Maresin1对高糖损伤肾小球系膜细胞的影响及其抗炎作用机制探讨

Effects of Maresin 1 on mesangial cells injured by high glucose and its anti-inflammatory effects

目的观察Maresin1对高糖刺激小鼠肾小球系膜细胞炎症反应的影响及其对核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体激活分泌炎症因子的抑制作用。方法培养小鼠肾小球系膜细胞株,NC组培养于低糖DMEM培养基中;OP组在低糖DMEM培养基中加入甘露醇;HG组培养于高糖DMEM培养基中;M1+HG组、M2+HG组、M3+HG组分别用1、10、100 nmol/L的Maresin1预处理后在高糖培养基中培养;M3+NC组用100nmol/L的Maresin 1预处理30 min后在低糖DMEM培养基中培养;另设N-乙酰半胱氨酸(NAC)+HG组作为活性氧抑制作用的阳性对照。用全光谱分光光度计和荧光显微镜观察活性氧(ROS)表达情况;采用RT-PCR法检测NLRP3、Caspase-1、IL-1βmRNA表达;采用Western blotting法检测NLRP3、pro-Caspase-1、Caspase-1、pro-IL-1β、IL-1β蛋白表达。结果与NC组相比,HG组细胞内ROS水平与NLRP3、Caspase-1、IL-1β蛋白及mRNA表达均增加,pro-Caspase-1、pro-IL-1β蛋白表达降低(P均<0.05);与HG组相比,M1+HG组、M2+HG组、M3+HG组ROS水平与NLRP3、Caspase-1、IL-1β蛋白及mRNA表达均降低,pro-Caspase-1、pro-IL-1β蛋白表达均增加(P均<0.05),以M3+HG组作用最明显。与HG组相比,M3+HG组NLRP3、Caspase-1、IL-1β蛋白及mRNA表达均降低,proCaspase-1、pro-IL-1β表达均增加(P均<0.05)。结论 Maresin1可抑制高糖刺激导致的小鼠肾小球系膜细胞氧化应激反应及炎症反应,其机制可能与降低ROS水平、抑制NLRP3炎症小体及相关炎症因子的表达有关。

Objective To observe the effects of Maresin1 on the inflammation of mouse glomerular mesangial cells（ GMCs） stimulated by high glucose,and its inhibitory effect on the NLR family pyrin domain-containing 3（ NLRP3） inflammasome activation. Methods We cultured GMCs and categorized as follows： normal control group（ NC group） was cultured in low glucose DMEM medium,osmotic pressure control group（ OP group） was added with mannitol in low glucose DMEM medium,high glucose group（ HG group） was cultured in high glucose DMEM medium and Maresin 1 intervention groups（ M1 ＋ HG group,M2 ＋ HG group,and M3 ＋ HG group） were treated with different concentrations of Maresin 1（ 1,10,and 100 nmol/L） for 30 min before being exposed to high glucose,M3 ＋ NC group was treated with 100 nmol/L maresin 1 for 30 min before being exposed to high glucose; meanwhile,the N-acetylcysteine（ NAC） intervention group（ NAC ＋ HG group） was taken as a positive control of reactive oxygen species inhibition. We used spectrophotometer and fluorescence microscopy to detect the level of ROS. RT-PCR was used to detect the mRNA expression of NLRP3,Caspase-1,and IL-1β. Western blotting was used to detect the protein expression of NLRP3,pro-Caspase-1,Caspase-1,pro-IL-1β,and IL-1β. Results Compared with the NC group,the ROS level and the protein and mRNA expression levels of NLRP3,Caspase-1,and IL-1β increased,but the protein expression levels of pro-Caspase-1 and pro-IL-1β decreased in the HG group（ all P〈0. 05）. Compared with the HG group,the ROS level and the protein and mRNA expression levels of NLRP3,Caspase-1,and IL-1β decreased,but the protein expression of pro-Caspase-1 and pro-IL-1β increased in the M1＋ HG group,M2 ＋ HG group,and M3 ＋ HG group,especially in the M3 ＋ HG group（ all P〈0. 05）. Compared with the HG group,the protein and mRNA expression levels of NLRP3,Caspase-1,and IL-1β decreased but the expression of proCaspase-1 and pro-IL-1β increased in the M3 ＋ HG group（ all P〈0. 05）. Conclusion Maresin 1 can inhibit the oxidative stress and inflammatory response of GMCs stimulated by high glucose,and its mechanism may be related to the decrease of ROS level,inhibition of NLRP3 inflammasome and its related inflammatory factors.