To observe the effect of Ligusticum wallichii-containing serum on the expressions of Toll-like receptor 4 and myeloid differentiation factor 88 in hepatic stellate cells. Clean-grade SD rats were randomly divided into 5 groups and orally given L. wallichii decoction, colchicine and normal saline for 7 d to prepare L. wallichii-containing serums. Except for the blank group, all of the remaining groups were stimulated with LPS 1 mg x L(-1) for 24 h. After being intervened, the L. wallichii-containing serums were cultured in 5% CO2 incubator at 37 degrees C for 24 hours. The expression of TLR4 and MyD88 were detected by RT-PCR and Western blot. After HSC was stimulated with LPS, TLR4 and MyD88 mRNA and protein expressions were significantly higher than the blank control group (P < 0.01). After being intervened with L. wallichii-containing serum, TLR4 and MyD88 mRNA and protein expressions were notably lower than the model group (P < 0.05 or P < 0.01). In conclusion, L. wallichii-containing serum could regulate the TLR4 signaling pathway and show the anti-fibrosis effect by inhibiting the expression of TLR4 and MyD88 in LPS-induced HSCs.
Animals ; Female ; Hepatic Stellate Cells ; drug effects ; metabolism ; Ligusticum ; Lipopolysaccharides ; pharmacology ; Liver Cirrhosis, Experimental ; drug therapy ; Myeloid Differentiation Factor 88 ; genetics ; physiology ; Phytotherapy ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Toll-Like Receptor 4 ; genetics ; physiology

PURPOSE: The degradation of the extracellular matrix has been shown to play an important role in the treatment of hepatic cirrhosis. In this study, the effect of thalidomide on the degradation of extracellular matrix was evaluated in a rat model of hepatic cirrhosis. MATERIALS AND METHODS: Cirrhosis was induced in Wistar rats by intraperitoneal injection of carbon tetrachloride (CCl4) three times weekly for 8 weeks. Then CCl4 was discontinued and thalidomide (100 mg/kg) or its vehicle was administered daily by gavage for 6 weeks. Serum hyaluronic acid, laminin, procollagen type III, and collagen type IV were examined by using a radioimmunoassay. Matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1), and alpha-smooth muscle actin (alpha-SMA) protein in the liver, transforming growth factor beta1 (TGF-beta1) protein in cytoplasm by using immunohistochemistry and Western blot analysis, and MMP-13, TIMP-1, and TGF-beta1 mRNA levels in the liver were studied using reverse transcriptase polymerase chain reaction. RESULTS: Liver histopathology was significantly better in rats given thalidomide than in the untreated model group. The levels of TIMP-1 and TGF-beta1 mRNA and protein expressions were decreased significantly and MMP-13 mRNA and protein in the liver were significantly elevated in the thalidomide-treated group. CONCLUSION: Thalidomide may exert its effects on the regulation of MMP-13 and TIMP-1 via inhibition of the TGF-beta1 signaling pathway, which enhances the degradation of extracellular matrix and accelerates the regression of hepatic cirrhosis in rats.
Actins ; Animals ; Carbon Tetrachloride/toxicity ; Collagen Type III/metabolism ; Down-Regulation ; Extracellular Matrix/metabolism ; Immunohistochemistry ; Immunosuppressive Agents/*pharmacology ; Liver Cirrhosis, Experimental/chemically induced/*metabolism/pathology/*prevention & control ; Male ; RNA, Messenger/analysis/metabolism ; Rats ; Rats, Wistar ; Thalidomide/*pharmacology ; Tissue Inhibitor of Metalloproteinase-1/biosynthesis/*drug effects ; Transcription Factor RelA/biosynthesis/drug effects ; Transforming Growth Factor beta1/biosynthesis/*drug effects ; Transforming Growth Factors/metabolism

OBJECTIVETo study the inhibitory effect of flavonoids from Glycyrrhiza uralensis on thioacetamide-induced chonic hepatic fibrosis in rats and the effect on the protein expressions of transforming growth factor-β1 (TGF-β1) and Caspase-3 in livers.

METHODMale Sprague-Dawley rats were randomly divided into totally seven groups: the normal control group, the model group, LF groups s (400, 200, 100, 50 mg · kg(-1) · d(-1)) and the silymarin positive control group (30 mg · kg(-1) · d(-1)). The hepatic fibrosis model was induced in the rats through intraperitoneal injection with 3% thioacetamide (TAA) at a dose of 150 mg · kg(-1) body weight twice a week for 12 weeks. During the course, the control group and the model group were orally administered with saline (1 mL · kg(-1) · d(-1)). After the modeling and drug intervention, the pathologic changes and fibrosis in liver tissues were observed by HE staining and Masson's Trichrome staining. The serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and liver hydroxyproline (HYP) contents were assayed by biochemical process. The serum hyaluronic acid (HA) was assessed by radioimmunoassay. In addition, the protein expressions of liver TGF-β1 and Caspase-3 were examined by immunohistochemical method. The mRNA expression of TGF-β1 in hepatic tissues was examined by quantitative Real-time PCR analysis.

RESULTCompared with the model group, flavonoids can protect the integrity of the structure of liver tissues, significantly reduce the hepatic cell degeneration and necrosis and the proliferation of fibrous tissues, notably reduce the serum AST, ALT, ALP and HA and HYP in hepatic tissues and down-regulate the protein expressions of liver TGF-β1 and Caspase-3 and the mRNA expression of TGF-β1 in hepatic tissues.

CONCLUSIONThe licorice flavonoids can resist the thioacetamide-induced hepatic fibrosis in rats. Its mechanism may be related to the down-regulation of the protein expressions of TGF-β1 and Caspase-3.

Animals ; Caspase 3 ; analysis ; Flavonoids ; pharmacology ; Glycyrrhiza uralensis ; chemistry ; Hyaluronic Acid ; blood ; Liver ; pathology ; Liver Cirrhosis, Experimental ; chemically induced ; metabolism ; prevention & control ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Thioacetamide ; Transforming Growth Factor beta1 ; analysis ; genetics

BACKGROUND/AIMS: The Wnt/beta-catenin signaling pathway has been reported to play an important role in liver fibrosis. This study was designed to investigate whether mesoderm-specific transcript homologue (Mest), a strong negative regulator of Wnt/beta-catenin signaling, could inhibit liver fibrosis. METHODS: pcDNA-Mest was transfected into hepatic stellate cells (HSCs) and rats. Rats were randomly divided into four groups: normal group (normal saline), treatment group (pcDNA-Mest+CCl4), control group (pcDNA-neo+CCl4), and model group (normal saline+CCl4). Changes in liver pathology were evaluated by hematoxylin and eosin and Masson's trichrome staining. The levels of alanine transaminase, aspartate transaminase, lactic dehygrogenase, hyaluronic acid, and laminin in the serum and hydroxyproline in the liver were detected by biochemical examination and radioimmunoassay, respectively. The expression and distribution of beta-catenin, alpha-smooth muscle actin (alpha-SMA), Smad3, and tissue inhibitor of metalloproteinase type I were determined, and the viability of the HSCs was tested. RESULTS: Our data demonstrate that Mest alleviated CCl4-induced collagen deposition in liver tissue and improved the condition of the liver in rats. Mest also significantly reduced the expression and distribution of beta-catenin, alpha-SMA and Smad3 both in vivo and in vitro, in addition to the viability of HSCs in vitro. CONCLUSIONS: We found that Mest attenuates liver fibrosis by repressing beta-catenin expression, which provides a new therapeutic approach for treating liver fibrosis.
Animals ; Carbon Tetrachloride/toxicity ; Cells, Cultured ; Hepatic Stellate Cells/physiology ; Liver Cirrhosis, Experimental/*physiopathology ; Male ; Proteins/*physiology ; Random Allocation ; Rats, Wistar ; Transfection ; Wnt Signaling Pathway/*physiology ; beta Catenin/metabolism

OBJECTIVETo study the protective effect of alcohol extract of Plumula Nelumbini (AEPN) on carbon tetrachloride (CCl4) induced hepatic fibrosis rats and to explore its possible mechanism.

METHODSTotally 32 male SD rats were randomly divided into four groups, i.e., the normal control group, the model group, the high dose AEPN group, and the low dose AEPN group, 8 in each group. 1,000 mg/kg AEPN was given to rats in the high dose AEPN group by gastrogavage at 10 mL/kg, once daily, while 500 mg/kg AEPN was given to rats in the low dose AEPN group by gastrogavage at 10 mL/kg, once daily. Hepatic fibrosis was induced by intraperitoneal injection of CCl4. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB) were examined using automatic biochemical analyzer. Activities of superoxide dismutase (SOD), contents of malondialdehyde (MDA) and hydroxyproline (Hyp) in the hepatic tissue were determined using colorimetry. The degree of liver fibrosis was observed by HE staining and Masson staining. The expression of α-smooth muscle actin (α-SMA) was detected using immunohistochemistry.

RESULTS(1) Compared with the normal control group, serum levels of ALT and AST obviously increased and the serum ALB level obviously decreased in the model group (all P < 0.05). After treated by AEPN, serum levels of ALT and AST were lowered. and the serum ALB level was higher (all P < 0.05). (2) Compared with the normal control group, collagen deposition was obviously seen in rats' livers of the model group, and pseudolobule had formed; inflammatory activities and fibrosis degrees were serious; contents of Hyp also increased (P < 0.05).After treated by AEPN, collagen deposition was obviously reduced with no obvious pseudolobule; inflammatory activities and fibrosis degrees were alleviated; contents of Hyp were also lowered (P < 0.05). (3) Compared with the normal control group, contents of MDA in the liver tissue obviously increased, while activities of SOD obviously decreased (P < 0.05) in the model group. After treated by AEPN, contents of MDA in the liver tissue decreased and the serum SOD level significantly increased (all P < 0.05). (4) Compared with the normal control group, the expression of α-SMA was obviously elevated in the model group (P < 0.05). After treated by AEPN, its expression was obviously lowered (P < 0.05).

CONCLUSIONSAEPN could fight against CCl4 induced liver fibrosis in rats. Fighting against lipid peroxidation and inhibi- ting activation and proliferation of hepatic stellate cells might be possibly main mechanism.

Alanine Transaminase ; metabolism ; Animals ; Carbon Tetrachloride ; Collagen ; Drugs, Chinese Herbal ; pharmacology ; Ethanol ; Hepatic Stellate Cells ; Hydroxyproline ; metabolism ; Lipid Peroxidation ; Liver Cirrhosis, Experimental ; drug therapy ; Male ; Malondialdehyde ; metabolism ; Rats ; Superoxide Dismutase ; metabolism

OBJECTIVETo investigate the effect of Fuzhenghuayu compound (FZHc) on expression of nuclear factor E2-related factor 2 (Nrf2) in hepatocytes under conditions of hepatic fibrosis using a mouse model.

METHODSMice were randomly assigned to a control group and a hepatic fibrosis model group. The control group was further divided into three subgroups for use as normal controls (A1), mineral oil-treated controls (A2), and FZHc-treated controls (A3); the hepatic fibrosis model group was administered carbon tetrachloride (CC14 dissolved in mineral oil and injected intraperitoneally) and further divided into four subgroups for use as 6-weeks models (B1), 10-weeks models (B2), low-dose (L)-FZHc models (C1), and high-dose (H)-FZHc models (C2). The FZHc (capsule powder diluted with double-distilled water to 0.1 g/mL) was administered via gastric perfusion to groups A3, C1, and C2 starting at week 7 of the experiment. At the end of week 6 and 10, hepatic specimens were collected and evaluated for degree of hepatic fibrosis and inflammation using routine haematoxylin-eosin staining and Masson staining. Immunohistochemical analysis was performed to measure the hepatocyte expression of Nrf2, NAD(P)H quinine oxidoreductase 1 (Nqol), a-smooth muscle actin (a-SMA) and fibronectin (FN). Real-time fluorescence quantitative PCR was used to measure Nrf2 mRNA expression. Western blotting was used to detect Nrf2 and Nqol total protein expression and Nrf2 nuclear translocation. F test, LSD test and ridit test were used for statistical analyses.

RESULTSCompared with the B2 group (ridit value: 0.09), the model groups treated with FZHc showed significantly lower degrees of hepatic inflammation and fibrosis for both the low (C1 group, ridit value: 0.32) and high doses (C2 group, ridit value: 0.40) (F =82.927, P less than 0.05). In addition, compared with the B2 group, the model groups treated with FZHc showed significantly decreased expression of a-SMA and FN proteins, with a dose-dependent trend (by immunohistochemistry: C 1 group at the end of 10 weeks, F =77.421, 118.262, P less than 0.05; C2 group, P =0.002, 0.013) and significantly increased expression of Nrf2 and Nqol proteins (by immunohistochemistry:C1 and C2 groups at the end of 10 weeks, F =182.537, 75.615, P less than 0.05 and by westen blotting: F =45.664, 127.673, P less than 0.05), which also showed a dose-dependent trend (C2 group, P =0.000, 0.014; 0.005, 0.014). Western blotting also indicated that the amount of nuclear transported Nrf2 was higher in the C1 and C2 groups at the end of 10 weeks (vs. B2 group, F =94.787, P less than 0.05), and the amount of nuclear transported Nrf2 was significantly higher in the C2 group (vs. C1 group, P =0.044). Nrf2 mRNA expression was significantly higher in the C1 group than in the B2 group (F =3230.105, P less than 0.05), and the C2 group had more substantially increased expression (P =0.001); there was no statistical difference found between groups B1 and B2 (P =0.094).

CONCLUSIONFuzhenghuayu compound increased the expression of Nrf2 mRNA and protein under conditions of hepatic fibrosis in mice and stimulated Nrf2 nuclear transport, as well as increased expression of the Nrf2 target gene Nqol that is known to suppress activation of hepatic stellate cells and decrease the deposition of FN. Therefore, Fuzhenghuayu compound may ameliorate hepatocyte injury in hepatic fibrosis in mice by exerting an antihepatic fibrosis effect.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hepatocytes ; drug effects ; metabolism ; Liver Cirrhosis, Experimental ; metabolism ; Mice ; Mice, Inbred Strains ; NAD(P)H Dehydrogenase (Quinone) ; metabolism ; NF-E2-Related Factor 2 ; metabolism

OBJECTIVETo determine the role of zinc finger protein 1 (ZEB 1) in liver fibrosis and in regards to expression of the tumor growth factor-beta (TGFb) signaling factor using a rat model system.

METHODSSprague-Dawley rats were randomly divided into a normal (control) group, liver fibrosis (model) group and a liver fibrosis + therapy (ZEB1 intervention) group. The model group and the ZEB1 intervention group were given intraperitoneal injections of dimethylnitrosamine (DMN) for the first 3 days of each week over a 7-week period; starting at week 5, the ZEB 1 intervention group was started on a routine of every other day tail vein injections of recombinant ZEB1. During this 7-week period, the control group was given intraperitoneal injections of 0.9% NaC1 alone on the DMN schedule. Liver tissues were collected for pathological examination (with hematoxylin-eosin and Masson staining) and for detection of TGFb1 and ZEB 1 expression (by RT-PCR and western blotting). Measurement data were compared between groups using the single-factor analysis of variance test, followed by the least significant difference LSD test. Count data were analyzed by Fisher's exact test.

RESULTSThe model group's liver tissues showed degeneration and necrosis, as well as obvious fibrous septa accompanied by pseudo lobules. The ZEB 1 intervention group's liver tissues showed a significantly higher degree of fibrosis (x²=21.63, P=0), with more coarse fiber cords. The expression of ZEB1 and TGFb1 was significantly higher in the model group than in the control group (both P less than 0.05). However, the ZEB 1 intervention group showed the highest levels of ZEB 1 and TGFb1 expression (vs. model group, P less than 0.05).

CONCLUSIONZEB 1 may promote the development of liver fibrosis in rats through a mechanism involving the TGFb/Smad signaling pathway.

Animals ; Homeodomain Proteins ; pharmacology ; Liver ; drug effects ; metabolism ; Liver Cirrhosis, Experimental ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Transcription Factors ; pharmacology ; Transforming Growth Factor beta1 ; metabolism ; Zinc Fingers

OBJECTIVETo investigate liver fibrosis-related changes of CD4⁺CD25⁺Foxp3+ regulatory T cells (Tregs) in peripheral blood and in liver-infiltrating lymphocytes (LILs) using a mouse model.

METHODSC57BL/6 mice were randomly divided into a model group and a control group. The model group received intraperitoneal injection of carbon tetrachloride (CC1₄) to induce liver fibrosis, and the control group received an equal volume of physiological saline. Serum was collected for detection of alanine aminotransferase level. Histopathological changes in liver were assessed by microscopic observation of tissues stained by hematoxylineosin and Masson. Frequencies of peripheral and intrahepatic Tregs, NK1.1⁺ cells, CD4⁺ and CD8⁺ cells were analyzed by flow cytometry. Intrahepatic Foxp3+ cells were detected by immunofluorescence. Liver expression of IL-6, IL-10, TGFb and Foxp3 was measured by RT-PCR detection of mRNA .Inter-group differences were evaluated by t-test.

RESULTSThe model group showed a significantly higher frequency of intrahepatic CD25⁺Foxp3⁺/CD4⁺ Tregs (10.63% ± 1.50% vs. control group:1.80% ± 0.66%; P less than 0.01) but only slightly higher frequency of peripheral Tregs (6.00% ± 0.62% vs. 5.33% ± 2.86%). The model group also showed significantly higher levels of intrahepatic Foxp3+ cells and of Foxp3 mRNA (both P less than 0.05), but significantly lower frequencies of NK1.1 cells in LILs (9.53% ± 2.25% vs. 19.80% ± 5.97%; P less than 0.05) and in peripheral blood (0.38% ± 0.13% vs. 1.06% ± 0.63%; P less than 0.05). The CD4⁺ cell frequency and the CD4⁺/CD8⁺ ratio were lower in LILs and peripheral blood of the model group, but none differed significantly from the control group. The intrahepatic expression of TGFb mRNA was significantly higher in the model group (P less than 0.01).

CONCLUSIONIn liver fibrosis, intrahepatic CD4⁺CD25⁺Foxp3+ Tregs are increased while NK1.1+ cells are decreased. Tregs may suppress NK1.1+ cells through a mechanism involving TGFb.

Animals ; Liver ; metabolism ; pathology ; Liver Cirrhosis, Experimental ; blood ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory

OBJECTIVETo observe the pharmacological effect of Cordyceps polysaccharide on dimethylnitrosamine (DMN)-induced liver fibrosis in rats.

METHODDMN rat liver fibrosis model was established and divided into the normal group (N, n = 6), the model group (M, n = 11), the Cordyceps polysaccharide group (C, n = 8) and the colchicine group (Q, n = 9). During the modeling for four weeks, Cordyceps polysaccharide (60 mg x kg(-1)) and colchicine (0.1 mg x kg(-1)) were orally administered for three weeks, while the model and normal groups were given disinfected water of the same amount.

OBSERVATIONserum ALT, AST, GGT and Alb, TBil content; content of hydroxyproline (Hyp) in liver tissues; liver pathology and collagen staining; SOD activity and MDA, GSH, GSH-Px in liver tissues; protein expression of proliferating cell nuclear antigen (PCNA) in liver tissues.

RESULTSerum ALT, AST, GGT, TBil significantly increased, and A1b decreased significantly in the model group. Hepatic Hyp significantly increased in the model group, whereas the index remarkably decreased in the Cordyceps polysaccharide group and the colchicine group. HE staining: the structure of normal hepatic lobules was damaged, with hepatocytes tumefaction and proliferation of connective tissues in portal tracts in the model group, while the Cordyceps polysaccharide group and the colchicine group recorded notable reduction in above pathological changes. Collagen staining: the model group showed hepatic lobule fibrous septum and many intact pseudolobules; while the Cordyceps polysaccharide group and the colchicine group witnessed decrease in collagen deposition. The model group showed significant decrease in SOD, GSH-Px and GSH and increase in MDA, whereas the Cordyceps polysaccharide group and the colchicine group recorded notable growth in GSH and GSH-Px. The model group showed significant decrease in protein expression of PCNA in liver tissues, while the Cordyceps polysaccharide group and the colchicine group showed significant reduction.

CONCLUSIONCordyceps polysaccharide can significantly inhibit DMN-induced liver fibrosis and lipid peroxidation in rats.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Blotting, Western ; Collagen ; metabolism ; Cordyceps ; chemistry ; Dimethylnitrosamine ; toxicity ; Drug Administration Schedule ; Glutathione ; metabolism ; Glutathione Peroxidase ; Hydroxyproline ; Immunohistochemistry ; Lipid Peroxidation ; drug effects ; Liver ; drug effects ; metabolism ; pathology ; Liver Cirrhosis, Experimental ; blood ; etiology ; prevention & control ; Male ; Malondialdehyde ; metabolism ; Phytotherapy ; Polysaccharides ; administration & dosage ; pharmacology ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Rats, Wistar ; Superoxide Dismutase ; metabolism ; gamma-Glutamyltransferase ; blood

OBJECTIVETo investigate the effect of Panax notoginseng saponins (PNS) on the expressions of matrix metalloproteinase (MMP)-13 and its tissue inhibitor of metalloproteinase (TIMP)-1 in carbon tetrachloride (CCl4)-induced hepatic fibrosis rats, and explore the possible mechanism of PNS's effect against hepatic fibrosis.

METHODThe rats were randomly divided into 6 groups: the normal group, the model group, PNS (50, 100, 200 mg x kg(-1)) treatment groups and the Col (0.1 mg x kg(-1)) group. Apart from the normal group, all of the remaining groups were subcutaneously injected with CCl4 twice a week for 18 weeks, in order to establish the hepatic fibrosis rat model. Since the 9th weeks, each treatment group was orally administered with corresponding drugs, and the normal group and the model group were orally administered with equal volume of normal saline for 10 weeks. After the end of the experiment, liver and spleen indexes were calculated; the levels of serum ALT and AST were measured by chromatometry. Liver tissues were collected to detect the pathological alteration HE staining; protein expressions of MMP-13 and TIMP-1 were determined with immuninochemistry. Moreover, MMP-13 and TIMP-1 mRNA expressions was detected by RT-PCR technology.

RESULTCompared with the model group, PNS (100, 200 mg x kg(-1)) significantly mitigated hepatic fibrosis in rats, reduced liver and spleen indexes, ALT and AST contents in serum, and TIMP-1 expression, and notably increased MMP-13 expression in rats with hepatic fibrosis.

CONCLUSIONP. notoginseng saponins have certain protective effect in rats with hepatic fibrosis. Its mechanism is related to up-regulating MMP-3, inhibiting TIMP-1 expression and improving collagen degradation.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Gene Expression Regulation ; drug effects ; Liver Cirrhosis, Experimental ; drug therapy ; metabolism ; Male ; Matrix Metalloproteinase 13 ; genetics ; metabolism ; Panax notoginseng ; chemistry ; RNA, Messenger ; genetics ; Rats ; Saponins ; administration & dosage ; pharmacology ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism