OBJECTIVE Hepatic fibrosis is a wound-healing response for injury.Activated hepatic stellate cells(HSCs)are the preferred targets of anti-hepatic fibrotic therapies.cucurbitacin E(CuE)is,one well-known natural compoun...OBJECTIVE Hepatic fibrosis is a wound-healing response for injury.Activated hepatic stellate cells(HSCs)are the preferred targets of anti-hepatic fibrotic therapies.cucurbitacin E(CuE)is,one well-known natural compound derived from traditional Chinese medicine,used in Asian countries for the prevention and treatment of hepatic disease.Therefore,the present study elucidated the mechanism of CuE on inducing apoptosis and attenuating hepatic fibrosis towards activated HSCs.METHODS The murine HSC(tHSC/Cl-6)cell line were incubated in 96-well plates and treated with TNF-αand CuE at various concentrations and indicated times.Cell viability was assessed with MTT assay.Another,t-HSC/Cl-6 were incubated in 6-well plates and also treated with TNF-α,CuE,AICAR or metformin for the indicated time and concentration.Cell protein and mRNA were prepared using kit and relevant signaling were detected by Western blotting and RT-PCR.RESULTS CuE inhibited cell proliferation of activated HSC/T-6cells in concentration-and time-dependent manner.CuE triggered the activation of caspase-3,cleaved the PARP,ration of bcl-2/bax,and cytochrom c protein in a time-and concentration-dependent manner.CuE decreased p-Erk/MAPK without effects on p-p38 and p-JNK.CuE inhibited the protein and mRNA expressions ofα-SMA,TIMP-1 and collagenⅠ in activated HSC-T6.CuE broadly blocked p-PI3 K,p-Akt,p-mTOR and p-p70S6 Kexpressions.CuE significantly increased phosphorylated AMPK expression as well as AICAR and metoformin.And metformin showed significantly higher activation of AMPK than AICAR.Ability of CuE on activation of AMPK was between AICAR and metformin.It′s also found that CuE significantly decreased p-mTOR as well as AICAR and metformin.CONCLUSION CuE could modulate HSC survival and activation as a potential anti-fibrotic agent for liver fibrosis treatment.The findings demonstrate that CuE induced HSC apoptosis via ERK/MAPK and PI3K/Akt-AMPK-mTOR signaling.展开更多
OBJECTIVE To investigate the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells(HSCs).METHODS Normal human Chang liver cells and human hepatic stellate cell line,LX-2 cells were t...OBJECTIVE To investigate the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells(HSCs).METHODS Normal human Chang liver cells and human hepatic stellate cell line,LX-2 cells were treated with SRT1720(10μmol·L^(-1))and AICAR(500μmol·L^(-1))prior to ethanol(50 mmol·L^(-1)) for 24 and 48 h.Cell viability was analyzed by methyl thiazolyl tetrazolium assay.SIRT1,AMPK and p-AMPK m RNA levels for 24 h and 48 h were analyzed by RT-PCR,SIRT1,AMPK and p-AMPK protein expressions in the supernatant at 24 and 48 h was detected by Western blot.RESULTS SRT1720 and AICAR effectively decreased LX-2 cell viabilities and exhibited scarcely little toxicity in human Chang liver cells.SRT1720 and AICAR attenuated collagen-I,α-smooth muscle actin(α-SMA)levels,activated liver kinase B-1(LKB1)and AMPK phosphorylation in ethanol treated LX-2 cells.Meanwhile,SRT1720 and AICAR enhanced SIRT1 expression mediated by ethanol both in Chang liver cells and LX-2 cells.Furthermore,SRT1720 and AICAR suppressed the expression of sterol regulatory element-binding protein-1(SREBP-1)to regulate fatty acid synthesis.CONCLUSION SIRT1 agonist and AMPK agonist blocked the crosstalk between hepatocytes and HSCs via SIRT1/AMPK signaling pathway to modulate hepatocytes accumulation of lipid and HSCs activation.展开更多
OBJECTIVE The pathological characteristics of nonalcoholic steatohepatitis(NASH)include liver steatosis,inflammation,and fibrosis.Fibrosis is the most severe and significant pathological feature in NASH.Effective drug...OBJECTIVE The pathological characteristics of nonalcoholic steatohepatitis(NASH)include liver steatosis,inflammation,and fibrosis.Fibrosis is the most severe and significant pathological feature in NASH.Effective drug treatment could reverse early liver fibrosis and is of significance to prevent NASH from progressing into cirrhosis and liver cancer.Identification of drug targets for NASH treatment has been an active research area and is essential for the development of anti-NASH medications.Naringenin(NGN)is a flavonoid compound rich in citrus fruits.Our preliminary data demonstrated that NGN reduced diet-induced lipid accumulation and inflammation in the mouse liver,but whether NGN can attenuate liver fibrosis of NASH is not known.METHODS To study the effect of NGN on NASH fibrosis.WT mice were fed with high fat diet(HFD)and injected intraperitoneally 20%carbon tetrachloride at the same time for 8 weeks to induce NASH,and NGN was administrated by gavage in the meantime.In vitro,LO2 cells and LX2 cells were stimulated by oleic acid(OA)combined with lipopolysaccharide(LPS),respectively.RESULTS Treating the WT mice with NGN 100 mg·kg^(-1)·d-1 significantly attenuated hepatic lipid accumulation,hepatic fibrosis,plasma ALT and AST levels,inhibited protein expression of p-ERK,p-FoxO3a in the mouse livers.In vitro,on OA and LPS stimulated LO2 or LX2 cells,NGN significantly promoted apoptosis of activated hepatic stellate cells while inhibited apoptosis of hepatocytes.Mechanism study indicated that NGN inhibited MAPK pathway and promoted activation of FoxO3a,consequently promoted apoptosis of the activated LX2 cells and inhibited liver fibrosis.CONCLUSION NGN preventes NASH fibrosis via regulating MAPK/FoxO3a pathway,thus promoting apoptosis of the activated hepatic stellate cells.展开更多
Hepatic fibrosis is a wound-healing response for injury. Activated hepatic stellate cells （HSCs） are the preferred targets of anti-hepatic fibrotic therapies. Cucurbitacin E （CUE） is, one well-known natural compou...Hepatic fibrosis is a wound-healing response for injury. Activated hepatic stellate cells （HSCs） are the preferred targets of anti-hepatic fibrotic therapies. Cucurbitacin E （CUE） is, one well-known natural compound de- rived from Traditional Chinese Medicine, used in Asian countries for the prevention and treatment of hepatic dis- ease. Therefore, the present study elucidated the mechanism of CuE on inducing apoptosis and attenuating hepatic fibrosis towards activated HSCs. The murine hepatic stellate cells （t-HSC/C1-6） cell line were incubated in 96-well plates and treated with TNF-α and CuE at various concentrations and indicated times. Cell viability was assessed with MTT assay. Another, t-HSC/C1-6 were incubated in 6-well plates and also treated with TNF-α, CuE, AICAR or metformin for the indicated time and concentration. Cell protein and mRNA were prepared using kit and relevant signaling were detected by Western blot and RT-PCR. CuE inhibited cell proliferation of activated HSC/T-6 cells in concentration- and time-dependent manner. CuE triggered the activation of caspase-3, cleaved the PARP, ration of bcl-2/bax, and cytochrom C protein in a time- and concentration-dependent manner. CuE decreased p-Erk/MAPK without effects on p-p38 and p-JNK. CuE inhibited the protein and mRNA expressions of oL-SMA, TIMP-1 and col- lagen I in activated HSC-T6. CuE broadly blocked p-PI3K, p-Akt, p-roTOR and p-p70S6K expressions. CuE sig- nificantly increased phosphorylated AMPK expression as well as AICAR and metoformin. And metformin showed significantly higher activation of AMPK than AICAR. Ability of CuE on activation of AMPK was between AICAR and metformin. It＇s also found that CuE significantly decreased p-roTOR as well as AICAR and metformin. CuE could modulate HSC survival and activation as a potential anti-fibrotic agent for liver fibrosis treatment. The find- ings demonstrate that CuE induced HSC apoptosis via Erk/MAPK and PI3IC/Akt-AMPK-mTOR signaling.展开更多
OBJECTIVE To explore the therapeutic effects and underlying mechanisms of velvet antler polypeptides(VVAPs)in CCl4-induced experimental hepatic fibrosis in rats.METHODS Anti-hepatic fibrosis properties of VAPs were te...OBJECTIVE To explore the therapeutic effects and underlying mechanisms of velvet antler polypeptides(VVAPs)in CCl4-induced experimental hepatic fibrosis in rats.METHODS Anti-hepatic fibrosis properties of VAPs were tested by Subcutaneous injection(SC)into male Wistar rats of CCl4- induced experimental hepatic fibrosis.After SC injections for 45 consecutive days at doses of 5mg·kg-1(low dose,VAPsL),10mg·kg-1(mid-dose,VAPsM)and 20mg·kg-1(high-dose,VAPsH),the rats were sacrificed and the various indicators were evaluated and tested.Observed hepatic cells degeneration and necrosis,inflammatory infiltration and levels of serum enzymes to assess treatment of VAPs;The expression levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),MDA,and hydroxyproline(HYP)in liver tissue were analyzed;RT-PCR analysis was carried out to detect the expression levels of matrix metalloproteinases2(MMP-2)and tissue inhibitor of metalloproteinases 1(TIMP-1)in liver tissue.RESULTS VAPs has obvious anti-hepatic fibrosis effects.Hepatocyte swelling,fatty degeneration was significantly reduced,reducing infiltration of inflammatory cells.Release of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)decreased significantly,reduction of hyaluronic acid(HA)and laminin(LN)obviously,at the same time,the content of total protein and albumin increased significantly in serum.Activity of SOD and GSH-Px was significantly raised and the content of MDA and HYP was reduced significantly in liver tissue.Expression levels of MMP-2and TIMP-1 mRNA in liver were decreased significantly.These improvements were more significant in high-dos and mid-dose groups(P<0.05 or P<0.01 vs model group).CONCLUSION These findings suggest VAPs can significant treat the hepatic fibrosis,which may be due to protect liver cells and improve liver functions by hydroxyl radical scavenging activity and great effect of antioxidation,and decrease the gene expression of MMP-2,improving exist-environment of liver cells and decreasing the gene expression of TIMP-1,prompting degradation of extracellular matrix.展开更多
Fibrosis is a phenomenon of fibrous connective tissue increase in the organs. Activated hepatic stellate cells （HSCs） are the preferred targets of anti-hepatic fibrotic therapies. Ginseng and Platycodon grandiflorum...Fibrosis is a phenomenon of fibrous connective tissue increase in the organs. Activated hepatic stellate cells （HSCs） are the preferred targets of anti-hepatic fibrotic therapies. Ginseng and Platycodon grandiflorum are impor- tant traditional Chinese medicine herbs. The study was to investigate the anti-fibrosis effects of ginseng extract and Platycodon grandiflorum extract in HSCs. Extract include Ginseng water extract and alcohol extract, Platycodon gran- diflorum water extract and alcohol extract, combination of both water extract and alcohol extract. Activated hepatic stellate cells were treated with Ginseng and Platycodon grandiflorum extracts for 24h. The maximum concentration of the drug is 2000 mg · L^-1, the lowest concentration of 7.8 mg · L^-1 , with concentration gradient dilution. 3-（4 , 5- dimethylthiazol-2-yl）-2, 5-diphenylte-trazolium bromide （MTT） can evaluate the survival rate of cells. The expres- sions of SIRT1, NF-KB and p53 were detected by western blot. The results showed that extract effectively decreased the HSC cells viability in concentration dependent manner. Among them, the effect of combination of both extract is best. Ginseng and Platycodon grandiflorum activated the protein expressions of SIRT1 in activated HSCs. The expres- sion of NF-KB and p53 were significantly decreased by Ginseng and Platycodon grandiflorum. This study demonstrated that ginseng extract and Platycodon grandiflorum extract showed obviously anti-fibrosis effect, and decreased expres-sion of NF-KB and p53 via SIRT1 signaling pathway.展开更多
OBJECTIVE To investigate the potency of Urena lobata leaves extract on the inhibition of hepatic complication on diabetic rats.METHODS This study uses control group post test only with male Sprague dawley rats.Diabeti...OBJECTIVE To investigate the potency of Urena lobata leaves extract on the inhibition of hepatic complication on diabetic rats.METHODS This study uses control group post test only with male Sprague dawley rats.Diabetic rats was induced by high fructose diet(HFD)and single dose streptozotocin 25mg·kg-1 bw intra peritoneal.The rat was administrated orally with water extract of U.lobataleaves in concentrations of 250,500 and 1000mg·kg-1 bw for 4 weeks.After scarifying,liver organ and blood were collected and then superoxyde dismutase(SOD)hepar level,malondialdehyda(MDA),tumor necrosis factor-alpha(TNF-α),serum glutamic oxaloacetic transaminase(SGOT)and serum glutamic piruvic transaminase(SGPT)were examined.The data was analyzed using ANOVA test continued with LSD test(P<0,05).RESULTS The oral administration of U.lobataleaves extract 250,500 and 1000mg·kg-1 bw were able to increase SOD hepar level about 90%,100% and 120%respectively compared to diabetic group(P<0.05),while the MDA hepar level was decreased by 40%,50% and 70% respectively(P<0.05),whereas the TNF-αhepar level was decreased by 30%,50% and 70%respectively(P<0.05).The supplementation of water extract from U.lobatain dose of 250,500 and 1000mg·kg-1 bw decrease SGOT level approximately10%,30% and 50% compared to control group(P<0.05),while the SGPT level was decreased by 10%,20% and 40%respectively(P<0.05).In diabetic groups,SOD heparlevel was decreased compared to normal group(P<0.05)whereas the MDA and TNF-αwere increased(P<0.05).Meanwhile SGOT level and SGPT were increased in diabetic group(P<0.05).CONCLUSION U.lobataleaves extract could inhibit hepatic complication on diabetic rats by increasing of SOD hepar level,decreasing of MDA hepar level,TNF-α,SGOT and SGPT.This effect may be related to active compounds that act as an antioxidant and anti-inflammatory in U.lobata extract.展开更多
Congenital choledochal cyst is frequently seen ininfants and children in China. The extra-hepatic ductalchanges have been well described, but the changes in theintra-hepatic ducts in choledochal cyst are less well kno...Congenital choledochal cyst is frequently seen ininfants and children in China. The extra-hepatic ductalchanges have been well described, but the changes in theintra-hepatic ducts in choledochal cyst are less well known.This report is composed of 2 groups of patients: thirty-four cases who were operated upon between 1971 and1982 were followed post-operatively by plain abdominalfilm and barium meal examination and a group of 26cases receiving PTC between 1982 and 1984. In this lattergroup, both extra-and intra-hepatic ducts were shownsatisfactorily by PTC. In 23 cases (88.5%) besides thedilatation of the extra-hepatic duct, there were differentdegree and different forms of dilatation of the intra-hepatic ducts. In 10 cases(38.5%)the branches peripheralto the dilated main intra-hepatic ducts were found to benormal, in the other 10 cases (38.5%) the more peripheralbranches abruptly narrowed or were obliterated. In one case(3.8%) the entire intra-hepatic ductal system were dilatedwith a bead-like appearance. In only 3 cases were theintra-hepatic ducts normal (11.5%). Moreover the dilata-tion of the intra-hepatic ducts were found to persist longafter the obstruction was relieved in 28 cases (82.3%) ofthe 34 post-operative cases. Due to the frequent associationof intra-hepatic ductal dilatation with choledochal cyst,the various forms of dilatation and persistance of dilata-tion after relief of the obstruction, the term congenital biliary dilatation is suggested for this disease.展开更多
OBJECTIVE To explore the protective effects of 3′-daidzein sulfonate sodium on chronic hepatic injury induced by carbon tetrachloride(CCl4)in mice and investigate the mechanism about regulating the T lymphocyte subse...OBJECTIVE To explore the protective effects of 3′-daidzein sulfonate sodium on chronic hepatic injury induced by carbon tetrachloride(CCl4)in mice and investigate the mechanism about regulating the T lymphocyte subsets.METHODS Healthy Kunming male mice were randomly divided in 5groups:control group,model group,bifendate positive control group(2.5mg·kg-1),low and high dose 3′-daidzein sulfonate sodium groups(0.1and 0.3mg·kg-1).The chronic hepatic injury mice were made by intraperitoneal injection of 10% CCl4 plant oil solution twice a week,and sustained for six weeks.At the same time,the mice were treated with normal saline,bifendate(2.5mg·kg-1)and 3′-daidzein sulfonate sodium(0.1and 0.3mg·kg-1),respectively by ig administration once a day and continued for six weeks.After the last administration,the mice blood and liver were taken.Using automatic biochemical analyzer survey the activity of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in serum.Flow cytometry was used to detect T lymphocyte subsets,enzymes analysis technique was used to observe the liver function(CD3+,CD4+and CD8+),HemateinEosin stain was used to explore the changes in liver morphology.RESULTS The level of ALT and AST in the serum in model group mice increased significantly.While the level of ALT and AST in the serum in 3′-daidzein sulfonate sodium(0.1 or 0.3mg·kg-1,ig)groups mice decreased compared with the model group(P<0.05).And compared with the model group,the ratios of CD3+,CD4-CD8-and CD4+/CD8+ decreased,and the ratios of CD8+ increased in 3′-daidzein sulfonate sodium groups(0.1 and 0.3mg·kg-1,ig;P<0.05).CONCLUSION 3′-daidzein sulfonate sodium have a protective effect on CCl4-induced liver injury mice,and it can result in the changes of T lymphocyte subsets,which may be one of the factors leading to hepatic injury by CCl4.展开更多
P2X7 receptor （P2X7r） is important in inflammation and fibrosis. The aim of the present study was to investigate the effect of P2X7r inhibition, using a specific inhibitor （A438079） to prevent the development of l...P2X7 receptor （P2X7r） is important in inflammation and fibrosis. The aim of the present study was to investigate the effect of P2X7r inhibition, using a specific inhibitor （A438079） to prevent the development of liver fibrosis on human hepatic stellate cells, LX-2. The supernatant from lipopolysaccharide （LPS）-stimulated RAW 264.7 mouse macrophages was supplemented to LX-2 cells for 24 h. LX-2 cells were primed with LPS for 4 h and subsequently stimulated for 30 rain with 3 mmol · L^-1 of adenosine 5＇-triphosphate （ATP）. A438079 （ 10 μmol · L^-1） was supplemented to LX-2 cells 10 rain prior to ATP. Directly treated with LPS on LX-2 cells, mRNA ex- pressions of IL-1β, IL-18 and IL-6 were increased, as well as P2X7r. And caspase-1, ASC and NLRP3 mRNA ex- pressions were increased with LPS stimulation. LPS stimulation also increased oL-SMA and collagen I mRNA expres- sions. Interestingly treatment of LX-2 cells with mediums from LPS-primed RAW 264.7 mouse macrophages exhibi- ted greater increase of mRNA expressions of above genes than those in LX-2 directly treated with LPS. Pretreatment of directly or indirectly LPS-stimulated LX-2 cells with A438079 both suppressed IL-1β mRNA expression. In addi- tion treatment of LPS-primed LX-2 cells with 3 mM ATP induced the significant increase of IL-1β, IL-6, caspase- 1, pannexin-1, α-SMA and collagen I mRNA expression, the increasing of oL-SMA protein expression and cleavage of IL-1β. These events were significantly suppressed by pretreatment with P2X7r antagonist A438079. P2XTr blockade also significantly reduced the protein expression of oL-SMA. Our results suggest that the involvement of the P2X7r-NLRP3 inflammasome pathway in the secretion of IL-1β from extracellular ATP/LPS-stimulated human he- patic stellate cells. This study demonstrated that repression of the P2XTr represents a novel potential therapeutic ap- proach to control liver fibrosis.展开更多
OBJECTIVE To investigate the effect of P2X7receptor(P2X7r)inhibition,using a specific inhibitor(A438079)to prevent the development of liver fibrosis on human hepatic stellate cells,LX-2.METHODS The supernatant from li...OBJECTIVE To investigate the effect of P2X7receptor(P2X7r)inhibition,using a specific inhibitor(A438079)to prevent the development of liver fibrosis on human hepatic stellate cells,LX-2.METHODS The supernatant from lipopolysaccharide(LPS)-stimulated RAW264.7 mouse macrophages was supplemented to LX-2 cells for 24 h.LX-2cells were primed with LPS for 4h and subsequently stimulated for 30 min with 3mmol·L-1 of adenosine 5′-triphosphate(ATP).A438079(10μmol·L-1)was supplemented to LX-2 cells 10 min prior to ATP.RESULTS Directly treated with LPS on LX-2 cells,mRNA expressions of IL-1β,IL-18 and IL-6 were increased,as well as P2X7 r.And caspase-1,ASC and NLRP3 mRNA expressions were increased with LPS stimulation.LPS stimulation also increasedα-SMA and collagenⅠ mRNA expressions.Interestingly treatment of LX-2cells with mediums from LPS-primed RAW264.7mouse macrophages exhibited greater increase of mRNA expressions of above genes than those in LX-2directly treated with LPS.Pretreatment of directly or indirectly LPS-stimulated LX-2 cells with A438079 both suppressed IL-1βmRNA expression.In addition treatment of LPS-primed LX-2 cells with 3mmol·L-1 ATP induced the significant increase of IL-1β,IL-6,caspase-1,pannexin-1,α-SMA and collagenⅠ mRNA expression,the increasing ofα-SMA protein expression and cleavage of IL-1β.These events were significantly suppressed by pretreatment with P2X7 rantagonist A438079.P2X7 rblockade also significantly reduced the protein expression ofα-SMA.CONCLUSION Our results suggest that the involvement of the P2X7r-NLRP3 inflammasome pathway in the secretion of IL-1βfrom extracellular ATP/LPS-stimulated human hepatic stellate cells.This study demonstrated that repression of the P2X7 rrepresents a novel potential therapeutic approach to control liver fibrosis.展开更多
OBJECTIVE To investigate the mechanism underlying valproate(VPA)-induced hepatic hepatotoxicity.METHODS C57B/6J mice were given VPA at500 mg·kg-1·d-1by intragastric administration for 14 consecutive days,whi...OBJECTIVE To investigate the mechanism underlying valproate(VPA)-induced hepatic hepatotoxicity.METHODS C57B/6J mice were given VPA at500 mg·kg-1·d-1by intragastric administration for 14 consecutive days,while the control group received the same volume of physiologic saline by intragastric administration for same days.Mice were sacrificed 24 h after the last administration.Blood samples were collected for plasma biochemical assays.Liver was fixed in 10%neutral buffered formalin for histopathological analysis,and RNA extraction.mR NA for selected genes expression encoding proteins key to fatty acid synthesis,triglyceride synthesis,fatty acid oxidation,phosphatidylcholine synthesis,and lipid uptake were measured using quantitative real-time PCR.RESULTS We found that VPA treatment induced hepatic injury in mice as evidenced by increased ALP,ATP,ASP,GGT,and LDH.Histopathological analysis of the liver in mice treated with VPA showed increased microvesicular steatosis in cytoplasm.More importantly,VPA treatment increased the m RNA expressions of sterol regulatory element binding protein(SREBP)-1c,peroxisome proliferator-activated receptor(PPAR)γ,diacylgycerol acyltransferase(DGAT)2,and cluster of differentiation(CD)36,while the mR NA levels of stearoyl-Co A desaturase(SCD)1,DGAT1,liver X receptorsα(LXRα),carnitine palmitoyltransferase(CPT)1,malonyl coenzyme A decarboxylase(MCD),uncoupling protein(UCP)2,phosphatidylethanolamine N-methyltransferase(PEMT),and pregnenolone X receptor(PXR)displayed significant decrease.CONCLUSION Our data showed that VPA induced disruption of hepatic lipid homeostasis,which could be helpful for a better under-standing of the mechanism underlying VPA-induced hepatotoxicity and for a better use of VPA.展开更多
OBJECTIVE To describe a highly sensitive LC-ESI MSnmethod that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites in rat plasma for microdosing cocktail study.METHODS After ad...OBJECTIVE To describe a highly sensitive LC-ESI MSnmethod that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites in rat plasma for microdosing cocktail study.METHODS After administration of a mixture of six probes(i.e.,a cocktail approach with caffeine 100μg·kg-1,tolbutamide100μg·kg-1,omeprazole 500μg·kg-1,dextromethorphan 500μg·kg-1,chlorzoxazone 50μg·kg-1and midazolam 100μg·kg-1)to SD rats.The plasma samples were extracted using ethyl acetate with diazepam and gliclazide as the IS.The assay was performed on an Agilent Eclipse Plus C18 column(2.1×50 mm,3.5μm).The mobile phase consisted of 0.01%formic acid(1 mmol·L-1ammonium formate)and acetonitrile.The flow rate was0.3 m L·min-1.The samples were analyzed by LC-20A&5500Qtrap ESI MSnin MRM mode.The MS/MS reaction selected 181.2/124.0 m/z ions for caffeine,195.2/138.2m/z for paraxanthine,269.1/170.0 m/z for tolbutamide,285.1/186.0 m/z for 4-hydroxytolbutamide,346.1/198.1m/z for omeprazole,362.2/214.2 m/z for 5-hydroxyomeprazole,272.3/147.1 m/z for dextromethorphan,258.2/157.0 m/z for dextrorphan,168.1/132.1 m/z for chlorzoxazone,326.1/291.2 m/z for midazolam,and 342.1/324.2m/z for 1′-hydroxymidazolam.RESULTS The datashowed that the method was with good linearity in the range of 0.2-200 ng·m L-1for caffeine,0.1-25 ng·m L-1for paraxanthine,0.05-100 ng·m L-1for omeprazole,0.01-25 ng·mL-1for 5-hydroxyomeprazole,0.1-200 ng·mL-1for dextromethorphan,0.05-12.5 ng·mL-1for dextrophan,0.2-200 ng·mL-1for midazolam,and 0.2-25 ng·mL-1for 1′-hydroxymidazolam,respectively.The stability%RSD for al probes was less than 15%and matrix effects in plasma on the ionization were negligible.CONCLUSION This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10-100 times lower than typical therapeutic doses.The established LCMS/MS method was suitable for pharmacokinetic study of this mixture cocktail probe group and could be applied deeply to CYP isoforms(1A2,2C9,2C19,2D6,2E1and 3A)research.展开更多
用RT-PCR方法检测辽东湾几个重点沿岸海域表层海水和几种经济贝类样品中甲肝病毒的分布。结果表明,6个重点沿岸海域表层海水中均检出甲肝病毒(Hepatitis A virus,HAV),6种经济贝类样品中有4个样品检出甲肝病毒。检测结果显示,辽东湾某...用RT-PCR方法检测辽东湾几个重点沿岸海域表层海水和几种经济贝类样品中甲肝病毒的分布。结果表明,6个重点沿岸海域表层海水中均检出甲肝病毒(Hepatitis A virus,HAV),6种经济贝类样品中有4个样品检出甲肝病毒。检测结果显示,辽东湾某些重点沿岸海域受陆源生活污水影响严重,有关部门应加强海洋环境和海产贝类卫生安全监测和管理,以避免引发甲型肝炎暴发流行。展开更多
OBJECTIVE To explore the role of gecko crude peptides(GCPs)in the proliferation,apoptosis,migration and lymphangiogenesis of human hepatocellular carcinoma cells(Hep G2)and human lymphaticendothelial cells(HLECs)in vi...OBJECTIVE To explore the role of gecko crude peptides(GCPs)in the proliferation,apoptosis,migration and lymphangiogenesis of human hepatocellular carcinoma cells(Hep G2)and human lymphaticendothelial cells(HLECs)in vitro.METHODS The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay was used to evaluate the anti-proliferative effect of GCPs and si RNA-VEGF-C on Hep G2 cells,Hoechst 33258 staining and flow cytometry were performed to analyze cycle and apoptosis.The migration and invasion ability of cells were assayed by transwell chamber experiment and wound-healing assay.The protein and m RNA expressions of vascular endothelial growth factor-C(VEGF-C)and CXC chemokine receptor-4(CXCR4)were detected by q-PCR,immunofluorescence,Western blot.The protein expressions of the extracellular signal regulated kinase(ERKI/2),c-Jun N-terminal kinase(JNK),p38-mitogen activated protein kinases(p38 MAPK),serine/threonine kinase(Akt)and phosphatidylinositol-3-kinase(PI3K)were detected by western blot.The anti-lymphangiogenesis effect of GCPs on the HLECs was analyzed using an in vitro tube-formation assay.The protein and m RNA expressions of vascular endothelial growth factor receptor-3(VEGFR-3)and stromal cell-derived factor-1(SDF-1)were detected by q-PCR,Western blot.RESULTS GCPs and si RNA-VEGF-C inhibited Hep G2 proliferation,invasion and migration,and the most obvious inhibitory effect was both synergistic effects.Thus,GCPs suppressed HLECs proliferation,migration and tubelike structure formationin a dose-dependent manner,and had inhibitory effect of tumor-induced lymphangiogenesis in vitro.Additionally,we found that GCPs and si RNA-VEGF-C decreased the expressions of MMP-2,MMP-9,VEGF-C,CXCR4,phospho-ERK1/2,phospho-P38,phospho-JNK and PI3K in Hep G2 cells.Moreover,GCPs had a dose-dependent depressive effecton the expressions of VEGFR-3,SDF-1 in HLECs.CONCLUSION The low expression of VEGF-C mediated by si RNA-VEGF-C and GCPs inhibit tumor proliferation,invasion and migrationby suppressing the MAPK signaling pathway through reduced levels of VEGF-C,and GCPs inhibit tumor lymphangiogenesis by suppressing the CXCR4/SDF-1 signaling pathway through suppressed VEGF-C/VEGFR-3.展开更多
A simple and effective route for the synthesis of mibolerone was described starting from the estr-5(10)-en-3,17-dione in four steps with the overall yield of 47.0 %.Thus,two methods for key intermediate methylnorandro...A simple and effective route for the synthesis of mibolerone was described starting from the estr-5(10)-en-3,17-dione in four steps with the overall yield of 47.0 %.Thus,two methods for key intermediate methylnorandrost were investigated:one(method A)starting from estr-4-en-3,17-dione underwent 3-keto group protected with ethyl orthoformate to give 3-ethoxy-3,5-dien-estr-17-one,the other(method B)from estr-5(10)-en-3,17-dione and protected 3-keto group to give 3,3-dimethoxy-estr-5(10)-7-one in a mild acidic condition.Then,two intermediates were subsequently reacted with methyllithium followed by a mild hydrolytic procedure and gave methylnorandrost with total yield 25.0% and 86.0 %,respectively.In the preparation of 6-dehydrogenation product of methylnorandrost,two procedures(method C and method D)were investigated:one was the protected 17α-methyl-17β-hydroxy 3,5-enol ethers estrendiene brominated and the resulting 6-bromo-19-methylnortestosterone was then immediately dehydrohaloenated to give 6-dehydro-19-methylnortestosterone,the total yield only reaches 36.0%;the other was directly dehydrogenated with chloranil and the yield reaches 75.6% under the optimum conditions:in refluxing tetrahydrofuran,the molar ratio of methylnorandrost to chloranil is 0.66 and reaction time of 5 h.The titled compound and intermediates were characterized by 1H and 13C NMR,IRMS and elemental analysis.展开更多
基金The project supported by National Natural Science Foundation of China(81260497,81460564)Science and Technology Department of Jilin Province Youth Scientific Research Fund Project(201201075)
文摘OBJECTIVE Hepatic fibrosis is a wound-healing response for injury.Activated hepatic stellate cells(HSCs)are the preferred targets of anti-hepatic fibrotic therapies.cucurbitacin E(CuE)is,one well-known natural compound derived from traditional Chinese medicine,used in Asian countries for the prevention and treatment of hepatic disease.Therefore,the present study elucidated the mechanism of CuE on inducing apoptosis and attenuating hepatic fibrosis towards activated HSCs.METHODS The murine HSC(tHSC/Cl-6)cell line were incubated in 96-well plates and treated with TNF-αand CuE at various concentrations and indicated times.Cell viability was assessed with MTT assay.Another,t-HSC/Cl-6 were incubated in 6-well plates and also treated with TNF-α,CuE,AICAR or metformin for the indicated time and concentration.Cell protein and mRNA were prepared using kit and relevant signaling were detected by Western blotting and RT-PCR.RESULTS CuE inhibited cell proliferation of activated HSC/T-6cells in concentration-and time-dependent manner.CuE triggered the activation of caspase-3,cleaved the PARP,ration of bcl-2/bax,and cytochrom c protein in a time-and concentration-dependent manner.CuE decreased p-Erk/MAPK without effects on p-p38 and p-JNK.CuE inhibited the protein and mRNA expressions ofα-SMA,TIMP-1 and collagenⅠ in activated HSC-T6.CuE broadly blocked p-PI3 K,p-Akt,p-mTOR and p-p70S6 Kexpressions.CuE significantly increased phosphorylated AMPK expression as well as AICAR and metoformin.And metformin showed significantly higher activation of AMPK than AICAR.Ability of CuE on activation of AMPK was between AICAR and metformin.It′s also found that CuE significantly decreased p-mTOR as well as AICAR and metformin.CONCLUSION CuE could modulate HSC survival and activation as a potential anti-fibrotic agent for liver fibrosis treatment.The findings demonstrate that CuE induced HSC apoptosis via ERK/MAPK and PI3K/Akt-AMPK-mTOR signaling.
基金supported by National Natural Science Foundation of China(81700523)
文摘OBJECTIVE To investigate the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells(HSCs).METHODS Normal human Chang liver cells and human hepatic stellate cell line,LX-2 cells were treated with SRT1720(10μmol·L^(-1))and AICAR(500μmol·L^(-1))prior to ethanol(50 mmol·L^(-1)) for 24 and 48 h.Cell viability was analyzed by methyl thiazolyl tetrazolium assay.SIRT1,AMPK and p-AMPK m RNA levels for 24 h and 48 h were analyzed by RT-PCR,SIRT1,AMPK and p-AMPK protein expressions in the supernatant at 24 and 48 h was detected by Western blot.RESULTS SRT1720 and AICAR effectively decreased LX-2 cell viabilities and exhibited scarcely little toxicity in human Chang liver cells.SRT1720 and AICAR attenuated collagen-I,α-smooth muscle actin(α-SMA)levels,activated liver kinase B-1(LKB1)and AMPK phosphorylation in ethanol treated LX-2 cells.Meanwhile,SRT1720 and AICAR enhanced SIRT1 expression mediated by ethanol both in Chang liver cells and LX-2 cells.Furthermore,SRT1720 and AICAR suppressed the expression of sterol regulatory element-binding protein-1(SREBP-1)to regulate fatty acid synthesis.CONCLUSION SIRT1 agonist and AMPK agonist blocked the crosstalk between hepatocytes and HSCs via SIRT1/AMPK signaling pathway to modulate hepatocytes accumulation of lipid and HSCs activation.
文摘OBJECTIVE The pathological characteristics of nonalcoholic steatohepatitis(NASH)include liver steatosis,inflammation,and fibrosis.Fibrosis is the most severe and significant pathological feature in NASH.Effective drug treatment could reverse early liver fibrosis and is of significance to prevent NASH from progressing into cirrhosis and liver cancer.Identification of drug targets for NASH treatment has been an active research area and is essential for the development of anti-NASH medications.Naringenin(NGN)is a flavonoid compound rich in citrus fruits.Our preliminary data demonstrated that NGN reduced diet-induced lipid accumulation and inflammation in the mouse liver,but whether NGN can attenuate liver fibrosis of NASH is not known.METHODS To study the effect of NGN on NASH fibrosis.WT mice were fed with high fat diet(HFD)and injected intraperitoneally 20%carbon tetrachloride at the same time for 8 weeks to induce NASH,and NGN was administrated by gavage in the meantime.In vitro,LO2 cells and LX2 cells were stimulated by oleic acid(OA)combined with lipopolysaccharide(LPS),respectively.RESULTS Treating the WT mice with NGN 100 mg·kg^(-1)·d-1 significantly attenuated hepatic lipid accumulation,hepatic fibrosis,plasma ALT and AST levels,inhibited protein expression of p-ERK,p-FoxO3a in the mouse livers.In vitro,on OA and LPS stimulated LO2 or LX2 cells,NGN significantly promoted apoptosis of activated hepatic stellate cells while inhibited apoptosis of hepatocytes.Mechanism study indicated that NGN inhibited MAPK pathway and promoted activation of FoxO3a,consequently promoted apoptosis of the activated LX2 cells and inhibited liver fibrosis.CONCLUSION NGN preventes NASH fibrosis via regulating MAPK/FoxO3a pathway,thus promoting apoptosis of the activated hepatic stellate cells.
文摘Hepatic fibrosis is a wound-healing response for injury. Activated hepatic stellate cells （HSCs） are the preferred targets of anti-hepatic fibrotic therapies. Cucurbitacin E （CUE） is, one well-known natural compound de- rived from Traditional Chinese Medicine, used in Asian countries for the prevention and treatment of hepatic dis- ease. Therefore, the present study elucidated the mechanism of CuE on inducing apoptosis and attenuating hepatic fibrosis towards activated HSCs. The murine hepatic stellate cells （t-HSC/C1-6） cell line were incubated in 96-well plates and treated with TNF-α and CuE at various concentrations and indicated times. Cell viability was assessed with MTT assay. Another, t-HSC/C1-6 were incubated in 6-well plates and also treated with TNF-α, CuE, AICAR or metformin for the indicated time and concentration. Cell protein and mRNA were prepared using kit and relevant signaling were detected by Western blot and RT-PCR. CuE inhibited cell proliferation of activated HSC/T-6 cells in concentration- and time-dependent manner. CuE triggered the activation of caspase-3, cleaved the PARP, ration of bcl-2/bax, and cytochrom C protein in a time- and concentration-dependent manner. CuE decreased p-Erk/MAPK without effects on p-p38 and p-JNK. CuE inhibited the protein and mRNA expressions of oL-SMA, TIMP-1 and col- lagen I in activated HSC-T6. CuE broadly blocked p-PI3K, p-Akt, p-roTOR and p-p70S6K expressions. CuE sig- nificantly increased phosphorylated AMPK expression as well as AICAR and metoformin. And metformin showed significantly higher activation of AMPK than AICAR. Ability of CuE on activation of AMPK was between AICAR and metformin. It＇s also found that CuE significantly decreased p-roTOR as well as AICAR and metformin. CuE could modulate HSC survival and activation as a potential anti-fibrotic agent for liver fibrosis treatment. The find- ings demonstrate that CuE induced HSC apoptosis via Erk/MAPK and PI3IC/Akt-AMPK-mTOR signaling.
基金The project supported by National Natural Science Foundation of China(U1204826)
文摘OBJECTIVE To explore the therapeutic effects and underlying mechanisms of velvet antler polypeptides(VVAPs)in CCl4-induced experimental hepatic fibrosis in rats.METHODS Anti-hepatic fibrosis properties of VAPs were tested by Subcutaneous injection(SC)into male Wistar rats of CCl4- induced experimental hepatic fibrosis.After SC injections for 45 consecutive days at doses of 5mg·kg-1(low dose,VAPsL),10mg·kg-1(mid-dose,VAPsM)and 20mg·kg-1(high-dose,VAPsH),the rats were sacrificed and the various indicators were evaluated and tested.Observed hepatic cells degeneration and necrosis,inflammatory infiltration and levels of serum enzymes to assess treatment of VAPs;The expression levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),MDA,and hydroxyproline(HYP)in liver tissue were analyzed;RT-PCR analysis was carried out to detect the expression levels of matrix metalloproteinases2(MMP-2)and tissue inhibitor of metalloproteinases 1(TIMP-1)in liver tissue.RESULTS VAPs has obvious anti-hepatic fibrosis effects.Hepatocyte swelling,fatty degeneration was significantly reduced,reducing infiltration of inflammatory cells.Release of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)decreased significantly,reduction of hyaluronic acid(HA)and laminin(LN)obviously,at the same time,the content of total protein and albumin increased significantly in serum.Activity of SOD and GSH-Px was significantly raised and the content of MDA and HYP was reduced significantly in liver tissue.Expression levels of MMP-2and TIMP-1 mRNA in liver were decreased significantly.These improvements were more significant in high-dos and mid-dose groups(P<0.05 or P<0.01 vs model group).CONCLUSION These findings suggest VAPs can significant treat the hepatic fibrosis,which may be due to protect liver cells and improve liver functions by hydroxyl radical scavenging activity and great effect of antioxidation,and decrease the gene expression of MMP-2,improving exist-environment of liver cells and decreasing the gene expression of TIMP-1,prompting degradation of extracellular matrix.
文摘Fibrosis is a phenomenon of fibrous connective tissue increase in the organs. Activated hepatic stellate cells （HSCs） are the preferred targets of anti-hepatic fibrotic therapies. Ginseng and Platycodon grandiflorum are impor- tant traditional Chinese medicine herbs. The study was to investigate the anti-fibrosis effects of ginseng extract and Platycodon grandiflorum extract in HSCs. Extract include Ginseng water extract and alcohol extract, Platycodon gran- diflorum water extract and alcohol extract, combination of both water extract and alcohol extract. Activated hepatic stellate cells were treated with Ginseng and Platycodon grandiflorum extracts for 24h. The maximum concentration of the drug is 2000 mg · L^-1, the lowest concentration of 7.8 mg · L^-1 , with concentration gradient dilution. 3-（4 , 5- dimethylthiazol-2-yl）-2, 5-diphenylte-trazolium bromide （MTT） can evaluate the survival rate of cells. The expres- sions of SIRT1, NF-KB and p53 were detected by western blot. The results showed that extract effectively decreased the HSC cells viability in concentration dependent manner. Among them, the effect of combination of both extract is best. Ginseng and Platycodon grandiflorum activated the protein expressions of SIRT1 in activated HSCs. The expres- sion of NF-KB and p53 were significantly decreased by Ginseng and Platycodon grandiflorum. This study demonstrated that ginseng extract and Platycodon grandiflorum extract showed obviously anti-fibrosis effect, and decreased expres-sion of NF-KB and p53 via SIRT1 signaling pathway.
基金The project supported by Ministry Education of Indonesia
文摘OBJECTIVE To investigate the potency of Urena lobata leaves extract on the inhibition of hepatic complication on diabetic rats.METHODS This study uses control group post test only with male Sprague dawley rats.Diabetic rats was induced by high fructose diet(HFD)and single dose streptozotocin 25mg·kg-1 bw intra peritoneal.The rat was administrated orally with water extract of U.lobataleaves in concentrations of 250,500 and 1000mg·kg-1 bw for 4 weeks.After scarifying,liver organ and blood were collected and then superoxyde dismutase(SOD)hepar level,malondialdehyda(MDA),tumor necrosis factor-alpha(TNF-α),serum glutamic oxaloacetic transaminase(SGOT)and serum glutamic piruvic transaminase(SGPT)were examined.The data was analyzed using ANOVA test continued with LSD test(P<0,05).RESULTS The oral administration of U.lobataleaves extract 250,500 and 1000mg·kg-1 bw were able to increase SOD hepar level about 90%,100% and 120%respectively compared to diabetic group(P<0.05),while the MDA hepar level was decreased by 40%,50% and 70% respectively(P<0.05),whereas the TNF-αhepar level was decreased by 30%,50% and 70%respectively(P<0.05).The supplementation of water extract from U.lobatain dose of 250,500 and 1000mg·kg-1 bw decrease SGOT level approximately10%,30% and 50% compared to control group(P<0.05),while the SGPT level was decreased by 10%,20% and 40%respectively(P<0.05).In diabetic groups,SOD heparlevel was decreased compared to normal group(P<0.05)whereas the MDA and TNF-αwere increased(P<0.05).Meanwhile SGOT level and SGPT were increased in diabetic group(P<0.05).CONCLUSION U.lobataleaves extract could inhibit hepatic complication on diabetic rats by increasing of SOD hepar level,decreasing of MDA hepar level,TNF-α,SGOT and SGPT.This effect may be related to active compounds that act as an antioxidant and anti-inflammatory in U.lobata extract.
文摘Congenital choledochal cyst is frequently seen ininfants and children in China. The extra-hepatic ductalchanges have been well described, but the changes in theintra-hepatic ducts in choledochal cyst are less well known.This report is composed of 2 groups of patients: thirty-four cases who were operated upon between 1971 and1982 were followed post-operatively by plain abdominalfilm and barium meal examination and a group of 26cases receiving PTC between 1982 and 1984. In this lattergroup, both extra-and intra-hepatic ducts were shownsatisfactorily by PTC. In 23 cases (88.5%) besides thedilatation of the extra-hepatic duct, there were differentdegree and different forms of dilatation of the intra-hepatic ducts. In 10 cases(38.5%)the branches peripheralto the dilated main intra-hepatic ducts were found to benormal, in the other 10 cases (38.5%) the more peripheralbranches abruptly narrowed or were obliterated. In one case(3.8%) the entire intra-hepatic ductal system were dilatedwith a bead-like appearance. In only 3 cases were theintra-hepatic ducts normal (11.5%). Moreover the dilata-tion of the intra-hepatic ducts were found to persist longafter the obstruction was relieved in 28 cases (82.3%) ofthe 34 post-operative cases. Due to the frequent associationof intra-hepatic ductal dilatation with choledochal cyst,the various forms of dilatation and persistance of dilata-tion after relief of the obstruction, the term congenital biliary dilatation is suggested for this disease.
基金The project supported by National Natural Science Foundation of China(81160399)
文摘OBJECTIVE To explore the protective effects of 3′-daidzein sulfonate sodium on chronic hepatic injury induced by carbon tetrachloride(CCl4)in mice and investigate the mechanism about regulating the T lymphocyte subsets.METHODS Healthy Kunming male mice were randomly divided in 5groups:control group,model group,bifendate positive control group(2.5mg·kg-1),low and high dose 3′-daidzein sulfonate sodium groups(0.1and 0.3mg·kg-1).The chronic hepatic injury mice were made by intraperitoneal injection of 10% CCl4 plant oil solution twice a week,and sustained for six weeks.At the same time,the mice were treated with normal saline,bifendate(2.5mg·kg-1)and 3′-daidzein sulfonate sodium(0.1and 0.3mg·kg-1),respectively by ig administration once a day and continued for six weeks.After the last administration,the mice blood and liver were taken.Using automatic biochemical analyzer survey the activity of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in serum.Flow cytometry was used to detect T lymphocyte subsets,enzymes analysis technique was used to observe the liver function(CD3+,CD4+and CD8+),HemateinEosin stain was used to explore the changes in liver morphology.RESULTS The level of ALT and AST in the serum in model group mice increased significantly.While the level of ALT and AST in the serum in 3′-daidzein sulfonate sodium(0.1 or 0.3mg·kg-1,ig)groups mice decreased compared with the model group(P<0.05).And compared with the model group,the ratios of CD3+,CD4-CD8-and CD4+/CD8+ decreased,and the ratios of CD8+ increased in 3′-daidzein sulfonate sodium groups(0.1 and 0.3mg·kg-1,ig;P<0.05).CONCLUSION 3′-daidzein sulfonate sodium have a protective effect on CCl4-induced liver injury mice,and it can result in the changes of T lymphocyte subsets,which may be one of the factors leading to hepatic injury by CCl4.
文摘P2X7 receptor （P2X7r） is important in inflammation and fibrosis. The aim of the present study was to investigate the effect of P2X7r inhibition, using a specific inhibitor （A438079） to prevent the development of liver fibrosis on human hepatic stellate cells, LX-2. The supernatant from lipopolysaccharide （LPS）-stimulated RAW 264.7 mouse macrophages was supplemented to LX-2 cells for 24 h. LX-2 cells were primed with LPS for 4 h and subsequently stimulated for 30 rain with 3 mmol · L^-1 of adenosine 5＇-triphosphate （ATP）. A438079 （ 10 μmol · L^-1） was supplemented to LX-2 cells 10 rain prior to ATP. Directly treated with LPS on LX-2 cells, mRNA ex- pressions of IL-1β, IL-18 and IL-6 were increased, as well as P2X7r. And caspase-1, ASC and NLRP3 mRNA ex- pressions were increased with LPS stimulation. LPS stimulation also increased oL-SMA and collagen I mRNA expres- sions. Interestingly treatment of LX-2 cells with mediums from LPS-primed RAW 264.7 mouse macrophages exhibi- ted greater increase of mRNA expressions of above genes than those in LX-2 directly treated with LPS. Pretreatment of directly or indirectly LPS-stimulated LX-2 cells with A438079 both suppressed IL-1β mRNA expression. In addi- tion treatment of LPS-primed LX-2 cells with 3 mM ATP induced the significant increase of IL-1β, IL-6, caspase- 1, pannexin-1, α-SMA and collagen I mRNA expression, the increasing of oL-SMA protein expression and cleavage of IL-1β. These events were significantly suppressed by pretreatment with P2X7r antagonist A438079. P2XTr blockade also significantly reduced the protein expression of oL-SMA. Our results suggest that the involvement of the P2X7r-NLRP3 inflammasome pathway in the secretion of IL-1β from extracellular ATP/LPS-stimulated human he- patic stellate cells. This study demonstrated that repression of the P2XTr represents a novel potential therapeutic ap- proach to control liver fibrosis.
基金The project supported by National Natural Science Foundation of China(81260664,81160538)
文摘OBJECTIVE To investigate the effect of P2X7receptor(P2X7r)inhibition,using a specific inhibitor(A438079)to prevent the development of liver fibrosis on human hepatic stellate cells,LX-2.METHODS The supernatant from lipopolysaccharide(LPS)-stimulated RAW264.7 mouse macrophages was supplemented to LX-2 cells for 24 h.LX-2cells were primed with LPS for 4h and subsequently stimulated for 30 min with 3mmol·L-1 of adenosine 5′-triphosphate(ATP).A438079(10μmol·L-1)was supplemented to LX-2 cells 10 min prior to ATP.RESULTS Directly treated with LPS on LX-2 cells,mRNA expressions of IL-1β,IL-18 and IL-6 were increased,as well as P2X7 r.And caspase-1,ASC and NLRP3 mRNA expressions were increased with LPS stimulation.LPS stimulation also increasedα-SMA and collagenⅠ mRNA expressions.Interestingly treatment of LX-2cells with mediums from LPS-primed RAW264.7mouse macrophages exhibited greater increase of mRNA expressions of above genes than those in LX-2directly treated with LPS.Pretreatment of directly or indirectly LPS-stimulated LX-2 cells with A438079 both suppressed IL-1βmRNA expression.In addition treatment of LPS-primed LX-2 cells with 3mmol·L-1 ATP induced the significant increase of IL-1β,IL-6,caspase-1,pannexin-1,α-SMA and collagenⅠ mRNA expression,the increasing ofα-SMA protein expression and cleavage of IL-1β.These events were significantly suppressed by pretreatment with P2X7 rantagonist A438079.P2X7 rblockade also significantly reduced the protein expression ofα-SMA.CONCLUSION Our results suggest that the involvement of the P2X7r-NLRP3 inflammasome pathway in the secretion of IL-1βfrom extracellular ATP/LPS-stimulated human hepatic stellate cells.This study demonstrated that repression of the P2X7 rrepresents a novel potential therapeutic approach to control liver fibrosis.
基金The project supported by National Natural Science Foundations of China(81573658,81102886)
文摘OBJECTIVE To investigate the mechanism underlying valproate(VPA)-induced hepatic hepatotoxicity.METHODS C57B/6J mice were given VPA at500 mg·kg-1·d-1by intragastric administration for 14 consecutive days,while the control group received the same volume of physiologic saline by intragastric administration for same days.Mice were sacrificed 24 h after the last administration.Blood samples were collected for plasma biochemical assays.Liver was fixed in 10%neutral buffered formalin for histopathological analysis,and RNA extraction.mR NA for selected genes expression encoding proteins key to fatty acid synthesis,triglyceride synthesis,fatty acid oxidation,phosphatidylcholine synthesis,and lipid uptake were measured using quantitative real-time PCR.RESULTS We found that VPA treatment induced hepatic injury in mice as evidenced by increased ALP,ATP,ASP,GGT,and LDH.Histopathological analysis of the liver in mice treated with VPA showed increased microvesicular steatosis in cytoplasm.More importantly,VPA treatment increased the m RNA expressions of sterol regulatory element binding protein(SREBP)-1c,peroxisome proliferator-activated receptor(PPAR)γ,diacylgycerol acyltransferase(DGAT)2,and cluster of differentiation(CD)36,while the mR NA levels of stearoyl-Co A desaturase(SCD)1,DGAT1,liver X receptorsα(LXRα),carnitine palmitoyltransferase(CPT)1,malonyl coenzyme A decarboxylase(MCD),uncoupling protein(UCP)2,phosphatidylethanolamine N-methyltransferase(PEMT),and pregnenolone X receptor(PXR)displayed significant decrease.CONCLUSION Our data showed that VPA induced disruption of hepatic lipid homeostasis,which could be helpful for a better under-standing of the mechanism underlying VPA-induced hepatotoxicity and for a better use of VPA.
基金The project supported by National Natural Science Foundation of China(81473579,81273654,81102879)the National Science and Technology Major Projects for"Major New Drugs Innovation and Development"(2013ZX09103002-022)
文摘OBJECTIVE To describe a highly sensitive LC-ESI MSnmethod that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites in rat plasma for microdosing cocktail study.METHODS After administration of a mixture of six probes(i.e.,a cocktail approach with caffeine 100μg·kg-1,tolbutamide100μg·kg-1,omeprazole 500μg·kg-1,dextromethorphan 500μg·kg-1,chlorzoxazone 50μg·kg-1and midazolam 100μg·kg-1)to SD rats.The plasma samples were extracted using ethyl acetate with diazepam and gliclazide as the IS.The assay was performed on an Agilent Eclipse Plus C18 column(2.1×50 mm,3.5μm).The mobile phase consisted of 0.01%formic acid(1 mmol·L-1ammonium formate)and acetonitrile.The flow rate was0.3 m L·min-1.The samples were analyzed by LC-20A&5500Qtrap ESI MSnin MRM mode.The MS/MS reaction selected 181.2/124.0 m/z ions for caffeine,195.2/138.2m/z for paraxanthine,269.1/170.0 m/z for tolbutamide,285.1/186.0 m/z for 4-hydroxytolbutamide,346.1/198.1m/z for omeprazole,362.2/214.2 m/z for 5-hydroxyomeprazole,272.3/147.1 m/z for dextromethorphan,258.2/157.0 m/z for dextrorphan,168.1/132.1 m/z for chlorzoxazone,326.1/291.2 m/z for midazolam,and 342.1/324.2m/z for 1′-hydroxymidazolam.RESULTS The datashowed that the method was with good linearity in the range of 0.2-200 ng·m L-1for caffeine,0.1-25 ng·m L-1for paraxanthine,0.05-100 ng·m L-1for omeprazole,0.01-25 ng·mL-1for 5-hydroxyomeprazole,0.1-200 ng·mL-1for dextromethorphan,0.05-12.5 ng·mL-1for dextrophan,0.2-200 ng·mL-1for midazolam,and 0.2-25 ng·mL-1for 1′-hydroxymidazolam,respectively.The stability%RSD for al probes was less than 15%and matrix effects in plasma on the ionization were negligible.CONCLUSION This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10-100 times lower than typical therapeutic doses.The established LCMS/MS method was suitable for pharmacokinetic study of this mixture cocktail probe group and could be applied deeply to CYP isoforms(1A2,2C9,2C19,2D6,2E1and 3A)research.
文摘用RT-PCR方法检测辽东湾几个重点沿岸海域表层海水和几种经济贝类样品中甲肝病毒的分布。结果表明,6个重点沿岸海域表层海水中均检出甲肝病毒(Hepatitis A virus,HAV),6种经济贝类样品中有4个样品检出甲肝病毒。检测结果显示,辽东湾某些重点沿岸海域受陆源生活污水影响严重,有关部门应加强海洋环境和海产贝类卫生安全监测和管理,以避免引发甲型肝炎暴发流行。
基金supported by Medical Science and Technology Research Project of Henan Province(142102310031)
文摘OBJECTIVE To explore the role of gecko crude peptides(GCPs)in the proliferation,apoptosis,migration and lymphangiogenesis of human hepatocellular carcinoma cells(Hep G2)and human lymphaticendothelial cells(HLECs)in vitro.METHODS The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay was used to evaluate the anti-proliferative effect of GCPs and si RNA-VEGF-C on Hep G2 cells,Hoechst 33258 staining and flow cytometry were performed to analyze cycle and apoptosis.The migration and invasion ability of cells were assayed by transwell chamber experiment and wound-healing assay.The protein and m RNA expressions of vascular endothelial growth factor-C(VEGF-C)and CXC chemokine receptor-4(CXCR4)were detected by q-PCR,immunofluorescence,Western blot.The protein expressions of the extracellular signal regulated kinase(ERKI/2),c-Jun N-terminal kinase(JNK),p38-mitogen activated protein kinases(p38 MAPK),serine/threonine kinase(Akt)and phosphatidylinositol-3-kinase(PI3K)were detected by western blot.The anti-lymphangiogenesis effect of GCPs on the HLECs was analyzed using an in vitro tube-formation assay.The protein and m RNA expressions of vascular endothelial growth factor receptor-3(VEGFR-3)and stromal cell-derived factor-1(SDF-1)were detected by q-PCR,Western blot.RESULTS GCPs and si RNA-VEGF-C inhibited Hep G2 proliferation,invasion and migration,and the most obvious inhibitory effect was both synergistic effects.Thus,GCPs suppressed HLECs proliferation,migration and tubelike structure formationin a dose-dependent manner,and had inhibitory effect of tumor-induced lymphangiogenesis in vitro.Additionally,we found that GCPs and si RNA-VEGF-C decreased the expressions of MMP-2,MMP-9,VEGF-C,CXCR4,phospho-ERK1/2,phospho-P38,phospho-JNK and PI3K in Hep G2 cells.Moreover,GCPs had a dose-dependent depressive effecton the expressions of VEGFR-3,SDF-1 in HLECs.CONCLUSION The low expression of VEGF-C mediated by si RNA-VEGF-C and GCPs inhibit tumor proliferation,invasion and migrationby suppressing the MAPK signaling pathway through reduced levels of VEGF-C,and GCPs inhibit tumor lymphangiogenesis by suppressing the CXCR4/SDF-1 signaling pathway through suppressed VEGF-C/VEGFR-3.
基金Project(50573019) support by the National Natural Science Foundation of China
文摘A simple and effective route for the synthesis of mibolerone was described starting from the estr-5(10)-en-3,17-dione in four steps with the overall yield of 47.0 %.Thus,two methods for key intermediate methylnorandrost were investigated:one(method A)starting from estr-4-en-3,17-dione underwent 3-keto group protected with ethyl orthoformate to give 3-ethoxy-3,5-dien-estr-17-one,the other(method B)from estr-5(10)-en-3,17-dione and protected 3-keto group to give 3,3-dimethoxy-estr-5(10)-7-one in a mild acidic condition.Then,two intermediates were subsequently reacted with methyllithium followed by a mild hydrolytic procedure and gave methylnorandrost with total yield 25.0% and 86.0 %,respectively.In the preparation of 6-dehydrogenation product of methylnorandrost,two procedures(method C and method D)were investigated:one was the protected 17α-methyl-17β-hydroxy 3,5-enol ethers estrendiene brominated and the resulting 6-bromo-19-methylnortestosterone was then immediately dehydrohaloenated to give 6-dehydro-19-methylnortestosterone,the total yield only reaches 36.0%;the other was directly dehydrogenated with chloranil and the yield reaches 75.6% under the optimum conditions:in refluxing tetrahydrofuran,the molar ratio of methylnorandrost to chloranil is 0.66 and reaction time of 5 h.The titled compound and intermediates were characterized by 1H and 13C NMR,IRMS and elemental analysis.
