Effect Of Nrf2/HO-1 Signaling Pathway On Which TGR5 Alleviated Oxidative Stress Injury In Mice Cardiomyocytes

Objective:To investigate whether activation of TGR5 could activate Nrf2/HO-1 signaling pathway and reduce oxidative stress injury induced by glucose/glucose oxidase(G/GO)in mice cardiomyocytes.Methods:1.primary culture and identification of neonatal mice cardiomyocytes: Two-step enzymatic digestion and differential adherence method for primary culture of mice cardiomyocytes and the cardiomyocytes were identified by immunofluorescence.2.To detect the effect of TGR5-specific agonist INT777 on mice cardiomyocytes:1)MTT method was used to detect the effects of different concentrations of INT777(0,1,10,20,30,40,50,100uM)on the activity of cardiomyocytes in mice;2)The effects of different concentrations of INT777(0,10,20,30,40uM)on ROS levels in cardiomyocytes markered with DCFH-DA probe were detected by flow cytometry.3.To establish a model of oxidative stress induced by glucose / glucose oxidase(G/GO)in mouse cardiomyocytes.Groups:(1)Control group: the cardiomyocytes were cultured with 10%FBS LOW/DMEM medium;(2)Experimental group: the cardiomyocytes were treated with glucose(33mmol/L)and glucose oxidase at different concentrations for 6 hours Blanking group:(3)only 10%FBS LOW/DMEM medium without cardiomyocytes.Cell viabilities were detected by MTT assay.4.To detect the effect of INT777 on oxidative stress injury induced by G/GO model.Groups:(1)Control group;(2)G/GO group: the cardiomyocytes were treated with G(33mmol/L)/GO(20mU/mL)for 6h;(3)G/GO+INT777 group: the cardiomyocytes of G/GO group were pretreated with INT777(30uM)for 3 hours;(4)Blank group:only 10%FBS LOW/DMEM medium without cardiomyocytes.(blanking group only for MTT detection).MTT method was used to detect and calculate the cell viability of every group.DCFH-DA probe and flow cytometry were used to detect the level of ROS in every group.Hoechst 33342-PI double staining method was used to detect the apoptosis of every group.5.To establish a model of the cardiomyocytes silenced the expression of TGR5 by TGR5 shRNA adenovirus.According to the instructions,the cardiomyocytes were infected with TGR5 shRNA adenovirus(MOI=100)by the method of 1/2 volume 4h infection.The fluorescence expressions of infected cardiomyocytes were detected by fluorescence microscopy.The expressions of TGR5 and GAPDH mRNA were detected by RT-PCR.The expressions of TGR5 and GAPDH protein were detected by Western Blot.6.To detect the effect of activated TGR5 on the activation of Nrf2/HO-1 signaling pathway in cardiomyocytes.(1)To detect the effect of INT777 on cardiomyocytes at different time points on Nrf2 nuclear transfer.Groups:(1)Control group;(2)Experimental group:The cardiomyocytes were treated with INT777(30uM)for 1h,2h,3h,6h,8h.The protein of Nrf2 and H3 in the nucleus of every group were detected by Western blot.(2)To detect the effect of INT777 on the expression of HO-1 protein at different time points.Groups:(1)Control group;(2)Experimental group:The cardiomyocytes were treated with INT777(30uM)for 2h,4h,6h,8h,10 h.The protein of HO-1 and GAPDH in the cell of every group were detected by Western blot.(3)To detect the effect of actived TGR5 on Nrf2 nuclear transfer in cardiomyocytes.Groups:(1)Control group;(2)INT777 Group:the cardiomyocytes were treated with INT777(30uM)for 2h;(3)INT777+TGR5 shRNA group: The INT777 group was pre-infected with TGR5 shRNA for 48h;(4)INT777+SQ22536 Group:The INT777 group was pretreated with SQ22536(cAMP inhibitor)100umol/L for 3h.The protein of Nrf2 and H3 in the nucleus of every group were detected by Western blot.(4)To establish a model of the cardiomyocytes silenced the expression of Nrf2 by Nrf2 siRNA lentivirus.According to the instructions,the cardiomyocytes were infected with Nrf2 siRNA lentivirus(MOI=20)by the method of 1/2 volume 4h infection.The expressions of Nrf2 and GAPDH protein were detected by Western Blot.(5)To detect the effect of actived TGR5 on the expression of HO-1 protein in cardiomyocytes.Groups:(1)Control group;(2)INT777 Group:the cardiomyocytes were treated with INT777(30uM)for 6h;(3)INT777+TGR5 shRNA group: The INT777 group was pre-infected with TGR5 shRNA for 48h;(4)INT777+SQ22536 Group:The INT777 group was pretreated with SQ22536(cAMP inhibitor)100umol/L for 3h.(5)INT777+Nrf2 siRNA group: The INT777 group was pre-infected with Nrf2 siRNA for 48 h.The protein of HO-1 and GAPDH in the cell of every group were detected by Western blot.reduce oxidative stress injury induced by G/GO in mice cardiomyocytes.7.To detect the effect of Nrf2/HO-1 signaling pathway on which TGR5 alleviated oxidative stress injury induced by G/GO in mice cardiomyocytes.Groups:(1)Control group;(2)G/GO group: the cardiomyocytes were treated with G(33mmol/L)/GO(20mU/mL)for 6h;(3)G/GO+INT777 group: The G/GO group was pretreated with INT777(30uM)for 3h;(4)G/GO+INT777+TGR5 shRNA group: The G/GO+INT777 group was pre-infected with TGR5 shRNA for 48h;(5)G/GO+INT777+ZNPP group: The G/GO + INT777 group was pretreated with ZNPP(HO-1 inhibitor)10uM for 1h.DCFH-DA probe and flow cytometry were used to detect the level of ROS in every group.Western blot was used to detect the expression of apoptotic protein Caspase 3 and GAPDH in every group.Results:1.The cardiomyocytes were cultured successfully and identified by immunofluorescence staining.The purity was more than 95%.2.The impact of INT777 on the cardiomyocytes:(1)The effect of INT777 on cardiomyocytes activity: Compared with control group,INT777 had no obvious effect on the growth activity of cardiomyocytes at 0-50uM(P>0.05),but INT777 had a toxic effect on cardiomyocytes at 100 uM concentration(P<0.05);(2)The effect of INT777 on ROS levels in cardiomyocytes: Compared with control group,INT777 had no significant effect on ROS production in cardiomyocytes at 10-40uM(P>0.05).Combined with the above results and related literatures,30 uM INT-777 was selected for subsequent experiments.3.INT777 could attenuate G/GO model-induced oxidative stress injury in cardiomyocytes:(1)Establishment of G/GO oxidative stress damage model:Compared with the control group,with the increasing of GO concentration,the rate of cardiomyocyte activity decreased.The activity of cardiomyocytes decreased significantly by nearly 50% at G(33mmol/L)/GO(20mU/mL)(P<0.05).Therefore,the model of cardiomyocytes damaged by G/GO oxidative stress was established by using G(33mmol/L)/GO(20mU/mL)for 6h of cardiomyocytes for follow-up experiments.(2)INT777 could alleviate the reduction of activity of cardiomyocytes in the G/GO model.Compared with control group,the cell viability of G/GO group was significantly decreased(P<0.05).Compared with G/GO group,the cell viability of G/GO+INT777 group was increased(P<0.05).(3)INT777 could reduce the elevation of ROS of cardiomyocytes in G/GO model.Compared with control group,the levels of ROS in G/GO group were significantly increased(P<0.05).Compared with G/GO group,the levels of ROS in G/GO+INT777 group significantly decreased(P<0.05).(4)INT777 could attenuate the apoptosis of cardiomyocytes in G/GO model.Hoechst 33342-PI double staining was observed under fluorescence microscope:Control group: Cardiomyocytes were stained with light blue,without strong red;G/GO group: most of the cardiomyocytes were stained with strong blue,and even a few cells were showed strong red color;G/GO+INT777 group: most of the cardiomyocytes were returned to normal,and only a few cells were stained with strong blue,without strong red.4.The model of cardiomyocytes silenced the expression of TGR5 by TGR5 shRNA adenovirus was successfully established.TGR5 shRNA adenovirus(MOI = 100)infected primary cardiomyocytes with a red fluorescence rate of more than 90%.Compared with control group,the relative expression of TGR5 mRNA and TGR5 protein in TGR5 shRNA group was significantly decreased(P <0.05).5.Activation of the TGR5 receptor on cardiomyocytes could activate the Nrf2/HO-1 signaling pathway(1)the expressions of Nrf2 nuclear transfer and HO-1 protein at different time points when cardiomyocytes were treated with INT777 :Nrf2 nuclear transfer and HO-1 protein expression level increased with the prolongation of the time of cardiomyocytes were treated with INT777.Nrf2 nuclear transfer reached the peak at about 2h,and the expression level of HO-1 protein reached the peak at about 6h.Therefore,the above-mentioned time was selected as the optimum detection time of the corresponding protein.(2)INT777 could activate Nrf2 nuclear transfer via TGR5-cAMP signaling pathway.Compared with control group,Nrf2 nuclear transfer significantly increased in INT777 group(P<0.05).However,compared with INT777 group,the Nrf2 nuclear transfer were significantly decreased in INT777+TGR5 shRNA group and INT777+SQ22536 group(P<0.05).(3)The model of Nrf2 siRNA lentivirus silencing Nrf2 expression on cardiomyocytes was successfully established.Nrf2 siRNA lentivirus(MOI=20)infected primary cardiomyocytes for 48 h.Compared with control group,the relative expression of Nrf2 protein in Nrf2 siRNA group significantly decreased(P<0.05).(4)INT777 could activate HO-1 via TGR5-cAMP-Nrf2 signal path.Compared with control group,the relative expression of HO-1 protein was significantly increased(P<0.05).However,compared with INT777 group,the relative expression of HO-1 protein in INT777+TGR5 shRNA group,INT777+SQ22536 and INT777+Nrf2 siRNA group was significantly decreased(P<0.05).6.Activation of TGR5 could attenuate oxidative stress injury in cardiomyocytes by Nrf2 / HO-1 signaling pathway(1)Activation of the TGR5-Nrf2-HO-1 signaling pathway could reduce G/GO-induced ROS levels in cardiomyocytes.Compared with control group,the level of ROS in G/GO group was significantly increased(P<0.05).Compared with G/GO group,the level of ROS in G/GO+INT777 group significantly decreased(P<0.05).However,Compared with G/GO+INT777 group,the levels of ROS in G/GO+INT777+TGR5 siRNA group and G/GO+INT777+ZNPP group increased again(P<0.05).(2)Activation of the TGR5-Nrf2-HO-1 signaling pathway could attenuate G/GO-induced cardiomyocyte apoptosis.Compared with control group,the relative expression of Caspase3 in G/GO group was significantly increased(P<0.05).Compared with G/GO group,the relative expression of Caspase3 in G/GO+INT777 group was significantly decreased(P<0.05).However,Compared with G/GO+INT777 group,the relative expression of Caspase3 in G/GO+INT777+TGR5 shRNA group and G/GO+INT777+ZNPP group increased again(P<0.05).Conclusion:1.Activation of TGR5 may reduce G/GO model-induced oxidative stress injury in cardiomyocytesthe.2.Nrf2/HO-1 signaling pathway may be involved in the mechanism of TGR5 attenuating oxidative stress injury in cardiomyocytes.