摘要
目的探究舒洛地特(SDX)通过调节NLRP3表达对视网膜Müller细胞凋亡的影响。方法将24只清洁级C57BL/6小鼠随机数字表法分为4组,正常组、DR组、DR+DMSO组、DR+SDX组,各6只。除正常组小鼠外,均采用腹膜内注射链脲佐菌素(STZ)诱导实验性糖尿病视网膜病变(DR)模型。正常组小鼠,标准饲料喂养;DR组模型小鼠42%高脂肪饮食;DR+DMSO组模型小鼠2%高脂肪饮食+1 mL•kg^(-1)•d^(-1)0.1%DMSO,DR+SDX组模型小鼠42%高脂肪饮食+1 g•kg^(-1)•d^(-1) SDX,连续干预21 d。通过蛋白质印迹法检测NLRP3炎症小体(免疫受体NLRP3、接头蛋白ASC和蛋白酶caspase-1)和促炎因子白细胞介素(IL)-1β的表达水平。然后从小鼠眼组织分离原代视网膜Müller细胞,分别在正常葡萄糖(NG组,5 mmol/L)或高糖葡萄糖(HG组,30 mmol/L)培养,用NLRP3炎症小体抑制剂MCC950(10μmol/L)处理24 h(HG+MCC950组),并用5 mmol/L NLRP3激动剂三磷酸腺苷(ATP)处理30 min(HG+SDX+ATP组)或使用1 mmol/L活性氧清除剂N-乙酰半胱氨酸(NAC)处理24 h(HG+NAC组),并用活性氧200μmol/L激动剂叔丁基过氧化氢(TBHP)处理最后3 h(HG+SDX+TBHP组),通过ELSIA检测氧化应激标志物活性氧、丙二醛、超氧化物歧化酶(SOD)和Müller细胞培养上清液中的血管内皮生长因子(VEGF)、造血生长因子(HGF)的表达水平。结果(1)与正常组相比,DR组NLRP3蛋白表达水平升高,差异有统计学意义(P<0.05);与DR组和DR+DMSO组相比,DR+SDX组NLRP3蛋白表达水平降低,差异有统计学意义(P<0.05)。(2)与正常组相比,DR组ASC、cleaved caspase-1/pro-caspase-1、cleaved IL-1β/pro-IL-1β水平均显著增加,差异均有统计学意义(P<0.05);与DR组和DR+DMSO组相比,DR+SDX组NLRP3以上指标表达均显著降低,差异均有统计学意义(P<0.05)。(3)与NG组相比,HG组和HG+DMSO组Müller细胞和培养液中VEGF和HGF均显著增加,差异均有统计学意义(P<0.05);与HG和HG+DMSO组相比,HG+MCC950和HG+SDX组细胞和培养液中VEGF和HGF均显著降低,而HG+SDX+ATP则显著高于HG+SDX组,差异均有统计学意义(P<0.05)。(4)与NG组相比,HG组和HG+DMSO组Müller细胞活性氧活性和丙二醛含量均显著增加,SOD活性则显著降低,差异均有统计学意义(P<0.05);与HG和HG+DMSO组相比,HG+NAC和HG+SDX组细胞中活性氧活性和丙二醛含量均显著降低,SOD活性则显著增加,差异均有统计学意义(P<0.05);与HG+SDX组相比,HG+SDX+TBHP组活性氧活性和丙二醛含量均显著增加,SOD活性则显著降低,差异均有统计学意义(P<0.05)。结论舒洛地特可通过抑制活性氧/NLRP3炎症小体信号转导来减轻高血糖诱导的Müller细胞增殖和促血管生成因子的产生。
Objective To explore the effect of sulodexide(SDX)on the apoptosis of retinal Müller cells by regulating the expression of NLRP3.Methods Twenty-four clean-grade C57BL/6 mice were used as the research objects,and the STZ experimental diabetes model was injected intraperitoneally,and they were randomly divided into 4 groups:normal group(normal mice,fed with standard chow),DR group(DR model mice),42%high-fat diet,DR+DMSO group(DR model mice,mice were given 2%high-fat diet+1 mL•kg^(-1)•d^(-1)0.1%DMSO by gavage)and DR+SDX group(DR model mice,mice were given 42%high-fat diet+1 g•kg^(-1)•d^(-1) SDX)by gavage,6 mice each,and were continuously intervened for 21 d.The expression levels of NLRP3 bodies(immunoreceptor NLRP3,adaptor protein ASC and protease caspase-1)and pro-inflammatory factor IL-1βwere verified by Western blotting.Primary retinal Müller cells were then isolated from mouse eye tissue,cultured in normal glucose(NG group,5 mmol/L)or high glucose glucose(HG group,30 mmol/L),and treated with NLRP3 inflammasome inhibitor MCC950(10μmol/L)for 24 h(HG+MCC950 group),and treated with 5 mmol/L NLRP3 agonist adenosine triphosphate(ATP)for 30 min(HG+SDX+ATP group)or treated with 1 mM ROS scavenger N-acetylcysteine(NAC)for 24 h(HG+NAC group),and treated with ROS 200μmol/L agonist tert-butyl hydroperoxide(TBHP)for the last 3 h(HG+SDX+TBHP group),and the expression levels of oxidative stress markers reactive oxygen species ROS,malondialdehyde MAD,superoxide dismutase SOD and Müller cell culture supernatants were detected by ELSIA.Results(1)Compared with the normal group,the expression level of NLRP3 protein in the DR group was increased,and the difference was statistically significant(P<0.05);compared with the DR group and the DR+DMSO group,the NLRP3 protein expression level in the DR+SDX group was decreased,and the difference was statistically significant(P<0.05).(2)Compared with the normal group,the levels of ASC,cleaved caspase-1/pro-caspase-1 and cleaved IL-1β/pro-IL-1βin the DR group were significantly increased,and the differences were statistically significant(P<0.05);compared with the DR group and the DR+DMSO group,the expression of NLRP3 and above in the DR+SDX group were significantly decreased,and the differences were statistically significant(P<0.05).(3)Compared with NG group,VEGF and HGF in Müller cells and culture medium in HG group and HG+DMSO group were significantly increased,and the differences were statistically significant(P<0.05);compared with HG and HG+DMSO group,VEGF and HGF in cells and culture medium of HG+MCC950 and HG+SDX group were significantly decreased,while HG+SDX+ATP was significantly higher than that of HG+SDX group,and the differences were statistically significant(P<0.05).(4)Compared with the NG group,the ROS activity and MDA content of Müller cells in the HG group and the HG+DMSO group were significantly increased,while the SOD activity was significantly decreased,and the differences were statistically significant(P<0.05);compared with HG and HG+DMSO group,ROS activity and MDA content in HG+NAC and HG+SDX group were significantly decreased,while SOD activity was significantly increased,and the differences were statistically significant(P<0.05);compared with the HG+SDX group,the ROS activity and MDA content in the HG+SDX+TBHP group were significantly increased,while the SOD activity was significantly decreased,and the differences were statistically significant(P<0.05).Conclusion Sulodexide can reduce hyperglycemia-induced Müller cell proliferation and pro-angiogenic factor production by inhibiting ROS//NLRP3 inflammasome signal transduction,thereby exerting a therapeutic effect on DR,therefore,it has certain application prospects in the treatment of this disease.
作者
尹晓艳
董永孝
刘彦章
薛涛
YIN Xiao-yan;DONG Yong-xiao;LIU Yan-zhang(Cataract Refractive Center,First People's Hospital of Xianyang City,Xianyang Shaanxi 712000,China;Department of Comprehensive Ophthalmology,First People's Hospital of Xianyang City,Xianyang Shaanxi 712000,China)
出处
《临床和实验医学杂志》
2022年第10期1013-1017,共5页
Journal of Clinical and Experimental Medicine
基金
陕西省卫生科研项目(编号:2018E12)。
