MCC950对线粒体损伤相关分子模式诱导大鼠肺损伤的保护作用

Protective effect of MCC950 against lung injury induced by mitochondrial damage-associated molecular patterns

目的严重创伤诱发的急性呼吸窘迫综合征目前尚无特异性治疗策略,探讨MCC950对线粒体损伤相关分子模式(MTDs)诱导肺损伤的影响,初步分析其作用机制。方法将40只SD大鼠采用随机数字表法分为对照组、MTDs组、MCC950组和MTDs+MCC950组4组,MTDs+MCC950组采用腹腔注射MCC950(20 mg/kg)预处理SD大鼠1 h后,经尾静脉注射MTDs(5%体积肝[5])到大鼠体内;对照组注射等渗盐水;MTDs组腹腔注射等渗盐水,尾静脉注射MTDs;MCC950组腹腔注射MCC950,尾静脉注射等渗盐水,12 h后麻醉,腹主动脉放血处死。采用酶联免疫吸附试验(ELISA)检测BALF中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和IL-18含量,BCA法检测BALF中蛋白含量,称重以计算肺湿重/体重比值(LWW/BW),采用HE染色后光镜观察组织病理学改变,Smith肺损伤评分评价肺损伤情况,Western blot检测Pro-Caspase-1和Caspase-1蛋白表达。结果与对照组相比,MTDs组大鼠BALF中TNF-α、IL-1β和IL-18含量显著增多(P<0.05),MCC950组差异无统计学意义(P>0.05),MTDs+MCC950组中TNF-α含量显著增多(P<0.05),而IL-1β和IL-18差异无统计学意义(P>0.05);与MTDs组相比,MCC950+MTDs组BALF中IL-1β和IL-18含量显著减少(P<0.05)。与对照组相比,MTDs组LWW/BW比值明显增大[(4.19±0.36)mg/g vs(6.32±0.54)mg/g,P<0.05],BALF中蛋白含量明显增多[(0.12±0.03)g/L vs(0.79±0.07)g/L,P<0.05];与MTDs组相比,MCC950组、MCC950+MTDs组LWW/BW比值[(4.35±0.29)mg/g、(4.47±0.46)mg/g]明显降低(P<0.05),BALF中蛋白含量[(0.12±0.06)g/L、(0.15±0.06)g/L]明显减少(P<0.05)。Smith肺损伤评分统计分析表明,与对照组相比,MTDs组肺损伤评分明显增高(1.00±0.00 vs 8.33±0.58,P<0.05);与MTDs组相比,MCC950+MTDs组肺损伤评分明显降低(8.33±0.58 vs 3.67±0.58,P<0.05)。与对照组相比,MTDs组Pro-Caspase-1和Caspase-1蛋白水平明显增高(P<0.05);与MTDs组相比,MCC950+MTDs组Caspase-1蛋白条带灰度降低,蛋白表达水平降低(P<0.05),Pro-caspase 1无明显变化(P>0.05)。结论 MCC950可能通过抑制NLRP3炎性体活化对MTDs诱导的肺损伤发挥保护作用。

Objective There is still no specific immunotherapy to acute respiratory distress syndrome induced by severe trauma. The article aimed to investigate the effect of MCC950 on lung injury induced by mitochondrial damage-associated molecular patterns（MTDs） and preliminarily evaluate its molecular mechanism.Methods 40 SD rats were randomly devided into control group, MTDs group, MCC950 group, MTDs＋MCC950 group. The rats were were taken MCC950 （20mg/kg） by peritoneal injection pretreatment for 1 hour, followed by tail vein injection of MTDs （5% liver volume） and were killed 12 hours later. ELISA were applied to detect tumor necrosis factor （TNF-α）, interleukin-1β（ IL-1β） and IL-18 in broncho-alveolar lavage fluid （BALF）, and BCA method to assess the content of total protein. Lung tissues were weighed to calculate lung wet weight/body weight（ LWW/BW） ratio, and stained by HE staining to observe the pathological changes through light microscope. Smith lung injury score was used to assess histological lung injury. Western blot was employed to evaluate the protein expression of Pro-Caspase-1 and Caspase-1.Results ①Compared with control group, TNF-α, IL-1β and IL-18 in BALF of MTDs group were significantly increased（ all P〈0.05）, but not in MCC950 group（P〉0.05）, TNF-α in BALF of MTDs ＋ MCC950 group were significantly increased（ all P〈0.05）, IL-1β and IL-18 were not（all P〉0.05）. Compared with MTDs group, IL-1β and IL-18 in BALF of MTDs ＋ MCC950 group were in serious decline （all P〈0.05）. Compared with control group, the LWW/BW ratio ／[（4.19±0.36）mg/g vs （6.32±0.54）mg/g, P〈0.05／] and the content of total protein ／[（0.12±0.03）g/L vs （0.79±0.07）g/L, P〈0.05／] were dramatically increased. Compared with MTDs group, the LWW/BW ratio ／[（4.35±0.29）mg/g, （4.47±0.0.46）mg/g, P〈0.05／] and the content of total protein ／[（0.12±0.06）g/L, （0.15±0.06）g/L, P〈0.05／] were in serious decline. Smith lung injury score revealed that compared with control group the score of MTDs group was elevated （1.00±0.00 vs 8.33±0.58, P〈0.05）, and the score of MTDs＋ MCC950 group was significantly decreased than MTDs group （8.33±0.58 vs 3.67±0.58, P〈0.05）. Compared with control group, the protein expression of Pro-caspase-1 and caspase-1 were markedly improved （all P〈0.05）. However, the expression of caspase-1 was significantly milder than that in MTDs group （P〈0.05）, the protein expression of Pro-caspase-1 was comparable （P〉0.05）.Conclusion MCC950 exerts protective effect against lung injury induced by MTDs probably via the inhibition of NLRP3 inflammasome activation.