Novel insights into the role of quercetin and kaempferol from Carthamus tinctorius L. in the management of nonalcoholic fatty liver disease via NR1H4-mediated pathways.

This study investigates the novel therapeutic potential of quercetin and kaempferol, two bioactive compounds derived from Carthamus tinctorius L., in treating nonalcoholic fatty liver disease (NAFLD) by modulating the bile acid receptor NR1H4 (Nuclear Receptor Subfamily 1 Group H Member 4) and its associated metabolic pathways. A rat model of NAFLD was established, and RNA sequencing and proteomics were carefully employed to identify differential gene expressions associated with the disease. The active components of Carthamus tinctorius L. were screened, followed by the construction of a comprehensive network that maps the interactions between these components, NR1H4 and NAFLD-related pathways. Both in vitro (using HepG2 cells) and in vivo experiments were conducted to evaluate the effects on NR1H4 expression levels through Western blot and RT-qPCR analyses. Our findings identify NR1H4 as a pivotal target in NAFLD. Network pharmacology analysis indicates that quercetin and kaempferol play crucial roles in combating NAFLD, with in vitro and in vivo experiments confirming their ability to mitigate hepatocyte steatosis by enhancing NR1H4 expression. Notably, the protective effects of these compounds were inhibited by the NR1H4 antagonist guggulsterone, highlighting the importance of NR1H4 upregulation. This study demonstrates the novel therapeutic efficacy of quercetin and kaempferol from Carthamus tinctorius L. in treating NAFLD through NR1H4 upregulation. This mechanism contributes to the regulation of lipid metabolism, improvement of liver function, reduction of inflammation, and alleviation of oxidative stress, offering a promising direction for future NAFLD treatment strategies.

NR1H4 ameliorates Parkinson's disease via inhibiting astrocyte activation and neuroinflammation in a CEBPß/NF-κB dependent manner.

Parkinson's Disease (PD) is a degenerative disease driven by neuroinflammation. Nuclear receptor subfamily 1 group H member 4 (NR1H4), a nuclear receptor involved in metabolic and inflammatory regulation, is found to be widely expressed in central nervous system. Previous studies suggested the protective role of NR1H4 in various diseases related to inflammation, whether NR1H4 participates in PD progression remains unknown. To investigate the role of NR1H4 in neuroinflammation regulation, especially astrocyte activation during PD, siRNA and adenovirus were used to manipulate Nr1h4 expression. RNA-sequencing (RNA-seq), quantitative real-time PCR, enzyme-linked immunosorbent assay, Chromatin immunoprecipitation and western blotting were performed to further study the underlying mechanisms. We identified that NR1H4 was down-regulated during PD progression. In vitro experiments suggested that Nr1h4 knockdown led to inflammatory response, reactive oxygen species generation and astrocytes activation whereasNr1h4 overexpressionhad the opposite effects. The results of RNA-seq on astrocytes revealed that NR1H4 manipulated neuroinflammation in a CEBPß/NF-κB dependent manner. Additionally, pharmacological activation of NR1H4 via Obeticholic acid ameliorated neuroinflammation and promoted neuronal survival. Our study first proved the neuroprotective effects of NR1H4against PD via inhibiting astrocyte activation and neuroinflammation in a CEBPß/NF-κB dependent manner.

Bletilla striata polysaccharide alleviates chronic obstructive pulmonary disease via modulating gut microbiota and NR1H4 expression in mice.

Bletilla striata polysaccharide (BSP) is the main component of Bletilla striata and has been revealed to enhance immune responses. Chronic obstructive pulmonary disease (COPD) results from the chronic inhalation of toxic particles and gases, which initiates innate and adaptive immune responses in the lungs. This study aimed to evaluate whether the effects of BSP on COPD were related to the abundance of gut microbiota and explored the underlying mechanism. COPD mice were induced with cigarette smoke and human bronchial epithelial cells (HBEC) were subjected to cigarette smoke extract (CSE) for in vitro studies. BSP alleviated the inflammatory response and the inflammatory cell infiltration in lung tissues and promoted the recovery of respiratory function in COPD mice. BSP mitigated CSE-induced HBEC injury by repressing inflammation and oxidative stress. 16s rRNA sequencing revealed that BSP increased the abundance of Bacteroides intestinalis. Bacteroides intestinalis colonization enhanced the therapeutic effect of BSP in COPD mice by upregulating NR1H4 and its encoded protein FXR. Reduction of NR1H4 impaired the therapeutic impact of BSP and Bacteroides intestinalis in COPD. These data demonstrate that BSP inhibits COPD by upregulating NR1H4 through Bacteroides intestinalis, which underpins the application of BSP as a therapeutic agent for COPD.

NR1H4 disease: rapidly progressing neonatal intrahepatic cholestasis and early death.

BACKGROUND: Clinical studies on progressive familial intrahepatic cholestasis (PFIC) type 5 caused by mutations in NR1H4 are limited. METHODS: New patients with biallelic NR1H4 variants from our center and all patients from literature were retrospectively analyzed. RESULTS: Three new patients were identified to be carrying five new variants. Liver phenotypes of our patients manifests as low-γ-glutamyl transferase cholestasis, liver failure and related complications. One patient underwent liver transplantation (LT) and survived, and two other patients died without LT. Nine other patients were collected through literature review. Twelve out of 13 patients showed neonatal jaundice, with the median age of onset being 7 days after birth. Reported clinical manifestations included cholestasis (13/13, 100%), elevated AFP (11/11, 100%), coagulopathy (11/11, 100%), hypoglycemia (9/13, 69%), failure to thrive (8/13, 62%), splenomegaly (7/13, 54%), hyperammonemia (7/13, 54%), and hepatomegaly (6/13, 46%). Six of 13 patients received LT at a median age of 6.2 months, and only one patient died of acute infection at one year after LT. Other 7 patients had no LT and died with a median age of 5 months (range 1.2-8). There were 8 patients with homozygous genotype and 5 patients with compound heterozygous genotype. In total, 13 different variants were detected, and 5 out of 12 single or multiple nucleotides variants were located in exon 5. CONCLUSIONS: We identified three newly-diagnosed patients and five novel mutations. NR1H4-related PFIC typically cause progressive disease and early death. LT may be the only lifesaving therapy leading to cure.

NR1H4 mutation and rapid progressive intrahepatic cholestasis in infancy: A case report and literature review.

Farnesoid X receptor (FXR) is a nuclear bile acid receptor encoded by the NR1H4 gene, a vital regulator of bile acid homeostasis. Pathogenic mutations of NR1H4 manifest as low gamma-glutamyl transferase (GGT) cholestasis with rapid progression to liver failure, which is referred to as progressive familial intrahepatic cholestasis 5 (PFIC-5). Herein, we present a case with rapid progressive cholestasis, liver failure in early infancy with the NR1H4 termination mutation.

FXR overexpression prevents hepatic steatosis through inhibiting AIM2 inflammasome activation in alcoholic liver disease.

BACKGROUND AND PURPOSE: Alcoholic liver disease (ALD), a metabolic liver disease caused by excessive alcohol consumption, has attracted increasing attention due to its high prevalence and mortality. Up to date, there is no effective and feasible treatment method for ALD. This study was to investigate whether Farnesoid X receptor (FXR, NR1H4) can alleviate ALD and whether this effect is mediated by inhibiting absent in melanoma 2 (AIM2) inflammasome activation. METHODS: The difference in FXR expression between normal subjects and ALD patients was analyzed using the Gene Expression Omnibus (GEO) database. Lieber-DeCarli liquid diet with 5% ethanol (v/v) (EtOH) was adopted to establish the mouse ALD model. Liver histopathological changes and the accumulation of lipid droplets were assessed by H&E and Oil Red O staining. Quantitative real-time PCR, Western blotting analysis and immunofluorescence staining were utilized to evaluate the expression levels of related genes and proteins. DCFH-DA staining was adopted to visualize reactive oxidative species (ROS). RESULTS: FXR was distinctly downregulated in liver tissues of patients with steatosis compared to normal livers using the GEO database, and in ethanol-induced AML-12 cellular steatosis model. FXR overexpression ameliorated hepatic lipid metabolism disorder and steatosis induced by ethanol by inhibiting the expression of genes involved in lipid synthesis and inducing the expression of genes responsible for lipid metabolism. Besides, FXR overexpression inhibited ethanol-induced AIM2 inflammasome activation and alleviated oxidative stress and ROS production during ethanol-induced hepatic steatosis. However, when FXR was knocked down, the results were completely opposite. CONCLUSIONS: FXR attenuated lipid metabolism disorders and lipid degeneration in alcohol-caused liver injury and alleviated oxidative stress and inflammation by inhibiting AIM2 inflammasome activation.

Farnesoid X receptor overexpression prevents hepatic steatosis through inhibiting AIM2 inflammasome activation in nonalcoholic fatty liver disease.

Oxidative stress-mediated activation of inflammasome has a significant effect on the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Farnesoid X receptor (NR1H4, FXR) has been implicated in biological function and many diseases, including NAFLD. The regulatory effect of FXR on oxidative stress and whether this process is related with the activation of absent melanoma 2 (AIM2) inflammasome in NAFLD remain unclear. In the present research, we confirmed that FXR in the livers of steatosis patients is significantly reduced compared with normal liver tissue by using the Gene Expression Omnibus (GEO) database and a palmitic acid (PA) - mediated steatosis model in AML-12 cells. Under the premise of ensuring the same food intake as the control group, overexpression of FXR in mice attenuated HFD-mediated weight gain and liver steatosis, facilitated lipid metabolism, improved fatty acid ß-oxidation, lipolysis, and reduced fatty acid synthesis and intake, which also inhibited the activation of AIM2 inflammasome. Overexpression of FXR alleviated PA-induced triglyceride (TG) accumulation, imbalance of lipid homeostasis, and the activation of AIM2 inflammasome in hepatic steatosis cells, while FXR knockdown appeared the opposite effects. FXR overexpression suppressed PA- and HFD-induced oxidative stress, but FXR siRNA demonstrated the opposite influence. The decreased ROS generation may be the reason why FXR weakens AIM2 activation when a fatty acid overload occurs. In conclusion, our results confirm that other than regulating lipid homeostasis and blocking NLRP3 inflammasome activation, FXR improves hepatic steatosis by a novel mechanism that inhibits oxidative stress and AIM2 inflammasome activation.

Study on the role of NR1H4 in chronic atrophic gastritis and drug prediction based on bioinformatics / 中国现代医生

Objective To explore the differential gene expression profile and small molecule drugs for chronic atrophic gastritis(CAG)by bioinformatics technology.Methods Two gene expression samples of CAG chips(GSE27411,GSE116312)were obtained through the Gene Expression Synthesis(GEO)database,screen the differentially expressed genes(DEGs)of CAG by R language,and CAG immune-related genes were obtained for gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.Protein-protein interaction(PPI)network was constructed using STRING database to screen out core genes,further study on immune invasion of core genes based on GSE27411 dataset,small molecular compounds interacting with core genes were predicted,molecular docking was carried out by MOE2022,and survival analysis was carried out by GEPIA2 website.Results A total of 517 DEGs were screened out based on GEO database.GO function enrichment analysis found that it mainly involved in granulocyte chemotaxis、leukocyte chemotaxis and neutrophil chemotaxis biological processes.KEGG pathway enrichment analysis showed that it mainly involved in cytokine-cytokine receptor interaction、nuclear factor kappa B signaling pathway、interleukin-17 signaling pathway.Six key genes of NR1H4、CCK、CCL20、CXCL1、LCN2、SAA1 were obtained by PPI network,through relevant verification,NR1H4 was regarded as the core gene.Immune cell infiltration analysis showed that central memory CD8 T cell、effector memeory CD4 T cell、gamma delta T cell、natural killer T cell、neutrophil and other immune cells may be involved in the development of CAG,and the neutrophil was positively correlated with NR1H4.It was predicted that six small molecular drugs,corilagin,stigmasterol,geniposide,tangeretin,chenodeoxycholic acid and epigallocatechin 3-gallate,have good binding force with NR1H4.Conclusion The potential mechanism of CAG is preliminarily explored in this study,the key gene of NR1H4 and neutrophil may play an important role in the"inflammatory cancer transformation"process of CAG,which can provide a certain reference for the study of the"inflammatory cancer transformation"mechanism of CAG.

Yinchenhao Tang alleviates high fat diet induced NAFLD by increasing NR1H4 and APOA1 expression.

Background and aim: Traditional Chinese medicine Yinchenhao Tang (YCHT) demonstrated benefits when treating nonalcoholic fatty liver disease (NAFLD), but the dose effects and potential targets are still ambiguous. In this study, different concentrations of YCHT were employed to treat NAFLD and the underlying therapeutic targets were investigated. Experimental procedure: Kunming mice were fed with high fat diet (HFD) for 8 weeks to induce NAFLD, then treated with 3 different concentrations of YCHT. Hepatic pathological changes and serum lipid levels were examined. Network pharmacology was applied to screen the potential targets of YCHT for NAFLD modulation. NR1H4 and APOA1 expression was evaluated by QPCR and western blotting. Immunohistochemistry (IHC) staining was conducted to visualize the localization pattern of NR1H4 and APOA1 in the liver. Results: YCHT significantly reduced liver lipid storage and improved the liver pathological status of NAFLD mice. The serum lipid levels, as well as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, were remarkably reduced by the middle and high dose YCHT. There are 35 potential targets for YCHT to regulate NAFLD. HFD suppressed both RNA and protein expression of NR1H4 and APOA1, while YCHT elevated NR1H4 and APOA1 expression. IHC staining indicated that NR1H4 was mainly located in the cell nucleus and the APOA1 signal was observed at the liver sinusoid or cytoplasm. Conclusion: YCHT can effectively ameliorate HFD induced NAFLD by modulating the promising targets of NR1H4 and APOA1.

NR1H4-mediated circRHOBTB3 modulates the proliferation, metastasis, and Warburg effects of cervical cancer through interacting with IGF2BP3.

Globally, cervical cancer (CC) ranks as the fourth most common cancer and the most lethal malignancy among females of reproductive age. The incidence of CC is increasing in low-income countries, with unsatisfactory outcomes and long-term survival for CC patients. Circular RNAs (CircRNAs) are promising therapeutics that target multiple cancers. In this study, we investigated the tumorigenic role of circRHOBTB3 in CC, showing that circRHOBTB3 is highly expressed in CC cells and circRHOBTB3 knockdown also repressed CC proliferation, migration, invasion, and the Warburg effects. CircRHOBTB3 interacted with the RNA-binding protein, IGF2BP3, to stabilize its expression in CC cells and is putatively transcriptionally regulated by NR1H4. In conclusion, this novel NR1H4/circRHOBTB3/IGF2BP3 axis may provide new insights into CC pathogenesis.

Reduced peroxisome proliferator-activated receptor-α and bile acid nuclear receptor NR1H4/FXR may affect the hepatic immune microenvironment of biliary atresia.

Background: Biliary atresia (BA) is a childhood liver disease characterized by fibrous obstruction and obstruction of the extrahepatic biliary system and is one of the most common and serious biliary disorders in infants. Significant inflammation and fibrosis of the liver and biliary tract are the most prominent features, regardless of the initial damage to the BA. Abnormalities in innate or adaptive immunity have been found in human patients and mouse models of BA. We previously reported that children with BA had abnormal lipid metabolism, including free serum carnitine. Objective: To study gene and protein expression levels of the hepatic peroxisome proliferator-activated receptor-α (PPARα) signaling pathway and farnesoid X receptor (FXR) in BA and BA fibrosis, and assess their clinical values. Methods: Low expression of PPARα and NR1H4 (FXR) in BA were validated in the Gene Expression Omnibus database. Functional differences were determined by gene set enrichment analysis based on of PPARα and NR1H4 expression. BA patients from GSE46960 were divided into two clusters by using consensus clustering according to PPARα, NR1H4, and SMAD3 expression levels, and immunoinfiltration analysis was performed. Finally, 58 cases treated in our hospital were used for experimental verification. (IHC: 10 Biliary atresia, 10 choledochal cysts; PCR: 10 Biliary atresia, 14 choledochal cysts; WB: 10 Biliary atresia, 4 choledochal cysts). Results: Bioinformatics analysis showed that the expression of PPARα, CYP7A1 and NR1H4 (FXR) in the biliary atresia group was significantly lower than in the control group. More BA-specific pathways, including TGFß signaling pathway, P53 signaling pathway, PI3K-AKT-mTOR signaling pathway, etc., are enriched in BA patients with low PPARα and NR1H4 expression. In addition, low NR1H4 expression is abundant in inflammatory responses, IL6/STAT3 signaling pathways, early estrogen responses, IL2 STAT5 signaling pathways, and TGFß signaling pathways. The TGFß signaling pathway was significant in both groups. According to the expression of PPARα, NR1H4 and SMAD3, a key node in TGFß pathway, BA patients were divided into two clusters using consensus clustering. In cluster 2, SMAD3 expression was high, and PPARα and NR1H4 expression were low. In contrast to cluster 1, immune cell infiltration was higher in cluster 2, which was confirmed by immunohistochemistry. The mRNA and protein levels of PPARα and NR1H4 in BA patients were lower than in the control group by immunohistochemistry, Western blot analysis and real-time PCR. Conclusions: The downregulation of PPARα and NR1H4 (FXR) signaling pathway may be closely related to biliary atresia.

Clinical significance and oncogenic function of NR1H4 in clear cell renal cell carcinoma.

BACKGROUND: Nuclear receptor subfamily 1 group H member 4 (NR1H4) have been reported in various cancer types, however, little is known about the clinical values and biological function in clear cell Renal cell carcinoma (ccRCC). METHODS: The expression pattens of NR1H4 in ccRCC were investigated in clinical specimens, cell lines and publicly­available databases. Cell Counting Kit-8 (CCK-8), colony formation, 5-ethynyl-2' -deoxyuridine (EdU), transwell and cell wound healing assays were performed to assess the biological functions of NR1H4 in 786-O ccRCC cells. Gene set enrichment analysis (GSEA), Flow Cytometry, quantitative real-time PCR (qRT-PCR), western blot and immunofluorescence were performed to explore the molecular mechanism of NR1H4 in ccRCC. We explored the early diagnostic value, prognostic value, genetic mutation and DNA methylation of NR1H4 by a comprehensive bioinformatics analysis based on the data published in the following databases: The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Kaplan-Meier Plotter, Gene Expression Profiling Interactive Analysis (GEPIA), UNIVERSITY OF CALIFORNIA SANTA CRUZ Xena (UCSC Xena), cBio Cancer Genomics Portal, MethSurv, SurvivalMeth and The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN). Its correlation with tumor-infiltrating immune cells in ccRCC was analyzed by Tumor Immune Estimation Resource 2.0 (TIMER2.0) and Tumor Immune System Interactions Database (TISIDB). RESULTS: In this study, NR1H4 was found to be highly expressed in ccRCC tissues and ccRCC cell lines. Knockdown of NR1H4 significantly suppressed cancer cell proliferation, migration and invasion. Mechanistically, tumor-associated signaling pathways were enriched in the NR1H4 overexpression group and si-NR1H4 could induce the downregulation of Cyclin E2 (CCNE2). By bioinformatics analysis, NR1H4 was identified as highly expressed in stage I ccRCC with a high diagnostic accuracy (area under the receiver operating characteristic curve > 0.8). Genetic alteration and DNA methylation of NR1H4 were significantly associated with prognosis in ccRCC patients. Moreover, NR1H4 expression associated with immune cell infiltration levels in ccRCC, which provides a new idea for immunotherapy. CONCLUSIONS: Our study indicated that NR1H4 might be a potential tumor biomarker and therapeutic target for ccRCC which could promote cancer cell proliferation, migration and invasion via regulating CCNE2.

NR1H4 rs35724 G>C variant modulates liver damage in nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Farnesoid X receptor (FXR) plays a key role in bile acid and lipid homeostasis. Experimental evidence suggests that it can modulate liver damage related to nonalcoholic fatty liver disease (NAFLD). We examined the impact of the NR1H4 rs35724 G>C, encoding for FXR, on liver damage in a large cohort of patients at risk of steatohepatitis. METHODS: We considered 2,660 consecutive individuals at risk of steatohepatitis with liver histology. The rs35724 G>C polymorphisms were genotyped by TaqMan assays. Gene expression was evaluated by RNASeq in a subset of patients (n = 124). RESULTS: The NR1H4 rs35724 CC genotype, after adjusting for clinic-metabolic and genetic confounders and for enrolling centre, was protective against severity of steatosis (GG vs CC OR 0.77, 95% CI 0.62-0.95; P = .01), steatohepatitis (GG vs CC OR 0.62, 95% CI 0.47-0.83; P = .001) and severity of fibrosis (GG vs CC OR 0.83, 95% CI 0.67-0.98; P = .04). The C allele was associated with higher total circulating cholesterol (P = .01). Patients carrying the NR1H4 rs35724 C allele had significantly higher hepatic mRNA levels of FXR and were associated with higher hepatic FGFR4 and Cyp39A1 that are in turn involved in bile acid synthesis. CONCLUSIONS: Increased hepatic FXR expression due to the NR1H4 rs35724 C allele is linked to higher serum cholesterol but protects against steatosis, steatohepatitis and liver fibrosis. The translational relevance of these results for patient risk stratification and FXR-targeted therapy warrants further investigation.

Epidemiology and burden of progressive familial intrahepatic cholestasis: a systematic review.

BACKGROUND: Progressive familial intrahepatic cholestasis is a rare, heterogeneous group of liver disorders of autosomal recessive inheritance, characterised by an early onset of cholestasis with pruritus and malabsorption, which rapidly progresses, eventually culminating in liver failure. For children and their parents, PFIC is an extremely distressing disease. Significant pruritus can lead to severe cutaneous mutilation and may affect many activities of daily living through loss of sleep, irritability, poor attention, and impaired school performance. METHODS: Databases including MEDLINE and Embase were searched for publications on PFIC prevalence, incidence or natural history, and the economic burden or health-related quality of life of patients with PFIC. Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed. RESULTS: Three systematic reviews and twenty-two studies were eligible for inclusion for the epidemiology of PFIC including a total of 2603 patients. Study periods ranged from 3 to 33 years. Local population prevalence of PFIC was reported in three studies, ranging from 9.0 to 12.0% of children admitted with cholestasis, acute liver failure, or splenomegaly. The most detailed data come from the NAPPED study where native liver survival of >15 years is predicted in PFIC2 patients with a serum bile  acid concentration below 102 µmol/L following bile diversion surgery. Burden of disease was mainly reported through health-related quality of life (HRQL), rates of surgery and survival. Rates of biliary diversion and liver transplant varied widely depending on study period, sample size and PFIC type, with many patients have multiple surgeries and progressing to liver transplant. This renders data unsuitable for comparison. CONCLUSION: Using robust and transparent methods, this systematic review summarises our current knowledge of PFIC. The epidemiological overview is highly mixed and dependent on presentation and PFIC subtype. Only two studies reported HRQL and mortality results were variable across different subtypes. Lack of data and extensive heterogeneity severely limit understanding across this disease area, particularly variation around and within subtypes.

The Role of Nuclear Receptor Subfamily 1 Group H Member 4 (NR1H4) in Colon Cancer Cell Survival through the Regulation of c-Myc Stability.

Nuclear receptor subfamily group H member 4 (NR1H4), also known as farnesoid X receptor, has been implicated in several cellular processes in the liver and intestine. Preclinical and clinical studies have suggested a role of NR1H4 in colon cancer development; however, how NR1H4 regulates colon cancer cell growth and survival remains unclear. We generated NR1H4 knockout (KO) colon cancer cells using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (CAS9) technology and explored the effects of NR1H4 KO in colon cancer cell proliferation, survival, and apoptosis. Interestingly, NR1H4 KO cells showed impaired cell proliferation, reduced colony formation, and increased apoptotic cell death compared to control colon cancer cells. We identified MYC as an important mediator of the signaling pathway alterations induced by NR1H4 KO. NR1H4 silencing in colon cancer cells resulted in reduced MYC protein levels, while NR1H4 activation using an NR1H4 ligand, chenodeoxycholic acid, resulted in time- and dose-dependent MYC induction. Moreover, NR1H4 KO enhanced the anti-cancer effects of doxorubicin and cisplatin, supporting the role of MYC in the enhanced apoptosis observed in NR1H4 KO cells. Taken together, our findings suggest that modulating NR1H4 activity in colon cancer cells might be a promising alternative approach to treat cancer using MYC-targeting agents.

Panel-Based Next-Generation Sequencing for the Diagnosis of Cholestatic Genetic Liver Diseases: Clinical Utility and Challenges.

OBJECTIVE: To test the application of a target enrichment next-generation sequencing (NGS) jaundice panel in genetic diagnosis of pediatric liver diseases. STUDY DESIGN: We developed a capture-based target enrichment NGS jaundice panel containing 42 known disease-causing genes associated with jaundice or cholestasis and 10 pathway-related genes. During 2015-2017, 102 pediatric patients with various forms of cholestasis or idiopathic liver diseases were tested, including patients with initial diagnosis of cholestasis in infancy, progressive familial intrahepatic cholestasis, syndromic cholestasis, Wilson disease, and others. RESULTS: Of the 102 patients, 137 mutations/variants in 44 different genes were identified in 84 patients. The genetic disease diagnosis rate was 33 of 102 (32.4%). A total of 79 of 102 (77.5%) of patients had at least 1 heterozygous genetic variation. Those with progressive intrahepatic cholestasis or syndromic cholestasis in infancy had a diagnostic rate of 62.5%. Disease-causing mutations, including ATP8B1, ABCB11, ABCB4, ABCC2, TJP2, NR1H4 (FXR), JAG1, AKR1D1, CYP7B1, PKHD1, ATP7B, and SLC25A13, were identified. Nine patients had unpredicted genetic diagnosis with atypical phenotype or novel mutations in the investigational genes. We propose an NGS diagnosis classification categorizing patients into high (n = 24), moderate (n = 9), or weak (n = 25) levels of genotype-phenotype correlations to facilitate patient management. CONCLUSIONS: This panel enabled high-throughput detection of genetic variants and disease diagnosis in patients with a long list of candidate causative genes. A NGS report with diagnosis classification may aid clinicians in data interpretation and patient management.

An FXR Agonist Reduces Bile Acid Synthesis Independently of Increases in FGF19 in Healthy Volunteers.

Bile acid (BA) synthesis is regulated through suppression of hepatic cholesterol 7α-hydroxylase via farnesoid X receptor (FXR) activation in hepatocytes and/or enterocytes; in enterocytes, this process requires FGF19 signaling. To study these pathways, we quantified markers of BA synthesis (7α-hydroxy-4-cholesten-3-one [C4]) and cholesterol production (lathosterol), fibroblast growth factor (FGF)19, and BAs in serum from healthy male volunteers given 1 oral dose of the nonsteroidal FXR agonist Px-102 (0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg, 1.12 mg/kg, 2.25 mg/kg, 3.38 mg/kg, or 4.5 mg/kg). After 8 hours, serum levels of C4 decreased by 80% in volunteers given 0.15 mg/kg, whereas serum levels of FGF19 were unchanged. Serum levels of FGF19 increased significantly, in a dose-dependent manner, in volunteers given >0.3 mg/kg Px-102, up to as much as 1600%, whereas C4 levels remained significantly reduced (by >80%). For all doses, FGF19 levels returned to normal 24 hours after administration of Px-102. Serum levels of C4 decreased before levels of FGF19 levels increased, and were still reduced by 95% 24 hours after the highest dose (4.5 mg/kg) of Px-102, even though levels of FGF19 had returned to baseline. Our findings indicate that activation of hepatic FXR is able to suppress BA synthesis, independent of FGF19.

Ciliogenesis is reciprocally regulated by PPARA and NR1H4/FXR through controlling autophagy in vitro and in vivo.

The primary cilia are evolutionarily conserved microtubule-based cellular organelles that perceive metabolic status and thus link the sensory system to cellular signaling pathways. Therefore, ciliogenesis is thought to be tightly linked to autophagy, which is also regulated by nutrient-sensing transcription factors, such as PPARA (peroxisome proliferator activated receptor alpha) and NR1H4/FXR (nuclear receptor subfamily 1, group H, member 4). However, the relationship between these factors and ciliogenesis has not been clearly demonstrated. Here, we present direct evidence for the involvement of macroautophagic/autophagic regulators in controlling ciliogenesis. We showed that activation of PPARA facilitated ciliogenesis independently of cellular nutritional states. Importantly, PPARA-induced ciliogenesis was mediated by controlling autophagy, since either pharmacological or genetic inactivation of autophagy significantly repressed ciliogenesis. Moreover, we showed that pharmacological activator of autophagy, rapamycin, recovered repressed ciliogenesis in ppara-/- cells. Conversely, activation of NR1H4 repressed cilia formation, while knockdown of NR1H4 enhanced ciliogenesis by inducing autophagy. The reciprocal activities of PPARA and NR1H4 in regulating ciliogenesis were highlighted in a condition where de-repressed ciliogenesis by NR1H4 knockdown was further enhanced by PPARA activation. The in vivo roles of PPARA and NR1H4 in regulating ciliogenesis were examined in greater detail in ppara-/- mice. In response to starvation, ciliogenesis was facilitated in wild-type mice via enhanced autophagy in kidney, while ppara-/- mice displayed impaired autophagy and kidney damage resembling ciliopathy. Furthermore, an NR1H4 agonist exacerbated kidney damage associated with starvation in ppara-/- mice. These findings indicate a previously unknown role for PPARA and NR1H4 in regulating the autophagy-ciliogenesis axis in vivo.

MicroRNA-194 inhibition improves dietary-induced non-alcoholic fatty liver disease in mice through targeting on FXR.

Non-alcoholic fatty liver disease (NAFLD) affects obesity-associated metabolic syndrome, which exhibits hepatic steatosis, insulin insensitivity and glucose intolerance. Previous studies indicated that hepatic microRNAs (miRs) play critical roles in the development of NAFLD. In this study, we aim to explore the pathophysiological role of miR-194 in obesity-mediated metabolic dysfunction. Our findings show that the high fat diet or palmitic acid treatment significantly increase hepatic miR-194 levels in vivo and in vitro. Silence of miR-194 protects palmitic acid-induced inflammatory response in cultured hepatocytes, and attenuates structural disorders, lipid deposits and inflammatory response in fatty liver. MiR-194 inhibitor also improves glucose and insulin intolerance in obese mice. Through dual luciferase assay, we demonstrate that miR-194 directly binds to FXR/Nr1h4 3'-UTR, and inhibits gene expression of FXR/Nr1h4. Furthermore, overexpression of miR-194 downregulates FXR/Nr1h4 in cultured hepatocytes, but miR-194 inhibitor reversely increases FXR/Nr1h4 expression in obese mouse liver tissues. On the contrast, silence of FXR/Nr1h4 abolishes the hepatic benefits in obese mice treated with miR-194 inhibitor. Present study provides a novel finding that suppression of miR-194 attenuates dietary-induced NAFLD via upregulation of FXR/Nr1h4. The findings suggest miR-194/FXR are potential diagnostic markers and therapeutic targets for NAFLD.