Remote Ischemic Conditioning Protects Diabetic Retinopathy in Streptozotocin-induced Diabetic Rats via Anti-Inflammation and Antioxidation
Ren Changhong1,5, Wu Hang1,2, Li Dongjie1,2, Yang Yong3, Gao Yuan1,2, Jizhang Yunneng4, Liu Dachuan2, Ji Xunming1,5, Zhang Xuxiang1,2,*
1Beijing Key Laboratory of Hypoxia Conditioning Translational Medicine, Beijing, China. 2Department of Ophthalmology, Xuanwu Hospital, Capital Medical University, Beijing, China. 3Department of Herbal Formula Science Medicine, Chinese Medicine College, Beijing University of Chinese Medicine, Beijing, China. 4Center of Cerebrovascular Disease Research, University of Pittsburgh School of Medicine, Pittsburgh, USA. 5Center of Stroke, Beijing Institute for Brain Disorder, Beijing 100069, China
Ischemic conditioning inhibits oxidative stress and inflammatory response in diabetes. However, whether limb remote ischemic conditioning (LRIC) has beneficial effects on diabetic retinopathy (DR) remains unknown. This study aims to investigate the protective effects of LRIC in retinal ganglion cell in streptozotocin (STZ) induced Type 1 diabetic rats. A total of 48 healthy male Sprague-Dawley (200-220g) rats were randomly assigned to the normal group, normal+LRIC group, diabetes mellitus (DM) group and DM+LRIC group. Streptozotocin (STZ, 60 mg/kg) was intraperitoneally injected into the rats to establish the diabetic model. LRIC was conducted by tightening a tourniquet around the upper thigh and releasing for three cycles daily (10 mins x 3 cycles). Retinas were harvested after 12 weeks of LRIC treatment for histopathologic, Western blot and ELISA analysis. Plasma were collected at the same time for ELISA analysis. LRIC alleviated diabetic retinopathy symptoms as evidenced by the increased number of retinal ganglion cells (P<0.01) and decreased glial fibrillary acidic protein (GFAP) expression level (P<0.01) in the rat retina. LRIC in DM rats exhibited anti-inflammatory and antioxidative effects as confirmed by the down-regulation of pro-inflammatory cytokine: interleukin-6 (IL-6), and the up-regulation of antioxidants: superoxide dismutase (SOD), and glutathione (GSH)/oxidized glutathione (GSSG). Furthermore, LRIC significantly downregulated VEGF protein expression in the retina (P<0.01). These results suggest that the antioxidative and anti-inflammatory activities of LRIC may be important mechanisms involved in the protective effect of LRIC in STZ-induced diabetic rats.
Figure 1. Effect of LRIC on blood glucose levels and body weight at 12 weeks after onset of diabetes. (A) Representative sketches of the experiment. (B) Quantification of blood glucose levels. (C) Quantification of body weight. Data are expressed as mean±SD, * P<0.05 (DM vs. Normal group). N=10 each group.
Figure 2. LRIC treatment ameliorated Brn3a+ retinal ganglion cell loss in diabetic rats. Immunohistochemical analysis of retinas of normal control group (A), normal+LRIC group (B), DM group (C) and DM+LRIC group (D). Arrows indicate Brn3a+ RGCs, scale bar=50 μm. (E) Bar graphs depicting the average number of Brn3a+ RGCs in each group. Data are expressed as mean±SD, * P<0.05. N=5 each group.
Figure 3. LRIC treatment ameliorated retinal Müller cell activation in diabetic rats. There was a significant increase in the level of GFAP expression in the diabetic retina (C) compared with the control retina (A). After 12 weeks of LRIC treatment, GFAP immunostaining decreased significantly in the diabetic retina (D). However, GFAP expression in the control retina was not affected by LRIC (B). Scale bar=50 μm. (E) Bar graphs depicting the the density of GFAP in each group. Data are expressed as mean±SD, ** P<0.01, *** P<0.001. N=5 each group.
Figure 4. LRIC treatment attenuated oxidative stress induced by hyperglycemia. ROS production in the retina was evaluated at 12 weeks after LRIC treatment. The graph showed the relative reactive oxygen species levels in each group. *P<0.05, **P<0.01, N=7 each group.
Figure 5. LRIC altered antioxidant enzyme levels in diabetic rats. Antioxidant enzyme levels in the retina were analyzed at 12 weeks after LRIC treatment. (A) Analysis of SOD activity. Data represent mean±SD of six independent experiments. (B) The analysis of total glutathione level. Data represent mean±SD of three independent experiments. (C) Analysis of SOD/CAT ratio. (D) Analysis of GSH/GSSG ratio. Data represent mean±SD of six independent experiments. *P< 0.05, ** P<0.01, N=7 each group.
Figure 6. LRIC treatment attenuated retinal inflammation in diabetic rats. The inflammatory markers were analyzed in the diabetic rat retina and plasma at 12 weeks after LRIC treatment by using ELISA. (A) Inflammatory cytokines TNF-α, IL-1β and IL-6 levels in the rat retina. (B) Inflammatory cytokines TNF-α, IL-1β, IL-6 levels in the rat plasma. Data represent mean±SD of six independent experiments. *P<0.05, **P<0.01, N=7 each group.
Figure 7. LRIC treatment decreased the level of VEGF in diabetic rats. The expression of VEGF was analyzed in the diabetic rat retina and plasma at 12 weeks after LRIC treatment. (A) The expression of VEGF in retinas was detected by ELISA for the di?erent groups. (B) The expression of VEGF in retinas was detected by Western blot for the di?erent groups. Data represent mean±SD of six independent experiments. *P<0.05, **P<0.01, N=7 each group.
Figure 8. Hypothesis regarding the mechanism of LRIC’s protective effects against diabetic retinopathy.
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