BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-inducible transcription factors that govern various essential metabolic activities in the liver and other organs. Recently, berberine (BBR) has been characterized as a modulator of PPARs; however, the matter of whether PPARs are involved in the inhibitory effect of BBR on hepatocellular carcinoma (HCC) is not well understood. PURPOSE: This study aimed to investigate the role of PPARs in the suppressive effect of BBR on HCC and to elucidate the relative mechanism. METHODS: We studied the role of PPARs in the anti-HCC effects of BBR both in vitro and in vivo. The mechanism whereby BBR regulated PPARs was studied using real-time PCR, immunoblotting, immunostaining, luciferase, and a chromatin immunoprecipitation coupled PCR assay. Additionally, we used adeno-associated virus (AAV)-mediated gene knockdown to address the effect of BBR more effectively. RESULTS: We demonstrated that PPARdelta played an active role in the anti-HCC effect of BBR, rather than PPARalpha or PPARgamma. Following a PPARdelta-dependent manner, BBR increased BAX, cleaved Caspase 3, and decreased BCL2 expression to trigger apoptotic death, thereby suppressing HCC development both in vitro and in vivo. It was noted that the interactions between PPARdelta and the apoptotic pathway resulted from the BBR-induced upregulation of the PPARdelta transcriptional function; that is, the BBR-induced activation of PPARdelta could mediate the binding with the promoters of apoptotic genes such as Caspase 3, BAX, and BCL2. Moreover, gut microbiota also contributed to the suppressive effect of BBR on HCC. We found that BBR treatment restored the dysregulated gut microbiota induced by the liver tumor burden, and a functional gut microbial metabolite, butyric acid (BA), acted as a messenger in the gut microbiota-liver axis. Unlike BBR, the effects of BA suppressing HCC and activating PPARdelta were not potent. However, BA was able to enhance the efficacy of BBR by reducing PPARdelta degradation through a mechanism to inhibit the proteasome ubiquitin system. Additionally, we found that the anti-HCC effect of BBR or a combination of BBR and BA was much weaker in mice with AAV-mediated PPARdelta knockdown than those in the control mice, suggesting the critical role of PPARdelta. CONCLUSION: In summary, this study is the first to report that a liver-gut microbiota-PPARdelta trilogy contributes to the anti-HCC effect of BBR. BBR not only directly activated PPARdelta to trigger apoptotic death but also promoted gut microbiota-derived BA production, which could reduce PPARdelta degradation to enhance the efficacy of BBR. CI - Copyright (c) 2023. Published by Elsevier GmbH.