1,25(OH)_2D_3对人肾小球系膜细胞凋亡活性的影响及机制探讨

Effect of 1,25(OH)_2D_3 on apoptosis of human mesangial cells and its mechanism

目的探讨1,25(OH)2D3对人肾小球系膜细胞(HMC)凋亡活性及凋亡相关因子表达的影响,并探讨其机制。方法体外培养HMC,随机分为空白对照组(N组)、表皮生长因子(EGF)组(E组)、1,25(OH)2D3组(V组)、EGF联合1,25(OH)2D3组(EV组)。N组在5%胎牛血清的DMEM培养基中培养,E组在DMEM培养基中加入含10 ng/m L EGF的培养液,V组加入含10-8mol/L 1,25(OH)2D3的培养液,EV组加入含10 ng/m L EGF及10-8mol/L 1,25(OH)2D3的培养液,各组均干预48 h。应用半胱氨酸天冬氨酸蛋白酶3(Caspase-3)活性试剂盒检测各组Caspase-3活性,Western blot法检测各组凋亡相关因子Akt、磷酸化Akt(p-Akt)、Caspase-3、Caspase-8、Caspase-9蛋白表达,荧光实时定量PCR法检测各组Caspase-3、Caspase-8、Caspase-9 mRNA表达。结果 E组Caspase-3活性低于N组,V组及EV组Caspase-3活性高于N组,EV组Caspase-3活性高于E组(P均<0.05)。E组p-Akt/Akt值高于N组,V组p-Akt/Akt值低于N组,EV组p-Akt/Akt值低于E组(P均<0.05)。E组Caspase-3、Caspase-9蛋白及mRNA表达均低于N组,V组Caspase-3、Caspase-9蛋白及mRNA表达均高于N组,EV组Caspase-3、Caspase-9蛋白及mRNA表达均高于E组(P均<0.05)。结论 1,25(OH)2D3可增强HMC的凋亡活性,从而发挥促HMC凋亡的作用,其机制可能为通过抑制Akt磷酸化,上调Caspase-3、Caspase-9的表达。

Objective To investigate the effects of 1,25-dihydroxyvitamin D3[1,25（OH）2D3 ]on apoptosis and expression of apoptosis-related factors in human mesangial cells（ HMC）. Methods The cultured HMC were randomly divided into four groups： normal control group（ group N,cultured in DMEM medium with 5% fetal bovine serum）,epidermal growth factor（ EGF） group（ group E,cultured in DMEM medium plus 10 ng/m L EGF）,1,25（OH）2D3 group（ group V,cultured in DMEM medium plus 10-8mol/L 1,25（OH）2D3 ）,and the EGF ＋ 1,25（OH）2D3 group（ group VE,cultured in DMEM medium plus 10 ng/m L EGF and 10-8mol/L 1,25（OH）2D3 ）,they were all treated for 48 h. The Caspase-3activity was detected by the Casapase-3 kits. The protein expression of Akt,phosphorylated Aakt（ p-Akt） Caspase-3,Caspase-8,and Caspase-9 was detected by Western blotting. Meanwhile,the mRNA expression of Caspase-3,Caspase-8,and Caspase-9 mRNA was detected by RT-PCR. Results The activity of Caspase-3 in the group E was lower than that in the group N,the activity of Caspase-3 in the groups V and EV was higher than that in the group N,and the activity of Caspase-3 in the group EV was higher than that in the group E（ all P 〈0. 05）. The ratio of p-Akt/Akt in the group E was higher than that in the group N,and the ratio of p-Akt/Akt in the group V was lower than that in the group N,and the ratio of p-Akt/Akt in the group EV was lower than that in the group E（ all P 〈0. 05）. The protein and mRNA expression of Caspase-3 and Caspase-9 in the group E was lower than that in the group N,the expression of Caspase-3 and Caspase-9 in the group V was higher than that in the group N,and the expression of Caspase-3 and Caspase-9 in the group EV was higher than that in the group E（ all P〈 0. 05）. Conclusion 1,25（OH）2D3 may enhance the apoptosis activity of HMC and thus promote the apoptosis of HMC by up-regulating the expression of Caspase-3/9 and down-regulating the expression of pAkt.