白桦脂醇对AGEs诱导H9C2心肌细胞自噬的影响

Effect of betulin on autophagy of H9C2 myocardial cells induced by AGEs

目的:探讨白桦脂醇(Betulin)对晚期糖基化终末产物(AGES)诱导的H9C2心肌细胞自噬的影响。方法:将心肌细胞随机分成5组:正常H9C2组,AGEs组,低剂量加药组(5μmol/L),中剂量加药组(10μmol/L),高剂量加药组(20μmol/L)。CCK8测定0、1、2、3、4d细胞活力。试剂盒检测肌红蛋白(Mb)、肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白(cTnⅠ)的表达。Western blot检测Beclin1、P62、LC3Ⅱ/LC3Ⅰ、PI3K和AKT的表达。免疫荧光染色检测LC3的表达量。结果:AGEs组与正常H9C2组相比,H9C2细胞增殖倍数明显降低;Mb、CK-MB和cTnⅠ的表达显著上调;Beclin1表达显著上调,LC3Ⅱ/LC3Ⅰ明显上升,P62的表达显著下调;免疫荧光检测LC3光点明显增加;同时磷酸化激活的PI3K和AKT的表达水平显著增加(P<0.01)。加药组与AGEs组相比,H9C2心肌细胞的增殖倍数明显增加;Mb、CK-MB和cTnⅠ的表达水平显著降低;Beclin1表达显著下调,LC3Ⅱ/LC3Ⅰ明显下降,P62的表达显著上升;另外LC3免疫荧光结果显示LC3光点明显减少;同时,磷酸化PI3K和磷酸化AKT的表达水平显著下降(P<0.01)。结论:白桦脂醇通过抑制PI3K/AKT的活性进而降低AGEs诱导的H9C2心肌细胞自噬水平。

Objective: To investigate the effect of betulin on the autophagy of H9C2 myocardial cells induced by AGES. Methods: Myocardial cells were randomly divided into five groups:normal H9C2 group,AGEs group,low dose administration group(5 μmol/L),medium dose administration group(10 μmol/L),and high dose administration group(20 μmol/L).The cell activity was determined by CCk8 kit.The expression level of Myoglobin(Mb),Creatine kinase isoenzyme(CK-MB),myocardial troponin(cTnⅠ)were detected by related kits.The expression level of Beclin1,P62,LC3Ⅱ/LC3Ⅰ,PI3K and AKT were detected by Western blot.The expression of LC3 was detected by immunofluorescence staining. Results: Compared with the H9C2 group,in the AGEs group the growth rate of H9C2 cells decreased significantly.The expression of Mb,CK-MB and cTnⅠ was significantly increased.The expression level of Beclin1 was significantly up-regulated,LC3Ⅱ/LC3Ⅰ was significantly increased,and the expression of P62 decreased significantly.In addition,the LC3 puncta increased significantly.At the same time,the content of phosphorylated PI3K and phosphorylated AKT increased significantly.Compared with the AGEs group,in the administration group Betulin promoted the proliferation of H9C2 myocardial cells.The concentration of Mb,Ck-MB and cTnⅠ decreased significantly.The expression of Beclin1 was significantly down-regulated,the LC3Ⅱ/LC3Ⅰ decreased significantly,and the expression of P62 increased significantly.In addition,immunofluorescence results showed a significant decrease of LC3 puncta.Meanwhile,the expression of phosphorylation of PI3K and AKT was significantly decresed. Conclusion: Betulin could reduce the effect of AGEs on H9C2 myocardial cells autophagy by inhibiting the activation of PI3K/AKT to reduce the formation of autophagosome and increase the proliferation of cells.