沉默HMGB1通过下调NF-κB信号来改善滋养细胞增殖侵袭及胰岛素抵抗和炎症反应

Silencing HMGB1 Improves Trophoblast Cell Proliferation Invasion Insulin Resistance and Inflammation by Downregulating NF-κB Signaling

目的:探讨沉默高迁移率族蛋白1(HMGB1)对胰岛素抵抗(IR)条件下滋养细胞的增殖、侵袭能力及胰岛素敏感性和炎症反应的影响和相关机制研究。方法:培养人绒毛膜滋养细胞系HTR-8,使用shRNA瞬时转染技术沉默细胞中HMGB1表达;建立滋养细胞的体外IR模型,并将细胞分为四组,Control组、胰岛素抵抗组(IR组)、IR+shRNA-NC组和IR+shRNA-HMGB1;葡萄糖含量测定试剂盒验证IR模型,使用Western blot和细胞免疫荧光染色检测HMGB1在各组滋养细胞中的表达;CCK-8、Transwell和ELISA实验分别检测沉默HMGB1对滋养细胞增殖、侵袭能力及炎症因子TNF-α、IL-1β、IL-6分泌的影响;Western blot检测沉默HMGB1对滋养细胞中胰岛素敏感性相关蛋白IRβ和PI3K的磷酸化水平及ISR-1、ISR-2和葡萄糖转运蛋白GLUT4与GLUT1表达的影响,以及NF-κB信号通路中蛋白IκBα和p65磷酸化水平。结果:与Control组相比,IR模型中滋养细胞的葡萄糖消耗量,细胞的增殖、侵袭能力和蛋白GLUT-1表达水平均明显降低(P<0.05),HMGB1蛋白表达及荧光活性和炎症因子TNF-α、IL-1β、IL-6的分泌水平,蛋白IRβ、PI3K、IκBα和p65的磷酸化水平及ISR-1、ISR-2和GLUT4表达水平均明显增加(P<0.05);而与IR组相比,IR+shRNA-HMGB1细胞的葡萄糖消耗量,细胞的增殖、侵袭能力和蛋白GLUT-1表达水平均明显增加(P<0.05),HMGB1的蛋白表达及荧光活性及炎症因子TNF-α、IL-1β、IL-6的分泌水平,蛋白IRβ、PI3K、IκBα和p65的磷酸化水平以及ISR-1、ISR-2和GLUT4表达均明显减少(P<0.05)。结论:HMGB1表达水平在IR条件下的滋养细胞中明显升高,沉默其表达能够通过NF-κB信号通路促进滋养细胞的增殖与侵袭能力,提高细胞对胰岛素的敏感性,并降低其对促炎因子的分泌。

Objective:To investigate the effects of silencing high mobility group 1(HMGB1)on trophoblast proliferation,invasion,insulin resistance and inflammatory response in insulin resistance(IR).Methods:Human chorionic trophoblast cell line htr-8 was cultured and the expression of HMGB1 was silenced by shRNA transient transfection;the IR model of trophoblast cells was established in vitro,and the cells were divided into four groups:control group,insulin resistance group(IR group),IR+shRNA-NC group and IR+shrna-HMGB1 group.Western blot and immunofluorescence staining were used to detect the expression of HMGB1 in trophoblasts;CCK-8,Transwell and ELISA were used to detect the effects of HMGB1 silencing on the proliferation and invasion of trophoblasts and the expression of TNF-α,IL-1βand IL-6;Western blot was used to detect the effects of HMGB1 silencing on the phosphorylation of insulin sensitivity related proteins IRβand PI3K,the expression of ISR-1,ISR-2,GLUT4 and GLUT1,and the phosphorylation levels of IκBαand p65 in NF-κB signaling pathway.Results:Compared with the control group,the glucose consumption,cell proliferation,invasion and protein GLUT-1 expression level of trophoblast in IR model were significantly decreased(P<0.05),the protein expression and fluorescence activity of HMGB1,the secretion levels of inflammatory factors TNF-α,IL-1β,IL-6,phosphorylation levels of proteins IRβ,PI3K,IκBαand p65,and the expressions of ISR-1,ISR-2 and GLUT4 were significantly increased(P<0.05).Compared with IR group,the glucose consumption,cell proliferation,invasion ability and GLUT-1 expression level of IR+shRNA-HMGB1 cells were significantly increased(P<0.05),protein expression and fluorescence activity of HMGB1,secretion levels of inflammatory factors TNF-α,IL-1β,IL-6,phosphorylation levels of proteins IRβ,PI3K,IκBαand p65,and expressions of ISR-1,ISR-2 and GLUT4 were significantly decreased(P<0.05).Conclusion:It found that HMGB1 expression level was significantly increased in the trophoblasts under IR condition.Silencing HMGB1 expression can promote the proliferation and invasion of trophoblasts through NF-κB signaling pathway,improve the sensitivity of trophoblasts to insulin,and reduce the secretion of pro-inflammatory factors.