Attenuation of diabetic retinopathy by enhanced inhibition of MMP-2 and MMP-9 using aspirin and minocycline in streptozotocin-diabetic rats

Abstract

Interruptions of Matrix Metalloproteinase-2 (MMP-2) and Matrix Metalloproteinase-9 (MMP-9) have been shown to reduce the ensuing threatening risk factors of vascular complications of diabetes by alteration in Extracellular Matrix (ECM). We hypothesized that minocycline induced MMP-2 and MMP-9 inhibition can be enhanced by aspirin, a non-selective COX and tPA inhibitor and this combination can reduce progression of diabetic retinopathy. Diabetes was induced in male Wistar rats by streptozotocin (55 mg/kg i.p.). Four weeks after diabetes induction rats were treated with minocycline (50 mg/kg, p.o.) per se, aspirin (50 mg/kg, p.o.) per se, or minocycline in combination with aspirin for a period of four weeks. At the end of eighth week rats were anesthetized and electroretinograms were recorded. B-wave latency, B-wave amplitude and retinal permeability were measured. Histology was done and retinal thickness was measured. Zymography was carried out for MMP-2 and MMP-9 level determinations. B-wave amplitude was significantly decreased while B- wave latency was significantly increased in diabetic group when compared with normo-glycemic rats. Treatment with combination of minocycline and aspirin significantly reversed B-wave amplitude and latency compared with vehicle-treated diabetic controls. Blood retinal permeability and retinal thickness were also significantly attenuated by the treatment of minocycline in combination with aspirin. Results of the present study suggest that MMP-2 and MMP-9 inhibition in presence of COX inhibitor prevents the development of experimental diabetic retinopathy in rats and can be a potential approach for the treatment.

Keywords: Matrix metalloproteinase 2; diabetic retinopathy; extracellular matrix; matrix metalloproteinase 9.

Figure 1

Effect of four week treatment with MINO, ASP and MINO in combination with ASP on B wave amplitude. ** p<0.01 vs vehicle treated diabetic rats, (n = 6 for all groups). DB + DW (Diabetic group treated with distilled water), DB + MINO (Diabetic group treated with Minocycline per se), DB + ASP (Diabetic group treated with Aspirin per se), DB + ASP + MINO (Diabetic group treated with Minocycline and Aspirin), DB + 0.05 % CMC (Diabetic group treated with 0.5 % Carboxymethyl Cellulose solution)

Figure 2

Effect of four week treatment with MINO, ASP and MINO in combination with ASP on B wave latency. * p<0.05 vs vehicle treated diabetic rats, (n = 6 for all groups) DB + DW (Diabetic group treated with distilled water), DB + MINO (Diabetic group treated with Minocycline per se), DB + ASP (Diabetic group treated with Aspirin per se), DB + ASP + MINO (Diabetic group treated with Minocycline and Aspirin), DB + 0.05 % CMC (Diabetic group treated with 0.5 % Carboxymethyl Cellulose solution).

Figure 3

Effect of four week treatment with MINO, ASP and MINO in combination with ASP on retinal thickness. * p<0.05, ** p<0.01 vs vehicle treated diabetic rats. (n = 6 for all groups) OLM -ILM (Outer Limiting Membrane to Inner Limiting Membrane), ONL (Outer Nuclear Layer), DB + DW (Diabetic group treated with distilled water), DB + MINO (Diabetic group treated with Minocycline per se), DB + ASP (Diabetic group treated with Aspirin per se), DB + ASP + MINO (Diabetic group treated with Minocycline and Aspirin), DB + 0.05 % CMC (Diabetic group treated with 0.5 % Carboxymethyl Cellulose solution).

Figure 4

Effect of three week treatment with MINO, ASP and MINO in combination with ASP on retinal protein (72 KDa) level. (a) Normal (b) Diabetic + DW (Diabetic group treated with distilled water), (c) Diabetic + 0.5 % CMC (Diabetic group treated with 0.5 % Carboxymethyl Cellulose solution) (d) Diabetic + ASP (Diabetic group treated with Aspirin per se) (e) Diabetic + MINO (Diabetic group treated with Minocycline per se) (f) Diabetic + ASP + MINO (Diabetic group treated with Minocycline and Aspirin)

Figure 5

Effect of three week treatment with MINO, ASP and MINO in combination with ASP on retinal protein (92 KDa) level. (a) Normal (b) Diabetic + DW (Diabetic group treated with distilled water), (c) Diabetic + 0.5 % CMC (Diabetic group treated with 0.5 % Carboxymethyl Cellulose solution) (d) Diabetic + ASP (Diabetic group treated with Aspirin per se) (e) Diabetic + MINO (Diabetic group treated with Minocycline per se) (f) Diabetic + ASP + MINO (Diabetic group treated with Minocycline and Aspirin).

Figure 6

Effect of four week treatment with MINO, ASP and MINO in combination with ASP on retinal permeability. * p<0.05, ** p<0.01 vs vehicle treated diabetic rats. (n = 6 for all groups) DB + DW (Diabetic group treated with distilled water), DB + MINO (Diabetic group treated with Minocycline per se), DB + ASP (Diabetic group treated with Aspirin per se), DB + ASP + MINO (Diabetic group treated with Minocycline and Aspirin), DB + 0.05 % CMC (Diabetic group treated with 0.5 % Carboxymethyl Cellulose solution)