阿魏酸对糖尿病小鼠视网膜和高糖诱导的人RPE细胞损伤的抑制作用及其机制

Inhibitory effect of ferulic acid on the retina of diabetic mice and high glucose-induced human retinal pigmentepithelium cell injury and the mechanism

目的探讨阿魏酸对糖尿病小鼠视网膜和高糖诱导的人视网膜色素上皮(RPE)细胞损伤的抑制作用及其机制。方法选取SPF级雄性8周龄2型糖尿病db/db小鼠30只,采用随机数字表法将小鼠分为模型组和阿魏酸组,每组15只,另选取15只同周龄db/m小鼠作为对照组。模型组和对照组每日采用生理盐水灌胃(5 ml/kg),阿魏酸组采用阿魏酸溶液灌胃(0.05 g/kg),治疗后2个月处死各组小鼠并摘除眼球。采用苏木精-伊红染色观察小鼠视网膜组织形态学变化;采用免疫荧光染色法和Western blot法检测各组小鼠视网膜组织线粒体钙离子单向转运蛋白(MCU)、p38丝裂原活化蛋白激酶(p38MAPK)、磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)蛋白荧光强度和表达水平。取人RPE细胞,将其分为对照组、二甲基亚砜(DMSO)组、高糖组和高糖+阿魏酸组,其中对照组不做任何处理,其余各组用相应试剂培养24 h。采用活性氧簇(ROS)检测试剂盒检测各组RPE细胞ROS水平;采用线粒体膜电位检测试剂盒(JC-1)检测各组RPE细胞线粒体膜电位水平;采用微丝绿色荧光探针检测各组RPE细胞MCU和微丝荧光强度;通过慢病毒转染技术沉默和过表达MCU蛋白水平探讨MCU与p38 MAPK、p-p38MAPK间的调控关系;采用免疫荧光染色法和Western blot法检测各组RPE细胞MCU、p38 MAPK、p-p38MAPK蛋白荧光强度和表达水平。结果与对照组比较,模型组小鼠视网膜组织外核层、内核层和神经节细胞层细胞间隙增大、排列紊乱,阿魏酸组小鼠视网膜组织明显改善。与对照组比较,模型组、阿魏酸组小鼠视网膜组织MCU、p-p38 MAPK和MCU+p-p38 MAPK蛋白荧光强度显著升高,差异均有统计学意义(均P<0.05);与模型组比较,阿魏酸组小鼠视网膜组织MCU、p-p38 MAPK和MCU+p-p38 MAPK蛋白荧光强度显著降低,差异均有统计学意义(均P<0.05)。与对照组比较,模型组小鼠视网膜组织MCU、p38 MAPK和p-p38 MAPK蛋白相对表达量显著升高,差异均有统计学意义(均P<0.05);与模型组比较,阿魏酸组小鼠视网膜组织MCU、p38 MAPK和p-p38 MAPK蛋白相对表达量显著降低,差异均有统计学意义(均P<0.05)。对照组、DMSO组、高糖组和高糖+阿魏酸组细胞ROS荧光强度分别为0.22±0.02、0.22±0.03、0.30±0.02和0.24±0.02,总体比较差异均有统计学意义(F=7.845,P<0.01),其中高糖组细胞ROS荧光强度明显高于对照组和DMSO组,高糖+阿魏酸组细胞ROS荧光强度明显低于高糖组,差异均有统计学意义(均P<0.05)。高糖组、高糖+阿魏酸组细胞线粒体膜电位水平明显低于对照组和DMSO组,高糖+阿魏酸组细胞线粒体膜电位水平明显高于高糖组,差异均有统计学意义(均P<0.05)。与对照组、DMSO组比较,高糖组MCU荧光强度较高,并伴随着细胞微丝减少和变细;高糖+阿魏酸组MCU蛋白荧光强度明显下降,细胞微丝数量明显增加。与对照组、DMSO组比较,高糖组细胞MCU、p38 MAPK和p-p38 MAPK蛋白荧光强度和相对表达量显著升高,差异均有统计学意义(均P<0.05);与高糖组比较,高糖+阿魏酸组细胞MCU、p38 MAPK和p-p38 MAPK蛋白荧光强度和相对表达量显著降低,差异均有统计学意义(均P<0.05)。与对照组和空载体组比较,MCU过表达组细胞MCU、p38 MAPK和p-p38 MAPK蛋白相对表达量显著升高,MCU shRNA组、MCU过表达+阿魏酸组细胞MCU、p38 MAPK和p-p38 MAPK蛋白相对表达量显著降低,差异均有统计学意义(均P<0.05);与MCU过表达组比较,MCU shRNA组、MCU过表达+阿魏酸组细胞MCU、p38 MAPK和p-p38 MAPK蛋白相对表达量显著降低,差异均有统计学意义(均P<0.05)。结论阿魏酸能够调控氧化应激和线粒体功能障碍,进而改善糖尿病小鼠视网膜和高糖诱导的RPE细胞损伤,其可能通过MCU及p38MAPK信号通路发挥保护作用。

Objective To investigate the inhibitory effect of ferulic acid on the retina of diabetic mice and high glucose-induced human retinal pigment epithelium(RPE)cell injury and the mechanism.Methods Thirty 8-weekold SPF male type 2 diabetic db/db mice were selected and divided into a model group and a ferulic acid group by the random number table method,with 15 mice in each group.Another 15 db/m mice of the same age were selected as a control group.The model and control groups received normal saline(5 ml/kg)by gavage daily,and the ferulic acid group received ferulic acid solution(0.05 g/kg)by gavage daily.After two months of treatment,the mice were sacrificed and the eyeballs were removed.The morphological changes of mouse retinal tissues were observed by hematoxylin-eosin staining.The fluorescence intensity and expression levels of mitochondrial calcium uniporter(MCU),p38 mitogen-activated protein kinase(p38 MAPK)and phosphorylated p38 MAPK(p-p38 MAPK)in mouse retinal tissues were detected by immunofluorescence staining and Western blot.Human RPE cells were divided into control group,dimethyl sulfoxide(DMSO)group,high glucose group and high glucose+ferulic acid group.The control group received no treatment,and the other cell groups were cultured with the corresponding reagents for 24 hours.The reactive oxygen(ROS)level of RPE cells in each group was detected with the ROS detection kit.The mitochondrial membrane potential level of RPE cells was detected with the a mitochondrial membrane potential detection kit(JC-1).The MCU and microfilament fluorescence intensity of RPE cells were detected with the a microfilament green fluorescent probe.To explore the regulatory relationship between MCU,p38 MAPK and p-p38 MAPK,the MCU protein level was silenced and overexpressed by lentivirus transfection technology.The fluorescence intensity and expression levels of MCU,p38 MAPK and p-p38 MAPK proteins in RPE cells were detected by immunofluorescence staining and Western blot.The use and feeding of experimental animals followed the 3R principle and the Statement of the Association for Research in Vision and Ophthalmology on the Use of Animals in Ophthalmology and Vision Research.This study protocol was approved by the Ethics Committee of Ningxia Eye Hospital,People's Hospital of Ningxia Hui Autonomous Region(No.2019085).Results The intercellular space of the outer nuclear layer,inner nuclear layer and ganglion cell layer of the retinal tissue in the model group was increased and the cell arrangement was disordered compared with the control group,and the retinal tissue in the ferulic acid group was significantly improved.Compared with the control group,the fluorescence intensity of MCU,p-p38 MAPK and MCU+p-p38 MAPK protein of mouse retinal tissue in model group and ferulic acid group was significantly increased(all at P<0.05).Compared with the model group,the fluorescence intensity of MCU,p-p38 MAPK and MCU+p-p38 MAPK protein of mice retinal tissue in ferulic acid group was significantly decreased(all at P<0.05).Compared with the control group,the relative expression levels of MCU,p38 MAPK and p-p38 MAPK proteins of mouse retinal tissue in model group were significantly increased(all at P<0.05).Compared with the model group,the relative expression levels of MCU,p38 MAPK and p-p38 MAPK proteins of mice retinal tissue in ferulic acid group were significantly decreased(all at P<0.05).The ROS fluorescence intensities in the control group,DMSO group,high glucose group and high glucose+ferulic acid group were 0.22±0.02,0.22±0.03,0.30±0.02 and 0.24±0.02,respectively,and the overall difference was statistically significant(F=7.845,P<0.01).The ROS fluorescence intensity was significantly higher in the high glucose group than in the control and DMSO groups,and it was significantly lower in the high glucose+ferulic acid group than in the high glucose group(all at P<0.05).The mitochondrial membrane potential was significantly lower in high glucose group and high glucose+ferulic acid group than in control and DMSO groups,and significantly higher in high glucose+ferulic acid group than in high glucose group(all at P<0.05).Compared with the control group and DMSO group,the fluorescence intensity of MCU was higher in the high glucose group,accompanied by the decrease and thinning of cell microfilaments,and the fluorescence intensity of MCU protein was significantly decreased in high glucose+ferulic acid group,with the number of microfilaments increased significantly.Compared with the control group and DMSO group,the fluorescence intensity and relative expressions of MCU,p38 MAPK and p-p38 MAPK proteins were significantly increased in the high glucose group(all at P<0.05).Compared with the high glucose group,the fluorescence intensity and relative expressions of MCU,p38 MAPK and p-p38 MAPK proteins were significantly decreased in the high glucose+ferulic acid group(all at P<0.05).Compared with the control group and the empty vector group,the relative expressions of MCU,p38 MAPK and p-p38 MAPK proteins were significantly increased in the MCU overexpression group and significantly decreased in the MCU shRNA group and the MCU overexpression+ferulic acid group(all at P<0.05).Compared with MCU overexpression group,the relative expressions of MCU,p38 MAPK and p-p38 MAPK proteins were significantly decreased in MCU shRNA group and MCU overexpression+ferulic acid group,and the differences were statistically significant(all at P<0.05).Conclusions Ferulic acid can regulate oxidative stress and mitochondrial dysfunction,thereby ameliorating retinal damage and high glucose-induced RPE cell injury in diabetic mice,which may play a protective role through MCU and p38MAPK signaling pathways.