R406对人RASFs炎性因子分泌及生物学功能调节、关节炎小鼠关节炎症缓解作用观察  

R406 inhibits secretion of inflammatory factors in rheumatoid arthritis synovial fibroblasts,improves bio⁃logical function and alleviates joint inflammation in arthritic mice

作　　者：张玲 徐鑫睿 王林 潘继红 ZHANG Ling;XU Xinrui;WANG Lin;PAN Jihong(School of Biomedical Sciences,Shandong First Medical University,Jinan 250014,China)

机构地区：[1]山东第一医科大学生物医学科学院暨山东省医药生物技术中心生物化学教研室,山东济南 [2]山东第一医科大学第一附属医院类风湿关节炎科 [3]国家卫健委生物医药重点实验室 [4]山东省罕见病防治重点实验室生物化学教研室

出　　处：《山东医药》2022年第15期49-54,共6页Shandong Medical Journal

基　　金：国家自然科学基金(81671624)。

摘　　要：目的探讨R406(free base)对人类风湿关节炎关节滑膜成纤维细胞(RASFs)炎性因子分泌的抑制及生物学功能(侵袭、迁移和凋亡)的调节作用,观察R406对关节炎小鼠关节炎症的缓解作用。方法取3~7代生长的RASFs,分为实验组、阴性对照组和阳性对照组。实验组分别用1、10、50、100 ng/mL R406和10 ng/mL IL-1β干预,阴性对照组加入DMSO,阳性对照组加入DMSO和10 ng/mL IL-1β干预。干预24 h,采用qRT-PCR法检测各组炎性因子IL-6、IL-8、CCL2 mRNA。选择最适浓度(50 ng/mL)的R406作用于RASFs(实验组)不同时间(6、12、24、48 h),阴性对照组及阳性对照组处理方法同上,采用qRT-PCR检测各组IL-6、IL-8、CCL2 mRNA,ELISA法检测各组IL-6、IL-8、CCL2蛋白,Trans Well检测各组RASFs的侵袭能力;划痕法检测细胞迁移能力;流式细胞术检测细胞凋亡率。建立Ⅱ型胶原诱导型关节炎(CIA)模型鼠,将18只8周龄的DBA 1/J小鼠平均分为实验组、阴性对照组和阳性对照组。实验组诱发CIA后腹腔注射R406,200 mg/kg,每两天注射1次;阴性对照组不诱发CIA;阳性对照组诱发CIA后腹腔注射等量DMSO,每两天注射1次。R406治疗30 d后,ELISA法检测各组小鼠血清中炎性因子IL-6、IL-8、IL-1α、COMP。结果与阳性对照组比较,实验组中不同剂量R406作用后RASFs中炎性因子IL-6、IL-8、CCL2 mRNA表达被抑制,且在50 ng/mL时抑制效果最为显著(P均<0.01)。与阳性对照组比较,实验组中50 ng/mL R406作用于RASFs不同时间,细胞中炎性因子IL-6、IL-8、CCL2蛋白的表达均被抑制,且在24 h抑制效果最显著(P均<0.01);与阳性对照组比较,实验组RASFs上清液中炎性因子IL-6、IL-8、CCL2水平降低(P均<0.01)。与阳性对照组比较,实验组RASFs侵袭和迁移数均降低,凋亡率增加(P均<0.05)。与阳性对照组比较,实验组小鼠血清中炎性因子IL-6、IL-8、IL-1α、COMP水平降低(P均<0.01)。结论R406可以抑制RASFs中炎性因子的分泌,调节RASFObjective To investigate the effects of R406 on the secretion of inflammatory factors of rheumatoid arthritis synovial fibroblasts(RASFs),and its regulatory effects on biological functions(cell invasion,migration and apoptosis),and to observe the alleviating effect of R406 on joint inflammation in collagen Ⅱ-induced arthritis(C.IA) mice.Methods RASFs from 3 to 7 generations were divided into the experimental group,negative control group,and positive control group.The RASFs in the experimental group were treated with 1,10,50,and 100 ng/mL R406 and 10 ng/mL IL-1β,respectively.The RASFs in the negative control group were added with DMSO,and the positive control group with DMSO and 10 ng/mL IL-1β,respectively.Real-time fluorescence quantitative PCR(qRT-PCR) was used to detect the inflammatory factors IL-6,IL-8,and CCL2 mRNAs in RASFs after 24-hour intervention,and the optimal dose(50 ng/mL)of R406 was used to treat RASFs at different time(6,12,24,and 48 h) by qRT-PCR.The treatment methods of the negative control group and the positive control group were the same as above.The qRT-PCR was used to detect the IL-6,IL-8 and CCL2 mRNAs in each group;ELISA was used to detect the IL-6,IL-8 and CCL2 proteins in each group.The invasive ability of RASFs in each group was detected by Trans Well;the cell migration ability was detected by Scratch test;the apoptosis rate was detected by flow cytometry.CIA model mice were established,18 eight-week-old DBA 1/J mice(20-25 g) were divided into the experimental group,negative control group,and positive control group.The mice in the experimental group were treated with R406 after arthritis induction(200 mg/kg,intraperitoneal injection,once every two days);in the negative control group,we did not induce arthritis;mice in the positive control group were treated with DMSO(intraperitoneal injection,once every two days) after arthritis induction.After 30 days of R406 treatment,the serum inflammatory factors IL-6,IL-8,IL-1α and COMP were detected by ELISA.Results Compared with the positiv

关 键 词：脾酪氨酸蛋白激酶抑制剂 R406 类风湿关节炎 滑膜成纤维细胞 白细胞介素6 白细胞介素8 趋化因子配体2 细胞生物学功能 小鼠 

分 类 号：R34[医药卫生—基础医学]