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

李雅倩, 安旭亮, 白倚菲, 刘洋, 高学敏, 蔡文臣, 徐洪, 杨方

李雅倩, 安旭亮, 白倚菲, 刘洋, 高学敏, 蔡文臣, 徐洪, 杨方. 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[J]. 环境与职业医学, 2023, 40(12): 1444-1450. DOI: 10.11836/JEOM23117
引用本文: 李雅倩, 安旭亮, 白倚菲, 刘洋, 高学敏, 蔡文臣, 徐洪, 杨方. 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[J]. 环境与职业医学, 2023, 40(12): 1444-1450. DOI: 10.11836/JEOM23117
LI Yaqian, AN Xuliang, BAI Yifei, LIU Yang, GAO Xuemin, CAI Wenchen, XU Hong, YANG Fang. Regulatory effect of integrated stress response inhibitors on endoplasmic reticulum stress signals in macrophages in silicotic mice[J]. Journal of Environmental and Occupational Medicine, 2023, 40(12): 1444-1450. DOI: 10.11836/JEOM23117
Citation: LI Yaqian, AN Xuliang, BAI Yifei, LIU Yang, GAO Xuemin, CAI Wenchen, XU Hong, YANG Fang. Regulatory effect of integrated stress response inhibitors on endoplasmic reticulum stress signals in macrophages in silicotic mice[J]. Journal of Environmental and Occupational Medicine, 2023, 40(12): 1444-1450. DOI: 10.11836/JEOM23117

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

基金项目: 国家自然科学基金项目(82204006);河北省自然科学基金项目(H2020209292);河北省高等学校科学技术研究项目(QN2022170);河北省研究生创新资助项目(CXZZBS2023121);河北省省属高校基本科研业务费研究项目(JQN2020006)
详细信息
    作者简介:

    李雅倩(1992—),女,博士生;E-mail:lyqewbar@163.com

    通讯作者:

    杨方,E-mail:fangyang@ncst.edu.cn

  • 中图分类号: R114

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

Funds: This study was funded.
More Information
  • 摘要:
    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50 μL 200 mg·mL−1 SiO2混悬液构建矽肺小鼠模型。灌注SiO2一周(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200 μL 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(1 μg·mL−1)、SiO2诱导组(100 μg·mL−1)和ISRIB干预组(先给予1 μg·mL−1 ISRIB处理1 h,再给予100 μg·mL−1 SiO2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SiO2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SiO2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SiO2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。

     

    Abstract:
    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 50 μL 200 mg·mL−1 SiO2 suspension. After one week of perfusion with SiO2 (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 200 μL 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, p-inositol-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 (1 μg·mL−1) group, a SiO2 induction group (100 μg·mL−1), and an ISRIB treatment group (1 μg·mL−1 ISRIB treatment for 1 h, followed by 100 μg·mL−1 SiO2 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 SiO2 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 SiO2 stimulation in vivo and in vitro; compared with the SiO2 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.

     

  • 矽肺是由于长期暴露于职业粉尘环境中吸入可吸入结晶二氧化硅(respirable crystalline silica, RCS)导致的以矽结节形成和弥漫性肺间质纤维化为主要病理特征的慢性肺疾病[1],在我国是最严重的职业病之一。近年来,发达国家由于职业防护不到位,相继出现了打磨人造石等引起新尘肺病种产生的职业[2]。矽肺发病隐匿,周期长,疾病持续进展无法逆转,尚无有效治疗手段,给患者、家庭和社会带来沉重的负担[34]。因此,迫切需要进一步探索矽肺发病机制,为其治疗提供新的靶点和思路。

    课题组前期研究结果发现,肺泡巨噬细胞受到呼吸性结晶型二氧化硅的刺激后,蛋白质折叠出现错误,内质网应激信号被激活[56],内质网应激通过激活促凋亡通路,诱导上皮-间质转化,促进炎症反应,进一步介导肺纤维化重塑[78]。抗纤维化短肽N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸(N-acetyl-serine-aspartyl-lysine-proline, Ac-SDKP)能抑制矽肺纤维化进程中内质网应激信号通路的激活从而延缓纤维化进展[9],但由于其半衰期短、易失活,进入临床试验阶段还面临许多困难。综合应激反应抑制剂(integrated stress response inhibitor, ISRIB)是研发于2013年的一种小分子药物[10],是蛋白激酶RNA样ER激酶(protein kinase RNA-like ER kinase, PERK)信号通路抑制剂,可逆转真核翻译启动因子2α(eukaryotic initiation factor 2 alpha, eIF-2α)磷酸化的作用,从而抑制活化转录因子4(activating transcription factor 4, ATF4)的激活。由于eIF-2α磷酸化与记忆巩固有关,目前针对ISRIB的研究主要集中在阿尔茨海默病、脑震荡、认知障碍等脑部疾病,发现ISRIB在小鼠神经退行性病变和创伤性脑损伤模型中起到重要的保护作用[1112]。在肺纤维化中,ISRIB能够通过修复肺泡上皮细胞的损伤从而延缓肺纤维化的进展[13]。目前对于ISRIB对矽肺的作用机制仍未所知,内质网应激是触发综合应激反应的机制之一,因此,本研究拟通过体内外实验观察ISRIB能否通过对肺泡巨噬细胞内质网应激信号的调节从而发挥拮抗矽肺纤维化的作用及其机制。

    8周龄雄性C57BL/6J小鼠(20±2)g 40只,SPF级,购于北京维通利华动物技术有限公司[SCXY(京)2016-0008],饲养于华北理工大学实验动物中心清洁级动物房[SYXK(冀)2020-0007],饲养环境明暗各12 h循环,恒温26 ℃,自由饮食饮水。本研究方案经华北理工大学伦理委员会批准,批准编号为LX2019033,符合动物研究伦理学要求。小鼠肺泡巨噬细胞株MH-S细胞购自中国科学院细胞库。

    小动物CT仪(NEMO-II NMC-20,中国平生),光学显微镜(DP 80,日本OLYMPUS),荧光显微镜(U-HGLGPS,日本OLYMPUS),显影仪(ChemiScope 6100 EXP,上海勤翔)。

    SiO2粉尘(美国Sigma);罗斯威尔公园纪念研究所(Roswell Park Memorial Institute, RPMI)1640培养基(以色列BI);胎牛血清(中国李记);β-巯基乙醇(中国瑞帕特);ISRIB(CAS号:1597403-47-8,纯度≥95%,美国cayman);异氟烷(中国RWD);苏木素-伊红(Hematoxylin eosin, HE)染液以及Van Gieson(VG)染液(中国贝索);蛋白提取试剂盒(中国英文特);I型胶原蛋白(collagen I, Col I)(中国博士德);肌醇需求蛋白-1α(inositol-requiring enzyme-1α, IRE-1α)(中国ABclonal);磷酸化肌醇需求蛋白-1α(p-inositol-requiring enzyme-1α, p-IRE-1α)(英国Abcam);PERK(中国华安);磷酸化PERK(p-PERK)(美国Affinity);eIF-2α(中国Abclonal);磷酸化eIF-2α(p-eIF-2α)(中国华安);ATF4(中国华安);α-微管蛋白(α-tubulin, α Tub)(中国Abclonal);NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3, NLRP3)(中国华安);荧光二抗(美国Novex);含二脒基苯基吲哚(diamidine phenyl indole, DAPI)的封片剂(美国Cell Signaling);免疫印迹二抗(美国KPL);增强型化学发光试剂(enhanced chemiluminescence, ECL)(中国庄盟)。

    将小鼠随机分为4组(每组10只)。(1)对照组:气管一次性灌注50 μL 0.9%氯化钠溶液,一周后每天腹腔注射200 μL 0.9%氯化钠溶液;(2)ISRIB对照组:灌注方法同对照组,一周后每天腹腔注射200 μL 2.5 mg·kg−1 ISRIB溶液;(3)矽肺模型组:根据课题组前期实验结果,气管一次性灌注50 μL 200 mg·mL−1 SiO2混悬液[14],一周后每天腹腔注射200 μL 0.9%氯化钠溶液;(4)ISRIB治疗组:灌注方法同矽肺模型组,一周后每天腹腔注射2.5 mg·kg−1的ISRIB溶液200 μL。各组小鼠均饲养至4周。用1%戊巴比妥钠麻醉小鼠,取小鼠肺组织,4%多聚甲醛固定左肺,用于HE染色、VG染色和免疫荧光染色,将其余肺组织冻于−80 ℃冰箱保存备用。

    MH-S细胞在37 ℃、5% CO2的孵育箱中培养,用含15%胎牛血清和0.1%β-巯基乙醇的RPMI 1640培养基培育细胞。本实验选用第3代细胞进行实验,用空白培养基将细胞同步化24 h后分为4组。(1)对照组:用不含血清的培养基培养;(2)ISRIB对照组:无血清培养条件下,给予1 μg·mL−1 ISRIB处理;(3)SiO2诱导组:无血清培养条件下,给予100 μg·mL−1 SiO2诱导;(4)ISRIB干预组:无血清培养条件下,先给予1 μg·mL−1 ISRIB处理1 h,再给予100 μg·mL−1 SiO2诱导。

    造模结束后饲养小鼠至4周取材前,用异氟烷麻醉小鼠,采用小动物CT仪拍摄小鼠活体肺CT图像,小鼠采用俯卧位,拍摄期间确保动物舱门关闭,挡板归位,并使用PadCam软件视频监控观察小鼠状态,使用cruiser 1.7.40软件采集拍摄数据,使用recon daemon 1.7.40软件离线重建拍摄数据,使用avatar3 1.7.40软件分析拍摄数据和重建肺结构。

    肺组织经4%多聚甲醛溶液固定24 h,流水过夜去除组织中的甲醛,在梯度乙醇溶液脱水,最后浸泡于石蜡溶液中6 h,用包埋机将肺组织包埋。将蜡块切成4 μm厚的肺切片。切片常规脱蜡至水,用苏木素染液染色3 min,2%盐酸酒精溶液中分化数秒,再将切片置于水中返蓝15 min,使用伊红染液染色2 min后将切片放入90%乙醇溶液5 min,依次将切片置于无水乙醇和二甲苯中浸泡,彻底透明后用中性树胶封片。待切片干燥后镜下观察肺组织形态。

    脱蜡至水步骤同HE染色,将铁苏木素A和铁苏木素B等比例混合后滴于组织表面染色3 min,然后在2%盐酸酒精中分化2 min,在自来水中水化10 min,再将VG染液覆盖于组织表面染色10 min,再放入90%乙醇溶液5 min,余下步骤同HE染色。

    石蜡切片脱蜡至水,细胞爬片洗去固定液,加入乙二胺四乙酸(ethylenediamine tetraacetic acid, EDTA)溶液后,采用高压修复80 s使抗原暴露,滴加p-PERK(1∶100),4 ℃孵育过夜,次日用磷酸盐缓冲液将切片洗3次后,将荧光二抗充分覆盖于组织和细胞上,于37 ℃恒温箱孵育90 min,用含DAPI的封片剂封片。荧光显微镜下观察阳性细胞染色。

    使用总蛋白提取试剂盒提取小鼠肺组织和MH-S细胞总蛋白,比辛可宁酸(bicinchonininc acid, BCA)试剂盒测定蛋白浓度后以每个泳道15 μg的蛋白量进行常规电泳电转。按1∶1000稀释一抗Col I、p-PERK、PERK、p-IRE-1α、IRE-1α、p-eIF-2α、eIF-2α、ATF4、NLRP3、α Tub,置于4 ℃冰箱孵育过夜,次日含吐温的缓冲盐水(triethanolamine-buffered saline +Tween, TBST)洗三次后,二抗(1∶5000)室温孵育40 min,采用ECL发光试剂盒显色。后期采用Image-Pro-plus 6.0图像处理软件对条带进行定量分析。

    所有实验至少重复3次,数据以平均数±标准差($ \bar x \pm s $)表示,采用SPSS 20.0统计分析软件将数据进行完全随机设计的单因素方差分析;两两比较方差齐采用最小显著性差异法(least significant difference, LSD)检验,方差不齐则采用Tamhane’s检验。P<0.05表示差异具有统计学意义。

    各组小鼠均无死亡情况出现。取材前,对照组体重(24.56±0.23)g,ISRIB对照组(25.35±0.43)g,矽肺模型组(20.79±0.67)g,ISRIB治疗组(22.39±0.42)g。小鼠肺部CT显示,对照组以及ISRIB对照组小鼠双肺纹理清晰,走向和分布正常,肺野内未见明显异常密度影;矽肺模型组小鼠肺纹理增粗,肺野内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(P<0.05)。结果见图1

    图  1  CT观察活体小鼠肺成像及肺纤维化体积定量比较
    A:CT横截面,B:CT冠状面,C:CT矢状面;1:对照组,2:ISRIB对照组,3:矽肺模型组,4:ISRIB治疗组。D:肺纤维化体积定量图;a:与对照组比较,P<0.05;b:与矽肺模型组比较,P<0.05。
    Figure  1.  Quantitative comparison of pulmonary fibrosis volume of living mice lung imaging by micro CT

    HE染色结果显示,对照组和ISRIB对照组组肺泡结构完整、肺泡壁薄,无炎症及渗出;矽肺模型组可见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;而和矽肺模型组相比,ISRIB治疗组矽结节面积和个数明显减少,矽结节范围被局限。结果见图2A1-A4。VG染色结果显示,矽肺模型组有较多胶原沉积,肺组织胶原纤维面积占比为21.47%±2.59%;而和矽肺模型组相比,ISRIB治疗组胶原沉积面积显著减少,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),结果见图2B1-B4图2C。同样,免疫印迹结果显示,和对照组相比,矽肺模型组Col I和NLRP3的表达上调,而给予ISRIB干预后,肺组织Col I和NLRP3的表达下调,差异具有统计学意义(P<0.05),结果见图3

    图  2  ISRIB对矽肺小鼠胶原沉积的调节作用(HE染色和VG染色)(20×)
    A:HE染色,B:VG染色;1:对照组,2:ISRIB对照组,3:矽肺模型组,4:ISRIB治疗组;红色箭头所指为矽结节。C:矽结节面积定量图;a:与对照组比较,P<0.05;b:与矽肺模型组比较,P<0.05。
    Figure  2.  The regulatory effect of ISRIB on collagen deposition in silicotic mice (HE staining and VG staining) (20×)
    图  3  ISRIB对各组小鼠胶原沉积的调节作用(免疫印迹法)
    A:各指标免疫印迹图,B:Col I和NLRP3表达量定量图;a:与对照组比较,P<0.05;b:与矽肺模型组比较,P<0.05。
    Figure  3.  The regulatory effect of ISRIB on collagen deposition in each group of mice (immunoblotting)

    免疫荧光结果显示,p-PERK在矽肺模型组小鼠肺结节中表达明显增强,并且和巨噬细胞标记物CD68共定位;而与矽肺模型组相比,ISRIB治疗组p-PERK的表达明显降低。HE所示组织结构与前述结果一致。结果见图4。免疫印迹结果显示,和对照组相比,内质网应激信号相关蛋白p-PERK、p-IRE-1α、p-eIF-2α和ATF4在矽肺模型组中上调;而与矽肺模型组相比,上述因子在ISRIB治疗组中的表达下调,差异具有统计学意义(P<0.05)。结果见图5

    图  4  p-PERK和CD68在小鼠肺组织中的共定位(免疫荧光染色)(20×)
    A:DAPI,B:CD68,C:P-PERK,D:组合图,E:HE染色;1:对照组,2:ISRIB对照组,3:矽肺模型组,4:ISRIB治疗组。
    Figure  4.  The colocalization of p-PERK and CD68 in mice (immunofluorescence staining) (20×)
    图  5  ISRIB对各组小鼠p-PERK、p-IRE-1α、p-eIF-2α和ATF4蛋白水平的影响(免疫印迹法)
    A:各指标免疫印迹图;B:p-PERK、p-IRE-1α、p-eIF2α和ATF4表达量定量图;a:与对照组比较,P<0.05;b:与矽肺模型组比较,P<0.05。
    Figure  5.  Effects of ISRIB on the expressions of p-PERK, p-IRE-1α, p-eIF-2α, and ATF4 in each group of mice (immunoblotting)

    取第3代细胞进行实验,镜下观察细胞密度为70%时按照前述分组进行细胞诱导实验。细胞免疫荧光染色结果显示,SiO2诱导组中可见p-PERK的强表达,而给予ISRIB干预后,ISRIB干预组中p-PERK的荧光强度明显减少,结果见图6。免疫印迹结果显示,与对照组相比,SiO2诱导组中内质网应激信号相关蛋白p-PERK、p-IRE-1α、p-eIF-2α和ATF4的表达上调;与SiO2诱导组相比,ISRIB干预组中的p-PERK、p-IRE-1α、p-eIF-2α和ATF4表达下调,差异具有统计学意义(P<0.05)。结果见图7

    图  6  p-PERK在MH-S细胞中的表达(免疫荧光染色)(20×)
    A:DAPI,B:P-PERK,C:组合图。
    Figure  6.  Expression of p-PERK in MH-S cells (immunofluorescence staining) (20×)
    图  7  ISRIB对SiO2诱导的MH-S细胞中p-PERK、p-IRE-1α、p-eIF-2α和ATF4蛋白水平的影响(免疫印迹法)
    A:各指标免疫印迹图,B:p-PERK、p-IRE-1α、p-eIF2α和ATF4表达量定量图;a:与对照组比较,P<0.05;b:与SiO2诱导组组比较,P<0.05。
    Figure  7.  Effects of ISRIB on the expressions of p-PERK, p-IRE-1α, p-eIF-2α, and ATF4 in SiO2-induced MH-S cells (immunoblotting)

    为探寻矽肺治疗的新方法,本研究采用气管一次性灌注法构建小鼠矽肺模型,给予ISRIB治疗后,小鼠纤维化指标Col I显著下调,肺功能明显改善,内质网应激信号相关因子的表达显著下调,表明ISRIB具有一定抗矽肺纤维化的效应。

    由于职业健康覆盖率较低,目前实际患矽肺病人数可能远大于已知病例数,且临床使用的抗纤维化药物吡非尼酮、汉防己甲素等在预防疾病进展和改善患者生活质量方面疗效有限[1516],因此仍需进一步探寻治疗矽肺的有效治疗靶点和治疗药物。内质网的功能旨在促进蛋白质的正确折叠和运输,在正常情况下,葡萄糖相关肽78(glucose-related peptide 78, GRP78)与内质网应激传感器PERK、活化转录因子6 (activating transcription factor 6, ATF6)和IRE1α结合,并抑制其信号转导。随着错误折叠的蛋白积累,GRP78与传感器的结合减少,PERK在与GRP78解离后发生自磷酸化,其下游的eIF-2α的Ser51位点被磷酸化,导致mRNA的翻译减少,这一系列反应激活ATF4,从而促进抗氧化反应等基因的表达[7]。课题组前期研究结果证实SiO2在大鼠体内外激活GRP78从而激活内质网应激信号[17],本研究观察到一次性灌注SiO2后,免疫荧光结果显示小鼠矽结节内p-PERK强表达,且定位于巨噬细胞,免疫印迹结果显示小鼠肺组织内质网应激信号相关蛋白p-PERK、p-IRE-1α、p-eIF-2α和ATF4表达显著上调。说明ISRIB能抑制二氧化硅诱导的小鼠体内外内质网应激信号的激活。

    在肺纤维化进展过程中,内质网应激是重要的驱动因素,巨噬细胞发生内质网应激后可通过外泌体诱导肌成纤维细胞分化,驱动纤维化进展[18]。靶向线粒体的抗氧化剂也能抑制内质网应激信号的激活,从而抑制炎症反应以及随后的肺纤维化进展[19]。因此阻断内质网应激信号通路对于阻抑矽肺纤维化进展起着至关重要的作用。本实验结果显示,给予小分子药物ISRIB干预后,首先在小鼠体内观察到无论是矽结节面积还是胶原沉积情况均有显著改善,证实了ISRIB在SiO2诱导的矽肺纤维化模型中有抑制胶原沉积,延缓肺纤维化进展的效应。为进一步探讨ISRIB抑制胶原沉积的作用机制,本实验观察了内质网应激信号通路相关因子的表达,结果显示ISRIB的干预在矽肺小鼠肺内阻断了PERK以及eIF-2α的磷酸化,从而阻止了其下游ATF4的激活。作为综合应激反应抑制剂[20],ISRIB能拮抗p-eIF-2α破坏细胞内蛋白质稳态的效应,直接结合于真核起始因子2B(eukaryotic initiation factor 2B, eIF2B),维持eIF2B的正常功能,启动被抑制的蛋白质翻译,使细胞恢复蛋白质稳态,从而恢复正常的细胞状态与功能[21]。此外,本实验研究结果还显示ISRIB同样阻抑了内质网应激另一传感器IRE-1α的磷酸化,提示ISRIB可能还通过抑制内质网应激IRE-1α通路,从而达到抑制内质网应激的效应。

    本研究结果显示巨噬细胞标记物CD68和p-PERK发生共定位,提示巨噬细胞发生内质网应激。另外,体外培养小鼠肺泡巨噬细胞(MH-S)细胞,给予SiO2刺激后,内质网应激信号被激活,而ISRIB显著抑制内质网应激信号的激活。巨噬细胞受SiO2刺激后发生极化是矽肺纤维化进展的驱动环节之一[22],改变巨噬细胞极化状态可减轻肺纤维化[23]。而PERK及其下游信号是巨噬细胞免疫抑制功能的代谢中枢[24],此外,内质网应激传感器的缺失,会导致巨噬细胞无法发生M1极化[25]。ISRIB作为内质网应激的抑制剂,可能通过抑制巨噬细胞发生极化从而发挥抗纤维化作用。

    本研究尚存在一定的局限性,例如只选择雄性小鼠作为实验对象,未能排除性别因素的影响;此外,可以结合前人的研究结论,同时在上皮细胞以及成纤维细胞上研究ISRIB的效应以拓展ISRIB可能的作用机制。

    矽肺纤维化的治疗目前仍然是难点,已批准上市的药物吡非尼酮和尼达尼布由于药物的耐受性和副作用等问题,使用范围十分受限[26]。ISRIB在动物实验中即使达到饱和浓度也未出现副作用,并且在起效浓度时保留急性综合应激反应的细胞保护作用[27],ISRIB能通过修复受损的肺泡II型上皮细胞从而改善石棉和博来霉素所致小鼠肺纤维化[13],十分适合进入临床试验,为矽肺的治疗提供新思路。

    综上所述,本研究通过体内外实验验证了ISRIB可通过抑制巨噬细胞内质网应激信号的激活拮抗矽肺纤维化的进展。

  • 图  1   CT观察活体小鼠肺成像及肺纤维化体积定量比较

    A:CT横截面,B:CT冠状面,C:CT矢状面;1:对照组,2:ISRIB对照组,3:矽肺模型组,4:ISRIB治疗组。D:肺纤维化体积定量图;a:与对照组比较,P<0.05;b:与矽肺模型组比较,P<0.05。

    Figure  1.   Quantitative comparison of pulmonary fibrosis volume of living mice lung imaging by micro CT

    图  2   ISRIB对矽肺小鼠胶原沉积的调节作用(HE染色和VG染色)(20×)

    A:HE染色,B:VG染色;1:对照组,2:ISRIB对照组,3:矽肺模型组,4:ISRIB治疗组;红色箭头所指为矽结节。C:矽结节面积定量图;a:与对照组比较,P<0.05;b:与矽肺模型组比较,P<0.05。

    Figure  2.   The regulatory effect of ISRIB on collagen deposition in silicotic mice (HE staining and VG staining) (20×)

    图  3   ISRIB对各组小鼠胶原沉积的调节作用(免疫印迹法)

    A:各指标免疫印迹图,B:Col I和NLRP3表达量定量图;a:与对照组比较,P<0.05;b:与矽肺模型组比较,P<0.05。

    Figure  3.   The regulatory effect of ISRIB on collagen deposition in each group of mice (immunoblotting)

    图  4   p-PERK和CD68在小鼠肺组织中的共定位(免疫荧光染色)(20×)

    A:DAPI,B:CD68,C:P-PERK,D:组合图,E:HE染色;1:对照组,2:ISRIB对照组,3:矽肺模型组,4:ISRIB治疗组。

    Figure  4.   The colocalization of p-PERK and CD68 in mice (immunofluorescence staining) (20×)

    图  5   ISRIB对各组小鼠p-PERK、p-IRE-1α、p-eIF-2α和ATF4蛋白水平的影响(免疫印迹法)

    A:各指标免疫印迹图;B:p-PERK、p-IRE-1α、p-eIF2α和ATF4表达量定量图;a:与对照组比较,P<0.05;b:与矽肺模型组比较,P<0.05。

    Figure  5.   Effects of ISRIB on the expressions of p-PERK, p-IRE-1α, p-eIF-2α, and ATF4 in each group of mice (immunoblotting)

    图  6   p-PERK在MH-S细胞中的表达(免疫荧光染色)(20×)

    A:DAPI,B:P-PERK,C:组合图。

    Figure  6.   Expression of p-PERK in MH-S cells (immunofluorescence staining) (20×)

    图  7   ISRIB对SiO2诱导的MH-S细胞中p-PERK、p-IRE-1α、p-eIF-2α和ATF4蛋白水平的影响(免疫印迹法)

    A:各指标免疫印迹图,B:p-PERK、p-IRE-1α、p-eIF2α和ATF4表达量定量图;a:与对照组比较,P<0.05;b:与SiO2诱导组组比较,P<0.05。

    Figure  7.   Effects of ISRIB on the expressions of p-PERK, p-IRE-1α, p-eIF-2α, and ATF4 in SiO2-induced MH-S cells (immunoblotting)

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出版历程
  • 收稿日期:  2023-04-13
  • 录用日期:  2023-10-11
  • 网络出版日期:  2023-12-28
  • 刊出日期:  2023-12-01

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