Tubastatin a Mitigates Lipotoxicity, Inflammation and Fibrosis Through Downregulation of HDAC6 and Modulation of AMPK-FGF21 Axis in Metabolic Dysfunction-Associated Steatohepatitis

Metabolic dysfunction- associated steatohepatitis (MASH) represents the progressive form of metabolic dysfunction- associated steatotic liver disease (MASLD). Tubastatin A (TA), a selective HDAC6 inhibitor, possesses anti-inflammatory and epigenetic modulatory properties, but its therapeutic potential in MASH remains unexplored. The MASH model was developed using a high-fat, high-fructose (HFFrD) diet in C57BL/6J animals for 12 weeks. After model development, HFFrD animals were randomised into three groups: MASH, MASH + TA (10 mg/kg), and MASH + TA (20 mg/kg). Furthermore, control animals were randomised into two groups: control and TA_Per Se (20 mg/kg), and TA was administered via the i.p. route for 6 weeks. Further, we have performed network pharmacology analysis to identify hub genes. TA treatment significantly improved morphometric parameters and attenuated metabolic abnormalities and liver injury markers. Additionally, TA treatment significantly reduced MASH-associated oxidative stress by restoring levels of malondialdehyde, nitrite, glutathione, and superoxide dismutase. The histopathological assessment demonstrated TA's hepatoprotective effects as evidenced by significant reductions in steatosis, hepatocellular ballooning, inflammatory infiltrates, and collagen deposition. Moreover, histopathological analysis of colon tissue revealed that TA treatment improved intestinal architecture and mucosal integrity. Mechanistically, TA downregulated HDAC6 and restored α-tubulin expression, suppressed lipogenic and gluconeogenic genes, restored FGF-21 expression, and reduced inflammatory and fibrotic gene expression. At the protein level, TA modulates the expression of AMPK, NF-κB, and Nrf2, thereby attenuating MASH. Further, the network pharmacology analysis also supported the role of TA in modulating epigenetic mechanisms in MASH by regulating HDACs. Overall, TA attenuates MASH by targeting multiple interconnected pathways, including lipogenesis, metabolic stress, oxidative damage, inflammation, and fibrosis, via HDAC6 inhibition, thereby modulating AMPK and FGF21 signalling, supporting its potential as a therapeutic candidate for MASH with fibrosis.