The Effect Of "Si Miao Yong An" Decoction On JNK Signaling Pathway In Cardiac Fibrolasts Of Neonatai Rats

In the 1980 s,Xing Yuepeng,a famous doctor of Chinese medicine in Shijiazhuang traditional Chinese Medicine Hospital,initially put forward a theory that acute myocardial infarction is "carbuncle of the heart ",the pathogenesis is that blood stasis and toxin exuberance block the heart vessels and so we shall remove the stasis and detoxify in the treatment by choosing the “Si Miao Yong An” decoction.Clinical experience demonstrated “Si Miao Yong An” decoction showed good effects on improving ventricular remodeling after acute myocardial infarction.In addition,the pathological basis of the ventricle remodeling is myocardial fibrosis.Based on the above facts,we speculated whether “Si Miao Yong An” decoction is able to inhibit myocardial fibrosis and the mechanism.Therefore,we started the experiments.In the experiments,the method of differential adhesion was used to collect primary cardiac fibrolasts.And the cardiac fibrolasts were identified by morphology and immunestochemical methods.The model was made by Ang and andⅡ the cells were randomly divided into five groups: the model control group,the normal control group,the high-dose group,the middle-dose group,and low-dose group.MTT assay could detect the cell viability.ELISA method was used to observe the collagen secretion ability of the type I and type Ⅲ in cell supernatant.Cell cycle and apoptosis rate were examined by Flow cytometry.And Western Blot detected the protein expression of JNK.Through the experiments,we found that the model control group had obvious cell proliferation changes(P<0.05)compared with the normal group.And compared with the model control group,“Si Miao Yong An” decoction different dose groups could significantly inhibit the proliferation of myocardial fibroblasts(P<0.05),and showed a certain degree of concentration and time dependence.Furthermore,we found that the collagenⅠand Ⅲ in the model control group had increased significantly compared with the normal group(P<0.05);and compared with the model control group,the synthesis and secretion of collagen I and Ⅲ were inhibited in the high-dose group and middle-dose group(P<0.05).And collagen Ⅲ was inhibited in the low-dose group,while no significant difference was found in the low-dose group for collagen I(P>0.05).In addition,Ang Ⅱ decreased the percentage of the cardiac fibrolasts in the early stage of DNA synthesis(G1 phase)significantly(P<0.05),increased the percentage in the stage of DNA synthesis(S phase)+ the late stage of DNA synthesis(G2 phase)(P<0.05),and reduced the rate of apoptosis compared with the normal control group(P<0.05).The high-dose group and the middle-dose group showed the opposite results(P<0.05).Compared with the model control group,the low dose group could increase the apoptosis rate(P<0.05),but it had no significant difference in the percentage of G1 phase and S phase+G2 phase(both P>0.05).Lastly,compared with the normal group,the model control group could significantly reduce the expression of JNK protein(P<0.05).“Si Miao Yong An” decoction different dose groups could up-regulate the expression of JNK than the model control group(P<0.05),and showed a concentration-dependent trend.The regulatory effect was stronger by increasing the dose.Based on the experimental results above,we concluded that “Si Miao Yong An” decoction could inhibit the proliferation and activity of myocardial fibroblasts.It also inhibited the synthesis and secretion of collagen I and Ⅲ.Furthermore,“Si Miao Yong An” decoction increased the percentage of myocardial fibroblasts in G1 phase,decreased the percentage in the stage of S phase+G2 phase,and increased the rate of apoptosis.These results showed that “Si Miao Yong An” decoction had the efect of alleviating myocardial fibrosis.The mechanism may be related to the JNK signaling pathway,and there was a quantitative effect relationship.