Circulating Hepatocyte Nuclear Factor-4α is Associated with Metabolic Dysfunction and Hepatic Remodeling in Fructose-Induced Metabolic Syndrome: An Immunomorphometric Study

Abstract

Metabolic syndrome (MS) driven by excessive dietary fructose is associated with progressive hepatic injury including steatosis, inflammation, fibrosis, and apoptosis. Hepatocyte nuclear factor-4α (HNF4α), a master transcriptional regulator of hepatic metabolism, is typically down-regulated in metabolic dysfunction-associated steatotic liver disease (MASLD). To characterize hepatic histopathological, immunohistochemical, and morphometric alterations in fructose-induced MS in Wistar rats and evaluate serum HNF4α levels and their correlations with biochemical and metabolic parameters. Twenty male Wistar rats were allocated to control and MS groups (10% fructose in drinking water, 8 weeks; n = 10 each). Liver sections were stained with H&E and Masson’s trichrome and immunostained for α-SMA, caspase-3, and PCNA. Morphometric analysis used Image-Pro Plus v6.0 and ImageJ. Biochemical parameters, serum HNF4α (quantified by commercial quantitative sandwich ELISA; R&D Systems, sensitivity 9.0 pg/mL), and oxidative stress markers were measured and analyzed using unpaired Student’s t-test with Cohen’s d effect sizes and Pearson correlation. MS rats showed significant increases in body weight, liver weight, hepatic index, fasting glucose, insulin, HOMA-IR, lipid profile, and serum HNF4α (771.6 ± 120.33 vs. 195.35 ± 62.3 pg/ml; p < 0.001; Cohen’s d = 6.0), with reduced HDL and total antioxidant capacity. Histology revealed macrovesicular steatosis, inflammation, and periportal, perisinusoidal, and pericentrilobular fibrosis. Morphometric analysis confirmed significant increases in collagen area%, α-SMA, caspase-3, and PCNA labeling index (all p < 0.001). Serum HNF4α correlated strongly with metabolic, lipid, and oxidative parameters (|r|=0.52–0.87), particularly malondialdehyde ( r = 0.87), body weight ( r = 0.85), and HDL ( r = − 0.86). Paradoxically, circulating HNF4α was elevated despite its typical hepatic down-regulation in MASLD; it is presented as a candidate biomarker requiring independent validation. Comprehensive morphometric analysis of five staining modalities provides objective, zone-specific quantification confirming the coordinated nature of the injury–fibrosis–regeneration axis. This elevation may reflect hepatocellular leakage or a transient compensatory response to early-stage metabolic stress, highlighting its potential utility as a non-invasive biomarker candidate requiring independent validation. However, because hepatic HNF4α protein and gene expression were not measured and conventional liver-injury markers were not assayed, these data demonstrate association rather than mechanistic involvement.