薄荷酮对内毒素致炎症模型小鼠的保护作用研究

The protective effects of menthone on endotoxin-induced infalmmation in mice

目的观察薄荷酮对内毒素(LPS)致炎症模型小鼠的保护作用,并初步探讨与NLRP3炎症小体激活相关的调控作用机制。方法♂C57BL/6J小鼠按体质量分层随机分为空白对照组、模型对照组、地塞米松组(5 mg·kg^(-1))、薄荷酮0.25 g·kg^(-1)及0.5 g·kg^(-1)剂量组。除地塞米松组实验当日腹腔注射给药1次外,其余各组小鼠连续灌胃给药5 d,每天1次。各组小鼠末次给药30 min后,除空白组小鼠外,其余各组小鼠腹腔注射LPS(15 mg·kg^(-1),10 m L·kg^(-1))制备炎症反应模型,造模12 h后,小鼠取血分离血清,采用ELISA及液相蛋白芯片技术测定炎症因子IL-18、IL-1β、IL-5、TNF-α、IFN-γ、粒细胞集落刺激因子(G-CSF)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)及巨噬细胞炎性蛋白-1β(MIP-1β)的水平;取小鼠全血进行白细胞(WBC)计数;剖取肺组织计算肺脏指数,并测定肺组织中NO含量,进行肺组织病理组织学检查;RT-PCR法检测肺组织中NLRP3、caspase-1及IFN-α mRNA表达;免疫组化法观察肺组织中cathepsin B、P2X7R蛋白表达。结果 0.5 g·kg^(-1)薄荷酮明显降低内毒素致炎症模型小鼠血清IL-18、IL-1β、IL-5、TNF-α、IFN-γ、G-CSF、GM-CSF、MIP-1β的水平(P<0.05或P<0.01),下调模型小鼠肺组织NLRP3、IFN-α mRNA表达及cathepsin-B、P2X7R蛋白表达(P<0.05或P<0.01),明显降低模型小鼠肺组织中NO的含量(P<0.05);0.25 g·kg^(-1)薄荷酮明显降低模型小鼠血清中IL-1β、IL-5、TNF-α、MIP-1β含量(P<0.05),下调肺组织中P2X7R蛋白表达及NO水平(P<0.05或P<0.01);病理组织学检查显示0.5、0.25 g·kg^(-1)薄荷酮能明显减少肺组织切片中嗜中性粒细胞数量(P<0.01),减轻肺泡膈的增厚;薄荷酮对模型小鼠全血白细胞计数与肺指数的升高表现出降低趋势。结论薄荷酮对内毒素致炎症模型小鼠有保护作用,能抑制血清多种炎性细胞因子的释放而减轻肺部炎性损伤,该作用可能与干扰NLRP3炎症小体的激活有关。

Aim To study the effects of menthone on LPS-induced inflammation in mice and to explore the mechanisms of activating of NLRP3 inflammasome. Methods C57BL/6J male mice were randomly divid-ed into five groups,namely the normal group,model group,dexamethasone group （5 mg·kg^-1 ）and men-thone（0.25,0.5 g·kg^-1 ）groups.The dexametha-sone group was given the drugs once by intraperitoneal injection on 5th day,while the other mice were given drugs by oral administration once a day for 5 d.Then, the normal group was injected with the saline and the other groups were injected LPS （1 5 mg·kg^-1 ,0.01 mL·g^ -1 ）after 30 min of the last administration.1 2 h after LPS injection,the blood,serum,and lung tissue of mice were collected.The levels of IL-1 8,IL-1 β,IL-5,TNF-α,IFN-γ,G-CSF,GM-CSF and MIP-1 βwere measured in serum by ELISA and LuminexMagpix. The white cell in blood （WBC）were counted.The lung index and the levels of NO in lung tissue was cal-culated.The lung histopathology was performed.The relative levels of NLRP3,caspase-1 and IFN-αmRNA in lung tissue were determined by RT-PCR.The ex-pressions of cathepsin B and P2X7R in lung tissue were quantified by immunohistochemical analysis.Re-sults 0.5 g·kg^-1 menthone significantly reduced the levels of IL-1 8,IL-1 β,IL-5,TNF-α,IFN-γ,G-CSF, GM-CSF,MIP-1 βin serum （P 〈0.05 or P 〈0.01 ）, and the expression of NLRP3,IFN-α,cathepsin B, P2X7R,NO in lung tissue （P 〈0.05 or P 〈0.01 ）.0.25 g ·kg^-1 menthone inhibited the IL-1 β,IL-5, TNF-α,MIP-1 βproduction in serum and the expres-sion of P2X7R,NO in lung tissue（P 〈0.05）.0.25, 0.5 g ·kg^-1 menthone could attenuate pathological change of the lung tissues by reducing neutrophil infil-tration and thickening of alveolar septa.And it slightly reduced the lung index and the level of white cells in blood.Conclusion Menthone has a protective effect on LPS-induced inflammation mice,and its mechanism is related to inhibiting the release of varies of inflam-matory cytokines,reducing the inflammation reaction and inhibiting the activation of NLRP3 inflammasome.