Lin28对血管紧张素Ⅱ诱导的心室重构细胞模型的影响

Effect of Lin28 on AngiotensinⅡInduced Ventricular Remodeling Cell Model

目的探讨Lin28对血管紧张素Ⅱ(AngⅡ)诱导的心室重构细胞模型的影响。方法本实验时间为2022年10月—2023年8月。从出生1~3 d的C57BL/6小鼠的心室分离并培养乳鼠原代心室肌细胞(NMVCMs),将其分为阴性对照组、AngⅡ对照组、AngⅡ+Lin28过表达组、AngⅡ+Lin28下调组。阴性对照组加入磷酸盐缓冲液(PBS);AngⅡ对照组加入AngⅡ(10μmol/L)并孵育24 h以构建心室重构模型;AngⅡ+Lin28过表达组转染Lin28-cDNA腺病毒36 h,之后加入AngⅡ(10μmol/L)并孵育24 h;AngⅡ+Lin28下调组传染Lin28-siRNA腺病毒36 h,之后加入AngⅡ(10μmol/L)并孵育24 h。采用酶联免疫吸附试验(ELISA)检测各组肿瘤坏死因子α(TNF-α)、白介素(IL)-6,TUNEL法检测各组细胞凋亡率,免疫荧光法检测各组GFP-LC3荧光点数,Western blotting法检测各组P62、Beclin 1表达水平。结果AngⅡ对照组、AngⅡ+Lin28过表达组、AngⅡ+Lin28下调组TNF-α、IL-6高于阴性对照组,AngⅡ+Lin28过表达组TNF-α、IL-6低于AngⅡ对照组,AngⅡ+Lin28下调组TNF-α、IL-6高于AngⅡ对照组、AngⅡ+Lin28过表达组(P<0.05)。AngⅡ对照组、AngⅡ+Lin28过表达组、AngⅡ+Lin28下调组细胞凋亡率高于阴性对照组,AngⅡ+Lin28过表达组细胞凋亡率低于AngⅡ对照组,AngⅡ+Lin28下调组细胞凋亡率高于AngⅡ对照组、AngⅡ+Lin28过表达组(P<0.05)。AngⅡ对照组、AngⅡ+Lin28过表达组GFP-LC3荧光点数、Beclin 1表达水平高于阴性对照组,P62表达水平低于阴性对照组(P<0.05);AngⅡ+Lin28过表达组GFP-LC3荧光点数、Beclin 1表达水平高于AngⅡ对照组,P62表达水平低于AngⅡ对照组(P<0.05);AngⅡ+Lin28下调组GFP-LC3荧光点数、Beclin 1表达水平低于阴性对照组、AngⅡ对照组、AngⅡ+Lin28过表达组,P62表达水平高于阴性对照组、AngⅡ对照组、AngⅡ+Lin28过表达组(P<0.05)。结论Lin28可减轻AngⅡ诱导的心室重构细胞模型炎症反应、减少心肌细胞凋亡、增强心肌细胞自噬。

Objective To investigate the effect of Lin28 on angiotensinⅡ(AngⅡ)induced ventricular remodeling cell model.Methods The experimental period of this study was from October 2022 to August 2023.Neonatal mouse ventricular cardiomyocytes(NMVCMs)were isolated from the ventricles of C57BL/6 mice born 1-3 days ago and cultured.NMVCMs were divided into negative control group,AngⅡcontrol group,AngⅡ+Lin28 overexpression group,and AngⅡ+Lin28 downregulated group.The negative control group was added to phosphate buffer saline(PBS);the AngⅡcontrol group was added to AngⅡ(10μmol/L)and incubated for 24 hours to construct a ventricular remodeling;the AngⅡ+Lin28 overexpression group was transfected with Lin28-cDNA adenovirus for 36 hours and then added to AngⅡ(10μmol/L)and incubated for 24 hours;the AngⅡ+Lin28 down-regulated group was transfected with Lin28-siRNA adenovirus for 36 hours and then added to AngⅡ(10μmol/L)and incubated for 24 hours.Tumor necrosis factor-α(TNF-α)and interleukin 6(IL-6)were detected by enzyme linked immunosorbent assay(ELISA),cell apoptosis rate was detected by TUNEL method,GFP-LC3 fluorescence points was detected by immunofluorescence,and P62 and Beclin 1 expression levels were detected by Western blotting method.Results The TNF-αand IL-6 in AngⅡcontrol group,AngⅡ+Lin28 overexpression group and AngⅡ+Lin28 down-regulated group were higher than those in negative control group,the TNF-αand IL-6 in AngⅡ+Lin28 overexpression group were lower than those in AngⅡcontrol group,the TNF-αand IL-6 in AngⅡ+Lin28 down-regulated group were higher than those in AngⅡcontrol group and AngⅡ+Lin28 overexpression group(P<0.05).The cell apoptosis rate in AngⅡcontrol group,AngⅡ+Lin28 overexpression group and AngⅡ+Lin28 down-regulated group was higher than that in negative control group,the cell apoptosis rate in AngⅡ+Lin28 overexpression group was lower than that in AngⅡcontrol group,the cell apoptosis rate in AngⅡ+Lin28 down-regulated group was higher than that in AngⅡcontrol group and AngⅡ+Lin28 overexpression group(P<0.05).The GFP-LC3 fluorescence points and Beclin 1 expression level in AngⅡcontrol group and AngⅡ+Lin28 overexpression group were higher than those in negative control group,the P62 expression level was lower than that in negative control group(P<0.05);the GFP-LC3 fluorescence points and Beclin 1 expression level in AngⅡ+Lin28 overexpression group were higher than those in AngⅡcontrol group,the P62 expression level was lower than that in AngⅡcontrol group(P<0.05);the GFP-LC3 fluorescence points and Beclin 1 expression level in AngⅡ+Lin28 down-regulated group were lower than those in negative control group,AngⅡcontrol group and AngⅡoverexpression group,the P62 expression level was higher than that in negative control group,AngⅡcontrol group and AngⅡoverexpression group(P<0.05).Conclusion Lin28 can reduce inflammatory response of AngⅡinduced ventricular remodeling cell model,reducing cardiomyocyte apoptosis,and enhancing cardiomyocyte autophagy.