| Part 1 Regulation of Na+-H+ exchanger 1 on Calpain and Glycogen synthase kinase 3?(GSK-3p)in myocardium of type 2 diabetic db/db miceObjective To explore the regulation-of Na+-H+ exchanger 1 inhibitor(Carporide,CAR)on cardiac Calpain and GSK-3? in type 2 diabetic db/db mice.Methods Sixteen 7 week old male C57BL/KS db/db mice and 16 wild-type mice were selected and divided into 4 groups:Control group(WT),CAR group(WT+CAR),Model group(db/db)and CAR treated group(db/db+CAR).The mice were sacrificed after administration with CAR for 10 weeks and the changes of cardiachemodynamics were measured.Fasting plasma glucose(FPG),triglyceride(TG)and total cholesterol(TC)were determined by using biochemical test.The level of plasma insulin was measured by enzyme linked immunosorbent assay(ELISA)and insulin resistance index(HOMA-IR)was calculated.The level of Cardiac tissue Tumor necrosis factor(TNF-?)and Interleukin-1?(IL-1?)was measured by ELISA.The myocardial coenzyme ?/reduced coenzyme ?(NADP+/NADPH)ratio and the concentration of myocardial Nitrotyrosine(NT),3-nitrotyrosine(3-NT)as well as 4-hydroxynonenal(4-HNE)were also measured respectively.The mRNA expressions of Nicotinamide-adenine dinucleotide phosphate(NADPH)oxidase p22phox and gp9lphox,transforming growth factor-?1(TGF-?1),matrix metalloproteinase 2(MMP-2),matrix metalloproteinase 9(MMP-9)and osteopontin(OPN)were detected by Reverse Transcription-Polymerase Chain Reaction.The protein expressions of OPN,Calpain-1,Calpain-2,Calcineurin,protein kinase B(Serine/threonine Kinase,AKT)and GSK-3? were detected by Western blotting.Results(1)Compared with the WT group,FPG,TC,TG and HOMA-IR in the db/db group increased significantly(5.78±1.14 vs 21.21±4.12,2.29±0.52 vs 4.28±0.74,0.86±0.36 vs 1.65±0.19,0.65±0.18 vs 23.75±9.39,t=10.35,6.228,5.428,6.957,P<0.05).Compared with db/db group,FPG,TC,TG and HOMA-IR decreased after CAR treatment in db/db mice(t=2.111,2.112,1.213,1.651,P>0.05).(2)Under light microscope,the myocardium in db/db mice showed obvious irregular arrangement of cardiac fiber and hypertrophy of cardiac muscle cells which were improved obviously by treatment with CAR.(3)Compared with WT mice,the LVEDP of mice in db/db group increased and the absolute value of ±1dp/dtmax decreased(t=9.973,8.929,7.174,P<0.05)which were significantly reversed by administration of CAR(t=6.404,2.865,4.531,P<0.05).(4)Compared with the WT group,the ratio of NADP+/NADPH,the mRNA level of p22phox,gp91phox,and the content of NT,3-NT and 4-HNE significantly increased in the db/db group(t=6.988,3.953,3.901,8.088,10.89,5.375,P<0.05)which were significantly reversed by administration of CAR(t=4.845,2.519,2.565,2.803,4.131,2.581,P<0.05).(5)Compared with the WT group,the level of TNF-? and IL-1? were not obviously chang in the CAR group(t=0.345,0.409,P>0.05),however,the level of TNF-? and IL-1? significantly increased in the db/db group(t=8.608,9.950,P<0.05).Compared with db/db group,TNF-? and IL-1? decreased after CAR treatment in db/db mice(t=2.682,2.804,P<0.05).(6)Compared with the WT group,the collagen content and the TGF-?1 and MMP-2 mRNA levels in the db/db group increased significantly whereas the level of MMP-9 mRNA decreased significantly(t=4.905,3.695,3.098,4.346,P<0.05);Administration of CAR significantly reversed the changes of collagen content,TGF-Pi,MMP-2 and MMP-9 mRNA in the myocardium.(t=2.914,2.925,2.249,2.732,P<0.05).(7)Compared with the WT group,the mRNA and protein content of OPN myocardial tissues increased significantly in the db/db group.Administration of CAR significantly reversed the changes of mRNA and protein content of OPN in myocardium.(8)Compared with the WT group,Calpain,CaN activity and protein content of myocardial tissues significantly increased in db/db mice(t=3.70,9.738,3.492,3.353,3.871,P<0.05).Compared with the db/db group,CAR can reduce Calpain,CaN activity and protein content of myocardial tissues(t=2.935,2.792,2.634,3.253,2.887,P<0.05).(9)Compared with the WT group,the expressions of p-AKT and p-GSK-3?protein decreased significantly(t=3.492,6.400,P<0.05).Compared with the db/db group,CAR can increase the expressions of p-AKT and p-GSK-3?(t=2.827,4.090,P<0.05).Conclusions These results suggest that CAR exerts potent cardioprotective effects against heart injury in type 2 diabetic db/db mice and the mechanism is involved in preservation of insulin sensitivity,antioxidant enzymes,as well as Calpain and GSK-3? survival pathway.Part 2 Effects of Na+-H+ exchanger 1on neonatal rat cardiac myocytes exposure to high-glucoseObjective Through culturing cardiac myocytes of neonatal Sprague-Dawley rats in vitro,observing the effect of Na+-H+ exchanger 1 inhibitor(Carporide,CAR)on cardiac myocytes of neonatal rats induced by high-glucose damage,and then explore the possible meecchanisms.Methods Ventricular myocytes were isolated from neonatal rat and cultured,different concentrations of CAR(1-1000 mol/L)intervened myocardial cells,and cell viability was determined by CCK8 method to establish CAR security concentration.Then we selected safe concentration range of CAR(5-80 mol/L)on myocardial cells under high glucose conditions,cell viability was determined by CCK8 method to establish the best treatment for CAR concentration.The cell viablity were determined by CCK8 assay to establish the optimal high glucose damage model.The activity of lactic dehydrogenase(LDH)was measured by kit;The level of Cardiac tissue Tumor necrosis factor(TNF-a)and Interleukin-1?(IL-1? was measured by ELISA.Superoxide Dismutase(SOD)and malondialdehyde(MDA)were determined by using biochemical test.Flow Cytometry(FCM)detected the rate of apoptosis;Caspase-3,Bcl-2,Bax and GSK-3? protein expression we re measured by Westernblot.Results(1)The concentration gradient screening of CAR showed that 20 mol/L CAR could effectively protect cardiomyocytes against high glucose injuried.(2)Compared with the Control group,the SOD activity decreased significantly and MDA,TNF-?,IL-6 content increased significantly in neonatal rat cardiac myocytes exposure to high-glucose(t=3.931,3.928,4.069,6.148,P<0.05).Treatment of CAR significantly reversed the changes of SOD activity,MDA,TNF-a,IL-6 content in high glucose injuried(t=2.268,2.435,2.561,3.257,P<0.05).(3)The apoptosis rate of myocardial cells cultured in normal medium was very low,and the apoptosis rate of cardiomyocytes increased obviously after high glucose induction(24h)(P<0.05).Compared with the HG group,after treatment of CAR(20?mol/L),the rate of apoptosis was significantly lowed,the difference was statistically significant(P<0.05).These results suggest that CAR(20mol/L)can effectively resist high glucose injuried and reduce apoptosis of cardiomyocytes induced by high glucose.(4)Western blot showed that high glucose could induce the increase of Proapoptotic protein Bax in cardiomyocytes and decrease the expression of anti apoptotic protein Bcl-2.CAR(20mol/L)can reffectively upregulate Bcl-2 and down regulate Bax,thereby resisting high glucose damage and inhibiting cardiomyocyte apoptosis.(5)High glucose could induce the expression of Cleaved-Caspase-3 protein and Caspase-3 activity in Cardiomyocytes(t=8.735,P<0.05).CAR(20 mol/L)can effectively reduce the expression of Cleaved-Caspase-3 protein and Caspase-3 activity(t=2.326,P<0.05).(6)Compared with the Control group,the expressions of p-GSK-3? protein decreased significantly in cardiomyocytes exposure to high-glucose(P<0.05).Compared with the high glucse group,CAR can increase the expressions of p-GSK-3?induced by high gluese(P<0.05).(7)Compared with the Control group,Calpain,CaN activity significantly increased in cardiomyocytes exposure to high-glucose(t=10.13,9.126,P<0.05).Compared with the high group,CAR can reduce Calpain,CaN activity induced by high gluese(t=2.588,2.675,P<0.05).Conclusion CAR could reduce high-glucose induced neonatal rat cardiac myocytes apoptosis.The underlying mechanism may be result from its capacity of regulating oxidative stress and inflammatory factors,and upregulating the Bcl-2 and Bax ratio.Activation of GSK-3? and Calpain also play an important role in protection of myocardial injury induced by high glucose... |
PDF Full Text Request