Experimental Study On The Intervention Effect And Mechanism Of Jianpi Yiqi Method Compound On High Glucose-induced H9c2 Cell Inflammation

Purpose:In this study,high glucose-induced rat cardiomyocytes(H9c2)were used to simulate the inflammation model of diabetic cardiomyopathy,and Jianpi Yiqi traditional Chinese medicine compound was used as an intervention factor.The effects on TLR4/MyD88/NF-κB signaling pathway related factors were tested by in vitro experiments,and to verify the regulatory effect of the serum containing Jianpi Yiqi Recipe on inflammatory mediators,to explore the protective mechanism and target of the method of invigorating the spleen and replenishing qi on myocardial inflammatory injury in type 2 diabetic cardiomyopathy,which is the prevention and treatment of diabetic cardiomyopathy Provide new choices of Chinese medicine clinical drugs.Materials and Methods:1.Preparation of Medicine serumSixty SPF male SD rats,8 weeks old,were selected and randomly divided into normal group,Jianpi Yiqi group and metformin group,with 20 rats in each group.The normal group was given normal saline by gavage,the Jianpi Yiqi group and the metformin group were gavage with Jianpi Yiqi recipe and metformin,once a day for 4 weeks.One hour after the last gavage,blood was collected from the abdominal aorta of rats and separated to prepare drug-containing serum.2.Inhibition of Jianpi Yiqi Recipe on H9c2 Cardiomyocyte Inflammation Induced by High Glucose in VitroThe rat cardiomyocytes(H9c2)cultured and passaged in vitro were randomly divided into 4 groups:the normal group,the model group,the Jianpi Yiqi group and the metformin group.Except for the normal group,the other groups were stimulated with 30mmol/L high glucose medium to stimulate H9c2 cardiomyocytes for 56h.The normal group and the model group were added with normal serum,the Jianpi Yiqi group and the metformin group were added with the serum of Jianpi Yiqi prescription and metformin serum to intervene the cells.It was divided into three time gradients:12h,24h,48h(that is,adding drugs after 44h,32h,8h of modeling).After 56h,the cell supernatant was collected.ELISA method was used to detect the expression levels of pro-inflammatory factors IL-6 and TNF-α in the cell supernatant of each group,and to explore the effect of different intervention times on the efficacy.3.Study on the mechanism of Jianpi Yiqi Decoction in inhibiting the inflammatory changes of H9c2 cardiomyocytes induced by high glucoseThe rat cardiomyocytes(H9c2)cultured and passaged in vitro were randomly divided into 6 groups:normal group,model group,blocker group,Jianpi Yiqi group,Jianpi Yiqi recipe+blocker group and metformin group.Except for the normal group,all the other groups were stimulated with 30mmol/L high glucose medium to stimulate H9c2 cardiomyocytes for 56h.Normal group and model group add normal serum,blocking agent group,Jianpi Yiqi group,Jianpi Yiqi recipe + blocker group and metformin group,add normal serum+TAK blocker,Jianpi Yiqi recipe serum,Jianpi Yiqi recipe serum+TAK The blocking agent and metformin serum intervened on the cells for 24 hours(that is to add medicine 32h after modeling),and the cells were collected 56 hours later.Real-timePCR and Western-blot methods were used to detect the expression of TLR4,MyD88,NF-κB mRNA and protein.Results:1.Results of interleukin 6(IL-6)expression in H9c2 cardiomyocytesCompared with the normal group,the IL-6 expression of the model group was significantly increased,the difference was statistically significant(p<0.05);compared with the model group,the IL-6 expression of each treatment group was decreased,the difference was statistically significant(p<0.05 or p<0.01).In this experiment,the Jianpi Yiqi group was divided into three time echelons(12h,24h,48h)to intervene in the model cells.The results showed that compared with the model group,the 24h intervention in the Jianpi Yiqi group was more statistically significant than the intervention at 12h and 48h.2.Results of tumor necrosis factor(TNF-α)expression in H9c2 cardiomyocytesCompared with the normal group,the expression of TNF-α in the model group was significantly increased,and the difference was statistically significant(p<0.05);compared with the model group,the expression of TNF-α in each treatment group decreased,and the difference was statistically significant(p<0.05 or p<0.01).In this experiment,the Jianpi Yiqi group were divided into three time echelons(12h,24h,48h)to interfere with the model cells.The results showed that compared with the model group,the 24h intervention in the Jianpi Yiqi group was more statistically significant than the intervention at 12h and 48h.3.Expression of TLR4 mRNA and TLR4 protein in H9c2 cardiomyocytesCompared with the normal group,the expression of TLR4 mRNA and TLR4 protein in the model group increased significantly(p<0.01);compared with the model group,the expression of TLR4 mRNA and TLR4 protein in the blocker group decreased(p<0.05);Compared with the blocker group,the expression of TLR4 mRNA and TLR4 protein in the Jianpi Yiqi group,the Jianpi Yiqi prescription+blocker group,and the metformin group decreased(p<0.01).The comparative curative effect of each treatment group is as follows:Jianpi Yiqi prescription+blocker group>metformin group>Jianpi Yiqi group.4.Expression of MyD88 mRNA and MyD88 protein in H9c2 cardiomyocytesCompared with the normal group,the expression of MyD88 mRNA and MyD88 protein in the model group increased significantly(p<0.01);compared with the model group,the expression of MyD88 mRNA and MyD88 protein in the blocker group decreased(p<0.05);Compared with the blocker group,the MyD88 mRNA and MyD88 protein expression levels in the Jianpi Yiqi group,Jianpi Yiqi prescription+blockade group and metformin group decreased(p<0.01).The comparative curative effect of each treatment group is as follows:Jianpi Yiqi prescription+blocker group>metformin group>Jianpi Yiqi group.5.Expression of NF-κB mRNA and NF-κB protein in H9c2 cardiomyocytesCompared with the normal group,the expression of NF-κB mRNA and NF-κB protein in the model group increased significantly(p<0.01);compared with the model group,NF-κB mRNA and NF-κB in the blocker group Protein expression decreased(p<0.05);compared with the blocker group,the NF-κB mRNA and NF-κB protein expression levels in the Jianpi Yiqi group,Jianpi Yiqi prescription+blocker group,and metformin group decreased(p<0.01).The comparative curative effect of each treatment group is as follows:Jianpi Yiqi prescription+blocker group>metformin group>Jianpi Yiqi group.Conclusion:1.Overexpression of inflammatory factors IL-6 and TNF-α appeared in H9c2 cardiomyocytes induced by high glucose,and NF-κB mRNA and protein expressions were found to increase in each group of inflammation model cells.Indicating that the excessive activation of NF-κB signaling pathway and myocardial cell inflammatory damage may be a mechanism that promotes the occurrence and development of DCM.2.This experiment uses Jianpi Yiqi prescription for intervention treatment,which can inhibit the expression of pro-inflammatory factors TNF-α and IL-6 in the NF-κB pathway,It shows that Jianpi Yiqi Recipe has anti-inflammatory effects on H9c2 cardiomyocytes induced by high glucose.3.Jianpi Yiqi Recipe can inhibit the expression of TLR4/MyD88/NF-κB signaling pathway,reduce the inflammatory damage of diabetic cardiomyopathy cardiomyocytes,improve cardiac function,and thus have a protective effect on DCM model rat cardiomyocytes.