Yanghua Decoction In Antagonizing Cerebral Ischemia/Reperfusion Injury Via PI3K/AKT1 Pathway Based On The Theory Of "Yang Transforming Qi And Yin Forming"

Objective:The theoretical connotation of"Yang transforming Qi and Yin forming"（YTQ）was discussed,and the formula of Yanghua decoction（YHT）was formed on this basis.To explore the antagonistic mechanism of YHT against cerebral ischemia/reperfusion injury through p-p70S6K mediated PI3K/AKT1 pathway from the level of post-translational modification,laying a solid foundation for the clinical application of YHT.Methods:1.In this study,literature research and literature retrieval were used to collect relevant materials,sort out the relevant contents of Traditional Chinese medicine classics such as Huangdi Neijing,and explore the connotation of YTQ theory and its relationship with the ontology structure theory of Yin and Yang.On this basis,the content of stroke YTQ theory was discussed to provide theoretical guidance for the development of this study.Understand the current research trends and shortcomings,and provide research ideas for the development of this study.2.A total of 70 SPF healthy male SD rats were selected to establish middle cerebral artery occlusion/reperfusion（MCAO/R）Model,and 60successfully modeled SD rats were randomly divided into Sham operation group（Sham）,Model group（Model）,and Model+YHT group,with 20 rats in each group.Another 10 normal rats were set as Blank group.YHT group was given 14 g·kg^-1 YHT twice a day for 72h after 1h MCAO/R.Sham group and Model group were given equal volume of normal saline by gavage.Then the following tests are performed:Immunohistochemical staining（IHC）was used to detect the expression of p-AKT.Protein expression levels of p-p70S6K,p-AKT1,p70S6K,AKT1 and Cleaved caspase-3 in rat brain tissues were detected by western blot.3.Based on the phosphorylated proteome,we carried out multidimensional in-depth analysis,including the differentially expressed phosphorylated peptides and phosphorylated sites of each group,subcellular localization analysis,GO and KEGG functional enrichment analysis of differentially expressed phosphorylated peptides,weighted gene co-expression network analysis（WGCNA）analysis of co-expression clusters,clustering trend pattern analysis.Motif analysis of phosphorylated proteins,kinase activity presumption,integrated network analysis and screening of key phosphorylated proteins.Results:1.The neurological deficit score showed that the neurological deficit score of YHT group was significantly lower than that of model group（P<0.001）;The neurological deficit score of model group was significantly higher than that of sham group（P<0.001）.There was no obvious cerebral infarction in model group and model group;Compared with sham group,the infarct volume of model group was significantly higher（P<0.001）;Compared with model group,YHT significantly reduced the infarct volume of brain tissue after intervention（P<0.01）.HE staining showed that compared with sham group,the ischemic brain tissue of model group showed disordered cell arrangement,obvious necrosis of nerve cells,pyknosis and deep staining of nucleus;Compared with the model group,the nucleus of ischemic brain tissue in YHT group was clearer and the damage of nerve cells was reduced.Immunohistochemical results showed that p-AKT was expressed in nucleus and cytoplasm;The expression of p-AKT protein in sham group was significantly higher than that in model group,and the expression of p-AKT protein in YHT group was significantly higher than that in model group.2.The results of phosphoproteomics showed that a total of 9002phosphorylated peptides,3254 proteins and 3041 phosphorylated proteins were identified.Compared with sham group,1188 phosphorylated peptides were differentially expressed in model group,662 were up-regulated and 526were down regulated;Compared with model group,there were 1951differentially expressed phosphorylated peptides,979 up-regulated and 972down regulated in YHT group.Subcellular localization showed that most phosphorylation events occurred in cytoplasm and nucleus.The analysis of phosphorylation modification sites showed that the relative abundance of serine（S）residue phosphorylation modification accounted for 85.8%,threonine（T）residue phosphorylation modification accounted for 13.5%,and tyrosine（Y）residue phosphorylation modification accounted for 0.8%.The biological function of YHT after intervention involves synaptic protein localization,regulation of presynaptic cytoplasmic calcium concentration,positive regulation of GTPase activity,positive regulation of excitatory postsynaptic potential,etc;The regulated signals include calcium signaling pathway,c AMP signaling pathway,Ras signaling pathway,glutamatergic synapse,erb B signaling pathway,m TOR signaling pathway,MAPK signaling pathway,PI3K/AKT signaling pathway and cholinergic synapse.3.At the stage of cerebral ischemia-reperfusion injury,five co-expression clusters were identified by weighted gene co-expression network analysis（WGCNA）and trend analysis,including two up-regulated patterns and three down-regulated patterns;Three co expression clusters were screened in the intervention stage of YHT,including one up-regulated mode and two down-regulated modes.In the intervention stage of YHT,the down-regulated co-expression cluster molecular function is closely related to kinase activity;KEGG and hallmark signal pathway analysis showed that the intervention of YHT was related to the activation of MAPK,m TOR,PI3K/AKT1 and c AMP signal pathway.The inference results of kinase activity showed that after the intervention of YHT,the down-regulation of P70S6K,a negative regulator of PI3K,released its inhibition on PI3K,driving the increase of AKT1 activation,and the phosphorylation of the latter can promote the expression of m TOR.Motif analysis showed that after the intervention of YHT,the types of kinases mainly included protein kinase C（PKC）and Akt of AGC kinase family,casein kinase II（ck II）,cyclin dependent protein kinases（CDKs）,mitogen activated protein kinases（MAPKs）,etc.It shows that there are differences in kinase activation tendency and substrate recognition preference after YHT intervention.Comprehensive network analysis shows that after the intervention of YHT,the key kinases m TOR and AKT1 are involved in the regulation of neuroprotection.YHT may play a neuroprotective role through S6K phosphorylation mediated PI3K/AKT1 pathway.4.Western blot analysis showed that the expression of p70S6K and p-p70S6K were lower in the ischemic brain tissue of YHT group than that of model group（P<0.05）;Compared with the model group,p-p70S6K/p70S6K were significantly down regulated in the ischemic brain tissue of YHT group（P<0.05）;Compared with the model group,AKT1 and p-AKT1 were highly expressed in the ischemic brain tissue of YHT group（P<0.05）;Compared with the model group,p-AKT1/AKT1 was significantly up-regulated in the ischemic brain tissue of YHT group（P<0.05）;Compared with YHT group,cleaved caspase-3 increased significantly in the model group（P<0.05）..Conclusions:1.YHT based on YTQ theory plays a neuroprotective role by improving neurological deficit,reducing infarct volume and reducing neuronal injury.2.YHT down regulates p-p70S6K phosphorylation and cleaved caspase-3 expression and up regulates p-AKT1 protein expression.Its neuroprotective effect may be related to the PI3K/AKT1 signal pathway mediated by p70S6K phosphorylation.