高糖通过白介素1β促进1型糖尿病大鼠原代主动脉平滑肌细胞迁移参与内膜新生的研究

High glucose promotes the migration of aortic smooth muscle cells in type 1 diabetic rats through IL-1β in neointima hyperplasia

目的探讨在动脉损伤后内膜新生中高糖促进血管平滑肌迁移的机制。方法采用组织块贴壁法分离大鼠原代主动脉平滑肌细胞,免疫荧光染色法进行细胞鉴定。大鼠原代主动脉平滑肌细胞分为正常对照组(Con)、高糖组(HG)、HG+半胱氨酸蛋白水解酶1(Caspase-1)抑制剂组(HG+VX-765)、Con+Caspase-1抑制剂组(VX-765)。大鼠随机分别为假手术组(C)、正常颈动脉球囊损伤组(I)、糖尿病假手术组(DM)、糖尿病球囊损伤组(DMI)。RT-PCR检测含NLR家族pyrin域蛋白3(NLRP3)、Caspase-1、消皮素D(GSDMD)、IL-1βmRNA水平表达。术后24 h并2周后免疫荧光染色法检测IL-1β表达,HE染色检测血管损伤后内膜新生情况,ELISA法检测IL-1β水平,划痕实验测定平滑肌细胞迁移能力。结果分离的细胞经免疫荧光染色确定为大鼠原代主动脉平滑细胞。与Con组比较,HG组IL-1β水平、细胞迁移率、NLRP3、Caspase-1、GSDMD、IL-1βmRNA表达水平升高(P<0.05或P<0.01)。与HG组比较,HG+VX-765组IL-1β水平、细胞迁移率降低(P<0.05或P<0.01)。24 h和2周后,与C组比较,DM组IL-1β表达水平、内膜面积/中膜面积升高(P<0.05或P<0.01);与I组比较,DMI组IL-1β表达升高(P<0.01)。结论高糖诱导细胞焦亡分泌的IL-1β促进大鼠原代主动脉平滑肌细胞迁移,参与动脉损伤后的内膜新生过程。

Objective To explore the mechanism of high glucose in promoting the migration of rat aortic smooth muscle cells(RASMCs)in intimal hyperplasia after vessel injury.Methods Primary rat aortic smooth muscle cells were isolated by tissue adhesion method and identified by immunofluorescence staining. Primary rat aortic smooth muscle cells were divided into normal control group(Con),high glucose group(HG),high glucose group +Caspase-1 inhibitor(HG+VX-765),and normal control group+Caspase-1 inhibitor(VX-765). The rats were randomly divided into sham operation group(C),normal carotid balloon injury group(I),diabetic sham operation group(DM)and diabetic balloon injury group(DMI). The m RNA expressions of NLRP3,Caspase-1,GSDMD and IL-1β were detected by RT-PCR. The expression of IL-1β was detected by immunofluorescence staining at 24 h and 2 weeks after operation,and the neointimal formation after vascular injury was detected by HE staining. The level of IL-1β was detected by ELISA,and the migration ability of smooth muscle cells was measured by scratch test.Results The isolated cells were identified as primary smooth aortic cells by immunofluorescence staining. Compared with Con group,the levels of IL-1β,cell mobility,NLRP3,Caspase-1,GSDMD and IL-1β m RNA were higher in HG group(P<0. 05 or P<0. 01). Compared with HG group,IL-1β level and cell mobility decreased in HG+VX-765 group(P<0. 05 or P<0. 01). After 24 h and 2 weeks,compared with C group,the expression level of IL-1β and intima/media area increased in DM group(P<0. 05 or P<0. 01). Compared with I group,the expression of IL-1β increased after 24 h and 2 weeks in DMI group(P<0. 01).Conclusion IL-1β secreted by cell scorch induced by high glucose promotes the migration of rat primary aortic smooth muscle cells and participates in the neointimal process after arterial injury.