E3泛素连接酶RNF121对脂多糖诱导的巨噬细胞表达促炎性细胞因子的影响

Effect of E3 ubiquitin ligase RNF121 on expression of proinflammatory cytokines in LPS-treated macrophages

目的探讨E3泛素连接酶RNF121对脂多糖(LPS)诱导的小鼠腹腔巨噬细胞表达促炎性细胞因子的调控作用。方法 LPS刺激小鼠腹腔巨噬细胞,Western blot检测RNF121的表达。用RNA干扰(si-RNF121)降低RNF121的表达,小鼠腹腔巨噬细胞经LPS刺激后,实时荧光定量PCR法检测TNF-α和IL-6的mRNA水平,ELISA检测细胞培养上清中TNF-α和IL-6的浓度,Western blot检测小鼠腹腔巨噬细胞中P65及磷酸化P65(p-P65)的表达水平,利用双荧光素酶报告基因法检测NF-κB的活性。结果 LPS诱导小鼠腹腔巨噬细胞后,RNF121的蛋白水平显著降低(P<0.05),蛋白酶抑制剂MG132能够显著增加RNF121的蛋白水平。si-RNF121降低RNF121的表达后,给予LPS刺激小鼠腹腔巨噬细胞,IL-6和TNF-α的表达水平均显著下降(P<0.05),转录因子P65的磷酸化(p-P65)水平及NF-κB的活性均显著降低(P<0.05)。结论 E3泛素连接酶RNF121通过调控NF-κB的活性从而影响小鼠巨噬细胞促炎性细胞因子TNF-α及IL-6的表达。

Objective To estimate the effect of E3 ubiquitin ligase RNF121 on the expression of proinflammatory eytokines in LPS-stimulated macrophages. Methods The level of RNF121 in mouse peritoneal maerophages treatment with LPS was detected by Western blot. RNF121 expression was reduced by RNA interference （si-RNF121）. After LPS stimulation ,the mRNA level of TNF-α and IL-6 in mouse peritoneal macrophages was detected by realtime quantitive PCR （q-PCR）. The level of TNF-α and IL-6 in supernatants was detected by ELISA. The level of phosphorylated P65 （p-P65） was detected by Weston blot. The NF-κB activity was measured by dual-luciferase assay. Results After treatment with LPS, the RNF121 protein level was significantly decreased （P 〈 0.05 ） in macrophages. However,protease inhibitor MG132 can significantly increase the RNF121 protein level. Decreased the expression of RNF121 by si-RNF121 and stimulated by LPS in macrophages, TNF-α and IL-6 expression （ mRNA and protein levels） was significantly decreased （ P 〈 0. 05 ） , and the transcription factor phosphorylatedP65 （p-P65） was also significantly decreased （P 〈 0. 05 ）. In addition, NF-κB activity was also decreased （ P 〈 0.05 ）. Conclusions The E3 ubiquitin ligase RNF121 is involved in the regulation of the expression of TNF-α and IL-6 in innate immune responses in mouse macrophages by NF-κB pathway.