Alcoholic liver disease (ALD) poses a significant health concern stemming from prolonged and heavy alcohol consumption. The progression of ALD from alcoholic fatty liver (AFL) to alcoholic steatohepatitis (ASH), alcoholic fibrosis, alcoholic cirrhosis, and potentially hepatocellular carcinoma (HCC) is closely linked to escalating alcohol consumption [1]. In 2018, the World Health Organization (WHO) reported that alcohol consumption contributes to 3.3 million deaths annually, representing approximately 5.9% of global mortality [2,3]. In addition, excessive alcohol consumption accounted for 47.9% of all cirrhosis-related deaths [4]. Hepatic steatosis emerges as the initial and prevalent pathological consequence of alcohol consumption, characterized by aberrant triglyceride accumulation within hepatocytes which further exacerbates liver damage by disrupting fat metabolism and promoting subsequent inflammation responses within hepatocytes [5]. Thus, AFL serves as an initial and reversible stage of ALD, underscoring the importance of timely intervention in enhancing liver function and averting the progression of ALD.

For AFL, ethanol can disrupt fatty acid synthesis in hepatocytes by impacting hepatocyte endoplasmic reticulum (ER) function and resulting in aberrant triglyceride storage in hepatocytes [6]. The ER, crucial for protein and lipid synthesis, is a highly responsive organelle with a rapid “detection, response, and adaption” mechanism to maintain protein synthesis, Ca2+ signaling, redox balance, and lipid metabolism homeostasis [[7], [8], [9]]. A variety of factors such as anoxia, oxidative stress, alcohol metabolism, and virus infection break the homeostasis of the ER and cause unfolded or misfolded protein accumulation in the ER lumen, which induces endoplasmic reticulum stress (ERs) and subsequently activates the unfolded protein response (UPR) [[10], [11], [12]]. Under physiological conditions, UPR as a protective mechanism, can increase the level of intracellular autophagy and promote lipid accumulation in hepatocytes while avoiding apoptosis. In turn, when the ER homeostasis cannot be restored timely, UPR can activate apoptosis signals which aggravate disease progression. Protein kinase RNA-like ER kinase (PERK), inositol-requiring kinase 1α (IRE1α), and activating transcription factor 6 (ATF6) are three transmembrane receptors that comprise the UPR signaling pathway [13], which is activated by the ERs to restore ER homeostasis [14]. Previous studies indicate that the knockdown of PERK in the ERs ERK-eukaryotic initiation factor 2α (eIF2α)-ATF4 pathway can suppress the expression of fatty acid synthase (FAS), carboxylase (ACC), and stearoyl coenzyme-A desaturase-1 (SCD1), leading to the mitigation of hepatic steatosis [15]. Therefore, targeting ERs to regulate lipid metabolism disorders represents a promising approach to enhance hepatic steatosis.

Folic acid also known as vitamin B9, the active form in vivo is 5-methyltetrahydrofolate (5-MTHF), which is an important one-carbon unit transporter in the body, involved in nucleic acids, neurotransmitter synthesis, homocysteine (Hcy) transmethylation metabolism and regulation of insulin sensitivity [16]. Silva and coworkers reported that folic acid deficiency can upregulate lipid metabolism-related genes like ACSL1 and lipin 1, potentially contributing to hepatic steatosis [17]. Notably, low serum folic acid levels independently increase the risk of nonalcoholic liver disease (NAFLD) and exhibit a negative correlation with hepatic steatosis [18]. Furthermore, folic acid intervention has been shown to diminish fat synthesis in primary chicken hepatocytes by inhibiting the PI3K/AKT/SREBP pathway [19]. Interestingly, by adjusting the abundance of folate-producing bacteria in the human gut and increasing the levels of serum folate, the hepatic signaling of fatty acid synthesis was downregulated while the β-oxidation capacity of hepatocyte mitochondria was enhanced [20]. Analogously, our previous study also indicates that folic acid facilitated the Hcy transmethylation process, and restored the mitochondrial function which reduced the mitophagy, while also correcting alcohol-induced swelling and vesicular changes in the hepatocyte endoplasmic reticulum [21]. Despite folic acid has been proven to alleviate hepatic steatosis by improving mitochondrial damage, insulin resistance, and DNA methylation [22], whether folic acid is involved in ERs-mediated hepatic fatty acid metabolism remains unclear. Thus, in this study, the potential interactions among folic acid and potential targets in AFL were analyzed by pharmacological network analysis, and the binding mode and affinity were explored through molecular docking technology to obtain a clearer mechanism.

In the present study, we aimed to evaluate how folic acid improves ethanol-induced hepatic steatosis and whether this protective mechanism is mediated through the modulation of the ERs-related signaling pathway.