Based On RhoA And MAPK Pathway, The Mechanism Of Action Of Qishen Granule On Myocardial Fibrosis Was Studied

Object:Myocardial fibrosis Huanshen granules are effective drugs for the clinical treatment of heart failure in traditional Chinese medicine and have previously been found to have anti-myocardial fibrosis effects.However,the mechanism is not yet clear.In this experiment,we want to investigate whether Ginseng particles act by inhibiting the ras homologous family member A(RhoA)pathway and the mitogen-activated protein kinase(MAPK)pathway and the RhoA and MAPK pathways for cell proliferation and migration behavior.Methods:1.Male SPF grade Sprague-Dawley(SD)rats 92 weeks old,of which 80 were prepared by ligation of the left anterior descending coronary artery myocardial fibrosis model,and the other 12 rats only threading without ligation as a sham operation group.After modeling,rats were randomly divided into model group,Chinese medicine group and western medicine group.Rats in the traditional Chinese medicine group were given Qishen granules by gavage.The dose of raw drugs was 18.66g/kg,which was twice the clinical equivalent dose.Western medicine group used fosinopril gavage at a dose of 1.2 mg/kg.The sham operation group and the model group were given an equivalent amount of normal saline instead of gavage.Each group was intragastrically administered once a day.The drugs were water-soluble and were intragastrically administered in an amount of 1 ml/100 g for 4 weeks.Heart function was used to measure cardiac left ventricular end-diastolic volume(LVEDd)and left ventricular end-systolic volume(LVEDs),and ejection fraction(EF)and short axis shortening(FS)were calculated.Hematoxylin and eosin(HE)staining was used to observe the arrangement of cardiomyocytes,Masson trichrome staining was used to measure collagen volume fraction(CVF),and immunohistochemistry was used to measure the average optical density(IOD)of type I and type III collagen.4.Serum Ang II was measured by radioimmunoassay.The serum levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),creatine kinase(CK),and muscle were measured using a biochemical analyzer.Acid kinase isozyme(CK-MB),alpha-hydroxybutyrate dehydrogenase(a-HBDH).5.Real-time quantitative PCR and Western blot were used to detect mRNA and protein expression of MAPK and RhoA pathway key molecules.6.The neonatal rats were used to extract fibroblasts for culture and stimulated with Ang? to establish a myocardial fibrosis myofibroblast model and identify.The cells were divided into five groups:blank control group,model group,Qishen granule group,PD98059/fasudil group and salvianolic acid B group.The concentration of Angll in the model group was 10-7 mol/L,the concentration of Qishen particles was 1000 ?g/ml,and the concentration of salvianolic acid B was 100 ?mol/L.The content of type I and type ? collagen in the cell supernatant was measured by enzyme-linked immunosorbent assay.7.Test cell proliferation ability by MTT assay and soft agar colony formation assay.The cell migration ability was measured by scratch test and Transwell assay.8.Real-time quantitative PCR was used to detect mRNA expression of MAPK and RhoA pathway key molecules.9.Use SPSS20.0 software for statistical analysis.Results:1.After 4 weeks of administration,the LVEDd and LVEDs of the model group increased significantly(P<0.01),EF and FS decreased significantly(P<0.01),and the EF and FS of the Chinese herb group decreased significantly(P<0.01).).HE staining showed that myocardial cells in the sham operation group were arranged neatly,while cells in the model group were disordered and there was inflammatory cell infiltration.Qishen granules have a more regular structure than the model group,with less inflammatory cell infiltration.Masson staining showed that there were a lot of collagen deposition in the model group,and there was less collagen deposition in the Qishen granule group and fosinopril group.The CVF of the model group increased significantly(P<0.01),and the CVF of the Chinese medicine group decreased significantly(P<0.01)compared with the model group.Immunohistochemistry results showed that the IOD of type I and type III collagen in the model group increased significantly(P<0.01,P<0.01).Collagen type I and III collagen deposition in the traditional Chinese medicine group was significantly lower than that in the model group(P<0.01,P<0.01).4.Serum test results showed that the Ang II,AST,LDH,CKMB,CK and a-HBDH levels in the model group increased significantly(P<0.01,P<0.01,P<0.01,P<0.01,P=0.016,P= 0.018).Compared with the model group,serum levels of Ang ?,AST,LDH,CKMB,CK and a-HBDH in the Chinese medicine group were significantly decreased(P<0.01,P<0.01,P=0.021,P=0.013,P=0.145,P= 0.089).RT-qPCR results showed that the mRNA levels of ERK,JNK,RhoA,ROCK1,ROCK2 and MLC in the model group increased significantly(P<0.01,P<0.05,P<0.01,P<0.01,P<0.05,P<0.01).).The ERK,JNK,RhoA,ROCK1 and ROCK2 in Qishen Granule group were significantly lower than those in the model group(P<0.01,P<0.01,P<0.05,P<0.05,P<0.05).Western blotting results showed that the expression levels of ERK,p-ERK/ERK,p-JNK/JNK,RhoA,ROCK1,ROCK2 and p-MLC/MLC in the model group increased significantly(P<0.01,P<0.01,P=0.025,P<0.01,P<0.01,P<0.01,P<0.01).The levels of ERK,p-ERK/ERK,p-JNK/JNK,RhoA,ROCK1,ROCK2,and p-MLC/MLC in Qishen granule group were significantly lower than those in the model group(P<0.01,P<0.05,P<0.05,P.<0.01,P<0.01,P<0.01,P<0.01).7.The fibroblasts exhibit a long spindle shape under an optical microscope.Vimentin staining showed that the cytoplasm showed green fluorescence.There was no green fluorescence in a-SMA staining of fibroblasts,and cytoplasmic green fluorescence was observed in a-SMA staining of myofibroblasts,suggesting that myofibroblasts express a-SMA.ELISA results showed that type I and type III collagen in the model group increased significantly(p<0.05,p<0.05).Collagen type I and type III collagen in the Chinese medicine group were significantly lower than those in the model group(p<0.01,p<0.01).Salvianolic acid B group also decreased significantly compared with the model group(p<0.05,p<0.05).MTT assay results showed that MTT values of the model group increased significantly at 48h and 72h(p<0.05,p<0.01).The MTT value of Qishen granule group was significantly lower than that of the model group(p<0.05,p<0.01).The MTT value of salvianolic acid B group was also significantly lower than that of the model group(p<0.05,p<0.05).Plate cloning results showed that the number of clones in the model group increased significantly(p<0.05).The number of clones in the Qishen granule group was significantly lower than that in the model group(p<0.01).The number of clones in the salvianolic acid B group was also significantly lower than that of the model group(p<0.05).9.Scratch test results showed that the scratch width at 48h and 72h of the model group decreased significantly(p<0.05,p<0.05).The scratch width of Qishen granules at 48h and 72h was significantly higher than that of the model group(p<0.05,p<0.01,p<0.01).The scratch width at 48h and 72h in salvianolic acid B group was also significantly higher than that in the model group(p<0.05,p<0.05).Transwell experimental results show that the number of crossings in the model group increased significantly(p<0.05).The number of crossings in the Qishen granule group was significantly lower than that in the model group(p<0.01).The number of crossings in the salvianolic acid B group was also significantly lower than that in the model group(p<0.01).10.RT-qPCR results showed that the mRNA levels of ERK,RhoA,and ROCK1 in the model group increased significantly(p<0.01,p<0.01,p<0.01,p<0.01,p<0.05).The levels of ERK,RhoA and ROCK1 in Qishen granule group were significantly lower than those in the model group(p<0.01,p<0.05,p<0.05).The levels of ERK and RhoA in salvianolic acid B group were also significantly lower than those in the model group(p<0.01,p<0.01).Conclusion:1.Qishen Granules play a role in protecting the heart by inhibiting fibrosis in an animal model of myocardial fibrosis.Its antifibrotic mechanism may be through inhibition of RhoA/ROCK and MAPK signaling pathways.Key molecules targeting the Angll-ATIR-RhoA and MAPK pathways provide an alternative treatment for myocardial fibrosis.2.Qishen Granules play a role in inhibiting the proliferation and migration of cardiac fibroblasts by inhibiting fibrosis in the myocardial fibrosis cell model,thereby preventing the progression of fibrosis.Its anti-fibrosis mechanism may be through the inhibition of RhoA/ROCK and MAPK signaling pathways.3.There is a close relationship between the RhoA pathway and the MAPK pathway.They affect each other and cooperate with each other,not only in cell signal transduction,but also in biological behavior such as cell proliferation and migration.