Mechanism of Hou's Black Powder in treating cerebral ischemia reperfusion injury based on lipid and atherosclerosis pathways
Objective To verify and analyze the ingredients,targets,and signaling pathways of Hou's Black Powder(HBP)from Jin Gui Yao Lve(Essentials from the Golden Cabinet)in treating cerebral ischemia reperfusion injury(CIRI)by network pharmacology and animal experiments,and to investigate its mechanism of action through lipid and atherosclerosis pathways.Methods The intersection targets of HBP and CIRI were obtained from TCM Integrated Pharmacology Research Platform(TCMIP)V2.0,GeneCards,and OMIM disease databases,and their protein-protein interaction(PPI)network was constructed using the STRING website.Cytoscape was used to screen the core targets and ingredients and to construct an ingredient-target network.R language was used for GO and KEGG enrichment analyses,and AutoDock Vina was employed to verify the docking ability between ingredients and targets.In animal experiments,sixty SPF-grade SD rats were randomized into sham-operated group,model group,western medicine group,and low-,medium-,and high-dose HBP groups.A MCAO/R rat model was prepared by suture-occluded method.Seven days after modeling,the rat neurological function was evaluated by modified neurological severity score(mNSS).The volume of cerebral infarction was determined by TTC staining and the pathological changes of the brain tissue were observed by HE staining.The serum content of low density lipoprotein cholesterol(LDL-C),high density lipoprotein cholesterol(HDL-C),total cholesterol(TC),and triglycerides(TG)was measured by fully automatic biochemical analyzer.The serum levels of interleukin 1β(IL-1β),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)were examined by ELISA.The protein and mRNA expressions of HSP90AA1,NF-κB1,and SRC in the brain tissue were measured by immunohistochemistry and RT-qPCR.Results A total of 508 active ingredients,549 drug targets and 1,592 CIRI disease targets were screened from HBP.A total of 153 intersection targets were found,with HSP90AA1,SRC,and NF-κB1 identified as the core targets.These core targets exhibited a strong binding affinity with core ingredients such as palmitic acid,adenosine triphosphate,clionasterol,and adenosine.The biological processes of GO enrichment analysis mainly involved the response to exogenous stimuli,location maintenance,and inflammatory response regulation.KEGG pathway analysis was primarily enriched in lipid and atherosclerosis pathways,cAMP signal pathway,and arachidonic acid metabolism.Compared with the model group,the mNSS score,cerebral infarction rate,serum content of IL-1β,IL-6,and TNF-α,as well as HSP90AA1 protein expression level and mRNA expression levels of HSP0AA1,NF-κB1,and SRC in the brain issue of each dose HBP group decreased(P<0.05,P<0.01);the levels of LDL-C,TC,and TG decreased in high-dose HBP group(P<0.05,P<0.01),while the HDL-C level increased(P<0.05);the TG level in medium-dose HBP group decreased(P<0.01);the protein expression levels of NF-κB1 and SRC decreased in western medicine group and medium-and high-dose HBP groups(P<0.05,P<0.01).Compared with low-dose HBP group,the mNSS scores,cerebral infarction rates,serum levels of TG,serum content of IL-1β,IL-6,and TNF-α,and mRNA expression levels of NF-κB1 in the brain tissue of the medium-and high-dose HBP groups decreased significantly(P<0.01);the protein expression levels of HSP90AA1,NF-κB1,and SRC and the mRNA expression levels of HSP90AA1 and NF-κB1 in the brain tissue of high-dose HBP group decreased(P<0.05,P<0.01).Compared with medium-dose HBP group,the high-dose HBP group showed reduced mNSS score,cerebral infarction rate,and serum content of IL-1β,IL-6,and TNF-α,as well as decreased expression levels of HSP90AA1 protein and NF-κB1 mRNA in the brain tissue(P<0.05,P<0.01).Conclusion Hou's Black Powder treats CIRI with a multi-ingredient,multi-target and multi-pathway approach.A key mechanism of its therapeutic effect involves suppressing the mRNA and protein expressions of the core targets HSP90AA1,NF-κB1,and SRC,as well as regulating the lipid and atherosclerosis pathways.
Hou's Black Powdercerebral ischemia reperfusion injurylipid and atherosclerosis pathwaysnetwork pharmacologyanimal experiment
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