人参二醇诱导3T3-L1脂肪前体细胞凋亡及其作用机制

Effect of apoptosis of 3T3-L1 adipocytes induced by panaxadiol and its mechanism

目的:探讨人参二醇(PD)诱导3T3-L1脂肪前体细胞凋亡作用,并阐明其可能的作用机制。方法:将3T3-L1脂肪前体细胞分为空白对照组和不同浓度PD处理组,采用CCK-8法测定0~100μmol·L^(-1)PD作用后未分化3T3-L1脂肪前体细胞及分化后成熟脂肪细胞的细胞活性。不同浓度(0、5、25和50μmol·L^(-1))PD作用于3T3-L1脂肪前体细胞,采用DAPI染色观察细胞凋亡的形态表现,流式细胞术检测3T3-L1脂肪前体细胞凋亡率和不同细胞周期细胞百分率,Western blotting法检测各组细胞中磷酸肌醇3激酶(PI3K)、磷酸化磷酸肌醇3激酶(p-PI3K)、蛋白激酶B(AKT)、磷酸化蛋白激酶B(p-AKT)、B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)和裂解的天冬氨酸特异性半胱氨酸蛋白酶3(cleaved-Caspase 3)蛋白表达水平,计算p-PI3K/PI3K和p-AKT/AKT比值。结果:CCK-8实验,与空白对照组比较,不同浓度PD处理组3T3-L1脂肪前体细胞的细胞活性降低(P<0.05或P<0.01),成熟脂肪细胞的细胞活性差异无统计学意义(P>0.05);DAPI染色实验,与空白对照组比较,不同浓度PD处理组3T3-L1脂肪前体细胞轮廓变得不规则,出现核碎裂,观察到凋亡小体与染色质碎片;流式细胞术检测,与空白对照组比较,不同浓度PD处理组3T3-L1脂肪前体细胞凋亡率升高(P<0.01),S期细胞百分率明显升高(P<0.05或P<0.01);Western blotting法检测,与空白对照组比较,不同浓度PD处理组细胞中Bax和cleaved-Caspase 3蛋白表达水平升高(P<0.05或P<0.01),Bcl-2蛋白表达水平降低(P<0.05或P<0.01),p-PI3K/PI3K和p-AKT/AKT比值降低(P<0.05或P<0.01)。结论:PD能够明显诱导3T3-L1脂肪前体细胞发生凋亡,其作用机制可能与其影响凋亡相关蛋白表达水平有关。

Objective:To investigate the induction effect of panaxadiol(PD)on the apoptosis of 3T3-L1 preadipocytes,and to elucidate its possible mechanism.Methods:The cells were divided into blank control group and different concentrations of PD treatment groups.The cell viabilities of undifferentiated 3T3-L1 adipocytes and differentiated mature adipocytes after treated 0-100μmol·L^(-1)with PD were determined by CCK-8 method.After the 3T3-L1 preadipocytes were treated with different concentrations(0,5,25,and 50μmol·L^(-1))of PD,DAPI staining was used to observe the morphology of apoptosis.Flow cytometry was used to detect the apoptotic rates and the percentages of 3T3-L1 preadipocytes in different cell cyeles.The protein expression levels of phosphatidylinositol 3-kinase(PI3K),phosphorylated phosphatidylinositol 3-kinase(p-PI3K),protein kinase B(AKT),phosphorylated protein kinase B(p-AKT),B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax)and cleaved-Caspase 3 in each group were detected by Western blotting method,and the ratios of p-PI3K/PI3K and p-AKT/AKT were calculated.Results:The CCK-8 results showed that compared with blank control group,the cell viabilities of 3T3-L1 preadipocytes in treatment groups were decreased(P<0.05 or P<0.01),but the cell activity of mature adipocytes had no significant differences(P>0.05).The DAPI staining results showed that compared with blank control group,the contour of 3T3-L1 preadipocytes in different concentrations of PD treatment became irregular and nuclear fragmentation was observed;the apoptotic bodies and chromatin fragments were observed.The results of flow cytometry showed that compared blank control group,the apoptotic rates of 3T3-L1 preadipocytes in different concentrations of PD treatment groups(P<0.01),and the percentages of cells in S phase were increased(P<0.05 or P<0.01).The Western blotting results showed that compared with blank control group,the expression levels of Bax and cleaved-Caspase 3 in different concentrations of PD treatment groups were increased(P<0.05 or P<0.01),and the expression levels of Bcl-2 were decreased(P<0.05 or P<0.01);the ratios of p-PI3K/PI3K and p-Akt/Akt were decreased(P<0.05 or P<0.01).Conclusion:PD can significantly induce the apoptosis of 3T3-L1 preadipocytes,and its mechanism may be related to its influence in the expression levels of apoptosis-related proteins.