p38 MAPK抑制剂对高糖诱导神经母细胞瘤细胞凋亡的作用及机制

Effect of p38 MAPK antagonist on high glucose-induced apoptosis of neuroblastoma

目的探讨丝裂原活化蛋白激酶p38(p38 MAPK)抑制剂对高糖诱导神经母细胞瘤细胞SH-SY5Y凋亡的保护作用及机制。方法将培养好的SH-SY5Y细胞随机分为6组,A、B、C组分别加入45、90、135 mmol/L高糖进行处理; D、E组分别加入10、25μmol/L的p38 MAPK抑制剂PD169316预处理30 min,之后加入135 mmol/L高糖溶液进行培养;对照组常规培养不做干预;另随机选部分细胞用135 mmol/L高糖处理3 h作为高糖对照组。采用Western blotting法检测细胞内磷酸化p38 MAPK及凋亡相关蛋白,用Annexin V-PI染色法检测细胞凋亡。结果高糖处理1. 5、3 h,与对照组比较,A、B、C组磷酸化p38 MAPK蛋白相对表达量呈浓度依赖性升高、抗凋亡蛋白BCL-2相对表达量低、促凋亡蛋白BAX、BID、BAD相对表达量高(P均<0. 05)。高糖处理6 h,A组BCL-2蛋白相对表达量低,BAX、BID、BAD蛋白相对表达量高(P均<0. 05),而B、C组促凋亡蛋白未见明显变化(P均> 0. 05)。与高糖对照组比较,D、E组磷酸化p38 MAPK蛋白相对表达量低(P均<0. 05),BCL-2蛋白蛋白相对表达量高(P均<0. 05),BAD蛋白及BID蛋白相对表达量低(P均<0. 05),E组以上指标变化最明显(P均<0. 05)。与对照组比较,C组细胞凋亡重;与C组比较,E组细胞凋亡轻。结论 p38 MAPK抑制剂可抑制高糖诱导的SH-SY5Y细胞凋亡,其作用机制可能与调控抗凋亡蛋白及促凋亡蛋白表达有关。

Objective To explore the protective effect and the possible mechanism of p38 mitogen-activated protein kinase (MAPK)antagonist on high glucose-induced apoptosis of neuroblastoma SH-SY5Y cells. Methods SH-SY5Y cells were randomly divided into 6 groups. The cells in the groups A,B,and C were cultured and treated with high glucose (45 mmol/L for group A,90 mmol/L for group B and 135 mmol/L for group C),the cells in the groups D and E were pretrea-ted with 10 and 25 μmol/ L p38 MAPK inhibitor PD169316 for half an hour,then followed by 135 mmol/ L high glucose so-lution for culture,while cells in the control group received routine culture. We randomly selected some cells and treated them with 135 mmol/ L high glucose for 3 h as the high glucose control group. The change of p38 MAPK phosphorylation and apoptosis-associated protein expression as well as the apoptosis was evaluated by Western blotting. The apoptosis was detected by Annexin V-PI staining. Results After treatment of high glucose for 90 min and 3 h,the phosphorylated p38 MAPK,BAX,BID and BAD increased significantly (all P <0.05),while the anti-apoptotic protein BCL-2 decreased sig-nificantly in the groups A,B and C (all P <0.05). After the treatment of high glucose for 6 h,the expression of BCL-2 protein in the group A was low,and the relative expression of BAX,BID,and BAD proteins was high (all P < 0.05), while there was no significant change in the proapoptotic proteins in the groups B and C (all P >0.05). Compared with the high glucose control group,the phosphorylated p38 MAPK protein in the groups D and E was low (P <0.05),the relative expression of BCL-2 protein was high (P <0.05),the relative expression of BAD protein and BID protein was low (both P <0.05),and the changes in the group E were the most significant (all P <0.05). Compared with the control group,the apoptosis level of group C was high,and compared with group C,the apoptosis level of group E was lower. Conclusion P38 MAPK inhibitor inhibits high glucose-induced apoptosis of SH-SY5Y cells and the mechanism may be related to the regulation of anti-apoptotic proteins and pro-apoptotic protein expression.