晚期糖基化终末产物通过氧化应激诱导大鼠软骨细胞损伤∗

摘要

目的：：探讨晚期糖基化终末产物( advanced glycation end products,AGEs)能否通过氧化应激引起大鼠软骨细胞损伤。方法：原代培养SD大鼠软骨细胞,对细胞表型进行鉴定；应用CCK-8法检测软骨细胞生存率；DCFH-DA染色荧光显微镜下检测胞内活性氧簇( reactive oxygen species,ROS)的水平；Hoechst 33342核染色法及Annexin V-FITC/PI流式细胞法测定软骨细胞的凋亡率；RT-PCR法检测软骨细胞中Bax、Bcl-2、caspase-3、MMP3、MMP13和COL2的mRNA水平；Western blotting法检测软骨细胞中cleaved caspase-3、MMP3、MMP13和COL2的蛋白水平。结果：与对照组相比,AGEs可显著上调胞内ROS水平(P<0.05),但经抗氧化剂N-乙酰半胱氨酸(NAC)抑制后ROS的生成明显减少(P<0.05)；另外,NAC可抑制 AGEs 引起的软骨细胞凋亡相关分子 Bax/Bcl-2和caspase-3水平的上调,并减少MMP3和MMP13表达及COL2的丢失(P<0.05)。结论： AGEs可通过氧化应激诱导大鼠软骨细胞损伤。%AIM: To explore the possibility that advanced glycation end products (AGEs) induces rat chon-drocyte injury by modulating oxidative stress. METHODS:Primarily cultured rat chondrocytes were identified. The viabil-ity of the chondrocytes was measured by CCK-8 assay. The intracellular levels of reactive oxygen species ( ROS) were de-tected by DCFH-DA staining. The number of apoptotic cells was determined by Hoechst 33342 nuclear staining and flow cytometry. RT-PCR was performed to measure the mRNA levels of Bax, Bcl-2, caspase-3, MMP3, MMP13 and COL2 in the chondrocytes. Western blotting was used to evaluate the protein levels of cleaved caspase-3, MMP3, MMP13 and COL2. RESULTS:Compared with control group, the intracellular levels of ROS in the chondrocytes treated with AGEs were significantly increased (P<0. 05), and pretreatment with N-acetyl-L-cysteine (NAC) suppressed the formation of ROS (P<0. 05). Besides, NAC inhibited AGEs-induced apoptosis of the chondrocytes, as indicated by reduceing the levels of Bax/Bcl-2 and caspase-3, decreased the expression of MMP3 and MMP13, and reduced the loss of COL2. CON-CLUSION:AGEs induce chondrocyte injury by activating oxidative stress.