棕榈酸钠脂毒性对小鼠L929成纤维细胞线粒体功能障碍和凋亡的影响及其机制

doi:10.13481/j.1671-587X.20260502

摘要/Abstract

摘要：

目的探讨棕榈酸钠（PA）处理所致脂毒性对小鼠L929成纤维细胞线粒体功能和细胞凋亡的影响，并阐明其与线粒体融合蛋白2（MFN2）和线粒体相关内质网膜（MAMs）结构之间的关系。方法以小鼠L929成纤维细胞为研究对象，采用不同浓度（0.1、0.2、0.3、0.4、0.5和0.6 mmol·L^-1）PA处理细胞12、24、36和48 h，采用细胞计数试剂盒8（CCK-8）法检测处理后各组细胞存活率，筛选PA最佳干预条件。将细胞分为对照组和PA组。采用流式细胞术检测2组细胞凋亡率，油红O染色法观察2组细胞脂质沉积情况，透射电子显微镜（TEM）观察2组细胞线粒体和MAMs结构特征，流式细胞术检测2组细胞中活性氧（ROS）水平，JC-1法检测2组细胞线粒体膜电位，实时荧光定量PCR（RT-qPCR）法和Western blotting法检测2组细胞中MFN2 mRNA及蛋白表达水平。结果 CCK-8法，处理12、24、36和48 h后，与对照组比较，PA处理浓度≥0.2 mmol·L^-1的各组L929细胞存活率均明显降低（P<0.05或P<0.001），且呈浓度和时间依赖性；其中处理24 h后0.3 mmol·L^-1 PA组L929细胞存活率约为60%，以该浓度和时间作为后续PA干预条件。流式细胞术和油红O染色法，与对照组比较，PA组L929细胞凋亡率明显升高（P<0.001），细胞内脂质沉积水平明显升高（P<0.001）。TEM观察，PA组L929细胞线粒体出现肿胀、嵴结构破坏和碎片化，MAMs接触位点减少，偶联距离增大。流式细胞术、JC-1法和RT-qPCR法，与对照组比较，PA组L929细胞中ROS水平明显升高（P<0.001），细胞线粒体膜电位明显降低（P<0.01），细胞中MFN2 mRNA和蛋白表达水平均明显降低（P<0.01）。结论 PA脂毒性可下调MFN2表达并破坏MAMs结构，促进细胞氧化应激、线粒体去极化和细胞凋亡，提示该条件下MFN2表达下调和MAMs结构受损与线粒体稳态失衡及细胞凋亡之间存在关联。

关键词: 盆底功能障碍, 脂毒性, 线粒体融合蛋白2, 线粒体相关内质网膜, 线粒体功能障碍

Abstract:

Objective To investigate the effects of sodium palmitate （PA）-induced lipotoxicity on mitochondrial function and apoptosis in mouse L929 fibroblasts， and to clarify its associations with mitofusin 2 （MFN2） expression and mitochondria-associated endoplasmic reticulum membrane （MAMs） structure. Methods Mouse L929 fibroblasts were used as the model，which were treated with different concentrations of PA （0.1， 0.2， 0.3， 0.4， 0.5， and 0.6 mmol·L^-1） for 12， 24， 36， and 48 h. Cell counting kit-8 （CCK-8） method was used to detect the survival rates of the cells in various groups after treatment， so as to screen the optimal intervention condition of PA. The cells were then divided into control group and PA group. Flow cytometry method was used to detect the apoptotic rates in the cells in two groups. Oil red O staining method was used to observe the lipid deposition in the cells in two groups. Transmission electron microscopy （TEM） was used to observe the structural characteristics of mitochondria and MAMs in the cells in two groups. Flow cytometry was used to detect the levels of reactive oxygen species （ROS） in the cells in two groups. JC-1 method was used to detect the mitochondrial membrane potential in the cells in two groups. Real-time fluorescence quantitative PCR （RT-qPCR） method and Western blotting method were used to detect the expression levels of MFN2 mRNA and protein in the cells in two groups. Results The CCK-8 assay results showed that at 12， 24， 36， and 48 h post treatment， compared with control group， the survival rates of L929 cells in groups treated with PA at concentrations ≥0.2 mmol·L^-1 were significantly decreased （P<0.05 or P<0.001）， showing a concentration- and time-dependent effect. The survival rate of L929 cells was approximately 60% in 0.3 mmol·L^-1 PA group after 24 h of treatment， which was used as the subsequent PA intervention condition. The flow cytometry and Oil red O staining results showed that compared with control group， the apoptotic rate of L929 cells in PA group was significantly increased （P<0.001） and the level of lipid deposition in the cells was significantly increased （P<0.001）. The TEM results showed that the mitochondria in L929 cells in PA group exhibited swelling， disruption of cristae structure， and fragmentation， while the number of MAMs contact sites was reduced and the coupling distance was increased. The flow cytometry， JC-1 method， and RT-qPCR method results showed that compared with control group， the level of ROS in L929 cells in PA group was significantly increased （P<0.001），the mitochondrial membrane potential of the cells was significantly decreased （P<0.01）， and the expression levels of MFN2 mRNA and protein in the cells were significantly decreased （P<0.01）. Conclusion PA-induced lipotoxicity downregulates MFN2 expression and disrupts MAMs structure， thereby promoting oxidative stress， mitochondrial depolarization， and cell apoptosis. These findings suggest that under PA-induced lipotoxic conditions， the downregulation of MFN2 expression and the damage to MAMs structure are closely associated with mitochondrial homeostasis imbalance and cell apoptosis.

Key words: Pelvic floor dysfunction, Lipotoxicity, Mitofusin 2, Mitochondria-associated endoplasmic reticulum membrane, Mitochondrial dysfunction

中图分类号:

R711.4

盛莹,刘剑锋,洪莉. 棕榈酸钠脂毒性对小鼠L929成纤维细胞线粒体功能障碍和凋亡的影响及其机制[J]. 吉林大学学报(医学版), 2026, 52(5): 1206-1214.

Ying SHENG,Jianfeng LIU,Li HONG. Effects of sodium palmitate-induced lipotoxicity on mitochondrial dysfunction and apoptosis in L929 fibroblasts and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2026, 52(5): 1206-1214.

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