核转录因子-KB参与光气吸入性肺损伤NLRP3炎性小体的表达

摘要

Objective To observe the effect of signal transduction pathway of nuclear factor-kappa B (NF-κB) on Nod-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome and pyroptosis in rats with acute lung injury induced (ALI) by phosgene. Methods The rats were randomly(random number) divided into 3 groups: air exposure control group, phosgene exposure group and PDTC group with phosgene exposure after 100 mg/kg pyrrolidine dithiocarbamate (PDTC) administration. The specimens of serum, bronchoalveolar lavage fluid (BALF) and lung tissue were collected 6 h after exposure. Morphological changes were observed by HE staining. The expression of NLRP3 in the lung of three groups was detected by immunohistochemistry. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expressions of NF-κB p65, NLRP3, ASC and caspase-1 mRNA in the lung tissue. NF-κB p65,NLRP3, ASC and caspase-1 protein levels in the lung tissue were quantified by Western blot. The concentrations of IL-1β, IL-18 and IL-33 in the serum and BALF were measured by enzyme-linked immunosorbent assay (ELISA). Pyroptosis was observed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL). Results The model of phosgene-induced ALI was successfully established in rats. Morphological changes with inflammatory cell infiltration were observed in the lung tissues of phosgene group, in which NLRP3 positive cells also could be observed by immunohistochemical staining. The mRNA expressions and protein levels of NF-κB p65, NLRP3 and caspase-1 in lung tissues were significantly increased (P＜0.05) in phosgene group, compared with air control group. The mRNA expressions and protein levels of NF-κB p65,NLRP3 and caspase-1 in lung tissues were significantly decreased (P＜0.05) in PDTC group, compared with phosgene group. The IL-1β,IL-18 and IL-33 protein levels in serum and BALF were significantly increased (P＜0.05) in phosgene group, compared with air control group. The IL-1β,IL-18 and IL-33 protein levels in serum and BALF were significantly decreased (P＜0.05) in PDTC group, compared with phosgene group. TUNEL results showed that pyroptosis in the lung tissue obviously increased in phosgene group, while decreased in PDTC group. Conclusions NLRP3 inflammasome and lung cell pyroptosis were induced through NF-kB signal transduction pathway in rats with acute lung injury caused by phosgene inhalation. Blockade of NF-κB can alleviate acute lung injury by down-regulating the expression of NLRP3 inflammasome to inhibit pyroptosis.%目的 通过四氢化吡咯二硫代氨基甲酸脂(PDTC)阻断核转录因子-κB (NF-κB)信号传导通路,探讨NF-κB信号通路对光气吸入性急性肺损伤(ALI)大鼠NOD样受体热蛋白结构域相关蛋白3 (NLRP3)的表达和细胞焦亡的影响.方法 采用大鼠光气吸人性肺损伤动物模型.将30只大鼠随机(随机数字法)分为三组,空气对照组(吸人与光气染毒组同等流量的空气) 10只,光气染毒组(吸入8.33 mg/L纯度为100％的光气5 min) 10只,PDTC干预组(吸入8.33 mg/L纯度为100％的光气5 min,腹腔注射NF-κ B抑制剂PDTC 100 mg/kg) 10只.染毒6 h后收集血清,支气管肺泡灌洗液(BALF)和肺组织.测定中性粒细胞细胞数、蛋白含量和肺湿/干质量比(W/D).制作肺脏石蜡切片,HE染色观察形态学改变,免疫组织化学检测肺组织中NLRP3蛋白表达水平.RT-PCR方法对肺脏组织中NF-κBp65、NLRP3、凋亡相关斑点样蛋白(ASC)和天冬氨酸特异性半胱氨酸蛋白酶1 (caspase-1) mRNA的水平进行半定量研究.蛋白质免疫印迹试验(Western blot)技术检测肺脏组织中NF-κ B p65、NLRP3、ASC和caspase-1的蛋白含量.采用双抗体夹心酶标免疫分析法(ELISA法)测定各组血清和BALF中IL-1 β、IL-18和IL-33水平.采用原位末端转移酶标记技术(TUNEL)法检测肺脏组织的细胞焦亡情况.结果 成功复制光气吸人性肺损伤模型.染毒6h后HE染色形态学观察发现:光气染毒组肺组织中大量炎性细胞浸润,免疫组织化学染色结果显示:光气染毒组肺组织中可见较多NLRP3阳性细胞.与空气对照组相比,光气染毒组肺脏组织中NF-κ B p65、NLRP3和caspase-1 mRNA和蛋白表达量明显升高(P＜0.05);与光气染毒组比较,PDTC干预组NF-κ B p65、NLRP3和caspase-1 mRNA和蛋白表达量明显降低,差异有统计学意义(P＜0.05).与空气对照组相比,光气染毒组血清和BALF中IL-1 β、IL-18和 IL-33蛋白含量明显升高(P＜0.05);与光气染毒组比较,PDTC干预组血清和BALF中IL-1β、 IL-18和IL-33蛋白含量明显下降,差异有统计学意义(P＜0.05).TUNEL结果提示光气染毒组肺脏组织细胞焦亡明显增多,经PDTC干预后肺脏组织细胞焦亡明显降低.结论 NF-κB信号通路促进大鼠光气吸人性ALI中NLRP3炎性小体的表达和细胞焦亡的发生,通过阻断NF-κ B信号通路可下调肺脏NLRP3炎性小体的表达,抑制细胞焦亡进而减轻急性肺损伤.