1.He University;2.General hospital of northern theater command;3.He unversity;4.Dongguan Kanghua Hosipital
目的 探讨低氧诱导剂二氯化钴(CoCl2)调控3T3-L1脂肪细胞自噬活性从而改善脂肪细胞炎症反应和胰岛素抵抗的机制。方法 常规培养和诱导分化3T3-L1脂肪细胞成为成熟的脂肪细胞，CoCl2作为低氧诱导剂在不同时间、不同浓度条件下干预成熟的脂肪细胞，确认CoCl2干预脂肪细胞自噬活性的最佳时间和浓度，随后根据此时间和浓度分组，分为对照组（0h组）、12h、24h、48h CoCl2处理组，收集细胞样本进行相关指标测定。MTT法评价各组细胞存活情况；Western blot分析各组细胞HIF-1α及其下游蛋白葡萄糖转运蛋白Glut-1、自噬相关蛋白LC3Ⅱ和Beclin 1的表达情况；免疫荧光法检测各组细胞的自噬水平；ELISA法检测各组细胞上清液中炎症因子TNF-α和IL-6分泌情况。结果 150μmol/L CoCl2是调控3T3-L1脂肪细胞自噬水平的最佳干预浓度；150μmol/L CoCl2干预成熟的脂肪细胞24h时，自噬活性水平增高，细胞存活率无显著的减低，脂肪细胞中HIF-1α、LC3-Ⅱ、Beclin 1、Glut-1蛋白的表达水平也显著升高，但炎症因子TNF-α和IL-6分泌水平无明显增加；48h时自噬水平减低，细胞存活率出现显著的减低，脂肪细胞中HIF-1α、LC3-Ⅱ、Beclin 1、Glut-1蛋白的表达水平减低，炎症因子TNF-α和IL-6分泌水平出现增加趋势。结论 150μmol/L CoCl2可以调控脂肪细胞自噬水平增加，自噬水平的增加以依赖HIF-1α的方式活化，从而使自噬发挥保护脂肪细胞的作用使其免受炎症损伤和改善脂肪细胞胰岛素抵抗。
Objective: To investigate the effect of hypoxia inducer cobalt chloride (CoCl2) on autophagy in 3T3-L1 adipocytes. Methods: 3T3-L1 adipocytes were cultured and induced to become mature adipocytes. CoCl2 was used as an inducer of hypoxia in vitro. Mature adipocytes were divided into control group, CoCl2 intervention group with different concentrations and different time. According to the above results, 150μmol/L cobalt chloride was selected to intervene adipocytes for 0h,12h,24h and 48h, respectively. Then the cells were collected for related tests. MTT assay was used to detect cell survival in each group. The expressions of HIF-1α, autophagy-related protein LC3Ⅱ, Beclin 1 and glucose transporter Glut-1 were analyzed by Western blot. The level of autophagy activity was detected by immunofluorescence.The levels of TNF-α and IL-6 in the supernatants of adipocytes were measured by ELISA. Results: The survival rate of adipocytes was decreased after treatment with 150μmol/L CoCl2 for 24 hours. The expression levels of HIF-1α, LC3-Ⅱ, Beclin 1 and Glut-1 proteins were significantly increased in 24h. Autophagy activity level was significantly increased in 24h. The secretion levels of inflammatory cytokines TNF-α and IL-6 were not significantly increased in 24h. Conclusion: Autophagy can be moderately activated by 150μmol/L CoCl2 intervention in adipocytes for 24 hours. The activation of autophagy can be activated in a HIF-1α-dependent manner, which plays a role in protecting adipocytes from inflammatory damage and improving insulin resistance.