Introduction: Liver diseases cause ~2 million deaths annually; cholestasis, defined as impaired bile formation or flow, can progress to cirrhosis and liver failure. Excess Cu2+ catalyzes Fenton chemistry, generating hydroxyl radicals that induce lipid peroxidation (elevated malondialdehyde, MDA) and reduce glutathione (GSH). The resulting oxidative stress activates nuclear factor-kappa B, releasing pro-inflammatory cytokines (TNF-α, IL-6, IL-1β). Concurrently, copper disrupts BCL-2 family balance, increasing Bax, releasing cytochrome c, activating caspase-3, and executing mitochondrial apoptosis. Critically, Cu also suppresses canalicular transporters BSEP (bile salt export pump) and MDR3 (phosphatidylcholine floppase), causing intrahepatic accumulation of toxic bile acids and a vicious cholestatic cycle.

Current therapies (ursodeoxycholic acid, obeticholic acid) have limited efficacy and side effects. The polyphenol-rich plant extracts offer multi-target hepatoprotection. Black-peel pomegranate (Punica granatum L. var. Black), an Iranian variety, has exceptionally high anthocyanin and ellagitannin content.

Aims: This study evaluated the hepatoprotective effects of aqueous black-peel pomegranate peel extract (BPPE) against CuSO4-induced cholestatic liver injury in rats, examining serum cholestatic markers (ALP, GGT), hepatic oxidative stress (MDA, GSH), copper accumulation, and mRNA expression of inflammatory (NF-κB, TNF-α, IL-6, IL-1β), apoptotic (Bax, Bcl-2, cytochrome c, caspase-3), and canalicular transporter (BSEP, MDR3) genes.

Materials and methods: Thirty-six male Wistar rats (190-220 g) were divided into six groups (n=6): (1) Control (normal water); (2) CuSO4 (150 mg/kg/day in drinking water); (3) CuSO4 + vehicle (1% CMC gavage); (4-6) CuSO4 + BPPE at 50, 100, and 150 mg/kg/day by gavage. Treatments lasted 30 days. At the endpoint, blood and liver were collected. Serum ALP and GGT were measured using commercial kits. Hepatic MDA (Thiobarbituric acid method), GSH (DTNB assay), and copper content (atomic absorption spectrometry after acid digestion) were quantified. Relative mRNA expression of target genes was analyzed by real-time PCR with SYBR Green, normalized to GAPDH. Data (mean ± SEM) were compared by one-way ANOVA and Tukey test.

Results: CuSO4 significantly increased serum ALP (~3-fold) and GGT (~6-fold) vs. control. BPPE at all doses lowered these, with 100 mg/kg restoring levels to near-control. Hepatic MDA doubled, and GSH fell to 38% of control in the CuSO4 group. BPPE (100 mg/kg) normalized MDA and recovered GSH to ~80% of control, not differing from healthy animals. Copper content in liver increased 3.3-fold with CuSO4; 100 mg/kg BPPE reduced it by ~54%, reaching a level similar to controls.

Gene expression (100 mg/kg BPPE vs. CuSO4 alone): NF-kB was upregulated 3.32-fold by CuSO4 and normalized to 1.04-fold by BPPE. IL-6 (3.7-fold) reduced to 1.36-fold; IL-1β (2.4-fold) to 1.8-fold; TNF-α (1.75-fold) to 1.5-fold. The pro-apoptotic Bax, cytochrome c, and caspase-3 were upregulated 2.7-, 1.6-, and 1.2-fold by CuSO4, while anti-apoptotic Bcl-2 dropped to 0.33-fold. BPPE decreased Bax, cytochrome c, and caspase-3, and increased Bcl-2 to 1.5-fold above control, restoring the Bax/Bcl-2 ratio. BSEP and MDR3 were suppressed by CuSO4 to 0.51- and 0.43-fold of control; BPPE partially restored them to 0.79- and 0.82-fold, respectively.

Discussion: The extract normalization of ALP and GGT confirms anti-cholestatic efficacy. CuSO4-induced oxidative stress (MDA rise, GSH depletion) was counteracted by BPPE’s direct radical scavenging and metal chelation, evidenced by a 54% reduction in hepatic copper. The observed bell-shaped dose-response (slight decline at 150 mg/kg) may reflect the pro-oxidant behavior of excess polyphenols.

At the molecular level, BPPE nearly abolished NF-κB activation and downstream cytokines, breaking the inflammatory feed-forward loop. This anti-inflammatory effect likely contributed to the partial restoration of BSEP and MDR3 expression, as these transporters are suppressed by cytokines and positively regulated by FXR. By preserving canalicular transporters, the extract aids bile acid secretion and interrupts the cholestatic cycle. Concomitantly, BPPE suppressed the mitochondrial apoptotic cascade, lowering Bax, cytochrome c, and caspase-3 while upregulating Bcl-2, an effect attributable to its abundant anthocyanins. Therefore, BPPE acts through an integrated polypharmacology: chelating copper, scavenging free radicals, blocking NF-κB, inhibiting apoptosis, and reviving bile transporter expression.

Conclusion: Aqueous black-peel pomegranate peel extract at 100 mg/kg effectively protects against CuSO4-induced cholestatic liver injury by reducing oxidative stress, chelating copper, suppressing NF-kB-driven inflammation, blocking mitochondrial apoptosis, and partially restoring BSEP/MDR3 transporters. These findings identify this extract as a safe, cost-effective natural candidate for managing heavy-metal-associated cholestatic liver damage. Further studies on chronic models and clinical validation are warranted.