Recent studies have revealed that human sodium taurocholate cotransporting polypeptide （SLC10A1 or NTCP） is a functional cellular receptor for hepatitis B virus （HBV）. However, whether human NTCP can support HBV i...Recent studies have revealed that human sodium taurocholate cotransporting polypeptide （SLC10A1 or NTCP） is a functional cellular receptor for hepatitis B virus （HBV）. However, whether human NTCP can support HBV infection in mouse hepatocyte cell lines has not been clarified. Because an HBV-permissible mouse model would be helpful for the study of HBV pathogenesis, it is necessary to investigate whether human NTCP supports the susceptibility of mouse hepatocyte cell lines to HBV. The results show that exogenous human NTCP expression can render non-susceptible HepG2 （human）, Huh7 （human）, Hepal-6 （mouse）, AML-12 （mouse） cell lines and primary mouse hepatocyte （PMH） cells susceptible to hepatitis D virus （HDV） which employs HBV envelope proteins. However, human NTCP could only introduce HBV susceptibility in human-derived HepG2 and Huh7 cells, but not in mouse-derived Hepal-6, AML-12 or PMH cells. These data suggest that although human NTCP is a functional receptor that mediates HBV infection in human cells, it cannot support HBV infection in mouse hepatocytes. Our study indicated that the restriction of HBV in mouse hepatocytes likely occurs after viral entry but prior to viral transcription. We have excluded the role of mouse hepatocyte nuclear factors in the restriction of the HBV life cycle and showed that knockdown or inhibition of Sting, TBK1, IRF3 or IRF7, the components of the anti-viral signaling pathways, had no effect on HBV infection in mouse hepatocytes. Therefore, murine restriction factors that limit HBV infection need to be identified before a HBV-permissible mouse line can be created.展开更多
NTCP is specifically expressed on the basolateral membrane of hepatocytes, participating in the enterohepatic circulation of bile salts, especially conjugated bile salts, to maintain bile salts homeostasis. In additio...NTCP is specifically expressed on the basolateral membrane of hepatocytes, participating in the enterohepatic circulation of bile salts, especially conjugated bile salts, to maintain bile salts homeostasis. In addition, recent studies have found that NTCP is a functional receptor of HBV and HDV. Therefore, it is important to study the interaction between drugs and NTCP and identify the inhibitors/substrates of NTCP. In the present study, a LLC-PK1 cell model stably expressing human NTCP was established, which was simple and suitable for high throughput screening, and utilized to screen and verify the potential inhibitors of NTCP from 102 herbal medicinal ingredients. The results showed that ginkgolic acid(GA)(13 : 0), GA(15 : 1), GA(17 : 1), erythrosine B, silibinin, and emodin have inhibitory effects on NTCP uptake of TCNa in a concentration-dependent manner. Among them, GA(13 : 0) and GA(15 : 1) exhibited the stronger inhibitory effects, with IC_(50) values being less than 8.3 and 13.5 mmol·L^(-1), respectively, than the classical inhibitor, cyclosporin A(CsA)(IC_(50) = 20.33 mmol·L^(-1)). Further research demonstrated that GA(13 : 0), GA(15 : 1), GA(17 : 1), silibinin, and emodin were not substrates of NTCP. These findings might contribute to a better understanding of the disposition of the herbal ingredients in vivo, especially in biliary excretion.展开更多
Sodium taurocholate co-transporting polypeptide(NTCP)is encoded by the solute carrier family 10 member 1(SLC10A1)gene and is predominantly expressed on the sinusoidal membrane of hepatocytes with the function of hepat...Sodium taurocholate co-transporting polypeptide(NTCP)is encoded by the solute carrier family 10 member 1(SLC10A1)gene and is predominantly expressed on the sinusoidal membrane of hepatocytes with the function of hepatic uptake of bile salt,steroid hormones,thyroid hormones,and various bile-acid-conjugated drugs.Almost 10 years ago,NTCP was identified as a functional cellular entry receptor of hepatitis B and hepatitis D viruses(HBV/HDV,respectively),1 which are among the major etiological factors leading to cirrhosis,liver failure,and hepatocellular carcinoma and affect around 250 million people worldwide.Previous studies have demonstrated that the binding site of HBV with NTCP is within the first 48 amino acid residues of the N-myristoylated preS1(myrpreS1)domain of the large envelope glycoprotein.Genetic variations,such as amino acids 84–87 and 157–165 in the NTCP sequences,are responsible for susceptibility to HBV in different species.Individuals who carry the NTCP p.S267F polymorphism on both alleles are associated with increased resistance to chronic HBV infection.Biochemical and virology studies have shown common molecular determinants as well as function separation on the transporting of bile acids and mediating HBV/HDV infection.However,important questions remain unanswered:for example,the binding mode of bile acid or the preS1 lipopeptide to NTCP on the hepatocyte surface,how to differentiate the pathway of substrate uptake and virus infection,and the dynamics of substrate uptake and the molecular mechanisms of virus infection are all unknown。展开更多
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 i...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.展开更多
文摘Recent studies have revealed that human sodium taurocholate cotransporting polypeptide （SLC10A1 or NTCP） is a functional cellular receptor for hepatitis B virus （HBV）. However, whether human NTCP can support HBV infection in mouse hepatocyte cell lines has not been clarified. Because an HBV-permissible mouse model would be helpful for the study of HBV pathogenesis, it is necessary to investigate whether human NTCP supports the susceptibility of mouse hepatocyte cell lines to HBV. The results show that exogenous human NTCP expression can render non-susceptible HepG2 （human）, Huh7 （human）, Hepal-6 （mouse）, AML-12 （mouse） cell lines and primary mouse hepatocyte （PMH） cells susceptible to hepatitis D virus （HDV） which employs HBV envelope proteins. However, human NTCP could only introduce HBV susceptibility in human-derived HepG2 and Huh7 cells, but not in mouse-derived Hepal-6, AML-12 or PMH cells. These data suggest that although human NTCP is a functional receptor that mediates HBV infection in human cells, it cannot support HBV infection in mouse hepatocytes. Our study indicated that the restriction of HBV in mouse hepatocytes likely occurs after viral entry but prior to viral transcription. We have excluded the role of mouse hepatocyte nuclear factors in the restriction of the HBV life cycle and showed that knockdown or inhibition of Sting, TBK1, IRF3 or IRF7, the components of the anti-viral signaling pathways, had no effect on HBV infection in mouse hepatocytes. Therefore, murine restriction factors that limit HBV infection need to be identified before a HBV-permissible mouse line can be created.
基金supported by National Nature Scientific Foundation of China(No.8117312)International Science&Technology Cooperation Program of China(No.2014DFE30050)+1 种基金Program for Zhejiang Leading Team of S&T Innovation Team(No.2011R50014)Fundamental Research Funds for the Central Universities of China Ministry of Education(2016XZZX001-08)
文摘NTCP is specifically expressed on the basolateral membrane of hepatocytes, participating in the enterohepatic circulation of bile salts, especially conjugated bile salts, to maintain bile salts homeostasis. In addition, recent studies have found that NTCP is a functional receptor of HBV and HDV. Therefore, it is important to study the interaction between drugs and NTCP and identify the inhibitors/substrates of NTCP. In the present study, a LLC-PK1 cell model stably expressing human NTCP was established, which was simple and suitable for high throughput screening, and utilized to screen and verify the potential inhibitors of NTCP from 102 herbal medicinal ingredients. The results showed that ginkgolic acid(GA)(13 : 0), GA(15 : 1), GA(17 : 1), erythrosine B, silibinin, and emodin have inhibitory effects on NTCP uptake of TCNa in a concentration-dependent manner. Among them, GA(13 : 0) and GA(15 : 1) exhibited the stronger inhibitory effects, with IC_(50) values being less than 8.3 and 13.5 mmol·L^(-1), respectively, than the classical inhibitor, cyclosporin A(CsA)(IC_(50) = 20.33 mmol·L^(-1)). Further research demonstrated that GA(13 : 0), GA(15 : 1), GA(17 : 1), silibinin, and emodin were not substrates of NTCP. These findings might contribute to a better understanding of the disposition of the herbal ingredients in vivo, especially in biliary excretion.
文摘Sodium taurocholate co-transporting polypeptide(NTCP)is encoded by the solute carrier family 10 member 1(SLC10A1)gene and is predominantly expressed on the sinusoidal membrane of hepatocytes with the function of hepatic uptake of bile salt,steroid hormones,thyroid hormones,and various bile-acid-conjugated drugs.Almost 10 years ago,NTCP was identified as a functional cellular entry receptor of hepatitis B and hepatitis D viruses(HBV/HDV,respectively),1 which are among the major etiological factors leading to cirrhosis,liver failure,and hepatocellular carcinoma and affect around 250 million people worldwide.Previous studies have demonstrated that the binding site of HBV with NTCP is within the first 48 amino acid residues of the N-myristoylated preS1(myrpreS1)domain of the large envelope glycoprotein.Genetic variations,such as amino acids 84–87 and 157–165 in the NTCP sequences,are responsible for susceptibility to HBV in different species.Individuals who carry the NTCP p.S267F polymorphism on both alleles are associated with increased resistance to chronic HBV infection.Biochemical and virology studies have shown common molecular determinants as well as function separation on the transporting of bile acids and mediating HBV/HDV infection.However,important questions remain unanswered:for example,the binding mode of bile acid or the preS1 lipopeptide to NTCP on the hepatocyte surface,how to differentiate the pathway of substrate uptake and virus infection,and the dynamics of substrate uptake and the molecular mechanisms of virus infection are all unknown。
基金supported by National Science and Technology Major Project of the Ministry of Science and Technology of China(No.2018ZX09735005)the National Natural Science Foundation of China(Nos.81922064,81874290,81673290,81803347 and 81903502)+1 种基金the Natural Science Foundation of Guangdong Province(No.2018A030313707)Post-Doctor Research Project,West China Hospital,Sichuan University(No.2019HXBH034)。
文摘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.
