胞外酸化对大鼠关节软骨细胞焦亡的影响及可能机制

Effects of extracellular acidosis on pyroptosis of rat articular chondrocytes and its possible mechanisms

目的观察胞外酸化对大鼠关节软骨细胞焦亡的影响,研究该过程中可能机制。方法利用酶消化法获得原代大鼠关节软骨细胞。软骨细胞分为不同pH处理组(pH7.4、pH 7.0、pH 6.5和pH 6.0)以及pH6.0酸化不同时间组(0、6、12、24、48 h),经ROS还原剂NAC预处理的酸化组和正常组,Real-time PCR和Western blot观察各组促炎细胞因子IL-1β、IL-18和炎症小体组成成分ASC、NLRP3、caspase-1基因和蛋白的表达情况,ELISA检测各组细胞培养基中的IL-1β、IL-18含量,AO/EB染色和LDH细胞毒性检测试剂盒分析细胞的死亡情况,ROS测定试剂盒观察细胞内ROS荧光强弱。结果与pH 7.4组比较,酸化组的促炎细胞因子IL-1β、IL-18和炎症小体组成成分ASC、NLRP3、caspase-1基因和蛋白的表达明显增加,荧光显微镜观察到ROS荧光明显增强,AO/EB染色和LDH检测实验发现酸化可以诱导软骨细胞死亡,且pH 6.0酸化处理组效果最明显;ROS还原剂NAC预处理可以明显下调IL-1β、IL-18、ASC、NLRP3、caspase-1基因和蛋白的表达,并且明显减弱软骨细胞内ROS荧光,同时明显下调细胞死亡率。结论胞外酸化可能通过上调细胞内ROS的含量,促使软骨细胞发生焦亡。

Aim To study the effects of extracellular acidosis on articular chondrocytes pyroptosis and its possible mechanisms. Methods Primary articular chondrocytes were incubated in different pH and NAC.The expression of proinflammatory cytokines IL-1β,IL-18, ASC, NLRP3, caspase-1 were detected by Western blot and real-time PCR. The state of pyroptosis was identified by AO/EB staining and LDH con-tents. The expression of ROS was observed by DCFHDA,and ELISA was used to detect the IL-1β,IL-18 in cultured supernatants. Results Compared with the normal cell,extracellular acidosis could increase the expression of IL-1β, IL-18, ASC, NLRP3 and caspase-1,upregulate the fluorescence intensity of intercellular ROS, accompanied with the promoted release of LDH. Moreover,it is observed that extra-cellular acidosis could also induce chondrocytes death by AO/EB staining. NAC,the scavenger of ROS could inhibit these effects of extracellular acidosis on chondrocytes. Conclusion Extracellular acidosis may in-duce chondrocyte pyroptosis via upregulating the intracellular ROS content.