Modulation of Kupffer cells on hepatic drug metabolism

AIM:To observe the effects of Kupffer cells on hepaticdrug metabolic enzymes.METHODS:Kunming mice were ip injected with GdCl_310,20,40mg/kg to decrease the number and block the functionof kupffer cells selectively.The contents of drug metabolicenzymes,cytochrorne P450,NADPH-cytochrom C redutase(NADPH-C),aniline hydroxylase (ANH),aminopyrine N-demethylase (AMD),erythromycin N-demethylase (EMD),and glutathione s-transferase (mGST) in hepatic microsomeand S9-GSTpi,S9-GST in supernatant of 9000g wereaccessed 1d after the injection.The time course ofalteration of drug metabolic enzymes was observed on d1,3,and 6 treated with a single dose GdCl_3.Mice weretreated with Angelica sinensis polysaccharides (ASP) of30,60,120mg/kg,ig,qd×6d,respectively and the sameassays were performed.RESULTS:P450 content and NADPH-C,ANH,AMD,andEMD activities were obviously reduced 1d after Kupffercell blockade.However,mGST and S9-GST activities weresignificantly increased.But no relationship was observedbetween GdCl_3 dosage and enzyme activities.With singledose GdCl_3 treatment,P450 content,NADPH-C,and ANHactivities were further decreased following Kupffer cellblockade lasted for 6d,by 35.7%,50.3%,36.5% after3d,and 57.9%,57.9%,63.2% after 6d,respectively.Onthe contrary,AMD,EMD,mGST,and S9-GST activitieswere raised by 36.5%,71.9%,23.1%,35.7% after 3d,and 155%,182%,21.5%,33.7% after 6d,respectively.Furthermore,the activities of drug metabolic enzymeswere markedly increased after 30mg/kg ASP treatment,and decreased significantly after 120mg/kg ASP treatment.No change in activity of S9-GSTpi was observed in thepresent study.CONCLUSION:Kupffer cells play an important role in themodulation of drug metabolic enzymes.The changes ofdrug metabolic enzyme activities depend on the time ofkupffer cell blockade and on the degree of Kupffer cellsactivated.A low concentration of ASP increases the activitiesof drug metabolic enzymes,but a high concentration ofASP decreases the activities of drug metabolic enzymes.     

Effects of lipopolysaccharides stimulated Kupffer cells on activation of rat hepatic stellate cells

AIM:To study the effects of Kupffer cell-conditioned medium(KCCN)derived from lipopolysaccharide(LPS)treatment onproliferation of rat hepatic stellate cells(HSC).METHODS:HSC and Kupffer cells were isolated from theliver of Wistar rats by in situ perfusion with pronase andcollagenase and density gradient centrifugation with Nycodenzand cultured.KCCM was prepared and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)colorimetricassay was used to detect HSC proliferation.The content oftype Ⅳ collagen and laminin secreted by HSC in the HSC-conditioned medium was determined by radioimmunoassay.TGF-β_1 production in the KCCM was detected by enzyme-linked immunosorbent assay(ELISA).RESULTS:HSC and Kupffer cells isolated had high purity.One microgram per mililiter LPS-activated KCCM andunstimulated KCCM could significantly promote HSCproliferation[0.132±0.005 and 0.123±0.008 vscontrol group(0.100±0.003),P<0.01],and there was a difference betweenthem(P<0.05).Ten microgram per mililiter LPS-activatedKCCM(0.106±0.010)was unable to promote HSC proliferation(P>0.05).Adding anti-TGF-β_1 antibodies could suppress theproliferation promoted by unstimulated KCCM and LPS(1μg/ml)-activated KCCM(0.109±0.009 vs 0.123±0.008,0.115±0.008 vs 0.132±0.005,P<0.01).LPS(1μg/ml or 10μg/ml)could not promote HSC proliferation immediately(0.096±0.003 and 0.101±0.004 vs 0.100±0.003,P>0.05).Therewas a parallel behavior between HSC proliferation and increasedECM level.One microgram per mililiter LPS-activated KCCNcontained a larger amount of TGF-β_1 than unstimulated KCCM.CONCLUSION:The technique for isolation of HSC andKupffer cells described here is simple and reliable.KCCMstimulated by LPS may promote HSC proliferation and collagenaccumulation,which are associated with hepatic fibrogenesis.     

The interaction of colon carcinoma cells with rat hepatic sinusoidal cells during early stages of metastasis

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TGF-β dependent regulation of oxygen radicals during transdifferentiation of activated hepatic stellate cells to myofibroblastoid cells

Background

The activation of hepatic stellate cells (HSCs) plays a pivotal role during liver injury because the resulting myofibroblasts (MFBs) are mainly responsible for connective tissue re-assembly. MFBs represent therefore cellular targets for anti-fibrotic therapy. In this study, we employed activated HSCs, termed M1-4HSCs, whose transdifferentiation to myofibroblastoid cells (named M-HTs) depends on transforming growth factor (TGF)-β. We analyzed the oxidative stress induced by TGF-β and examined cellular defense mechanisms upon transdifferentiation of HSCs to M-HTs.

Results

We found reactive oxygen species (ROS) significantly upregulated in M1-4HSCs within 72 hours of TGF-β administration. In contrast, M-HTs harbored lower intracellular ROS content than M1-4HSCs, despite of elevated NADPH oxidase activity. These observations indicated an upregulation of cellular defense mechanisms in order to protect cells from harmful consequences caused by oxidative stress. In line with this hypothesis, superoxide dismutase activation provided the resistance to augmented radical production in M-HTs, and glutathione rather than catalase was responsible for intracellular hydrogen peroxide removal. Finally, the TGF-β/NADPH oxidase mediated ROS production correlated with the upregulation of AP-1 as well as platelet-derived growth factor receptor subunits, which points to important contributions in establishing antioxidant defense.

Conclusion

The data provide evidence that TGF-β induces NADPH oxidase activity which causes radical production upon the transdifferentiation of activated HSCs to M-HTs. Myofibroblastoid cells are equipped with high levels of superoxide dismutase activity as well as glutathione to counterbalance NADPH oxidase dependent oxidative stress and to avoid cellular damage.     

Effect of natural interferon α on proliferation and apoptosis of hepatic stellate cells

Inhibition of the proliferation of hepatic stellate cells (HSC) is clinically important for the control of liver fibrosis and cirrhosis. Interferons are now frequently used for chronic viral hepatitis because of their anti-viral activity. However, patients treated with interferons exhibit a regression of liver fibrosis even if viral eradication is not achieved, indicating that interferon itself has anti-fibrotic activity. Herein, we show the anti-proliferation and pro-apoptotic activity of natural interferon α against HSC. We found that interferon α inhibited serum-stimulated [³H]thymidine incorporation of HSC in a dose-dependent manner, with a significant reduction at more than 100 U/ml. Interferon α also attenuated PDGF-BB-stimulated DNA synthesis of HSC. Although the molecular mechanism behind these phenomena has not been defined, we found that interferon α triggers the apoptosis of HSC treated with low-dose tumor necrosis factor α, as determined by the Alamar blue assay, morphology, and DNA ladder formation. Furthermore, interferon α decreased inhibitor of caspase-activated DNase (ICAD) levels, which may augment tumor necrosis factor α-induced cell death signals. Thus, interferon α regulates the number of myofibroblastic hepatic stellate cells and may clinically contribute to the regression of human liver fibrosis.     

Antiproliferative and proapoptotic effects of somatostatin on activated hepatic stellate cells

AIM:To assess the effects of somatostatin on proliferationand apoptosis of activated rat hepatic stellate cells (HSCs).METHODS:HSCs isolated from the livers of adult Sprague-Dawley rats (weighing 400-500 g) by in situ perfusion andpurified by single-step density gradient centrifugation withNycodenz,became activated after 10 days' cultivation.Thenthe apoptotic rate of HSCs treated with different doses ofsomatostatin for 72 h,was assayed by acridine orange/ethidium bromide fluorescent staining,terminaldeoxynucleotidyl transferase-mediated dUTP nick endlabeling,transmission electron microscopy and flowcytometry,while the proliferation of HSCs was measured byMTT assay.Furthermore,the mechanisms of somatostatinwere investigated by cytodynamic analysis.RESULTS:Somatostatin at the concentration of 10~(-6)-10~(-9) mol/Lcould decrease the proliferative rate,and promote theapoptosis of activated rat HSCs in a dose-dependent way.Its action was most significant when the concentrationreached 10~(-6) mol/L or 10~(-7) mol/L (P<0.05-0.01).An obviouscell-cycle arrest (G_0/G_1 arrest) was the important way forsomatostatin to exert its action.CONCLUSION:Antiproliferative and proapoptotic effects oflow-dose somatostatin on activated rat HSCs can be obtained.These findings reveal its potential antifibrotic action.     

Characterization and enrichment of hepatic progenitor cells in adult rat liver

AIM: To detect the markers of oval cells in adult rat liver andto enrich them for further analysis of characterization in vitro.METHODS: Rat model for hepatic oval cell proliferation wasestablished with 2-acetylaminofluorene and two third partialhepatectomy (2-AAF/PH).Paraffin embedded rat liversections from model (11 d after hepatectomy) and controlgroups were stained with HE and OV6,cytokeratin19 (CK19),albumin,alpha fetoprotein (AFP),connexin43,and c-kitantibodies by immunohistochemistry.Oval cell proliferationwas measured with BrdU incorporation test.C-kit positiveoval cells were enriched by using magnetic activated cellsorting (MACS).The sorted oval cells were cultured in a lowdensity to observe colony formation and to examine theircharacterization in vitro by immunocytochemistry and RT-PCR.RESULTS: A 2-AAF/PH model was successfully establishedto activate the oval cell compartment in rat liver.BrdUincorporation test of oval cell was positive.The hepatic ovalcells coexpressed oval cell specific marker OV6,hepatocyte-marker albumin and cholangiocyte-marker CK19.They alsoexpressed AFP and connexin 43.C-kit,one hematopoieticstem cell receptor,was expressed in hepatic oval cells athigh levels.By using c-kit antibody in conjunction with MACS,we developed a rapid oval cell isolation protocol.The sortedcells formed colony when cultured in vitro.Cells in the colonyexpressed albumin or CK19 or coexpressed both and BrdUincorporation test was positive.RT-PCR on colony showedexpression of albumin and CK19 gene.CONCLUSION: Hepatic oval cells in the 2-AAF/PH modelhad the properties of hepatic stem/progenitor cells.UsingMACS,we established a method to isolate oval cells.Thesorted hepatic oval cells can form colony in vitro whichexpresses different combinations of phenotypic markers andgenes from both hepatocytes and cholangiocyte lineage.     

Effect of ligand of peroxisome proliferator-activated receptor γ on the biological characters of hepatic stellate cells

AIM: To study the effect of rosiglitazone, which is a ligand of peroxisome proliferator-activated receptor gamma (PPARγ), on the expression of PPARγ in hepatic stellate cells (HSCs) and on the biological characteristics of HSCs. METHODS: The activated HSCs were divided into three groups: control group, 3 μmol/L rosiglitazone group, and 10 μmol/L rosiglitazone group. The expression of PPARγ, α-smooth muscle actin (α-SMA), and type Ⅰ and Ⅲ collagen was detected by RT-PCR, Western blot and immunocytochemiccal staining, respectively. Cell proliferation was determined with methylthiazolyltetrazolium (MTT) colorimetric assay. Cell apoptosis was demonstrated with flow cytometry. RESULTS: The expression of PPARy at mRNA and protein level markedly increased in HSCs of 10 umol/L rosiglitazone group (t value was 10.870 and 4.627 respectively, P<0.01 in both). The proliferation of HSCs in 10 μmol/L rosiglitazone group decreased significantly (t=5.542, P<0.01), α-SMA expression level and type I collagen synthesis ability were also reduced vs controls (t value = 10.256 and 14.627 respectively, P<0,01 in both). The apoptotic rate of HSCs significantly increased in 10 μmol/L rosiglitazone group vs control (X2=16.682, P<0,01). CONCLUSION: By increasing expression of PPARγ in activated HSCs, rosiglitazone, an agonist of PPARγ, decreases α-SMA expression and type I collagen synthesis, inhibits cell proliferation, and induces cell apoptosis.     

Effect of ligand of peroxisome proliferator-activated receptor γ on the biological characters of hepatic stellate cells

AIM: To study the effect of rosiglitazone, which is a ligand of peroxisome proliferator-activated receptor gamma (PPARgamma), on the expression of PPARgamma in hepatic stellate cells (HSCs) and on the biological characteristics of HSCs. METHODS: The activated HSCs were divided into three groups: control group, 3 micromol/L rosiglitazone group, and 10 micromol/L rosiglitazone group. The expression of PPARgamma, alpha-smooth muscle actin (alpha-SMA), and type I and III collagen was detected by RT-PCR, Western blot and immunocytochemical staining, respectively. Cell proliferation was determined with methylthiazolyltetrazolium (MTT) colorimetric assay. Cell apoptosis was demonstrated with flow cytometry. RESULTS: The expression of PPARgamma at mRNA and protein level markedly increased in HSCs of 10 micromol/L rosiglitazone group (t value was 10.870 and 4.627 respectively, P<0.01 in both). The proliferation of HSCs in 10 micromol/L rosiglitazone group decreased significantly (t = 5.542, P<0.01), alpha-SMA expression level and type I collagen synthesis ability were also reduced vs controls (t value = 10.256 and 14.627 respectively, P<0.01 in both). The apoptotic rate of HSCs significantly increased in 10 micromol/L rosiglitazone group vs control (chi(2) = 16.682, P<0.01). CONCLUSION: By increasing expression of PPARgamma in activated HSCs, rosiglitazone, an agonist of PPARgamma, decreases alpha-SMA expression and type I collagen synthesis, inhibits cell proliferation, and induces cell apoptosis.     

Effect of rat serum containing Biejiajian oral liquid on proliferation of rat hepatic stellate cells

AIM:Liver fibrosis is a common pathological process ofchronic liver diseases.Activation of hepatic stellate cells(HSCs) is the key issue in the occurrence of liver fibrosis.Inthis study,we observed the inhibitory action of rat serumcontaining Biejiajian oral liquid (BOL),a decoction of turtleshell,on proliferation of rat HSCs,and to explore the anti-hepatofibrotic mechanisms of BOL.METHODS:A rat model of hepatic fibrosis was induced bysubcutaneous injection of CCl_4.Serum containing low,medium and high dosages of BOL was prepared respectively.Normal and fibrotic HSCs were isolated and cultured.Theeffect of sera containing BOL on proliferation of HSCs wasdetermined by ~3H-TdR incorporation.RESULTS:The inhibitory rate of normal rat HSC proliferationcaused by 100 mL/mL sera containing medium and highdosages of BOL showed a remarkable difference as comparedwith that caused by colchicine (medium dosage group:34.56±4.21% vs29.12±2.85%,P<0.01;high dosage group:37.82±1.32% vs29.12±2.85%,P<0.01).The inhibitory rateof fibrotic rat HSC proliferation caused by 100 mL/L serumcontaining medium and high dosages of BOL showed aremarkable difference as compared with that caused by colchicine(medium dosage group:51.31±3.14% vs 38.32±2.65%,P<0.01;high dosage group:60.15±5.36% vs38.32±2.65%,P<0.01).The inhibitory rate of normal rat HSC proliferationcaused by 100 mL/L and 200 mL/L sera containing a mediumdosage of BOL showed a significant difference as comparedwith that caused by 50 mL/L (100 mL/L group:69.02±9.96%vs 50.82±9.28%,P<0.05;200 mL/L group:81.78±8.92%vs 50.82±9.28%,P<0.01).The inhibitory rate of fibrotic rat HSCproliferation caused by 100 mL/L and 200 mL/L sera containinga medium dosage of BOL showed a significant difference ascompared with that caused by 50 mL/L (100 mL/L group:72.19±10.96% vs 61.38±7.16%,P<0.05;200 mL/L group:87.16±8.54% vs 61.38±7.16%,P<0.01).CONCLUSION:Rat serum containing BOL can inhibitproliferation of rat HSCs,and the inhibition depends on thedosage and concentration of BOL.The inhibitory effect on HSC proliferation is one of the main anti-hepatofibroticmechanisms of BOL.     

Effect of lipid on proliferation and activation of rat hepatic stellate cells (Ⅰ)

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Role of free fatty acids in proliferation of rat hepatic stellate cells(Ⅱ)

ＩＮＴＲＯＤＵＣＴＩＯＮＡｌｔｈｏｕｇｈｔｈｅｒｏｌｅｏｆｈｅｐａｔｏｃｙｔｅｓｉｎｆａｔｙａｃｉｄｓｍｅｔａｂｏｌｉｓｍａｎｄｔｒａｎｓｐｏｒｔｈａｖｅｂｅｅｎｄｉｓｃｕｓｅｄｉｎｔｅｎｓｉｖｅｌｙ，ｆｅｗｏｆｔｈａｔｏｆＨＳＣｗａｓｒｅｐｏｒｔｅ...     

Inhibitory effects of prostaglandin E_1 on activation of hepatic stellate cells in rabbits with schistosomiasis

BACKGROUND: Liver fibrosis is the result of an imbalance between synthesis and degradation of extracellular matrix proteins of the liver. At the cellular and molecular levels, this progressive process is mainly characterized by activation of hepatic stellate cells (HSCs). Schistosoma japonica is one of the most prevalent causes of liver fibrosis in China. It is characterized by hepatocyte damage, inflammation, and chronic parasite egg-induced granuloma formation leading to fibrosis. This study aimed to investigate the inhibitory effects of prostaglandin E1 (PGE1) on activation of HSCs and the alteration of type Ⅰ and Ⅲ collagen in rabbits with schistosomiasis. The study may promote the clinical application of praziquantel and PGE1 as a combined therapy to reverse hepatic fibrosis caused by schistosomiasis. METHODS: Rabbits were percutaneously infected with cercaria of S. japonicum. Seven rabbits were subjected to intravenous injections of PGE1 (2.5 μg/kg daily) from days 60 to 120 after infection. The ultrastructural changes in activated HSCs were observed under transmission electron microscopy. The expression of α-smooth muscle actin (α-SMA) was detected by immunohistochemistry. Fibril-forming collagens were detected by picrosirius staining. RESULTS: Activation of HSCs was a characteristic alteration in schistosome-induced hepatic fibrosis. The expression of contraction-related α-SMA and thecontent of collagens were increased. Exogenous PGE1 markedly inhibited the activation of HSCs and reduced the expression of α-SMA around the hepatic sinusoids (P<0.01). The contents of type Ⅰ and Ⅲ collagens were significantly attenuated. The ratio of staining area to the whole field (10×3.3) under a polarized light microscope in the untreated and treated groups was 37.25±9.71 vs. 13.38±4.24 (P<0.01) and 9.66±3.52 vs. 6.23±1.81 (P<0.05), respectively. CONCLUSIONS: Activation of HSCs may play a key role in the progress of schistosome-induced hepatic fibrosis. PGE1 effectively protects rabbit liver from fibrosis, at least in part by inhibiting the activation of HSCs.     

Depletion of β-catenin from mature hepatocytes of mice promotes expansion of hepatic progenitor cells and tumor development

Depletion of β-catenin impairs regeneration of the rapid turn-over gut epithelial cells, but appears dispensable for that of the slow turn-over mature hepatocytes in mice until 1 y of age. As the life span of mature murine hepatocytes is about 400 d, we studied conditional β-catenin knockout mice (Alb-Cre;Ctnnb1(flx/flx)) until 20 mo of age to determine the function of β-catenin in the postnatal liver. β-catenin was absent from the hepatocytes of β-catenin knockout mice 4 wk after delivery. From 9 mo of age, hepatocytes were gradually replaced by newly formed β-catenin-positive hepatocytes, which constituted about 90% of hepatocytes at 18-20 mo of age. This process was accompanied by active proliferation of bile duct/ductule cells. β-catenin-positive hepatocytes exhibited elevated proliferation activity and expression of progenitor cell markers, but lower albumin and Cre. This might explain their intact β-catenin protein, and suggest their origins from hepatic progenitor cells. Liver tumors arose spontaneously from β-catenin-positive cells, and tumorigenesis was accelerated by hepatitis B X protein. These results indicate β-catenin critical for the regeneration of mature hepatocytes. Failure to regenerate mature hepatocytes results in proliferation of hepatic progenitor cells that are able to maintain liver function but are predisposed to form liver tumors.     

HNF-4α determines hepatic differentiation of human mesenchymal stem cells from bone marrow

AIM: To investigate the differentiation status and key factors to facilitate hepatic differentiation of human bone-marrow-derived mesenchymal stem cells (MSCs). METHODS: Human MSCs derived from bone marrow were induced into hepatocyte-like cells following a previously published protocol. The differentiation status of the hepatocyte-like cells was compared with various human hepatoma cell lines. Overexpression of hepatocyte nuclear factor (HNF)-4α was mediated by adenovirus infection of these hepatocyte-like...