Objective: To investigate the effects of methylprednisolone on NOD-like receptor hot protein domain-associated protein 3 (NLRP3) inflammasome in phosgene-induced acute lung injury. Methods: Rats were randomly divided into four groups, 10 rats in Air group (inhalation of air of the same volume as the phosgene group) , 10 rats in Phosgene group (inhalation of 8.33 mg/L with 100% purity phosgene for 5 min) , 10 rats in Saline control group (inhalation of the same dose of phosgene and 2 mg/kg saline via tail vein injection one hour later) , 10 rats in MP group (inhalation of the same dose of phosgene and 2 mg/kg MP via tail vein injection one hour later) . The specimens of serum, bronchoalveolar lavage fluid (BALF) and lung tissue were collected after 6h. Morphological changes were observed by HE staining. The expression of NLRP3 in the lung of four groups was detected by immunohistochemistry. NLRP3、ASC and caspase-1 expression in the lung tissue was quantified by Western blot. Reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression of NLRP3、ASC and caspase-1 mRNA in the lung tissue. The concentrations of IL-1β、IL-18 and IL-33 in the serum and BALF were measured by enzyme-linked immunosorbent assay. Results: We successfully replicated the model of phosgene-induced ALI in rats. Morphological of HE staining after phosgene exposure to 6 h observed inflammatory cell infiltration in lung tissue in Phosgene group. Immunohistochemical staining results showed that there were many NLRP3 positive cells in lung tissue in Phosgene group. The levels of NLRP3, caspase-1 mRNA and protein expression in lung were significantly increased (P<0.05) in Phosgene group compared with Air group; compared with Phosgene group, The levels of NLRP3 and caspase-1 mRNA and protein expression in MP group were significantly decreased (P<0.05) . Compared with Air group, The levels IL-1β、IL-18 and IL-33 mRNA protein expression in the serum and BALF were significantly increased (P<0.05) in Phosgene group. Compared with Phosgene group, The levels IL-1β、IL-18 and IL-33 mRNA protein expression in the serum and BALF were significantly decreased (P<0.05) in MP group. Conclusion: Methylprednisolone can effectively protect the rats from phosgene-induced acute lung injury by inhibiting the expression of the NLRP3 inflammasome and reducing the release of inflammatory factors such as interleukin-1β (IL-1β) mediated by it.
目的： 探讨甲基强的松龙（MP）对光气吸入性急性肺损伤中NOD样受体热蛋白结构域相关蛋白3（NLRP3）炎症小体及其下游炎症因子的影响。 方法： 将40只大鼠随机分为4组，空气对照组（吸入与光气染毒组同等流量的空气）、光气染毒组（吸入8.33 mg/L纯度为100%的光气5 min）、生理盐水对照组（吸入同等剂量的光气1 h后尾静脉注入2 mg/kg生理盐水）、MP干预组（吸入同等剂量的光气1 h后尾静脉注入2 mg/kg甲基强的松龙），每组10只。不同处理后6 h收集血清，支气管肺泡灌洗液（BALF）和肺组织。制作肺组织石蜡切片，HE染色观察形态学改变，免疫组化检测肺组织中NLRP3炎症小体相关蛋白表达水平。蛋白质免疫印迹试验（Western blot）技术和RT-PCR方法检测肺组织中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和蛋白的表达。采用双抗体夹心酶标免疫分析法（ELISA法）测定各组血清和BALF中白细胞介素（IL）-1β、IL-18和IL-33水平。 结果： 染毒6 h后HE染色形态学观察可见，光气染毒组肺组织中有大量炎性细胞浸润。免疫组化染色结果显示，光气染毒组大鼠肺泡间隔增厚，肺组织中可见较多NLRP3、ASC和caspase-1阳性细胞；MP干预组肺组织中有炎性细胞浸润较光气染毒组明显减少，同时NLRP3和caspase-1阳性细胞较光气染毒组减少，而ASC阳性细胞无明显减少。与空气对照组比较，光气染毒组肺组织中NLRP3、ASC和caspase-1mRNA和蛋白表达量明显升高，差异有统计学意义（P<0.05）;与光气染毒组比较，MP干预组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），而ASC mRNA和蛋白表达量无明显减少。与空气对照组比较，光气染毒组血清和BALF中IL-1β、IL-18和IL-33蛋白含量明显升高，差异均有统计学意义（P<0.05）；与光气染毒组比较，MP干预组血清和BALF中IL-1β、IL-18和IL-33蛋白含量明显降低，差异有统计学意义（P<0.05）。 结论： MP可通过抑制NLRP3炎症小体的表达进而减少其介导的IL-1β等炎症因子释放来有效地保护大鼠光气吸入性急性肺损伤。.
Keywords: Acute Lung Injury; Inflammatory Response; Methylprednisolone; NLRP3 Inflammasome; Phosgene.