Protective effects of berberine and its metabolites on high glucose-induced H9c2 cardiomyocytes injury
Objective To investigate the protective effect of berberine metabolites on H9c2 cardiomyocytes injury induced by high glucose.Methods H9c2 cardiomyocytes were divided into control group,model group,berberine metabolites-treated group and high glucose together with berberine metabolites-treated group.The control group was cultured in serum-free DMEM,the normal treatment group was treated with medication in serum-free DMEM,and the model group was cultured in serum-free DMEM containing 50 mmol·L-1 glucose(modeling dose screening experiments were set at 25,50,100,and 200 mmol·L-1).The model treatment group was treated in high glucose serum-free DMEM with concentrations of berberine(BBR)1.25,2.50,5.00,and 10.00 μmol·L-1,dihydroberberine(DHB)0.5,1.0,2.0,and 4.0 μmol·L-1,berberine(BRB)1.25,2.50,5.00,and 10.00 μmol·L-1,columbamine(COL)1.25,2.50,5.00,and 10.00 μmol·L-1,palmatine(PAL)3.125,6.250,12.500,and 25.000 μmol·L-1,jatrorrhizine(JAT)6.25,12.50,25.00,and 50.00 μmol·L-1,demethyleneberberine(DEM)6.25,12.50,25.00,and 50.00 μmol·L-1.After 48 hours of treatment,the cell survival rate was measured by cell proliferation count(CCK-8)method.Detection of classical metabolic regulatory pathway silencing regulatory protein 1(Sirt1)and peroxisome proliferator activated receptors-y Co-activation factor 1 α(PGC1α),peroxisome proliferator activated receptor α(PPARα)Gene level,expression levels of glucose metabolism related genes pyruvate dehydrogenase kinase 4(PDK4),glucokinase(GCK),hexokinase(HK2),glucose transporter 4(Glut4),and mitochondrial dynamics related genes mitochondrial fusion protein 2(Mfn2),optic atrophy protein 1(OPA1),dynamics related protein 1(Drp1)and apoptosis related genes Caspase-3,Caspase-9,Bcl-2 related X protein(Bax),and B lymphomatoma-2(Bcl-2)by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Detection of PGC1α,Glut4,mitochondrial oxidative phosphorylation system(OXPHOS)protein expression through Western blotting experiment.Results Compared with the high glucose model group,the cell survival rate significantly increased in BBR 2.5,5.0,10.0 μmol·L-1,DHB 1,2 μmol·L-1,COL 5,10 μmol·L-1,PAL 12.5 μmol·L-1,JAT 25,50 μmol·L-1,and DEM 12.5,25.0 μmol·L-1 group after 48 hours of treatment(P＜0.05,0.01,0.001).The levels of PDK4 were significantly increased in cardiomyocytes treated with BBR,DHB,JAT,and DEM(P＜0.05,0.001),GCK levels were significantly increased in cardiomyocytes treated with BBR,DHB,and DEM(P＜0.01,0.001),HK2 levels were significantly increased in cardiomyocytes treated with BBR,DHB,COL,PAL,and DEM(P＜0.05,0.01),and Glut4 levels were significantly increased in cardiomyocytes treated with DEM(P＜0.001).The levels of OPA1 were significantly increased in cardiomyocytes treated with JAT and DEM(P＜0.05,0.001),while the levels of Drpl were significantly reduced in cardiomyocytes treated with BBR,DHB,COL,PAL,JAT,and DEM(P＜0.01,0.001).The levels of Mfn2 were significantly increased in cardiomyocytes treated with BBR,DHB,JAT,and DEM(P＜0.05,0.001).The levels of Caspase-3 in cardiomyocytes treated with BBR,DHB,and COL were significantly reduced(P＜0.01,0.001),while the levels of Caspase-9 in cardiomyocytes treated with BBR,BRB,COL,and PAL were significantly reduced(P＜0.01,0.001).The levels of Bax in cardiomyocytes treated with BBR,DHB,BRB,COL,PAL,JAT,and DEM were significantly reduced(P＜0.05,0.001),while the levels of Bcl-2 in cardiomyocytes treated with BBR,DHB,and PAL were significantly increased(P＜0.01,0.001).The expression of PGClα,Glut4 and OXPHOS in cardiomyocytes treated with BBR and its metabolites was significantly increased.Conclusion BBR metabolites can ameliorate high glucose induced H9c2 cardiomyocyte injury,and its mechanism may be through regulating Sirt1/PGC1 α/PPAR α signaling pathway,promoting glucose metabolism of H9c2 cardiomyocytes,improving mitochondrial function,and inhibiting apoptosis.
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