| Part 1 Role of GLP-1 analogue liraglutide in autoimmune myocarditis mice Objective: To investigate the effect of liraglutide on myocardial inflammation in mice with autoimmune myocarditis.Methods: 18-20 g male Balb/c mice were randomly divided into normal group,liraglutide group,experimental autoimmune myocarditis(EAM)group,and EAM+ liraglutide group.Construction of experimental autoimmune myocarditis(EAM)mouse model by using mouse cardiac protein heavy chain alpha(MyHC-α)polypeptide,the liraglutide group and the EAM + liraglutide group were given liraglutide(200 μg /kg/day)by subcutaneous injection.The changes of body weight and blood glucose in each group were observed.The color and volume of heart in each group were compared and the weight/body weight(HW/BW)ratio was calculated.H&E staining was used to detect the pathological changes of myocardial tissue in each group.The expression of inflammatory factors TNF-α,IL-1β and chemokine MCP-1 in myocardial tissue of each group of mice were detected by RT-PCR.Results: Compared with the normal group,liraglutide attenuated the body weight of mice in liraglutide goup,but had no effect on the body weight in EAM group.Meanwhile,liraglutide did not affect the blood glucose level in EAM group.The hearts of EAM group were soft and ruddy,the hearts of EAM group were significantly larger,harder and whiter than those of normal group;the hearts of EAM + liraglutide group were significantly smaller,softer and lower in HW / BW ratio than those of EAM group.The results of H&E staining showed that the myocardial cells of EAM group were enlarged and necrotic,accompanied by a large number of inflammatory cell infiltration;the myocardial cells of EAM + liraglutide group had a small amount of edema and necrosis,less inflammatory cell infiltration,and the area of myocardial necrosis and the score of myocarditis were significantly reduced.Myocardial tissue RT-PCR results showed that compared with the normal group,EAM group TNF-alpha,IL-1beta and MCP-1 were significantly increased,while the expression of inflammatory and chemokines was significantly decreased in EAM + liraglutide group.Conclusion: Liraglutide attenuates myocardial inflammation and improves myocardial damage in mice with autoimmune myocarditis.Part 2 Immunomodulation of GLP-1 analogue liraglutide in autoimmune myocarditis mice Objective: To investigate the effect of liraglutide on Th17/Treg cells in mice with autoimmune myocarditis Methods: 18-20 g male Balb/c mice were randomly divided into normal group,experimental autoimmune myocarditis(EAM)group,and EAM+ liraglutide group.Construction of experimental autoimmune myocarditis(EAM)mouse model by using mouse cardiac protein heavy chain alpha(MyHC-α)polypeptide,the EAM + liraglutide group were given liraglutide(200 μg /kg/day)by subcutaneous injection.RT-PCR was used to detect the expression of Th17-related factors RORγt,IL-6,IL-17 A and Treg cell-related factors IL-10 and TGF-beta in myocardial tissue of each group.Flow cytometry was used to detect the proportion of Th17 and Treg cells in spleen and the levels of Th17 cell-related factors IL-6,TNF-α and IFN-γ in peripheral blood,as well as the expression of Treg cell-related factors TGF-βand IL-10.The spleen cells of Balb/c mice were cultured in vitro and treated with liraglutide(100n M).The effects of liraglutide on the proliferation of CD4+T cells was measured by flow cytometry.Results: Compared with the EAM group,the expression of Th17/Treg cell-associated factors were decreased in the myocardial tissue in EAM + liraglutide group;the proportion of Th17 cells decreased and Treg cells were increased in spleen.Expression of Th17 cell-associated factors in peripheral blood was decreased in EAM + liraglutide group.In vitro spleen cell proliferation experiments showed that liraglutide intervention had no inhibitory effect on CD4+ T cell proliferation.Conclusion: Liraglutide inhibits the differentiation and function of Th17 cells in EAM mice,and increases the differentiation and function of Treg cells.Liraglutide regulates inflammation in EAM mice indirectly.Part 3 Effect of GLP-1 analogue liraglutide on intestinal flora of autoimmune myocarditis in mice Objective: To investigate the effect of liraglutide on the intestinal flora in mice with autoimmune myocarditis.Methods: 18-20 g male Balb/c mice were randomly divided into normal group,liraglutide group,experimental autoimmune myocarditis(EAM)group,and EAM+ liraglutide group.Construction of experimental autoimmune myocarditis(EAM)mouse model by using mouse cardiac protein heavy chain alpha(MyHC-α)polypeptide,the liraglutide group and EAM + liraglutide group were given liraglutide(200 μg /kg/day)by subcutaneous injection.Collected the feces of four groups of mice,extracted intestinal flora,then detected the intestinal flora of each group by 16S rRNA high throughput sequencing.The expression of TLR4 in myocardium was detected by RT-PCR and Western blot.Results: Compared with the normal group,the enrichment of the intestinal flora was increased in the liraglutide group,without diversity change;the relative abundance of the Firmicutes was decreased,resulting decreased of the F/B ratio.The composition of intestinal flora in the two groups of mice was different.In the liraglutide group,the Bacteroides is an important biomarker,and the Firmicutes is the important biomarker in the normal group.Compared with the EAM group,the richness and diversity of intestinal flora in EAM+lilastin group were decreased,and the relative abundance of the Bacteroides was increased,resulting significantly decreased of the F/B ratio.There was a significant difference in the composition of the intestinal flora of two goups.Compared with the normal group,in addition,the expression of TLR4 in myocardium was significantly increased in the EAM group,and decreased in the EAM+lilarutide group.Conclusion: Liraglutide improves the intestinal flora composition in normal Balb/c mice,and reduces myocardial inflammation injury by regulating intestinal flora and TLR4 expression in mice with autoimmune myocarditis. |
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