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

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-week-old 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.

Ferulic acid; Diabetes mellitus; Retina; Retinal pigment epithelial cell; Mitochondrial calcium uniporter; Oxidative stress; Mitochondrial dysfunction