen free radical damage (Sebastian Zimmer et al., 2021), reduces neuronal and hippocampal The expression of pro-apoptotic proteins (An-rong Wang et al., 2021) protects brain tissue, which provides modern medical basis for the “kidney brain correlation” theory of TCM.

Professor Yuan Zhenyi of our school prepared the “Guishen Decoction”, which is composed of three TCMs: mulberry, yam and dodder seed. It is very effective in treating kidney Qi deficiency. The kidneys are the foundation of innateness. Kidney yang transpires and transforms into Qi, guiding the passage of water; kidney yin communicates with the liver and relieves the liver's strong Qi; kidney essence inherits the innate nature and is nourished by acquired nutrients. In Guishen Decoction, mulberry nourishes yin and blood, and also benefits moisture; yam nourishes the lungs, spleen, and kidneys, which can nourish the kidneys and astringent essence, improve intelligence and calm the mind; dodder seeds nourish the liver and kidneys, warm the kidneys, solidify essence, and reduce urination. The three medicines work together to nourish kidney Qi, kidney essence, kidney yin and kidney yang, diuretic, astringent essence, soothe the nerves and nourish blood. The properties of the three medicines are nourishing and greasy, which is consistent with the nature of the kidney as a water organ. It nourishes the kidneys, replenishes essence and nourishes vitality, and then nourish the brain. At present, the research on the treatment of stroke with kidney is gradually deepening and extensive. Ling et al (Ling et al., 2022) used Ruyi Zhenbao Pills to treat stroke, evaluation found that the patient's motor and sensory functions were restored; Li et al (L. Li et al., 2023) verified that Zuogui Pill's treatment for ischemic stroke by means of network pharmacology and in vitro experimental verification therapeutic effect of ischemic stroke; Tang et al. (Tang et al., 2012) found that kidney essence deficiency syndrome is the most common among the TCM syndrome types in patients with post-stroke dementia, and is commonly used in clinical practice. Among the kidney-based prescriptions for treating ischemic stroke, Zuogui Pills, Liuwei Dihuang Decoction, Ruyi Zhenbao Dan, etc. all contain the shadow of Guishen Decoction. For example, Zuogui Pills contain yam and dodder seeds, while wolfberry and cooked rehmannia root are included, deer antler glue has the same effect as mulberry; Liuwei Dihuang Pills contain yam, while Rehmannia glutinosa, wine cornus and Alisma have the same effect as dodder and mulberry; Ruyi Zhenbao Dan contains long pepper, clove, galangal, fragrant celery, Cinnamon and cardamom dispel wind and cold, warm and unblock the life vessels, and have the same effect as Guishen Decoction. It can be seen that the formulation idea of Guishen Decoction has important implications in the clinical cure process of ischemic stroke building upon the “kidney brain correlation” theory. Wide range of applications. In modern medical research, mulberry could reverse the disorder of redox system in brain conduced by rotenone (Saji et al., 2016), chronic stress (Nade & Yadav, 2010), halopeidol (Nade et al., 2010), D-galactose (Turgut et al., 2016), Schistosoma mansoni infection (Bauomy, 2014), aging (Shih et al., 2010), glyphosate (Rebai et al., 2017), Alzheimer (El-Baz et al., 2016) and cholinotoxins (Wattanathorn et al., 2012), and increase the levels of antioxidant enzymes in the body including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GRd), and the contents of reduced glutathione (GSH), decrease the levels of lactate dehydrogenase (LDH) activity, nitrite (NO), and malonyldialdehyde (MDA) levels, which are formed by the oxidation. Sheeba S. S. et al (Samuel et al., 2016) Comparison of mulberry leaf extract MLE-AR-14 and the effect of resveratrol on CIRI mice. Both mulberry extract and resveratrol can remarkably deplete the cerebral infarction mice’s volume, and mulberry extract can reduce malondialdehyde (a kind of malondialdehyde) in serum, increase glutathione (an endogenous antioxidant) level, and exert a protective effect on nerve cells by suppressing the activity of oxygen free radicals. Studies by Kang T. H. (Kang et al., 2006), Kaewkaen P. (Kaewkaen et al., 2012) and others have proven mulberry exerts protective effects on the brain tissue of stroke rats (Tam et al., 2021). In the study of Hou et al. (Hou et al., 2023), Cuscutae can increase brain Nrf-2 and inflammatory responses caused by oxidative stress. HO-1 protein levels, reduce neuroinflammatory response in mice, and inhibit the activation of microglia and astrocytes; Ju et al (Ju et al., 2019) found that dodder it can inhibit synaptic loss in the hippocampus of Alzheimer's mice and inhibit neuronal apoptosis by inhibiting Caspase-3 activation, thereby exerting neuronal protection. Although there are few studies on yam and ischemic stroke, current research shows that yam has immunomodulatory effects and can effectively inhibit the inflammatory response (Kim et al., 2012; Lee et al., 2002).

With the advancement of bioinformatics technology and the improvement of related databases, network pharmacology technology has become one of the main tools in drug research to reveal the relationship between drugs, targets, pathways and related diseases (S. Li & Zhang, 2014), molecular docking technology has also become an effective means to verify the potential association between active ingredients and target genes analyzed by network pharmacology (C. Liu et al., 2021). Therefore, this study uses Professor Yuan Zhenyi’s self-prepared “Guishen Decoction” to conduct network pharmacology research on ischemic stroke, and conducts in-depth research on the molecular correlation between nourishing kidney and filling essence recipe and ischemic stroke through network pharmacology methods, explore the modern medical connotation of the “kidney brain correlation” theory of TCM, broaden strategies for stroke prevention and treatment using TCM, and further carry out research on the “kidney essence” mechanism.

In this study, the author plans to employ network pharmacology approaches to study the bioactive compound targets and signaling pathways of the pharmaceutical ingredients of Guishen Decoction to provide more complete TCM ideas for the ischemic stroke prevention and management. In this study, the author examined the intricate interplay among diseases, medications, and targets utilizing diverse databases and system predictions. The focus was on elucidating the pivotal targets and potential mechanisms of Guishen Decoction in managing ischemic stroke. Molecular docking technology was employed to validate the potential target of Guishen Decoction for ischemic stroke. The entire study is illustrated in figure 1..