羊踯躅含药血清对TNF-α诱导的类风湿关节炎成纤维样滑膜细胞增殖及炎症因子分泌的影响被引量：2

Effects of medicated serum containing Rhododendron molle G.Don on the proliferation and inflammatory factors secretion of fibroblast-like synoviocytes in rheumatoid arthritis induced by tumor necrosis factor

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摘要目的探究羊踯躅含药血清对肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)诱导的类风湿关节炎成纤维样滑膜细胞(rheumatoid arthritis fibroblast-like synoviocytes,RA-FLS)增殖和炎症因子分泌的影响和作用机制,为临床治疗RA提供理论及实验依据。方法将细胞分为空白组、模型组、雷公藤多苷组、5%和10%空白血清组及5%和10%羊踯躅含药血清组。RA-FLS经TNF-α(10 ng/mL)处理24 h以构建RA细胞模型。CCK-8检测细胞增殖情况。采用ELISA检测炎症因子白细胞介素-1β(interleukin-1 beta,IL-1β)、白细胞介素-17A(interleukin-17A,IL-17A)、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-2(interleukin-2,IL-2)和γ干扰素(interferon-γ,IFN-γ)的含量。Western blot检测蛋白激酶B(protein kinase B,AKT1)、磷酸化AKT1(phosphorylated AKT1,p-AKT1)、原癌基因c-JUN、p-c-JUN、p65、p-p65、表皮生长因子受体(epidermal growth factor receptor,EGFR)蛋白表达水平。结果与未用羊踯躅含药血清处理的正常RA-FLS相比,用5%和10%羊踯躅含药血清处理的细胞增殖活性在48 h内没有明显的变化(P>0.05),而用20%和30%羊踯躅含药血清处理的细胞增殖活性明显降低(P<0.05)。与空白组相比,模型组的细胞增殖活性显著上升(P<0.05);与5%和10%空白血清组相比,5%和10%羊踯躅含药血清组的细胞增殖活性均显著降低(P<0.05)。与空白组相比,模型组的IL-1β、IL-17A、IL-6、IL-2和IFN-γ水平均明显升高(P<0.05);与5%和10%空白血清组相比,5%和10%羊踯躅含药血清组的IL-1β、IL-17A、IL-6、IL-2和IFN-γ水平明显下降(P<0.05)。与空白组相比,模型组的EGFR表达量以及AKT1、c-JUN和p65的磷酸化水平均明显升高(P<0.05);与5%和10%空白血清组相比,5%和10%羊踯躅含药血清组的EGFR表达量以及AKT1、c-JUN和p65的磷酸化水平显著降低(P<0.05)。此外,10%羊踯躅含药血清的效果比5%羊踯躅含药血清更为显著(P<0.05)。结论羊踯躅含药血清能抑制TNF-α诱导的RA-FLS增殖及促炎因子的分泌,其机制可能与调控EGFR、核因子-κB(nuclear factor-κB,NF-κB)、AKT和c-Jun N末端激酶(c-Jun N-terminal kinase,JNK)通路有关。 Objective To explore the effects and mechanism of action of medicated serum containing Rhododendron molle G.Don on the proliferation and inflammatory factors secretion of tumor necrosis factor-α(TNF-α)-induced rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS),so as to provide theoretical and experimental basis for clinical treatment of RA.Methods RA-FLS cells were divided into blank group,model group,tripterygium glycosides group,blank serum(5%,10%)groups,and medicated serum(5%,10%)groups.RA-FLS were treated with TNF-α(10 ng/mL)for 24 h to construct RA cell models.Cell Counting Kit-8(CCK-8)was utilized to examine cell proliferation.Enzyme-linked immunosorbent assay(ELISA)was used to determine the content of inflammatory factors including interleukin-1 beta(IL-1β),interleukin-17A(IL-17A),interleukin-6(IL-6),interleukin-2(IL-2),and interferon-gamma(IFN-γ).Western blot was used to determine the protein expression levels of protein kinase B(AKT1),phosphorylated AKT1(p-AKT1),c-JUN,p-c-JUN,p65,p-p65,and epidermal growth factor receptor(EGFR).Results Compared with the groups without medicated serum containing Rhododendron molle G.Don,there was no significant change in cell proliferation activity within 48 h in the 5%and 10%medicated serum containing Rhododendron molle G.Don groups(P>0.05),but cell proliferation activity significantly reduced in the 20%and 30%medicated serum containing Rhododendron molle G.Don groups(P<0.05).Compared with the blank group,cell proliferation activity in the model group was significantly higher(P<0.05).Compared with the 5%and 10%blank serum groups,cell proliferation activity in the medicated serum groups containing different doses of Rhododendron molle G.Don was significantly lower(P<0.05).Compared with the blank group,the levels of IL-1β,IL-17A,IL-6,IL-2,and IFN-γin the model group were significantly higher(P<0.05).Compared with the blank serum groups,the levels of IL-1β,IL-17A,IL-6,IL-2,and IFN-γin the serum groups containing different doses of Rhododendron molle G.Don were significantly lower(P<0.05).Compared with the 5%and 10%blank group,the expression level of EGFR as well as the phosphorylation levels of AKT1,c-JUN,and p65 in the model group were significantly higher(P<0.05).Compared with the blank serum groups,the expression levels of EGFR as well as the phosphorylation levels of AKT1,c-JUN,and p65 in the medicated serum groups containing different doses of Rhododendron molle G.Don were significantly lower(P<0.05).Additionally,the effects of the 10%serum containing Rhododendron molle G.Don were more significant than those of the 5%medicated serum containing Rhododendron molle G.Don(P<0.05).Conclusion The medicated serum containing Rhododendron molle G.Don can inhibit cell proliferation and the secretion of pro-inflammatory factors of RA-FLS induced by TNF-α,which may be related to the regulation of EGFR,nuclear factorκB(NF-κB),AKT,and c-JUN N-terminal kinase(JNK)pathways.

作者刘笑蓉李硕夫梅文亚湛欢刘平安周日宝 LIU Xiaorong;LI Shuofu;MEI Wenya;ZHAN Huan;LIU Ping'an;ZHOU Ribao(Hunan University of Chinese Medicine,Changsha,Hunan 410208,China;The First Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410007,China;Hunan Academy of Chinese Medicine,Changsha,Hunan 410006,China)

机构地区湖南中医药大学湖南中医药大学第一附属医院湖南省中医药研究院

出处《湖南中医药大学学报》 CAS 2023年第10期1809-1814,共6页 Journal of Hunan University of Chinese Medicine

基金湖南省自然科学基金项目(2022JJ80086) 湖南省卫健委科学研究项目(D202302078705) 湖南省教育厅科学研究项目(19C1384) 湖南省大学生创新创业训练计划项目(2022-5313) 湖南省中医药管理局科研计划项目(2021161) 湖南中医药大学中药学一级学科开放基金项目(2020ZYX01) 湖南中医药大学青苗计划(校行人字[2017]25) 湖南省一流学科中药学(校行科字[2018]3) 2020年湖南省一流本科专业建设点(湘教通[2020]248号):中药资源与开发2020年国家级一流本科专业建设点(教高厅函[2021]7号):中药学专业。

关键词类风湿关节炎成纤维样滑膜细胞羊踯躅含药血清细胞增殖炎症因子 rheumatoid arthritis fibroblast-like synoviocyte medicated serum containing Rhododendron molle G.Don cell proliferation inflammatory factor

分类号 R285.5 [医药卫生—中药学]

作者简介第一作者:刘笑蓉,女,讲师,硕士,研究方向:中药资源与药效物质研究,E-mail:364059345@qq.com。