短叶松素-3-乙酸酯对佐剂性关节炎大鼠成纤维样滑膜细胞增殖及炎性因子表达的影响  

Effect of Pinobanksin-3-acetate on Proliferation of Fibroblast-like Synoviocytes and Expression of Inflammatory Factors in Rats with Adjuvant Induced Arthritis

作　　者：王启海[1,2] 张智 WANG Qihai;ZHANG Zhi(Central Laboratory,Anhui College of Traditional Chinese Medicine,Wuhu 241000 Anhui,China;School of Life Sciences,University of Science and Technology of China,Hefei 230026 Anhui,China)

机构地区：[1]安徽中医药高等专科学校中心实验室,安徽芜湖241000 [2]中国科学技术大学生命科学学院,安徽合肥230026

出　　处：《中药新药与临床药理》2022年第4期454-460,共7页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家杰出青年科学基金资助项目(32025017);安徽省卫生健康委科研项目(AHWJ2021b061);安徽中医药高等专科学校自然科学研究重点项目(ZRKXZ202103)。

摘　　要：目的探讨短叶松素-3-乙酸酯(Pinobanksin-3-acetate,PB3A)对体外培养佐剂性关节炎(AIA)大鼠成纤维样滑膜细胞(FLSs)的增殖及脂多糖(LPS)诱导的炎性因子表达的影响。方法采用灭活分枝杆菌+弗氏完全佐剂(CFA)建立AIA大鼠模型,分离并原代培养AIA-FLSs。PB3A作用于细胞后,采用CCK-8法检测细胞增殖抑制率;流式细胞术检测细胞凋亡率;Western Blot法检测细胞中Bcl-2、Bax、Caspase-3蛋白表达。LPS诱导AIA-FLSs后,以PB3A干预,采用ELISA法检测细胞培养上清中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素6(IL-6)的含量;RT-qPCR法检测细胞IL-1β、iNOS、MCP-1、MMP3 mRNA表达;Western Blot法检测细胞MMP3蛋白表达。结果与空白对照组比较,2.5~40μg·mL^(-1)的PB3A对AIA-FLSs细胞的增殖抑制率均有明显升高(P<0.05,P<0.01),且具有时间和浓度依赖性;PB3A 5、10、20μg·mL^(-1)组的AIA-FLSs细胞凋亡率明显升高(P<0.05,P<0.01);10、20μg·mL^(-1)PB3A均能明显下调Bcl-2蛋白表达(P<0.01),上调Bax、Caspase-3蛋白表达(P<0.01)。与LPS组比较,10、20μg·mL^(-1)PB3A能明显降低细胞培养上清中TNF-α、IL-1β、IL-6的分泌量(P<0.01),下调细胞IL-1β、iNOS、MCP-1、MMP3 mRNA表达及MMP3蛋白表达(P<0.05,P<0.01)。结论PB3A可通过调控凋亡相关蛋白表达抑制AIA-FLSs的细胞增殖,促进细胞凋亡,并通过下调TNF-α、IL-1β、IL-6、iNOS、MCP-1及MMP3的表达来抑制LPS诱导的炎性反应。Objective To investigate the effects of Pinobanksin-3-acetate(PB3A)on the proliferation of fibroblastlike synoviocytes in rats with adjuvant induced arthritis rats(AIA-FLSs)and the expression of inflammatory factors induced by Lipopolysaccharide(LPS). Methods A rat adjuvant induced arthritis(AIA)model was established by inactivated Mycobacterium and Freund complete adjuvant(CFA),and AIA-FLSs was isolated and primary cultured.CCK-8 assay was used to detect the proliferation inhibition rate of PB3A treated cells. Cell apoptosis rate was detected by flow cytometry. Western Blot was used to detect the protein expression of Bcl-2,Bax and Caspase-3.The contents of TNF-α,IL-1β and IL-6 in the supernatance of cells cultured with PB3A were detected by ELISA after LPS induction of AIA-FLSs. The mRNA expression of IL-1β,iNOS,MCP-1 and MMP3 were detected by RTqPCR. The protein expression of MMP3 was detected by Western Blot method. Results Compared with the blank control group,the proliferation inhibition rate of AIA-FLSs cells was significantly increased by PB3A at 2.5-40 μg·mL^(-1)(P<0.05, P<0.01), which was time-and concentration-dependent;the apoptosis rate of AIA-FLSs cells in PB3A 5,10 and 20 μg·mL^(-1)groups was significantly increased(P<0.05,P<0.01);both PB3A 10 and 20 μg·mL^(-1)significantly down-regulated Bcl-2 protein expression(P<0.01) and up-regulated Bax and Caspase-3 protein expression(P<0.01). Compared with the LPS group,PB3A 10 and 20 μg·mL^(-1)significantly decreased the secretion of TNF-α,IL-1β and IL-6 in cell culture supernatant(P<0.01),down-regulated cellular IL-1β,iNOS,MCP-1 and MMP3 m RNA expression(P<0.05, P<0.01) and down-regulated cellular MMP3 protein expression(P<0.05, P<0.01). Conclusion PB3A inhibited the proliferation of AIA-FLSs cells and promoted apoptosis by regulating the expression of apoptosis-related proteins,and also inhibited the LPS-induced inflammatory response by down-regulating the expression of TNF-α,IL-1β,IL-6,i NOS,MCP-1 and MMP3.

关 键 词：短叶松素-3-乙酸酯 佐剂性关节炎 大鼠成纤维样滑膜细胞 增殖 凋亡 炎性因子 

分 类 号：R285.5[医药卫生—中药学]