Hormophysa cuneiformis protects against methamphetamine-induced hepatotoxicity via antioxidant, anti-inflammatory, and autophagy-regulatory mechanisms: histological and biochemical studies

Abstract

Methamphetamine (METH) abuse is linked to significant hepatic injury, driven by oxidative stress, inflammatory pathway activation, autophagic dysregulation, and apoptosis induction. This study explored the hepatoprotective and anti‐inflammatory potential of the brown seaweed Hormophysa cuneiformis extract against METH‐induced liver damage in adult male Wistar rats. Extracts prepared using methanol, ethanol, hexane, and water were screened for antioxidant activity. The methanolic extract, selected for further investigation, was characterized by phytochemical analysis and GC–MS profiling. Molecular docking was performed to evaluate the interactions of the identified bioactive compounds with TNF‐α and LC3B‐II. The cytotoxicity and selectivity of the methanolic extract were assessed using HepG‐2 hepatocellular carcinoma and WI‐38 normal cells. Its hepatoprotective effects were further evaluated in vivo in METH‐treated adult male Wistar rats using biochemical, Western blot, immunohistochemical, and histopathological analyses. The methanolic extract showed the greatest antioxidant activity, achieving 57.16% ± 1.96% DPPH radical scavenging and 54.71% ± 0.84% ferric‐reducing antioxidant power. Although these values were lower than those of ascorbic acid (97.23 ± 0.48%), the methanolic extract exhibited higher antioxidant potential than the other extracts. Secondary metabolites, including alkaloids, saponins, tannins, and total phenolic compounds, were detected in notable amounts. GC‐MS analysis identified oleic acid, palmitic acid, linoleic acid, and arachidonic acid as the major constituents. The extract exhibited dose-dependent cytotoxicity against HepG–2 cells, with an IC50 of 77.14 ± 1.63 µg mL −1 , while showing lower toxicity toward WI-38 cells (CC50 = 141.99 ± 3.54 µg mL −1 ), indicating selective antiproliferative activity and a favorable safety margin. In vivo, METH administration significantly elevated serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels. Oral co-treatment with the H. cuneiformis methanolic extract (15 mg kg −1 ) significantly attenuated METH-induced hepatocellular injury, lowering serum ALT from 89.33 to 54.33 U L −1 , AST from 294.33 to 57.33 U L −1 , and ALP from 264.67 to 189.00 U L −1 . Western blot analysis showed that METH markedly increased LC3B-II and Beclin-1 expression, whereas co-administration of the extract significantly reduced both proteins. Immunohistochemical analysis also demonstrated increased caspase-3- and NF-κB-positive cells in the METH-treated group, which were significantly reduced following H. cuneiformis treatment. Collectively, these findings suggest that H. cuneiformis exerts hepatoprotective effects against METH-induced liver injury through antioxidant, anti-inflammatory, anti-apoptotic, and autophagy-modulating mechanisms.