Lu Jin-Ping, Ren Jiang-Hua, Chen Ling, Li Xia, Chen Hui-Li
Department of Internal Medicine & Geriatrics, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China.
J Huazhong Univ Sci Technolog Med Sci. 2015 Feb;35(1):42-47. doi: 10.1007/s11596-015-1386-5. Epub 2015 Feb 12.
In order to clarify the potential role of calcium sensing receptor (CaSR), a typical G protein coupled receptor (GPCR), in hyperglacemia-induced macroangiopathy, experimental hyperglycemia models in vivo and in vitro were prepared. Firstly, SD rats were divided into control group (n=10) and diabetes group (n=10), and diabetic model was induced via high-fat diet feeding and streptozotocin (STZ, 30 mg/kg) injection. Hydroxyproline level, determined via Choramnie T oxidation method, in vessel wall in diabetic rats was 30% more than that in control group. The gene transcription and expression levels were detected by real-time PCR and Western blotting, respectively. Both of collagen I and III mRNA levels in diabetic aorta were nearly twice those in normal aorta. The cleaved caspase-3 and -9 were elevated 1.5 and 2.5 times respectively in diabetic vascular cells. As compared with controls, mRNA and protein levels of CaSR in aorta were increased by 3 and 1.5 times in diabetes group. The expression levels of Bax as well as pro-apoptotic kinases (phospho-p38 and phosphor-JNK) were also increased 2, 0.5 and 0.5 times respectively in diabetic rats. To further validate the involvement of CaSR in cell apoptosis and explore the potential mechanism, the endothelial cell line (human umbilical vascular endothelial cells, HUVECs) was stimulated with high concentration of glucose (33 mmol/L) to mimic hyperglycemia in vitro. Cell-based assays also showed that the CaSR level and key apoptotic proteins (cleaved caspase-3 and -9, Bax, phospho-p38 and phosphor-JNK) were elevated in response to stimulation, and inhibition of CaSR by using specific inhibitor (NPS-2143, 10 μmol/L) could protect cells against apoptosis. Our results demonstrated that CaSR might take important part in the development of diabetic macroangiopathy through promoting cell apoptosis induced by hyperglycemia.
为了阐明钙敏感受体（CaSR）这一典型的G蛋白偶联受体（GPCR）在高血糖诱导的大血管病变中的潜在作用，制备了体内和体外实验性高血糖模型。首先，将SD大鼠分为对照组（n = 10）和糖尿病组（n = 10），通过高脂饮食喂养和注射链脲佐菌素（STZ，30 mg/kg）诱导糖尿病模型。采用氯胺T氧化法测定，糖尿病大鼠血管壁中的羟脯氨酸水平比对照组高30%。分别通过实时PCR和蛋白质免疫印迹法检测基因转录和表达水平。糖尿病主动脉中I型和III型胶原蛋白的mRNA水平均几乎是正常主动脉的两倍。糖尿病血管细胞中裂解的半胱天冬酶-3和-9分别升高了1.5倍和2.5倍。与对照组相比，糖尿病组主动脉中CaSR的mRNA和蛋白质水平分别增加了3倍和1.5倍。糖尿病大鼠中Bax以及促凋亡激酶（磷酸化p38和磷酸化JNK）的表达水平也分别增加了2倍、0.5倍和0.5倍。为了进一步验证CaSR参与细胞凋亡并探索潜在机制，用高浓度葡萄糖（33 mmol/L）刺激内皮细胞系（人脐静脉血管内皮细胞，HUVECs）以在体外模拟高血糖。基于细胞的实验还表明，CaSR水平和关键凋亡蛋白（裂解的半胱天冬酶-3和-9、Bax、磷酸化p38和磷酸化JNK）在受到刺激后升高，使用特异性抑制剂（NPS-2143，10 μmol/L）抑制CaSR可保护细胞免于凋亡。我们的结果表明，CaSR可能通过促进高血糖诱导的细胞凋亡在糖尿病大血管病变的发展中起重要作用。
