Bear bile powder alleviates Parkinson’s disease-like behavior in mice by inhibiting astrocyte-mediated neuroinflammation

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.

Bear Bile Powder(熊胆粉) Induces Apoptosis of Human Hepatocellular Carcinoma Cells via Mitochondrion-Dependent Pathway

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.

Bear Bile Powder Inhibits Angiogenesis In Vivo and In Vitro

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.

Bear bile powder attenuates senecionine-induced hepatic sinusoidal obstruction syndrome in mice

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.

Therapeutic Effects of Different Animal Bile Powders on Lipid Metabolism Disorders and Their Composition Analysis

Objective: To compare the therapeutic effect of different animal bile powders on lipid metabolism disorders induced by high-fat diet in rats, and analyze the bioactive components of each animal bile powder. Methods: Sixty Sprague-Dawley rats were randomly divided into 6 groups(n=10): normal diet control group, high-fat diet model group, high-fat diet groups orally treated with bear, pig, cow and chicken bile powders, respectively. Serum biochemical markers from the abdominal aorta in each group were analyzed. Changes in the body weight and liver weight were recorded. Pathohistological changes in the livers were examined. High performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry was used to determine the composition of bioactive components in each animal bile powder. Results: Treatment with different types of animal bile powders had different inhibitory effects on high-fat diet-induced increase of body weight and/or liver weight in rats, most notably in bear and pig bile powders(P<0.05). High-fat diet induced lipid metabolism disorder in rats, which could be reversed by treatment with all kinds of bile powders. Bear bile and chicken bile showed the most potent therapeutic effect against lipid metabolism disorder. Cow and bear bile effectively alleviated high-fat diet induced liver enlargement and discoloration, hepatocyte swelling, infiltration of inflammatory cells and formation of lipid vacuoles. Bioactive component analysis revealed that there were significant differences in the relative content of taurocholic acid, taurodeoxycholic acid and ursodeoxycholic acid among different types of animal bile. Interestingly, a unique component with molecular weight of 496.2738 Da, whose function has not yet been reported, was identified only in bear bile powder. Conclusions: Different animal bile powders had varying therapeutic effect against lipid metabolism disorders induced by high-fat diet, and bear bile powder demonstrated the most effective benefits. Bioactive compositions were different in different types of animal bile with a novel compound identified only in bear bile powder.