宁心痛颗粒抑制HSP60诱导MyD88^(-/-)THP-1细胞炎症反应  

Inhibitory effect of the serum containing Ningxintong Granule on the inflammation induced by extracellular human heat shock protein60 in the MyD88^(-/-)THP-1 cells

作　　者：高颖[1,2] 陈蕊[3] 顾宁[4] 何小丽[4] 

机构地区：[1]南京中医药大学博士后流动站,江苏南京210046 [2]第二军医大学附属公利医院,上海200135 [3]南京中医药大学第一临床医学院,江苏南京210029 [4]南京中医药大学第三附属医院,江苏南京210001

出　　处：《时珍国医国药》2016年第12期2821-2825,共5页Lishizhen Medicine and Materia Medica Research

基　　金：国家自然科学基金面上项目(No.81173399);中国博士后科学基金面上项目(No.2015M571792);江苏省博士后科研资助项目(No.1401137C)

摘　　要：目的利用siRNA沉默THP-1源性巨噬细胞(Mφ)的髓样分化因子88(MyD88)基因表达,论证MyD88在介导重组人热休克蛋白60(rhHSP60)诱导Mφ炎症反应中的作用及益气活血方宁心痛颗粒含药血清的抗炎机制。方法 THP-1细胞诱导分化为Mφ,分为空白对照组、模型组、阴性对照组、RNAi组(MyD88^(-/-)Mφ)、含药血清组、RNAi+含药血清组共6组,荧光定量PCR法测定MyD88基因表达,免疫印迹法测定MyD88及磷酸化核因子κB(p-NF-κB)的蛋白表达,酶联免疫法测定TNF-α、IL-6的含量。结果 1模型组MyD88基因表达升高,与空白对照组相比,差异有统计学意义(P<0.01);RNAi组、含药血清组分别与模型组相比表达均下降,差异有统计学意义(P均<0.01);含药血清可增强siRNA的抑制作用,RNAi+含药血清组与RNAi组相比,差异有统计学意义。2模型组MyD88及p-NF-κB蛋白表达升高,与空白对照组相比,差异有统计学意义(P均<0.01);RNAi组二者表达均下降,与模型组相比,差异有统计学意义;含药血清可增强siRNA的抑制作用,RNAi+含药血清组与空白对照组相比,差异无统计学意义。3模型组TNF-α、IL-6含量升高,与空白对照组相比,差异有统计学意义(P均<0.01);转染MyD88 siRNA可抑制二者升高,RNAi组与模型组相比,差异具有统计学意义(P均<0.01);含药血清单独使用可抑制二者升高,并可增强siRNA的抑制效果,RNAi+含药血清组与RNAi组相比差异有统计学意义(P均<0.01)。结论 MyD88/NF-κB是人热休克蛋白60诱导THP-1源性Mφ发生炎症反应的关键信号通路,宁心痛颗粒含药血清可通过调控该信号通路发挥抗炎作用。Objective To investigate the inhibitory effect of the serum containing Ningxintong Granule on the inflammation in- duced by extracellular heat shock protein60（HSP60） in the myeloid differentiation factor 88 （MyD88） ＂knocked down＂ THP - 1 cells. Methods The human acute monocytic leukemia cell line THP - 1 cells were differentiated into macrophages by phorbol - 12 -myristate- 13 -acetate（PMA）. Then the macrophages were divided into six groups：blank control group, model group, negative control group, the serum group containing Ningxintong Granule （NXT） , RNAi group and RNAi ＋ NXT group. The last two groups were transfected with MyD88 small interfering RNA （ siRNA ）. 48 hours before adding of the recombinated human HSP60 （ rhHSP60 ）, the serum group containing Ningxintong Granule was added into the NXT group and the RNAi ＋ NXT group. 54 hours after the siRNA transfection, rhHSP60 was added to the five groups except for the blank control group. After incubating for the in- dicated times with rhHSP60 ,the macrophages or the culture supertenants were collected for real -time PCR, western blot a（ 1 ） The gene expression of MyD88 was elevated in the model group which was decreased significantly in the RNAi group（ P 〈 0.01 ）. The gene expression in the NXT group was also decreased compared with that in the model group. In the RNAi ＋ NXT group thegene expression was further inhibited. （2）Both of the protein expressions of MyD88 and p - NF - κB were elevated in the model group which were decreased significantly in the RNAi group（ MyD88,1. 199 ±0. 084 vs 0. 450 ± 0. 050 P 〈 0.01 ; p - NF - κB, 0.798±0.027 vs 0.409±0.039 P 〈 0.01 ）. The two protein expressions in the NXT group were decreased compared with those in the model group too. In the RNAi ＋ NXT group the protein expressions were further inhibited. （ 3 ） The contents of TNF -α and IL - 6 were lifted in the model group, while in the RNAi group they were significantly decreased （ TNF - α, 307.03 ±

关 键 词：宁心痛颗粒 髓样分化因子88 人重组热休克蛋白60 RNA干扰 炎性反应 

分 类 号：R285.5[医药卫生—中药学]