摘要
目的探讨miR-483-3p对脂多糖(LPS)诱导的小鼠Ⅱ型肺泡上皮细胞损伤的影响及可能机制。方法LPS诱导小鼠Ⅱ型肺泡上皮细胞后,实时荧光定量PCR(RT-qPCR)检测细胞中miR-483-3p和磷酸酪氨酸衔接蛋白1(APPL1)mRNA表达水平,酶联免疫吸附法检测细胞中丙二醛(MDA)和超氧化物歧化酶(SOD)水平,流式细胞术检测细胞凋亡,蛋白印迹(Western Blot)法检测细胞中APPL1、B淋巴细胞瘤-2(Bcl-2)和B淋巴细胞瘤-2相关蛋白(Bax)蛋白表达水平。转染miR-483-3p抑制剂或APPL1过表达载体构建miR-483-3p表达干扰或APPL1过表达小鼠Ⅱ型肺泡上皮细胞。LPS诱导干扰miR-483-3p表达或过表达APPL1的小鼠Ⅱ型肺泡上皮细胞后,上述相同方法检测细胞中MDA和SOD水平、细胞凋亡及Bcl-2和Bax蛋白表达水平。双荧光素酶报告基因实验验证miR-483-3p与APPL1调控关系。结果LPS诱导细胞后,细胞中miR-483-3p和MDA表达、细胞凋亡率和Bax蛋白表达升高(P<0.05),APPL1的mRNA和蛋白、SOD活性及Bcl-2蛋白表达降低(P<0.05)。干扰miR-483-3p表达或过表达APPL1后,LPS诱导的细胞中MDA含量、凋亡率和Bax蛋白表达降低(P<0.05),SOD活性升高(P<0.05)。miR-483-3p在小鼠Ⅱ型肺泡上皮细胞中靶向负调控APPL1表达。抑制APPL1表达逆转了干扰miR-483-3p对LPS诱导的细胞中MDA和SOD水平、细胞凋亡及Bcl-2和Bax蛋白表达的影响。结论干扰miR-483-3p表达可能通过负调控抑制APPL1表达抑制LPS诱导的小鼠Ⅱ型肺泡上皮细胞氧化应激和凋亡,减轻细胞损伤。
Objective To investigate the effects of miR-483-3p targeting APPL1 on the typeⅡalveolar epithelial cells injury induced by lipopolysaccharide(LPS)in mice,and to explore possible action mechanism.Methods After the typeⅡalveolar epithelial cells of mice were induced by LPS,the expression levels of miR-483-3p and APPL1 mRNA in cells were detected by RT-qPCR,and the expression levels of MDA and SOD in cells were measured by ELISA,and the cells apoptosis was detected by flow cytometry,and the expression levels of APPL1,Bcl-2 and Bax protein were detected by Western Blot.The miR-483-3p inhibitor or APPL1 overexpression vector was transferred to typeⅡalveolar epithelial cells to construct the typeⅡalveolar epithelial cells that interfered with miR-483-3p expression or overexpression of APPL1,after that,the above methods were used to detect the expression levels of MDA and SOD,cells apoptosis,and the expression levels of Bcl-2 and Bax protein.The dual luciferase reporter gene experiment verified the regulatory correlation between miR-483-3p and APPL1.Results After LPS induced cells,the expression levels of miR-483-3p and MDA,apoptosis rate and the expression levels of Bax protein in cells were significantly increased(P<0.05),however,the expression levels of mRNA and protein of APPL1,SOD activity and the expression levels of Bcl-2 protein were significantly decreased(P<0.05).After interfering with miR-483-3p expression or overexpressing APPL1,the MDA content,apoptosis rate and the expression levels of Bax protein in LPS-induced cells were significantly decreased(P<0.05),however,the SOD activity and the expression levels of Bcl-2 protein were significantly increased(P<0.05).The miR-483-3p negatively regulated APPL1 expression in cells,and inhibiting APPL1 expression reversed the effects of interfering with miR-483-3p on the expression levels of MDA and SOD levels,apoptosis,and the expression level of Bcl-2 and Bax protein.Conclusion Interfering with miR-483-3p expression may inhibit LPS-induced oxidative stress and apoptosis of typeⅡalveolar epithelial cells of mice by negatively regulating APPL1 expression,so as to relieve the cell damage.
作者
于智轩
高映春
YU Zhixuan;GAO Yingchun(Department of ICU,Liaoyang Central Hospital,Liaoning,Liaoyang 111000,China)
出处
《河北医药》
CAS
2022年第3期325-329,335,共6页
Hebei Medical Journal
