摘要
目的 探究沉默信息调节因子3(SIRT3)对心肌细胞的保护作用是否与叉头框蛋白O亚型(FoxO)信号通路相关。方法 2024年2月至2024年10月,将AC16人心肌细胞按照随机数字表法分为6组:空白对照(Blank)、对照组(Control)、SIRT3阴性对照(si-SIRT3)、SIRT3敲低组(LV-SIRT3)、SIRT3敲低加FoxO激活剂组(LV-SIRT3+LOM)、SIRT3过表达组(OE-SIRT3)。除Blank组外,其余各组均构建缺氧复氧损伤模型。检测指标包括:qRT-PCR测SIRT3 mRNA;流式细胞术测细胞凋亡;酶联免疫吸附试验(ELISA)检测细胞上清丙二醛(MDA)和超氧化物歧化酶(SOD);蛋白质免疫印迹法(Western blot)测SIRT3、FoxO1、FoxO3、B细胞淋巴瘤-2相关X蛋白(bax)及裂解型胱天蛋白酶-3(cleaved Caspase-3)蛋白水平。组间比较采用独立样本t检验,多组比较采用方差分析,两两比较用Tukey’s或Games-Howell法。结果 LV-SIRT3组的SOD水平低于Blank组[(42.81±6.84)U/L比(145.71±12.46)U/L,t=12.539,P<0.05],MDA水平高于Blank组[(14.74±1.36)μmol/L比(3.51±1.06)μmol/L,t=11.280,P<0.05],凋亡率高于Blank组[(59.27±2.71)%比(5.05±1.12)%,t=32.026,P<0.05],凋亡蛋白cleaved Caspase-3和bax的表达高于Blank组(0.77±0.06比0.34±0.03,t=11.103,P<0.05;0.81±0.05比0.41±0.05,t=9.798,P<0.05),SIRT3、FoxO1和FoxO3的蛋白表达低于Blank组(0.26±0.06比0.77±0.09,t=8.167,P<0.05;0.33±0.04比0.69±0.08,t=6.971,P<0.05;0.34±0.05比0.64±0.03,t=8.911,P<0.05)。FoxO激活剂LOM612显著逆转了LV-SIRT3引起的SOD降低、MDA升高、凋亡率升高以及cleaved Caspase-3和bax蛋白表达升高。结论 SIRT3通过抑制细胞凋亡来保护受损的心肌细胞,该过程可能是通过激活FoxO信号通路来实现的。
Objective To investigate whether the protection of Sirtuin 3(SIRT3)on cardiomyocytes is related to the forkhead homeobox type O(FoxO)signaling pathway.MethodsThe study period was from February 2024 to October 2024.AC16 human cardiomyocytes were randomly divided into 6 groups:blank group,control group,si-SIRT3(SIRT3 negative control)group,LV-SIRT3(lentiviral vector-mediated SIRT3 knockdown)group,LV-SIRT3+LOM group,and OE-SIRT3(SIRT3 overexpression)group.Except for the blank group,H/R injury models were established in the rest groups.Quantitative real-time PCR(qRT-PCR)was used to measure SIRT3 mRNA.Cell apoptosis was detected by flow cytometry.The levels of malondialdehyde(MDA)and superoxide dismutase(SOD)in cell supernatant were measured by enzyme linked immunosorbent assay(ELISA).The protein levels of SIRT3,FoxO1,FoxO3,bax and cleaved Caspase-3 were detected by Western blotting.Between-group comparisons were conducted using independent samples t-tests,while multiple group comparisons were performed using ANOVA,with pairwise comparisons conducted using Tukey’s or Games-Howell methods.ResultsThe LV-SIRT3 group exhibited lower SOD levels[(42.81±6.84)U/L vs.(145.71±12.46)U/L,t=12.539,P<0.05],higher MDA levels[(14.74±1.36)μmol/L vs.(3.51±1.06)μmol/L,t=11.280,P<0.05],higher apoptosis rates[(59.27±2.71)%vs.(5.05±1.12)%,t=32.026,P<0.05],and higher expression of cleaved Caspase-3 and bax(0.77±0.06 vs.0.34±0.03,t=11.103,P<0.05;0.81±0.05 vs.0.41±0.05,t=9.798,P<0.05),while SIRT3,FoxO1,and FoxO3 protein expression was lower(0.26±0.06 vs.0.77±0.09,t=8.167,P<0.05;0.33±0.04 vs.0.69±0.08,t=6.971,P<0.05;0.34±0.05 vs.0.64±0.03,t=8.911,P<0.05).LOM612,a FoxO activator,significantly reversed the decrease of SOD,increase of MDA,increase of apoptosis rate,and increase of cleaved Caspase-3 and bax protein expression induced by LV-SIRT3.ConclusionSIRT3 protects injured cardiomyocytes by inhibiting apoptosis,which may be achieved by activating FoxO signaling pathway.
作者
唐耀
张巍
贾毅
韩静
Tang Yao;Zhang Wei;Jia Yi;Han Jing(Shanxi Cancer Hospital,Chinese Academy of Medical Sciences Cancer Hospital Shanxi Branch,Shanxi Medical University Affiliated Cancer Hospital,Cardiovascular and Pulmonary Function Examination Room,Taiyuan 030013,China;Shanxi Cancer Hospital,Chinese Academy of Medical Sciences Cancer Hospital Shanxi Branch,Shanxi Medical University Affiliated Cancer HospitalThoracic Surgery,Taiyuan 030013,China;Shanxi Cancer Hospital,Chinese Academy of Medical Sciences Cancer Hospital Shanxi Branch,Shanxi Medical University Affiliated Cancer Hospital,Hepatobiliary and Pancreatic Surgery,Taiyuan 030013,China;Shanxi Cancer Hospital,Chinese Academy of Medical Sciences Cancer Hospital Shanxi Branch,Shanxi Medical University Affiliated Cancer Hospital Department of Anesthesiology,Taiyuan 030013,China)
出处
《中华实验外科杂志》
2025年第9期1758-1761,共4页
Chinese Journal of Experimental Surgery
基金
山西省中医药科研课题(2024ZYY2C078)。
