The Analysis Of GPR35/ UCP2 In The Cardiac Injury Induced By Abnormal Glucose And Fat Metabolism

Objective: 1.To determine the concrete role of GPR35 in cardiac injury induced by abnormal glucose and fat metabolism.2.To investigate whether UCP2 is a potential downstream of GPR35.3.To clarify whether oxidative stress is an intrinsic mechanism of GPR35/UCP2 signaling against diabetic cardiomyopathy.Methods: 1.The male mice with SPF C57BL/6 were randomly divided into two groups: the control group(n=15)and the experimental group(n=40).The control group was fed with normal diet for 3 weeks at first,and then combined with intraperitoneal injection of sodium citrate buffer solution(50 mg/kg/d)for 5 consecutive days,with continuous normal diet;the experimental group was fed with high fat diet for 3 weeks in advance,then intraperitoneally injected STZ(50 mg/kg/d)for 5 consecutive days with continuous high fat diet.After the models were established successfully,(1)the weight,fasting blood-glucose,glucose tolerance test,insulin tolerance test in mice were measured.(2)The serum level of TG,TC,FFA were tested and the contents of serum insulin was detected by ELISA kits.(3)The serum BNP level was tested.(4)The cardiac systolic function was obtained by echocardiography.(5)The interstitial fibrosis was detected by Masson staining and Sirius Red staining.(6)Transmission electron microscopy was used to measure the mitochondrial morphologic alterations.(7)The expression of GPR35 and UCP2 were measured by q RT-PCR and Western blot.2.The experimental groups were randomly divided into four groups: Vehicle group(n=8),GPR35 agonist(Zaprinast)group(n=8),GPR35 inhibitor(CID-2745687)Group(n=8)and UCP2 inhibitor(Genipin)Group(n=8).(1)The expression of UCP2 in myocardium was measured by q RT-PCR and Western blot.(2)The interstitial fibrosis was detected by Masson staining.(3)The morphologic alterations of mitochondria were assessed by transmission electron microscopy.3.The mouse myocardial cell lines(MCMs)were divided into three groups: the high glucose +0 ?mol/l palmitic acid sodium group,high glucose+200 ?mol/l palmitic acid sodium group,high glucose +300 ?mol/l palmitic acid sodium group,and were intervened in 9 hours and 18 hours respectively.(1)The expression of GPR35 was measured by q RT-PCR and Western blot.(2)The alterations of myocardial apoptosis were tested by flow cytometry.(3)The changes of ROS in high glucose+0 ?mol/l palmitic acid sodium Group,high glucose +300 ?mol/l palmitic acid sodium group were detected by DHE staining and ELISA kit.The high glucose +300 ?mol/l palmitic acid sodium group were further divided into five groups: Vehicle group,Zap group(Zaprinast group),CID group(CID-2745687 group),Zap+NAC group(Zaprinast+NAC group),and Gen group(genipin group).Results: 1.Compared with the control group,the mice in experimental group had significantly elevated weight,fasting blood-glucose,serum levels of insulin,with impaired glucose tolerance and insulin tolerance.In addition,TG,TC,LDL-c and FFA of the experimental group were significantly increased.2.By flow cytometry,the proportion of myocardial apoptosis increased with the concentration of sodium palmitate(0,200,300 ?mol/L)and intervention time(9,18 hours).The optimal intervention condition was 300?mol/L sodium palmitate for 18 hours,using in the subsequent cell experiments.3.Compared with the control group,diabetes mellitus with hyperlipidemia elevated serum BNP,increased myocardial fibrosis,decreased E/A ratio and LVEDD,and mitochondrial morphology destruction.4.The results showed that the expression of GPR35 and ROS generation were significantly increased in the experimental group;Inhibition of GPR35 reduced ROS content and apoptosis of cells;Antioxidant(NAC)could reverse the increase of ROS production and apoptosis induced by GPR35 activation.5.The expression of UCP2 in the experimental group significantly decreased.The reduction of GPR35 enhanced UCP2 expression.Conversely, GPR35 activation inhibited the level of UCP2.Moreover,we also found that UCP2 downregulation aggravated the glucolipid metabolic myocardial injury,evidenced by increased myocardial fibrosis,damaged mitochondrial morphology,decreased membrane potential and ATP production,and aggravated ROS generation and apoptosis.Conclusions: The antagonism of GPR35 and UCP2 influences cardiac injury induced by abnormal glucose and fat metabolism via modulating oxidative stress.