炎调方对巨噬细胞中HSP70表达及其效应的影响  

Influence of “Yantiao Prescription”on Expression and Action of HSP70 in Macrophages

作　　者：何淼[1] 计高荣[1] 熊旭东[1] 沈晓红[1] 周钱梅[2] 

机构地区：[1]上海中医药大学附属曙光医院,上海200021 [2]上海中医药大学,上海201203

出　　处：《上海中医药大学学报》2017年第6期76-79,共4页Academic Journal of Shanghai University of Traditional Chinese Medicine

基　　金：教育部高等学校博士学科点专项科研基金项目(20113107110003)

摘　　要：目的:观察炎调方药物血清对离体巨噬细胞中热休克蛋白70(HSP70)及其他相关细胞因子的调控效应,以验证炎调方治疗脓毒症的作用途径和位点。方法:培养大鼠巨噬细胞RAW 264.7,将培养后的巨噬细胞RAW 264.7分为空白对照组(RAW 264.7+培养液)、正常血清组(RAW 264.7+正常大鼠血清)、炎调方药物血清组(RAW 264.7+炎调方药物血清),每组设4个平行孔。采用MTT比色法测定细胞增殖,确定炎调方药物血清的无毒性剂量范围;采用qRT-PCR方法检测细胞HSP70、核因子-κB(NF-κB)、肿瘤坏死因子(TNF)-α、白介素(IL)-1β、IL-8的mRNA表达;采用Western blot方法检测细胞HSP70、NF-κB蛋白表达,以ELISA方法检测细胞TNF-α、IL-1β、IL-8蛋白表达。结果:炎调方药物血清的无毒性浓度范围包括10%~30%,故后续实验选取20%浓度的炎调方药物血清进行。正常血清组的各项指标与空白对照组比较,其差异均无统计学意义(P>0.05);与空白对照组和正常血清组比较,炎调方药物血清组RAW 264.7细胞的HSP70 mRNA和蛋白表达均明显升高(P<0.01),NF-κB、TNF-α、IL-1β、IL-8的mRNA和蛋白表达均明显降低(P<0.01)。结论:炎调方治疗脓毒症的作用机制可能是上调HSP70的表达,进而抑制NF-κB的活化和相关细胞因子的表达。Objective： To observe the regulatory effects of drug medicated serum of ＂Yantiao Prescription＂ on the heat shock protein 70 （ HSP70 ） and other relevant cytokines in macrophages in vitro, and validate the pathway and sites of ＂Yantiao Prescription＂ in the treatment of sepsis. Methods： The rat macrophages RAW 264.7 were cultured and divided into the blank control group treated with culture medium, normal serum group treated with serum from normal rats, and ＂ Yantiao Prescription＂ drug medicated serum group treated with the drug medicated serum of ＂Yantiao Prescription＂, with 4 parallel- hole for each group. The cell proliferation was tested by MTI＇ colorimetric method to determine the dose ranges of drug medicated serum of ＂Yanfiao Prescription＂ with no toxicity. The mRNA expressions of HSP70, NF-κB, TNF-α, IL-1β and IL-8 were detected by qRT-PCR, the protein expressions of HSP70 and NF-KB were detected by Western blot, and the expressions of TNF-α, IL-1β and IL-8 protein were detected by ELISA. Results： The dose ranges of drug medicated serum of ＂Yantiao Prescription＂ with no toxicity included 10%-30%, so the 20% concentration of ＂Yantiao Prescription＂ drug medicated serum was selected for the further experiment. There were no statistically significant differences on all indexes between the blank control group and normal serum group （ P 〉 0.05 ）. Compared with the blank control group and normal sermn group, the mRNA and protein expressions of HSP70 in the ＂Yantiao Prescription＂ drug medicated serum group were significanfly increased （P 〈 0.01 ）, and the mRNA and protein expressions of NF-KB, TNF-α, IL-1β and IL-8 were obviously decreased （P 〈0.01 ）. Conclusion： The mechanisms of ＂Yantiac Prescription＂ on sepsis may be associated with the up- regulation of HSP70 expression, which further inhibit the activation of NF-κB and the expressions of relevant cytokines.

关 键 词：炎调方 巨噬细胞 热休克蛋白70 核因子-ΚB 

分 类 号：R285.5[医药卫生—中药学]