烟碱通过N1受体促进热应激对RAW264.7细胞HSP70的诱导表达

Nicotine enhanced the inducible expression of HSP70 under heat stress through N1 receptor in RAW264. 7 macrophages

目的:本研究探讨在巨噬细胞中,低剂量烟碱联合温和热应激对热休克因子1(heat shock factor 1,HSF1)和热休克蛋白70(heat shock proteins70,HSP70)表达的影响,并揭示其受体机制。方法:用低剂量烟碱(浓度1 mmol/L)联合温和热应激(39℃)刺激RAW264.7巨噬细胞,采用Western-blot观察HSP70的表达,免疫荧光细胞化学观察HSF1的入核,凝胶滞留实验(EMSA)观察HSF1与热休克元件(heat shock element,HSE)的结合;接着用不同的阻断剂预处理RAW264.7巨噬细胞,再给予烟碱和热应激刺激,观察HSP70的表达及HSF1的入核。结果:单纯低剂量烟碱(浓度1 mmol/L)刺激不能诱导HSP70的表达,但HSF1入核明显增多,HSF1与HSE的结合增强;在39℃时,再加入烟碱能增强HSP70的表达、HSF1的入核、HSF1与HSE的结合;六烃季铵预处理,HSF1的入核较单纯烟碱处理明显减少,且能明显阻断烟碱在39℃时诱导的HSP70表达的作用。结论:低剂量烟碱(浓度1 mmol/L)刺激可以促进HSF1入核并与HSE结合,但不能诱导HSP70的表达;烟碱通过N1受体诱导HSF1入核并促进温和热应激(39℃)对RAW264.7巨噬细胞中HSP70的诱导表达。

AIM：To explore whether low doses of nicotine enhances the HSF1 nuclear translocation and expression of HSP70 in the RAW264.7 macrophages treated under lower heat stress （〈 40 ℃）,and to uncover its receptor mechanism.METHODS：After stimulating with nicotine at doses of 1 mmol/L and its antagonists prior to nicotine treatment at 37 ℃ or 39 ℃,the expression of HSP70 in RAW264.7 macrophages was detected by Westernblot,translocation of HSF1 was detected by immunofluorescence and the combination of HSF1 and heat shock element （HSE） was detected by EMSA,respectively.RESULTS：Western-blot showed that nicotine （1 mmol/L） alone could not induce HSPs expression,but was able to enhance the HSP70 expression at 39 ℃ in RAW246.7.Immunofluorescence showed nicotine （1 mmol/L） was able to induce translocation of HSF1 from cytoplasm to nucleus,which was blocked by the pre-treating with hexamethonium apparently;EMSA showed that nicotine （1 mmol/L） could promote the binding of HSF1 to HSE at 37℃.Furthermore,nicotine （1 mmol/L） induced HSF1 binding to HSE greatly at 39℃.CONCLUSION：Nicotine （1 mmol/L） alone can induce HSF1 nuclear translocation and its DNA-binding activity to HSE,but not the expression of HSP70;nicotine （1 mmol/L） can enhance the expression of HSP70 under mild heat stress （39℃） through N1-receptor in the RAW264.7 macrophages.