Ischemic stroke is one of the leading causes of death and disability worldwide. Studies have shown that the injury mechanism of ischemic stroke includes apoptosis, necrosis, inflammation, immune regulation, oxidative stress, etc[17]. However, no effective treatment has been found to prevent damage to the brain in such cases except tissue plasminogen activator. This single compound or single target drug has limited effect on ischemic stroke. Therefore, a promising treatment approach for ischemic stroke is utilization of multiple-component agents with multiple targets[18].
Buyang huanwu decoction has the functions of supplementing qi, activating blood circulation and clearing collaterals. As a classic prescription for treating qi deficiency and blood stasis, it has been attached great importance by doctors of all generations. Traditional Chinese medicine cures disease through multi-approach, multi-target and integerconcept. It has unique advantages for thetreatment of complex diseases, but because of the complex composition of traditional Chinese medicine compound to make its modernization process slow. Furthermore, network pharmacology has provided a new way for exploring pharmacological mechanisms of TCMs. many scholars have been made an effort to apply network pharmacology to explore the complex of ingredients, targets, and mechanisms of herbal formulas[19].
In this study, network pharmacology was used to investigate the pharmacological mechanism of BYHWD related to cerebral ischemia, which improved the accuracy of targeted prediction to some extent. Network pharmacological analysis of BYHWD identified seven herbs, 42 compounds, and 79 target gene-regulated major pathways related to cerebral ischemia. In particular, the 16 compounds (baicalein, beta-carotene, Baicalin, kaempferol, luteolin, quercetin, hydroxysafflor yellow A, isorhamnetin, Bifendate, formononetin,Calycosin, AstragalosideIV, Stigmasterol, sitosterol, Z-ligustilide, Dihydrocapsaicin) were linked to more than five genes. A number of studies have reported that baicalein has potent neuroprote ctive properties under in vitro as well as in vivo systems[20]. Beta-carotene serve as an antioxidant, inhibiting free radical production. it may regulate cell growth anddeath[21]. Baicalin inhibited microglial cell activation and reduced inflammation, oxidative damage, and brain edema[22]. Additionally, Kaempferol has strong anti-inflammatory and antioxidant effects. Numerous scientific reports showed that it has beneficial role on different inflammatory-related diseases such as cardiovascular, and neurode generative diseases. Luteolin suppresses inflammation in the brain tissues and regulates different cell signaling pathways[23]. Quercetin has antioxidant stress and neuroprotective effects[24]. moreover, Hydroxysafflor yellow A protects brain microvascular endothelial cells (BMECs) against oxygen glucose deprivation/reoxygenation(OGD/R) induced injury by inhibiting autophagy via the Class IPI3K/Akt/mTOR signaling pathway[25]. Treatment of experimental stroke mice with isorhamnetin attenuated cerebral edema, improved blood-brain barrier function, and upregulated gene expression of tight junction proteins including occludin, ZO-1, and claudin-5[26]. Furthermore, calycosin protected the brain against ischemic injury through inhibiting calpain activation[27]. Dihydrocapsaicin treated cerebral I/R rats attenuate cerebral and BBB damage through inhibition of oxidative stress and inflammatory pathways[28]. In a word, these findings suggest that the main components of BYHWD are effective for treating cerebral ischemia.
Genes with high degrees of differential articulation were actually acquired depending on PPI system analysis, IL6, TNF, VEGFA, HIF1A, MAPK1, MAPK3, JUN, STAT3, IL1B and IL10 were recognized as center genes. Interleukin-6 (IL-6) is a multi-functional cytokine with a wide range of biological activities such as regulation of the immune system, generation of acute phase reactions[29]. Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor that is crucially involved in neurovascular remodeling in the ischemic brain. VEGF promotes angiogenesis, protects ischemic neurons from injury, has potent anti-inflammatory actions, and promotes brain plasticity[30]. HIF1A regulates the expression of genes encoding molecules that participate in erythropoiesis, cell proliferation, and energy metabolism, and is closely associated with the regulation of neuronal survival in ischemia[31, 32]. HIF1A can up-regulate the gene expression of proteins related to the vascular system and promote the angiogenesis of VEGF and its receptors to increase blood flow and reduce ischemic injury[33]. TNF is a typical cytokine involved in the acute phase of systemic inflammation and is closely related to the severity of cerebral ischemia[34]. IL-10 is a potent anti-inflammatory mediator, and, if overexpressed, can suppress neuronal degeneration[35].
GO and KEGG pathway analysis to better comprehend the interaction and action pathway of target genes. In results, GO analyses revealed that target genes were majorly associated with the biological processes of positive regulation of transcription from RNA polymerase II promoter, inflammatory response, transcription, DNA-templated, negative regulation of apoptotic process, positive regulation of transcription, DNA-templated, angiogenesis, response to hypoxia, response to hypoxia. The enriched molecular function ontologies were dominated through DNA binding transcription factor activity, sequence-specific DNA binding,sequence-specific DNA binding, identical protein binding, heparin binding and heme binding so on. KEGG pathway analysis were primarily pertaining to TNF, IL-17, Apoptosis, PI3K-Akt, Toll-like receptor, MAPK, NF-kappa B and HIF-1 signaling pathway. Our results suggest that these pathways might interact to exert their combined effects against cerebral ischemia, which could explain the apparent effects of BYHWD.
Cerebral ischemia results in decreased cerebral blood flow, decreased oxygen supply, induced activation of the HIF-1 signaling pathway, and upregulated HIF1A expression, which contributes to the recovery of blood circulation in the penumbra after cerebral ischemia and the transport of glucose, and mediates hypoxia adaptation after hypoxia, and plays a protective role in promoting cell survival and inhibiting apoptosis in brain tissues[36]. MAPK and PI3K/AKT signaling pathways are the main signaling pathways related to apoptosis after cerebral ischemia. Currently, it is believed that MAPK signaling plays a dual role in the process of cell apoptosis, while PI3K/Akt signaling is an important cell survival signaling pathway. Multiple neurotrophic factors inhibit apoptosis by activating the PI3K/Akt signaling pathway, thus playing a protective role in the brain[37]. The PI3K/Akt signaling pathway is involved in the regulation of various intracellular signaling pathways and plays a key role in promoting cell survival and proliferation, anti-apoptosis, regulating glucose metabolism and protein synthesis[38]. Toll-like receptors (TLRS), as inflammatory signal receptors, play an important role in the inflammatory cascade reaction triggered by cerebral ischemia and are closely related to the expression of various inflammatory mediators. Therefore, it is of great significance to intervene the TLRs signaling pathway in the initial stage of inflammatory response to effectively reduce the inflammatory injury in the acute stage of ischemic stroke[39].
In addition, we verified three of the key targets (IL6, VEGFA and HIF1A) that predicted in the network by using western blot analysis. Therefore, the pharmacological mechanism of BYHWD in the treatment of cerebral ischemia can be more clearly verified. The results showed that compared with the control group (no treatment), BYHWD could significantly inhibit the expression of IL6 and increased the expression of HIF1A and VEGFA.