DAIa2GIP抑制软骨细胞线粒体凋亡的机制

Mechanism of DAIa2GIP inhibiting mitochondrial apoptosis in chondrocytes

背景:课题组前期研究表明,葡萄糖依赖性促胰岛素多肽新型类似物DAIa2GIP对软骨细胞有保护作用,但其作用机制尚不清楚。目的:观察葡萄糖依赖性促胰岛素多肽新型类似物DAIa2GIP拮抗白细胞介素1β诱发软骨细胞线粒体凋亡的效应,同时探究其分子生物学机制。方法:提取SD大鼠幼鼠肋软骨细胞,SABC免疫组化染色法鉴定后,取传代培养第3代细胞不同处理分为6组:①正常对照组;②白细胞介素1β诱导组;③白细胞介素1β+DAla2GIP孵育组;④白细胞介素1β+DAla2GIP+pro3GIP(GIPR拮抗剂)孵育组;⑤DAla2GIP孵育组;⑥pro3GIP孵育组。药物处理48 h后进行线粒体膜电位及Annexin-V FITC kit(磷脂酰丝氨酸外翻分析)细胞凋亡检测、激光共聚焦显微镜细胞钙超载测定及ELISA法测定细胞色素C含量。实验方案经山西医科大学动物实验伦理委员会批准。结果与结论:①SABC免疫组化染色法显示,所提取细胞Ⅱ型胶原可显影,为软骨细胞;②线粒体膜电位保持程度:白细胞介素1β+DAla2GIP孵育组明显高于白细胞介素1β诱导组,均低于正常对照组;软骨细胞凋亡率:白细胞介素1β+DAla2GIP孵育组明显低于白细胞介素1β诱导组,均高于正常对照组;钙超载程度以荧光强度进行比较:白细胞介素1β+DAla2GIP孵育组明显低于白细胞介素1β诱导组,均高于正常对照组;软骨细胞线粒体细胞色素C的释放:白细胞介素1β+DAla2GIP孵育组显著低于白细胞介素1β诱导组(P<0.01),均高于正常对照组(P<0.01);上述4项指标,白细胞介素1β诱导组与白细胞介素1β+DAla2GIP+pro3GIP孵育组比较差异无显著性意义,两组均低于正常对照组;正常对照组、DAla2GIP孵育组及pro3GIP孵育组比较均差异无显著性意义;③结果说明,DALa2GIP孵育可以有效拮抗白细胞介素1β诱导的大鼠软骨细胞线粒体功能障碍,从而拮抗其所致的软骨细胞凋亡,这一过程中DALa2GIP可以有效降低细胞钙超载程度,并且减少细胞色素C等的释放。

BACKGROUND:The previous research of our group has shown that DAIa2GIP,a new analogue of glucose dependent insulin stimulating polypeptide,has a protective effect on chondrocytes,but its mechanism is not clear.OBJECTIVE:To observe the effect of DAIa2GIP on interleukin-1β(IL-1β)induced apoptosis of chondrocytes,and to explore its molecular biological mechanism.METHODS:The costal chondrocytes of Sprague-Dawley rats were extracted and identified by SABC immunohistochemistry.The third generation cells were divided into six groups:(1)normal control group;(2)IL-1βinduction group;(3)DAIa2GIP+IL-1βinduction group;(4)DAIa2GIP+IL-1β+pro3GIP(GIPR antagonists)induction group;(5)DAIa2GIP induction group;(6)pro3GIP induction group.After 48 hours of drug treatment,mitochondrial membrane potential was tested,cell apoptosis was detected using Annexin-V FITC Kit(phosphatidylserine eversion analysis),calcium overload of cells was determined under laser confocal microscope,and cytochrome C content were measured by ELISA.The study protocol was approved by the Animal Ethics Committee of Shanxi Medical University.RESULTS AND CONCLUSION:SABC immunohistochemical staining showed that collagen II could be developed as chondrocytes;the maintenance degree of mitochondrial membrane potential was significantly higher in the IL-1β+DAIa2GIP incubation group than the IL-1βinduction group,but the potential in both groups was lower than that in the normal control group.The apoptotic rate of chondrocytes in the IL-1β+DAIa2GIP incubation group was significantly lower than that in the IL-1βinduction group,but the apoptotic rate in both groups was higher than that in the normal control group.The degree of calcium overload was compared with fluorescence intensity,and the result showed that the IL-1β+DAIa2GIP incubation group had a significantly higher intensity than the IL-1βinduction group,and the fluorescence intensity in both groups was higher than that in the normal control group.The release of chondrocyte mitochondrial cytochrome C in the IL-1β+DAIa2GIP incubation group was significantly lower than that in the IL-1βinduction group(P<0.01),and the release amount in both groups was significantly higher than that in the normal control group(P<0.01).The above four indicators showed no significant difference between the IL-1βinduction group and the DAIa2GIP+IL-1β+pro3GIP group,but the values in both groups were lower than those in the control group.There was no significant difference among the normal control group,DAIa2GIP induction group,and pro3GIP induction group.To conclude,DALa2GIP can effectively antagonize IL-1βinduced mitochondrial dysfunction of rat chondrocytes,thus antagonizing chondrocyte apoptosis.In this process,DALa2GIP can effectively reduce the degree of calcium overload and the release of cytochrome C.