6-甲酰基吲哚并[3,2-b]咔唑抑制内质网应激改善脂多糖诱导的急性肺损伤

6-Formylindolo[3,2-b]carbazole alleviates lipopolysaccharide-induced acute lung injury via suppressing endoplasmic reticulum stress

目的探讨6-甲酰基吲哚并[3,2-b]咔唑(FICZ)对脂多糖(LPS)诱导小鼠急性肺损伤(ALI)的作用及机制。方法采用简单随机抽样方法将8~12周龄雄性C57BL/6J小鼠分为4组,每组8只。以腹腔注射LPS 5 mg/kg制备脓毒症致ALI小鼠模型(LPS组);磷酸盐缓冲液(PBS)对照组(PBS组)注射等体积PBS。LPS+FICZ组制模后1 h腹腔注射FICZ 1μg进行干预,而FICZ对照组(FICZ组)腹腔注射PBS后1 h给予等量FICZ。制模后24 h取血清及肺组织,经苏木素-伊红(HE)染色、肺组织湿/干重(W/D)比值分析肺组织病理改变;用酶联免疫吸附试验(ELISA)检测血清及肺组织中炎性因子表达;实时荧光定量聚合酶链反应(RT-qPCR)和蛋白质免疫印迹试验(Western blotting)检测内质网应激信号通路相关分子的表达水平。结果与PBS组比较,LPS组肺组织炎性细胞浸润增加,可见肺泡萎陷及明显的肺泡内渗出性病变,肺组织W/D比值显著升高,血清白细胞介素-6(IL-6)水平、肺组织IL-6 mRNA表达和内质网应激信号通路葡萄糖调节蛋白78(GRP78)、蛋白激酶R样内质网激酶(PERK)、CCAAT/增强子结合蛋白同源蛋白(CHOP)的mRNA表达以及肺组织GRP78、PERK、活化转录因子6(ATF6)、CHOP的蛋白表达水平均明显升高;而FICZ组各指标则不受影响。与LPS组比较,LPS+FICZ组肺组织炎性细胞浸润较少,肺泡结构相对完整,肺组织W/D比值显著下降(5.38±0.10比6.60±0.30,P<0.01),血清IL-6水平(ng/L:15.55±3.77比32.22±3.84)和肺组织IL-6 mRNA表达(2^(-ΔΔCt):0.79±0.21比6.89±0.92)显著降低(均P<0.01),内质网应激信号通路GRP78、PERK、CHOP的mRNA表达显著降低〔GRP78 mRNA(2^(-ΔΔCt)):1.90±0.16比7.55±1.29,PERK mRNA(2^(-ΔΔCt)):1.68±0.20比4.54±0.89,CHOP mRNA(2^(-ΔΔCt)):1.13±0.24比4.44±1.13,均P<0.05〕,GRP78、PERK、ATF6、CHOP的蛋白表达水平也显著降低(GRP78/GAPDH:0.59±0.02比0.77±0.01,PERK/GAPDH:0.48±0.03比1.04±0.05,ATF6/GAPDH:0.51±0.03比0.65±0.01,CHOP/GAPDH:0.91±0.05比1.11±0.07,均P<0.05)。结论FICZ对LPS诱导的小鼠ALI具有保护作用,其机制与抑制内质网应激、下调血清和肺组织IL-6水平有关。

Objective To investigate the effect and mechanism of 6-formylindolo[3,2-b]carbazole(FICZ)on lipopolysaccharide(LPS)-induced acute lung injury(ALI)in mice.Methods Male C57BL/6J mice aged 8-12 weeks were divided into 4 groups with 8 mice in each group,according to the method of simple random sampling.Sepsis-induced ALI mice model was established by intraperitoneal injection of LPS 5 mg/kg(LPS group),and phosphate buffer saline(PBS)control group(PBS group)was injected with equal volume of PBS.The LPS+FICZ group was intervened by intraperitoneal injection of 1μg FICZ 1 hour after LPS stimuli,while the FICZ control group(FICZ group)was given the same amount of FICZ 1 hour after intraperitoneal injection of PBS.Serum and lung tissue were collected 24 hours after LPS stimuli,and the pathological changes of lung tissue were analyzed by hematoxylin-eosin(HE)staining and wet/dry weight(W/D)ratio of lung tissue.The concentrations of inflammatory factors in serum and lung tissue were detected by enzyme linked immunosorbent assay(ELISA).The expression levels of endoplasmic reticulum stress signaling pathway related molecules were detected by real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)and Western blotting.Results Compared with PBS group,inflammatory cell infiltration,alveolar collapse and obvious alveolar exudative lesions had increased,lung tissue W/D ratio was significantly increased,serum interleukin-6(IL-6)level,lung tissue IL-6 mRNA expression,and the mRNA expressions of glucose-regulated protein 78(GRP78),protein kinase R-like endoplasmic reticulum kinase(PERK),CCAAT/EBP homologous protein(CHOP),and the protein expressions of GRP78,PERK,activating transcription factor 6(ATF6),CHOP in lung tissue were significantly increased in LPS group.However,the indexes of FICZ group were not affected.Compared with LPS group,LPS+FICZ group had less inflammatory cell infiltration,relatively intact alveolar structure.Lung W/D weight ratio in LPS+FICZ group was significantly decreased(5.38±0.10 vs.6.60±0.30,P<0.01),so as serum IL-6(ng/L:15.55±3.77 vs.32.22±3.84)and lung IL-6 mRNA expression(2^(-ΔΔCt):0.79±0.21 vs.6.89±0.92,both P<0.01).The mRNA expressions of GRP78,PERK and CHOP were also significantly decreased[GRP78 mRNA(2^(-ΔΔCt)):1.90±0.16 vs.7.55±1.29,PERK mRNA(2^(-ΔΔCt)):1.68±0.20 vs.4.54±0.89,CHOP mRNA(2^(-ΔΔCt)):1.13±0.24 vs.4.44±1.13,all P<0.05],and the protein expressions of GRP78,PERK,ATF6 and CHOP were significantly decreased(GRP78/GAPDH:0.59±0.02 vs.0.77±0.01,PERK/GAPDH:0.48±0.03 vs.1.04±0.05,ATF6/GAPDH:0.51±0.03 vs.0.65±0.01,CHOP/GAPDH:0.91±0.05 vs.1.11±0.07,all P<0.05).Conclusion FICZ protects LPS-induced ALI possibly via suppressing endoplasmic reticulum stress and reducing IL-6 expression in blood and lung tissue.