银杏叶提取物对高糖诱导的人视网膜色素上皮细胞凋亡的影响

目的:观察银杏叶提取物对高糖诱导的人视网膜色素上皮细胞ARPE-19细胞凋亡的影响并初步探讨可能机制。方法:体外培养ARPE-19细胞株,分为正常组(5.5 mmol·L-1葡萄糖),高糖组(30 mmol·L-1葡萄糖),银杏叶提取物组(30mmol·L-1葡萄糖+12.5,25,50,100 mg·L~(-1)银杏叶提取物)。噻唑蓝(MTT)比色法检测细胞活性;流式细胞术测定细胞凋亡率;采用实时荧光定量PCR(Real-time PCR)和蛋白免疫印迹法(Western blot)测定B细胞白血病/淋巴瘤相关抗原-2(Bcl-2),白血病/淋巴瘤相关抗原相关X蛋白(Bax),半胱氨酸天冬氨酸蛋白酶-3(Caspase-3),凋亡相关因子(Fas),Fas受体(Fas L)mRNA和蛋白的表达。Western blot检测c-Jun氨基末端激酶(JNK)磷酸化及核JNK表达变化。结果:高糖组吸光度A明显低于正常组(P0.05),12.5,25,50,100 mg·L~(-1)银杏叶提取物组比高糖组A明显升高(P0.05);高糖组细胞凋亡率明显高于正常组,12.5,25,50,100 mg·L~(-1)银杏叶提取物组比高糖组细胞凋亡率明显降低(P0.05);与正常组比较,高糖组Bax和Caspase-3,Fas和Fas L表达升高(P0.05),12.5,25,50,100 mg·L~(-1)银杏叶提取物组能降低Bax和Caspase-3的表达,升高Bcl-2表达水平,降低Fas和Fas L表达(P0.05);与正常组比较,高糖组JNK磷酸化及核JNK蛋白水平升高(P0.05),12.5,25,50,100 mg·L~(-1)银杏叶提取物降低JNK磷酸化及核JNK表达(P0.05)。结论:银杏叶提取物能通过JNK通路,抑制细胞凋亡,改善糖尿病视网膜病变。

Effect of ginkgo leaves extract on Apoptosis in High Glucose-induced Human Retinal Pigment Epithelial Cells

Objective: To investigate the effect of ginkgo leaves extract on the apoptosis in high-glucose-induced human retinal pigment epithelial cells, in order to explore possible mechanisms.Method: In vitro, ARPE-19 cells was divided into normal control group (5.5 mmol · L^-1 glucose), high-glucose group(30 mmol · L^-1 glucose) and ginkgo leaves extract group (30 mmol · L^-1 glucose+12.5, 25, 50, 100 mg · L^-1 ginkgo leaves extract). MTT assay was used to detect cell viability. Flow cytometry was used to determine the apoptotic rate. Bcl-2, Bax, Caspase-3, Fas and FasL mRNA and protein expressions were measured by Real-time PCR and Western blot. Western blot was used to detect the phosphorylation of JNK and the expression of nuclear JNK.Result: A value of the high-glucose group was significantly lower than that of the control group. Those in 12.5, 25, 50,100 mg · L^-1 ginkgo leaves extract groups were significantly higher than those of the high-glucose group (P<0.05). The apoptotic rate of the high-glucose group was significantly higher than that of the control group. Those in 12.5, 25, 50,100 mg · L^-1 ginkgo leaves extract groups were significantly lower than those of the high-glucose group (P<0.05). Compared with the control group, expressions of Bax, Caspase-3, Fas and FasL in the high-glucose group were increased. And expressions of Bax, Caspase-3, Fas and FasL were decreased by 12.5, 25, 50, 100 mg · L^-1 ginkgo leaves extract (P<0.05). JNK phosphorylation and nuclear JNK protein were increased in the high-glucose group, compared with the control group; and JNK expression was decreased in 12.5, 25, 50, 100 mg · L^-1 ginkgo leaves extract groups (P<0.05).Conclusion: ginkgo leaves extract could alleviate diabetic retinopathy and inhibit apoptosis through the JNK pathway.