基于HIF-1α/自噬途径探讨二氯化钴调控3T3-L1脂肪细胞炎症反应及胰岛素抵抗的机制

Effect of cobalt dichloride regulating the inflammatory response and insulin resistance in 3T3-L1 adipocytes through the HIF-1α/autophagy pathway

目的探讨低氧诱导剂二氯化钴(CoCl_(2))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,CoCl_(2)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CoCl_(2)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0 h(对照组)、12 h、24 h、48 h CoCl_(2)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LC3-Ⅱ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 CoCl_(2)是调控3T3-L1脂肪细胞自噬水平的最佳干预浓度;150μmol/L CoCl_(2)干预成熟的脂肪细胞24 h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;48 h时自噬水平减低,细胞存活率出现显著减低,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 CoCl_(2)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。

ObjectiveTo investigate the effect of hypoxia inducer cobalt dichloride(CoCl_(2))on autophagy of 3T3-L1 adipocytes.Methods3T3-L1 adipocytes were cultured and induced to mature adipocytes.CoCl_(2) was used as an inducer of hypoxia in vitro.Mature adipocytes were divided into control and CoCl_(2) treatment groups with various concentrations and times.In accordance with the result,150μmol/L cobalt dichloride was selected to treat adipocytes for 0,12,24 and 48 h.Then,the cells were collected for related tests.MTT assays were used to assess cell survival.Expression of HIF-1α,autophagy-related protein LC3-Ⅱ,Beclin-1 and Glut-1 was analyzed by Western blot.The level of autophagy was measured by immunofluorescence.TNF-αand IL-6 levels in culture supernatants of adipocytes were measured by ELISA.Results150μmol/L CoCl_(2) is the optimal intervention concentration to regulate the autophagy level of 3T3-L1 adipocytes.The levels of autophagic activity increased without reduction in cell viability after 150μmol/L CoCl_(2) intervention in mature adipocytes for 24 h.The expression protein levels of HIF-1α,LC3-Ⅱ,Beclin-1 and Glut-1 were significantly increased in adipocytes.However,the secretion level of inflammatory factors TNF-αand IL-6 did not increased significantly.The level of autophagic activity and cell viability were decreased at 48 h.The expression protein levels of HIF-1α,LC3-Ⅱ,Beclin-1 and Glut-1 were significantly decreased in adipocytes.However,the increased secretion of inflammatory cytokines TNF-αand IL-6 were observed.ConclusionsAutophagy was moderately activated in adipocytes by 150μmol/L CoCl_(2) treatment for 24 hours.Autophagy was activated in a HIF-1α-dependent manner,which plays a role in protecting adipocytes from inflammatory damage and improving insulin resistance.