Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease globally, characterized by the accumulation of lipid droplets (LDs) in more than 5 % of hepatocytes [1]. Closely linked to metabolic syndrome, obesity, and type 2 diabetes, NAFLD affects approximately 25 % of the global population and constitutes a major public health challenge [2]. The disease spectrum ranges from simple steatosis to non-alcoholic steatohepatitis (NASH), fibrosis, and ultimately hepatocellular carcinoma (HCC) [3]. Lipotoxicity induced by free fatty acids (FFAs), particularly saturated fatty acids, plays a critical role in triggering hepatocyte apoptosis [4]. Although apoptosis is essential for maintaining tissue homeostasis, its dysregulation can exacerbate the progression of liver diseases [5].

Saturated fatty acids, such as palmitate, and unsaturated fatty acids, such as oleate, exert distinct effects on hepatocytes. Palmitate induces endoplasmic reticulum (ER) stress and generates reactive oxygen species (ROS), thereby activating apoptotic pathways. For example, Cho et al. demonstrated that palmitate upregulates the expression of CHOP and Xbp-1 genes, contributing to ER stress [6]. In contrast, oleate, due to its unsaturated structure, exhibits relatively protective effects against apoptosis, as reported by Zeng et al. [5]. The palmitate/oleate (P/O) ratio used in this study mimics the composition of modern dietary patterns, such as Western diets, which are rich in saturated fatty acids (e.g., palmitate from animal fats and processed foods) and unsaturated fatty acids (e.g., oleate from olive oil and nuts). This ratio is critical for modeling NAFLD, as it captures the synergistic interplay of these fatty acids in promoting lipid accumulation and cellular stress, key drivers of NAFLD progression[7,8]. Investigating their combined effects provides insights into how dietary fatty acid profiles contribute to lipotoxicity and hepatocyte apoptosis, addressing a critical gap in understanding NAFLD pathogenesis.

Key genes regulating apoptosis include p53, Bcl-2, Bax, and FasL. The p53 gene, a central regulator, is activated in response to cellular stress and induces both intrinsic and extrinsic apoptotic pathways [9]. Mutations in p53 have been reported in over 50 % of HCC cases, highlighting its pivotal role in liver diseases [10]. Proteins of the Bcl-2 family, including anti-apoptotic Bcl-2 and pro-apoptotic Bax, regulate mitochondrial membrane permeability and cytochrome C release [11]. Additionally, FasL, by binding to the Fas death receptor, activates caspases and accelerates the extrinsic apoptotic pathway [12].

This study aims to investigate the apoptosis process in HepG2 cells treated with palmitate, oleate, and a combination of palmitate and oleate. By evaluating the expression of p53, Bcl-2, Bax, and FasL, this research enhances the understanding of cell death mechanisms under lipotoxic conditions and lays the foundation for identifying novel therapeutic targets for NAFLD-related liver diseases.