The Inhibition Of Calcium Sensing Receptor On Smooth Muscle Cells Proliferation In Diabetic Rats Through Regulating Cystathionineγ-lyase/hydrogen Sulfide Pathway

Objective To investigate the effect of calcium-sensing receptor(CaSR) on cystathionineγ-lyase/hydrogen sulfide(CSE/H2S) pathway and the mechanism of CaSR regulating on mesenteric vascular smooth muscle cells proliferation in diabetic rats.Methods To build diabetes rat models, the rats were injected a low dose of streptozotocin(STZ, 50 mg/kg), dissolved in 0.1 M citrate buffer(pH 4.4), through intraperitoneal. The rats with blood glucose level>16.7 mmol/L were considered to be diabetic rats, which were divided into six groups at random: 4, 8, 12 weeks diabetes model groups and 4, 8, 12 diabetes treatment with NaHS groups. The control and diabetes groups were maintained on standard rat chow while the diabetes+NaHS groups were treated with NaHS(80 umol/L/100 g/d) by intraperitoneal injection for 24 h. Morphology and ultrastructure alteration of mesenteric secondary arteries in diabetic rats were detected by HE staining and electron transmission microscope; The collagen deposition around artery walls was observed by Masson staining; The relaxation of mesenteric secondary artery loop of diabetic rats was detected by microvessel perfusion system in vitro; Western blotting analyzed the expression of CaSR, CSE, Cyclin D1 and HB-EGF of mesenteric artery in diabetic rats. The cultured vascular smooth muscle cells of rat thoracic aorta A7r5 were divided into five groups: Control group(5.56 mmol/L glucose), High glucose group(HG, 25 mmol/L glucose), HG+NaHS group(80 μmol/L NaHS), HG+Calindol group(2 mmol/L) and HG+A23187 group(4 μmol/L). The intracellular calcium concentration of VSMCs([Ca2+]i) treatment by CaCl2 and Calindol was detected by laser-scanning confocal microscope; Western blotting analyzed the expression of CaSR, CSE and ERK in different times of A7r5 which were treated by HG and other treated groups; The concentration of endogenous H2 S was detected with the ultraviolet spectroscopy detected; The expression of PCNA and p21 was measured by immunofluorescence; The proliferation of VSMCs were measured by flow cytometry and ELIASA.Results Compared to the control group, the water and food intake, the concentration of blood glucose were significantly increased in diabetes groups; Diabetic rats displayed abnormal mesenteric secondary artery structure(SMC layer thickening and collagen deposited around artery wall obviously); The relaxation of mesenteric secondary artery loop weakened, the expression of CaSR, CSE were decreased while the expression of Cyclin D1 and HB-EGF were increased. Compared to the diabetes group, the SMC layer thickness, collagen deposition significantly increased with the treatment of Na HS and the relaxation of mesenteric secondary artery loop were relieved after treatment with NaHS; The expression of CaSR, CSE increased, The expression of Cyclin D1 and HB-EGF was down-regulated. [Ca2+]i of VSMCs increased with treatment of CaCl2 and calindol while decreased by calhex231, U73122, 2-APB and TG; Compared to the control group, the expression of CaSR and CSE of A7r5 were decreased while the expression of ERK increased with the treatment of high glucose, the expression of CaSR and CSE was upregulated by Calindol/NaHS/A23187 administration; Compared to the control group, the endogenous H2 S concentration dereased in HG while the concentration of H2 S increased with treatment of CaSR activators. Compared to the activator groups, pre-treatment with BAPTA and 2-APB, the concentration of H2 S decreased; The expression of PCNA increased, whereas the expression of p21 decreased in HG group, while the expression of PCNA decreased and the expression of p21 increased with the treatment of Calindol and NaHS. The cell viability and proliferation increased in the HG group compare to the control group, while those were decreased with the treatment of Calindol and NaHS.Conclusion(1) Diabetic rats mesenteric secondary artery smooth muscles layer thickness, SMCs proliferation, and the expression of CaSR and CSE decreased while the expression of HB-EGF increased.(2) Calindol and NaHS increased the relaxation rate of rats mesenteric loop.(3) The expression of CaSR regulated the expression of CSE in both diabetic rats mesenteric artery and high glucose cultured VSMCs.(4) CaSR influenced [Ca2+]i stability and the concentration of endogenous H2 S through PLC-IP3 R pathway, the increase of [Ca2+]i inhibited the proliferation of VSMCs by endogenous H2 S.(5) H2 S inhibited the proliferation of VSMCs in diabetes through inhibiting HB-EGF expression.