| Objective: The progression of viral myocarditis(VMC)can develop into dilated cardiomyopathy.Myocardial fibrosis is a key factor in influencing the prognosis of viral myocarditis.Studying the mechanism of myocardial fibrosis in viral myocarditis could provide a theoretical basis for the treatment of viral myocarditis.Cordyceps sinensis(CS)is a traditional Chinese medicine,which can regulate immunity and prevent kidney fibrosis,but can it inhibit myocardial fibrosis? This is our choice of Cordyceps sinensis as the original idea of inhibition of myocardial fibrosis drugs.The mammalian target of rapamycin(mTOR)is an important signal transduction molecule that regulates the production of protein,affects cell growth and cell cycle progression and is closely related to cell growth and proliferation.However,is the inhibitor rapamycin(Rap),mTOR related to myocardial fibrosis? In this study,we constructed myocardial fibroblasts and Balb / c mice model to study the expression and function of mTOR in myocardial fibrosis of viral myocarditis from cell level and animal level,and to evaluate the mechanism and function of Cordyceps sinensis antiviral myocarditis.The experiment is divided into two parts:(1)To study the Expression of mTOR in Viral Cardiac Fibroblasts and Effect of Cordyceps sinensis on Antiviral Cardiac Fibroblast FibrosisMaterials and Methods: Cardiac fibroblasts were divided into control group,CVB3 group,Rap + CVB3 group,different dose of CS + CVB3 group,CS + Rap + CVB3 group.The morphology of the cells was observed by immunofluorescence microscopy.The expression of CVB3-m RNA,mTOR-m RNA,collagen I-m RNA,collagen III-m RNA and TGF-?1 m RNA were detected by RT-PCR.Western Blot were used to detect mTOR and mTOR downstream markers P-P70S6 K,P-4EBP1,and TGF-?1,Smad protein and collagenI and collagen-III.Results:1)CVB3 can infect cardiac fibroblasts.2)RT-PCR could detect the optical density value of cardiac fibroblasts mTOR / ?-actin.CVB3 group was higher than the control group,CVB3 + Rap group was lower than CVB3 group,the difference was significant(P<0.05).MTOR,P-P70S6 K,P-4EBP1 and CVB3 were detected by western blot.P-P70S6 K in CVB3 group was higher than that in control group,and P-P70S6 K in CVB3 + Rap group was lower than that in CVB3 group(P <0.05);P-4EBP1 in the CVB3 group was lower than that in the control group while it is higher in CVB3+Rap group than that in the CVB3 group,and the difference was significant(P <0.05).3)The optical density of type I collagen measured by RT-PCR in cardiac fibroblasts of CVB3 group was significantly higher than that in control group while it is lower in CVB3+Rap group than that in CVB3 group(P<0.05),and the difference was statistically significant.Collagen I grayscale value was detected by Western blot.CVB3 group was higher than control group.CVB3 + Rap group was lower than CVB3 group,the difference was significant(P<0.05).4)The level of TGF-?1 in myocardium fibroblasts was detected by RT-PCR.The levels of TGF-?1 and Smad3 were measured by Western Blot.The levels of TGF-?1 and Smad3 in CVB3 group were higher than that of control group.The levels of TGF-?1 and Smad3 in CVB3+Rap group were lower than that of CVB3 group(P <0.05).5)RT-PCR was used to detect the CVB3 m RNA density in myocardium fibroblasts.CVB3 m RNA expression in CS group was lower than that in CVB3 group(P <0.05).6)The expression of CVB3,mTOR,TGF-?1,collagen-I and collagen m RNA in cardiac fibroblasts and Cordyceps sinensis were lower than those in CVB3 group.The levels of PP70S6 K,TGF-?1,Smad protein and collagen-I and collagen-III were lower than those in CVB3 group while the expression of P-4EBP1 was higher than that in CVB3 group,and the difference was significant(P<0.05).The effect of the above-mentioned is dose-dependent,and the effect of high-dose Cordyceps sinensis group is more obvious.(2)To study the Expression of MTOR in Mice with Viral Myocarditis and the Effect of Cordyceps sinensis on Myocardial Fibrosis in Chronic Mice with CVB3Materials and Methods: A chronic phase model of viral myocarditis was administered by using CVB3(made by twice injecting virus in a conventional way from the abdominal cavity).Divide into normal control group,model group,high and low dose CS group and captopril control group and calculate statistical mortality.At the end of the experiment,the myocardial collagen volume fraction(CVF)was detected by MASSON staining.The mTOR m RNA was detected by RT-PCR.MTOR,mTOR downstream marker P-P70S6 K,P-4EBP1,and TGF,Smad protein and collagen-I and collagen-III collagen in myocardium were detected by Western blot.Results:1)CVB3 viral myocarditis mice chronic phase animal model was established successfully.2)The mortality of mice in model group was the highest,and the mortality of mice in the CS high dose group was the least,and there was no death in the normal control group(P <0.05).3)The pathology of cardiomyopathy showed that the myocardial fibrosis in the model group was significantly higher than that in the control group.The fibrosis of the mice in the Cordyceps sinensis group was less than that in the model group and there is dose-dependent dependency.4)MTOR m RNA and downstream markers P-P70S6 K,P-4EBP1 express in the myocardium.The expression of mTOR m RNA and downstream marker P-P70S6 Kin the viral group were significantly higher than those in the normal control group while the expression of P-4EBP1 in the viral group was lower than that in the normal control group(P <0.01),and the difference was significant(P<0.01).Compared with the virus model group,the expression of mTOR m RNA and P-P70S6 K was significantly decreased and the expression of P-4EBP1 was significantly increased(P<0.01)in the CS high dose group and Captopril group and there was a significant difference(P<0.01).Compared with the mTOR m RNA level in the CS high dose group and the low dose group,the dose-dependent dependency was significantly different(P<0.01).5)The expression of TGF-?1 m RNA and Smads in myocardium: The expression of TGF-?1m RNA and Smads in the virus model group was significantly higher than that in the normal control group(P<0.01).Compared with the small dose group,the small dose group and the virus model group,the expression of collagen-1 was significantly decreased in the Captopril group(P<0.05).The expression of collagen-1 in high dose CS group was significantly lower than that in low dose CS group(P<0.05).Conclusion:1)Cardiac fibroblasts can be used as CVB3-infected VMC cell models.2)CVB3 viral myocarditis chronic phase mouse model can be used as a study of VCM myocardial fibrosis animal model.3)Activation of mTOR signaling pathway is one of the mechanisms of CVB3-induced cardiomyocyte proliferation,and rapamycin is the target of mTOR.4)The cell and animal levels show that mTOR signaling pathway is involved in VCM myocardial fibrosis,and may interact with TGF-?1,Smad signaling pathway.5)Cordyceps sinensis has the effect of anti-CVB3,inhibiting the growth of CVB3 cardiac fibroblasts,anti-VMC mouse myocardial fibrosis and reducing the mortality of VMC mice.These effects are dose-dependent and high dose effect is more obvious,One of its mechanisms may be inhibiting mTOR signaling pathway,and may interact with TGF-?1,Smad signaling pathway to inhibit myocardial fibrosis.Therefore,this study from the CVB3 cardiac fibroblast cell model and CVB3 viral myocarditis mouse model experiments indicates that mTOR pathway is involved in myocardial fibroblast proliferation and myocardial fibrosis in viral myocarditis,Cordyceps sinensis has the effect of anti-VM fibrosis and dose-dependency.One of its mechanisms is inhibiting mTOR pathway and interacting with TGF-?,Smads pathway,which participate in myocardial fibrosis.Previous studies have shown that VM myocardial fibrosis is actually a complex biological process involving multiple factors.From another point of view,the results of this study indicate that rapamycin and Cordyceps sinensis have the effect of inhibiting the mTOR pathway involved in anti-myocardial fibrosis,and this study is to provide theoretical basis and new ideas in search for VM myocardial fibrosis drugs. |
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