基于NLRP3/Caspase-1通路探讨PM_(2.5)短期暴露对大鼠肺损伤机制

Mechanism of acute exposure to PM_(2.5)on lung injury in rats via NLRP3/Caspase-1 pathway

目的采用气管滴注法对大鼠进行PM_(2.5)短期暴露,研究其对不同性别大鼠的肺损伤及其机制。方法48只SD大鼠随机分为生理盐水对照组和PM_(2.5)高、中、低剂量(13.5、4.5和1.5 mg/kg)组,气管滴注连续7 d后解剖,观察肺大体变化情况;苏木素-伊红(HE)染色观察肺组织病理变化;支气管肺泡灌洗检查(BALF)肺上皮损伤指标[乳酸脱氢酶(LDH)和碱性磷酸酶(AKP)]变化情况;ELISA检测肺组织IL-1β和IL-18的表达水平;RT-PCR和Western blot检测NLRP3、Caspase-1 mRNA和蛋白表达水平。结果与对照组相比,各剂量组均出现不同程度的大片黑色斑块或者散在黑色斑点,且病变处质感相对较硬,病变程度呈现出剂量依赖性。HE染色发现,随着染毒剂量增加,肺泡壁逐渐增厚,中和高剂量组出现大面积的肺泡实变,且雌鼠病变较雄鼠严重。与对照组相比,雌鼠中和高剂量组、雄鼠高剂量组AKP及LDH含量均升高(F_(AKP(雌))=8.193、F_(AKP(雄))=9.121、F_(LDH(雌))=8.193、F_(AKP(雄))=9.121,P<0.05),雌鼠、雄鼠高剂量组IL-1β及雌鼠中和高剂量组、雄鼠高剂量组IL-18水平均出现明显升高(F_(IL-1β(雌))=19.523、F_(IL-1β(雄))=12.127、F_(IL-18(雌))=14.195、F_(IL-18(雄))=6.474,P<0.05),雌鼠中和高剂量组NLRP3、Caspase-1 mRNA及雄鼠高剂量组Caspase-1 mRNA表达水平上调(F_(NLRP3 mRNA(雌))=3.131、F_(Caspase-1 mRNA(雌))=6.072、F_(Caspase-1 mRNA(雄))=3.033,P<0.05),雌鼠各剂量组及雄鼠低、中剂量组NLRP3蛋白均上调(F_(NLRP3蛋白(雌))=7.810、F_(NLRP3蛋白(雄))=3.490,P<0.05),雌鼠中和高剂量组Caspase-1蛋白表达均出现上调(F_(Caspase-1蛋白(雌))=12.447、F_(Caspase-1蛋白(雄))=3.137,P<0.01);与同剂量组雌鼠比较,雄鼠中剂量组Caspase-1 mRNA表达水平较低(t=-2.349,P<0.05)。结论PM_(2.5)短期暴露会对大鼠肺组织造成形态损伤,并且造成大鼠肺组织炎症反应,使大鼠肺泡上皮损伤,可能与激活NLRP3/Caspase-1信号通路有关,并且这些损伤可能具有性别差异性,表现为雌鼠损伤比雄鼠严重。

Objective To explore the lung injury and mechanism of fine particulate matter(PM_(2.5))in rats by tracheal drip method.Methods Forty-eight SD rats were randomly divided into normal saline control group,PM_(2.5)high,medium and low dose(13.5,4.5,1.5 mg/kg)groups,and dissected after seven consecutive days of tracheal infusion to observe the general changes of the lungs.The pathological changes of lung tissue were observed by HE staining.Changes in lung epithelial injury indexes(LDH,AKP)in BALF were detected.ELISA was used to detect the expression levels of NLRP3 inflammasome effector inflammatory factors IL-1βand IL-18 in lung tissue;RT-PCR and Western blot were used to detect NLRP3/Caspase-1 signaling pathway gene NLRP3,Caspase-1 mRNA,and protein expression levels in lung tissue.Results Compared with the control group,all dose groups had different degrees of large black patches or scattered black spots,and the texture of the lesions was relatively hard,and the degree of lesions showed a dose-dependence.HE staining showed that with the increase of the dose of infection,the alveolar wall gradually thickened,and a large area of alveolar consolidation appeared in the medium and high dose groups,and the lesions of female mice were more serious than those of male mice.Compared with the control group,the contents of AKP and LDH in the medium-and high-dose groups of female mice and high-dose groups and high-dose groups of male mice were increased(F_(AKP(雌))=8.193、F_(AKP(雄))=9.121、F_(LDH(雌))=8.193、F_(AKP(雄))=9.121,P<0.05),IL-1βin the high-dose group of female mice and male rats,IL-1βin the medium-and high-dose groups of female mice and high-dose groups and high-dose groups of male mice(F_(IL-1β(雌))=19.523、F_(IL-1β(雄))=12.127、F_(IL-18(雌))=14.195、F_(IL-18(雄))=6.474,P<0.05)were significantly increased,NLRP3 and Caspase-1 mRNA in the medium-and high-dose groups of female mice and Caspase-1 in the high-dose group of male mice were significantly increased.The expression level of mRNA was upregulated(F_(NLRP3 mRNA(雌))=3.131、F_(Caspase-1 mRNA(雌))=6.072、F_(Caspase-1 mRNA(雄))=3.033,P<0.05),the NLRP3 protein in the female and low and intermediate dose groups was upregulated(F_(NLRP3蛋白(雌))=7.810、F_(NLRP3蛋白(雄))=3.490,P<0.05),and the expression of Caspase-1 protein in the medium and high dose groups of female mice was upregulated(F_(Caspase-1蛋白(雌))=12.447、F_(Caspase-1蛋白(雄))=3.137,P<0.01),and compared with the female mice in the same dose group,the expression of Caspase-1 mRNA in the male mouse medium dose group was lower(t=-2.349,P<0.05).Conclusion PM_(2.5)can cause morphological damage to rat lung tissue,and cause inflammatory response of rat lung tissue,therefore resulting in alveolar epithelial damage in rats,which may be due to the activation of NLRP3/Caspase-1 signaling pathway,resulting in inflammatory response of the body,resulting in a series of reactions such as tissue damage,and these injuries are sex-different,showing that female mice are more severely injured than male mice.