SP600125对1型糖尿病小鼠颈动脉内皮功能及单核细胞趋化蛋白1表达的影响

The Effect of SP600125 on Endothelial Function and Monocyte Chemotactic Protein-1 Expression in Carotid Artery in Type 1 Diabetic Mice

目的探讨c-Jun氨基末端激酶(JNK)特异性抑制剂SP600125对1型糖尿病小鼠颈动脉内皮功能及单核细胞趋化蛋白1表达的影响。方法本研究分5组。第1组为C57BL/6野生型雄性小鼠,喂养8周;第2组为INS2AK ITA雄性小鼠,每日腹腔注射生理盐水50μL,共8周;第3、4、5组为INS2AK ITA雄性小鼠,每日各腹腔注射SP600125 10、20和30 mg/kg;连续8周。8周后麻醉处死动物,取静脉血检测血清髓过氧化物酶、丙二醛、一氧化氮、一氧化氮合酶;取双侧颈总动脉分别行凝胶迁移或电泳迁移率实验检测颈动脉信号转导子和转录激活子1活性,行W estern b lot检测p-JNK、JNK、单核细胞趋化蛋白1蛋白表达,行免疫组化检测颈动脉内皮单核细胞趋化蛋白1表达。结果与野生型雄性小鼠相比,1型糖尿病小鼠血清髓过氧化物酶、丙二醛明显增高,而血清一氧化氮合酶、一氧化氮明显减低(P<0.01)。给予1型糖尿病小鼠SP600125不同剂量干预后,髓过氧化物酶浓度较1型糖尿病组明显减低,而一氧化氮、一氧化氮合酶浓度明显增高,且SP600125剂量越高,一氧化氮、一氧化氮合酶浓度增加越明显(P<0.05)。同时,1型糖尿病小鼠p-JNK/JNK、单核细胞趋化蛋白1蛋白表达、颈动脉信号转导子和转录激活子1活性以及颈动脉内皮单核细胞趋化蛋白1表达比正常小鼠明显增高(P<0.05)。而经过3种剂量SP600125干预后,单核细胞趋化蛋白1蛋白表达分别下降21.82%、39.09%、68.18%;信号转导子和转录激活子1活性分别下降25.70%、33.20%、61.29%;颈动脉内皮单核细胞趋化蛋白1表达分别下降19.51%、39.14%、59.28%。结论 JNK特异性抑制剂可通过抑制信号转导子和转录激活子1,从而升高糖尿病血清一氧化氮及一氧化氮合酶水平,改善血管舒张功能,并且降低单核细胞趋化蛋白1、髓过氧化物酶表达水平,并具有剂量依赖性。进一步表明JNK通过信号转导子和转录激活子1参与了糖尿病颈动脉内皮功能紊乱及炎症因子表达,并为开发预防1型糖尿病大血管并发症的药物提供了一个新的理论依据。

Aim The incidence of stroke in diabetes is increasing seriously,which is associated with endothelial dysfunction and increased inflammation in carotid artery.Our study is to investigate the effect of c-Jun amino-terminal kinase（JNK） specific inhibitor SP600125 on carotid endothelial function and monocyte chemotactic protein-1（MCP-1） expression in type 1 diabetes. Methods Animals were divided into five groups in this study.The 1st group was C57BL/6 wild type male mice;the 2nd group was INS2AKITA male mice,intraperitoneal injection with 0.9% normal saline（NS） each day for 8 weeks;the 3rd,4th,5th group were INS2AKITA male mice,intraperitoneal injection with SP600125 10 mg/kg,20 mg/kg,30 mg/kg respectively each day for consecutive 8 weeks.Then the mice were killed and sreum myeloperoxidase（MPO）,malondialdehyde（MDA）,nitric oxide（NO）,total nitric oxide synthase（TNOS） were measured.HE staining was performed in carotid artery and immunohistochemistry was performed to investigate MCP-1 protein expression in endothelial cells of carotid artery.P-JNK,JNK,MCP-1 protein expression in carotid artery was measured by Western blot,signal transducer and activator of transcription-1（STAT-1） DNA binding activity was assayed by electrophoretic mobility shift assay（EMSA）. Results Compared with wild type mice,serum MPO and MDA expression increased prominently（P0.05）,whereas TNOS,NO decreased and p-JNK/JNK,MCP-1 expression increased significantly in INS2AKITA mice（P0.05）;STAT-1 DNA binding activity increased prominently in type 1 diabetic group compared with that in control group（P0.05）.Compared with type 1 diabetic mice,after treatment with SP600125,MPO decreased and NO,TNOS increased and p-JNK/JNK,endothelial MCP-1 expression decreased（P0.05）.The effects of SP600125 on MPO,NO,TNOS,MCP-1 were increased accordingly as the dose of SP600125 increased.As the dose of SP600125 increased,MCP-1 protein expression decreased 21.82%,39.09%,68.18% respectively;STAT-1 DNA binding activity decreased 25.70%,33.20%,61.29% respectively compared with type 1 diabetic mice. Conclusions JNK specific inhibitor SP600125 could inhibit STAT-1 DNA binding activity,thus increase serum NO and TNOS,and improve carotid diastolic function.SP600125 could also decrease MCP1 and MPO in a dose-dependent manner.The results also showed JNK signal pathway is associated with carotid endothelial dysfunction and inflammatory expression via activation of STAT-1 in type 1 diabetes.Our study provides a novel pharmacologic agent to prevent the promotion of macrovascular complication in type 1 diabetes.