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OBJECTIVE To establish the method for determination of bile acid active constituents （cholic acid， hyodeoxycholic acid and sodium taurocholate） in Babao jingfeng powder， and to compare the differences of 3 constituents in Babao jingfeng powder from different manufacturers. METHODS UPLC-MS method was adopted to determine the contents of cholic acid， hyodeoxycholic acid and sodium taurocholate in 9 batches of Babao jingfeng powder from 3 manufacturers. The separation was performed on a Phenomenex Luna® C18 column with mobile phase consisted of acetonitrile-water （gradient elution） at a flow rate of 0.5 mL/min. The column temperature was 40 ℃ and the sample size was 2 μL. MS system was operated in a negative ion single quadrupole mode， and the m/z of cholic acid， hyodeoxycholic acid and sodium taurocholate were 407.2， 392.2 and 514.2， respectively. With the contents of three components as the index， cluster analysis and principle component analysis were performed on 9 batches of samples. RESULTS The linear ranges of cholic acid， hyodeoxycholic acid and sodium taurocholate were 5.48- 548.40， 5.38-538.40， 4.74-474.05 ng/mL， respectively （r≥ 0.999 3）. RSDs of precision， repeatability and stability tests（24 h） were all lower than or equal to 3.7% （n＝6）， and the average recoveries were 103.3%， 103.3% and 101.6% with RSDs of 3.3%， 3.4%， 4.2% （n＝6）， respectively. The contentsof cholic acid ranged 0.702-1.711 mg/g， those of deoxycholic acid ranged 0.599-2.049 mg/g， and those of sodium taurocholate ranged 0.664-1.752 mg/g in 9 batches of samples. The average content of 3 components in samples from manufacturer A was high， and the difference between batches was the smallest； the average content of 3 components from manufacturer C was the lowest， and the difference between batches was large. Cluster analysis could basically distinguish samples from different manufacturers； cluster analysis was conducted by using the comprehensive score of principal components as an indicator， the results of which were basically consistent with those of cluster analysis for content. CONCLUSIONS The method is established successfully for the content determination of the 3 bile acid active constituents of artificial bezoar in Babao jingfeng powder. The contents of 3 components in Babao jingfeng powder from 3 manufacturers are significantly different.
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Sodium taurocholate cotransporting polypeptide (NTCP) is not only an important transporter for bile acid absorption into the liver, but also a functional receptor for HBV and HDV, and extensive studies have been performed for its structure, function, gene characteristics, and expression and regulation mechanisms. NTCP is also associated with chronic viral hepatitis, nonalcoholic fatty liver disease, liver fibrosis, primary biliary cholangitis, and hepatocellular carcinoma. This article elaborates on the role of NTCP in various hepatobiliary diseases, so as to provide new direction for the diagnosis and treatment of related diseases.
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Objective To investigate the clinical and gene mutation features of sodium taurocholate cotransporting polypeptide (NTCP) deficiency. Methods A total of 10 children, aged 50%). Second-generation gene sequencing showed that all 10 children had a homozygous mutation of the SLC10A1 gene, i.e., c.800C > T(p.Ser267Phe, chr14∶70245193). Conclusion Although NTCP deficiency often has no symptoms, some of the children may manifest as infant cholestasis in the early stage. The possibility of NTCP deficiency should be considered when there is persistent hypercholanemia and the changing trend of serum TBA is not consistent with that of other liver function parameters.
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Bile acids (BAs) are a major component of bile salt, which plays a vital role in the metabolism of lipids in humans. Ninety-five percent of bile acids are recycled by the enterohepatic circulation (EHC), and therefore EHC is essential for bile acid homeostasis. There are four transporters that mediate the transmembrane transport of bile acids, each of which plays an important role in the enterohepatic circulation. Gene defects in bile acid transporters can lead to disorders of the enterohepatic circulation, ultimately leading to clinical phenotypes such as metabolic diseases and even death. Bile transporter expression is altered in patients with various metabolic disease states, suggesting that disruption of bile acid transporters may be a pivotal pathological mechanism for the development of metabolism diseases. Thus, many drugs targeting bile acid transporters are being developed. We provide a concise overview of the progress of bile acid transporters research, discuss the relationship between different bile acid transporters and disease development, and summarize the current progress in drug development targeting bile acid transporters.
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Sodium-taurocholate cotransporting polypeptide (NTCP) deficiency is a new hereditary bile acid metabolic disease due to biallelic mutations of the SLC10A1 gene and is not rare in China. Marked and persistent hypercholanemia in childhood is the major clinical feature of NTCP deficiency, and this condition might be involved in the development of neonatal hyperbilirubinemia, cholestasis in early infancy, and cholestasis in pregnancy. At present, there lack specific therapies for NTCP deficiency, but such patients tend to have good prognosis. SLC10A1 gene detection may facilitate the timely and definite diagnosis of this disease and thus avoid unnecessary examinations and interventions.
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ObjectiveThe transient expression of the functional receptor sodium taurocholate cotransporting polypeptide (NTCP) leads to the inefficiency and instability of the hepatitis B virus (HBV)-associated hepatocellular carcinoma cell model. The aim of this study was to construct NTCP-Huh7 cell lines by transfecting GV358-NTCP into Huh7 cells and identify their susceptibility to HBV.MethodsThe recombinant plasmid GV358-NTCP was obtained by ligation of GV358 and NTCP, and then transfected into Huh7 cells for the construction of NTCP-Huh7 cells. The NTCP-Huh7 cells (NTCP-Huh7 group) and Huh7 cells (Huh7 group) were infected with HBV and co-incubated with HBV for 18 hours, and the co-incubation was continued after change of the culture medium. At 2, 4, 6, 8, 10, and 12 days of incubation, the supernatant and cells were collected for measurement of the contents of HBsAg, HBeAg, HBcAg and HBV DNA in the supernatant and HBV cccDNA in the cells as well as for determination of HBV susceptibility of the NTCP-Huh7 recombinant cells.ResultsWestern blot showed stably expressed NTCP proteins in the NTCP-Huh7 cells. At 8 days of incubation, the levels of HBsAg and HBeAg were significantly higher in the NTCP-Huh7 group (0.866±0.040 and 0.603±0.053) than in the Huh7 group (0.237±0.063 and 0.209±0.112) (P<0.05), reaching the peak at 8 days and also remarkably higher in the former than in the latter group at 4, 6 and 10 days (P < 0.05). So were the levels of HBV DNA and HBV cccDNA in the NTCP-Huh7 than in the Huh7 group at 4, 6, 8, 10 and 12 days (P < 0.05). Immunofluorescence assay revealed the core antigen of HBV in the NTCP-Huh7 but not in Huh7 cells.ConclusionNTCP-Huh7 cells obtained by transfection of the GV358-NTCP recombinant plasmid into Huh7 cells are susceptible to HBV infection.
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Aim To study the anti-inflammatory activity of the Channa argus bile.Methods The bile was isolated and purified by extraction and silica gel column chromatography.Then the compounds were identified by hydrogen and carbon spectra.The spleen lymphocytes proliferation assay and Lipopolysaccharide(LPS) induced mouse macrophage RAW264.7 releasing Nitrogen Monoxide(NO) experiment were used to evaluate the anti-inflammatory activity.Results Compound(C1) of sodium taurocholate and compound(C2) of sodium taurochenodeoxycholate were isolated by activity tracing.The cell relative viabilities of the two compounds on Concanavalin A(Con A) induced spleen lymphocytes proliferation assay were 65.9%±11.7% and 60.5%±9.4%, which were significantly different from the result of model group (P<0.01), respectively.The NO production of LPS-induced RAW264.7 release of NO was (16.4±1.9) μmol·L-1 and (15.5±1.7) μmol·L-1, which were significantly different from the result of model group(P<0.01).Conclusion Sodium taurocholate and sodium taurochenodeoxycholate from Channa argus perform the anti-inflammatory activities but have no cytotoxic effect on spleen lymphocytes and macrophage.
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OBJECTIVE:To study the effects of grape seed proanthocyanidin(GSP)on the transmembrane transport of sodium glycocholate (GA) and sodium taurocholate (TA) in colon glandular cell Caco-2. METHODS:Caco-2 model was used,and RP-HPLC was conducted to determine the contents of GA and TA in cell culture medium. The test was divided into GSP group, GA group,TA group,GSP+GA group and GSP+TA group,the transport volumes of transporting GA and TA from Transwell apical (AP)side to basolateral(BL)side by Caco-2 cell at 0,2,4,8 h were detected,respectively. RESULTS:The linear ranges of GA and TA were 0.05-1.2 mmol/L(R2=0.9999). With the time passing,transport volumes of GA and TA in BL site in GA group and TA group were sharply increased;while the transport volumes were obviously decreased after adding GSP,with statistical signifi-cance(P<0.01). CONCLUSIONS:GSP has inhibitory effect on the transmembrane transport of GA and TA in Caco-2 cell.
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Cholestasis is defined as a conjugated bilirubin level >1 mg/dL(17.1 μmol/L)if total serum bilirubin is ≤5 mg/dL(85.5 μmol/L),or conjugated bilirubin fraction >20％of total bilirubin when the total bilirubin is >5 mg/dL(85.5 μmol/L).In the recent years,the diagnosis and management of genetic cholestasis have caused considerable attention in the pediatric world,in pace with the development,maturation,and clinical application of the theories and techniques in genomics as well as molecular genetics.With a diversity of causative genes,genetic cholestasis usually demonstrates nonpathognomonic clinical manifestations.The etiology diagnosis such a disease relies on genetic tests,the treatment is often difficult,and the prognosis varies disparately,usually causing tremendous pain and burden on the affected patient and the family as well.Taking citrin deficiency,mitochondrial DNA depletion syndrome,microvi-llus inclusion disease and sodium taurocholate cotransporting polypeptide deficiency as samples,the recent advances in the diagnosis and treatment of genetic cholestasis are addressed.
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According to World Health Organization, more than 200 million people suffer with chronic hepatitis caused by Hepatitis B virus (HBV) infection worldwide. Chronic hepatitis B causes various complications including liver cirrhosis and hepatocellular carcinoma and approximately 0.5~4.2 million deaths occur annually due to HBV infection. Current therapies such as antivirals and vaccine are often hampered by drug intolerance, side effects, and long-time medication, therefore, the development of powerful anti-HBV drugs is demanded. Recently, sodium taurocholate co-transporting polypeptide (NTCP) receptor was revealed to play a pivotal role in HBV entry into hepatocytes. Cell lines transfected with NTCP receptor enables to analyze HBV life cycle by inducing HBV infection stably, but in vivo models still have some limitations such as high costs, restrictive differentiation, and unveiled cofactors related to human NTCP. Therefore, it requires well-established in vivo models to develop and evaluate novel therapeutic agents targeting NTCP receptor, and viral entry inhibitors that inhibit the early step of viral infection are potent sufficient to substitute for existing antivirals.
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Humans , Antiviral Agents , Carcinoma, Hepatocellular , Cell Line , Hepatitis B virus , Hepatitis B, Chronic , Hepatitis, Chronic , Hepatocytes , Life Cycle Stages , Liver Cirrhosis , Taurocholic Acid , World Health OrganizationABSTRACT
Objective To investigate the association of restriction fragment length polymorphisms (RFLP) in p.S267F of SLC10A1 gene with clinical outcomes of hepatitis B virus (HBV) infection. Methods Clinical data of 1 268 patients with HBV infections admitted in Public Health Clinical Center Affiliated to Fudan University during July 2014 and February 2015 were collected.Polymerase chain reaction-restriction fragment length polymorphism ( PCR-RFLP) method was used to genotype the p .S267F of SLC10A1 gene in all patients, and the potential association between variants in p .S267F of SLC10A1 gene and the clinical outcomes of HBV infection was analyzed .Results Among 1 268 patients with HBV infections, 1 226 were of genotype CC, and 42 were of genotype CT, so the variation rate in p.S267F was 3.31%(42/1 268).Compared with patients with genotype CC , patients with genotype CT had a higher incidence of acute HBV infections (13.6%vs.28.6%,χ2 =19.819, P<0.05) and a lower incidence of HBV-related liver cirrhosis or hepatocellular carcinoma (13.9% vs.4.8%, χ2 =18.945, P <0.05). Conclusion RFLP in p.S267F of SLC10A1 gene may be associated with chronicity and aggravation of HBV infection, and genotype CT is possibly a protective factor .
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Objective to provide the evidence for inducing the SAP model in rats with proper concentration of sodium taurocholate.Methods 60 SD rats were divided into sham operated group, group of 1.5% in concentration, group of 3.5% in concentration and group of 5% in concentration randomly, while the SAP model was induced by the sodium taurocholate concentration of 1.5%,3.5% and 5% with the method of retrograde injection into the biliopancreatic duct.To calculate the mortality of different groups, measure the serum amylase, tumor necrosis factor -α(TNF -α) and interleukin -6 (IL -6),and to observe the pancreatic pathological scores of HE staining in rats.Results The mortality in group of 5% in concentration has a significant ascending compared with group of 1.5% in concentration, while the serum amylase, tumor necrosis factor -α(TNF -α) , interleukin -6( IL -6), pathological score of hemorrhage and acinar necrosis in group of 5% in concentration have a significant ascending compared with group of 1.5% in concentration and group of 3.5% in concentration.Conclusions A better SAP model may be induced by sodium taurocholate with the concentration of 5% by the method of retrograde injection into the biliopancreatic duct, which may accord with the physiological and pathological manifestation of SAP.
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Chronic hepatitis B affects 400 million people worldwide and is one of the leading causes of liver-related morbidity and mortality. All clinically available hepatitis B virus (HBV) drugs are nucleoside or nucleotide analogs that inhibit viral reverse transcriptase (RT) activity. Resistance to these HBV drugs has been widely reported, and is due to specific mutations in the viral RT domain. Therefore, the development of new, non-polymerase targeting anti-HBV agents is urgently needed. A potential drug target, the HBV receptor that mediates the viral entry process, has been recently identified using human primary hepatocytes, northern tree shrew (Tupaia belangeri) hepatocytes, and HepaRG cells. A transporter of bile acids, sodium taurocholate cotransporting polypeptide (NTCP), was identified as the receptor for HBV and hepatitis D virus, and the transport function of NTCP was correlated with HBV entry. Therefore, functional inhibitors of NTCP may inhibit HBV infection, and viral entry was blocked by several NTCP receptor-targeting compounds. The HBV receptor is an attractive target for development of entry inhibitors, and serves as a model for the mechanistic study of HBV entry and infection. This review will summarize the characteristics and clinical importance of NTCP, and will discuss the potential therapeutic use of NTCP inhibitors to prevent HBV entry.
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Humans , Bile Acids and Salts , Hepatitis B virus , Hepatitis B , Hepatitis B, Chronic , Hepatitis Delta Virus , Hepatocytes , Mortality , RNA-Directed DNA Polymerase , Taurocholic Acid , TupaiidaeABSTRACT
Hepatitis B virus (HBV) is the prototype of hepatotropic DNA viruses (hepadnaviruses) infecting a wide range of human and non-human hosts. Previous studies with duck hepatitis B virus (DHBV) identified duck carboxypeptidase D (dCPD) as a host specific binding partner for full-length large envelope protein, and p120 as a binding partner for several truncated versions of the large envelope protein. p120 is the P protein of duck glycine decarboxylase (dGLDC) with restricted expression in DHBV infectible tissues. Several lines of evidence suggest the importance of dCPD, and especially p120, in productive DHBV infection, although neither dCPD nor p120 cDNA could confer susceptibility to DHBV infection in any cell line. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a binding partner for the N-terminus of HBV large envelope protein. Importantly, knock down and reconstitution experiments unequivocally demonstrated that NTCP is both necessary and sufficient for in vitro infection by HBV and hepatitis delta virus (HDV), an RNA virus using HBV envelope proteins for its transmission. What remains unclear is whether NTCP is the major HBV receptor in vivo. The fact that some HBV patients are homozygous with an NTCP mutation known to abolish its receptor function suggests the existence of NTCP-independent pathways of HBV entry. Also, NTCP very likely mediates just one step of the HBV entry process, with additional co-factors for productive HBV infection still to be discovered. NTCP offers a novel therapeutic target for the control of chronic HBV infection.
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Animals , Carboxypeptidases/genetics , Gene Products, pol/genetics , Heparan Sulfate Proteoglycans/metabolism , Hepatitis B virus/physiology , Hepatocytes/metabolism , Organic Anion Transporters, Sodium-Dependent/antagonists & inhibitors , RNA Interference , Symporters/antagonists & inhibitors , Viral Envelope Proteins/metabolism , Virus InternalizationABSTRACT
OBJECTIVE: To investigate the regulation effect of isoniazid on the hepatobiliary membrane transporters multidrug resistance protein 2 (Mrp2), bile salt export pump (Bsep), P-glycoproteins (P-gp), sodium taurocholate cotransporting plypeptide (Ntcp), and which would lay the foundation for the studies of the mechanism on isoniazid-induced liver injury from the level of transporters. METHODS: Following an oral dose of 30, 60, 120 mg · kg-1 · d-1 for 1, 2 and 3 months in mouse respectively, the biochemical indicator of serum were determined; the liver were removed for hepatic pathology; the protein mass of Bsep, Mrp2, Ntcp and P-gp were analyzed by Western Blotting. RESULTS: After high/middle/low dose isoniazid administration, the expression of Mrp2, Bsep, P-gp and Ntcp were all changed, especially the high/middle dose group. In addition, the biochemical and pathological were significantly lagged behind the expression change of the transporters. CONCLUSION: The hepatotoxicity of isoniazid may be associated with excessive hepatic accumulation of the related exogenous substances that medicated by Mrp2, Bsep, P-gp and Ntcp.
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OBJETIVO: Avaliar os efeitos do uso de cloreto de gadolínio como pré-tratamento e tratamento em um modelo experimental de pancreatite em ratos induzida por tauracolato de sódio a 3 por cento. MÉTODOS: Ratos Wistar foram divididos em cinco grupos: grupo SF - controle com solução fisiológica intra-ductal e IV; grupo TS - controle com PA induzida por tauracolato de sódio a 3 por cento e solução fisiológica a 0,9 por cento IV; grupo GD - controle com SF intra-ductal e cloreto de gadolínio IV; grupo GDTS - pré-tratamento com GD (24h antes da indução da PA) e grupo TSGD - tratamento com GD (1h após a indução da PA). Foi realizado dosagem sérica de amilase, transaminases e TNF-á; determinação da atividade da MPO no tecido pulmonar; histologia pancreática e pulmonar. RESULTADOS: O número de animais mortos antes do término previsto do experimento foi significativamente maior no grupo TSGD (p=0,046). Os escores de pancreatite e de dano pulmonar foram mais elevados nos grupos que utilizaram tauracolato em comparação aos grupos com infusão intra-ductal de solução salina. Não houve diferenças nas demais variáveis estudadas na comparação entre os grupos TS; GDTS e TSGD. CONCLUSÃO: Não foram demonstrados benefícios com o uso de cloreto de gadolínio de forma profilática e terapêutica.
OBJECTIVE: To evaluate the effects of the use of gadolinium chloride before and after induction of acute pancreatitis with sodium taurocholate 3 percent in rats. METHODS: Wistar rats were divided into five groups: SF - control with saline intra-ductal and IV; GD control with saline intra-ductal and gadolinium chloride IV; TS - with AP control induced by sodium taurocholate 3 percent and saline IV; GDTS - pre-treatment with GD (24 hours before the induction of AP) and TSGD - treatment with GD (1 hour after the induction of AP). Analysis was made in serum amylase, transaminases and TNF-á; determination of the MPO activity in lung tissue, lung and pancreatic histology. RESULTS: The number of dead animals before the end of the experiment was significantly higher in TSGD (P = 0.046). The scores of pancreatitis and lung damage were higher in the groups that used sodium taurocholate compared to groups with intra-ductal infusion of saline solution. There were no differences in other variables studied when comparing TS, GDTS and TSGD groups. CONCLUSION: The benefits with the use of gadolinium chloride as a prophylactic and therapeutic drug were not demonstrated.
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Animals , Male , Rats , Gadolinium/therapeutic use , Pancreatitis/drug therapy , Contrast Media , Pancreatitis/chemically induced , Rats, Wistar , Taurocholic AcidABSTRACT
A stable and reliable infected necrotizing pancreatitis (INP) model in rats was established in order to study the pathophysiological mechanism and pathological development rule of INP and explore the new therapeutic methods for the diseases. Forty-six SD rats were randomly divided into 5 groups. The animals in group A received the injection of 5% sodium taurocholate into the pancreatic duct and those in group B underwent that of E. Coli into the pancreatic duct. The rats in groups C, D and E were subjected to the injection of 5% sodium tanrocholate in combination with different con-centrations of E. Coli (103, 104, 105/mL, respectively) into the pancreatic duct. The dose of injection was 0.1 mL/100 g and the velocity of injection was 0.2 mL/min in all the 5 groups. Eight h after the injection, the survival rate of animals was recorded and the surviving rats were killed to determine the serum content of amylase and perform pathological examination and germ cultivation of the pancre-atic tissue. The results showed that acute necrotizing panereatitis model was induced by injection of 5% sodium taurocholate into the pancreatic duct. The positive rate of germ cultivation in group A was 12.5%. The acute necrotizing pancreatitis model was not induced by injection of E. Coli into the pan-creatic duct and the positive rate of germ cultivation in group B was 0. The INP model was estab-lished in groups C to E. The positive rate of germ cultivation was 60%, 100% and 100% and 8-h sur-vival rate 100%, 100% and 70% in groups C, D and E, respectively. It was concluded that a stable and reliable model of INP was established by injection of 5% sodium taurocholate in combination with 104/mL E. Coli into the pancreatic duct with a dose of 0.1 mL/100 g and a velocity of 0.2 mL/min. The pathogenesis of INP might he that the hemorrhage and necrosis of pancreatic tissue in-duced by sodium taurocholate results in weakness of pancreatic tissue in fighting against the germs.Meanwhile, the necrotic pancreatic tissue provides a good proliferative environment for the germs.