JiaGaSongTang improves chronic cholestasis via enhancing FXR-mediated bile acid metabolism.

BACKGROUND: Bile acid (BA) enterohepatic circulation disorders are a main feature of chronic cholestatic diseases. Promoting BA metabolism is thus a potential method of improving enterohepatic circulation disorders, and treat enterohepatic inflammation, oxidative stress and fibrosis due to cholestasis. PURPOSE: To investigate the effect of JiaGaSongTang (JGST) and its blood-absorbed ingredient 6-gingerol on α-naphthylisothiocyanate (ANIT)-induced chronic cholestasis, as well as elucidate the underlying regulatory mechanism. METHODS: Chronic cholestasis was induced in mice via subcutaneous injection of ANIT (50 mg/kg) every other day for 14 d. Treatment groups were administered JGST orally daily. Damage to the liver and intestine was observed using histopathological techniques. Biochemical techniques were employed to assess total BA (TBA) levels in the serum, liver, and ileum samples. Liquid chromatograph-mass spectrometry/mass spectrometry (LC-MS/MS) was used to analyze fecal BA components. Bioinformatic methods were adopted to screen the core targets and pathways. The blood-absorbed ingredients of JGST were scrutinized via LC-MS/MS. The effects of the major JGST ingredients on farnesoid X receptor (FXR) transactivation were validated using dual luciferase reporter genes. Lastly, the effects of the FXR inhibitor, DY268, on JGST and 6-gingerol pharmacodynamics were observed at the cellular and animal levels. RESULTS: JGST ameliorated pathological impairments in the liver and intestine, diminishing TBA levels in the serum, liver and gut. Fecal BA profiling revealed that JGST enhanced the excretion of toxic BA constituents, including deoxycholic acid. Bioinformatic analyses indicated that JGST engaged in anti-inflammatory mechanisms, attenuating collagen accumulation, and orchestrating BA metabolism via interactions with FXR and other pertinent targets. LC-MS/MS analysis identified six ingredients absorbed to the bloodstream, including 6-gingerol. Surface plasmon resonance (SPR) and dual luciferase reporter gene assays confirmed the abilities of 6-gingerol to bind to FXR and activate its transactivation. Ultimately, in both cellular and animal models, the therapeutic efficacy of JGST and 6-gingerol in chronic cholestasis was attenuated in the presence of FXR inhibitors. CONCLUSION: The findings, for the first time, demonstrated that 6-gingerol, a blood-absorbed ingredient of JGST, can activate FXR to affect BA metabolism, and thereby attenuate ANIT-induced liver and intestinal injury in chronic cholestasis mice model via inhibition of inflammation, oxidative stress, and liver fibrosis, in part in a FXR-dependent mechanism.

Timosaponin Bâ ¡ reduces colonic inflammation and alleviates DSS-induced ulcerative colitis by inhibiting NLRP3.

ETHNOPHARMACOLOGICAL RELEVANCE: The Timosaponin BⅡ (TBⅡ) is one of the main active components of the traditional Chinese medicine Anemarrhena asphodeloides, and it is a steroidal saponin with various pharmacological activities such as anti-oxidation, anti-inflammatory and anti-apoptosis. However, its role in acute ulcerative colitis remains unexplored thus far. AIM OF THE STUDY: This study aims to investigate the protective effect of TBⅡ against dextran sulfate sodium (DSS)-induced ulcerative colitis in mice and elucidate its underlying mechanisms. METHODS: Wild-type (WT) and NLRP3 knockout (NLRP3-/-) mice were applied to evaluate the protective effect of TBⅡ in DSS-induced mice colitis. Pharmacological inhibition of NLRP3 or adenovirus-mediated NLRP3 overexpression in bone marrow-derived macrophages (BMDM) from WT mice and colonic epithelial HCoEpiC cells was used to assess the role of TBⅡ in LPS + ATP-induced cell model. RNA-seq, ELISA, western blots, immunofluorescence staining, and expression analysis by qPCR were performed to examine the alterations of colonic NLRP3 expression in DSS-induced colon tissues and LPS + ATP-induced cells, respectively. RESULTS: In mice with DSS-induced ulcerative colitis, TBⅡ treatment attenuated clinical symptoms, repaired the intestinal mucosal barrier, reduced inflammatory infiltration, and alleviated colonic inflammation. RNA-seq analysis and protein expression levels demonstrated that TBⅡ could prominently inhibit NLRP3 signaling. TBⅡ-mediated NLRP3 inhibition was associated with alleviating intestinal permeability and inflammatory response via the blockage of communication between epithelial cells and macrophages, probably in an NLRP3 inhibition mechanism. However, pharmacological inhibition of NLRP3 by MCC950 or Ad-NLRP3 mediated NLRP3 overexpression significantly impaired the TBⅡ-mediated anti-inflammatory effect. Mechanistically, TBⅡ-mediated NLRP3 inhibition may be partly associated with the suppression of NF-κB, a master pro-inflammatory factor for transcriptional regulation of NLRP3 expression in the priming step. Moreover, co-treatment TBⅡ with NF-κB inhibitor BAY11-7082 partly impaired TBⅡ-mediated NLRP3 inhibition, and consequently affected the IL-1ß mature and secretion. Importantly, TBⅡ-mediated amelioration was not further enhanced in NLPR3-/- mice. CONCLUSION: TBⅡ exerted a prominent protective effect against DSS-induced colitis via regulation of alleviation of intestinal permeability and inflammatory response via the blockage of crosstalk between epithelial cells and macrophages in an NLRP3-mediated inhibitory mechanism. These beneficial effects could make TBⅡ a promising drug for relieving colitis.

Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury.

The excessive accumulation of bile acids (BA) in hepatocytes can trigger inflammatory response and recruit macrophages, thereby accelerating cholestatic liver injury. The crosstalk between hepatocytes and macrophages has been recently implicated in the pathogenesis of cholestasis; however, the underlying mechanisms remain unclear. Here, we demonstrated that BA initiate NLRP3 inflammasome activation in hepatocytes to release proinflammatory cytokines and promote the communication between hepatocytes and macrophages, thus enhancing liver inflammation in an NLRP3-dependent manner. NLRP3-inhibition by geniposidic acid (GPA), a novel NLRP3-specific covalent inhibitor that directly interacts with NLRP3, in hepatocytes and macrophages abated BA-induced inflammation. Moreover, NLRP3-deletion or its inhibition mitigated ANIT-induced cholestatic inflammation, whereas disrupting the crosstalk between hepatic macrophages and hepatocytes attenuated the hepatoprotective effect of GPA against ANIT-induced cholestatic inflammation. Therefore, blocking this crosstalk by suppressing NLRP3 inflammasome activation may represent a novel therapeutic strategy for cholestasis.