Nogo receptor knockdown and ciliary neurotrophic factor attenuate diabetic retinopathy in streptozotocin-induced diabetic rats

Abstract

Diabetic retinopathy (DR) is a common complication of diabetes mellitus (DM). We investigated whether Nogo receptor (NgR) knockdown and ciliary neurotrophic factor (CNTF) treatment, either alone or in combination, ameliorated diabetic retinopathy (DR) in diabetic rat model. STZ‑induced diabetic rats were administrated for a total of 12 weeks with 3 µM siRNA (5 µl) once every 6 weeks and/or 1 µg CNTF weekly. The retinal tissues were excised. We measured cell number in ganglion cell layer (GCL) using H&E staining and cell apoptosis using TUNEL assay. Bax, Bcl‑2, Caspase‑3, F‑actin, GAP‑43, NgR, RhoA and Rock1 levels were then analyzed by Western blotting, Immunohistochemistry or Real‑time PCR. We found that NgR siRNA or CNTF injection alone significantly increased cell count in GCL in diabetic rats, inhibited ganglion cell apoptosis, elevated Bcl‑2, F‑actin and GAP‑43, and decreased Bax, Caspase‑3, NgR, RhoA and Rock1 levels. Combination treatment further prevented retinal ganglion cell loss, enhanced growth cone cytoskeleton and axonal regeneration, and suppressed NgR/RhoA/Rock1. Our results indicate that combination therapy has therapeutic potential for the treatment of DR.

Figure 1.

Effect of NgR siRNA and/or CNTF injection on the cell number in GCL in vivo. Diabetes was induced by STZ injection (65 mg/kg) in male SD rats. The diabetic rats were administrated with siRNA and/or CNTF. After animal experiments, the retina was excised from the sacrificed rats and subjected to H&E staining. Scale bars 50 µm. Number of cells in GCL was measured. **P<0.01 vs. STZ group. ^&&P<0.01 vs. STZ+control siRNA group.

Figure 2.

Effect of NgR siRNA and/or CNTF injection on cell apoptosis in the retina in vivo. (A) Apoptosis was detected by TUNEL assay. Scale bars 50 µm. (B) Bcl-2, Bax and Caspase-3 protein levels were examined by western blotting. *P<0.05 and **P<0.01 vs. STZ group. ^&P<0.05 and ^&&P<0.01 vs. STZ+control siRNA group. ^$$P<0.01 vs. STZ+NgR siRNA group. ^##P<0.01 vs. STZ+CNTF group.

Figure 3.

Effect of NgR siRNA and/or CNTF injection on F-actin and GAP-43 in vivo. (A) Western blot analysis of F-actin and GAP-43. (B) qPCR analysis of F-actin and GAP-43. (C) F-actin and GAP-43 were examined by IHC. Scale bars 50 µm. **P<0.01 vs. STZ group. ^&&P<0.01 vs. STZ+control siRNA group. ^$P<0.05 vs. STZ+NgR siRNA group. ^##P<0.01 vs. STZ+CNTF group.

Figure 4.

Effect of NgR siRNA and/or CNTF injection on NgR/RhoA/Rock1 signaling pathway in vivo. (A) NgR, RhoA and Rock1 levels were measured by western blotting. (B) NgR, RhoA and Rock1 levels were measured by qPCR. **P<0.01 vs. STZ group. ^&&P<0.01 vs. STZ+control siRNA group. ^$P<0.05 and ^$$P<0.01 vs. STZ+NgR siRNA group. ^##P<0.01 vs. STZ+CNTF group.