Conjugated bile acids alleviate acute pancreatitis through inhibition of TGR5 and NLRP3 mediated inflammation | Journal of Translational Medicine

Journal of Translational Medicine volume 22, Article number: 1124 (2024 ) Cite this article

Severe acute pancreatitis (SAP) is a crucial gastrointestinal disease characterized by systemic inflammatory responses and persistent multiple organ failure. The role of bile acids (BAs) in diverse inflammatory diseases is increasingly recognized as crucial, but the underlying role of BA conjugation remains elusive. mirabegron 223673 61 8

Our study aim to investigate the potential role of conjugated bile acids in SAP and reveal the molecular mechanisms underlying its regulatory effects. We hypothesized that taurochenodeoxycholic acid (TCDCA) and glycochenodeoxycholic acid (GCDCA) could protect SAP through inhibiting the activation of NLRP3 inflammasomes via the TGR5 pathway in macrophages.

To test our hypothesis, we used BA-CoA: amino acid N-acyltransferase knockout (Baat−/−) mice and established SAP mouse models using caerulein- and sodium taurocholate- induced. We utilized a range of methods, including pathology sections, qRT-PCR, immunofluorescence, Western blotting, and ELISA, to identify the mechanisms of regulation.

BA-CoA: Amino acid N-acyltransferase knockout (Baat−/−) mice significantly exacerbated pancreatitis by increasing pancreatic and systemic inflammatory responses and pancreatic damage in SAP mouse models. Moreover, the serum TCDCA levels in Baat−/− mice were lower than those in wild-type (WT) mice with or without SAP, and GCDCA and TCDCA showed stronger anti-inflammatory effects than chenodeoxycholic acid (CDCA) in vitro. TCDCA treatment alleviated SAP in a Takeda G protein-coupled receptor 5 and NOD-like receptor family, pyrin domain containing 3—dependent manner in vivo. Reinforcing our conclusions from the mouse study, clinical SAP patients exhibited decreased serum content of conjugated BAs, especially GCDCA, which was inversely correlated with the severity of systemic inflammatory responses.

Conjugated bile acids significantly inhibit NLRP3 inflammasome activation by activating TGR5 pathway, thereby alleviating pancreatic immunopathology. The results provide new insights into the variability of clinical outcomes and paves the way for developing more effective therapeutic interventions for AP.

Acute pancreatitis (AP) arises from excessive activation of pancreatic enzymes leading to pancreatic autodigestion. Well-established etiologies such as gallstone-induced pancreatic ductal obstruction—recognized as the most common cause—alongside alcohol consumption, endoscopic retrograde cholangiopancreatography (ERCP), and certain pharmacological agents, disrupt cellular signaling and organelle function, ultimately manifesting as acinar cell death, along with both localized and systemic inflammation [1,2,3]. In some patients, this inflammatory cascade escalates into severe systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS), conditions that often necessitate prolonged intensive care unit (ICU) care due to their life-threatening nature [4].

The global incidence of AP has reached 34 cases per 100,000 person-years, with a consistent upward trend across regions [5]. Although the disease imposes a significant worldwide burden, no effective treatments or preventative measures have been identified thus far. Mounting evidence indicates a dysregulation of bile acids (BAs) homeostasis in AP patients. BAs, a diverse group of cholesterol-derived amphipathic molecules, play dual roles: acting as detergents in lipid digestion and absorption, while also serving systemic endocrine functions [6,7,8].

Enterohepatic circulation recycles bile acids, with the majority being reabsorbed while a minor fraction is transformed into secondary BAs by gut microbiota [9]. Primary BAs, such as CA and CDCA, undergo intricate biochemical conjugations under the influence of bile acyl-CoA synthetase (BACS) and BA-CoA: amino acid N-acyltransferase (BAAT), resulting in compounds like tauro/glycocholic acid and tauro/glycochenodeoxycholic acid (T/GCA or T/GCDCA) [10, 11]. BACS activates bile acids by converting them into their CoA derivatives, while BAAT is responsible for conjugating activated BAs (CoA derivatives) with taurine or glycine as the final step in BA synthesis [12]. BAs are primarily conjugated with taurine in rodents, which can be contrasted with humans, where a mixture of both taurine and glycine conjugation occurs. While the role of BAs in pancreatitis has been extensively studied, the molecular mechanisms underlying BA-mediated effects remain incompletely defined [13, 14]. Xie et al. demonstrated that taurochenodeoxycholic acid (TCDCA) significantly attenuated the SFTSV-induced inflammatory response in mouse peripheral blood mononuclear cells, mitigated pathological organ damage, and reduced proinflammatory cytokine expression. They further identified that harmaline, a metabolite of A. muciniphila, enhanced BAAT expression, increased TCDCA levels, and modulated the host's inflammatory response, providing resistance against systemic viral infection [15]. Chenodeoxycholic acid (CDCA) shows a dynamic pattern, with levels decreasing during the acute phase and rising in the recovery phase, correlating with pancreatic necrosis [16].

Utilizing targeted metabolomic mass spectrometry analysis in both murine and human cohorts, this study sought to characterize disruptions in BA metabolism, with a focus on conjugated BAs in AP. G protein-coupled bile acid receptor 1 (GPBAR1, aka TGR5), a key mediator of immune signaling, plays a significant role in modulating immune responses. LCA suppresses NOD-like receptor family, pyrin domain containing 3 inflammasome (NLRP3) inflammasome activation through the TGR5-cAMP-PKA pathway, resulting in enhanced control of inflammation [17, 18]. This raises a pertinent question: Do functional distinctions exist between conjugated and unconjugated BAs, and what implications do they hold for AP pathogenesis? To address this, comprehensive in vivo and in vitro experiments were conducted using AP murine models and macrophage cells, focusing on the biological roles of the primary conjugated BAs, TCDCA and GCDCA.

The following antibodies were used for immunoblotting analysis: anti-Caspase-1 (ab179515; Abcam), anti-NLRP3 (ab263899; Abcam), anti-TGR5 (YT7569; Immunoway).

The following reagents were used for this study: caerulein (HY-A0190, MCE); alpha-amylase determination kit (BIOSINO, Beijing, China), LPS from Escherichia coli O55: B5 (Sigma, Germany), Nigericin (N1495, Invitrogen).Cholic acid (81–25-4), Chenodeoxycholic Acid (474–25-9), Taurodeoxycholate sodium salt (1180–95-6), Taurocholic acid (81–24-3), Taurochenodeoxycholic acid (516–35-8), Deoxycholic acid sodium salt (302–95-4), Lithocholic acid (434–13-9), Taurolithocholic acid sodium salt (6042–32-6), Ursodeoxycholic acid (128–13-2) and Glycoursodeoxycholic acid (64,480–66-6) are all from MCE.

THP-1 cells were purchased from ATCC American Type Culture Collection and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin solution (Gibco, cat.15140122). THP-1 cells were incubated with 100 ng·ml−1 phorbol-12-myristate 13-acetate (PMA; Sigma Aldrich) for 1 day to differentiate before utilization.

Murine BMDMs were prepared from 6-week-old C57BL/6 J mice. Briefly, marrow from both femurs and tibiae was collected in DMEM and further filtered through a 70 μm filter. The dispersed cells were cultured in DMEM supplemented with 10% FBS, 30% L929 supernatant and 1% penicillin–streptomycin solution. After 3 days of incubation at 37 °C with 5% CO2, the medium was replaced with fresh medium containing 10% FBS, 20% L929 supernatant and 1% penicillin–streptomycin solution. On the sixth day, the positive rate of macrophages was detected by flow cytometry. The resulting cells were shown as purified macrophages up to 95% based on extracellular double staining with anti-CD11b and anti-F4/80.

C57BL/6 J WT mice, homozygous Baat- deficient (referred to as Baat−/−) mice, and homozygous NLRP3- deficient (referred to as NLRP3−/−) mice, were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China) and GemPharmatech Co., Ltd. For the TGR5 conditional knockout (CKO) mouse protection experiment, the mice were intraperitoneally injected with 1 mg of tamoxifen per every 10 g of body weight, daily for one week. All the conducted research involved mice aged between 6 to 8 weeks with a weight bracket of 18 to 23 g. All specific-pathogen-free (SPF) mice were housed in contaminant-free settings with an average temperature of 22 °C, following a standard 12-h light/dark cycle (from 7:00 am to 7:00 pm) at the Animal Experimentation Center of Zhejiang University.

The mice were marked with ear tags, and randomized table was used. The mice were divided into groups using a completely randomized design. The animal groupings were blinded to the pathologists but not to the experiment operators.

A study was conducted with 10 Acute Pancreatitis (AP) patients, ranging in age from 25 to 91. These patients, who presented symptoms within 7 days of onset, were recruited from the Department of Intensive Care Unit at Sir Run Run Shaw Hospital affiliated with Medical College of Zhejiang University. All patients met the criteria for SAP according to the revised Atlanta classification. AP is frequently associated with complications, among which liver injury is observed in 40.1% to 56.6% of cases. The incidence of liver injury increases significantly in SAP, reaching up to 88.9%. Hepatic dysfunction typically persists throughout the course of the disease and correlates with the severity of pancreatitis [19]. Among the ten clinical samples collected for this study, four patients were administered Ursodeoxycholic Acid Capsules (Ursofalk®) after ruling out common bile duct obstruction, aimed at managing cholestatic liver disease. Following 1 to 2 cycles of UDCA therapy, bilirubin levels normalized, and the pancreatitis resolved following stabilization. The Healthy Control (HC) group was selected based on certain criteria: they must not have any chronic metabolic, cardiovascular, or gastrointestinal illnesses; they should not be pregnant; and they should not be on any medication that affects intestinal function. Both patients and healthy individuals who had taken antibiotics or probiotics four weeks prior to the collection of samples were not included in the study. Every participant provided a written informed consent, adhering to protocols approved by the ethical committees of Sir Run Run Shaw Hospital. Information about the participants' demographics and clinical statuses was gathered during their visits to the clinic.

In the MAP model setup, groups of mice were administered with 7 hourly intraperitoneal (i.p.) injections with a dosage of 50 μg/kg of caerulein, which was sourced from MCE. The model of SAP, stimulated by caerulein in combination with LPS, was developed by administering 10 hourly i.p. injections of caerulein at a dosage of 50 μg/kg together with a single i.p. injection of LPS at a dosage of 7.5 mg/kg.

A surgical SAP model was established by retrograde injection of 125 μg/kg sodium taurocholate (5%) (86,339, Sigma) into the pancreatic-bile duct. Briefly, a combination of nembutal and buprenorphine was used as the anesthetic/analgesic agents. The mouse was allowed to breath spontaneously and no intubation/ventilation was used.After the procedures, the mouse was returned to its cage placed on a heated pad (37 °C) until it recovered and survival kinetics were observed for 72 h as described previously [20]. Serum samples and tissue (pancreas, spleen and liver) are collected at 12 h or 72 h after SAP modeling.

Briefly, 3 mM TCDCA (516–35-8, MCE) was dissolved in drinking water, sterilized, freshly prepared, and changed every 3 days. Age- and sex-matched WT and knockout mice littermates originating from the same breeders received normal drinking or TCDCA supplementation for 4 weeks before AP induction as described previously [15].

100 μL serum samples were treated with a 500 μL solution mixture of acetonitrile and methanol in the ratio of 8:2. These samples were then centrifuged at a force of 12,000 g for a duration of 20 min. The resultant supernatant was then left to dry under the influence of a nitrogen blower. The residual precipitates were re-dissolved with 100 μL of a solution containing water and acetonitrile in an 8:2 ratio, also including 0.1% formic acid. A thorough vortexing and subsequent centrifugation were carried out for complete dissolution. Finally, the supernatant (2 μL) was injected into the LC–MS/MS system for analysis. A Triple Quadrupole LC/MS System (Agilent 6470) was used to quantitate bile acids. The concentration of the standard was used as the abscissa, and the ratio of the internal standard peak area was used as the ordinate to investigate the linearity of the standard solution.

Serum samples collected from animal experiments were extracted serum. Briefly, the samples were resuspended in prechilled 80% methanol by vortexing. The samples were then incubated on ice for 5 min and centrifuged at 15,000 g and 4 °C for 15 min. The supernatant was injected into the LC–MS/MS system for analysis. Ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS) analyses were performed using a Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher) by Novogene. The raw data files generated by UHPLC–MS/MS were processed using Compound Discoverer 3.1 (CD3.1, Thermo Fisher) to perform peak alignment, peak picking and quantitation for each metabolite.

The pancreas was treated with 4% paraformaldehyde and embedded in paraffin. 5-μm sections were cut and stained with hematoxylin and eosin (H&E) and semi-quantitatively scored using Schmidt’s criteria by two board-certified veterinary pathologists in a double-blinded manner. The final pathology score expressed is the average of these two values (Table S1).

For immunofluorescence assays (IFA), pancreatic tissue was collected from mice at 12 h post-caerulein injection, fixed in 4% paraformaldehyde, and embedded in paraffin. Dewaxed pancreatic sections were incubated with 3% BSA for 30 min to block nonspecific binding. The slides were then incubated with specific primary antibodies at 4 °C overnight. Antibodies used: anti-CD11b (2185–1-ap; Proteintech), and anti-F4/80 (cst70076; Cell Signaling Technology) monoclonal antibodies, and anti-Ly6G monoclonal antibody (65,140–1-ig; Proteintech). After incubation with the primary antibody overnight at 4 °C, the secondary antibody (Proteintech) was added and incubated at room temperature for 30 min. Stained sections were imaged with a Nikon Eclipse C1 vertical fluores-cence microscope. IFA was conducted as described previously [21].

To extract total RNA from tissues or cells, TRIzol reagent (provided by Invitrogen) was used as per the instructions provided by the manufacturer. Tissue (pancreas, liver, spleen), was collected from mice at 12 h post-caerulein injection or at 72 h surgical SAP model and preserved in liquid nitrogen. Cells were collected after bile acids treatment. The determination of gene expression levels was carried out through qRT-PCR and normalized to GAPDH expression. qRT–PCR was performed with the HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme, cat. Q221). The specific primers used for the assay are listed under Table S2.The final results are presented in relation to a fold change of gene expression in AP mice, comparing it to gene expression in the control or mock animals (2−ΔΔCT).

Tissues and cells that were treated as directed were lysed using RIPA lysis buffer from Beyotime. The lysates underwent a 12% SDS–Polyacrylamide Gel Electrophoresis procedure and were then moved to Polyvinylidene Difluoride membranes, which were manufactured by Millipore. The protein bands were probed using an enhanced chemiluminescence kit from Vazyme with a ChemiDoc Touch Gel Imaging System from Bio-Rad.

After being left undisturbed for 4 h, the blood was subjected to centrifugation at 3000 rpm at room temperature for a period of 15 min. The serum was then harvested from the supernatant. Serum amylase concentration was determined by the Alpha-amylase Determination Kit (BIOSINO, Beijing, China). Serum lipase levels were detected using the fully automatic biochemistry analyzer (IDEXX Catalyst One) in the laboratory department of the Zhejiang University Veterinary Teaching Hospital.

The protein levels of IL-1β, IL-6, and TNF-α in the serum samples were determined using the appropriate enzyme-linked immunosorbent assay (ELISA) kits, provided by MultiSciences.

The statistical analysis of the experiment was conducted using Prism GraphPad software version 9.0. Error bars represent standard errors of the means in all figures and. Student’s t-test between two groups or one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test among multiple groups. R2 was estimated using a nonlinear regression model for correlation analysis. The results are expressed as the mean ± SEM. Data were considered significant when p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***). All the experiments were replicated, and the number of replicates was listed in the figure legends. Representative images for western blots were from at least 3 independent sample preparations.

In Baat−/− mice, taurine conjugation dysfunction predominantly manifests as previously indicated [22]. To assess the pathological metabolic consequences, serum bile acid concentrations were first measured using targeted metabolomics in these mice (Table S3). As anticipated, taurine-conjugated BA levels were significantly reduced in Baat−/− mice compared to wild-type C57BL/6 J (WT) controls (Fig. 1a). Subsequently, the involvement of BAAT in acute pancreatitis (AP) was further evaluated using Baat−/− mice. Mild acute pancreatitis (MAP) and severe acute pancreatitis (SAP) were induced via caerulein and/or LPS administration in WT and Baat−/− mice (Fig. 1b). Histological analyses, considered the benchmark for pancreatic injury evaluation in AP models [23], demonstrated that Baat−/− mice experienced exacerbated pancreatic edema, necrosis, and inflammatory infiltration, with significantly elevated histopathological scores compared to WT counterparts in both MAP and SAP conditions (Fig. 1c, d). Furthermore, Baat−/− mice exhibited markedly increased serum amylase and lipase levels (Fig. 1e, f), alongside heightened serum concentrations of proinflammatory cytokines IL-1β, IL-6, and TNF-α (Fig. 1g). Corresponding upregulation of IL1b, IL6, and Tnfa expression was observed in the pancreas, spleen, and liver (Fig. 1h, S1a, S1b). Immunofluorescence assays (IFA) identified a significantly higher presence of macrophages (CD11b+/F4/80+), as well as IL-1β and IL-6 positive signals, in pancreatic tissues from Baat−/− mice within both caerulein-induced MAP and SAP models (Fig. 1i, Fig. S1c).

Acute pancreatitis was exacerbated in Baat−/− mice. a Heatmap showing different bile acids in serum of individual animals between Wildtype (WT) and Baat−/− mice (n = 6 individuals/group). b–i MAP and SAP models induced by caerulein injection, including PBS. Samples were at 12 h post MAP or SAP modeling (n = 6 individuals/group). b Schematic illustration of experiments in MAP and SAP models induced by caerulein injection. c Pancreatic pathological sections and d pathology scores from mice from b. Histopathological changes were scored by HE staining. n = 3 biologically independent samples. The pathology was semi-quantitatively scored using Schmidt’s criteria by two board-certified veterinary pathologists in a double-blinded manner. Blue arrows indicate acinar necrosis, pink arrows indicate inflammatory cell infiltration, and black arrows indicate edema. e Serum amylase activity and f serum lipase activity of SAP mice with species of WT and Baat−/− mice at 12 hpt. n = 3 biologically independent samples. g Serum expression (ELISA) of IL-1β (left), IL-6 (mid) and TNF-α (right) in WT and Baat−/− SAP mice. n = 3 biologically independent samples. h Relative mRNA expression of Il1b (left), Il6 (mid), and Tnfa (right) in the pancreas of WT or Baat−/− SAP mice. Fold change is relative to respective mock mice. n = 3 biologically independent samples. i Infiltration of macrophages and expression of proinflammatory cytokines IL-1β in the pancreas of WT or Baat−/− SAP mice shown via IFA. Results are representative of data generated in at least two independent experiments and are expressed as mean ± s.d. The two-sided P values were examined using Student’s t-test for comparison of variables between two groups. *: P < .05; **: P < .01; ***: P < .001; ****: P < .0001

In a more severe surgical AP model, induced via retrograde injection of 5% sodium taurocholate into the pancreatic-bile duct (Fig. 2a), Baat−/− mice exhibited reduced survival rates (Fig. 2b). Compared to wild-type counterparts, Baat−/− mice displayed elevated serum amylase and lipase activities, alongside more extensive pancreatic damage, as evidenced by higher pathological scores (Fig. 2c–f). Through qRT-PCR analysis, a significant increase in the expression levels of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α was identified in both the pancreatic (Fig. 2h) and splenic tissues (Fig. 2i) of Baat−/− mice. The protein levels of IL-1β, IL-6, and TNF-α in the serum samples were determined using the appropriate ELISA kits (Fig. 2g). IFA analysis demonstrated an increased macrophage presence in the pancreas of Baat−/− mice (Fig. 2j). Collectively, results across various AP models suggest that Baat−/− mice exhibit aggravated pancreatitis pathology.

Acute pancreatitis was exacerbated in Baat−/− mice. a Schematic illustration of experiments in SAP surgical models induced by 5% sodium taurocholate injection with species of WT and Baat−/− mice. b Survival kinetics in surgical SAP model. Survival was observed for 72 h following retrograde injection of 5% sodium taurocholate into the pancreatic-bile duct in WT or Baat−/− SAP mice (n = 25 individuals/group). c—(j) Samples were collected at 72 h post-retrograde injection in Surgical SAP model (n = 4—6). c Serum amylase (n = 6) and d serum lipase (n = 4) level of mice from a at 72 h post-retrograde injection with species of WT and Baat−/− mice. n = 3 biologically independent samples. e Pancreatic pathological sections and f pancreatic pathology scores from a. Histopathological changes were scored by HE staining. The pathology was semi-quantitatively scored using Schmidt’s criteria by two board-certified veterinary pathologists in a double-blinded manner. The final pathology score is expressed as the average of these two values. Blue arrows indicate acinar necrosis, pink arrows indicate inflammatory cell infiltration, and black arrows indicate edema. n = 3 biologically independent samples. g Serum expression (ELISA) of IL-1β (left), IL-6 (mid) and TNF-α (right) in WT or Baat−/− SAP mice. n = 3 biologically independent samples. Relative mRNA expression of Il1b (left), Il6 (mid), and Tnfa (right) in the h pancreas and i spleen of WT or Baat−/− SAP mice (n = 6). Fold change is relative to respective mock mice. n = 3 biologically independent samples. j Infiltration of macrophages (CD11b+/F4/80+) in the pancreas of WT or Baat−/− SAP mice shown via IFA. Results are representative of data generated in at least two independent experiments and are expressed as mean ± s.d. The two-sided P values were examined using Student’s t-test for comparison of variables between two groups. *: P < .05; **: P < .01; ***: P < .001; ****: P < .0001

Serum-targeted bile acid metabolomics were employed to distinguish the pathological variances between Baat−/− and WT mice in AP (Table S4). A total of 22 common BA components were quantified in both control and model groups, and these data were analyzed using SIMCA-P + software for multivariate assessments. The OPLS-DA scatter plots (Fig. 3a) illustrated a clear separation between Baat−/− and WT mice with SAP, with distinct clustering of data points according to the OPLS-DA model. To assess the risk of overfitting in this supervised approach, the PLS-DA model underwent validation, involving 200 permutation tests. When the final Q2 ordinate remained below 0.05, it confirmed the model's robustness and absence of overfitting. Our results from the 200 permutation tests indicate that the PLS-DA model is reliable and free from overfitting (Fig. 3b).

The decrease of TCDCA may be a key factor in aggravating acute pancreatitis. Serum was collected in caerulein or PBS injection mice at 12 h post caerulein-induced SAP modeling for bile acids quasi-targeted metabolomics (n = 6). a OPLS-DA scatter plots of serum bile acids in WT or Baat−/− mice of control and b SAP model and the Permutation analysis of it. c Heatmap showing different bile acids in serum of individual animals between WT and Baat−/− mice of control and caerulein-induced SAP model. Univariate analysis of major serum bile acids in WT d and e mice of control and SAP model. Volcano map of comparing the control group with the model group of WT f and Baat−/− g mice. Each dot indicates an individual metabolite. Data are presented as mean ± s.d. The two-sided P values were examined using Student’s t-test for comparison of variables between two groups. One-way ANOVA followed by Tukey’s multiple-comparison test was used for comparison of continuous variables among multiple groups. *: P < .05; **: P < .01; ***: P < .001; ****: P < .0001

In Baat−/− and WT mice with AP, the serum concentrations of taurine-conjugated BAs, such as TCDCA and TUDCA, were markedly lower compared to those in their counterparts without AP (Fig. 3c). This observation indicates that AP triggers a decline in taurine-conjugated BAs. Specifically, TCDCA levels in WT mice with AP were found to be approximately 15-fold lower than in WT mice without AP, with Baat−/− mice displaying even lower levels of TCDCA regardless of the presence of AP (Fig. 3d, e). A volcano plot comparison between control and AP model groups in WT mice highlighted a pronounced reduction in TCDCA linked to pancreatitis, a shift absent in Baat−/− mice due to their inherently low basal levels of conjugated BAs (Fig. 3f, g). This reduction in taurine-conjugated BAs, particularly TCDCA, may play a significant role in the progression of AP.

Macrophages are integral to modulating both local and systemic inflammatory responses in AP, with NLRP3 inflammasome acting as a central mediator of macrophage-induced inflammation. Primary BAs (cholic acid, CA; chenodeoxycholic acid, CDCA), secondary BAs (deoxycholic acid, DCA; lithocholic acid, LCA), and their tauro-conjugated counterparts were evaluated for their impact on NLRP3 inflammasome activation in in lipopolysaccharide (LPS)- and nigericin-stimulated bone-marrow-derived macrophages (BMDMs). All BAs tested markedly reduced nigericin-induced IL1b, IL6, and Tnfa production in BMDMs (Fig. 4a). Among them, LCA and tauro-conjugated lithocholic acid (TLCA) demonstrated the strongest inhibition of NLRP3 inflammasome activation, consistent with previous findings [18]. Additionally, TCDCA displayed a more pronounced anti-inflammatory effect compared to CDCA in BMDMs (Fig. 4a).

Taurine or glycine-conjugated forms of CDCA show stronger anti-inflammatory effects. a LPS-primed BMDMs stimulated with nigericin (2 μM) for 3 h were treated with indicated BAs at 50 μM for 24 h. Relative mRNA expression of Il1b (left), Il6 (mid), and Tnfa (right) in BMDMs treated with 50 μM BAs (n = 6). b LPS-primed BMDMs stimulated with nigericin (2 μM) for 3 h were treated with CDCA or TCDCA at 5–100 μM for 24 h. Relative mRNA expression of Il1b (left) and Il6 (right)in BMDMs treated with BAs of gradient concentration from 5 to 100 μM (n = 6). c LPS-primed Peritoneal macrophages stimulated with nigericin (2 μM) for 3 h were treated with CDCA or TCDCA at 5–100 μM for 24 h. Relative mRNA expression of Il1b (left) and Il6 (right) in peritoneal macrophages treated with BAs of gradient concentration from 5 to 100 μM (n = 6). Data are presented as mean ± s.d. The two-sided P values were examined using Student’s t-test for comparison of variables between two groups. One-way ANOVA followed by Tukey’s multiple-comparison test was used for comparison of continuous variables among multiple groups. *: P < .05; **: P < .01; ***: P < .001; ****: P < .0001

qRT-PCR analysis of IL1b and IL6 gene expression in BMDMs demonstrated a dose-dependent suppression of inflammatory cytokines by TCDCA and CDCA. Notably, the concentrations required to inhibit inflammasome activation aligned with physiological BA levels in the portal vein (10–80 mM) and peripheral circulation (2–10 mM) of humans and mice. At lower concentrations (5 μM), CDCA lost its efficacy in reducing IL1b expression in macrophages, whereas TCDCA maintained its anti-inflammatory effect (Fig. 4b). This observation indicates that TCDCA exerts a more potent anti-inflammatory action at lower in vivo concentrations compared to CDCA. Consistent results were observed in murine peritoneal macrophages (Fig. 4c), further affirming TCDCA's superior anti-inflammatory properties. In humans, CDCA predominantly conjugates with glycine (forming GCDCA) and less frequently with taurine (forming TCDCA). Consequently, glycine-conjugated chenodeoxycholic acid (GCDCA) was selected for similar treatment in THP-1 cells, revealing reduced IL1b transcription levels in response to GCDCA relative to CDCA (Fig. S2a). These data suggest that both glycine- and taurine-conjugated forms of CDCA exhibit more pronounced anti-inflammatory effects compared to the unconjugated form.

To investigate the protective effects of conjugated BAs in AP, Baat−/− mice were treated with TCDCA via drinking water for four weeks to assess its impact on inflammatory responses in the SAP model (Fig. 5a). This treatment markedly elevated TCDCA serum levels (Fig. S3a), reaching near-physiological concentrations after four weeks. A notable reduction in serum amylase and lipase levels was observed following TCDCA supplementation (Fig. 5b and c). Additionally, TCDCA administration significantly diminished proinflammatory cell infiltration and mitigated pancreatic damage during caerulein-induced SAP (Fig. 5d and e). Serum levels of IL-1β, IL-6, and TNF-α were substantially lowered in TCDCA-treated mice (Fig. 5f). Consistently, IL1b, IL6, and Tnfa expression in the pancreas (Fig. 5g, Fig. S3b), as well as in spleen and liver tissues (Figs. S3c, d), was also significantly reduced, highlighting TCDCA's potential in ameliorating pancreatitis.

TCDCA treatment alleviated severe acute pancreatitis in mice. a-g 3 mM TCDCA was added to the drinking water for 4 weeks before caerulein injection SAP models. Samples were at 12 h post caerulein-induced SAP modeling (n = 6). a Schematic illustration of experiments in complement of TCDCA and SAP models induced by caerulein injection. b Serum amylase activity and c serum lipase activity of SAP mice. n = 3 biologically independent samples. d Pathological sections and e pancreatic pathology scores from mice from a. Histopathological changes were scored by HE staining. Blue arrows indicate acinar necrosis, pink arrows indicate inflammatory cell infiltration, and black arrows indicate edema. n = 3 biologically independent samples. f Serum expression (ELISA) of IL-1β (left), IL-6 (mid) and TNF-α (right) in WT or Baat−/− SAP mice. n = 3 biologically independent samples. g Relative mRNA expression of Il1b (left) and Il6 (right) in the pancreas in WT or Baat−/− SAP mice. Fold change is relative to respective mock mice. n = 3 biologically independent samples. Data are presented as mean ± s.d. The two-sided P values were examined using Student’s t-test for comparison of variables between two groups. One-way ANOVA followed by Tukey’s multiple-comparison test was used for comparison of continuous variables among multiple groups. *: P < .05; **: P < .01; ***: P < .001; ****: P < .0001

Previous research indicates that TCDCA mitigates pancreatitis. Notably, in patients with AP, substantial activation of the NLRP3 inflammasome has been observed, a key mediator of systemic immune responses during SAP. Li demonstrated that the BA receptor agonist INT-777 exerted protective effects against inflammatory diseases by inhibiting the ROS/NLRP3 pathway [24]. Additionally, BAs activate distinct receptors across various tissues and cell types.

TCDCA is proposed to inhibit NLRP3 inflammasome activation via stimulation of TGR5 signaling. Western blot analysis was employed to assess key proteins within the NLRP3 inflammasome signaling pathway in both Baat knockout and WT mice, with or without TCDCA treatment in the AP model, followed by grayscale quantification. Elevated levels of NLRP3, pro-Caspase1, and mature Caspase1 (P10) were observed in AP mice compared to controls, while TCDCA administration led to a reduction in these proteins and a concurrent increase in TGR5 expression (Fig. 6a and S4a-S4d). These findings indicate that TCDCA mitigates AP through TGR5 receptor activation.

TCDCA alleviates pancreatitis via TGR5 and NLRP3 signaling. a-g 3 mM TCDCA was added to the drinking water for 4 weeks before caerulein injection SAP models. Samples were at 12 h post caerulein-induced SAP modeling (n = 6). a Representative western blot showing expression of TGR5 and NLRP3 signaling pathway-related molecules (NLRP3, pro-CASP1 and CASP1 p10) in the pancreas. Densitometric analysis of band intensity was performed using ImageJ software. The grayscale intensity of the bands was normalized and the results are expressed as a fold change relative to the control group. b Pancreatic pathological sections and c pathology scores from different species (WT, TGR5−/− and NLRP3−/−). Histopathological changes were scored by HE staining. Blue arrows indicate acinar necrosis, pink arrows indicate inflammatory cell infiltration, and black arrows indicate edema. n = 3 biologically independent samples. d Serum amylase activity and e serum lipase activity of different species mice in SAP model. n = 3 biologically independent samples. f Serum expression (ELISA) of IL-1β (left), IL-6 (mid) and TNF-α (right) in WT, TGR5−/− and NLRP3−/− SAP mice. n = 3 biologically independent samples. g Relative mRNA expression of Il1b (left) and Il6 (right) in the pancreas in WT, TGR5−/− and NLRP3−/− SAP mice. Fold change is relative to respective mock mice. n = 3 biologically independent samples. Data are presented as mean ± s.d. The two-sided P values were examined using Student’s t-test for comparison of variables between two groups. One-way ANOVA followed by Tukey’s multiple-comparison test was used for comparison of continuous variables among multiple groups. *: P < .05; **: P < .01; ***: P < .001; ****: P < .0001. Representative images of three independent replicates were shown in immunoblotting analysis

The roles of TGR5 and NLRP3 in pancreatitis progression were examined using TGR5 and NLRP3 knockout mice. In the cerulein-induced pancreatitis model, TGR5−/− mice exhibited more pronounced pancreatic injury compared to WT controls, as indicated by histopathological analysis (Fig. 6b, c). Serum amylase and lipase levels were marginally elevated (Fig. 6d, e), while IL-1β, IL-6, and TNF-α serum protein levels remained unchanged (Fig. 6f). Increased expression of IL1b, IL6, and Tnfa in pancreatic tissues further suggested that TGR5 deficiency intensified the inflammatory response in pancreatitis. In contrast, NLRP3−/− mice exhibited significant alleviation of pancreatitis symptoms. Notably, TCDCA supplementation failed to mitigate the damage associated with TGR5 deficiency or improve the pathological condition in NLRP3−/− mice (Fig. 6b–g). Moreover, TCDCA supplementation in TGR5−/− mice did not substantially reduce NLRP3 expression (Fig. S4e, S4f). These results suggest that TCDCA alleviates pancreatitis through the activation of TGR5 signaling, which suppresses NLRP3 inflammasome activation.

To understand the clinical relevance of our findings, we analyzed the serum bile acids in ten hospitalized SAP patients and six healthy controls (HC) through targeted bile acids metabolism (Table S5). OPLS-DA scatter plots (Fig. 7a) showed a clear separation between knockout and WT mice in the SAP groups. Significant differences in BA concentrations were found between HC and AP patients. Metabolomics results indicated no significant change in TCDCA levels in pancreatitis patients; however, glycine-conjugated CDCA and UDCA levels were significantly reduced (Fig. 7b and c). Compared to HC, AP patients showed markedly higher serum IL-1β and IL-6 levels (Fig. 7d). Furthermore, serum IL-1β (Fig. 7e) and IL-6 (Fig. 7f) levels inversely correlated with GCDCA abundance in AP patients (For IL-1β: R2 = 0.7007, p = 0.0025 and for IL-6: R2 = 0.436, p = 0.037). These data support a role for conjugated BAs TCDCA or GCDCA in regulating systemic inflammatory responses during AP.

Patients with AP exhibit a decreased serum abundance of GCDCA that was inversely correlated with the severity of their systemic inflammatory responses. a OPLS-DA scatter plots of serum bile acids HC (n = 6) and AP patients (n = 10). b Permutation analysis of a serum bile acid OPLS-DA plot. c Heatmap showing different bile acids in serum of individual animals between HC and AP patients (n = 10). d Differential serum expression of IL-1β (left) and IL-6 (right) between HC (n = 6) and AP patients (n = 10), as determined by ELISA. Correlation between serum GCDCA abundance and serum concentration of IL-1β f or IL-6 e in AP patients (n = 10). R2 and exact two-sided P values calculated by Pearson’s test are shown d–f. Data are presented as mean ± s.d. The two-sided P values were examined using Student’s t-test for comparison of variables between two groups. One-way ANOVA followed by Tukey’s multiple-comparison test was used for comparison of continuous variables among multiple groups

This study elucidates the regulatory role of conjugated bile acids, such as TCDCA and GCDCA, in inhibiting macrophage function in AP and potentially other immune diseases. Our initial findings in BAAT knockout mice, which exhibit bile acid metabolism disorders, reveal that both cerulein-induced and retrograde infusion models of the bile duct exacerbate pancreatitis. Serum bile acids metabolites altered in SAP, and we found TCDCA maybe a key factor. Conjugated bile acids TCDCA or GCDCA inhibit NLRP3 inflammasome activation by activating TGR5 pathway, thereby alleviating pancreatic immunopathology (Fig. S5).

The primary pathogenesis of AP involves pathological calcium signal transduction [2, 25], mitochondrial dysfunction [26, 27], premature activation of trypsinogen in acinar cells and macrophages, endoplasmic reticulum stress [28], impaired unfolded protein response [29, 30], and impaired autophagy [31]. Additionally, increased pressure and exposure to bile acids in the pancreatic duct can indirectly contribute to these events. The interaction between acinar cells and the immune system is pivotal in pancreatitis, with the severity largely dependent on the immune response’s intensity [32,33,34]. Until now, the cerulein-induced pancreatitis model in rodents is commonly used to investigate early cellular events during acute pancreatitis [35]. Models of pancreatitis induced by cerulein combined with lipopolysaccharide are popular owing to their low cost and high reproducibility. The pancreatic duct ligation mainly mimics the pathogenesis of biliary acute pancreatitis, which accounts for more than 50% of acute pancreatitis [36]. Additionally, intraperitoneal injection of L-arginine is a model for establishing severe pancreatitis and is commonly used in acute pancreatitis research, but has a high mortality rate [37]. Alcohol and lipopolysaccharide can be administered to rodents to simulate a model relevant to alcohol-related pancreatitis, which is primarily used to study chronic pancreatitis [38].

Bile acids act as signaling molecules, regulating metabolism and maintaining host homeostasis by activating certain cell receptors. Bile acids function as natural ligands for bile acid receptors, which are primarily categorized into TGR5 and Farnesoid X receptor (FXR). CDCA is the most potent ligand of FXR while UDCA and MCA do not activate FXR [39]. In Chinese hamster ovary (CHO) cells transfected with human TGR5, LCA shows the best potency for activation, with taurine conjugates being more potent than glycine conjugates. TGR5 is identified as the receptor responsible for bile acid-mediated suppression of macrophage activation [40].Upon reviewing prior research, it has been observed that in the caerulein-induced AP model using FXR-deficient mice, the absence of FXR function does not influence the severity of AP [41]. Conversely, following retrograde TLCS injection in TGR5−/− mice, the severity of AP was alleviated, yet the use of the CCK analog cerulein did not affect its severity [42]. In contrast, the present findings indicate that in TGR5−/− mice, caerulein-induced AP is exacerbated, a result that diverges from previous studies, possibly due to differing model-building methodologies. Nevertheless, aligning with our findings, Perides et al. also proposed that TGR5 primarily regulates bile acid-induced pancreatitis [43]. Furthermore, research by Zhou et al. demonstrated that the secondary bile acids UDCA and LCA enhance barrier integrity and reduce inflammation in mouse colitis through TGR5 activation [44]. Endoplasmic reticulum (ER) stress is activated in SAP, leading to the phosphorylation of JNK mediated by nositol-requiring enzyme 1(IRE1), which in turn activates downstream transcription factors such as c-Jun and c-Fos. Meanwhile, by inhibiting the nuclear transport of p38 MAPK and ERK1/2, it plays a protective role and alleviates pancreatic inflammation [30, 45]. Compared to UDCA, TUDCA is more effective in protecting against ER stress, mitigating pro-inflammatory pathways, and reducing cellular damage in conditions like AP. Additionally, in a rat adjuvant arthritis model, TCDCA was found to activate the glucocorticoid receptor in a concentration-dependent manner, reversing the elevated expression of Il1b-induced c-Fos and phosphorylated c-Jun, and exhibiting a similar effect on the transactivation of activator protein-1 (AP-1) [46]. Although Zhu et al. found that CDCA and its receptor, FXR, may be a novel target for acinar cell necrosis treatment [16], and studies have shown TCDCA’s anti-inflammatory effects on immune cells across various mammals, including alveolar macrophages in rats and mice [39, 47]. Activated monocytes are central to systemic inflammation and worsening tissue injury in AP [20]. Our findings indicate significant changes in bile acid metabolism during AP in both mice and humans (Figs. 3 and 7). TCDCA influences various processes, including anti-inflammatory activity, immune activity, apoptosis, and lipid metabolism [48]. These effects are likely due to the activation of specific cell surface receptors. The pathological increase in Ca2⁺ concentration in acinar cells is central to AP, regulating pro-apoptotic and pro-inflammatory pathways such as premature trypsinogen activation, NF-κB activation, and mitochondrial dysfunction. Ryanodine receptors (RyRs), typically found in skeletal muscle, are also present in acinar cells. High TCA concentrations enhance RyR sensitivity to 3H-ryanodine binding, triggering intracellular Ca2⁺ signaling and exacerbating cell damage [49]. Taurine-conjugated bile acids like TLC induce significant Ca2⁺ increases, while TCA and TDCA have weaker effects [43, 50, 51]. In our in vitro cell experiments, TCDCA and TUDCA demonstrated comparable anti-inflammatory effects, both of which were more potent than their respective monomers (Fig. 4a). TUDCA shows protective effects in AP by mitigating enzyme activity and alleviating ER stress in both cell and animal studies [52, 53]. This compound has demonstrated significant potential for cell protection in exocrine glands.

Currently, we do not have direct evidence indicating the intricate mechanisms how TCDCA activates TGR5. Activation of the TGR5 receptor initiates several critical signal transduction pathways, including NF-κB, Protein Kinase B (AKT), Extracellular Regulated Kinases (ERK), and Signal Transducer and Activator of Transcription 3 (STAT3). However, a popular explanation is that TCDCA has an anti-inflammatory role via cAMP-PKA signaling pathway induced by the TGR5 receptor [54,55,56]. This may be an interpretation, but further studies are required to elucidate the molecular mechanisms involved in this activation. We verified that activation of TGR5 inhibits NLRP3 expression (Fig. 6a). This result aligns well with previous studies that demonstrated bile acids inhibited NLRP3 inflammasome activation via the TGR5-cAMP-PKA axis. Activation of protein kinase A by the bile acid receptor TGR5 leads to the ubiquitination and phosphorylation of NLRP3, thus suppressing NLRP3 inflammasome activation[18]. However, it must be pointed out that they used LCA, the strongest receptor for TGR5. Moreover, in vivo experiments demonstrate that bile acids activate TGR5 to block NLRP3-dependent inflammation, including lipopolysaccharide-induced systemic inflammation, inflammation associated with type 2 diabetes, and alum-induced peritonitis [18, 57]. Our study utilizing a panel of knockout mice deficient in key sensors and mediators, demonstrate the inhibitory effect of TCDCA on NLRP3 by activating the TGR5 receptor. Further supporting this, Qi et al. revealed the essential role of TCDCA in suppressing NLRP3 inflammasome activation via the macrophage TGR5-cAMP-PKA pathway [54]. Highlighting its significance in acute neuroinflammatory conditions in mice, TUDCA engages the TGR5 receptor to reduce microglial IFN-γ, TNF-α, and other pro-inflammatory agents, thereby orchestrating a controlled anti-inflammatory response [58]. Moreover, in CD14+ macrophages, both LCA and DCA have been observed to activate c-Fos through TGR5, suppressing the secretion of TNF-α and IL-6 [59]. These observations illustrate the remarkably intricate nature of the inhibitory effects of bile acids on NLRP3 inflammasome activation, underscoring the complex bio-regulatory mechanisms involved.

AP is a complex inflammatory disease with no specific approved therapy, and current treatment relies on supportive care. Key aspects of early management include fluid resuscitation, nutritional support, etiology identification, and analgesia. Recent mechanistic studies have identified potential therapeutic targets for SAP. Agents like CM4620 and GSK-7975A target calcium signaling, while Tocilizumab inhibits the IL-6 receptor. Lactated Ringer’s solution, preferred over other crystalloids, targets the NLRP3 inflammasome pathway [60]. NLRP3 inflammasome activation is a rapidly advancing area in immunology, with discoveries such as NEK7-NLRP3 interaction, GSDMD’s role in pyroptosis, and the involvement of ionic flux and mitochondrial dysfunction [61]. Several NLRP3 inhibitors, including IL-1 antagonists like Anakinra, have shown promise in reducing pancreatic inflammation in animal models, though not yet approved for AP treatment. Natural compounds, such as Emodin, have also demonstrated efficacy in blocking NLRP3 activation by inhibiting the P2X7/NLRP3 pathway, mitigating systemic inflammation. Further, PKA-induced phosphorylation and ubiquitination of NLRP3 suggest TGR5 as a potential therapeutic target [20].

Finally, the clinical data aligned with the findings from the mouse models. The level of CDCA decreases during the acute phase of pancreatitis, increases during the recovery phase, suggesting that targeting CDCA is a potential strategy for the treatment of acinar cell necrosis in AP [16]. Similarly, the abundance of serum GCDCA, a form of conjugated bile acid, has been found to be inversely proportional to the severity of AP. Additionally, there is a negative correlation between the increase in serum IL-1β and IL-6 levels and the physiological abundance of GCDCA. Targeting CDCA has been proposed as a novel therapeutic approach for SAP [16], and we hypothesize that targeting conjugated bile acids TCDCA and GCDCA may offer an additional strategy. Currently, the only bile acid drug used clinically for pancreatitis is UDCA, which has shown potential due to its anti-inflammatory, cytoprotective, and antioxidant effects [62]. While TCDCA or GCDCA at high doses, may have adverse effects on the liver and other organs, such as inducing apoptosis through mitochondrial pathways [63, 64]. However, the precise inhibitory mechanisms of bile acids are not yet fully understood, posing potential risks of off-target effects. Further research is required to clarify these mechanisms and optimize their therapeutic application. Cancer, as the second leading cause of mortality globally, presents significant challenges in drug development, with success rates falling below 10% due to its inherent complexity [65]. A promising strategy to overcome this barrier is drug repurposing, utilizing systems biology, multi-omics, and pathway analysis. For example, such as virus-related cancers, cervical cancers [66] and endometrial cancers [67], or for non-cancer diseases, like Tubulointerstitial Fibrosis [68], psoriasis [69] and Idiopathic Pulmonary Fibrosis [70], Artificial Intelligence assisted methodologies [71], including deep learning [72], have enabled network-based drug repurposing, identifying small molecules drugs. This paradigm also offers a novel avenue for therapeutic intervention in AP, highlighting the potential of leveraging existing foundational research to facilitate the development of new treatments or the repurposing of existing drugs.

One limitation of the clinical study is the small sample size of only ten clinical cases. This limited number restricts the generalizability of the findings and reduces the statistical power of the analysis and the ability to capture the full spectrum of variability within the condition, which is essential for a more comprehensive understanding of the disease process. In addition, we did not collect continuous blood samples from the same patient during the early days of onset, resulting in a lack of dynamic presentation of bile acid metabolism changes. Further research with a larger cohort is needed to validate and expand upon these initial observations. Animal-based experimental models (available mice strains with genetic deletions) have improved our understanding of the pathogenesis of acute pancreatitis. But due to obvious differences between human and rodent pancreatitis, animal study findings should be cautiously applied to humans [73]. However, ex vivo human studies have shown many mechanisms are still relevant [74].

Our comprehensive functional study reveals that conjugated bile acids significantly inhibit NLRP3 inflammasome activation by activating TGR5 pathway, thereby alleviating pancreatic immunopathology. The finding that TCDCA or GCDCA can ameliorate SAP provides new insights into the variability of clinical outcomes and paves the way for developing more effective therapeutic interventions for SAP and potentially other inflammatory disorders.

Nlr i strive for domain-comicing protein 3

Severe systemic inflammatory response syndrome

G protein-coupled bile acid receptor 1

Signal Transducer and Activator of Transcription 3

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We thank W. Han (Department of Medical Oncology, Sir Run Run Shaw Hospital, College of Medicine) for providing a valuable TGR5 knockout mouse strain.

This current work was supported by grants from the Natural Science Foundation of Zhejiang Province (No. LZ24H030002), Science and Technology Program of Zhejiang Province (2024C03201), Co-construction science and technology program of Zhejiang Traditional Chinese Medicine Administration (GZY-ZJ-KJ-24032).

Key Laboratory of Animal Virology of Ministry of Agriculture, Center for Veterinary Sciences, Zhejiang University, Hangzhou, People’s Republic of China

Zi-yi Zhang, Jing-tian-yi Liu, Xing-wei Ji & Shu Zhu

Department of Critical Care Medicine, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China

Xiu-liu Guo, Shu Zhu, Jin-yan Xie & Feng Guo

State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of China

Provincial Key Laboratory of Precise Diagnosis and Treatment of Abdominal Infection, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Zhejiang, People’s Republic of China

Jin-yan Xie & Feng Guo

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Z.Z., S.Z., F.G. and J.X. designed the experiments. Z.Z. and X.G. preformed experiments. J.L., Y.G. and W.J. collected samples and data. Z.Z and J.X. wrote the draft of the manuscript and edit it. F.G. and J.X. supervised research, coordination and strategy. All authors have read and approved the submitted manuscript.

Correspondence to Jin-yan Xie or Feng Guo.

All animal experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang University (#117113). All human experiments were conducted in accordance with a protocol (#20200224–31) approved by the Medical Ethics Committee of Sir Run Run Shaw Hospital.

The authors declare no competing interests.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Additional file 1: Figure S1. Acute pancreatitis was exacerbated in Baat−/− mice. (a) Relative mRNA expression of Il1b (left), Il6 (mid), and Tnfa (right) in the spleen of WT or Baat−/− SAP mice. Fold change is relative to respective mock mice. n = 3 biologically independent samples. (b) Relative mRNA expression of Il1b (left), and Il6 (right) in the liver of WT or Baat−/− SAP mice. Fold change is relative to respective mock mice. n = 3 biologically independent samples. (c) Infiltration of macrophages and expression of proinflammatory cytokines IL-6 in the pancreas of WT or Baat−/− SAP mice shown via IFA. Results are representative of data generated in at least two independent experiments and are expressed as mean ± s.d. The two-sided P values were examined using Student’s t-test for comparison of variables between two groups. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

Additional file 2: Figure S2. Taurine or glycine-conjugated forms of CDCA show stronger anti-inflammatory effects. (a) LPS-primed THP-1PMA stimulated with nigericin (2 μM) for 3 h were treated with CDCA or GCDCA at 5–100 μM for 24 h. Relative mRNA expression of Il1b and Il6 in Thp-1 treated with BAs of gradient concentration from 5–100 μM. Results are representative of data generated in at least two independent experiments and are expressed as mean ± s.d. The two-sided P values were examined using Student’s t-test for comparison of variables between two groups. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

Additional file 3: Figure S3. TCDCA treatment alleviated severe acute pancreatitis in mice. (a) Determination of TCDCA concentration in mouse serum using an LC–MS/MS system in Wt and Baat−/− mice. (b) Relative mRNA expression of Tnfa in the pancreas of WT or Baat−/− SAP mice. Relative mRNA expression of Il1b, Il6, and Tnfa in the (c) spleen and (d) liver of WT or Baat−/− SAP mice. Fold change is relative to respective mock mice. n = 3 biologically independent samples. Results are representative of data generated in at least two independent experiments and are expressed as mean ± s.d. The two-sided P values were examined using Student’s t-test for comparison of variables between two groups. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

Additional file 4: Figure S4. TCDCA alleviates pancreatitis via TGR5 and NLRP3 signaling. (a)-(g) (a)-(f) 3 mM TCDCA was added to the drinking water for 4 weeks before caerulein injection SAP models. Samples were at 12 h post caerulein-induced SAP modeling (n = 6). (a)-(d) Represent the grayscale analysis of proteins TGR5, NLRP3, pro-CASP1 and CASP1 p10, respectively, performed using ImageJ software. (e) Representative western blot showing expression protein NLRP3 in TGR5−/− mice and represent the grayscale analysis of proteins performed using ImageJ software (f). Densitometric analysis of band intensity was performed using ImageJ software. The grayscale intensity of the bands was normalized and the results are expressed as a fold change relative to the control group. n = 3 biologically independent samples. Results are representative of data generated in at least two independent experiments and are expressed as mean ± s.d. The two-sided P values were examined using Student’s t-test for comparison of variables between two groups. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

Additional file 5: Figure S5. Schematic illustration of conjugated bile acids suppress NLRP3 inflammasome through TGR5 activation, mitigating pancreatic immunopathology.

Additional file 6: Table S1. Comparison of serum bile acid metabolites altered between SAP patients (n = 10) and healthy control (n = 6).

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Zhang, Zy., Guo, Xl., Liu, Jty. et al. Conjugated bile acids alleviate acute pancreatitis through inhibition of TGR5 and NLRP3 mediated inflammation. J Transl Med 22, 1124 (2024). https://doi.org/10.1186/s12967-024-05922-0

DOI: https://doi.org/10.1186/s12967-024-05922-0

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Conjugated bile acids alleviate acute pancreatitis through inhibition of TGR5 and NLRP3 mediated inflammation | Journal of Translational Medicine | Full Text