综合应激反应抑制剂对实验性矽肺纤维化模型中肺泡巨噬细胞内质网应激信号的调节作用

Regulatory effect of integrated stress response inhibitors on endoplasmic reticulum stress signals in macrophages in silicotic mice

[背景]矽肺是我国最严重的职业病之一,需要新的治疗靶点和疗法,综合应激反应抑制剂(ISRIB)对矽肺的作用及机制仍未所知。[目的]研究ISRIB对矽肺纤维化的作用及可能的作用机制。[方法]研究分体内、外实验两部分。随机将40只SPF级雄性C57BL/6J小鼠分为对照组、ISRIB对照组、矽肺模型组、ISRIB治疗组,每组10只。采用气管一次灌注50μL 200 mg·mL^(–1)SiO_(2)混悬液构建矽肺小鼠模型。灌注SiO_(2)一周(对照组及ISRIB对照组灌注等量生理盐水)后,开始给ISRIB对照组及ISRIB治疗组小鼠每天腹腔注射200μL 2.5 mg·kg^(–1)ISRIB,其余组别注射等量生理盐水,至4周。采用小动物CT仪观察各组小鼠肺野清晰度及肺纹理;采用苏木素-伊红(HE)染色观察各组小鼠肺组织病理学形态和矽结节形成情况;采用Van Gieson(VG)染色观察结节胶原沉积情况;采用免疫荧光法检测肺组织磷酸化(p)-蛋白激酶RNA样ER激酶(p-PERK)的表达和定位,采用免疫印迹法检测I型胶原蛋白(Col I)及内质网应激信号相关蛋白p-PERK、磷酸化肌醇需求蛋白-1α(p-IRE-1α)、磷酸化真核翻译启动因子-2α(p-eIF-2α)、活化转录因子4(ATF4)和NOD样受体热蛋白结构域相关蛋白3(NLRP3)的表达情况。体外培养小鼠肺泡巨噬细胞(MH-S细胞),分为对照组、ISRIB对照组(1μg·mL^(–1))、SiO_(2)诱导组(100μg·mL^(–1))和ISRIB干预组(先给予1μg·mL^(–1)ISRIB处理1 h,再给予100μg·mL^(–1)SiO_(2)诱导)。采用免疫荧光法检测MH-S细胞p-PERK的表达;采用免疫印迹法检测内质网应激信号相关蛋白p-PERK、p-IRE-1α、p-eIF-2α和ATF4的表达情况。[结果]体内实验中,CT结果显示矽肺模型组小鼠肺纹理增粗,肺野内可见数个大小不等的高密度影,主要分布于支气管周围;和矽肺模型组相比,ISRIB治疗组高密度影数量和体积均减少。HE染色结果显示矽肺模型组部分肺组织失去正常肺结构,有矽结节形成,矽结节周围肺泡增厚,有炎症细胞浸润;ISRIB治疗组矽结节面积和个数明显减少,矽结节范围被局限。VG染色结果显示矽肺模型组肺组织胶原纤维面积占比为21.47%±2.59%;ISRIB治疗组中,肺组织胶原纤维面积占比降至9.34%±1.06%,差异具有统计学意义(P<0.05)。免疫荧光染色结果显示,小鼠矽结节肺内p-PERK强表达,且定位于巨噬细胞;体外实验的SiO_(2)诱导的MH-S细胞中p-PERK荧光表达明显增加;体内、外实验的ISRIB治疗或干预组中p-PERK表达强度均减弱。免疫印迹结果显示,与对照组相比,体内、外实验中SiO_(2)刺激后内质网应激信号相关蛋白p-PERK、p-IRE-1α、p-eIF-2α和ATF4表达上调;而与SiO_(2)诱导组相比,ISRIB治疗或干预组中p-PERK、p-IRE-1α、p-eIF-2α和ATF4的表达降低,差异均具有统计学意义(P<0.05)。[结论]ISRIB通过抑制巨噬细胞内质网应激信号的激活发挥拮抗矽肺纤维化的作用。

[Background]Silicosis is one of the most serious occupational diseases in China,requiring new treatment targets and therapies.The effects and mechanisms of integrated stress response inhibitors(ISRIB)on silicosis are still unknown.[Objective]To observe the effects of ISRIB on silicosis fibrosis and its possible mechanisms.[Methods]The study was divided into two parts:in vivo and in vitro experiments.For the in vivo part,40 SPF grade male C57BL/6J mice were randomly divided into four groups:control group,ISRIB group,silicotic model group,and ISRIB treatment group,with 10 mice in each group.A silicotic mouse model was established by using a single tracheal infusion of 50μL 200 mg·mL^(–1)SiO_(2)suspension.After one week of perfusion with SiO_(2)(the control group and the ISRIB group were perfused with an equal amount of sodium chloride solution),the ISRIB group and the ISRIB treatment group were intraperitoneally injected with 200μL 2.5 mg·kg^(–1)ISRIB for four weeks,and mice of other groups were injected with equal amounts of sodium chloride solution.A micro-CT instrument was used to observe the lung field clarity and lung texture of each group;hematoxylin eosin(HE)staining was used to observe the histopathology morphology of the lung and the formation of silicon nodules;Van Gieson(VG)staining was used to observe the deposition of collagen in silicotic nodules;immunofluorescence assay was used to detect the expression and localization of p-protein kinase RNA-like ER kinase(PERK)in lung tissue;immunoblotting was used to detect the expression of collagen I(Col I)and endoplasmic reticulum stress signal related proteins p-PERK,pinositol-requiring enzyme-1α(p-IRE-1α),p-eukaryotic initiation factor 2α(p-eIF-2α),activating transcription factor 4(ATF4),and NOD-like receptor thermal protein domain associated protein 3(NLRP3).For the in vitro part,mouse alveolar macrophages MH-S cells were cultured in vitro and divided into a control group,an ISRIB(1μg·mL^(–1))group,a SiO_(2)induction group(100μg·mL^(–1)),and an ISRIB treatment group(1μg·mL^(–1)ISRIB treatment for 1 h,followed by 100μg·mL^(–1)SiO_(2)induction).Immunofluorescence assay was used to detect the expression of p-PERK in MH-S cells;immunoblotting was used to determine expressions of endoplasmic reticulum stress signal related proteins p-PERK,p-IRE-1α,p-eIF-2α,and ATF4.[Results]The CT images showed that the lung markings of the silicotic model group mice were thickened,and several high-density shadows of varying sizes were observed in the lung field,mainly distributed around the bronchi.Compared with the silicotic model group,the ISRIB treatment group showed a decrease in the number and volume of high-density shadows.The HE staining results showed that lung tissues in the silicotic model group lost their normal structure,with the formation of silicon nodules,around which were thickened alveoli around the silicon nodules,and infiltration of inflammatory cells;the area and number of silicon nodules in the ISRIB treatment group were significantly reduced,and the range of silicon nodules was limited.The results of VG staining showed that the proportion of collagen fiber area in the lung tissue of the silicotic model group was 21.47%±2.59%,and it decreased to 9.34%±1.06%in the ISRIB treatment group,with a statistically significant difference(P<0.05).The immunofluorescence results showed that p-PERK was strongly expressed in the silicotic nodules and localized in macrophages;the expression of p-PERK was also significantly increased in the SiO_(2)induced MH-S cells,while the intensity of p-PERK was weakened in the ISRIB treatment groups both in vitro and in vivo.The results of immunoblotting showed that compared with the control group,the expressions of endoplasmic reticulum stress signal related proteins p-PERK,p-IRE-1α,p-eIF-2α,and ATF4 were upregulated after SiO_(2)stimulation in vivo and in vitro;compared with the SiO_(2)induction group,the expressions of p-PERK,p-IRE-1α,p-eIF-2α,and ATF4 were significantly downregulated in the ISRIB treatment group in vivo and in vitro,and the differences were statistically significant(P<0.05).[Conclusion]ISRIB antagonizes silicosis fibrosis by inhibiting the activation of macrophage endoplasmic reticulum stress signals.