MCC950对脂多糖诱导小鼠急性肺损伤的保护作用

Protective effect of MCC950 on lipopolysaccharide-induced acute lung injury in mice

目的探讨核苷酸结合寡聚化结构域受体-3(NLRP3)炎性体抑制剂MCC950对脂多糖(lipopolysaccharide,LPS)诱导小鼠急性肺损伤(acute lung injury,ALI)的保护作用和治疗价值.方法通过气管注入LPS制作ALI小鼠模型.取用30只清洁级C57BL6雄性小鼠,采用随机数字法随机分为三组:对照组(Control组)、LPS组和MCC950干预组(LPS+MCC950组),每组10只.LPS组小鼠气管内注入LPS溶液(3 mg/kg),LPS+MCC950组小鼠在LPS处理后1h经腹腔注射MCC950溶液(50 mg/kg).LPS处理后6h收集标本,苏木素-伊红(HE)染色观察肺组织病理学改变,检测肺脏湿干质量比值,酶联免疫吸附法(ELISA)检测支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中白细胞介素(IL)-1β和IL-18水平,蛋白免疫印迹法(Western blot)检测NLRP3、适应分子凋亡相关斑点样蛋白(ASC)、半胱氨酸天冬氨酸蛋白酶-1前体(pro-caspase-1)在肺组织中的表达及半胱氨酸天冬氨酸蛋白酶-1(caspase-1)裂解生成情况.另外60只小鼠用于观察各组小鼠的5d生存率,其中LPS气管注入剂量调整为10 mg/kg.结果与Control组比较,LPS组肺组织病理损伤(肺损伤评分:2.30±1.49 vs.11.20±1.87,P<0.05)和肺组织水肿(肺湿干质量比值:4.06±0.40 vs.6.73±0.52,P<0.05)均显著加重;BALF中细胞因子IL-1β(pg/mL:8.34±2.09 vs.200.40±18.22,P<0.05)、IL-18(pg/mL:13.51±3.57 vs.224.10±13.35,P<0.05)释放显著增加,肺组织中NLRP3、ASC和pro-caspase-1蛋白表达含量虽均未见显著增加(P>0.05),但pro-caspase-1裂解生成caspase-1 p10显著增加(P<0.05).给予MCC950干预后,与LPS组比较,LPS+MCC950组肺组织病理损伤(肺损伤评分:11.20±1.87 vs.6.90±1.66,P<0.05)和肺组织水肿(肺湿干质量比值:6.73±0.52 vs.5.14±0.43,P<0.05)均显著减轻;BALF中细胞因子IL-1β(pg/mL:200.40±18.22 vs.97.76±10.17,P<0.05)、IL-18(pg/mL:224.10±13.35 vs.124.70±10.49,P<0.05)释放显著减少,肺组织中NLRP3、ASC和pro-caspase-1蛋白表达含量虽均未见减少(P>0.05),但pro-caspase-1裂解生成caspase-1 p10显著减少(P<0.05).在LPS(10 mg/kg)诱导小鼠ALI模型中,与Control组比较,LPS组小鼠的5d生存率显著下降(100%vs.15%,P<0.05);与LPS组比较,LPS+MCC950组小鼠的5d生存率显著改善(15%vs.50%,P<0.05).结论MCC950对LPS诱导的ALI小鼠具有保护和治疗作用,其作用机制与特异性抑制NLRP3炎性体活性、减轻炎症反应有关.

Objective To investigate the effect of MCC950(a NLRP3 inflammasome inhibitor)on lipopolysaccharide(LPS)-induced acute lung injury(ALI)in mice.Methods ALI mice models were established by intratracheal injection of LPS.Thirty C57BL6 male mice were randomly divided into 3 groups:control group,LPS group and LPS+MCC950 group.Mice in the LPS group were intratracheally instilled with LPS solution(3 mg/kg),and mice in the LPS+MCC950 group were intraperitoneally injected with MCC950 solution(50 mg/kg)1 hour after LPS administration.The mice were sacrificed 6 hours after LPS instillation.Hematoxylin-eosin(HE)staining was used to observe pathological changes of lung tissue.The lung injury score and lung wet-dry weight ratio were measured,and IL-1 p and IL-1β levels in bronchoalveolar lavage fluid(BALF)were detected by enzyme-linked immunosorbent assay(ELISA).Pyrin domain-containing protein 3(NLRP3),apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),pro-cysteinyl aspartate specific proteinase-1(pro-caspase-1)and the production of caspase-1 in lung tissue were measured by Western blot.Additional 60 mice were randomly divided into the three groups and used to observe the 5-day survival rate of each group,mice in the LPS group and LPS+MCC950 group were intratracheally injected with a higher dose of LPS(10 mg/kg).Results Compared with the control group,the pulmonary pathology injury(lung injury score:2.3±1.49 vs.11.2±1.87,P<0.05)and lung edema(lung wet-dry weight ratio:4.06±0.40 vs.6.73±0.52,P<0.05)were both significantly aggravated by LPS stimulation.The levels of IL-1β(pg/mL:8.34±2.09 vs.200.40±18.22,P<0.05)and IL-1β(pg/mL:13.51±3.57 vs.224.10±13.35,P<0.05)in BALF were both significantly increased.The protein expression of NLRP3,ASC and pro-caspase-1 in lung tissue were all significantly increased by LPS induction(P<0.05),and the pro-caspase-1 cleavaged into caspase-1 p10 was markedly increased by LPS(P<0.05).Compared with LPS group,the pulmonary pathology injury(lung injury score:11.20±1.87 vs.6.90±1.66,P<0.05)and lung edema(lung wet-dry weight ratio:6.73±0.52 vs.5.14±0.43,P<0.05)were both obviously improved by MCC950 treatment.The levels of IL-1β(pg/mL:200.40±18.22 vs.97.76±10.17,P<0.05)and IL-1β(pg/mL:224.10±13.35 vs.124.70±10.49,P<0.05)in BALF were both markedly reduced in the LPS+MCC950 group.None of the protein expression of NLRP3,ASC and pro-caspase-1 in lung tissue was significantly decreased by MCC950 intraperitoneal injection(P>0.05),but the pro-caspase-1 cleavaged into caspase-1 p10 was dramatically reduced by MCC950(P<0.05).The 5-day survival rate of the LPS group was significantly lower than that of the control group(100%vs.15%,P<0.05).Compared with the LPS group,the 5-day survival rate of the LPS+MCC950 group was obviously improved(15%vs.50%,P<0.05).Conclusions MCC950 plays protective and therapeutic effects on LPS-induced ALI in mice,and involves in the specific inhibition of NLRP3 inflammasome activity and alleviation of inflammatory response.