补肾开窍方通过调控ERK1/2和铁死亡治疗糖尿病合并缺血性卒中机制研究OACSTPCD

Objective:To explore and verify the molecular mechanism of Bushen Kaiqiao Formula in the treatment of diabetes mellitus combined with ischemic stroke based on network pharmacology,molecular docking and animal experiments.Methods:48 male SD rats were randomly divided into a sham operation group of 12 and a modeling group of 36,and the modeling group rats were injected with 1%streptozotocin(STZ,50 mg·kg-1)intraperitoneally once on days 1 and 4,respectively.On day 14,the rats were subjected to middle cerebral artery occlusion(MCAO)to establish a rat model of diabetes mellitus combined with ischemic stroke.The modeling group rats were randomly divided into STZ+MCAO model group,high-dose group of Bushen Kaiqiao Formula(18.7 g·kg-1),and low-dose group of Bushen Kaiqiao Formula(9.4 g·kg-1).After the diabetes mellitus model was successfully established on day7,the rats were given the decoction of Bushen Kaiqiao Formula once a day by gavage for 8 consecutive days.The cerebral ischemic volume in rats was measured with the TTC method;the degree of neurological deficit was detected with the Garcia JH method;and the brain wa-ter content was determined by using the wet-dry weight method to clarify the effect of the Bushen Kaiqiao Formula in the treatment of diabetes mellitus combined with ischemic stroke.Then,the active components and targets of the Bushen Kaiqiao Formula were screened from the TCMSP,HERB,GeneCards,OMIM and Disgenet databases,and a component-target-pathway association network was con-structed using Cytoscape 3.8.0 software.The STRING database was used for protein-protein interaction(PPI)network analysis,the Metascape database was used for Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis,and molecular docking software was used to verify the the active ingredients as well as the core targets.Next,immu-nofluorescence staining was used to detect the expression of p-ERK1/2,and Western blot was used to detect the expression of p-ERK1/2,ERK1/2,GPX4 and SLC7A11 in the ischemic cortex of rats.Results:Animal experiment:compared with that of the sham op-eration group,the blood glucose levels,cerebral ischemic volume and brain water content of model group rats was significantly increased(P<0.01),while the neurological function scores were significantly lower(P<0.01).The results of high-dose and low-dose groups of Bushen Kaiqiao Formula were opposite to those above.A total of 436 active ingredients were screened with network pharmacol-ogy,and 219 potential targets of Bushen Kaiqiao Formula were predicted.PPI analysis showed that RELA,STAT3,ERK1,ERK2 and JUN were the core targets.GO and KEGG analysis showed that the mechanism of Bushen Kaiqiao Formula may be closely related to MAPK signaling pathway.Molecular docking results showed that ERK1 and ERK2 were well bound to the active ingredients.Verification experiment:compared with that of the sham operation group,the expression of p-ERK1/2 in the ischemic cortex of the STZ+MCAO group of rats was significantly increased(P<0.01),while the expression of GPX4 and SLC7A11 proteins was significantly decreased(P<0.01).Compared with that of the STZ+MCAO group,the p-ERK1/2 expression was significantly decreased(P<0.01),while the expression of GPX4 and SLC7A11 proteins was increased(P<0.05-0.01)of the high-dose and low-dose Bushen Kaiqiao Formula groups.Conclusion:Bushen Kaiqiao Formula has neuroprotective effects on rats model with diabetes mellitus and ischemic stroke,and its mechanism may be closely related to the regulation of ERK1/2 and the inhibition of ferroptosis.