Effect Of Activating TGR5 On Expression Of Inflammatory Cytokines In Neonatal Mice Cardiomyocytes Induced By High Glucose

Objective: To investigate the effect of G protein coupled receptor 1(TGR5)on the expression of inflammatory cytokines in neonatal mice cardi omyocytes induced by high glucose,and to explore the possible mechanism.Methods:experiment one:1.Culturing and identifying neonatal mice cardiomyocytes: neonatal mice cardiomyocytes were cultured by a two-step enzymatic digestion and differential adhesion cultured.Cardiomyocytes were identified by immunofluorescence.2.To detect the efficiency of TGR5 virus in cardiomyocytes,the groups were set as follows:(1)Control groups:medium;medium+Polybrene solution.(2)Experimental groups:medium+TGR5 shRNA;medium+TGR5 shRNA+Poly brene solution.TGR5 shRNA had the effect of silencing TGR5 gene expression,Polybrene was a virus infection enhancement solution.Each group set MOI =5,25,50,100,150 five subgroups,each group set up three holes(MOI = number of virus/total number of cells).3.RT-PCR and Western Blot were used to detect the expression of TGR5 mRNA and protein in neonatal mice cardiomyocytes.The neonatal mice cardio myocytes were isolated and incubated for 48 hours.The normal control group:the normal medium incubated 48h;TGR5 shRNA group: add in the normal medium containing TGR5 shRNA virus incubated for 4 ~ 6h and then add inthe same volume of normal mediumthe incubated for another 16 h,replace the virus-free medium and then incubated 24 h.4.The neonatal mice cardiomyocytes were isolated and incubated for48 h,and then intervented with different concentrations of 0,1,10,20,30,50 and 100umol/L INT-777.MTT assay was used to determine the effective concentra tion of TGR5-specific agonist INT-777 by detecting cell viability.Experiment 2: Effect of activating TGR5 on HG-induced expression of inflammatory cytokines and activation of NF-?B in cardiomyocytes1.To observe the effect of high glucose(HG)on the expression of IL-1?,IL-6 and TNF-? in cardiomyocytes at different times,set 0,1,2,4,8,16,24 h different High Glucose action time,the levels of IL-1?,IL-6 and TNF-?mRNA expression in myocardium were measured by RT-PCR.2.To observe the effect of high glucose(HG)on p-I?B? / NF-?B signaling pathway in cardiomyocytes at different times,isolating and culturing neonatal mice cardiomyocytes,after 48 h,then set 0,1,2,4,8,16,24 h different High Glu cose action time,the levels of p-I?B?/NF-?B in cardiomyocytes were measured by Western Blot.3.To observe the effect of activating TGR5 on p-I?B?/NF-?B signaling pathway in HG-induced cardiomyocytes,the experiment groups as follows :(1)normal control group;(2)HG;(3)HG+INT-777(The TGR5 specific agonist);(4)HG+INT-777+TGR5 shRNA;(5)HG+INT-777+SQ22536(cAMP inhibitor),the expression of p-I?B?/NF-?B protein were deter mined by Western Blot.4.To detect the effect of activating TGR5 on the expression of IL-1?,IL-6and TNF-? mRNA in myocardium of mice induced by HG,isolating neonatal mice cardiomyocytes which were incubated for 48 hours,then the cardiomyocy tes were divided into the following groups:(1)normal control group;(2)HG;(3)HG+INT-777;(4)HG+INT-777+TGR5 shRNA;(5)HG+INT-777+SQ22536;(6)HG+JSH-23.The levels of IL-1?,IL-6 and TNF-? mRNA in cardiomyocytes were measured by RT-PCR.Experiment 3: Effect of activating TGR5 on expression of inflammat ory cytokines and MAPKs signal activation in HG-induced neonatal mice cardiomyocytes1.To observe the effect of high glucose(HG)on MAPKs signaling pathway in cardiomyocytes at different times,isolating and culturing neonatal mice cardiomyocytes,after 48 h,then set 0,1,2,4,8,16,24 h different High Glucose action time,the levels of p-JNK,p-ERK,p-P38 in cardiomyocytes were measured by Western Blot.2.To observe the effect of activating TGR5 on MAPKs signaling pathway in HG-induced cardiomyocytes,the experiment groups as follows :(1)normal control group;(2)HG;(3)HG+INT-777;(4)HG+INT-777+TGR5 shRNA;(5)HG+INT-777+SQ22536,the expression of p-JNK,p-ERK,p-P38 protein were determ ined by Western Blot.3.To detect the effect of activating TGR5 on the expression of IL-1?,IL-6and TNF-? mRNA in myocardium of mice induced by HG,Isolating neonatal mice cardiomyocytes which were incubated for 48 hours,then the cardiomyocy tes were divided into the following groups:(1)normal control group;(2)HG;(3)HG+INT-777;(4)HG+PD98059(JNK inhibitor);(5)HG+SP600125(ERK inhi bitor).The levels of IL-1?,IL-6 and TNF-?mRNA in cardiomyocytes were measured by RT-PCR.Results:experiment 1:1.Cardiomyocytes cultured successfull y,the flu orescence immunoassay showed that the purity of Cardiomy ocytes was more than 90%.2.Under the fluorescence microscope to observe the TGR5 shRNA virus infecting cardiomyocyte,selected MOI=100 for the following experiments.After silencing TGR5 gene the experiment result showed that compared with the normal group,the expression of TGR5 protein and mRNA in TGR5 shRNA groups were significantly lower than that in normal group,differences were statistically significant(P<0.05).3.In the normal cultured cardiomyocytes with 1-50umol/L concentration of INT-777,the growth of myocardial cells showed no cytotoxicity.But compar ed with the normal control group,100umol/L of INT-777 significantly inhibited the growth of cardiomyocytes(P<0.05).According to the literature and the exp erimental results,30umol/L of INT-777 finally was made sured as the experime ntal concentration.Experiment 2:Effect of activating TGR5 on HG-induced expression of inflammatory cytokines and activation of NF-?B in cardiomyocytes1.The effects of high glucose(HG)at different time on the expression of IL-1?,IL-6 and TNF-? mRNA in cardiomyocytes were observed as follows :compared with the normal group,effect of HG on the express ion of variousinflammatory cytokines in cardiomyocytes were significantly different from that of the normal group,the expression of IL-1?and IL-6 mRNA at HG-indu ced 8h while TNF-?mRNA at HG-induced 2h was significantly higher than that in control group,so select above times to the following experiments.2.The effect of HG at different time on p-I?B? / NF-?B signaling pathway in cardiomyocytes was observed as follows:compared with the normal group,effect of HG on the activation of I?B? and NF-?B in cardiomyocytes were significantly different from that of normal group,the expression of p-I?B?and NF-? B at HG-induced 6h was significantly higher than that in control group,so it was selected to the following experiments.3.The effect of activating TGR5 on the activation of NF-?B and I?B?induced by HG in cardiomyocytes were as follows:(1)Compared with normal group,the protein expression of p-I?B?and NF-?B in HG,HG+INT-777+TGR5shRNA,HG+INT-777+SQ22536 were significantly increased(P<0.001),the protein expression of p-I?B?and NF-?B in HG+INT-777 group were signif icantly decreased(P<0.05);(2)Compa red with HG group,the protein expression of p-I?B and NF-?B in HG+INT-777 group were significantly decreased(P<0.05),p-I?B? and NF-?B in HG+INT-777+TGR5 shRNA were signifi cantly increased(P<0.05).The protein expression of NF-?B in HG+ INT-777+SQ22536 group was significantly increased(P<0.05),while the protein expre ssion of p-I?B? were no significant difference between HG and HG+SQ22536groups(P>0.05);(3)Compared with HG+INT-777 group,the protein expression of p-I?B? and NF-?B in HG+INT-777+TGR5 shRNA,HG+ INT-777+SQ22536 group were significantly increased(P<0.05).The results suggested that TGR5-specific agonist INT-777 might decrease the expression of p-I?B?/NF-?B in HG-induced cardiomyocytes by activating TGR5.4.The effect of activating TGR5 on the different expression of inflamma tory cytokines induced by HG in cardiomyocytes were as follows:(1)Compa red with normal group,the express ion of IL-1?,IL-6 and TNF-?mRNA in HG,HG+INT-777+TGR5 shRNA,HG+INT-777+SQ22536 groups were signifi cantly higher than that in normal group(P<0.05).The expression of IL-1? and IL-6 mRNA in HG+INT-777 group were not significantly different(P>0.05),the expression of IL-1?and TNF-?mRNA in HG+JSH-23 group were't significantly different(P>0.05),and the expression of IL-6 mRNA in HG+ JSH-23 group was significantly higher than that in normal group(P<0.05).(2)Compared with HG group,the expression of IL-1?,IL-6 and TNF-amRNA in HG+INT-777,HG+JSH-23 group were significantly lower than that in HG group(P<0.05),the expression of IL-1?,TNF-?mRNA in HG+INT-777+TGR5 shRNA had no different(P>0.05),while the expression of IL-6 mRNA in HG+INT-777+TGR5shRNA was significantly increased(P<0.05).The expression of IL-6 mRNA in HG+INT-777+SQ22536 group was significantly higher than that in HG group,and the expression of IL-1 mRNA was decreased(P<0.05),but the expression of TNF-?mRNA was no significant change(P>0.05);(3)Compared with HG+INT-777 group,the expression of IL-1?,IL-6 and TNF-?mRNA in HG+INT-777+TGR5 shRNA,HG+ NT-777+ SQ22536 groups were significantly higher than that in HG+INT-777 group(P<0.05),the expression of IL-1? mRNA inHG+JSH-23 group has no difference(P<0.05),the expression of IL-6 mRNA in HG+JSH-23 group was significantly higher than that in HG group(P<0.05),the expre ssion of TNF-? mRNA in HG+ JSH-23 group was significantly lower than that in HG group(P<0.05).The results suggested that TGR5-cAMPp-I?B?-NF-?B pathway might decrease the expression of IL-1?,IL-6 and TNF-? mRNA in HG-induced cardiomyocytes by activating TGR5.Experiment 3:Effect of activating TGR5 on expression of inflammat ory cytokines and MAPKs signal activation in HG-induced neonatal mice cardiomyocytes1.To observe the effect of HG on MAPKs signal activation in cardiomyo cytes as follows:compared with the normal group,the effect of HG on the activation of p-JNK,p-ERK and p-P38(MAPKs)in cardiomyocytes were signi ficantly different from that in the normal group.The p-ERK and p-P38 selected HG for 1h,p-JNK selected HG for 2h for the best time to the following experiments.2.The effect of activating TGR5 on the activation of p-JNK,p-ERK and p-P38(MAPKs)induced by HG in cardiomyocytes were follows:(1)Compared with normal group,the protein expression of p-JNK,p-ERK and p-P38 in HG,HG+INT-777+TGR5 shRNA,HG+INT-777+SQ22536 groups were significant increased(P<0.05),the expression of p-JNK and p-ERK protein in HG + INT-777 group were not significantly different(P>0.05),while the expression of p-P38 protein was significantly increased(P<0.05);(2)Compared with HG group,the protein expression of p-JNK and p-ERK in HG + INT-777 groupwere significantly decreased(P<0.05),while the expression of p-P38 protein was significantly increased(P<0.05),the expression of p-ERK in HG+ INT-777+TGR5 shRNA group was decreased(P<0.05),the expression of p-P38 in HG+INT-777+TGR5 shRNA group was increased(P<0.05),while p-JNK had no significant difference(P>0.05);(3)Compared with HG + INT-777 group,the protein expression of p-JNK and p-ERK in HG+INT-777+TGR5 shRNA,HG+INT-777+SQ22536 groups were significantly increased(P<0.05),but p-P38 had no difference(P>0.05).The results suggested that TGR5-specific agonist INT-777 might decrease the expression of p-ERK and p-JNK(MAPKs)in HGinduced cardiomyocytes by cAMP.3.The effect of activating TGR5 on the expression of different inflammatory cytokines in HG-induced cardiomyocytes were as follows:(1)Compared with the normal group,the expression of IL-1?,IL-6 and TNF-?mRNA in HG groups were significantly higher than that in normal group(P<0.05),and the expression of IL-1? mRNA in HG+PD98059,HG+SP600125,HG+INT-777 were significantly increased(P<0.05),while the expression of IL-6 and TNF-?mRNA were not significantly different(P>0.05);(2)Compared with HG group,the expression of IL-1?,IL-6 and TNF-?mRNA in HG+INT-777,HG+PD98059 and HG +SP600125 groups were significantly decreased(P<0.05);(3)Compared with HG+INT-777 group,the expression of IL-1?,IL-6and TNF-?mRNA had no significantly difference(P>0.05).The results suggest ed that TGR5-specific agonist INT-777 might through inhibiting the ERK and JNK reduce the expression of IL-1?,IL-6 and TNF-? mRNA in HG-inducedcardiomyocytes by activating TGR5.Conclusion:1.The expression of inflammatory cytokines in cardiomyo cyte induced by high glucose were through the NF-?B,ERK and JNK.2.Activation of TGR5 could reduce the expression of inflammatory cytokines in high glucose-induced cardiomyocytes.3.Activation of TGR5 might reduce the expression of inflammatory cytokines in cardiomyocytes induced by high glucose through inhibiting NF-?B,ERK and JNK signaling pathways.