Fibrinogen-like Protein 2 Gene Silencing Downregulated The Expression Of Toll-like Receptors-9,increased Cardiac Function In Autoimmune Myocarditis Rats And The Molecular Mechanism Involved

Objective: This study is aimed to establish rat model of autoimmune myocarditis and lentivirus mediated Fgl2 gene silencing model in rats.To observe the Toll-like receptors-9( TLR9) expression and the feasibility of the treatment of autoimmune myocarditis in rats using lentivirus mediated fibrinogen-like protein 2(Fgl2) as the target and its mechanisms.Methods: We produced and packed Fgl2 RNA interference lentivirus, and there were 32 6-8-week-old Lewis Rats, and 8 normol rat were selected as the control group. The remaining rats,which were immunized with purified cardiac myosin of pigs’ heart on day 1 and 8 days to build experimental autoimmune myocarditis model in rats, then the autoimmune myocarditis were randomly divided into three groups:myocarditis model group(EAM), the GFP empty carrier lentivirus transfection group(GFP) and Fgl2- RNAi- LV group(RNAi),and there were 8 rats in each group.Echocardiography was performed at 0 day after the intitial immune, 21 day after the initial immune,and 4 rats in each group were killed separately on day 21 and day 40 after the initial immunization. Then we observed the inflammatory situation of heart tissue using HE staining and serum IL-6, the INF-a and NF-k B level were detected using ELISA kit, CD4+CD25+T cells percentage of serum total T cell was detected using flow cytometry instrument, TLR9 and CLTA-4 m RNA levels was detected using RT-PCR, TLR9 and Fgl2 protein expression was detected using Western Blot.Results: Echocardiography showed that cardiac function of EAM group was significantly lower than the control group on 21 d after primary immune(P<0.05).Compared with EAM group,cardiac function improved in RNAi group,there was statistically significant(P<0.05),and and there was no difference between the EAM group with the GFP group(P>0.05). Serological examination showed the levels of INF-α and NF-κB of the EAM group reached peak in acute stage(21d after the first immunization), the levels of IL- 6 attained peak in chronic phase(40d after the first immunization),which were significantly higher than other groups(P<0.05),the inflammatory factor levels of RNAi group was lower than the EAM group(P<0.05), and and there was no difference between the EAM group with the GFP group.Compared with the control group, the inflammation score,TLR9 protein and m RNA expression in myocardial tissue of the EAM group were significantly higher than other groups on the day 21 after initial immunization(P<0.05), the inflammation score and TLR9 protein expression in RNAi group reduced compared with those in the EAM group(P < 0.05), and there was no difference between the EAM group with the GFP group. Serum CD4+,CD25+and CD4+CD25+T cells percentage, there was no statistically significant difference among these group on the day 21 after initial immunization(P > 0.05).Conclusion:TLR9 protein and m RNA upregulate in the heart of experimental autoimmune myocarditis rats,and lentivirus mediated Fgl2 gene silencing in autoimmune myocarditis rats can downregulate TLR9 signal pathway, which decreased TLR9 protein and m RNA expression, so as to alleviate inflammatory situation in myocardium.It suggests that signal pathways of TLR9 may be involved in the process of myocarditis in the experimental autoimmune myocarditis rats;lentivirus Fgl2 RNA can relieve myocardial inflammation, may have played to prevent and stop its chronic deferment, even effect for the treatment of myocarditis.