Effect of diabetes blood-stasis syndrome and Xuefu Zhuyu decoction on ROS-ERK1/2 signaling pathway in rat retina Müller cells

Abstract

This research aimed to investigate whether diabetic blood-stasis syndrome had a relationship with ROS-ERK1/2 signaling pathway in rat retina Müller cells and explore the effects of traditional Chinese drugs designed for promoting blood circulation to remove blood stasis on diabetic retinopathy (DR) treatment. Immunofluorescence was applied to determine purity of Müller cells. The diabetes was induced in rats by streptozotocin (STZ). Müller cells were stimulated by blood serum obtained from rats with blood-stasis syndrome and then treated by Xuefu Zhuyu decoction. Kits for reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione (GSH) were used for corresponding detection. Western blot analysis was used to determine the phosphorylation of ERK1/2. The results indicated that stimulation of Müller cells by blood serum of rats with diabetic blood-stasis syndrome increased the expression of ROS, inhibited SOD and GSH, and activated ERK1/2 signaling pathway. Treatment of Xuefu Zhuyu decoction could weaken this phenomenon. What's more, similar effects of ERK1/2 inhibitor U0126 with Xuefu Zhuyu decoction proved the involvement of ERK1/2 signaling pathway. Therefore, our results suggested that traditional Chinese drugs for promoting blood circulation to remove blood stasis would be an effective therapy to treat DR.

Keywords: Blood-stasis syndrome; Diabetic retinopathy; ERK1/2 signaling pathway; Müller cells; Reactive oxygen species.

Fig. 1

Results of determining the purity of Müller cells. Morphology of Müller cells a in good condition was photographed and then the immunofluorescence b was performed to detect the purity of Müller cells: a the DAPI staining for nucleus of Müller cells; (b, d) the green fluorescence of GFAP in Müller cells; (c) the DAPI staining for nucleus of Müller cells with the secondary antibodies conjugated-FITC; d the Müller cells just with the secondary antibodies conjugated-FITC as control

Fig. 2

Results of ROS expression in Müller cells treated with high mannitol or glucose. After treatment of Müller cells with high concentration of mannitol or glucose for 6 h or 12 h, the concentration of ROS was detected with flow cytometry

Fig. 3

Results of screening for diabetic blood stasis syndrome rats. After injection of STZ into the SD rats by intraperitoneal route, the blood glucose levels (a) and body weight (b) of all SD rats were measured every week. Then, to screen for diabetic rats with blood-stasis syndrome, the cataract-related symptoms (c) and blood serum (d) were observed and photographed. e The pancreas of some rats in normal (a, d), diabetes (b, e) and Xuefu Zhuyu decoction treatment (c, f) groups were acquired, the pancreas islets structure was inspected by H&E staining. The magnification is 100 for a–c; 200 for d–f. The data were expressed as mean ± SD, P value less than 0.05 was considered statistically significant, ‘ns’ indicates no significance, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Diabetic blood-stasis syndrome inhibited the activity of SOD and GSH in Müller cells. After stimulated by the serum from different groups of blood-stasis syndrome rats and followed by treatment of Xuefu Zhuyu decoction in different dosages, the expressions of SOD (a) and GSH (b) were detected in Müller cells. The data were expressed as mean ± SD

Fig. 5

U0126 blocked the activation of ERK1/2 signaling pathway in Müller cells. After stimulated by the serum from different groups of blood-stasis syndrome rats, U0126 was added into the culture medium of Müller cells and incubated for 24 h. Subsequently, western blot analysis was conducted to detect the expression levels of p-ERK1/2 and ERK1/2. The data were expressed as mean ± SD, P value less than 0.05 was considered statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Xuefu Zhuyu decoction and U0126 blocked the expression of ROS in Müller cells. After stimulated by the serum from different groups of blood-stasis syndrome rats and followed by treatment of Xuefu Zhuyu decoction in different dosages, ROS expression was detected. Subsequently, U0126 was added into the culture medium of Müller cells and incubated for 24 h, and ROS expression was detected. The data were expressed as mean ± SD