Astragalus polysaccharides suppresses high glucose-induced metabolic memory in retinal pigment epithelial cells through inhibiting mitochondrial dysfunction-induced apoptosis by regulating miR-195

Abstract

Background: Metabolic memory contributes to the development of diabetic retinopathy (DR), which is the complication of diabetes. But it's still unknown how to prevent the metabolic memory to treat the DR. In our study, we want to examine the function of Astragalus polysaccharides (APS) in the metabolic memory of retinal pigment epithelium (RPE) pretreated with high glucose (HG).

Methods: ARPE-19 and PRPE cells were exposed to HG followed by normal glucose (NG) treatment with or without APS. QPCR was used to examine the levels of miR-195 and Bcl-2. MDA and SOD detection assays were used to examine the oxidative stress level. Western blotting and immunostaining were applied to detect the protein level of mitochondrial damage and apoptotic signaling pathway. Flow cytometry and TUNEL staining were used to analyze cell apoptosis. Luciferase assay was used to examine the direct target of miR-195.

Results: APS treatment significantly decreased the expression of miR-195, while increased the expression of Bcl-2 with optimized dosages which were induced by HG treatment, even after replacing the HG with NG. And we found Bcl-2 was the direct target of miR-195. APS alleviated the oxidative stress, mitochondrial damage and cell apoptosis induced by HG and HG + NG treatments in RPE cells via regulating miR-195. Furthermore, we found overexpression of miR-195 abolished the alleviated effects of APS on the HG-treated RPE cells.

Conclusions: APS suppressed high glucose-induced metabolic memory in retinal pigment epithelial cells through inhibiting mitochondrial dysfunction-induced apoptosis by regulating miR-195.

Keywords: Apoptosis; Astragalus polysaccharides; Diabetic retinopathy; Metabolic memory; Mitochondrial damage; miR-195.

Fig. 1

The effects of APS on the survival of retinal pigment epithelium cells. a Different dosages of APS were applied to treat the cultured RPE cell line (ARPE-19) for different time course. b Different dosages of APS were applied to treat the cultured primary RPE cells (PRPE) for different time course. Error bars represented mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05

Fig. 2

APS inhibited the high glucose-induced upregulation of miR-195. a Treatment with APS significantly decreased the expression of miR-195 and increased the expression of Bcl-2 which were induced by HG or HG + NG treatment in ARPE-19 cell line. b Treatment with APS significantly decreased the expression of miR-195 and increased the expression of Bcl-2 which were induced by HG or HG + NG treatment in PRPE cells. c Sequence analysis result showed miR-195 directly targeted to the 3’UTR of Bcl-2. d Dual luciferase reporter assay showed miR-195 repressed the luciferase activity which contained the WT Bcl-2 3’UTR, but not the mutant Bcl-2 3’UTR. Error bars represented mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05

Fig. 3

APS reversed the oxidative stress and mitochondrial damage in RPE cells induced by HG. a Treatment with APS significantly decreased the MDA and increased SOD level which were induced by HG or HG + NG treatment in ARPE-19 cells. b Treatment with APS significantly decreased the MDA and increased SOD level which were induced by HG or HG + NG treatment in PRPE cells. c Western blotting showed treatment with APS significantly increased the ratio of mitochondrial to cytosol cyt-c which induced by HG and HG + NG treatment in ARPE-19 and PRPE cells. d The statistical results of western blotting. Error bars represented mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05

Fig. 4

APS ameliorated the mitochondrial membrane potential induced by HG in RPE cells. a Immunostaining showed treatment with APS significantly decreased the ratio of monomeric to aggregate JC-1 which was induced by HG or HG + NG treatment in ARPE-19 cells. b. Immunostaining showed treatment with APS significantly decreased the ratio of monomeric to aggregate JC-1 which was induced by HG or HG + NG treatment in PRPE. c The statistical results of the staining in A and B. Error bars represented mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05

Fig. 5

APS decreased the RPE cells apoptosis via mitochondrial apoptotic pathway. a Western blotting showed treatment with APS significantly decreased the expression of Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP which were induced by HG and HG + NG treatment, while increased the expression of Bcl-2 and uncleaved form of caspase-9, caspase-3 and PARP which were repressed by HG and HG + NG treatment in APRE-19 and PRPE cells. b and c. The statistical results of western blotting in APRE-19 and PRPE cells. Error bars represented mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05

Fig. 6

APS rescued the apoptosis in metabolic memory model by flow cytometry. a Flow cytometry showed treatment with APS significantly decreased the cell apoptosis induced by HG and HG + NG treatment in APRE-19 cells. b Flow cytometry showed treatment with APS significantly decreased the cell apoptosis induced by HG and HG + NG treatment in PRPE cells. Error bars represented mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05

Fig. 7

APS rescued the apoptosis in metabolic memory model by TUNEL staining. a TUNEL staining showed treatment with APS significantly decreased the TUNEL positive cells induced by HG and HG + NG treatment in APRE-19 cells. b TUNEL staining showed treatment with APS significantly decreased the TUNEL positive cells induced by HG and HG + NG treatment in PRPE cells. Error bars represented mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05

Fig. 8

Overexpression of miR-195 increased the oxidative stress and mitochondrial damage in ARPE-19 cells. a MiR-195 mimics significantly increased the miR-195 level and decreased the Bcl-2 level which were modulated by APS treatment in HG and HG + NG treated ARPE-19 cells. b MiR-195 mimics increased the MDA and decreased SOD levels which were regulated by APS in HG and HG + NG treated ARPE-19 cells. c Immunostaining showed miR-195 mimics significantly increased the ratio of monomeric (Green) to aggregate (Red) JC-1 which was decreased by APS in HG or HG + NG treated ARPE-19 cells. d The statistical results of mitochondrial membrane potential in C. e Western blotting showed miR-195 mimics significantly decreased the ratio of mitochondrial to cytosol cyt-c which was increased by APS in HG and HG + NG treated ARPE-19 cells. f The statistical results of ratio of mitochondrial to cytosol cyt-c. Error bars represented mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05

Fig. 9

MiR-195 activated mitochondrial apoptotic pathway in ARPE-19 cells. a Western blotting showed miR-195 mimics significantly increased the expressions of Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP while decreased the expressions of Bcl-2 and uncleaved form of caspase-9, caspase-3 and PARP compared to the APS treated ARPE-19 cells. bThe statistical results of western blotting in A. Error bars represented mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05

Fig. 10

Overexpression of miR-195 enhanced the cell apoptosis in ARPE-19 cells. a Flow cytometry showed miR-195 increased the cell apoptosis compared to the APS treated ARPE-19 cells. b TUNEL staining showed miR-195 increased the TUNEL positive cells compared to the APS treated ARPE-19 cells. c The statistical results of flow cytometry and TUNEL staining in A and B. Error bars represented mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05