Determination of the protective effects of boric acid against experimentally induced cholestasis with ANIT in rats.

Cholestasis is self-perpetuating: failure of canalicular bile salt export expands the intracellular bile acid pool, and retained acids progressively disrupt the systems and barriers sustaining bile secretion. α-Naphthylisothiocyanate (ANIT) is widely used to model intrahepatic cholestasis. Boron, internalized through the NaBC1 (SLC4A11) transporter, regulates redox balance and membrane integrity; however, its protective mechanisms in the cholestatic liver remain undefined. The explicit mechanism tested was that boric acid interrupts this circuit at its regulatory origin: by sustaining sirtuin-1 (SIRT1), which deacetylates the farnesoid X receptor (FXR) at Lys-217 and thereby maintains transcription of its canalicular targets - the bile salt export pump (BSEP), UDP-glucuronosyltransferase 1A1 (UGT1A1) and cholesterol 7α-hydroxylase (CYP7A1) - while phosphoinositide 3-kinase/protein kinase B (PI3K/AKT)-dependent preservation of the claudin-1/occludin/zonula occludens-1 (ZO-1) barrier and restraint of the protein kinase R-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4)-C/EBP homologous protein (CHOP) arm of the unfolded protein response (UPR) limit the downstream consequences. Forty male Sprague Dawley rats received vehicle, ANIT (50 mg/kg, intragastrically), boric acid (100 mg/kg/day, 7 days) before ANIT, or boric acid alone, and were sacrificed 24 h later. Transporters and the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/SIRT1 axis were analyzed by Western blot; the unfolded protein response and PI3K/p-AKT/mTOR signaling by immunohistochemistry; tight junction proteins and 8-hydroxy-2'-deoxyguanosine (8-OHdG) by immunofluorescence; and FXR, CYP7A1, and UGT1A1 by RT-qPCR, alongside histopathology. ANIT markedly suppressed BSEP and Na⁺/taurocholate cotransporting polypeptide (NTCP) while preserving multidrug resistance-associated protein (MRP)2, which exports the ANIT-glutathione conjugate, indicating selective impairment of bile salt-dependent secretion rather than canalicular export. FXR exhibited the greatest proportional reduction, with coordinated declines in CYP7A1 and UGT1A1, identifying the SIRT1-FXR node rather than any individual carrier as the level of regulatory failure. These alterations coincided with more than tenfold induction of glucose-regulated protein 78 (GRP78), eIF2α, ATF4, and CHOP, disruption of occludin, claudin-1 and ZO-1, 8-OHdG accumulation, suppression of Nrf2/HO-1 and PI3K/p-AKT/mechanistic target of rapamycin (mTOR) signaling and a tenfold expansion of fibrotic area. Boric acid attenuated these changes differentially: transporters, junctional integrity, redox balance and tissue architecture approached control levels, whereas the unfolded protein response and transcriptional bile acid regulation recovered only partially. Because NaBC1, SIRT1 and PI3K/AKT were not inhibited, this sequence is inferred from coordinated, pathway-consistent expression changes rather than established causally.