Department of Genetics and Genomics, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates.
Present Address: Indian Institute of Science, C V Raman Road, 560012, Bangalore, India.
Lipids Health Dis. 2023 May 29;22(1):69. doi: 10.1186/s12944-023-01835-x.
The accumulation of misfolded proteins, encoded by genetic variants of functional genes leads to Endoplasmic Reticulum (ER) stress, which is a critical consequence in human disorders such as familial hypercholesterolemia, cardiovascular and hepatic diseases. In addition to the identification of ER stress as a contributing factor to pathogenicity, extensive studies on the role of oxidized Low-Density Lipoprotein (oxLDL) and its ill effects in expediting cardiovascular diseases and other metabolic comorbidities are well documented. However, the current understanding of its role in hepatic insults needs to be revised. This study elucidates the molecular mechanisms underlying the progression of oxLDL and ER stress-induced cytotoxicity in HepG2.
HepG2 cells stably expressing wild-type Low-Density lipoprotein receptor (WT-LDLR) and missense variants of LDLR that are pathogenically associated with familial hypercholesterolemia were used as the in vitro models. The relative mRNA expression and protein profiles of ER stress sensors, inflammatory and apoptotic markers, together with cytotoxic assays and measurement of mitochondrial membrane potential, were carried out in HepG2 cells treated with 100 µg per ml oxLDL for 24 to 48 h. 1-way or 2-way ANOVA was used for statistical analyses of datasets.
ER stress responses are elicited along all three arms of the unfolded protein response (UPR), with adverse cytotoxic and inflammatory responses in oxLDL-treated conditions. Interestingly, oxLDL-treated ER-stressed HepG2 cells manifested intriguingly low expression of BiP- the master regulator of ER stress, as observed earlier by various researchers in liver biopsies of Non-Alcoholic Steatohepatitis (NASH) patients. This study shows that overexpression of BiP rescues hepatic cells from cytotoxic and inflammatory mechanisms instigated by ER stress in combination with oxLDL, along the ER and mitochondrial membrane and restores cellular homeostasis.
The data provide interesting leads that identify patients with familial hypercholesterolemia conditions and potentially other Endoplasmic Reticulum Associated Degradation (ERAD) diseases as highly susceptible to developing hepatic insults with molecular signatures like those manifested in Non-Alcoholic Fatty Liver Disease (NAFLD) and NASH.
Although the use of HepG2 cells as the model is a major caveat of the study, the findings of this research may be used as the pilot study to expand further investigations in primary hepatocytes or iPSC- derived cellular models.
功能基因的遗传变异导致错误折叠蛋白的积累，从而引发内质网（ER）应激，这是家族性高胆固醇血症、心血管和肝脏疾病等人类疾病的一个关键后果。除了将 ER 应激确定为致病因素之外，大量研究还充分证明了氧化低密度脂蛋白（oxLDL）及其在加速心血管疾病和其他代谢合并症中的不良作用的作用。然而，目前对其在肝损伤中的作用的理解需要修改。本研究阐明了 oxLDL 和 ER 应激诱导的 HepG2 细胞毒性进展的分子机制。
使用稳定表达野生型低密度脂蛋白受体（WT-LDLR）和与家族性高胆固醇血症相关的 LDLR 错义变体的 HepG2 细胞作为体外模型。用 100μg/ml oxLDL 处理 HepG2 细胞 24 至 48 小时，进行 ER 应激传感器、炎症和凋亡标志物的相对 mRNA 表达和蛋白谱分析，以及细胞毒性测定和线粒体膜电位测量。使用单因素或双因素方差分析对数据集进行统计分析。
未折叠蛋白反应（UPR）的三条途径都引发了 ER 应激反应，oxLDL 处理条件下表现出不良的细胞毒性和炎症反应。有趣的是，oxLDL 处理的 ER 应激 HepG2 细胞表现出 BiP 的表达明显降低，BiP 是 ER 应激的主要调节剂，这是早期的研究人员在非酒精性脂肪性肝炎（NASH）患者的肝活检中观察到的。本研究表明，BiP 的过表达可挽救 ER 应激与 oxLDL 共同引发的肝细胞的细胞毒性和炎症机制，并恢复细胞内稳态。
数据提供了有趣的线索，表明家族性高胆固醇血症患者和潜在的其他内质网相关降解（ERAD）疾病患者极易发生肝脏损伤，其分子特征类似于非酒精性脂肪性肝病（NAFLD）和 NASH 中表现出的特征。
尽管使用 HepG2 细胞作为模型是本研究的一个主要缺陷，但本研究的发现可以作为进一步在原代肝细胞或 iPSC 衍生的细胞模型中进行研究的初步研究。
