非奈利酮通过抑制小胶质细胞炎症反应减轻大鼠糖尿病视网膜病变

Finerenone alleviates diabetic retinopathy in rats by inhibiting inflammatory response of microglia

目的:探究非奈利酮(finerenone,FIN)对糖尿病视网膜病变(diabetic retinopathy,DR)大鼠的治疗作用及其可能的作用机制。方法:(1)取30只清洁级雄性SD大鼠,随机选取10只作为对照(control)组,其余大鼠构建DR模型,建模成功后随机分为DR组和DR+FIN组,每组10只。DR+FIN组大鼠每天灌胃给予10 mg/kg的FIN,DR组大鼠灌胃给予等体积的生理盐水,连续干预3个月。采用双免疫荧光细胞化学法检测视网膜深层小胶质细胞的激活情况;采用CellF系统分析视网膜周细胞和无细胞型毛细血管(acellular capillaries,AC)数量;使用多焦视网膜成像系统检测大鼠神经视网膜功能;采用RT-qPCR和Western blot法检测各组大鼠视网膜组织中相关因子的mRNA和蛋白表达水平。(2)体外培养HAPI大鼠小胶质细胞,并随机分为正常葡萄糖(normal glucose,NG)组、高葡萄糖(high glucose,HG)组和HG+FIN组。NG组细胞在NG培养液(含1 g/L葡萄糖)中培养,HG组细胞在HG培养液(含4.5 g/L葡萄糖)中培养,HG+FIN组细胞在HG培养液中培养24 h后使用10 mmol/L的FIN处理。采用划痕实验检测各组细胞迁移能力。结果:(1)与control组比较,DR组大鼠视网膜周细胞数量显著减少(P<0.05),a波和b波振幅均显著降低(P<0.05),AC数量和深血管层的离子钙结合衔接分子1(ionized calcium-binding adaptor molecule 1,Iba1)表达显著增加(P<0.05),空腹血糖(fasting blood glucose,FBG)水平显著升高(P<0.05),视网膜组织中白细胞介素1β(interleukin-1β,IL-1β)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、IL-8、血管细胞黏附分子1(vas⁃cular cell adhesion molecule 1,Vcam1)、转化生长因子β1(transforming growth factor-β1,TGF-β1)、C-C基序趋化因子配体2(C-C motif chemokine ligand 2,Ccl-2)和盐皮质激素受体(mineralocorticoid receptor,MR)表达水平均显著升高(P<0.05);与DR组比较,DR+FIN组大鼠视网膜周细胞数量显著增加(P<0.05),a波和b波振幅均显著升高(P<0.05),AC数量和深血管层的Iba1表达均显著减少(P<0.05),FBG水平显著降低(P<0.05),视网膜组织中IL-1β、TNF-α、IL-8、Vcam1、TGF-β1、Ccl-2和MR表达水平均显著降低(P<0.05)。(2)FIN处理可显著抑制HG诱导的HAPI细胞迁移。结论:FIN可能通过抑制小胶质细胞激活而减轻其炎症反应,从而缓解大鼠DR。

AIM:To investigate the therapeutic effect of finerenone(FIN)on diabetic retinopathy(DR)in rats,and to explore its possible mechanism.METHODS:(1)A total of 30 clean-grade male SD rats were selected.Ten rats were randomly selected as control group,and the remaining rats were DR rats.After successful modelling,the DR rats were randomly divided into DR group and DR+FIN group,with 10 rats in each group.The rats in DR+FIN group received 10 mg/kg FIN by intragastric administration every day,while those in DR group were given an equal volume of normal saline intragastrically for 3 months.Double immunofluorescence staining was used to examine the activation of deep retinal microglia,and the CellF system was used to analyze the numbers of retinal pericytes and acellular capillaries(AC).A multifocal retinal imaging system was used to examine the neuroretinal function of the rats,and RT-qPCR and Western blot were used to examine the mRNA and protein expression of related factors.(2)Rat microglia HAPI cells were cultured in vitro and randomly divided into normal glucose(NG)group,high glucose(HG)group,and HG+FIN group.The cells in NG group was cultured in NG nutrient solution(containing 1 g/L glucose),the cells in HG group was cultured in HG nutrient solution(containing 4.5 g/L glucose),and the cells in HG+FIN group were cultured in HG medium for 24 h and treated with 10 mmol/L FIN.The migration of the cells in each group was examined by wound healing assay.RESULTS:(1)Compared with control group,the number of retinal pericytes,a-wave amplitude and b-wave amplitude were significantly decreased in DR group(P<0.05),while the number of AC,ionized calcium-binding adaptor molecule 1(Iba1)expression in deep retinal vascular layer,fasting blood glucose(FBG)level and the relative expression levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),IL-8,vascular cell adhesion molecule 1(Vcam1),transforming growth factor-β1(TGF-β1),C-C motif chemokine ligand 2(Ccl-2)and mineralocorticoid receptor(MR)in the retinal tissue were significantly increased(P<0.05).Compared with DR group,the number of retinal pericytes,a-wave amplitude and b-wave amplitude of the rats in DR+FIN group were significantly increased(P<0.05),while the number of AC,Iba1 expression in deep retinal vascular layer,FBG level and the relative expression levels of IL-1β,TNF-α,IL-8,Vcam1,TGF-β1,Ccl-2 and MR in the retinal tissue were significantly decreased(P<0.05).(2)Treatment with FIN significantly inhibited HAPI cell migration induced by HG.CONCLUSION:Treatment with FIN attenuates DR in rats by inhibiting microglial activation and inflammatory response.