烟碱通过N1受体促进热应激对RAW264.7细胞HSP70的诱导表达

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。

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

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 Western-blot, translocation of HSF1 was detected by immunofluorescence and the combination of HSF1and 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.