Non-alcoholic fatty liver disease (NAFLD) is a prevalent metabolic disorder associated with obesity and insulin resistance. The increasing incidence of NAFLD necessitates the exploration of effective therapeutic strategies. Traditional medicine, particularly the use of natural compounds with fewer side effects compared to synthetic drugs, has gained attention. Flavonoids, abundant in various plants, have shown promise in reducing NAFLD risk. Previous studies indicated that Quzhou Fructus Aurantii, rich in flavonoids, ameliorates fatty liver and insulin resistance. Neohesperidin (NHP), a major component of Quzhou Fructus Aurantii, has demonstrated lipid accumulation inhibition in adipocytes and blood glucose/lipid lowering effects. However, the direct impact of NHP on hepatic steatosis remained unclear. This study aimed to investigate the effects of NHP on HFD-induced hepatic steatosis and insulin resistance in mice, elucidating its underlying mechanisms.

Existing literature highlights the crucial role of mitochondrial biogenesis in improving NAFLD. Studies show that flavonoids can reduce NAFLD risk, and previous research by the authors demonstrated that Quzhou Fructus Aurantii ameliorates fatty liver and insulin resistance in HFD-fed mice. Neohesperidin, a key component of this extract, has been shown to inhibit lipid accumulation in adipocytes and improve glucose and lipid profiles. However, its direct effect on hepatic steatosis was not established. Other studies link AMPK activation and PGC-1α expression to improved mitochondrial function and alleviation of NAFLD. The role of oxidative stress and inflammation in NAFLD progression is well-documented.

The study used 8-week-old male C57BL/6 mice, randomly divided into three groups (chow diet, HFD, HFD + NHP (50 mg/kg/day for 12 weeks)). Body weight, food intake, and fasting blood glucose were monitored weekly. Biochemical analyses included serum and hepatic ALT, AST, TG, NEFA, TC, MDA, ROS, SOD, CAT, and GSH levels. Oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT) were performed at week 8. Histopathological analysis (H&E and Oil Red O staining) assessed hepatic steatosis and NAFLD activity score (NAS). Immunohistochemical staining quantified MPO-positive neutrophils. HepG2 cells were used to establish an in vitro hepatic steatosis model using palmitic acid (PA). Cells were treated with DMSO, PA, PA + NHP, PA + NHP + SR-18292 (PGC-1α inhibitor), or PA + NHP + Compound C (AMPK inhibitor). Mitochondrial function (MTT assay, ATP content) and mitochondrial staining (Mito-Tracker Red) were performed. Real-time PCR analyzed mRNA expression of genes related to fatty acid synthesis, oxidation, mitochondrial biogenesis, inflammation, and antioxidant defense. Western blot analysis assessed PGC-1α and AMPK protein levels. Statistical analyses included Student's t-test and one-way ANOVA with Bonferroni's post hoc test.

NHP significantly reduced body weight gain and adipose tissue weight in HFD-fed mice. NHP treatment lowered serum and hepatic ALT and AST levels, indicating improved liver function. Hepatic steatosis was significantly alleviated by NHP, as evidenced by H&E staining and NAS scores. NHP decreased serum and hepatic TC, TG, and NEFA levels. Oil Red O staining confirmed reduced hepatic lipid accumulation. NHP improved fasting blood glucose, serum insulin, and HOMA-IR levels, and enhanced glucose tolerance and insulin sensitivity. NHP reduced the number of MPO-positive neutrophils and mRNA expression of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α). Hepatic MDA and ROS levels were reduced by NHP, while mRNA expression of antioxidant genes (Cat, Sod1, Gpx1, Ucp2) and antioxidant enzyme levels (SOD, CAT, GSH) were increased. NHP upregulated fatty acid oxidation genes (Ppara, Acaa2, Cpt-1, Acox1, Pdk4) and downregulated lipogenic genes. NHP significantly increased mtDNA copy number, ATP content, and mRNA expression of mitochondrial biogenesis genes (Nrf-1, Tfam). NHP increased PGC-1α protein and mRNA expression. In HepG2 cells, NHP increased mitochondrial mass, ATP generation, and succinate dehydrogenase activity, effects blocked by SR-18292. NHP also increased AMPK phosphorylation, and this effect was reversed by Compound C, which also reduced NHP-induced PGC-1α mRNA expression.

The study's findings demonstrate that NHP ameliorates hepatic steatosis and insulin resistance in HFD-fed mice by promoting mitochondrial biogenesis and fatty acid oxidation. This effect is mediated by the upregulation of PGC-1α expression via AMPK activation. The improvement in fatty acid oxidation is a crucial mechanism through which NHP reduces hepatic lipid accumulation. The simultaneous reduction of oxidative stress and inflammation by NHP counteracts the potential adverse effects of enhanced fatty acid oxidation. The results highlight the potential therapeutic role of NHP as a dietary supplement for NAFLD.

Neohesperidin effectively alleviates hepatic steatosis and insulin resistance in HFD-fed mice, primarily through PGC-1α-mediated mitochondrial biogenesis driven by AMPK activation. These findings suggest NHP's potential as a supplementary treatment for NAFLD. Further studies are needed to investigate the clinical efficacy and long-term effects of NHP in humans.

The study used a mouse model, which may not fully reflect human physiology. The sample size was relatively small. The study focused on the effects of NHP; further research is needed to explore its interaction with other components of Quzhou Fructus Aurantii. The study did not address the potential for adverse effects or long-term consequences of NHP administration.