Simultaneous quantification of chlorogenic acid and taurocholic acid in human plasma by LC-MS/MS and its application to a pharmacokinetic study after oral administration of Shuanghua Baihe tablets

An LC-MS/MS method was developed and validated for the simultaneous quantification of chlorogenic acid(CGA) and taurocholic acid(TCA) in human plasma using hydrochlorothiazide as the internal standard. The chromatographic separation was achieved on a Hedera ODS-2 column with a gradient elution using 10 mmol·L^(-1) of ammonium acetate buffer solution containing 0.5% of formic acid- acetonitrile as mobile phase at a flow rate of 300 μL·min-1. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring in negative ESI mode. The method was fully validated over the concentration ranges of 0.1–10 ng·m L^(-1) for CGA and 2–150 ng·m L^(-1) for TCA. It was successfully applied to a pharmacokinetic study of CGA and TCA in healthy Chinese volunteers after oral administration of Shuanghua Baihe tablets(SBTs). In the single-dose study, the maximum plasma concentration(C_(max)), time to reach C_(max)(T_(max)) and elimination half-life(t_(1/2)) of CGA were(0.763 8 ± 0.542 0) ng·m L^(-1),(1.0 ± 0.5) h, and(1.3 ± 0.6) h, respectively. In the multiple-dose study, the C_(max), T_(max) and t_(1/2) of CGA were(0.663 7 ± 0.583 3) ng·m L^(-1),(1.1 ± 0.5) h, and(1.4 ± 0.7) h, respectively. For TCA, no significant characteristic increasing plasma TCA concentration-time curve was found in the volunteers after oral administration of SBTs, indicating its complicated process in vivo as an endogenous ingredient.

De novo mutation loci and clinical analysis in a child with sodium taurocholate cotransport polypeptide deficiency: A case report

BACKGROUND Sodium taurocholate cotransport polypeptide(NTCP)deficiency disease is a genetic metabolic disorder due to mutations in the SLC10A1 gene and impaired bile acid salt uptake by the basolateral membrane transport protein NTCP in hepatocytes.A variety of clinical manifestations and genetic mutation loci have been reported for this disease.However,specific therapeutic measures are lacking,and the long-term effects are unknown.CASE SUMMARY An infant with elevated bile acids and behavioral neurodevelopmental delay failed to respond to bile acid-lowering therapy.Genetic testing for metabolic liver disease revealed that the child had NTCP deficiency due to the SLC10A1 mutation:c.422dupA(p.Y141X),which is a novel mutation site.The current followup revealed a gradual decrease in bile acid levels after 1 year of age,but the child still had behavioral neurodevelopmental delays.CONCLUSION The clinical manifestations,genetic characteristics,treatment and long-term prognosis due to NTCP deficiency remain poorly defined and need to be further confirmed by more studies and reports.

Elucidation of the early infection machinery of hepatitis B virus by using bio-nanocapsule

Currently, hepatitis B virus(HBV), upon attaching to human hepatocytes, is considered to interact first with heparan sulfate proteoglycan(HSPG) via an antigenic loop of HBV envelope S protein. Then, it is promptly transferred to the sodium taurocholate cotransporting polypeptide(NTCP) via the myristoylated N-terminal sequence of pre-S1 region(from Gly-2 to Gly-48, HBV genotype D), and it finally enters the cell by endocytosis. However, it is not clear how HSPG passes HBV to NTCP and how NTCP contributes to the cellular entry of HBV. Owing to the poor availability and the difficulty of manipulations, including fluorophore encapsulation, it has been nearly impossible to perform biochemical and cytochemical analyses using a substantial amount of HBV. A bio-nanocapsule(BNC), which is a hollow nanoparticle consisting of HBV envelope L protein, was efficiently synthesized in Saccharomyces cerevisiae. Since BNC could encapsulate payloads(drugs, genes, proteins) and specifically enter human hepatic cells utilizing HBV-derived infection machinery, it could be used as a model of HBV infection to elucidate the early infection machinery. Recently, it was demonstrated that the N-terminal sequence of pre-S1 region(from Asn-9 to Gly-24) possesses low p H-dependent fusogenic activity, which might play a crucial role in the endosomal escape of BNC payloads and in the uncoating process of HBV. In this minireview, we describe a model in which each domain of the HBV L protein contributes to attachment onto human hepatic cells through HSPG, initiation of endocytosis, interaction with NTCP in endosomes, and consequent provocation of membrane fusion followed by endosomal escape.

PROTECTIVE EFFECT OF MOXIBUSTION AT SHENQUE(RN 8)ON GASTRIC MUCOSA

Male S.D rats weighing 200g were used(as experimental animals).Moxibus-tion at Shenque(RN 8)was performed for 15 minutes each day.The course lasted for weeks.In ratsundergoing oral infusion of taurocholate,index of gastric mucosal injury were 9.0±6.1 and 4.6±2.5 in two and four week groups respectively.Compared with control group(index:16.8±7.6),Pwas less than 0.05 and 0.01.This indicated that moxibustion had protective effect on gastric mucosalinjury.Time-effect relationship also existed.In experiment of analysis about mechanism of moxibustion,we found that moxibustion.1.stim-ulated the secretion of gastric mucus(P【0.001);2.increased the PGE 2 content in gastric mucosa(P【0.05);3.increased the number of β-endorphin-like immunoactive cells in gastric mucosa(P【0.05);4.enhanced the transformation of lymphocytes(P【0.01);5.reinforced the function of an-tibody-producing cells in the spleen.The results suggested that protective effect of moxibustion might result from the increase in mu-cus,the PGE 2 and the endogenous β-endorphin.It might also be related to the reinforcing effect ofmoxibustion on transformation of lymphocytes and antibody-producing function of spleen cells.

Pancreatic blood perfusion in sodium taurocholate－induced pancreatitis in rats

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Establishment of an Infected Necrotizing Pancreatitis Model by Retrograde Pancreatic Duct Injection of Sodium Taurocholate and E. coli in Rats

A stable and reliable infected necrotizing pancreatitis （INP） model in rats was established in order to study the pathophysiological mechanism and pathological development role 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 taurocholate in combination with different concentrations of E. coli （10^3, 10^4, 10^5/mE, 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 pancreatic tissue. The results showed that acute necrotizing pancreatitis 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 pancreatic duct and the positive rate of germ cultivation in group B was 0. The INP model was established in groups C to E. The positive rate of germ cultivation was 60%, 100% and 100% and 8-h survival-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 10^4/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 be that the hemorrhage and necrosis of pancreatic tissue induced 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.

Basic Experimental Pancreatitis Models for Beginners

Efforts to find an ideal model for pancreatitis date back to 1960’s. Many models are suggested since then. Every model has its own advantages and disadvantages. Some of these models test etiology while others simulate the complications of pancreatitis. An ideal model which by itself demonstrates all aspects of pancreatitis including systemic changes is yet to be described. In this review we tried to gather the basic, easy to construct models.

Oral nano-formulation improves pancreatic islets dysfunction via lymphatic transport for antidiabetic treatment

Type 2 diabetes mellitus(T2DM)therapy is facing the challenges of long-term medication and gradual destruction of pancreatic isletβ-cells.Therefore,it is timely to develop oral prolonged action formulations to improve compliance,while restoringβ-cells survival and function.Herein,we designed a simple nanoparticle with enhanced oral absorption and pancreas accumulation property,which combined apical sodiumdependent bile acid transporter-mediated intestinal uptake and lymphatic transportation.In this system,taurocholic acid(TCA)modified poly(lactic-co-glycolic acid)(PLGA)was employed to achieve pancreas location,hydroxychloroquine(HCQ)was loaded to execute therapeutic efficacy,and 1,2-dilauroyl-sn-glycero-3-phosphocholine(DLPC)was introduced as stabilizer together with synergist(PLGA-TCA/DLPC/HCQ).In vitro and in vivo results have proven that PLGA-TCA/DLPC/HCQ reversed the pancreatic islets damage and dysfunction,thus impeding hyperglycemia progression and restoring systemic glucose homeostasis via only once administration every day.In terms of mechanism PLGA-TCA/DLPC/HCQ ameliorated oxidative stress,remodeled the inflammatory pancreas microenvironment,and activated PI3K/AKT signaling pathway without obvious toxicity.This strategy not only provides an oral delivery platform for increasing absorption and pancreas targetability but also opens a new avenue for thorough T2DM treatment.

Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived(Huh7) cells

Feasible and effective cell models for hepatitis B virus(HBV) infection are required for investigating the complete lifecycle of this virus, including the early steps of viral entry. Resistance to dimethyl sulfoxide/polyethylene glycol(DMSO/PEG), h NTCP expression, and a differentiated state are the limiting factors for successful HBV infection models. In the present study, we used a hepatoma cell line(Hu7^(hDNTCPh)) to overcome these limiting factors so that it exhibits excellent susceptibility to HBV infection. To achieve this goal, different hepatoma cell lines were tested with 2.5% DMSO/4%PEG8000, and one resistant cell line(Huh7 D) was used to construct a stable h NTCP-expressing cell line(Hu7^(hDNTCPh)) using a recombinant lentivirus system. Then, the morphological characteristics and differentiation molecular markers of Hu7^(hDNTCPh) cells with or without DMSO treatment were characterized. Finally, the susceptibility of Hu7^(hDNTCPh) cells to HBV infection was assessed. Our results showed that Huh7 D cells were resistant to 2.5% DMSO/4% PEG8000, whereas the others were not. Hu7^(hDNTCPh) cells were established to express a high level of h NTCP compared to liver extracts, and Hu7^(hDNTCPh) cells rapidly transformed into a non-dividing, well-differentiated polarized phenotype under DMSO treatment. Hu7^(hDNTCPh) cells fully supported the entire lifecycle of HBV infection. This cell culture system will be useful for the analysis of host-virus interactions, which should facilitate the discovery of antiviral drugs and vaccines.

Discovery of a novel sodium taurocholate cotransporting polypeptide(NTCP) inhibitor: Design, synthesis, and anti-proliferative activities

Sodium taurocholate cotransporting polypeptide(NTCP)is identified as the functional receptor for HBV entry,which is responsible for upregulated HBV transcription in the HBV life cycle.Besides,NTCP is also implicated in the progression of HBV-induced hepatocellular carcinoma(HCC).Thereby,NTCP-targeting entry inhibitors are proposed to suppress HBV infection and replication in HBV-induced hepatoma therapy.Herein,we integrated in silico screening and chemical synthesis to obtain a small-molecule NTCP inhibitor B7,which exhibited moderate anti-proliferative activities against HepG2 cells and anti-HBV activity in vitro.Additionally,CETSA assay,molecular docking,and MD simulation validated that B7 could bind to NTCP.Furthermore,western blot analysis demonstrated that B7 induced apoptosis with an increased expression of Bax and caspase 3 cleaving as well as a decreasing expression of Bcl-2 in HepG2 cells.Taken together,our study identified B7 as a novel NTCP inhibitor with anti-proliferation activities which might provide a new opportunity for HCC therapy.

The S267F variant of sodium taurocholate co-transporting polypeptide is strongly associated with resistance to chronic hepatitis B and high level of serum total bile acids

Background and aims:The sodium taurocholate co-transporting polypeptide(NTCP)is a functional receptor for the hepatitis B virus(HBV),and it is critical for bile acid homeostasis.Previous studies of the association between the S267F variant and chronic hepatitis B(CHB)have generated conflicting results.This study analyzed the correlation between the NTCP S267F variant and CHB susceptibility by using a large sample of participants classified by gender and age,and this study also analyzed the relationship between this variant and the level of serum total bile acids.Methods:In total,543 patients with CHB and 429 control subjects underwent S267F variant genotyping using SNaPshot technology.Logistic regression was utilized to evaluate any association of the NTCP S267F variant with CHB susceptibility.Results:The S267F variant was inversely correlated with the risk of chronic HBV infection in both the dominant model(GG genotype vs.AG genotype:odds ratio(OR)=0.46,95%confidence interval(CI)0.30 -0.71,P<0.001)and the allele model(G allele vs.A allele:OR=0.50,95%CI 0.33-0.76,P=0.001),and this correlation was not affected by gender and age stratification.The carriers of the heterozygous NTCP variant exhibited higher total bile acids levels than the carriers of wild-type NTCP,regardless of whether they were control subjects or patients with CHB.Heterozygous carriers exhibited reduced hepatitis B e antigen(HBeAg)-positivity rates and had lower ALT,AST,and lg DNA concentrations compared with wild-type carriers in patients with CHB.Conclusions:The S267F variant of NTCP is a protective factor that reduces the risk of chronic HBV infection and exhibits a higher total bile acids level.Patients with CHB who carry this variant may have a better prognosis than those carrying wild-type NTCP.