Our previous study shows that activation of pregnane X receptor (PXR) exerts hepatoprotection against lithocholic acid (LCA)-induced cholestatic liver injury. In this study we investigated whether PXR activation could inhibit hepatocyte pyroptosis, as well as the underlying mechanisms. Male mice were treated with mouse PXR agonist pregnenolone 16α-carbonitrile (PCN, 50 mg·kg-1·d-1, i.p.) for 7 days, and received LCA (125 mg/kg, i.p., bid) from D4, then sacrificed 12 h after the last LCA injection. We showed that LCA injection resulted in severe cholestatic liver injury characterized by significant increases in gallbladder size, hepatocellular necrosis, and neutrophil infiltration with a mortality rate of 68%; PCN treatment significantly inhibited hepatocyte pyroptosis during LCA-induced cholestatic liver injury, as evidenced by reduced serum lactic dehydrogenase (LDH) levels, TUNEL-positive cells and hepatocyte membrane damage. Furthermore, PXR activation suppressed both the NOD-like receptor protein 3 (NLRP3) inflammasome-induced canonical pyroptosis and the apoptosis protease activating factor-1 (APAF-1) pyroptosome-induced non-canonical pyroptosis. Inhibition of the nuclear factor kappa B (NF-κB) and forkhead box O1 (FOXO1) signaling pathways was also observed following PXR activation. Notably, dual luciferase reporter assay showed that PXR activation inhibited the transcriptional effects of NF-κB on NLRP3, as well as FOXO1 on APAF-1. Our results demonstrate that PXR activation protects against cholestatic liver injury by inhibiting the canonical pyroptosis through the NF-κB-NLRP3 axis and the non-canonical pyroptosis through the FOXO1-APAF-1 axis, providing new evidence for PXR as a prospective anti-cholestatic target.

Hepatocyte nuclear factor 4 alpha (HNF4α) and the pregnane X receptor (PXR) are involved in hepatocyte regeneration. It is not clear whether HNF4α is involved in hepatocyte regeneration through the regulation of PXR. This study aims to explore the regulatory relationship between HNF4a and PXR, and whether it affects hepatocyte regeneration. A mouse PXR gene reporter and an HNF4α overexpression plasmid were constructed and transfected into mouse hepatoma cells (Hepa1-6). Overexpression of HNF4α, detection of the PXR gene reporter fluorescence value, PXR gene, and protein expression analysis were conducted to explore the regulatory relationship between HNF4α and PXR. Apoptosis and cell cycle data were measured to verify whether HNF4α is involved in hepatocyte regeneration through PXR. The luciferase gene reporter assay results indicated when HNF4α was overexpressed, the fluorescence value of the PXR gene reporter was higher than that in the control at 24 h. With increasing HNF4α expression, the PXR gene and protein expression increased, indicating that HNF4α binds to the PXR promoter and upregulates PXR expression. Apoptosis and cell cycle analysis results demonstrated that when the expression of HNF4α increased, the expression of PXR increased, the apoptosis rate decreased, and the proliferation rate increased. Meanwhile, when the upward trend of PXR gene expression was inhibited by ketoconazole, the proliferation rate decreased. By inhibiting HNF4α and creating a partial hepatectomy (PHx), we demonstrated that HNF4α can upregulate PXR to promote liver regeneration in vivo. Therefore, HNF4α is shown to improve hepatocyte regeneration by upregulating PXR, which provides a reference for future research on the combined application of drugs for the treatment of liver injury.

Pregnane X receptor (PXR) has been reported to regulate glycolipid metabolism. The dysfunction of intestinal barrier contributes to metabolic disorders. However, the role of intestinal PXR in metabolic diseases remains largely unknown. Here, we show that activation of PXR by tributyl citrate (TBC), an intestinal-selective PXR agonist, improves high fat diet (HFD)-induced obesity. The metabolic benefit of intestinal PXR activation is associated with upregulation of β-1,3 galactosyltransferase 5 (B3galt5). Our results reveal that B3galt5 mainly expresses in the intestine and is a direct PXR transcriptional target. B3galt5 knockout exacerbates HFD-induced obesity, insulin resistance and inflammation. Mechanistically, B3galt5 is essential to maintain the integrity of intestinal mucus barrier. B3galt5 ablation impairs the O-glycosylation of mucin2, destabilizes the mucus layer, and increases intestinal permeability. Furthermore, B3galt5 deficiency abolishes the beneficial effect of intestinal PXR activation on metabolic disorders. Our results suggest the intestinal-selective PXR activation regulates B3galt5 expression and maintains metabolic homeostasis, making it a potential therapeutic strategy in obesity.

Activation of pregnane X receptor (PXR) by xenobiotics has been associated with metabolic diseases. This study aimed to reveal the impact of PXR activation on hepatic metabolome and explore novel mechanisms underlying PXR-mediated lipid metabolism disorder in the liver. Wild-type and PXR-deficient male C57BL/6 mice were used as in vivo models, and hepatic steatosis was induced by pregnenolone-16α-carbonitrile, a typical rodent PXR agonist. Metabolomic analysis of liver tissues showed that PXR activation led to significant changes in metabolites involved in multiple metabolic pathways previously reported, including lipid metabolism, energy homeostasis, and amino acid metabolism. Moreover, the level of hepatic all-trans retinoic acid (ATRA), the main active metabolite of vitamin A, was significantly increased by PXR activation, and genes involved in ATRA metabolism exhibited differential expression following PXR activation or deficiency. Consistent with previous research, the expression of downstream target genes of peroxisome proliferator-activated receptor α (PPARα) was decreased. Analysis of fatty acids by Gas Chromatography-Mass Spectrometer further revealed changes in polyunsaturated fatty acid metabolism upon PXR activation, suggesting inhibition of PPARα activity. Taken together, our findings reveal a novel metabolomic signature of hepatic steatosis induced by PXR activation in mice.

Human pregnane X receptor (PXR) is critical for regulating the expression of key drug-metabolizing enzymes such as CYP3A and CYP2C. Our recent study revealed that treatment with rodent-specific PXR agonist pregnenolone-16α-carbonitrile (PCN) significantly induced hepatomegaly and promoted liver regeneration after two-thirds partial hepatectomy (PHx) in mice. However, it remains unclear whether PXR activation induces hepatomegaly and liver regeneration and simultaneously promotes metabolic function of the liver. Here, we investigated the metabolism activity of CYP1A2, CYP3A1/2 and CYP2C6/11 during PXR activation-induced liver enlargement and regeneration in rats after cocktail dosing of CYP probe drugs. For PCN-induced hepatomegaly, a notable increase in the metabolic activity of CYP3A1/2 and CYP2C6/11, as evidenced by the plasma exposure of probe substrates and the AUC ratios of the characteristic metabolites to its corresponding probe substrates. The metabolic activity of CYP1A2, CYP3A1/2 and CYP2C6/11 decreased significantly after PHx. However, PCN treatment obviously enhanced the metabolic activity of CYP2C6/11 and CYP3A1/2 in PHx rats. Furthermore, the protein expression levels of CYP3A1/2 and CYP2C6/11 in liver were up-regulated. Taken together, this study demonstrates that PXR activation not only induces hepatomegaly and liver regeneration in rats, but also promotes the protein expression and metabolic activity of the PXR downstream metabolizing enzymes such as CYP3A1/2 and CYP2C6/11 in the body.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Pregnane X receptor (PXR), a xenobiotic-sensing nuclear receptor, plays a critical role in the metabolism of endogenous and exogenous substances in the liver. Here, we investigate whether PXR plays a role in pathogenesis of HCC. We show that liver tumors were developed in diethylnitrosamine (DEN)-treated in PXR knockout (KO) mice. Hepatic levels of prostaglandin F2α (PGF2α) and aldo-keto reductase family 1 member C18 (Akr1c18), a prostaglandin synthase of catalyzing reduction of PGH2 to PGF2α, were significantly elevated in DEN-treated PXR KO mice. Hepatic mRNA levels of alpha fetoprotein (AFP), cyclin D1 (Ccnd1), fibroblast growth factor 21 (FGF21), and inflammatory cytokine interleukin 6 (IL-6) were significantly increased in DEN-treated PXR KO mice. Other members of Akr1c family, liver metabolizing enzymes including Cyp1a2, Cyp2b10 and Cyp3a11, and bile acid synthesis enzyme Cyp7a1 mRNA levels were significantly decreased in DEN-treated PXR KO mice. Our findings revealed that PXR deficiency promoted DEN-induced HCC in mice via induction of Akr1c18 expression and PGF2α levels and the increased PGF2α levels synthetized by Akr1c18 enhanced hepatocytes proliferation and induced inflammatory cytokine production, which accelerated liver tumor development after DEN treatment, suggesting that PXR deficiency may create a microenvironment that is more prone to DEN-induced liver tumors and targeting PXR and Akr1c18 to reduce PGF2α biosynthesis may be a potential and novel therapeutic strategy for HCC.

The activation of pregnane X receptor (PXR) or peroxisome proliferator-activated receptor α (PPARα) can induce liver enlargement. Recently, we reported that PXR or PPARα activation-induced hepatomegaly depends on yes-associated protein (YAP) signaling and is characterized by hepatocyte hypertrophy around the central vein area and hepatocyte proliferation around the portal vein area. However, it remains unclear whether PXR or PPARα activation-induced hepatomegaly can be reversed after the withdrawal of their agonists. In this study, we investigated the regression of enlarged liver to normal size following the withdrawal of PCN or WY-14643 (typical agonists of mouse PXR or PPARα) in C57BL/6 mice. The immunohistochemistry analysis of CTNNB1 and KI67 showed a reversal of hepatocyte size and a decrease in hepatocyte proliferation after the withdrawal of agonists. In details, the expression of PXR or PPARα downstream proteins (CYP3A11, CYP2B10, ACOX1, and CYP4A) and the expression of proliferation-related proteins (CCNA1, CCND1, and PCNA) returned to the normal levels. Furthermore, YAP and its downstream proteins (CTGF, CYR61, and ANKRD1) also restored to the normal states, which was consistent with the change in liver size. These findings demonstrate the reversibility of PXR or PPARα activation-induced hepatomegaly and provide new data for the safety of PXR and PPARα as drug targets.

Ultrasound therapy is a method of applying ultrasonic energy to the stimulation produced by human body to change the function and tissue state of the body in order to achieve the purpose of treating diseases. Chronic venous ulcer is a common chronic skin ulcer. GSE222503 for ultrasound therapy of chronic venous ulcers was downloaded from gene expression omnibus database, which were used to identify differentially expressed genes. Weighted gene co-expression network analysis, functional enrichment analysis, gene set enrichment analysis, immune infiltration analysis and construction and analysis of protein-protein interaction network were performed. Draw gene expression heatmaps. Comparative toxicogenomics database analysis was performed. Two hundred thirty-five differentially expressed genes were obtained. According to gene ontology analysis, in biological process analysis, they were mainly enriched in positive regulation of cellular biosynthetic process, reproductive cell development, vasculogenesis, vascular morphogenesis, and inflammatory response. In cellular component analysis, they were mainly enriched in leading edge of growing cell, extracellular matrix binding organelle, F-actin capping protein complex. In molecular function analysis, they were mainly concentrated in receptor ligand activity, cytokine receptor binding. In Kyoto encyclopedia of genes and genomes analysis, they were mainly enriched in cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, HIF-1 signaling pathway, heme biosynthesis. In weighted gene co-expression network analysis, the soft threshold power was set to 9. Thirty modules were generated. PF4, NR1I2, TTC16, H3C12, KLRB1, CYP21A2 identified by 4 algorithms (MCC, EPC, closeness, stress). Heatmap of core gene expression showed that H3C12, KLRB1, PF4, NR1I2 were all underexpressed in samples of ultrasound-treated chronic venous ulcers and overexpressed in samples of untreated chronic venous ulcers. Comparative toxicogenomics database analysis showed that H3C12, KLRB1, PF4, NR1I2 are associated with thrombophlebitis, phlebitis, vascular malformations, metabolic syndrome, ulcers, and inflammation. In samples of chronic venous ulcer tissue treated with ultrasound, NR1I2 shows low expression, while in samples of chronic venous ulcer tissue without ultrasound treatment, it shows high expression. This finding suggests a potential role of NR1I2 in the process of ultrasound therapy for chronic venous ulcers, which may be related to the therapeutic effect of ultrasound therapy on chronic venous ulcers.

Pregnane X receptor (PXR) is essential in the regulation of liver homeostasis, and the gut microbiota is closely linked to liver physiologic and pathologic status. We previously found that activation of PXR significantly promotes liver enlargement through interaction with yes-associated protein (YAP). However, whether gut microbiota contributes to PXR-induced hepatomegaly and the involved mechanisms remain unclear. In this study, C57BL/6 mice were administered the mouse-specific agonist pregnenolone 16α-carbonitrile (PCN) for 5 days. Depletion of gut microbiota was achieved using broad-spectrum antibiotics (ABX) and fecal microbiota transplantation (FMT) was performed to restore the gut microbia. The composition of gut microbiota was analyzed by 16S rRNA sequencing, while the expression of PXR, YAP, and their downstream target genes and proteins were assessed. The results indicated that PCN treatment altered the composition and abundance of specific bacterial taxa. Furthermore, depletion of gut microbiota using ABX significantly attenuated PCN-induced hepatomegaly. FMT experiments further demonstrated that the fecal microbiota from PCN-treated mice could induce liver enlargement. Mechanistic studies revealed that ABX treatment impeded the PXR and YAP activation induced by PCN, as evidenced by decreased expression of PXR, YAP, and their downstream targets. Moreover, alterations in PXR and YAP activation were likely contributing to hepatomegaly in recipient mice following FMT from PCN-treated mice. Collectively, the current study demonstrated that gut microbiota is involved in PCN-induced hepatomegaly via regulating PXR and YAP activation, providing potential novel insights into the involvement of gut microbiota in PXR-mediated hepatomegaly. SIGNIFICANCE STATEMENT: This work describes that the composition of gut microbiota is altered in mouse pregnane X receptor (PXR) agonist pregnenolone 16α-carbonitrile (PCN)-induced hepatomegaly. Treatment with an antibiotic cocktail depletes the intestinal microbiota, leading to the impairment of liver enlargement caused by PCN. Additionally, fecal microbiota transplantation from PCN-treated mice induces liver enlargement. Further study revealed that gut microbiota is involved in hepatomegaly via regulating PXR and yes-associated protein activation.

Deoxynivalenol (DON) is a prevalent toxin causing severe liver damage through hepatocellular oxidative stress. However, the underlying mechanisms and effective therapeutic approaches remain unknown. Here, the unique role of the xenobiotic metabolism factor pregnane X receptor (PXR) in mediating DON-induced hepatocellular oxidative stress is investigated. Treatment with the PXR agonist 3-indole-propionic acid (IPA) alleviates DON-induced oxidative stress and liver injury both in vitro and in vivo. Mechanistically, it is discovered for the first time that PXR agonist IPA directly transactivates the m6A demethylase FTO expression, leading to site-specific demethylation and decreased abundance of YTHDC1-bound Malat1 lncRNA at single-nucleotide resolution. The diminished m6A modification of Malat1 lncRNA reduces its stability and augments antioxidant pathways governed by NRF2, consequently mitigating DON-induced liver injury. Furthermore, Malat1 knockout mice exhibit decreased DON-induced liver injury, emphasizing the role of Malat1 lncRNA in oxidative stress. Collectively, the findings establish that PXR-mediated m6A-dependent Malat1 lncRNA expression determines hepatocyte oxidative stress via m6A demethylase FTO, providing valuable insights into the potential mechanisms underlying DON-induced liver injury and offers potential therapeutic strategies for its treatment.