巴瑞替尼在脂多糖诱导的小鼠急性肺损伤中的作用及机制

Role and mechanism of baricitinib in mouse acute lung injury induced by lipopolysaccharide

目的:研究巴瑞替尼在脂多糖诱导的小鼠急性肺损伤中的作用及机制。方法:将40只成年雄性C57BL/6小鼠,随机分成3组:对照组(10只)、脂多糖组(15只)和脂多糖+巴瑞替尼组(15只)。腹腔注射脂多糖,建立小鼠脓毒症模型。脂多糖注射后,每12 h灌胃巴瑞替尼(10 mg/kg),观察并记录48 h内各组小鼠生存情况。脂多糖注射48 h后留取支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF),检测BALF中的细胞总数。HE染色和免疫组化检测小鼠肺组织大体形态和炎症细胞浸润情况。ELISA检测小鼠血浆和支气管肺泡灌洗液中炎症因子肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素1β(interleukin-1β,IL-1β)和IL-6的水平。Western blot检测小鼠肺组织中Janus激酶(Janus kinase,JAK)-信号转导及转录激活因子(signal transducer and activator of transcription,STAT)通路相关蛋白的表达水平。流式细胞术检测小鼠肺组织中巨噬细胞比率和分型。培养小鼠巨噬细胞,予以不同浓度巴瑞替尼干预,脂多糖孵育后,Western blot检测各组巨噬细胞JAK-STAT通路相关蛋白表达水平。结果:与对照组相比,脂多糖组小鼠48 h生存率33.3%,BALF中细胞总数显著增加(P<0.05),HE染色可见肺泡水肿和塌陷,肺泡间隔增厚且可见大量炎症细胞浸润,免疫组化可见肺泡间隔大量巨噬细胞浸润(P<0.05)。ELISA检测见脓毒症小鼠血浆和肺泡冲洗液TNF-α、IL-1β和IL-6表达水平显著升高(P<0.05),Western blot检测可见肺组织中JAK-STAT通路相关蛋白表达显著上调(P<0.05),流式检测可见肺组织M1型巨噬细胞比率显著上调(P<0.05)。与脂多糖组相比,脂多糖+巴瑞替尼组小鼠生存率提高,上述病理生理学改变得到改善(P<0.05)。细胞实验中,LPS诱导巨噬细胞JAK-STAT通路相关蛋白表达显著上调(P<0.05),而巴瑞替尼呈剂量依耐性地抑制JAKSTAT通路激活(P<0.05)。结论:巴瑞替尼通过抑制JAK-STAT通路,减轻小鼠肺部组织炎症,从而减轻脓毒症急性肺损伤。

AIM To study the role and mechanism of baricitinib in lipopolysaccharide(LPS)-induced acute lung injury.METHODS Forty male C57BL/6 mice were randomly divided into three groups:control group(n=10),LPS group(n=15)and LPS+baricitinib group(n=15).The model of sepsis was established by intraperitoneal injection of LPS.After LPS injection,the mice were given baricitinib(10 mg/kg)by gavage in every 12 h.The survival rates of mice in each group were observed and recorded within 48 h.Meanwhile,bronchoalveolar lavage fluid(BALF)from each mouse was undertaken to detect the total number of BALF cells.HE staining and immunohistochemistry were used to determine the gross morphology and inflammatory infiltration in the lung tissues in each mouse.Plasma and BALF cytokines such as tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and IL-6 were measured by ELISA.The expression levels of Janus kinase(JAK)-signal transducer and activator of transcription(STAT)signaling pathway-related proteins in the lung tissues were detected by Western blot.Flow cytometry was utilized to calculate the proportions of macrophages in the lung tissue from each mouse.In vitro,RAW264.7 cells were cultured,incubated with different concentrations of baricitinib and stimulated by LPS.The expression levels of JAK-STAT signaling pathway-related proteins in the macrophages from different groups were detected by Western blot.RESULTS Compared with control group,the survival rate of the mice in LPS group within 48 h was 33.3%.In LPS group,increased total number of cells in BALF(P<0.05),alveolar edema and collapse,thickening alveolar septa and substantial inflammatory cell infiltration in the lung tissues by HE staining were observed,characterized by the infiltration of macrophages by immunohistochemistry(P<0.05).The plasma and BALF levels of TNF-α,IL-1βand IL-6 increased significantly(P<0.05).The up-regulated expression levels of JAK-STAT signaling pathway-related proteins and increasing proportions of M1 macrophages were observed in the lung tissues from LPS-induced sepsis mice(P<0.05).Compared with LPS group,the survival rate of the mice in LPS+baricitinib group was improved and the above pathophysiological changes were ameliorated(P<0.05).In vitro,predominant up-regulation of JAK-STAT signaling pathway-related proteins in the RAW264.7 cells was induced by LPS,which was inhibited by treatment with baricitinib in a dose-dependent manner(P<0.05).CONCLUSION Baricitinib inhibits the inflammation by suppressing JAK-STAT signaling pathway,and consequently attenuates LPS-induced acute lung injury in mice.