【摘要】 目的:探讨细胞焦亡(pyroptosis)能否介导高糖(HG;45 mmol/L葡萄糖)引起的小鼠胚胎成骨细胞MC3T3-E1炎症和损伤。方法:应用细胞计数试剂盒8(CCK-8)检测成骨细胞活力;Western blot测定成骨细胞的核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)和胱天蛋白酶1(CASP1)的表达水平;ELISA法测定细胞培养上清液中白细胞介素18(IL-18)和IL-1β的水平;2’,7’-二氯二氢荧光素二乙酯染色荧光显微镜照相法检测胞内活性氧(ROS)水平;罗丹明123染色荧光显微镜照相法测定线粒体膜电位(MMP)水平;碱性磷酸酶(ALP)试剂盒测定成骨细胞早期标志物ALP的活性;茜素红染色观察成骨细胞晚期标志物矿化结节的形成。结果:HG处理MC3T3-E1细胞24 h可明显促进NLRP3和CASP1的表达,引起IL-18和IL-1β的分泌增多,同时可使细胞活力降低,ROS生成和MMP丢失增加,成骨细胞分化与矿化功能下降(表现为ALP活性降低和矿化结节数量减少)。利用siRNA沉默CASP1表达可显著减轻HG引起的上述成骨细胞炎症和损伤。结论:焦亡可介导HG引起的MC3T3-E1细胞炎症和损伤。更多还原

【Abstract】 AIM:To investigate whether pyroptosis contributes to the inflammation and injury in mouse embryonic osteoblastic cell line MC3 T3-E1 induced by high glucose(HG;45 mmol/L glucose).METHODS:The cell viability was measured by CCK-8 assay. The protein expression levels of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)and caspase-1(CASP1)were determined by Western blot. The secretion levels of interleukin-18(IL-18)and IL-1β were measured by ELISA. The intracellular level of reactive oxygen species(ROS)was detected by2’,7’-dichlorodihydrofluorescein diacetate staining followed by photofluorography. Mitochondrial membrane potential(MMP)was examined by rhodamine 123 staining followed by photofluorography. The alkaline phosphatase(ALP)activity was determined using the ALP kit,and the number of mineralized nodules was detected by alizarin red S staining.RESULTS:After the MC3T3-E1 osteoblasts were treated with HG for 24 h,the protein expression levels of NLRP3 and CASP1,and the secretion levels of IL-18 and IL-1β were significantly increased. The decrease in cell viability,and the increases in ROS generation and MMP loss were also observed. Moreover,the differentiation and mineralization of MC3T3-E1 osteoblasts were inhibited,evidenced by decreases in both ALP activity and mineralized nodule number. Knockdown of CASP1 by siRNA attenuated the HG-induced osteoblast inflammation and injury mentioned above.CONCLUSION:Pyroptosis mediates HG-induced inflammation and injury in MC3T3-E1 osteoblasts.更多还原