颗粒酶B通过SRGN/NF-kappaB信号通路调节类风湿关节炎成纤维细胞样滑膜细胞的增殖和炎症反应

GZMB regulates the proliferation and inflammatory response of fibroblast-like synovial cells in rheumatoid arthritis through the SRGN/NF-kappaB signaling pathway

目的 颗粒酶B(GZMB)在类风湿关节炎中发挥作用的机制尚不完全清楚。文章主要研究GZMB对类风湿关节炎成纤维细胞样滑膜细胞(RA-FLSs)的增殖和炎症的影响及其潜在的机制。方法 采用RT-qPCR和Western blot检测RA-FLSs细胞中GZMB的表达,构建GZMB以及丝甘蛋白聚糖(serglycin;SRGN)过表达载体(oe-GZMB;oe-SRGN)和GZMB干扰质粒(si-GZMB)并转染到RA-FLSs中,RT-qPCR和Western blot检测过表达和干扰效率。CCK-8实验以及EdU检测细胞增殖水平。使用ELISA试剂盒检测炎性因子IL-6、IL-8和IL-1β的表达水平。免疫共沉淀(CO-IP)实验检测GZMB和SRGN之间的相互作用。Western blot检测NF-kappaB信号通路相关蛋白表达水平。结果 与正常人的滑膜细胞(NC-FLSs)相比,GZMB在RA-FLSs细胞中表达上调(P<0.01);过表达GZMB能促进RA-FLSs的增殖和炎症反应(P<0.01),而敲除GZMB则显著抑制RA-FLSs的增殖和炎症反应(P<0.05);RT-qPCR和Western blot检测显示,与NC-FLSs相比,SRGN在RA-FLSs细胞中高表达(P<0.01)。CO-IP实验验证GZMB能够与SRGN结合(P<0.01),且过表达SRGN逆转了GZMB敲除对RA-FLSs的增殖和炎症反应的抑制作用(P<0.01)。此外,过表达SRGN也逆转了GZMB敲除对NF-kappaB信号通路相关蛋白(GZMB、SRGN、P65、p-P65和P50)的抑制作用(P<0.01)。结论 过表达GZMB能促进RA-FLSs的增殖和炎症反应,此外,GZMB能和SRGN互相作用并通过NF-kappaB信号通路调节RA-FLSs的增殖和炎症反应。

Objective The mechanism by which granulozyme B(GZMB) plays a role in rheumatoid arthritis is not fully understood. This paper aims to investigate the effect of GZMB on proliferation and inflammation of fibroblast-like synovial cellsin rheumatoid arthritis(RA-FLSs) and its potential mechanism. Methods The expression of GZMB in RA-FLSs cells was detected by RT-qPCR and Western blot. The GZMB and SRGN overexpression vectors(Oe-GZMB;Oe-SRGN) and GZMB interfering plasmid(si-GZMB) were constructed and transfected into RA-FLSs, and the over-expression and interfering efficiency were detected by RT-qPCR and Western blot. CCK-8 test and EdU assay were performed to detect the cell proliferation. The expression levels of inflammatory factors IL-6, IL-8 and IL-1β were detected by ELISA kit. Co-immunoprecipitation(CO-IP) assay was used to detect the interaction between GZMB and SRGN. The expression level of NF-kappaB signaling pathway-related protein was detected by Western blot. ResultsCompared with FLSs from normal controls(NC-FLSs), GZMB was up-regulated in RA-FLSs cells(P<0.01). Overexpression of GZMB promoted the proliferation and inflammatory response of RA-FLSs(P<0.01), while knockdown of GZMB significantly inhibited the proliferation and inflammatory response of RA-FLSs(P<0.05). RT-qPCR and Western blot assays showed high expression of SRGN in RA-FLSs cells compared with NC-FLSs(P<0.01). CO-IP experiment verified that GZMB could bind to SRGN(P<0.01), and overexpression of SRGN reversed the inhibition of GZMB knockout on the proliferation and inflammatory response of RA-FLSs(P<0.01). In addition, overexpression of SRGN also reversed the inhibition of GZMB knockdown on NF-kappaB signaling pathway-related proteins(GZMB, SRGN, P65, p-P65 and P50)(P<0.01). Conclusion The overexpression of GZMB can promote RA-FLSs proliferation and inflammation. In addition, GZMB can interact with SRGN and regulate RA-FLSs proliferation and inflammation by NF-kappaB signaling pathway.