薄荷脑通过miR-1247-3p/PI3K/Akt通路影响LPS诱导的Ⅱ型肺泡上皮细胞凋亡和炎症反应的分子机制研究

Study on molecular mechanism of menthol via miR-1247-3p/PI3K/Akt pathway influencing LPS-induced alveolar typeⅡepithelial cells apoptosis and inflammation

目的:探讨薄荷脑对脂多糖(LPS)诱导的Ⅱ型肺泡上皮(AT-Ⅱ)细胞凋亡和炎症反应的影响及分子机制。方法:以5、10、15 mg/L的LPS处理AT-Ⅱ细胞,CCK-8法检测细胞活性;将AT-Ⅱ细胞随机分为对照(NC)组、15 mg/L LPS组、1、5、10µmol/L薄荷脑+15 mg/L LPS组、anti-miR-NC+15 mg/L LPS组、anti-miR-1247-3p+15 mg/L LPS组、miR-NC+10µmol/L薄荷脑+15 mg/L LPS组、miR-1247-3p+10µmol/L薄荷脑+15 mg/L LPS组;采用流式细胞术检测细胞凋亡;ELISA检测TNF-α、IL-6、IL-1β水平;Western blot检测蛋白表达;RT-qPCR检测miR-1247-3p表达水平。结果:不同浓度LPS处理后AT-Ⅱ细胞活性降低(P<0.05)。LPS诱导的AT-Ⅱ细胞中Cleaved-caspase-3表达水平及细胞凋亡率升高,TNF-α、IL-6、IL-1β水平升高,miR-1247-3p表达水平升高,p-PI3K、p-Akt蛋白表达水平降低(P<0.05)。不同浓度薄荷脑处理及miR-1247-3p低表达后,Cleaved-caspase-3表达水平及细胞凋亡率降低,TNF-α、IL-6、IL-1β水平降低(P<0.05)。miR-1247-3p高表达逆转了薄荷脑对LPS诱导的AT-Ⅱ细胞凋亡、炎症反应及对p-PI3K、p-Akt表达水平的影响。结论:薄荷脑通过miR-1247-3p/PI3K/Akt通路抑制LPS诱导的AT-Ⅱ细胞凋亡和炎症反应。

Objective:To explore the effect of menthol on lipopolysaccharide(LPS)-induced apoptosis and inflammation of alveolar typeⅡepithelial(AT-Ⅱ)cells and its molecular mechanism.Methods:AT-Ⅱcells were treated with 5,10,15 mg/L LPS,CCK-8 method was used to detect cell viability;AT-Ⅱcells were randomly divided into control(NC)group,15 mg/L LPS group,1,5,10μmol/L menthol+15 mg/L LPS group,anti-miR-NC+15 mg/L LPS group,anti-miR-1247-3p+15 mg/L LPS group,miR-NC+10μmol/L menthol+15 mg/L LPS group,miR-1247-3p+10μmol/L menthol+15 mg/L LPS group;cell apoptosis was evaluated using flow cytometry;ELISA was used to detect TNF-α,IL-6,IL-1βlevels;Western blot was used to detect protein expression;RT-qPCR was used to detect miR-1247-3p expression.Results:AT-Ⅱcell activity was decreased after treatment with different concentrations of LPS(P<0.05).Expression level of Cleaved-caspase-3 and apoptosis rate were increased in LPS-induced AT-Ⅱcells,levels of TNF-α,IL-6 and IL-1βwere increased,and expression level of miR-1247-3p was increased,expression levels of p-PI3K and p-Akt protein were decreased(P<0.05).After treatment with different concentrations of menthol or low expression of miR-1247-3p,expression level of Cleaved-caspase-3 and rate of apoptosis were decreased,and levels of TNF-α,IL-6 and IL-1βwere decreased(P<0.05).miR-1247-3p overexpression reversed effects of menthol on LPS-induced apoptosis and inflammation of AT-Ⅱcells,as well as expression levels of p-PI3K and p-Akt.Conclusion:Menthol inhibits LPS-induced AT-Ⅱcell apoptosis and inflammation through regualting miR-1247-3p/PI3K/Akt pathway.