All animal care and experimental protocols for in vivo studies conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH (NIH publication no. 85–23, revised 1996). The animal studies were reported in compliance with the ARRIVE guidelines (McGrath and Lilley, 2015; Lilley et al., 2020; Percie du Sert et al., 2020). We decided sample size for animal studies based on a survey of data from published research or preliminary studies, and no mice were excluded for statistical analysis. Animal studies were approved by the Ethics Committee of the Second Clinical Medical College of Jinan University, Shenzhen People’s Hospital. Male C57BL/6J wild type mice (8 ± 0.5 weeks old) were purchased from Gempharmatech Co. Ltd (Nanjing, Jiangsu, China). These mice were maintained in SPF units of the Animal Center of Shenzhen People’s Hospital (with a 12-h light cycle from 8 a.m. to 8 p.m., 23 ± 1°C, 60–70% humidity) and maintained on a standard rodent diet with free access to water in plastic bottles. Mice were allowed to acclimatize to their housing environment for at least 7 days before experiments. Up to five mice were kept per plastic cage with corn cob bedding material. We conducted treatment to mice in a blinded fashion. The drugs used for treating animals were prepared by researchers who did not carry out the treatments. In addition, all animals were randomized before they received treatment. At the end of experiment, all mice were anesthetized and euthanized in a CO2 chamber followed by collection of liver and blood samples. According to previous study, the dose of 20 or 40 mpk PTE (oral gavage) treatment effectively improve liver fibrosis induced by high fructose feeding in rats (Song et al., 2019). Hence, in present study, the dose of 25, 50 and 100 mpk PTE (oral gavage) was selected as the concentrations in vivo experiment.

Male C57BL/6J wild type mice (8 ± 0.5 weeks old) were divided into six group (n = 6). And then mice were pretreated with different dose of PTE (oral gavage) or vehicle for five consecutive days. Subsequently, mice were then treated with either vehicle (corn oil) or ANIT (100 mpk; oral gavage) on day 5. All the animals were sacrificed 48 h after ANIT gavage.

Male C57BL/6J wild type mice (8 ± 0.5 weeks old) were divided into six group (n = 6). C57BL/6J mice fed with a diet (control diet; Research Diets Inc, United States) containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 2 weeks. After 1 week DDC treatment, mice were treated with different dose of PTE (oral gavage) for another week.

Primary hepatocyte isolation was performed using a modified 2-step collagenase perfusion technique as previously reported (Xiang et al., 2021). Briefly, the liver specimen was cannulated under sterile conditions and flushed once with 50 ml washing buffer containing 2.5 mM EGTA (SIGMA-Aldrich). This was followed by perfusion with 30 ml digestion buffer containing 0.03% w/v collagenase (Roche Diagnostics) allowing recirculation of the perfusate. Removed the liver and put it in a dish of 10% FBS DMEM after infusion. The resulting cell suspension was poured through a 100 μM Cell Strainer and centrifuged with subsequent washing of the cell pellet using Serum-free medium (50 g, 2 min, 4°C). Cells were then re-suspended in William’s medium E (Biochrom AG) containing 10% fetal bovine serum. Cell number and viability were determined by the Trypan blue exclusion test. Hepatocytes were cultured using 6-well plates precoated with a single layer of rat tail collagen. Cells were seeded at a concentration of 2.5 × 10⁶ viable cells per well. Sixteen to 18 hours after plating, culture medium was changed to remove dead and non-adherent cells.

At the end of experiment, Trizol reagent (Invitrogen) was used to extract total RNA as described in classic protocol. Chloroform was well mixed with homogenate and centrifuged at 13,300 rpm for 15 min at 4°C. The top aqueous phase was collected and mixed with isopropanol and stored at -20°C. Next day, RNA was centrifuged, and washed with 75% ethanol and 100% ethanol in sequence. Then RNA was dissolved in an appropriate amount of RNase water. The cDNA was obtained by a reverse transcription kit purchased from New England Biolab (lpswich, MA, USA). qRT–PCR was performed using the ABI StepOnePlus™ Real-time PCR system (Applied Biosystem) with specific primers (Table 1). The relative mRNA level of target genes was analyzed using equation 2–ΔCt (ΔCt = Ct of the target gene–Ct of β-actin) and normalized using the level detected in the control group as 1. After treatment, a piece of tissue or cells were lysed or homogenized in the lysis buffer (sigma-Aldrich; St. Louis, MO, United States), and total protein obtained according the classical protocol. 1:1000 diluted fresh primary antibody or anti-β-actin antibody (1:5000) in PBS containing 1% fresh dry fat-free milk, and 1:5000 diluted fresh HRP-conjugated anti-rabbit or mouse IgG in PBS containing 1% fresh dry fat-free milk. Western blotting experiments were performed using antibodies for SIRT1 (abcam; Cat:# ab110304), p53 (abcam; Cat:# ab32389), FXR (abcam; Cat:# ab187735), IκBα (abcam; Cat:# ab76429), p19Arf (abcam; Cat:# ab26696), p-IκBα (Ser32) (abcam; Cat:# ab92700), PUMA (abcam; Cat:# ab9643) and β-actin (CST; Cat:# 3700), as described previously (Yang et al., 2020). The levels of level of AST, ALT, bilirubin, and AP etc., were measured using a commercial ELISA kit (Abcam) according to the manufacturer’s protocol. The activity of SIRT1 was measured using a commercial SIRT1 Activity Assay Kit (Fluorometric) (ab156065) according to the manufacturer’s protocol.

Immunoprecipitation of ubiquitinylated proteins was performed using the UbiQapture-Q Kit (Enzo Life Sciences), following the manufacturer’s instructions. Briefly, protein samples were harvested from BMDM and protein concentration was measured by BCA assay. Ubiquitinylated proteins were captured from 25 μg of total proteins lysates using 40 μl UbiQapture-Q matrix. Captured and uncaptured fraction samples were analyzed by Western blotting, as detailed above.

Treatment of mice with ANIT or feeding 0.1% DDC are commonly accepted methods of establishing sclerosing cholangitis which is similar to PSC (Fickert et al., 2007; Fickert et al., 2014); we thus assessed hepatoprotective effects of PTE using such ANIT or DDC-induced CLD models. Various dosages of PTE alleviated ANIT-induced liver injury, as evidenced by reduced levels of serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (AP), and γ-GT (Figure 1A). Moreover, PTE intervention reduced the size and number of ANIT-induced necrosis (assessed using HE staining) and apoptotic cell death (assessed using TUNEL staining) (Figures 1B–E). ANIT treatment increased caspase-3 activity in mouse livers, which was reduced after PTE treatment (Figure 1F). Compared with the 25 or 100 mpk PTE tratment, 50 mpk ameliorated ANIT-induced liver injury more effectively. In vitro analyses of primary hepatocytes isolated from WT mice confirmed that PTE treatment desensitized liver cells to bile-acid–induced apoptotic cell death (Figure 1G).

To further confirm the role of PTE in the experimental CLD, C57BL/6J mice fed with PTE in a diet containing 0.1% DDC for 2 weeks. PTE treatments at 25, 50, and 100 mpk alleviated DDC-induced cholestasis in C7BL/6J mice, as shown by decreasing cholestasis-serum markers (AST, ALT, AP, and γ-GT) (Figure 2A). Consistent with this, the pathological analysis of liver was also supported our finding, PTE treatment improved liver damage induced by DDC fed (Figure 2B). In addition, hepatic fibrosis (assessed using Sirius red stain), α-SMA positive cells, and mRNA levels of Col1α1, Col4α1, and CK-19, compared with DDC-fed controls (Figures 2C–H). The 50 mpk PET treatment ameliorated DDC-induced liver damage more effectively than the 25 or 100 mpk treatments. Taken together, PTE (50 mpk) treatment attenuated liver damage caused by ANIT or DDC.

The recruitment of monoctyes and macrophages is significantly increased in the liver of patients with cholestasis (Ma and Chen, 20191979). Macrophage recruitment to the liver was decreased in PTE-treated CLD model mice, compared with mice treated with DDC only (Figures 3A,B), and it was significantly lower in the 50 mpk than in the 25 mpk PTE treatment (Figures 3A,B). Hepatic macrophage recruitment did not differ significantly between the 50 and 100 mpk PTE treatments (Figures 3A,B). F4/80 immunofluorescence staining produced comparable results (Figures 3C,D). Furthermore, PTE treatment inhibited macrophage activation (M1 macrophage polarization) in the liver of DDC-induced CLD mouse models, as evidenced by fewer CD80-positive cells in the liver after PTE treatment (Figures 3C,D). Liver macrophage activation was significantly reduced in the 50 and 100 mpk treatments, compared to the 25 mpk treatment; however, no significant difference was observed between the 50 and 100 mpk treatments. Previous studies highlight an anti-inflammatory function of p53 in a variety of pathophysiological conditions (Zheng et al., 2005; Liu et al., 20091950). Evidence suggests that SIRT1 activation increased p19, which inactivates MDM2 E3 ligase and sustains p53 activity (Alarcon-Vargas and Ronai, 2002; Nakamura et al., 2017). Western blotting of SIRT1, p19Arf, p53, and ubiquitinylated-p53 (Ub-p53) of protein levels showed that SIRT1 expression was slightly but not significantly decreased in the liver of mice treated with DDC, compared with those of controls, and DDC treatment significantly inhibited p53 and p19Arf expression but increased Ub-p53 (Figures 3E,F). Compared with mice treated with DDC only, PTE treatment at different dosages had no significant influence on SIRT1 expression but increased p19Arf and p53 expression and decreased Ub-p53. DDC treatment induced expression of macrophage activation markers (IL-1β, TNF-α, Ccr2, and Nos2) and reduced macrophage alternative activation (Supplementary Figure S1A). PTE treatment significantly inhibited macrophage activation gene expression and slightly increased expression of macrophage alternative activation genes. Similar changes were observed in the ANIT-induced CLD models (Supplementary Figure S1B). Taken together, PTE inhibited hepatic inflammation in DDC and ANIT-induced experimental intrahepatic cholestasis.

Western blotting of FXR protein levels in the liver showed that protein levels of FXR were significantly decreased and acetylation of FXR slightly but not significantly increased in ANIT-treated mice, compared to the controls (Figures 4A,B). ANIT-induced CLD model mice treated with 25 mpk PTE showed only slightly upregulated FXR expression, whereas the 50 and 100 mpk treatments produced significantly increased FXR expression; however, FXR expression was lower in the 100 mpk PTE treatment than in the 50 mpk treatment. Liver BA pool sizes were increased in mice treated with ANIT only, compared to the controls, whereas a significant decrease was observed in PTE-treated mice (Figure 4C). Lower expression of SHP, Bsep, ATP binding cassette subfamily C member 2 (Abcc2), organic anion transporting polypeptide (Oatp), and sodium taurocholate cotransporting polypeptide (Ntcp) and higher cholesterol seven hydroxylase (Cyp7A1) in mice treated with ANIT only but not with PTE confirmed impaired FXR signaling (Figures 4D,E). PTE treatment attenuated ANIT-induced effects on BA synthesis genes and transporters; however, 100 mpk PTE adversely affected the FXR signaling pathway, compared with the 50 mpk treatment. Similar changes were observed in DDC-induced CLD models (Figures 4F–J). Overall, our results demonstrate that PTE treatment improved bile metabolic dysfunction induced by ANIT or DDC.

Structural modeling of the PTE-SIRT1 interaction was performed using BIOVIA Discovery Studio (Figure 5A), and key interface residues in SIRT1 are shown in the stick representation and are labeled according to residue names and positions. In vitro protein kinase assays demonstrated that PTE activated biochemical activity of SIRT1 in primary hepatocytes isolated from wild type mice (Figure 5B). PTE treatment reduced FXR acetylation and protein levels in a dose-dependent manner (Figures 5C,D). Furthermore, mRNA of Bsep (an FXR regulated gene) was significantly increased at 25 μm PTE compared to that at 10 and 100 μm PTE (Figure 5D). To test whether PTE regulated FXR acetylation and activity through SIRT1, primary hepatocytes were treated with PTE and/or siSIRT1 in presence of LPS (Figures 5E,F), showing that PTE did not affect FXR acetylation and protein levels in absence of SIRT1. Furthermore, when SIRT1 was deficient, PTE treatment did not affect expression of Cyp7a1, Cyp8b1, Ntcp, Oatp, Abcc4, Bsep, and Abcc2 (Figure 5G). Taken together, PTE regulated bile metabolic genes through SIRT1-FXR signaling.

Next, we investigated whether PTE regulated p53 ubiquitination in BMDM cultures. PTE inhibited LPS-induced p53 ubiquitination in BMDMs (Figure 6A), whereas no significant difference occurred between the 25 and 100 μm treatment groups. Western blotting of LPS-stimulated BMDM cultures supplemented with PTE or siRNA-SIRT1 showed that PTE treatment increased expression of p19Arf, p53 and p53 downstream effector protein PUMA, and reduced macrophage activation markers (i.e., phosphorylation of IκBα and mRNA levels of TNF-α, IL-1β, Nos2, Ccl2, and Ccr2); this pattern that was reversed after SIRT1 silencing (Figures 6B–D). In addition, PTE increased SIRT1 activation in BMDMs, and was significantly inhibited in the si-SIRT1 treatment (Figure 6E).

Next, UDCA was used as positive control, and the efficacy of PTE on cholestasis induced by DDC or ANIT was compared with UDCA. According to our results, both UDCA (50 mpk) or PTE (50 mpk) treatment alleviated DDC-induced liver injury, as seen by downregulation of serum AST, ALT, γ-GT, the accumulation of TBA, and hepatic fibrosis (assessed using Sirius red stain) (Figures 7A–E). Additionally, compared with the 50 mpk UDCA tratment, 50 mpk PTE could furhter ameliorate DDC-induced liver injury (Figures 7A–E). The similar results were observed in ANIT-induced experimental cholestasis (Figures 7F–H).