摘要
目的:研究萆薢总皂苷(TSD)对尿酸钠(MSU)诱导THP^-1细胞Toll样受体/核转录因子-κB(TLR/NF-κB)信号通路的影响,探讨其抗痛风性关节炎的可能作用机制。方法:佛波酯(PMA)诱导THP^-1细胞分化为巨噬细胞,分为正常组,模型组,TSD低、中、高质量浓度组(1,3,10 mg·L^-1)和秋水仙碱组(0.2 mg·L^-1),除正常组外,其余各组均用400 mg·L^-1MSU刺激6 h,诱导体外炎症反应。噻唑蓝(MTT)比色法检测TSD对细胞活力影响;酶联免疫吸附测定法(ELISA)检测细胞上清液炎性因子如肿瘤坏死因子-α(TNF-α)和白细胞介素^-1β(IL^-1β)的分泌水平;蛋白免疫印迹法(Western blot)检测TLR4,髓样分化因子88(MyD88)及NF-κB蛋白表达;实时荧光定量PCR(Real-time PCR)检测TLR4,NF-κB及IL^-1β前体(Pro-IL^-1β)mRNA表达水平;免疫荧光法检测NF-κB p65的核位移情况。结果:0~32 mg·L^-1TSD对细胞活力基本无影响;与正常组比较,模型组炎性因子TNF-α及IL^-1β的分泌水平显著升高(P<0.01),通路关键蛋白(TLR4,MyD88及NF-κB)和基因(TLR4,NF-κB及Pro-IL^-1β)的表达显著升高(P<0.01);与模型组比较,1~30 mg·L^-1TSD可显著下调炎性因子TNF-α及IL^-1β的分泌(P<0.01),通路关键蛋白(TLR4,MyD88及NF-κB)和基因(TLR4,NF-κB及Pro-IL^-1β)的表达明显降低(P<0.05,P<0.01),细胞核内NF-κB p65叠加减少,部分转位至胞浆,抑制NF-κB p65核转位。结论:TSD可能通过下调TLR4,NF-κB及Pro-IL^-1βmRNA的表达,减少炎性因子TNF-α及IL^-1β分泌而发挥抗炎作用。
Objective:To investigate the effect of total saponin of Dioscoreae Collettii Rhizoma(TSD)on Toll-like receptor/nuclear factor-κB(TLR/NF-κB)signaling pathway induced by monosodium urate in THP^-1cells,in order to explore the possible mechanism of anti-gout arthritis.Method:Phorbol 12-myristate 13-acetate(PMA)-induced THP^-1 cells were differentiated into macrophages,divided into normal group,model group,low,medium and high-concentration TSD groups(1,3,10 mg·L^-1)and colchicine group(0.2 mg·L^-1).Except the normal group,the other groups were stimulated with 400 mg·L^-1monosodium urate to replicate an inflammation model in vitro.Cell viability was measured by methyl thiazolyl tetrazolium(MTT)assay,the levels of inflammatory factors tumor necrosis factor-α(TNF-α)and interleukin^-1β(IL^-1β)were detected by enzyme-linked immunosorbent assay(ELISA).The protein levels of Toll-like receptor 4(TLR4),myeloid differentiation factor88(MyD88)and NF-κB were detected by Western blot.The mRNA levels of TLR4,NF-κB and Pro-IL^-1βwere measured by real-time fluorescence quantitative PCR(Real-time PCR),and the nuclear shift of NF-κB p65 was detected by immunofluorescence.Result:0~32 mg·L^-1TSD has no effect on cell viability.Compared with the normal group,the secretion levels of inflammatory factors TNF-αand IL^-1βin the model group were significantly increased(P<0.01),and the expressions of key proteins(TLR4,MyD88 and NF-κB)and genes(TLR4,NF-κB and Pro-IL^-1β)were increased(P<0.01).Compared with the model group,1-30 mg·L^-1TSD significantly down-regulated the secretion of inflammatory factors TNF-αand IL^-1β(P<0.01),the expressions of key proteins(TLR4,MyD88 and NF-κB)and genes(TLR4,NF-κB and Pro-IL^-1β)were decreased(P<0.05,P<0.01),and the NF-κB p65 partially trans-located to the cytosol and the superposition in the nucleus were decreased,inhibiting the nuclear translocation of NF-κB p65.Conclusion:TSD may exert an anti-inflammatory effect by downregulating the expressions of TLR4,NF-κB and Pro-IL^-1βmRNA and reducing the secretion of inflammatory factors TNF-αand IL^-1β.
作者
李国莺
章维志
姜璐
陈光亮
LI Guo-ying;ZHANG Wei-zhi;JIANG Lu;CHEN Guang-liang(College of Integrated Chinese and Western Medicine,Anhui University of Chinese Medicine,Hefei 230012,China;Anhui Provincial Key Laboratory of Traditional Chinese Medicine Compound Research,Hefei 230012,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2020年第5期34-41,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81573670).
作者简介
第一作者:李国莺,在读硕士,从事中药防治代谢型疾病研究,E-mail:monica7215@163.com;通信作者:陈光亮,博士,教授,从事中药防治代谢型疾病研究,E-mail:chguangl@163.com
