| ObjectiveCalcitonin gene - related peptide ( CGRP) is a neuropeptide with potent vasodilator effect. Although there are reports about CGRP can promote normal endothelium cell but inhibit VSMC proliferation, there is no report about the effect of CGRP on the proliferation of diabetic VSMC. In order to definitude the effect of CGRP on diabetic angiopathy , we studied the function of CGRP in different concentration on proliferation and cell cycle of diabetic rat VSMC compared with normal control. The main potassium channel of VSMC is ATP - sensitive potassium channel ( KATP). Effect of CGRP is intimate with KATP channel which can active il through cAMP - PKA pathway. So we also studied the influence of potassium channel opener glibenclamide and potassium channel blocker pinacidil on the effect of CGRP.MethodsAfter measuring the fasting blood glucose, 8w male wistar rats (weighting 200 ~250g) were randomly distributed into diabetes group and control one according to the criterion of body weight balance. Diabetes were induced with a single i. p. injection of streptozotocin (STZ) (65mg/kg, dissolved in citrate buffer). The diabetic state (blood glucose higher than 10mmol/l) was then ensured after threedays by measurement of blood glucose. Control rats were injected with an equivalent volume of vehicle. Four weeks after induction of diabetes , the rats were killed and thoracic aortae were prepared for VSMC. Classic tissue expanding method was used to acquire VSMC. By the method of MTT colorimetric analysis, we examined the effect of CGRP (1 ~ 100nmol/l) on proliferation of cultured diabetic and control VSMC. We also studied the influence of KATP opener glibenclamide and KATP blocker pinacidil on the effect of CGRP. At last, we observed the effect of CGRP and pinacidil on the cell cycle of VSMC, cell cycle was determined by flow cytometry. The percentage of inhibition of cell growth was derived from the following formula: inhibition % = ( Acontrol - Adn]g)/Acontro, x 100 %. Data were expressed as and analyzed with t - test. In all cases, differences were considered significant if p < 0.05.Results4w STZ - induced diabetic rats had higher fasting blood glucose and lower mean body weight than age - matched control. The data from cell culture indicated that VSMC from both diabetes and control had similar pattern of growth curves , and their shapes had no distinct difference, both were fusiform and typically in the shape of peak and valley . But in same inoculate density, VSMC from diabetes grew faster than those from control. CGRP inhibited the cell proliferation In the method of time and concentration dependency. In diabetic states , VSMC had higher sensitivity to CGRP, and the antiproliferation of CGRP was enhanced, even in low concentration , CGRP (10-9mol/ 1) had higher effect of antiproliferation, VSMC from diabetes and con-trol incubated with CGRP for 48h, the inhibation rate was 21. 3 0. 6% and 47.2 1.4% respectively. In diabetic state pinacidil (10-6 mol/L) enhanced the antiproliferation of CGRP on VSMC, the percentage of inhibition rate changed from 49. 3 1.2% to 58. 3 3.9% ( P <0.01) and glibenclamide (10 ~5mol/L) partly inhibited the action of CGRP, made the inhibition rate from 49. 3 1. 2% to 35. 6 1. l%(p <0. 01). But to VSMC derived from normal control, both pinacidil and glibenclamide had no significant influence. Results from flow cytometer indicated that the number of diabetic VSMC in G0/G, phase decreased, however, the number in S and G2/M phase increased. CGRP had different inhibition to the cultured cells from diabetes and control, manifold the number in G0/Gl phase, but decrease the cell number in S and G2/M, when CGRP and pinacidil acted together, the cell in G0/G, phase was much more than that acted by CGRP alone.Conclusions1) CGRP had marked antiproliferatrion role to diabetic VSMC, increased the cell in G0/G, phase and decreased the cell in S and G2/ M phase.2) The antiproliferation role of CGRP may be partly related with the open of KATP channel. |
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