Peptide-immunized mice developed significantly higher levels of MyHC614C629-reactive total IgG compared to mock-immunized control mice ( 005), and there was no significant difference between the insulin and saline treatment groups in autoantibody production (Fig. in this process. Total ERK1/2 and phospho-ERK1/2 (p-ERK1/2) were both up-regulated in insulin-treated mice after immunization. We also found that insulin treatment promoted T cell recovery without changing the naive-to-memory T-cell ratio; in particular, CD3+ T cells in insulin-treated mice proliferated more vigorously than in control mice ( 005). We report here for the first time that insulin alleviates myocarditis in the EAM model. These data show that insulin has a direct effect on T cell proliferation in EAM. It is possible that GIK or insulin may assist T cell recovery towards normal in myocarditis, especially for diabetic or hyperglycaemic patients. 005) (Fig. 1b), but no significant differences in HW : BW were observed between insulin-treated mice and the normal mice. To avoid too large a drop in blood glucose, we chose the dose of 25 U/kg body weight of insulin on an alternate day. The results showed that there were no significant differences in blood glucose between each of the groups (Fig. 1c). Open in a separate window Fig. 1 Insulin-treated mice were protected from autoimmune myocarditis. (a) Histopathological score of hearts at day 21 after immunization. There was a tendency for insulin to decrease the myocarditis score (= 021). (b) Relative heart weight of saline-treated (open triangle) and insulin-treated (filled triangle) mice at day 21 of experimental autoimmune myocarditis (EAM). Comparison between saline-treated mice and normal control mice (filled square), * 005. The MannCWhitney = 7 in each group). (e,f) The serum cardiac troponin I (cTnI) level was assessed by ELISA on days 14 and 21. Comparison between the saline- and insulin-treated groups indicated less cTnI in serum from EAM mice treated with insulin, # 005 Gingerol (= 10 in each group). To test the antigen-specific autoantibody response, MyHC614C629 reactive serum antibodies were measured by ELISA at day 21 of EAM. Peptide-immunized mice developed significantly higher levels of MyHC614C629-reactive total IgG compared to mock-immunized control mice ( 005), and there was no significant difference between the insulin and saline treatment groups in autoantibody production (Fig. 1d). Meanwhile, we found that insulin significantly lowered the serum cTnI levels of EAM mice on day 14 ( 005) (Fig. 1e) and on day 21 ( 005) (Fig. 1f) compared with that of the saline group. Insulin did not decrease inflammatory cytokines significantly in heart BALB/c mice developed severe myocarditis which peaked at day 21, as described previously [13]. Marked inflammation in the myocardium was observed in the saline-treated group as a prominent feature of this myocarditis model, characterized by extensive mononuclear and polymorphonuclear cell infiltration. In contrast, the inflamed area in the 25 U/kg insulin group was smaller than that in Rabbit Polyclonal to NCAML1 the saline-treated group (Fig. 2aCc). IL-1 and TNF- are the key inflammatory cytokines in the pathogenesis of myocarditis; blockade of TNF- (or IL-) ameliorates myocarditis in Gingerol mice [13]. Thus, we analysed these two cytokines in the heart by qPCR at day 21 of EAM. We found that IL-1 and TNF- tended to be lower in hearts from insulin-treated mice at day 21 of EAM compared to saline-treated controls, but with no significant differences between each group (Fig. 2d). Open in a separate window Fig. 2 Inflammatory infiltrates were present in the control group but there were fewer infiltrates in the insulin-treated hearts at day 21 after immunization compared to control; (a) 5 original magnification, representative histopathology of -myosin heavy chain (MyHC)614C629 induced experimental autoimmune myocarditis (EAM) at day 21 from saline-treated (middle), insulin-treated (right) mice and normal mouse heart (left); (b,c) 20 original magnification. (d,e) Quantitative polymerase chain reaction (PCR) detected the proinflammatory cytokines interleukin (IL)-1 and Gingerol tumour necrosis factor (TNF)- in the heart. There was more IL-1 and TNF- in the saline-treated EAM group than in the normal control group. However, levels of IL-1 and.