Study On The Mechanism Of Buyang Huanwu Decoction On Diabetic Encephalopathy Through PI3K/AKT Pathway

Objective:Using R language data mining to explore the compatibility law of Huangqi prescription Buyang Huanwu decoction,through network pharmacology prediction analysis cooperate with in vitro and in vivo experimental verification,to explore the prevention and treatment mechanism of Buyang Huanwu Decoction on diabetic encephalopathy.Methods:1.Inquire Shanghan Zabing Lun,Qianjin Yaofang,Qianjin Yifang,Piwei Lun,Neiwai Shangbian Huolun,Lanshi Micang which contain Huangqi to establish a database of Huangqi prescription,use the Chinese Pharmacopoeia to standardize drug names.Using R language statistical analysis of data,Use the item Frequency function(perform frequency analysis),corrplot function(performs correlation analysis),extrafont function(visualization),arules and arules Viz function(analyzed for association rules).Cluster analysis of drugs and prescriptions was carried out by Q and R type clustering.2.The chemical components of Buyang Huanwu Decoction and their corresponding protein targets were searched TCMSP and TCMID databases.Using uniprot database,the protein target and target gene of Buyang Huanwu Decoction were exchanged.Search disgenet database for target gene of diabetic encephalopathy.Cytoscape3.8.0 software was used to construct the network diagram of Chinese materia medica,component and target.Venny2.1was used to draw the Venn diagram of the intersection of drug and disease target gene.STRING database was used to construct protein interaction network between drug and disease target.GO and KEGG analysis was performed by DAVID6.8.3.In vitro experiment: Diabetic encephalopathy in vitro model was established by PC12 cell damage induced by high glucose.CCK-8 detect the Buyang Huanwu Decoction cytotoxicity of PC12 and viability of HG-PC12,define the dosage.Flow cytometry was used to detect the apoptosis of HG-PC12 treated by Buyang Huanwu Decoction.Western blot was used to detect the expression levels of Bcl-2,Bax,Cleaved Caspase-3,Caspase-3 proteins and PI3K/AKT signaling pathway related proteins in PC12 cells.4.In vivo experiment: SD rats were fed high-fat-sugar diet and low dose of streptozotocin(STZ)for 4 weeks to construct T2 DM rat model.The experiment was divided into model group,Buyang Huanwu Decoction high-dose,medium-dose and low-dose and metformin group,with 13 rats in each group.In addition,10 blank control group were set.The rats in each group were given corresponding intervention,to observe the body weight and fasting glucose.Fasting glucose tolerance was measured at the end of 8th week.After 8 weeks of drug intervention,blood samples were collected through abdominal aorta and serum was separated.Serum glycosylated hemoglobin and blood lipid levels were detected.H&E staining was used to observe the histopathological changes of rat hippocampal tissue.The expression of Bcl-2,Bax,Caspase-3 in hippocampal tissues of rats were detected by immunohistochemistry.Western blot detect the expression of PI3K/AKT signaling pathway related proteins and Bcl-2,Bax,Cleaved Caspase-3 and Caspase-3 in the hippocampus.Results:1.A total of 295 Huangqi prescriptions were included,and 71% Huangqi prescriptions contained 6-15 herbs.Drug used more than 100 times included Huangqi,Danggui,ginseng,Baishao.Drug frequency of use > 30 times,most of the tonic drugs were found.Buyang Huanwu decoction contains Danggui and Chishao,which is line with the rule of Huangqi prescription.The positive correlation coefficients is Shihu-desertliving cistanche,and the negative correlation coefficients is Guizhi-Chaihu.Association analysis showed that{Danggui}=>{Huangqi},{Danggui,ginseng}=>{Huangqi},{Danggui,ginseng,Gancao}=>{Huangqi} were the combinations with the highest support among 2,3 and 4 drug association rules.R cluster analysis shows that Danggui-ginseng was the first to be classified.The tonic and deficiency structure of Huangqi prescription with Danggui,Shaoyao and Chuanxiong in Buyang Huanwu Decoction was consistent with the formula.2.Network pharmacology analysis finally screened 91 active compounds and 201 gene targets of Buyanghuwu Decoction,obtained 1148 disease targets of Diabetic encephalopathy,35 drug-disease common targets,PPI protein interaction network screened key targets such as AKT1,PYGM,MPO,etc.The biological processes in GO analysis mainly include cell response to proliferation,regulation of apoptotic signaling pathway and response to drugs,etc.Cell composition mainly includes cytoplasmic membrane complex,protein complex,cell solute,etc.Molecular functions include enzyme binding,DNA binding,transcription factor binding.KEGG analysis showed that the core targets of Buyang Huanwu Decoction were enriched in PI3K/AKT,MAPK and TNF signaling pathway.3.In vitro experiments :(1)The survival rate of PC12 cells treated with 100 m M high glucose medium for 48 h was significantly decreased(P < 0.01).HG-PC12 cells were treated with Buyang Huanwu Decoction(400,600,800μg/m L),The cell viability was significantly increased(P < 0.01).The cell viability was the highest when the concentration of Buyang Huanwu Decoction was 800μg/m L.(2)CCK-8 results: Compared with the high glucose group,the survival rate of HG-PC12 cells in the groups treated with Buyang Huanwu Decoction was significantly increased(P < 0.01).(3)Flow cytometry results: Compared with normal group,PC12 cells induced by high glucose showed early apoptosis(P < 0.01);The group of Buyang Huanwu Decoction significantly improved the apoptosis of PC12 cells induced by HG(P <0.01).These results indicated that Buyang Huanwu Decoction could enhance the activity of PC12 cells and inhibit the injury.(4)Western blot results: Compared with the normal group,Bax,Caspase-3,Cleaved Caspase-3 protein expression was significantly increased in the HG group(P < 0.01),and Bax/Bcl-2 ratio was significantly increased(P < 0.01);Compared with HG group,the expression of Bax and Caspase-3 protein in Buyang Huanwu Decoction highdose group and metformin group was significantly decreased(P < 0.01),and the expression of Bcl-2 was significantly increased in Buyang Huanwu Decoction high-dose group.Cleaved Caspase-3 protein expression was significantly decreased in Buyang Huanwu Decoction lowdose group(P < 0.01).(5)Compared with the normal group,the protein expression of p-AKT in the HG group was significantly decreased(P < 0.05);Compared with the HG group,the protein expression of PI3 K,AKT and p-AKT in the high dose Buyang Huanwu Decoction and metformin group was significantly increased(P < 0.05).4.In vivo experiments:(1)Compared with model group,body weight of Buyang Huanwu Decoction group increased,FBG and Hb A1 c decreased significantly(P < 0.05),serum TC,TG and LDL-C were significantly decreased,HDL-C was significantly increased(P < 0.05).OGTT result: Compared with model group,blood glucose levels in Buyang Huanwu Decoction and metformin group were significantly decreased(P < 0.01).(2)H&E result: Compared with model group,most neurons of Buyang Huanwu Decoction high-dose and metformin group were relatively intact,the cell morphological changes were lighter.(3)IHC result: Compared with model group,the positive expressions of Bax and Caspase-3 in Buyang Huanwu Decoction and metformin group were significantly decreased(P < 0.01),the positive expressions of Bcl-2 were significantly increased(P < 0.01).(4)Western blot result:Compared with model group,the expression of Bcl-2 protein in Buyang Huanwu Decoction and metformin groups was significantly increased(P < 0.01),the expression of Bax protein in Buyang Huanwu Decoction high-dose group was significantly decreased(P < 0.01);Caspase-3 protein expression was significantly decreased in Buyang Huanwu Decoction highdose group(P < 0.05);Cleaved Caspase-3 protein expression was significantly decreased in Buyang Huanwu Decoction high-dose and metformin group(P < 0.01).Compared with model group,the expression of PI3 K protein in Buyang Huanwu Decoction high-dose and metformin group was significantly increased(P < 0.05),the expression of p-PI3 K in Buyang Huanwu Decoction medium dose group was significantly increased(P < 0.01).Conclusion:1.Buyang Huanwu Decoction meets the compatibility rule of Huangqi prescription,and the compatibility of Huangqi with Danggui,Chishao and Chuanxiong in the formula may be a relatively mature complement structure of Huangqi prescription.2.Buyang Huanwu Decoction may play a therapeutic role in diabetic encephalopathy by regulating multiple signaling pathways including PI3K/AKT,and the core target molecule of regulation may be AKT1.3.Buyang Huanwu Decoction can promote the proliferation of PC12 cells and inhibit apoptosis of PC12 cells induced by HG in vitro model of diabetic encephalopathy.The mechanism may be related to the activation of PI3K-AKT/Bcl-2 signaling pathway.4.Buyang Huanwu Decoction can reduce fasting blood glucose,blood lipid,glycosylated hemoglobin,and inhibit hippocampal neuronal apoptosis in T2 DM rats,the mechanism may be related to the activation PI3K/AKT signaling pathway.