Nonalcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disease strongly associated with obesity and characterized by hepatic steatosis, inflammation, hepatocellular injury, and fibrosis. Its progression can lead to severe complications like cirrhosis and hepatocellular carcinoma. Current pharmacological treatments for NAFLD are limited, highlighting the urgent need for effective therapies. The pathogenesis of NAFLD involves complex interplay of factors, but chronic inflammation and oxidative stress are central to its development and progression. The "three-hit" hypothesis posits that steatosis, lipotoxicity, and inflammation drive disease progression. NF-κB activation plays a critical role in inflammation, leading to the production of pro-inflammatory cytokines like TNF-α, IL-6, and IL-1β. Lipotoxicity, resulting from excessive fat accumulation, impairs mitochondrial function and endoplasmic reticulum, triggering reactive oxygen species (ROS) production. Excessive ROS causes mitochondrial damage, hepatocyte apoptosis, and lipid peroxidation, further fueling inflammation and fibrogenesis. Alantolactone (Ala), a sesquiterpene lactone isolated from *Inula helenium* L., possesses anti-inflammatory properties demonstrated in various models. However, its therapeutic potential in NAFLD remained unexplored. This research aimed to evaluate Ala's hepatoprotective effects on NAFLD in a mouse model induced by a high-fat diet and in vitro using AML-12 cells.

The existing literature extensively documents the role of inflammation and oxidative stress in NAFLD pathogenesis. Studies have shown the involvement of NF-κB activation in the inflammatory process and the contribution of lipotoxicity and mitochondrial dysfunction to ROS generation. Previous research has demonstrated Ala's anti-inflammatory effects in models of diabetes and cigarette smoke-induced inflammation, primarily through the modulation of NF-κB and Nrf2/HO-1 pathways. However, prior to this study, there was a lack of research examining the effects of Ala specifically in NAFLD.

This study employed both in vivo and in vitro approaches to investigate the effects of Alantolactone (Ala) on NAFLD. In the in vivo study, male C57BL/6 mice were divided into five groups: a control group (CON) fed a low-fat diet, a group receiving Ala (10 mg/kg), a high-fat diet (HFD) group, and two HFD groups treated with Ala (5 mg/kg and 10 mg/kg). After 16 weeks on the respective diets, Ala treatment was initiated via oral gavage for an additional 8 weeks. At the study's conclusion, liver tissues and serum samples were collected for analysis. In vitro studies utilized AML-12 mouse liver cells, which were treated with Ala at varying concentrations to determine the optimal concentration for subsequent experiments. The cells were then challenged with palmitic acid (PA) to induce inflammation, oxidative stress and fibrosis and Ala's protective effect was assessed. Numerous assays were conducted, including liver function tests (AST, ALT), lipid profile analysis, histological examinations (H&E, Oil red O, Sirius red, Masson's trichrome), immunofluorescence (DHE), RNA transcriptome sequencing, RT-qPCR, and Western blotting to evaluate the effects of Ala on liver function, fibrosis, oxidative stress, and inflammatory responses. The RNA sequencing data was analyzed using BioPlanet 2019 to identify impacted pathways. Statistical analyses involved appropriate tests (Student's t-test, one-way ANOVA with Dunnett's post-hoc test) to compare groups.

Ala treatment significantly reduced serum levels of ALT and AST, indicative of hepatoprotection. It also reduced liver weight and serum levels of triglycerides (TG), total cholesterol (TCH), and low-density lipoprotein (LDL). Histological analyses showed a reduction in lipid droplet accumulation in liver tissues, confirmed by H&E and Oil red O staining. Furthermore, Ala significantly attenuated HFD-induced liver fibrosis, evidenced by decreased collagen deposition (Sirius red and Masson's trichrome staining) and reduced expression of fibrosis-associated proteins (COL1, TGF-β1, α-SMA). RNA sequencing and pathway analysis revealed that Ala modulated the Nrf2/Keap1 pathway, and Ala treatment increased Nrf2 and HO-1 protein levels, while reducing Keap1. DHE staining confirmed the reduction in ROS levels in Ala-treated mice. Ala also inhibited the HFD-induced activation of the NF-κB pathway, demonstrated by reduced phosphorylated P65 and increased IκBα levels, and decreased mRNA levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β). In vitro studies using PA-challenged AML-12 cells corroborated these findings: Ala reduced PA-induced oxidative stress, as measured by DHE staining, and modulated Nrf2/Keap1 pathway protein and mRNA expression. Ala also significantly suppressed PA-induced inflammation (reduced p65 phosphorylation, decreased NF-κB nuclear translocation, and decreased pro-inflammatory cytokine mRNA levels) and fibrosis (reduced COL1, TGF-β1, and α-SMA protein and mRNA levels). The use of an Nrf2 inhibitor suggested that Ala's hepatoprotective effects were at least partially mediated through the Nrf2 pathway.

This study demonstrated that Ala effectively attenuates HFD-induced liver injury in mice by reducing inflammation and oxidative stress. The mechanistic investigations revealed the key roles of Nrf2/Keap1 and NF-κB pathways. Ala's ability to activate the Nrf2 pathway enhanced antioxidant defenses and counteracted ROS-mediated damage. Simultaneously, its inhibition of the NF-κB pathway suppressed the inflammatory response, reducing liver damage. These findings provide strong preclinical evidence supporting Ala's therapeutic potential for NAFLD. The in vitro data using AML-12 cells further validated the effects observed in the animal model.

This study provides compelling evidence for the protective effects of Alantolactone against NAFLD. Its ability to modulate both inflammatory and oxidative stress pathways offers a promising therapeutic approach. Future research should focus on elucidating the precise molecular mechanisms of Ala's action, including identifying its direct targets and evaluating its efficacy in larger animal models and clinical trials. Further investigations into other pathways, such as the NOD-like receptor pathway, are warranted.

This study has several limitations. First, the precise molecular targets of Ala remain to be fully elucidated. Second, while glucose and insulin tolerance testing was not included, related findings from other studies indicate potential benefits in those areas, warranting further investigation. Third, the HFD-induced model used, while effective in inducing NAFLD, may not perfectly capture the complexities of human NAFLD. Fourth, the study did not investigate the impact on adipose tissue.