Molecular mechanism of tualang honey supplementation in the attenuation of 12% high cholesterol diet induced non-alcoholic steatohepatitis in rats

Abstract

The global surge in obesity, metabolic syndrome, and type 2 diabetes has driven a corresponding rise in non-alcoholic steatohepatitis (NASH), an advanced form of non-alcoholic fatty liver disease (NAFLD). Despite advancements in understanding its complex pathophysiology, NASH treatment options are limited. Current interventions primarily involve lifestyle changes, such as weight loss and exercise, and pharmacological agents which often present adverse effects, and no medication has been approved specifically for NASH. This highlights the urgent need for alternative therapeutic strategies. Tualang Honey (TH), a natural Malaysian product rich in antioxidants such as flavonoids and phenolic acids, represents a potential option for better NASH management. Previous studies have demonstrated TH's efficacy in improving liver enzymes and lipid profiles, particularly in animal models. However, the precise molecular mechanisms underlying its hepatoprotective properties remain poorly understood. This study investigated the molecular mechanisms underlying the attenuation of NASH through TH supplementation in a high-cholesterol diet (HCD)-induced NASH animal model. Twenty-five male Sprague Dawley rats were divided into five groups of 5 rats each: a normal control group (Group 1) fed with normal diet for 20 weeks and four groups fed a 12% HCD for 16 weeks to induce NASH, followed by four weeks of 12% HCD without TH (Group 2), 12% HCD and TH supplementation at doses of 1.2 g/kg/day (Group 3), moderate dose of 2.4 g/kg/day (Group 4), and high dose of 3.0 g/kg/day (Group 5). Histological examinations, biochemical assessment, immunohistochemical analyses, and gene expression profiling were conducted, followed by pathway analysis and validation of targeted gene expression. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey's least significant difference (LSD) post hoc test to identify group differences. The 12% HCD induced mild NASH, as evidenced by increased relative liver weight, presence of steatosis and inflammation in liver tissue samples, hypercholesterolaemia, hypertriglyceridaemia, as well as increased ?-SMA expression and TUNEL assay positive staining in the liver sections of Group 2. TH supplementation at 2.4 g/kg/day dose improved liver gross morphology, significantly reduced total triglycerides, activated hepatic stellate cells and apoptotic liver cells. Microarray analysis revealed ten most significant upregulated and 10 most downregulated genes involved in oxidative stress, immune response, lipid metabolism, and cellular injury repair in the TH-treated animals. Further pathway analysis demonstrated three key mechanisms underpinning TH's hepatoprotective effects: (1) lipid and cholesterol metabolism (2) antioxidant and anti-inflammatory pathways, and (3) glucose and energy metabolism. Notably, the significant upregulation of the aldehyde dehydrogenase (Aldh) gene at 2.4 g/kg/day compared to the HCD group indicates enhanced aldehyde detoxification processes with TH supplementation. Meanwhile, a significantly reduced suppression at 2.4g/kg/day of the interferon regulatory factor 2 (Irf2) gene aligns with an attenuated inflammatory response. Conversely, high-dose TH (3.0 g/kg/day) exhibited paradoxical effects, as the histology, biochemical profiles and immunohistochemical analysis demonstrate findings resembling NASH pathology. In conclusion, this study demonstrates that the hepatoprotective effects of TH in a 12% HCD-induced NASH animal model were most pronounced at the moderate dose of 2.4 g/kg/day. Lipid and cholesterol metabolism, antioxidant and anti-inflammatory mechanisms, and glucose and energy metabolism pathways were identified as the primary molecular pathways supporting these effects, highlighting the therapeutic potential of TH at optimal dosage.