Isolation of antifungal compound from Paeonia suffruticosa and its antifungal mechanism.

OBJECTIVE: To isolate antifungal compound from Paeonia suffruticosa, and to find the antifungal mechanisms by observing the ultrastructural modifications of yeasts in growth phase produced by 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG). METHODS: Peony (Paeonia suffruticosa) root bark (PRB) was separated by solvent extraction and purified by high performance liquid chromatography (HPLC) method using analytical and preparative reversed phase C18 column on the basis of bio-assay method. In order to investigate the antifungal mechanism of PGG, Yeasts were submitted to different concentrations [3 × minimum inhibition concentration (MIC), 0.3 × MIC] for 1 h under constant stirring at 30 °C, and transmission electron microscopy was performed. RESULTS: Based on the antifungal activity of PRB on Candida glabrata CBS138, the antifungal compound were isolated in ethyl acetate layer of PRB and identified as PGG by mass spectrometry, 1H nuclear magnetic resonance (NMR) analyses, with molecular weight of 940 and molecular formular as C41H32O26. Transmission electron microscopy showed that PGG degraded the cell wall envelope. CONCLUSION: The results suggest that PGG may be responsible for the antifungal activity of PRB by disrupting the structure of cell wall directly.

Effects of herbal compound 861 on collagen synthesis and degradation in rat mesangial cells exposed to high glucose.

OBJECTIVE: To investigate the effects of Herbal Compound 861 (Cpd 861) on collagen synthesis and degradation in rat mesangial cells exposed to high glucose. METHODS: The third to fifth passage of rat mesangial cells were exposed to high glucose and Cpd 861 at a concentration of 0.25-4.00 g/L for 24, 48 and 72 h, respectively. Benazepril (10(-7)-10(-3) mmol/L) was selected as positive control. The methyl thiazolyl tetrazolium colorimetric assay was used to evaluate the effect of Cpd 861 on cell proliferation. After incubation with Cpd 861 at a concentration of 2.00 g/L for 48 h, the protein secretions of collagen type IV, matrix metallopeptidase 9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), transforming growth factor beta 1 (TGF-ß1), and hepatocyte growth factor (HGF) were detected by enzyme-linked immunosorbent assay method. And rat mesangial cells were harvested to determine MMP-9, TIMP-1, TGF-ß1 and HGF mRNA expression by reverse transcription polymerase chain reaction. RESULTS: Cpd 861 inhibited cell proliferation induced by high glucose in a dose- and time-dependent manner. Compared with high glucose, collagen type IV production was decreased significantly by Cpd 861 (P<0.01). Cpd 861 increased the protein secretions and mRNA expressions of MMP-9 and HGF, whereas the protein secretions and mRNA expressions of TIMP-1 and TGF-ß1 were reduced markedly (P<0.05). The ratio of MMP-9 to TIMP-1 was enhanced by Cpd 861 significantly. There was no significant difference in all above-mentioned effects between Cpd 861 (2.00 g/L) and benazepril (10(-5) mmol/L). CONCLUSION: The anti-glomerulosclerosis mechanisms of Cpd 861 were partly attributed to its effects of inhibiting mesangial cell proliferation, decreasing collagen synthesis and enhancing collagen degradation.

Emodin improves lipopolysaccharide-induced microcirculatory disturbance in rat mesentery.

OBJECTIVE: Sepsis is a systemic inflammatory response syndrome. Emodin is a major ingredient of Rheum Palmatum, a Chinese herb that is widely used in China for treatment of endotoxemia-related diseases. This study intended to examine the effect of Emodin on LPS-induced rat mesenteric microcirculatory disturbance and the underlying mechanisms. METHODS: The male Wistar rats received LPS (5 mg/kg/hr) for 90 min, with or without administration of Emodin (10 mg/kg/hr) by enema 30 min before (pre-treatment) or after (post-treatment) LPS infusion, and the dynamics of mesenteric microcirculation were determined by inverted intravital microscopy. Expression of adhesion molecules and TLR4, NF-κB p65, ICAM-1, MPO, and AP-1 in mesentery tissue was evaluated by flow cytometry and Western-blot, respectively. RESULTS: Pre or post-treatment with Emodin significantly ameliorated LPS-induced leukocyte emigration, reactive oxygen species production and albumin leakage, and the expression of TLR4, NF-κB p65, ICAM-1, MPO and AP-1 in mesentery. CONCLUSIONS: These results demonstrate the beneficial role of Emodin in attenuating the LPS-induced microcirculatory disturbance, and support the use of Emodin for patients with endotoxemia.

Research on the relationship between thick greasy tongue fur formation and vascular endothelial cell permeability with the protein expression of zonula occludens-1.

OBJECTIVE: To determine the relationship of thick greasy tongue fur formation and permeability of vascular endothelial cells (ECs) with the protein expression of zonula occludens-1 (ZO-1). METHODS: Sprague Dawley rats were randomly divided into a model group of severe acute pancreatitis (SAP) and a sham-operated (SO) group. The SAP rats were further divided into two subgroups on the basis of tongue-coating status: a thick greasy tongue fur group (SAP-TGF) and a normal tongue fur group (SAP-NF). Six rats were chosen randomly from every group mentioned above for an Evans blue assay 5 days after model establishment. For the histomorphology analysis, the expressions of ZO-1 protein and mRNA were studied by hematoxylin-eosin (H&E) staining, transmission electron microscope, Western blot, and Q-PCR using blood and tongue tissues, which were collected from 8 rats randomly chosen from each group. RESULTS: The papillae density of the rat tongue surface and the caryocinesis frequency of the basal layer were significantly increased in the SAP-TGF group compared with the SO group (P<0.05). Evans blue levels in the tongue tissue of the SAP-TGF group were significantly higher than that of the SO and SAP-NF groups (P<0.05). Vascular ECs were wider and obviously fissured in the SAP-TGF group under transmission electron microscope observation. The protein and mRNA expression of ZO-1 in the SAP-TGF group were lower than those in the SAP-NF (P<0.05). CONCLUSIONS: Reproductive activity enhancement of glossal epithelial cells was one of the main characteristics of thick greasy tongue fur formation. An increase in vasopermeability was closely associated with thick greasy tongue fur formation. Tight junction structural variation of vascular ECs might play an important role in the pathological and physiological process of thick greasy tongue fur formation.

Primary isolated hepatic oval cells maintain progenitor cell phenotypes after two-year prolonged cultivation.

BACKGROUND & AIMS: Although expandable hepatic progenitors provide renewable cell sources for treatment of hepatic disorders, long-term cultivation of hepatic progenitors may affect proliferation and differentiation abilities, and even initiate the formation of malignant cancer stem cells. This study aims to determine characteristics of primary cultured hepatic oval cells after prolonged cultivation in vitro. METHODS: Hepatic oval cells isolated from rats fed with a choline-deficient, ethionine-supplemented diet were continuously propagated every 5-7 days, to 100 passages over two years. Hepatocytic differentiation was induced by sodium butyrate and characterized using western blot, periodic acid Schiff assays, albumin secretion and urea production. Proliferation capacity was evaluated using growth-curve and cell-cycle analysis; anchorage-independent growth and tumorigenicity were determined using soft agar and xenograft assay. RESULTS: After 2 years of serial passages, hepatic oval cells with typical epithelial morphology continuously expressed OV-6, BD-1, BD-2, and Dlk as markers for hepatic progenitors, cytokeratin 19 as a cholangiocyte marker, and alpha-fetoprotein and albumin as hepatocyte markers. Furthermore, sodium butyrate could induce these cells to become glycogen-storage cells with the functions of albumin secretion and ureagenesis from ammonia clearance, indicating hepatocytic differentiation. Although proliferation slightly accelerated after the 50th passage, hepatic oval cells stayed diploid cells with features of chromosomal stability, which did not acquire anchorage-independent growth capacity and caused no tumor in immunodeficient mice, suggesting no spontaneous malignant transformation. CONCLUSIONS: Hepatic oval cells retain the progenitor cell features without spontaneous malignant transformation after prolonged cultivation, and thus may serve as an expandable cell source for future exploitation of stem cell technology.

Inhibition of methicillin-resistant Staphylococcus aureus by the compound Qingre granules.

BACKGROUND: The infection rate of methicillin-resistant Staphylococcus aureus (MRSA) is increasing yearly due to the overprescription of antibiotics. Traditional Chinese compound medicines are less inclined to induce bacterial resistance in the clinical setting because of their multi-acting mechanisms. However, most current research is limited to bacteriostasis in vitro using single extracts or formulations. Plasma pharmacology is an in vitro method, using what is called "medicine serum". The aim of this study was to investigate whether the medicine serum of compound Qingre granules (QRKL) alone or in combination with antibiotics may treat MRSA infection in the clinic. METHODS: An animal model of MRSA resistance was created by injecting rabbits with the standard strain of MRSA ATCC43300. Infected rabbits were treated with QRKL by intragastric administration. Sixty minutes after the last intragastric administration, serum was obtained from the rabbits by heart puncture to obtain what is termed "medicine serum". The minimum inhibitory concentration (MIC) of QRKL, medicine serum alone, or serum combined with antibiotics was assessed by agar dilution. RESULTS: were compared with the growth of sixteen isolates of MRSA. RESULTS: The MIC of QRKL to the standard strain ATCC43300 was 10.00 mg/ml. The MIC(90) of vancomycin was 1.00 microg/ml, which, when combined with QRKL, dropped to 0.50 microg/ml. The MIC(90) of cefuroxime alone was 512.00 microg/ml. This level also decreased to 256.00 microg/ml when combined with QRKL. The addition of QRKL thus significantly reduced the MIC of both cefuroxime and vancomycin compared with antibiotics alone (P < 0.01). The MIC(90) of vancomycin with medicine serum decreased to 0.50 microg/ml, and the MIC of vancomycin with medicine serum also descended compared with using vancomycin alone (P < 0.01). CONCLUSIONS: The growth of MRSA can be inhibited by QRKL or medicine serum of QRKL in vitro. The addition of QRKL results in increased sensitivity of MRSA to vancomycin and this may provide a novel treatment for patients with MRSA infection.

Induction of lymphocyte apoptosis in a murine model of acute lung injury--modulation by lipid emulsions.

Acute lung injury (ALI) and sepsis are the major causes of mortality in intensive care units. Lymphocytes apoptosis is a hallmark feature of late detrimental sepsis. Parenteral nutrition in critically ill patients is based on lipid emulsions, but the impact of ALI and lipid emulsions on lymphocytes has not been defined. The effects of intravenously infused conventional soybean oil (SO)-based and new olive oil (OO)-based emulsions on splenic and blood lymphocytes were investigated in a murine model of endotoxin-induced ALI. After LPS challenge and infusion of lipid emulsions, apoptosis of lymphocytes and lung injury were assessed by flow cytometry, Western blot, and histology. Induction of ALI led to a time-dependent decline in splenic and circulating lymphocyte numbers and an increase in apoptosis, with engagement of the extrinsic apoptotic pathway. Both SO- and OO-based emulsions promoted the apoptosis of splenic lymphocytes before induction of ALI. The OO-based emulsions exhibited lower proapoptotic activity than did SO-based emulsions, an observation paralleled by the induction of survival factors. Induction of ALI increased the mortality of mice receiving SO-based emulsions compared with OO-based emulsions and normal saline. Splenic lymphocyte apoptosis is apparent in murine ALI, which may be linked to detrimental outcome. Infusion of lipid emulsions per se provoked splenic lymphocyte apoptosis. Infusion of SO-based emulsions further augmented the apoptosis of splenic and circulating lymphocytes in ALI and led to increased mortality in mice. These findings may be of relevance for patients experiencing ALI that require parenteral nutrition.

Pirfenidone inhibits carbon tetrachloride- and albumin complex-induced liver fibrosis in rodents by preventing activation of hepatic stellate cells.

1. Pirfenidone (PFD; 5-methyl-1-phenyl-2(1H)-pyridone) is an effective and novel agent with antifibrotic and anti-inflammatory properties. In the present study, we investigated the antifibrotic effects of PFD on experimental liver fibrosis models in rodents and the possible underlying molecular mechanisms. 2. Liver fibrosis was induced by carbon tetrachloride (CCl(4)) in BALB/c mice. Pirfenidone (250 mg/kg) and silymarin (50 mg/kg) were given to different groups of rats by gastric gavage for 4 weeks. Pirfenidone significantly attenuated fibrosis severity, as determined by histopathological scores and hydroxyproline levels in liver tissue, by 49.8 and 44.9%, respectively, compared with the CCl(4)-treated group. The antifibrotic effects of PFD were significantly greater than those of silymarin, as indicated by a decrease of 23.5 and 24.8% in histopathological scores and hydroxyproline levels, respectively. 3. Liver fibrosis was also induced by albumin antigen-antibody complex in Wistar rats, which were then treated with the same doses of PFD and silymarin for 8 weeks. Pirfenidone significantly reduced the degree of fibrosis compared with CCl(4)-treated rats (by 45.0 and 51.0% as determined by histopathological scores and hydroxyproline levels in liver tissue, respectively). The antifibrotic effects of PFD were comparable to those of silymarin. 4. The effects of PFD on the expression of extracellular matrix-associated genes in human hepatic stellate cells (the LX-2 cell line) were measured by real-time quantitative polymerase chain reaction. LX-2 cells were treated with or without 100 micromol/L or 1 mmol/L PFD for 24 h. Pirfenidone significantly inhibited the expression of a-smooth muscle actin and Type I collagen in 8 ng/mL transforming growth factor-beta1- or 5% fetal bovine serum-activated LX-2 cells in a dose-dependent manner. 5. In conclusion, the results of the present study demonstrate that PFD is effective in ameliorating fibrogenesis induced by CCl(4) in mice and by the albumin complex in rats. These effects were mediated mainly via inhibition of the activation of hepatic stellate cells, as well as antifibrotic actions (i.e. inhibition of collagen synthesis) of PFD.

[Protective effect of compound tongfu granule on intestinal mucosal barrier in patients with cirrhosis of decompensation stage].

OBJECTIVE: To explore the intestinal mucosal barrier protective effect of herbal medicine Compound Tongfu Granule (CTG) in patients with liver cirrhosis of decompensation stage. METHODS: Fifty patients enrolled were randomly assigned to the control group (26 cases) and the CTG group (24 cases), and 30 healthy adults were set up as normal control. After 2-week treatment, the intestinal permeability (IP, represented by urinary lactulose/mannitol excretion rate), plasma endotoxin (EDT) level, and change of enteric bacteria (EB) in patients were observed before and after treatment, and compared with those in the normal control. RESULTS: Before treatment, cirrhotic patients showed significantly higher levels of IP, EDT, and intestinal bacilli, but a lower amount of enteric bifidobacteria as compared with those the normal control. After 2-week treatment, levels of EDT and urinary excretion rate of lactulose in the CTG group were lowered more significantly than those in the control group (P < 0.05), while the amount of bifidobacteria in the CTG group increased accompanied with intestinal bacilli significantly lowered to near the levels in the normal control (P < 0.05, P < 0.01). CONCLUSION: CTG can improve the intestinal barrier function, correct the intestinal bacteria disturbance, and significantly reduce the entero-derived endotoxemia in cirrhotic patients of decompensation stage.

Down-regulation of transforming growth factor beta 1/activin receptor-like kinase 1 pathway gene expression by herbal compound 861 is related to deactivation of LX-2 cells.

AIM: To investigate the effect of herbal compound 861 (Cpd861) on the transforming growth factor-beta1 (TGF beta 1)/activin receptor-like kinase 1 (ALK1, type I receptor) signaling-pathway-related gene expression in the LX-2 cell line, and the inhibitory mechanism of Cpd861 on the activation of LX-2 cells. METHODS: LX-2 cells were treated with TGF beta 1 (5 ng/mL) Cpd861 (0.1 mg/mL), TGF beta 1 (5 ng/mL) plus Cpd861 (5 ng/mL) for 24 h to investigate the effect of Cpd861 on the TGF beta 1/ALK1 pathway. Real-time PCR was performed to examine the expression of alpha-SMA (alpha-smooth muscle actin), ALK1, Id1 (inhibitor of differentiation 1). Western blotting was carried out to measure the levels of alpha-SMA and phosphorylated Smad1, and immunocytochemical analysis for the expression of alpha-SMA. RESULTS: In LX-2 cells, TGF beta 1/ALK1-pathway-related gene expression could be stimulated by TGF beta 1, which led to excessive activation of the cells. Cpd861 decreased the activation of LX-2 cells by reducing the expression of alpha-SMA mRNA and protein expression. This effect was related to inhibition of the above TGF beta 1/ALK1-pathway-related expression of genes such as Id1 and ALK1, and phosphorylation of Smad1 in LX-2 cells, even with TGF beta 1 co-treatment for 24 h. CONCLUSION: Cpd861 can restrain the activation of LX-2 cells by inhibiting the TGF beta 1/ALK1/Smad1 pathway.

Herbal compound 861 regulates mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells.

AIM: To identify the role of herbal compound 861 (Cpd 861) in the regulation of mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells (HSCs). METHODS: mRNA levels of collagen types I and III, matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 2 (MMP-2), membrane type-1 matrix metalloproteinase (MT1-MMP), tissue inhibitor of metalloproteinase 1 (TIMP-1), and transforming growth factor beta1 (TGF-beta1) in cultured-activated HSCs treated with Cpd 861 or interferon-gamma (IFN-gamma) were determined by real-time PCR. RESULTS: Both Cpd 861 and IFN-gamma reduced the mRNA levels of collagen type III, MMP-2 and TGF-beta1. Moreover, Cpd 861 significantly enhanced the MMP-1 mRNA levels while down-regulated the TIMP-1 mRNA expression, increasing the ratio of MMP-1 to TIMP-1 to (6.3 + 0.3)- fold compared to the control group. CONCLUSION: The anti-fibrosis function of Cpd 861 may be mediated by both decreased interstitial collagen sythesis by inhibiting the transcription of collagen type III and TGF-beta1 and increased degradation of these collagens by up-regulating MMP-1 and down-regulating TIMP-1 mRNA levels.

[Experimental study of Qishen Yiqi Dropping Pills on liver fibrosis in rats].

OBJECTIVE: To investigate the anti-fibrotic effects of Qishen Yiqi Dropping Pills (QYDP) in rats with liver fibrosis (LF). METHODS: The LF model was induced by intraperitoneal injection with dimethylnitrosamine (DMN). Sixty Wistar rats were randomly divided into the normal group, the model group A, the QYDP intervened group , the model group B , and the QYDP treated group B. The degree of LF was evaluated according to 6-phase grading method. The expressions of collagen type I and III and tissue inhibitor of metalloproteinase-1 (TIMP-1) in liver tissues were determined by immunohistochemistry and the levels of collagen type I and III and TIMP-1 mRNA determined by semi-quantitive RT-PCR. RESULTS: Compared with the model group A and B, the degree of LF, the positive expressions of TIMP-1 mRNA and the content of collagen type I and III in liver tissue in the QYDP intervened and treated groups were significantly lower. CONCLUSION: QYDP could reduce the pathological changes and degree of LF in rats, which may be partially through inhibiting the expressions of collagen type I and III and TIMP-1.

[Effect of different fat emulsions on acute lung injury induced by endotoxin].

OBJECTIVE: To assess the effect of Clinoleic 20% (olive oil-based, n-9) and Lipoven 20% (soy bean-based, n-6) lipid emulsions on inflammatory parameters in a murine acute lung injury (ALI) model induced by lipopolysaccharide (LPS) of E. coli O111:B4. METHODS: Male Balb/C mice were infused for three days with 0.9% NaCl, Clinoleic 20%, or Lipoven 20% respectively, and sacrificed either at 8 hours or 24 hours after intra-tracheal introduction of LPS. Survival rate, lung wet/dry weight ratio (W/D), lung tissue myeloperoxidase (MPO) activity were determined, and tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-2 (MIP-2) in bronchoalveolar lavage fluid (BALF) were determined with enzyme linked immunosorbent assay (ELISA). Serum free fatty acids [arachidonic acid (AA), oleic acid, linoleic acid] were determined by gas chromatography. Leukocytes in BALF were counted under light microscope. RESULTS: Lipoven significantly decreased survival rate at 24 hours after intra-tracheal LPS challenge compared to corresponding controls (both P<0.01). No significant difference was observed between Clinoleic and NaCl groups. MPO activity was obviously increased in lipids groups than that in NaCl group at 24 hours (both P<0.01), and no difference was found between two lipids groups. LPS markedly induced an increase in leukocyte infiltration, W/D ratio, lung MPO activity, release of TNF-alpha as well as MIP-2 into alveolar space in both lipids and NaCl groups. Pre-infusion with Lipoven gave rise to heavier leukocyte infiltration at 24 hours, which was blunted in Clinoleic group and NaCl group (both P<0.01). In contrast to Clinoleic and NaCl groups, Lipoven increased production of TNF-alpha at 24 hours and MIP-2 at 8 hours in LPS-treated mice (all P<0.01). Notably, lipid emulsions increased LPS-induced MPO activity, but no difference in effects was found in both Lipoven and Clinoleic groups. Clinoleic significantly reduced free AA at 8 and 24 hours compared with Lipoven (both P<0.01). There were no differences in lung tissues edema, serum oleic acid and linoleic acid among three groups. CONCLUSION: In murine model of ALI, although LPS caused an increase in alveolar leucocytic infiltration, MPO activity, cytokine generation in both lipids and NaCl groups, Lipoven 20%, n-6 lipid emulsion induces a severer inflammatory reaction. It is speculated that by increasing AA, Lipoven 20% may aggravate ALI, whereas Clinoleic 20%, n-9 lipid emulsion possibly offers an alternative choice in producing less impact on inflammatory lung injury.

Treatment of severe post-kidney-transplant lung infection by integrative Chinese and Western medicine.

OBJECTIVE: To explore treatments of severe post-kidney-transplant lung infection by integrative Chinese and Western medicine (ICWM), in order to elevate the curing rate as well as to lower the death rate. METHODS: Based on conventional ways of Western medical treatments of 18 cases of severe post-kidney-transplant lung infection, such as putting the patients in single individual ward, antibiotics to prevent infection, respiratory machines, blood filtration, nutritional support, steroids, and maintaining electrolytes balance, we applied integrated Chinese medicinal treatments, like altering conventional prescription "pneumonia III", and conducted clinical observation of effectiveness, and indexes including white blood cell (WBC), neutrophilic granulocyte, blood urea nitrogen (BUN), blood creatinine (Cr), etc. RESULTS: Of the 18 cases studied, 7 were already cured, 8 proved the treatment effective, 3 died. All clinical indexes had statistically significant changes compared with those of before treatment (P < 0.01). CONCLUSION: ICWM can increase curing rate and lower death rate.

[Inhibitory effects of silymarin on hepatic fibrosis induced by dimethylnitrosamine: experiment with rats].

OBJECTIVE: To investigate the antifibrotic effects of silymarin on hepatic fibrosis. METHODS: Sixty-one male Wistar rats were randomly divided into three groups: control group (15 rats); DMN model group (23 rats), injected intraperitoneally with dimethylnitrosamine (DMN) 10 mg/kg twice per week for 8 weeks to induce hepatic fibrosis; and silymarin group (23 rats), injected intraperitoneally with DMN and given silymarin 50 mg/kg by gastric gavage daily for 8 weeks. Eight weeks late all rats were sacrificed. Blood samples were collected to measure the alanine transaminase (ALT), aspirate aminotransferase (AST), albumin, and total bilirubin (TBIL). The hydroxyproline (Hyp) content in the liver tissue was measured. The histopathological changes as well as the fibrosis stages and score were examined by microscopy. RESULTS: The levels of ALT, AST, and TBIL of the silymarin groups were 59 U/L +/- 19 U/L, 159 U/L +/- 39 U/L, and mean rank 24 respectively, all significantly lower than those of the DMN model group (128 U/L +/- 25 U/L, 246 U/L +/- 61 U/L, and mean rank 37 respectively, P < 0.01, P = 0.001, and P = 0.003). Compared with DMN rats, the level of Hyp of the silymarin was lower by 42.6%, the hepatic score of the silymarin was 6.2 +/- 2.4, significantly than that of the DMN model group (12.8 +/- 4.4, P = 0.001), and more cases in the silymarin group were at the lower stages. CONCLUSION: Silymarin markedly inhibits and reverse the progression of hepatic fibrosis induced by dimethylnitrosamine.

[Sodium butyrate induces rat hepatic oval cells differentiating into mature hepatocytes in vitro].

OBJECTIVE: To elucidate the effects of sodium butyrate on rat hepatic oval cell differentiation in vitro. METHODS: Hepatic oval cells were isolated from rats fed with a choline-deficient diet supplemented with 0.1% (w/w) ethonine for 4 to 6 weeks. The cultured hepatic oval cells were identified by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). After hepatic oval cells were treated with sodium butyrate, the morphological changes were studied through Giemsa staining and the albumin expression level was tested by Western blot. RESULTS: Immunohistochemical results showed the isolated cells were positive for both mature hepatocyte marker albumin and bile duct cell marker cytokeratin-19. Furthermore, RT-PCR results showed that the cells expressed stem cell marker c-kit, but not hematopoietic stem cell marker CD34. In short, the isolated cells were rat hepatic oval cells. 0.75 mmol/L sodium butyrate induced obvious phenotype changes of hepatic oval cells, including enlargement of the oval cells, a decrease in nucleus to cytoplasm ratio, and a 50% increase in the number of binucleated cells. Western blot results showed that 0.75 mmol/L sodium butyrate markedly raised the expression of albumin. CONCLUSION: Sodium butyrate, a differentiation promoting agent, can induce rat hepatic oval cells (liver progenitor cells) to differentiate into mature hepatocytes in vitro.

[The effect of Cpd 861 on chronic hepatitis B related fibrosis and early cirrhosis: a randomized, double blind, placebo controlled clinical trial].

OBJECTIVES: To further assess the clinical antifibrotic efficacy of Cpd 861 on chronic hepatitis B related fibrosis and early cirrhosis using a randomized, double blind, and placebo controlled clinical trial. METHODS: Total 136 patients with HBV-related fibrosis and early cirrhosis were allocated randomly into Cpd 861 treatment group and placebo group for 24 weeks treatment. Serum fibrosis markers including hyaluronic acid (HA), IV collagen (IV-C), amino terminal propeptide of type III procollagen (PIIIP), and laminin (LN) and serum MMP1, 2, 9, TIMP1, 2 level were determined before and after 24 weeks treatment. Liver biopsies before and after 24 weeks of treatment were assessed according to modified Scheuer and Chevallier's scoring system. RESULTS: Total 52 patients in Cpd 861 treatment group and 50 patients in placebo-controlled group completed the 6 months. ALT level decreased from 68.2 U/L+/-68.6 U/L to 45.9 U/L+/-26.1 U/L, AST level decreased from 60.4 U/L+/-62.6 U/L to 46.7 U/L+/-39.0 U/L (P < 0.05) after 24 weeks treatment, whereas there was no significant change in placebo group (ALT: 65.3 U/L+/-48.3 U/L to 85.4 U/L+/-115.5 U/L; AST: 60.4 U/L+/-44.6 U/L to 77.6 U/L+/-89.6 U/L, P > 0.05). Serum fibrosis markers, including HA, IV-C, PIIIP, and LN were decreased after treatment, but there is no statistically significant compared with placebo group. Compared with placebo group, serum TIMP1 and MMP9 level decreased significantly (TIMP1 172.0 ng/ml+/-79.6 ng/ml vs 133.5 ng/ml+/-66.8 ng/ml; MMP9 116.1 ng/ml+/-88.2 ng/ml vs 80.4 ng/ml+/-79.0 ng/ml), and the ratio of TIMP1/MMP1 (48.3+/-96.3 vs 19.9+/-28.0) were also decreased after 861 treatment. In patients treated with Cpd 861, hepatic inflammatory score (from 14.0+/-6.0 to 10.2+/-6.1), fibrosis score (from 11.9+/-6.5 to 8.2+/-4.5), and relative content of collagen (from 18.9%+/-9.5% to 14.9%+/-8.4%) decreased significantly. In contrast, there was no significant change in placebo group. The reversal (fibrosis score decrease > or = 2) rate of fibrosis in Cpd 861 group was 38.9% in S2, 53.3% in S3 (precirrhotic) and 78.6% in S4 (cirrhosis), significantly higher than those in placebo group (14.3%, 25.0%, and 41.7%, respectively). The overall reversal rate was 52.0% in Cpd 861 group, and 20.0% in placebo group (P < 0.05). No serious adverse effects were observed during Cpd 861 treatment. CONCLUSION: Liver fibrosis and early cirrhosis due to HBV infection in man could be definitely reversed by herbal remedy Cpd 861.