高糖及巨噬细胞共培养对成骨细胞氧化应激及自噬的影响

Effects of hyperglycemia and macrophage co-culture on oxidative stress and autophagy in osteoblasts

目的 探讨在高糖环境下,与巨噬细胞Raw264.7共培养的成骨细胞MC3T3-E1氧化应激、自噬及细胞凋亡的变化。方法 (1)根据培养基的葡萄糖浓度将Raw264.7细胞分为25、30、35 mmol/L 3组,流式细胞术检测细胞CD86表达;RT-qPCR检测IL-6、TNF-α、iNOS、NOX2、p47phox的mRNA表达水平,Western blot检测iNOS的蛋白表达水平,DCFH-DA探针检测ROS水平。(2)MC3T3-E1细胞分为正常糖单独培养组(5.5 mmol/L葡萄糖浓度,5.5-Mono组)、高糖单独培养组(35 mmol/L葡萄糖浓度,35-Mono组)和高糖共培养组(与Raw264.7细胞共培养24 h, 35-Co组)3组,DCFH-DA探针检测MC3T3-E1细胞ROS水平;透射电镜、Western blot检测MC3T3-E1细胞自噬水平;流式细胞术检测MC3T3-E1细胞凋亡水平。结果 高糖下Raw264.7细胞表面标志CD86表达增加,与25 mmol/L组相比,35 mmol/L组IL-6、TNF-α、iNOS的mRNA表达水平显著增加(P<0.05),iNOS蛋白表达水平显著增加(P<0.05),ROS水平增加,NOX2、p47phox的mRNA表达水平显著增加(P<0.05);与5.5-Mono组相比,35-Mono、35-Co组MC3T3-E1细胞ROS均增加且35-Co组增加更明显,透射电镜显示35-Co组自噬溶酶体数量明显增加,35-Co组MC3T3-E1细胞中LC3-II及SQSTM1/p62的蛋白表达水平明显升高(P<0.05),35-Co组的MC3T3-E1细胞凋亡率较35-Mono组明显升高(P<0.05)。结论 与高糖下M1向极化的Raw264.7细胞共培养会诱导MC3T3-E1细胞凋亡,该作用可能与MC3T3-E1细胞胞内ROS增加和自噬过强有关。

Objective To evaluate the effects of high glucose on oxidative stress, autophagy, and apoptosis in MC3 T3-E1 cells co-cultured with Raw264.7 cells. Methods(1)Raw264.7 cells were separated into 3 groups based on the glucose levels in the medium(25, 30, and 35 mmol/L). Flow cytometry was used to detect CD86 expression. RT-qPCR was employed to detect the mRNA expression levels of IL-6, TNF-α, iNOS, NOX2, and p47phox in Raw264.7 cells. Western blotting was used to detect the protein expression level of iNOS. The DCFH-DA probe was used to measure the level of reactive oxygen species(ROS) in Raw264.7 cells.(2)MC3 T3-E1 cells were divided into 3 groups: normal glucose group(5.5 mmol/L glucose, 5.5-Mono), high glucose mono-culture group(35 mmol/L glucose, 35-Mono) and high glucose co-culture group(co-cultured with Raw264.7 for 24 h, 35-Co). The ROS level of MC3 T3-E1 cells were detected by DCFH-DA probe. The autophagic level was assessed by transmission electron microscopy(TEM) and the expression of related proteins was detected with Western blotting. Flow cytometry was applied to determine the rate of apoptosis. Results Compared with the normal glucose group, the expression level of CD86 in Raw 264.7 cells was increased in other high glucose groups. Compared with the 25 mmol/L group, the mRNA levels of IL-6, TNF-α, iNOS, NOX2, and p47phox were increased(P<0.05), the protein level of iNOS was elevated(P<0.05) and the ROS level in the Raw264.7 cells was raised in the 35 mmol/L group as well. Compared with the normal glucose group, the ROS level of MC3 T3-E1 cells in the high glucose groups was increased, especially in high glucose co-culture group. The number of autophagolysosomes in the MC3 T3-E1 cells were increased sharply in the high glucose co-culture group. The protein levels of LC3-II and SQSTM1/p62 in MC3 T3-E1 cells were increased significantly in the high glucose co-culture group(P<0.05). The apoptotic rate of MC3 T3-E1 cells was significantly higher in the high glucose co-culture group than the high glucose mono-culture group(P<0.05). Conclusion Co-culture with M1-polarized Raw264.7 cells under high glucose induces apoptosis in MC3 T3-E1 cells, which may be related to the production of ROS and over-enhancement of autophagy in MC3 T3-E1 cells.