环脂蛋白20调控组蛋白H2B泛素化经NF-κB信号通路对巨噬细胞迁移的影响

Effect of cyclic finger protein 20 regulating histone H2B ubiquitination on macrophage migration through NF-κB pathway

目的探讨环指蛋白20(RNF20)调控组蛋白H2B泛素化(H2Bub)经核因子-κB(NF-κB)途径对巨噬细胞迁移的影响。方法将培养好的巨噬细胞分为7组,A组(对照组):培养48 h;B组(ox-LDL刺激组):50 mg/mL oxLDL刺激巨噬细胞48 h;C组(ox-LDL+oe-RNF20转染组):oe-RNF20转染巨噬细胞后用50 mg/mL ox-LDL刺激48 h;D组(ox-LDL+pcDNA转染对照组):pcDNA转染巨噬细胞后用50 mg/mL ox-LDL刺激48 h;E组(ox-LDL+SN50干预组):50 mg/mL ox-LDL刺激巨噬细胞24 h后再给予10μg/mL的SN50处理24 h,共干预48 h;F组(ox-LDL+LY294002干预组):50 mg/mL ox-LDL刺激巨噬细胞24 h后再给予10μg/mL的LY294002处理24 h,共干预48 h;G组(ox-LDL+p38干预组):50 mg/mL ox-LDL刺激24 h后再给予10μg/mL的p38,共干预48 h。采用Transwell法观察巨噬细胞迁移数量,Western blotting法检测RNF20、H2B、H2Bub、NF-κB及白细胞介素6(IL-6)表达。结果在oxLDL刺激的巨噬细胞中,与A组比较,B组RNF20蛋白表达、H2Bub/H2B降低,巨噬细胞迁移数量增多,NF-κB和IL-6蛋白表达增加(P均<0.01)。在RNF20过表达细胞模型中,与B组比较,C组RNF20蛋白表达、H2Bub/H2B增加,NF-κB、IL-6蛋白表达减少,巨噬细胞迁移数量减少(P均<0.01);D组RNF20蛋白表达、H2Bub/H2B、NF-κB、IL-6无明显改变,巨噬细胞迁移数量无明显改变(P均>0.05)。给予SN50(NF-κB抑制剂)、SB203580(p38抑制剂)和LY294002(PI3K抑制剂)处理巨噬细胞后,与B组比较,E、F、G组RNF20蛋白表达、H2Bub/H2B无明显改变(P均>0.05);E组NF-κB、IL-6蛋白表达及巨噬细胞迁移数量减少(P均<0.01);F、G组NF-κB、IL-6蛋白表达及巨噬细胞迁移数量无明显改变(P均>0.05)。结论RNF20减少,H2Bub减少,加剧巨噬细胞迁移及炎症反应,促进了动脉粥样硬化的形成,此过程经NF-κB信号通路起作用。

Objective To investigate the effect of cyclic finger protein-20(RNF20)regulating histone H2B ubiqui⁃tination(H2Bub)on macrophage migration through the NF-κB pathway.Methods The cells were divided into seven groups:group A(control group,the cells were cultured for 48 h),group B(ox-LDL stimulation group,using 50 mg/mL ox-LDL to stimulate macrophages for 48 h),group C(ox-LDL+oe-RNF20 transfection group,macrophages transfected with oe-RNF20 were stimulated with ox-LDL at a dose of 50 mg/mL for 48 h),group D(ox-LDL+pcDNA transfection con⁃trol group,macrophages were transfected with pcDNA and stimulated with 50 mg/mL ox-LDL for 48 h),group E(oxLDL+SN50 intervention group,after using 50 mg/mL ox-LDL to stimulate macrophages for 24 h,followed by 10μg/mL SN50 treatment for 24 h,and a total of 48 h),group F(ox-LDL+LY294002 intervention group,after using 50 mg/mL oxLDL to stimulate macrophages for 24 h,rats were treated with 10μg/mL LY294002 for 24 h,and a total of 48 h)and group G(ox-LDL+p38 intervention group,using 50 mg/mL ox-LDL to stimulate for 24 h,followed by 10μg/mL p38 for 24 h,and a total of 48 h).Transwell assay was used to observe the migration number of macrophages,and Western blot⁃ting was used to detect the expression levels of RNF20,H2B,H2Bub,nuclear factor-κB(NF-κB)and interleukin 6(IL6).Results In the ox-LDL stimulated macrophages,compared with group A,the expression of RNF20 protein and H2Bub/H2B ratio decreased,the number of migration macrophage increased,and the expression of NF-κB and IL-6 pro⁃tein increased in the group B(all P<0.01).In the RNF20 overexpression cell model,compared with group B,the expres⁃sion of RNF20 protein and H2Bub/H2B ratio increased,while the expression levels of NF-κB and IL-6 protein decreased,and the number of migration macrophage decreased in the group C(all P<0.01).In the group D,the expression of RNF20 protein,the ratio of H2Bub to H2B,NF-κB,IL-6 and the number of migration macrophage did not significantly change(all P>0.05).After treatment with SN50(NF-κB inhibitor),SB203580(p38 inhibitor),and LY294002(PI3K inhibi⁃tor),the expression of RNF20 protein and the ratio of H2Bub/H2B in the groups E,F,and G did not significantly change as compared with those of the group B(all P>0.05).In the group E,the expression levels of NF-κB and IL-6 proteins and the number of migration macrophage decreased(all P<0.01).The expression levels of NF-κB and IL-6 proteins and the number of migration macrophage in the groups F and G did not significantly change(all P>0.05).Conclusion De⁃creased RNF20 and H2Bub aggravated macrophage migration and inflammatory reaction,and promoted the formation of atherosclerosis,which worked through the NF-κB signaling pathway.