目的:以牛磺熊去氧胆酸和牛磺鹅去氧胆酸为指标,建立龙泽熊胆胶囊中熊胆粉的含量测定方法。方法:采用高效液相色谱串联蒸发光检测器,色谱柱为ChromCore AQ C 18(4.6 mm×250 mm,5μm),乙腈(A)-5 mmol•L^(-1)醋酸铵溶液(B)为流动相,...目的:以牛磺熊去氧胆酸和牛磺鹅去氧胆酸为指标,建立龙泽熊胆胶囊中熊胆粉的含量测定方法。方法:采用高效液相色谱串联蒸发光检测器,色谱柱为ChromCore AQ C 18(4.6 mm×250 mm,5μm),乙腈(A)-5 mmol•L^(-1)醋酸铵溶液(B)为流动相,梯度洗脱(0~40 min,25%A;40~50 min,25%A→29%A;50~80 min,29%A;80~100 min,29%A→40%A),流速1.0 mL•min^(-1),柱温30℃,ELSD漂移管温度110℃,氮流量2.5L•min^(-1)。结果:牛磺熊去氧胆酸进样量在1.069~9.57μg内、牛磺鹅去氧胆酸进样量在0.74046~7.40464μg内,进样量的对数与峰面积的对数呈良好的线性关系;仪器精密度、重复性、稳定性试验的RSD<2.0%;经低、中、高3个浓度的准确度试验考察,牛磺熊去氧胆酸的回收率为95.2%~97.7%,牛磺鹅去氧胆酸的回收率为91.9%~95.9%。测定样品42批次,牛磺熊去氧胆酸和牛磺鹅去氧胆酸的含量分别为0.18~0.43、0.10~0.44 mg•粒^(-1)。结论:本法适用于龙泽熊胆胶囊中熊胆粉的质量控制,可为完善龙泽熊胆胶囊的质量标准提供科学的依据。展开更多
Parkinson’s disease(PD)is a common neurodegenerative disease in middle-aged and elderly people.In particular,increasing evidence has showed that astrocyte-mediated neuroinflammation is involved in the pathogenesis of...Parkinson’s disease(PD)is a common neurodegenerative disease in middle-aged and elderly people.In particular,increasing evidence has showed that astrocyte-mediated neuroinflammation is involved in the pathogenesis of PD.As a precious traditional Chinese medicine,bear bile powder(BBP)has a long history of use in clinical practice.It has numerous activities,such as clearing heat,calming the liver wind and anti-inflammation,and also exhibits good therapeutic effect on convulsive epilepsy.However,whether BBP can prevent the development of PD has not been elucidated.Hence,this study was designed to explore the effect and mechanism of BBP on suppressing astrocyte-mediated neuroinflammation in a mouse model of PD.PD-like behavior was induced in the mice by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)(30 mg·kg−1)for five days,followed by BBP(50,100,and 200 mg·kg−1)treatment daily for ten days.LPS stimulated rat C6 astrocytic cells were used as a cell model of neuroinflammation.THe results indicated that BBP treatment significantly ameliorated dyskinesia,increased the levels of tyrosine hydroxylase(TH)and inhibited astrocyte hyperactivation in the substantia nigra(SN)of PD mice.Furthermore,BBP decreased the protein levels of glial fibrillary acidic protein(GFAP),cyclooxygenase 2(COX2)and inducible nitric oxide synthase(iNOS),and up-regulated the protein levels of takeda G protein-coupled receptor 5(TGR5)in the SN.Moreover,BBP significantly activated TGR5 in a dose-dependent manner,and decreased the protein levels of GFAP,iNOS and COX2,as well as the mRNA levels of GFAP,iNOS,COX2,interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α)in LPS-stimulated C6 cells.Notably,BBP suppressed the phosphorylation of protein kinase B(AKT),inhibitor of NF-κB(IκBα)and nuclear factor-κB(NF-κB)proteins in vivo and in vitro.We also observed that TGR5 inhibitor triamterene attenuated the anti-neuroinflammatory effect of BBP on LPS-stimulated C6 cells.Taken together,BBP alleviates the progression of PD mice by suppressing astrocyte-mediated inflammation via TGR5.展开更多
Objective： To evaluate the effect of Bear Bile Powder （熊胆粉, BBP） on the growth and apoptosis of HepG2 human hepatocellular carcinoma cells, and investigate the possible molecular mechanisms mediating its anti-ca...Objective： To evaluate the effect of Bear Bile Powder （熊胆粉, BBP） on the growth and apoptosis of HepG2 human hepatocellular carcinoma cells, and investigate the possible molecular mechanisms mediating its anti-cancer activity. Methods： HepG2 cells were treated with 0.4-1.0 mg/mL of BBP for 24, 48 and 72 h. The viability of HePG2 cells was determined by MTT assay. Cellular morphology was observed via phase-contrast microscopy. Fluorescence-activated cell sorting analysis with Annexin-V/propidium idodide and 5,5＇,6,6＇-tetrachloro- 1 ,1＇,3,3＇-tetraethyl-benzimidazol-carbocyanine iodide （JC-1） staining was performed to determine cell apoptosis and the loss of mitochondrial membrane potential, respectively. Activation of caspase-9 and -3 was evaluated by a colorimetric assay. Results： The treatment with 0.4-1 mg/mL of BBP for 24, 48, or 72 h respectively reduced cell viability significantly by 7%-60%, 20%-90% or 25%-98%, compared with the untreated control cells （P〈0.01）. In addition, BBP treatment induced morphological changes in HepG2 cells. Furthermore, after treated with 0, 0.4, 0.6, 0.8 and 1.0 mg/mL of BBP, apoptosis cells （including early and late apoptotic cells） were 18.0% ± 1.3%, 34.9% ± 2.2%, 33.9% ± 2.8%, 37.4% ± 2.8% and 46.0% ± 2.5%, respectively （P〈0.05）; and the percentage of cells with reduced JC-1 red fluorescence were 6.6% ± 0.8%, 8.5% ± 0.8%, 13.5% ± 1.6%, 17.6%± 2.3% and 46.7% ± 3.6%, respectively （P〈0.01）. Finally, BBP treatment significantly and dose-dependently induced activation of both caspase-9 and caspase-3 in HepG2 cells （P〈0.05）. Conclusions： BBP could inhibit the growth of HepG2 hepatocellular cancer cells through mitochondrion-mediated apoptosis, which may, in part, explain its anti-cancer activity. BBP may be a potential novel therapeutic agent for the treatment of hepatocellular carcinoma.展开更多
Objective： To evaluate the effect of bear bile powder （BBP） on angiogenesis, and investigate the underlying molecular mechanisms. Methods： A chick embryo chorioallantoic membrane （CAM） assay was used to evaluate...Objective： To evaluate the effect of bear bile powder （BBP） on angiogenesis, and investigate the underlying molecular mechanisms. Methods： A chick embryo chorioallantoic membrane （CAM） assay was used to evaluate the angiogensis in vivo. Human umbilical vein endothelial cells （HUVECs） were treated with 0, 0.25, 0.5, 0.75, and 1.0 mg/mL of BBP for 24, 48 and 72 h, respectively. The 3-（4, 5-dimethylthiazol-2-yl）-2,5- diphenyltetrazolium bromide assay was performed to determine the viability of HUVECs. Ceil cycle progression of HUVECs was examined by fluorescence-activated cell sorting （FACS） analysis with propidium iodide staining. HUVEC migration was determined by wound healing method. An ECMatrix gel system was used to evaluate the tube formation of HUVECs. The mRNA and protein expression of vascular endothelial growth factor （VEGF）-A in both HUVECs and HepG2 human cells were examined by reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay, respectively. ]Results： Compared with the untreated group, BBP inhibited angiogenesis in vivo in the CAM model （P〈0.01）. In addition, treatment with 0.25-1 mg/mL of BBP for 24, 48, or 72 h respectively reduced cell viability by 14%-27%, 29%-69% and 33%-91%, compared with the untreated control cells （P〈0.01）. Additionally, BBP inhibited the proliferation of HUVECs via blocking the cell cycle G1 to S progression, compared with the S phase of untreated cells 48.05% ± 5.00%, 0.25-0.75 mg/mL BBP reduced S phase to 40.38% ± 5.30%, 36.54 ±4.50% and 32.13 ± 3.50%, respectively （P〈0.05）. Moreover, BBP inhibited the migration and tube formation of HUVECs, compared with the tube length of untreated cells 100%± 12%, 0.25-0.75 mg/mL BBP reduced the tube length to 62% ± 9%, 43% ± 5% and 17% ± 3%, respectively （P〈0.01）. Furthermore, BBP treatment down-regulated the mRNA and protein expression levels of VEGF-A in both HepG2 cells and HUVECs. Conclusion： BBP could inhibit the angiogenesis by reducing VEGF-A expression, which may, in part, explain its anti-tumor activity.展开更多
Objective: To evaluate the inhibitory effect of bear bile powder(BBP) on hepatocellular carcinoma(HCC) growth in vivo and investigate the underlying mechanisms. Methods: A HCC xenograft mouse model was developed by pr...Objective: To evaluate the inhibitory effect of bear bile powder(BBP) on hepatocellular carcinoma(HCC) growth in vivo and investigate the underlying mechanisms. Methods: A HCC xenograft mouse model was developed by producing with huh7 cells. After 5 days following xenograft implantation, ten HCC xenograft mice were given intra-gastric administration with 10 mg/(kg·d) dose of BBP or saline for 3 weeks. Tumor growth in HCC xenograft mice was evaluated by measuring the tumor weight and volume. Cell apoptosis, proliferation or tumor angiogenesis were examined via immunohistochemical(IHC) staining for transferase-mediated deoxyuridine triphosphate-biotin nick end labeling(TUNEL), proliferating cell nuclear antigen(PCNA) or cluster of differentiation 31(CD31), respectively. Phosphorylation of signal transducer and activator of transcription 3(STAT3) were determined by Western blot. The m RNA and protein expressions of Bcl-2, Bax, Cyclin D1 and Cyclin-dependent kinase 4(CDK4) in HCC tumor tissues were respectively determined by reverse transcription polymerase chain reaction(RT-PCR) and Western blot. The protein expression of vascular endothelial growth factor A(VEGF-A) in tumor tissues was examined by IHC staining. Results: BBP treatment led to a significant decrease on tumor volume and tumor weight in HCC mice(P<0.05) and had no effect on the change of body weight. In addition, BBP profoundly promoted cell apoptosis, inhibited cell proliferation and intratumoral microvessel density in HCC tumor tissues(P<0.05). Moreover, BBP treatment remarkably suppressed the STAT3 phosphorylation and modulated the expression of critical target genes including Bcl-2, Bax, Cyclin D1, CDK4 and VEGF-A in HCC mice. Conclusion: BBP exerts its anti-cancer activities via suppressing STAT3 signaling pathway and affecting multiple intracellular targets.展开更多
Hepatic sinusoidal obstruction syndrome(HSOS)via exposure to pyrrolizidine alkaloids(PAs)is with high mortality and there is no effective treatment in clinics.Bear bile powder(BBP)is a famous traditional animal drug f...Hepatic sinusoidal obstruction syndrome(HSOS)via exposure to pyrrolizidine alkaloids(PAs)is with high mortality and there is no effective treatment in clinics.Bear bile powder(BBP)is a famous traditional animal drug for curing a variety of hepatobiliary diseases such as cholestasis,inflammation,and fibrosis.Here,we aim to evaluate the protective effect of BBP against HSOS induced by senecionine,a highly hepatotoxic PA compound.Our results showed that BBP treatment protected mice from senecionine-induced HSOS dose-dependently,which was evident by improved liver histology including reduced infiltration of inflammatory cells and collagen positive cells,alleviated intrahepatic hemorrhage and hepatic sinusoidal endothelial cells,as well as decreased conventional serum liver function indicators.In addition,BBP treatment lowered matrix metalloproteinase 9 and pyrrole-protein adducts,two well-known markers positively associated with the severity of PA-induced HSOS.Further investigation showed that BBP treatment prevents the development of liver fibrosis by decreasing transforming growth factor beta and downstream fibrotic molecules.BBP treatment also alleviated senecionine-induced liver inflammation and lowered the pro-inflammatory cytokines,in which taurours-odeoxycholic acid played an important role.What’s more,BBP treatment also decreased the accumulation of hydrophobic bile acids,such as cholic acid,taurocholic acid,glycocholic acid,as well.We concluded that BBP attenuates senecionine-induced HSOS in mice by repairing the bile acids homeostasis,preventing liver fibrosis,and alleviating liver inflammation.Our present study helps to pave the way to therapeutic approaches of the treatment of PA-induced liver injury in clinics.展开更多
基金the Organizational Key Research and Development Program of Shanghai University of Traditional Chinese Medicine(No.2023YZZ02).
文摘Parkinson’s disease(PD)is a common neurodegenerative disease in middle-aged and elderly people.In particular,increasing evidence has showed that astrocyte-mediated neuroinflammation is involved in the pathogenesis of PD.As a precious traditional Chinese medicine,bear bile powder(BBP)has a long history of use in clinical practice.It has numerous activities,such as clearing heat,calming the liver wind and anti-inflammation,and also exhibits good therapeutic effect on convulsive epilepsy.However,whether BBP can prevent the development of PD has not been elucidated.Hence,this study was designed to explore the effect and mechanism of BBP on suppressing astrocyte-mediated neuroinflammation in a mouse model of PD.PD-like behavior was induced in the mice by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)(30 mg·kg−1)for five days,followed by BBP(50,100,and 200 mg·kg−1)treatment daily for ten days.LPS stimulated rat C6 astrocytic cells were used as a cell model of neuroinflammation.THe results indicated that BBP treatment significantly ameliorated dyskinesia,increased the levels of tyrosine hydroxylase(TH)and inhibited astrocyte hyperactivation in the substantia nigra(SN)of PD mice.Furthermore,BBP decreased the protein levels of glial fibrillary acidic protein(GFAP),cyclooxygenase 2(COX2)and inducible nitric oxide synthase(iNOS),and up-regulated the protein levels of takeda G protein-coupled receptor 5(TGR5)in the SN.Moreover,BBP significantly activated TGR5 in a dose-dependent manner,and decreased the protein levels of GFAP,iNOS and COX2,as well as the mRNA levels of GFAP,iNOS,COX2,interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α)in LPS-stimulated C6 cells.Notably,BBP suppressed the phosphorylation of protein kinase B(AKT),inhibitor of NF-κB(IκBα)and nuclear factor-κB(NF-κB)proteins in vivo and in vitro.We also observed that TGR5 inhibitor triamterene attenuated the anti-neuroinflammatory effect of BBP on LPS-stimulated C6 cells.Taken together,BBP alleviates the progression of PD mice by suppressing astrocyte-mediated inflammation via TGR5.
基金Supported by Important Science&Technology Specific Projects of Fujian Province(No.2010YZ0001-1 and 2010Y2004)Developmental Fund of Chen Keji Integrative Medicine(No.CKJ2010019)
文摘Objective： To evaluate the effect of Bear Bile Powder （熊胆粉, BBP） on the growth and apoptosis of HepG2 human hepatocellular carcinoma cells, and investigate the possible molecular mechanisms mediating its anti-cancer activity. Methods： HepG2 cells were treated with 0.4-1.0 mg/mL of BBP for 24, 48 and 72 h. The viability of HePG2 cells was determined by MTT assay. Cellular morphology was observed via phase-contrast microscopy. Fluorescence-activated cell sorting analysis with Annexin-V/propidium idodide and 5,5＇,6,6＇-tetrachloro- 1 ,1＇,3,3＇-tetraethyl-benzimidazol-carbocyanine iodide （JC-1） staining was performed to determine cell apoptosis and the loss of mitochondrial membrane potential, respectively. Activation of caspase-9 and -3 was evaluated by a colorimetric assay. Results： The treatment with 0.4-1 mg/mL of BBP for 24, 48, or 72 h respectively reduced cell viability significantly by 7%-60%, 20%-90% or 25%-98%, compared with the untreated control cells （P〈0.01）. In addition, BBP treatment induced morphological changes in HepG2 cells. Furthermore, after treated with 0, 0.4, 0.6, 0.8 and 1.0 mg/mL of BBP, apoptosis cells （including early and late apoptotic cells） were 18.0% ± 1.3%, 34.9% ± 2.2%, 33.9% ± 2.8%, 37.4% ± 2.8% and 46.0% ± 2.5%, respectively （P〈0.05）; and the percentage of cells with reduced JC-1 red fluorescence were 6.6% ± 0.8%, 8.5% ± 0.8%, 13.5% ± 1.6%, 17.6%± 2.3% and 46.7% ± 3.6%, respectively （P〈0.01）. Finally, BBP treatment significantly and dose-dependently induced activation of both caspase-9 and caspase-3 in HepG2 cells （P〈0.05）. Conclusions： BBP could inhibit the growth of HepG2 hepatocellular cancer cells through mitochondrion-mediated apoptosis, which may, in part, explain its anti-cancer activity. BBP may be a potential novel therapeutic agent for the treatment of hepatocellular carcinoma.
基金Supported by Important Science and Technology Specific Projects of Fujian Province(No.2010YZ0001-1 and2010Y2004)the Developmental Fund of Chen Ke-ji Integrative Medicine(No.CKJ 2010019)
文摘Objective： To evaluate the effect of bear bile powder （BBP） on angiogenesis, and investigate the underlying molecular mechanisms. Methods： A chick embryo chorioallantoic membrane （CAM） assay was used to evaluate the angiogensis in vivo. Human umbilical vein endothelial cells （HUVECs） were treated with 0, 0.25, 0.5, 0.75, and 1.0 mg/mL of BBP for 24, 48 and 72 h, respectively. The 3-（4, 5-dimethylthiazol-2-yl）-2,5- diphenyltetrazolium bromide assay was performed to determine the viability of HUVECs. Ceil cycle progression of HUVECs was examined by fluorescence-activated cell sorting （FACS） analysis with propidium iodide staining. HUVEC migration was determined by wound healing method. An ECMatrix gel system was used to evaluate the tube formation of HUVECs. The mRNA and protein expression of vascular endothelial growth factor （VEGF）-A in both HUVECs and HepG2 human cells were examined by reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay, respectively. ]Results： Compared with the untreated group, BBP inhibited angiogenesis in vivo in the CAM model （P〈0.01）. In addition, treatment with 0.25-1 mg/mL of BBP for 24, 48, or 72 h respectively reduced cell viability by 14%-27%, 29%-69% and 33%-91%, compared with the untreated control cells （P〈0.01）. Additionally, BBP inhibited the proliferation of HUVECs via blocking the cell cycle G1 to S progression, compared with the S phase of untreated cells 48.05% ± 5.00%, 0.25-0.75 mg/mL BBP reduced S phase to 40.38% ± 5.30%, 36.54 ±4.50% and 32.13 ± 3.50%, respectively （P〈0.05）. Moreover, BBP inhibited the migration and tube formation of HUVECs, compared with the tube length of untreated cells 100%± 12%, 0.25-0.75 mg/mL BBP reduced the tube length to 62% ± 9%, 43% ± 5% and 17% ± 3%, respectively （P〈0.01）. Furthermore, BBP treatment down-regulated the mRNA and protein expression levels of VEGF-A in both HepG2 cells and HUVECs. Conclusion： BBP could inhibit the angiogenesis by reducing VEGF-A expression, which may, in part, explain its anti-tumor activity.
基金Supported by the Developmental Fund of CHEN Ke-ji Integrative Medicine(No.CKJ2013009)。
文摘Objective: To evaluate the inhibitory effect of bear bile powder(BBP) on hepatocellular carcinoma(HCC) growth in vivo and investigate the underlying mechanisms. Methods: A HCC xenograft mouse model was developed by producing with huh7 cells. After 5 days following xenograft implantation, ten HCC xenograft mice were given intra-gastric administration with 10 mg/(kg·d) dose of BBP or saline for 3 weeks. Tumor growth in HCC xenograft mice was evaluated by measuring the tumor weight and volume. Cell apoptosis, proliferation or tumor angiogenesis were examined via immunohistochemical(IHC) staining for transferase-mediated deoxyuridine triphosphate-biotin nick end labeling(TUNEL), proliferating cell nuclear antigen(PCNA) or cluster of differentiation 31(CD31), respectively. Phosphorylation of signal transducer and activator of transcription 3(STAT3) were determined by Western blot. The m RNA and protein expressions of Bcl-2, Bax, Cyclin D1 and Cyclin-dependent kinase 4(CDK4) in HCC tumor tissues were respectively determined by reverse transcription polymerase chain reaction(RT-PCR) and Western blot. The protein expression of vascular endothelial growth factor A(VEGF-A) in tumor tissues was examined by IHC staining. Results: BBP treatment led to a significant decrease on tumor volume and tumor weight in HCC mice(P<0.05) and had no effect on the change of body weight. In addition, BBP profoundly promoted cell apoptosis, inhibited cell proliferation and intratumoral microvessel density in HCC tumor tissues(P<0.05). Moreover, BBP treatment remarkably suppressed the STAT3 phosphorylation and modulated the expression of critical target genes including Bcl-2, Bax, Cyclin D1, CDK4 and VEGF-A in HCC mice. Conclusion: BBP exerts its anti-cancer activities via suppressing STAT3 signaling pathway and affecting multiple intracellular targets.
基金This work was supported by Shanghai Natural Science Foundation(20ZR1473300)Shanghai Talents Development Foundation(2020099)+1 种基金the Program of Shanghai Municipal Commission of Health and Family Planning(ZY(2018-2020)-CCCX-5002)the Xinglin Scholar Program of Shanghai University of Traditional Chinese Medicine(B1-GY21-409-04-06).
文摘Hepatic sinusoidal obstruction syndrome(HSOS)via exposure to pyrrolizidine alkaloids(PAs)is with high mortality and there is no effective treatment in clinics.Bear bile powder(BBP)is a famous traditional animal drug for curing a variety of hepatobiliary diseases such as cholestasis,inflammation,and fibrosis.Here,we aim to evaluate the protective effect of BBP against HSOS induced by senecionine,a highly hepatotoxic PA compound.Our results showed that BBP treatment protected mice from senecionine-induced HSOS dose-dependently,which was evident by improved liver histology including reduced infiltration of inflammatory cells and collagen positive cells,alleviated intrahepatic hemorrhage and hepatic sinusoidal endothelial cells,as well as decreased conventional serum liver function indicators.In addition,BBP treatment lowered matrix metalloproteinase 9 and pyrrole-protein adducts,two well-known markers positively associated with the severity of PA-induced HSOS.Further investigation showed that BBP treatment prevents the development of liver fibrosis by decreasing transforming growth factor beta and downstream fibrotic molecules.BBP treatment also alleviated senecionine-induced liver inflammation and lowered the pro-inflammatory cytokines,in which taurours-odeoxycholic acid played an important role.What’s more,BBP treatment also decreased the accumulation of hydrophobic bile acids,such as cholic acid,taurocholic acid,glycocholic acid,as well.We concluded that BBP attenuates senecionine-induced HSOS in mice by repairing the bile acids homeostasis,preventing liver fibrosis,and alleviating liver inflammation.Our present study helps to pave the way to therapeutic approaches of the treatment of PA-induced liver injury in clinics.
