lncRNA MALAT1调节miR-22-3p/NLRP3轴对LPS诱导的急性肺损伤的影响

LncRNA MALAT1 regulates miR-22-3p/NLRP3 axis on LPS-induced inflammatory injury and apoptosis of alveolar epithelial cells

目的探究长链非编码RNA MALAT1通过miR-22-3p/NLRP3信号轴促进脂多糖(lipopolysaccharide,LPS)诱导的急性肺损伤(acute lung injury,ALI)的分子机制。方法将SD大鼠随机分为假手术组、ALI组、sh-NC组、sh-MALAT1组,每组9只。通过气管内灌注LPS法建立ALI大鼠模型,假手术组气管内灌注等量的PBS作为阴性对照,sh-NC组、sh-MALAT1组在LPS诱导前48 h,通过静脉分别注射2×10^(7) TU/mL的sh-NC慢病毒或相同剂量的sh-MALAT1慢病毒。通过HE染色法观察肺组织的组织病理学改变;通过TUNEL染色法观察肺组织的细胞凋亡情况。通过双荧光素酶报告基因系统验证MALAT1与miR-22-3p、NLRP3与miR-22-3p的调控关系;通过实时荧光定量PCR技术和免疫印记技术检测肺组织和细胞中MALAT1、miR-22-3p、NLRP3、ASC、caspase-1的表达水平;通过酶联免疫吸附试验检测支气管肺泡灌洗液和细胞培养基中白细胞介素(IL)-1β、IL-18、肿瘤坏死因子-α(TNF-α)的分泌情况;通过CCK-8技术检测A549细胞增殖情况;通过流式细胞术检测A549细胞凋亡情况。结果与假手术组相比,LPS诱导的ALI大鼠肺组织和A549细胞中MALAT1、NLRP3、ASC、caspase-1的mRNA水平显著升高,miR-22-3p水平显著降低(P<0.05)。此外,与假手术组相比,LPS诱导的ALI大鼠肺组织的组织病理损伤水平、炎症反应和细胞凋亡率升高(P<0.05)。与ALI组、sh-NC组相比,抑制MALAT1表达可改善LPS诱导的ALI大鼠肺组织和细胞中的炎症反应和细胞凋亡(P<0.05)。双荧光素酶实验结果显示,MALAT1与miR-22-3p、NLRP3与miR-22-3p存在相互调控关系。与ALI组、sh-NC组相比,sh-MALAT1组肺组织中MALAT1、NLRP3、ASC、caspase-1 mRNA水平降低(P<0.05),miR-22-3p水平升高(P<0.05)。同时,BALF上清液中IL-1β、IL-18、TNF-α水平降低(P<0.05)。此外,下调MALAT1的同时抑制miR-22-3p表达后,细胞中miR-22-3p和Bcl-2表达水平显著降低,细胞增殖水平亦显著降低(P<0.05)。结论降低MALAT1的表达可通过促进miR-22-3p的表达,从而抑制NLRP3的蛋白水平,并最终抑制LPS诱导的ALI模型中的炎症反应和细胞凋亡。

Objective To investigate the molecular mechanism of long non-coding RNA MALAT1 promoting LPS-induced ALI through the miR-22-3p/NLRP3 signaling axis.Methods The ALI rat model was established by intratracheal infusion of LPS.The histopathological changes of lung tissue were observed by HE staining.The apoptosis of lung tissue was observed by TUNEL staining.The regulatory correlations between MALAT1 and miR-22-3p,NLRP3 and miR-22-3p were verified by dual luciferase reporter gene system.The expression levels of MALAT1,miR-22-3p,NLRP3,ASC,and caspase-1 in lung tissue and cells were assessed by real-time quantitative PCR and western blotting.The secretion of IL-1β,IL-18 and TNF-αin bronchoalveolar lavage fluid and cell culture medium were assessed by enzyme-linked immunosorbent assay.The proliferation and apoptosis of A549 cells were assessed by CCK-8 technology and flow cytometry,respectively.Results The ALI rat model was established by intratracheal infusion of LPS.The histopathological changes of lung tissue were observed by HE staining.The apoptosis of lung tissue was observed by TUNEL staining.The regulatory correlations between MALAT1 and miR-22-3p,NLRP3 and miR-22-3p were verified by dual luciferase reporter gene system.The expression levels of MALAT1,miR-22-3p,NLRP3,ASC,and caspase-1 in lung tissue and cells were assessed by real-time quantitative PCR and western blotting.The secretion of IL-1β,IL-18 and TNF-αin bronchoalveolar lavage fluid and cell culture medium were assessed by enzyme-linked immunosorbent assay.The proliferation and apoptosis of A549 cells were assessed by CCK-8 technology and flow cytometry,respectively.Conclusion Reducing the expression of MALAT1 can inhibit the protein level of NLRP3 by promoting the expression of miR-22-3p,and ultimately inhibit the inflammatory response and apoptosis in the LPS-induced ALI model.