| Objective:The rat model of experimental autoimmune myocarditis was established.Endothelin B receptor(ETBR)overexpression lentivirus was injected into tail vein of rats to interfere with autoimmune myocarditis,and the effect of ETBR overexpression on autoimmune myocarditis was observed.On this basis,to explore the mechanism of ETBR overexpression regulating T lymphocyte homing related molecules such as ICAM-1 in the treatment of autoimmune myocarditis.Methods:To prepare lentivirus with overexpression of ETBR gene.Six normal rats(NC group)were randomly selected from 24 male Lewis rats,and the remaining 18 rats were injected with porcine cardiac myosin on day 0(day of immune injection)and day 7 respectively to establish the rat model of experimental autoimmune myocarditis.Eighteen rats injected with cardiac myosin for the first time were randomly divided into three groups:experimental autoimmune myocarditis group(EAM group),ETBR gene overexpression lentivirus autoimmune myocarditis group(ETBR-oe group)and no-load lentivirus autoimmune myocarditis control group(GFP group).On the first day of immunization,1×10~8 TU lentivirus(200?L)was injected into the tail vein of rats in the ETBR-oe group,the same dose of no-load lentivirus was injected into the rats in the GFP group,and the same dose of normal saline was injected into the NC group and the EAM group.On the 21st day after the first immunization,the changes of cardiac function were detected by echocardiography.On the same day,the rats were killed to take blood and heart samples.The changes of serum BNP and AMA were detected by ELISA.The myocardial tissue was stained with HE,the expression of ETBR,JAM-C,E-selectin and ICAM-1 protein was detected by immunohistochemistry,and the mRNA expression of ETBR,IFN-?,IL-12,IL-17,IL-4,JAM-C,E-selectin,ICAM-1,MCP-1 and MIP-1?was detected by fluorescence quantitative PCR.The protein expressions of ETBR and ICAM-1 in rat myocardium were detected by Western blot.Results:1.On the 21st day after the first immunization,the cardiac LVEDd and LVEDs in EAM group were significantly higher than NC group(P<0.01),EF and FS was significantly lower than that in NC group(P<0.01).There was no significant difference in EF and FS between GFP group and EAM group(P>0.05).Compared with EAM group,LVEDd and LVEDs in ETBR-oe group decreased,while EF and FS increased significantly(P<0.01).2.HE staining showed that there were a large number of inflammatory cell infiltration dominated by lymphocytes in EAM group,and the score of myocardial inflammation in NC group was significantly higher than that in NC group(P<0.05).There was no significant difference in the score of myocardial inflammation between GFP group and EAM group(P>0.05).Compared with EAM group,myocardial inflammatory cell infiltration,histopathological changes and myocardial inflammation score decreased significantly in ETBR-oe group(P<0.05).3.Immunohistochemistry showed that the expression of ETBR protein in myocardial tissue of EAM group was higher than that of NC group,but that of ETBR-oe group was significantly higher than that of EAM group.There was no significant difference in color depth between GFP group and EAM group.The staining depth of ICAM-1 in myocardial tissue in EAM group was significantly higher than that in NC group,and decreased significantly in ETBR-oe group compared with EAM group.There was no significant difference in color depth between GFP group and EAM group.There was no significant difference in the staining of JAM-C and E-selectin among EAM group,ETBR-oe group and GFP group,but they were all higher than those in normal control group.4.ELISA detection showed that the relative levels of serum BNP and AMA in EAM group were significantly higher than those in normal control group on the 21st day after initial immunization,while the relative levels of serum BNP and AMA in ETBR-oe group were significantly lower than EAM group(P<0.05).There was no significant difference between GFP group and EAM group(P>0.05).5.The results of fluorescence quantitative PCR showed that on the 21st day after the first immunization,the mRNA of ETBR in EAM group was higher than that in NC group(P<0.05),and the expression of mRNA in ETBR-oe group was significantly higher than that in EAM group(P<0.01)).The mRNA of IFN-?,IL-12and IL-17 in EAM group was significantly higher than that in normal control group(P<0.01),while the mRNA of IFN-?,IL-12 and IL-17 in ETBR-oe group was significantly lower than that in EAM group(P<0.05).The IL-4 of myocardial tissue in EAM group was significantly lower than that in normal control group(P<0.05),and the mRNA of IL-4 in ETBR-oe group was significantly higher than that in EAM group(P<0.01).The mRNA expression levels of adhesion molecules JAM-C and E-selectin in EAM group were significantly higher than those in normal control group(P<0.05).There was no significant difference in mRNA expression among EAM group,ETBR-oe group and GFP group(P>0.05).The mRNA expression level of ICAM-1 in EAM group was significantly higher than that in normal control group(P<0.05),and the mRNA expression level of ICAM-1 in ETBR-oe group was significantly lower than that in EAM group(P<0.05).The mRNA expression levels of chemokine MCP-1 and MIP-1?in EAM group were significantly higher than those in normal control group(P<0.05).There was no significant difference in mRNA expression level among EAM group,ETBR-oe group and GFP group(P>0.05).6.Western blot detection showed that the protein expression level of ETBR in EAM group was higher than that in NC group(P<0.05),and the protein expression level of ETBR in ETBR-oe group was significantly higher than that in EAM group(P<0.01).The protein expression level of ICAM-1 in EAM group was significantly higher than that in normal control group(P<0.05),and the protein expression level of ICAM-1 in ETBR-oe group was significantly lower than that in EAM group(P<0.01).Conclusion:1.Lewis rats immunized with porcine cardiac myosin successfully induced the rat model of experimental autoimmune myocarditis.2.The overexpression of endothelin B receptor in autoimmune myocarditis can prevent T lymphocytes from homing,reduce the pathological injury of autoimmune myocarditis and improve cardiac function.3.The overexpression of endothelin B receptor prevents T lymphocytes from homing in autoimmune myocarditis,which is mainly due to the inhibition of the expression of T lymphocyte homing related molecules such as ICAM-1. |
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