The Early Intervenient Effects Of Recombinant Human Proinsulin C-peptide On Experimental Diabetic Retinopathy

Objective: To investigate the effects of equal mole of insulin and proinsulin C-peptide on the functions of cultured rat retinal Muller cells and bovine retinal microvascular endothelial cells. And to evaluate the effects of proinsulin C-peptide on the vascular endothelial growth factor (VEGF) expression, the nitric oxide (NO) synthesis of diabetic rat retina and its protective effect on the blood-retinal barrier (BRB) function. Methods: (1) Rat retinal Muller cells and bovine retinal microvascular endothelial cells were cultured by enzyme digest method. Immunocytochemistry and electronic microscopy were used for cell identification. Different concentrations of insulin, proinsuling C-peptide were added to the cells separately or combined 24 hours before measuring. The nitric oxide level of the supernatant was measured by nitrate reductase method and VEGF concentration by ELISA. (2) Diabetic rats were induced by streptozotocin intravenously injected of 50mg/Kg body weight. All the animals (diabetic group, C-peptide interfering diabetic group, and control group) were fed in a specific pathogen free environment for eight weeks. The dose of C-peptide was 130nmol/Kg body weight by subcutaneous injection twice a day. The blood glucose level and the body weight were measured every week. Immunohistochemistry method was used to qualitatively detect the VEGF reaction on the retinal section. The VEGF concentration was measured by enzyme-linked immunoabsorption assay (ELISA) method. The vitality of different type of nitric oxide synthase was measured separately and NO level was measured by nitrate reductase method. And the BRB function was measured by Evans blue quantitatively.Results: In vitro: (1) The retinal Muller cells NO content was increased by insulin (p<0.01) and decreased by C-peptide (P<0.05) in the concentration of 10^-5M. And the efficacy of C-peptide correlated with the concentration. (2) The VEGF expression was increased by insulin, and decreased to normal level by combined using equal mole of insulin and C-peptide in cultured Muller cells. While it was not changed by C-peptidesolely. (3) The NO over synthesis caused by insulin (t=4.922, P<0.001) in BREC could be corrected by combined with equal mole of proinsulin C-peptide (vs Insulin: t=2.318, P<0.05; vs control: t=2.806, P<0.02). There is no statistically significant difference among different concentration of C-peptide (P>0.05). In vivo: (1) The blood glucose was maintained in a very high level ranged from 24.14 to 33.3mol/L in the two diabetic groups (with/without C-peptide interfering) and the body weight was not increased with the age. While the blood glucose level was normal and the body weight was growing steadily in control group. (2) The VEGF immuno-reaction was detectable on the retinal ganglion cell layer and near the inner nuclei layer in all three groups. And the positive reaction was more distinctive in the diabetic without C-peptide interfering group than the other twos. The difference of VEGF concentrations among the three groups was statistically significant (F-13.604, P＜0.01). The C-peptide interfering diabetic group had the concentration that is lower than diabetic group (P<0.05) but higher than control group (P<0.05). (3) The C-peptide group had the vitality of eNOS, iNOS and NO content between the other two groups. (4) The Evans blue exudation were 186.74±16.00 in diabetic group, 141.47±33.48 in C-peptide group, and 32.02±11.45 in control group. The EB level in C-peptide group was lower than diabetic group (P<0.05) but still higher than control group (P<0.01).Conclusion: (1) The nitric oxide over synthesis in Muller cells caused by insulin can return to normal level by combined using equal mole of insulin and proinsulin C-peptide. (2) The VEGF over expression in Muller cells caused by insulin can also return to normal by combined using. (3) The NO over synthesis caused by insulin in BREC could be corrected by combined with equal mole of proinsulin C-peptide. (4) Early treatment of proinsulin C-peptide solely can partially inhibit the VEGF over expression, regulate NOS vitality, inhibit NO over synthesis, ameliorate the albumin exudation in diabetic rat retina, and restore the blood-retinal barrier function.