Study on mechanism of salidroside against liver fibrosis by regulating CXCL16 / 中国中药杂志

In order to investigate the effect of salidroside on inhibiting liver fibrosis and its relationship with CXC chemokine ligand 16(CXCL16) in vivo and in vitro, totally 45 C57 BL/6 J male mice were randomly divided into normal group, model group and salidroside group, with 15 mice in each group. The mice in model group and salidroside group were injected intraperitoneally with 15% carbontetrachloride(CCl_4) olive oil solution to establish liver fibrosis model, and the mice in normal group were injected intraperitoneally with the same dose of olive oil. Salidroside group was given with 100 mg·kg~(-1 )salidroside by gavage, while the normal group and model group received the same amount of double distilled water by gavage. All mice were sacrificed after 5 weeks of intragastric administration. The pathological changes of mouse liver were observed by hematoxylin-eosin(HE) staining, and the degree of liver fibrosis was observed by sirius red staining. The protein expressions of collagen Ⅰ(ColⅠ), α-smooth muscle actin(α-SMA), fibronectin(FN), CXCL16, phosphorylated Akt(p-Akt), Akt in liver tissues were detected by Western blot. Hepatic stellate cell line JS 1 was cultured in vitro and divided into control group, model group(100 μg·L~(-1) CXCL16) and salidroside group(100 μg·L~(-1) CXCL16+1×10~(-5) mol·L~(-1) salidroside). Cell migration was detected by cell scratch, the mRNA expressions of ColⅠ and α-SMA were detected by RT-PCR, and the protein expressions of p-Akt and Akt were detected by Western blot. As compared with the normal group, the protein expressions of ColⅠ, α-SMA, FN, CXCL16, and p-Akt in the model group were significantly increased, and salidroside could reduce the expression of these indicators(P<0.05 or P<0.01). In vitro, CXCL16 could promote the migration of JS 1, increase the mRNA expressions of ColⅠ and α-SMA in JS 1, and enhance Akt phosphorylation in JS 1(P<0.05 or P<0.01). As compared with the model group, salidroside could inhibit the migration of JS 1 induced by CXCL16(P<0.05), and reduce the high expression of ColⅠ and α-SMA mRNA and the phosphorylation of Akt in JS 1 induced by CXCL16(P<0.05). In conclusion, salidroside might attenuate CCl_4-induced liver fibrosis in mice by inhibiting the migration, activation and Akt phosphorylation of hepatic stellate cells induced by CXCL16.

Guidelines for diagnosis and treatment of hepatic fibrosis with integrated traditional Chinese and Western medicine (2019 edition) / 中西医结合学报

In 2006, the Hepatology Committee of Chinese Association of Integrative Medicine issued the "Guidelines for the Prevention and Treatment of Liver Fibrosis with Integrated Traditional Chinese and Western Medicine." In recent years, the fields of Chinese medicine, Western medicine, and integrative medicine have made rapid advances in basic and clinical research into chronic liver disease, and accumulated new evidence for the prevention and treatment of hepatic fibrosis. Therefore, in order to meet clinical needs, liver disease experts of integrated traditional Chinese and Western medicine were united to revise the previous guidelines in order to help physicians make correct and reasonable decisions in the diagnosis and treatment of hepatic fibrosis.

Effect of bone marrow mesenchymal stem cell transplantation on CCl₄-induced liver injury in rats and intervention effects of Yiguanjian / 中国中药杂志

The aim of this paper was to observe the function of bone marrow mesenchymal stem cell (BMSC) transplantation in process of liver injury induced by carbon tetrachloride (CCl₄) and the intervention effect of Yiguanjian (YGJ), a compound of Chinese herbal medicine. Wistar rats were randomly divided into five groups: normal group, model group, cell transplantation (CT) group, YGJ group and cell transplantation plus Yiguanjian (CTY) group. Liver injury was induced through subcutaneous injection with CCl₄ at a dose of 3 mL·kg⁻¹ body weight for 4 weeks, twice a week. They were injected for a total of 9 times. After the first injection with CCl₄, rats in the CT group and CTY group were injected with the third-generation BMSCs at dose 1×10⁶ (suspended in 1 mL saline solution) via tail vein. Rats in the YGJ and CTY groups were also intragastrically administered with Yiguanjian once a day. Rat serum ALT and AST activities were increased significantly on the second day after injection with CCl₄, while BMSC transplantation and Yiguanjian decreased their activities. After 4 weeks of injection with CCl₄, serum ALT, AST and -GT activities, and serum TNF- and IL-6 expressions were increased, while TBIL were decreased in model rats compared with normal rats. Meanwhile, liver cells edema, plasmatic loose, and numerous lipid droplets were observed in rats of the model group. BMSC transplantation aggravated liver injury compared with model rats, which was manifested by decreasing SOD activity, increased MDA, TG, TNF- and IL-6 levels, and aggravated necrosis level of hepatocytes, fusion of lipid droplets, and collagen deposition in liver tissue. Yiguanjian decreased liver injury induced by CCl₄ alone and CCl₄ plus BMSC transplantation. SRY gene hybridization method was used to detect the positive SRY expressions in heart, liver, spleen, lung and kidney, especially in liver, while Yiguangjian decreased liver SRY expression. Wnt and -catenin showed high expressions in rats of normal group, which were decreased significantly in rats of models group, while Yiguanjian increased their expressions. In conclusion, BMSC transplantation could exacerbate liver injury, while Yiguanjian could protect liver injury induced by CCl₄ and BMSC transplantation, which was related to decreasing the homing of BMSCs to liver and up-regulating Wnt/-catenin signaling pathway.

Study on inhibitory effect of calycosin on hepatic stellate cell activation in rats by up-regulating peroxisome proliferator-activated receptor γ / 中国中药杂志

To observe the effect of calycosin on the proliferation and activation of primary hepatic stellate cells (HSCs) in rats, and prove calycosin shows the effects through peroxisome proliferator-activated receptor γ(PPARγ) and farnesoid X receptor (FXR). The results indicated that calycosin could inhibit HSC proliferation and expressions of activation marker smooth muscle actin-α and type I collagen. With the increase in HSC activation time, FXR expression reduced, but with no notable impact from calycosin. Calycosin could up-regulate PPARγ expression and its nuclear transition in a concentration-dependent manner. Its prohibitory effect on HSC activation could be blocked by PPARγ antagonist. In conclusion, calycosin could inhibit HSC activation and proliferation, which may be related with the up-regulation of PPARγ signal pathway.

Traditional Chinese medicine for treatment of liver diseases: progress, challenges and opportunities / 中西医结合学报

Traditional Chinese medicine (TCM) is commonly used in treating liver diseases worldwide, especially in China. The advantages of using TCM for treatment of liver diseases include: protecting hepatocytes, inhibiting hepatic inflammation and antifibrosis in the liver. In this article, we introduce TCM herbal preparations from the Chinese materia medica (such as Fuzheng Huayu) that are typically used for the treatment of liver diseases. Literature surrounding the mechanisms of TCM therapy for treatment of liver diseases is presented and discussed. We propose that side effects of herbal compounds are often under-appreciated, and that more care should be taken in the prescription of potentially hepatotoxic medicines. Further, to deepen the understanding of TCM mechanisms, new techniques and methodologies must be developed. Future studies will lead to the enhancement of clinical outcomes of TCM. As complementary and alternative therapies, TCMs will play an expanding role in the future of liver disease treatment.

Effect of Yiguan Decoction on differentiation of bone marrow mesenchymal stem cells into hepatocyte-like cells: an experimental research / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the effect of Yiguan Decoction (YGD) on differentiation of bone marrow mesenchymal stem cells (BMSCs) into hepatocyte-like cells in vitro.</p><p><b>METHODS</b>Rat BMSCs were isolated using whole bone marrow adherent method. The properties of BMSCs were identified by analyzing the expression of surface cytokines by flow cytometry. The third passage cells were differentiated into fat cells to identify their features. BMSCs were incubated with hepatocyte growth factor (HGF) plus fibroblast growth factor 4 (FGF4) or YGD containing serum YGD for 21 days. The mRNA expression of alpha-fetoprotein (alphaAFP), albumin (Alb), and hepatocyte nuclear factor 4alpha (HNF4alpha) were detected by real time PCR. Expression of AFP and cytokeratin 18 (CK18) protein was detected by cell immunofluorescence. Glycogen synthesis was observed using periodic acid-Schiff stain (PAS). CK18, Wnt 3alpha, and alphacatenin protein expressions were detected by Western blot.</p><p><b>RESULTS</b>High expression of CD90, CD29, and CD44, and low expression of CD34 and CD11b were observed in BMSCs isolated by whole bone mar- row adherent method, and numerous lipid droplets were observed in BMSCs using oil red O staining. Both YGD containing serum and growth factor stimulated the expression levels of Alb, AFP, HNF4alpha mRNA and CK18 protein. The down-regulated expression of Wnt 3alpha and beta-catenin could be detected at 21 days after induction. The synthesized glycogen granule could be seen. Down-regulated Wnt 3alpha and beta-catenin expression could also be observed.</p><p><b>CONCLUSION</b>YGD could induce the differentiation of rat BMSCs into hepatocyte-like cells, which was related to down-regulating Wnt/beta-catenin signal pathway.</p>

Performance of FibroScan in evaluating the curative effects of traditional Chinese medicine on liver fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To assess the performance of FibroScan in evaluating the curative effects of traditional Chinese medicine (TCM) on liver fibrosis, and to analyze factors influencing the diagnostic accuracy.</p><p><b>METHODS</b>Data of FibroScan values, types of disease, use of drug, liver function indexes, prothrombin time (PT) and international normalized ratio (INR) were collected at both pre- (1 month prior) and post-FibroScan for 102 patients who underwent at least two FibroScan procedures. Patients were subgrouped according to presence of fibrosis, presence of cirrhosis, and TCM formulation and statistically analyzed.</p><p><b>RESULTS</b>The pre- and post-FibroScan mean liver stiffness measurements (LSMs) were significantly different when the variation of LSM was more than or equal to2 kPa for the non-fibrotic group (vs. the fibrotic group), or when the variation wasmore than or equal to4 kPa for the cirrhotic group (vs. the non-cirrhotic group). In addition, the three TCM formulation groups showed significant differences, with the most robust difference exhibited between the FuZheng HuaYu formulation group and the other treatment groups (P = 0.010). No significant differences were observed for the liver function indexes, PT, or INR. However, the post-FibroScan levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyltransferase (GGT) was significantly reduced in patients with reduced LSM.</p><p><b>CONCLUSION</b>FibroScan may be a useful non-invasive clinical tool for evaluating the comprehensive curative effect of treatments for chronic liver diseases, and its performance is not obviously impacted by ALT, AST, GGT, PT, and INR. The criteria for efficacy established by FibroScan are 2 kPa for the patients without liver fibrosis and 4 kPa for patients with liver cirrhosis.</p>

Effects of cordyceps acid and cordycepin on the inflammatory and fibrogenic response of hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects of cordyceps acid and cordycepin on the inflammatory phenotype and fibrogenic property of hepatic stellate cells (HSCs).</p><p><b>METHODS</b>An immortalized mouse HSC line (JS1) was stimulated with lippolysaccharide (LPS; 100 ng/ml) to induce an inflammatory response with or without co-administration of cordyceps acid or cordycepin in various concentrations (10, 50, or 200 mumol/L). Effects of the treatments on the chemokine monocyte chemotactic protein-1 (MCP-1) mRNA expression in the cells and the protein secretion in the cell culture supernatants were determined by reverse transcription and real-time quantitative PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. In addition, JS1 cells were treated with transforming growth factor-b1 (TGFb1; 10 ng/ml) to induce a fibrogenic response with or without co-administration of cordyceps acid or cordycepin in various concentrations (10, 50, or 200 mumol/L). Effects on the expression of fibrogenic proteins including collagen type I and a-smooth muscle actin (a-SMA), were investigated by Western blot.</p><p><b>RESULTS</b>High-concentration (200 mumol/L) treatments of both cordyceps acid and cordycepin significantly inhibited the LPS-induced up-regulation of MCP-1 transcription and secretion (mRNA: 2.07 +/- 0.29 vs. 3.35 +/- 0.26, t = 15.90 and 1.15 +/- 0.23 vs. 4.17 +/- 0.61, t = 8.93; protein: 1.88 +/- 0.06 vs. 2.33 +/- 0.06, t = 10.39 and 1.47 +/- 0.25 vs. 1.97 +/- 0.04, t = 4.60; all P less than 0.05). All concentrations of cordyceps acid and cordycepin inhibited the TGFb1-induced up-regulation of collagen type I and a-SMA protein expression. However, the effects were more robust with the 200 mumol/L concentrations (P less than 0.05).</p><p><b>CONCLUSION</b>Cordyceps acid and cordycepin ameliorate the LPS-induced inflammatory phenotype and TGFb1-induced fibrogenic response of cultured HSCs. These effects may contribute significantly to the drugs' therapeutic mechanisms to inhibit and resolve liver fibrosis.</p>

Effects of salvianolic-acid B on the p38MAPK signaling pathway and its transcriptional factor induced by transforming growth factor b1 in activated rat hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects of Salvianolic-acid B on p38MAPK signaling pathway and its transcriptional factor activated by Transforming growth factor b1 in rat hepatic stellate cells.</p><p><b>METHODS</b>Hepatic stellate cells were isolated from normal rat by in situ perfusion and Nycodenz density-gradient centrifugation method.TGFb1 (10 ng/ml), PD98059(50 mumol/L), SB203580(10 mumol/L) and SA-B (10-6 mol/L) were directly added to the medium of the isolated HSCs. Groups: (1)The detection of total p38, MKK3/6, MEF2A and MEF2C induced by TGFb1 in HSC: include control group, SA-B group, SA-B+TGFb1 group and TGFb1 group. (2)The detection of the phosphorylation of p38, MKK3/6 and a-SMA induced by TGFb1 in HSC: include control group, SA-B group, SA-B+TGFb1 group, TGFb1 group, PD98059 group, PD98059+SA-B group, PD98059+TGFb1 group and SA-B+PD98059+TGFb1 group. (3)The effects of SA-B on activity of MEF2 reporter and collagen a 1(I) reporter induced by TGFb1 in HSC: include mt group, wt group, TGFb1 group, SA-B+TGFb1 group, SA-B group, SB203580+TGFb1 group and SB203580 group. Total and phosphorylated p38 and MKK3/6, MEF2A, MEF2C and a-SMA were assayed by Western blot. HSCs were transfected with either MEF2 or collagen a1(I) luciferase reporter gene by Lipofectamine 2000 transfection method, Cellular extracts were assayed for both MEF2 and collagen a1(I) luciferase activities. Comparisons between groups were performed with Student-Newman-Keuls test.</p><p><b>RESULTS</b>The relative expression level of the phosphorylation of p38 of SA-B group is 0.33+/-0.05,obviously lower than control group(q=7.08, P less than 0.01); SA-B+TGFb1 group is 0.46+/-0.04, obviously lower than TGF b1 group(q=10.45, P less than 0.01); The relative expression level of the phosphorylation of MKK3/6 of SA-B group is 0.11+/-0.07, obviously lower than control group(q=3.944, P less than 0.05); SA-B+TGF b1 group is 0.28+/-0.07, obviously lower than TGFb1 group (q=7.91, P less than 0.01); The relative luciferase activity of MEF2 reporter of SA-B+TGFb1 group and SB203580+TGF b1 group is 2.93+/-0.09 and 2.50+/-0.05 respectively, both obviously lower than TGFb1 group(q=35.35 and 37.2, P less than 0.01); The relative expression level of MEF2C and MEF2A of SA-B group is 15.82+/-0.97 and 13.00+/-0.40 respectively, obviously lower than control group(q is 5.18 and 13.32, both P less than 0.01); SA-B+TGF b1 group is 13.40+/-0.72 and 20.47+/-0.83 respectively, obviously lower than TGFb1 group(q is 43.93 and 12.52,both P less than 0.01); The relative expression level of a-SMA of SA-B+TGFb1 group is 8.76+/-0.44, obviously lower than TGFb1 group(q=20.35, P less than 0.01); SA-B+SB203580+TGFb1 group is only 3.57+/-0.49, obviously lower than TGFb1 group(q=39.78, P less than 0.01); The relative luciferase activity of collagen a1(I) reporter of SA-B+TGF b1 group and SB203580+TGFb1 group is 1.61+/-0.05 and 1.42+/-0.07 respectively, obviously lower than TGFb1 group(q=26.4 and 27.62, both P less than 0.01).</p><p><b>CONCLUSION</b>SA-B could inhibit activation of HSC induced by TGFb1 through inhibiting p38MAPK signaling pathway in hepatic stellate cells.</p>

Effect and mechanism of salvianolic-acid B on inhibiting endothelin-1-induced contraction of human hepatic stellate cells / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the contraction effect of endothelin-1 (ET-1) on human hepatic stellate cells (HSCs) and the inhibition of salianic-acid B (SA-B) on ET-1, to explore the acting link and the possible mechanism.</p><p><b>METHODS</b>HSC were isolated from human normal liver tissue by enzyme digestion and Nycondenz density gradient centrifugation. The contraction of ET-1 on passage HSCs and the intervention of SA-B with three doses (low-, middle-, and high-) on the contraction were observed by collagen gel contraction. ET-1 and SA-B were directly added to the serum-free medium of HSCs, then calcium ion concentration was detected by laser scanning confocal microscope.</p><p><b>RESULTS</b>Collagen gel contraction experiments showed that ET-1 could induce the contraction of HSC directly (P < 0.01). Three doses of SA-B significantly inhibited the contraction effects of ET-1 on HSCs (all P < 0.01). After adding the ET-1, HSCs morphology changed obviously with the number of cells decreased. However, SA-B inhibited the changes. Laser scanning confocal microscope experiments revealed that ET-1 stimulated the transiently rapid increase of intracellular calcium ion concentration, and the effects was obviously inhibited when SA-B was added.</p><p><b>CONCLUSIONS</b>SA-B could inhibit the contraction of HSCs induced by ET-1, and its mechanism might be related to the lowing of free calcium ion concentration in HSCs. This anti-contraction effect of SA-B is perhaps one of the mechanisms of its anti-fibrosis and anti-portal hypertension effects.</p>

Effects of curcumin on peroxisome proliferator-activated receptor gamma expression and nuclear translocation/redistribution in culture-activated rat hepatic stellate cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The function of peroxisome proliferator-activated receptor gamma (PPARgamma) in hepatic fibrogenesis remains largely unknown. Curcumin is a natural substance extracted form Curcuma Longa Linn and has a variety of pharmacological effects. In this study, the effects of curcumin on the proliferation, activation and apoptosis of rat hepatic stellate cells (HSCs) through PPARgamma signaling were investigated.</p><p><b>METHODS</b>HSCs were isolated from the normal Sprague Dawley rats through in situ perfusion of the liver with Pronase E and density-gradient centrifugation with Nycodenz. Cells were treated with curcumin, troglitazone, salvianolic acid B or GW9662. The effect on HSCs proliferation was determined by MTT colorimetry. Total RNA was extracted by TRizol reagent and gene levels were determined by semi-quantitative RT-PCR. Total cellular and nuclear protein were isolated and separated by 10% sodium dodecy lsulfate polyacrylamide gel electrophoresis. Protein levels were determined by Western blot. Cell apoptosis was detected by Hoechst 33258 staining. PPARgamma subcellular distribution was detected by immunofluorescent staining. The activities of MMP-2 and 9 were measured by Gelatin zymograph assay.</p><p><b>RESULTS</b>Curcumin suppressed HSCs proliferation in a dose-dependent manner. As HSCs underwent gradual activation with culture prolongation the PPARgamma nuclear expression level decreased. Curcumin up-regulated PPARgamma expression and significantly inhibited the production of alpha-SMA and collagen I. PPARgamma is expressed in the cytoplasm and nucleus and is evenly distributed in HSCs, but accumulated in the nucleus of HSCs and disappeared from cytoplasm after curcumin treatment. Hoechst 33258 staining showed that curcumin induced the apoptosis of culture-activated HSCs and significantly increased pro-apoptotic Bax expression and reduced anti-apoptotic Bcl-2 expression. Cyclin D1 gene, activated NFkappaB p65 protein and TGFbetaR-I protein expression were down-regulated significantly by curcumin. The activities of MMP-2 and MMP-9 were enhanced significantly by curcumin.</p><p><b>CONCLUSIONS</b>Curcumin can inhibit the proliferation and activation of HSCs, induce the apoptosis of activated HSCs and enhance the activities of MMP-2 and MMP-9. The effects of curcumin are mediated through activating the PPARgamma signal transduction pathway and associated with PPARgamma nuclear translocation/redistribution.</p>

Effects of salvianolic acid B on signal transduction induced by transforming growth factor-beta1 and platelet-derived growth factor- BB in hepatic stellate cells of rats / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the anti-hepatic fibrosis mechanisms of salvianolic acid B (SA-B).</p><p><b>METHODS</b>Hepatic stellate cells (HSCs) isolated from rats were primarily cultured in uncoated plastic culture dish for 7 days, then were incubated with 10(-6) mmol/L SA-B and stimulated with 10 ng/ml transforming growth factor-beta1 (TGF-beta1) or platelet-derived growth factor-BB (PDGF-BB). Expressions of extracellular-regulated kinase (ERK) and its phosphorylation in HSC, and expressions of TGF beta1, receptor I (TbetaR I) and II (TbetaR II) and PDGF receptor beta (PDGFR-beta) on the surface of HSC induced by TGF-beta1 or PDGF-BB were detected with Western blot assay.</p><p><b>RESULTS</b>SA-B inhibited the phosphorylation of ERK1/2 in HSC primary normally cultivated for 9 days stimulated or un-stimulated by TGF-beta1, but could not affect the expressions of TbetaR I and TbetaR II on the HSC surface; it down-regulated the expression of PDGFR-beta, but had no obvious effect on the phosphorylation of ERK1/2 in those HSC stimulated or un-stimulated by PDGF-BB.</p><p><b>CONCLUSION</b>SA-B inhibits the ERK signal transduction induced by TGF-beta1 in HSC, which is independent of the expressions of TbetaR on HSC surface and also free from the ERK signal transduction stimulated by PDGF-BB. And its inhibition on PDGF-BB signal transduction in HSC is by way of restraining the expression of PDGFR in HSC.</p>

The value of serum markers in evaluating liver fibrotic changes / 中华肝脏病杂志

<p><b>OBJECTIVE</b>Serum fibrotic markers were investigated for diagnosing and prognosing liver fibrosis in chronic hepatitis B.</p><p><b>METHODS</b>Liver biopsy data of 93 patients before and after treatment were gathered from an experiment group (Fuzhenghuayu capsule, 36 cases) and a control group (Heluoshugan capsule, 57 cases) from multiple medical centers, using randomized and double blind strategies to evaluate the effectiveness of Fuzhenghuayu capsules against liver fibrosis. The patients were divided into 2 groups according to the treatment efficacy: an effectual group and a non-effectual group. The hepatic inflammation, liver function and serum fibrotic markers of the patients of the two groups were analyzed.</p><p><b>RESULTS</b>We found that (1) Liver fibrosis improved with hepatic inflammation improvement. (2) After the drug treatment, the serum HA and PIIIP levels of the effectual group decreased obviously (t = 3.34, t =3.17, P < 0.01), and the decreased degree was higher than that of the non-effectual group, but there were no changes for LN and IV-C levels. (3) Alb contents increased (t = 3.24, P < 0.01) and activities of GGT and AST and PT decreased significantly in the effectual group, but there was no change in the non-effectual group.</p><p><b>CONCLUSION</b>The serum GGT and AST activities, PT, Alb, HA and PIIIP contents in the chronic hepatitis B patients are good markers for evaluating the degree of liver fibrosis and the effectiveness of the drug action, but the values of LN and IV-C in the evaluation need to be studied more.</p>

Proteomic analysis of proliferation and apoptosis in carbon tetrachloride induced rat liver fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the proliferation and apoptosis in carbon tetrachloride induced rat liver fibrosis.</p><p><b>METHODS</b>Wistar rats were injected subcutaneously with 40% CCl4-olive oil twice weekly for 12 weeks. Liver tissues were obtained at the end of 4, 8, 12 and 16 weeks for histological examination, hydroxyproline (Hyp) assay and proteomic analysis. After two dimension electrophoresis (2-DE), the silver stained gels were analyzed with PDQUEST 2-DE. More than 30 differentially expressed proteins were identified by MALDI-TOF-MS.</p><p><b>RESULTS</b>The degree of collagen deposition and hydroxyproline content of the fibrotic livers increased continuously during the 12 weeks of CCl4 administration, peaked at the end of week 12 (P < 0.05) and declined significantly at week 16 (P < 0.05). Significant differences were observed in two parameters at each time point between the control and the model group. Meanwhile, dramatic change of hepatic proteome in the model group rats was also seen. Differentially expressed proteins identified by MALDI-TOF-MS were categorized as proliferation-related proteins/enzymes (proliferating cell nuclear antigen p120, p40 and cyclin F ubiquitin-conjugating enzyme 7 UBC7), and apoptosis-related proteins, mainly caspase-12 which was absent in the control rats.</p><p><b>CONCLUSION</b>Proliferation and apoptosis related proteins are expressed dynamically in different stages of rat liver fibrosis induced by CCl4.</p>

Salianic-acid B inhibits MAPK signaling in activated rat hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the inhibiting effect of salianic-acid B (SA-B) on mitogen-activated protein kinase (MAPK) Signaling in activated rat hepatic stellate cells (HSCs).</p><p><b>METHODS</b>HSCs were isolated from normal rat by in situ perfusion and Nycodenz density-gradient centrifugation method. HSCs were primarily cultured on uncoated plastic for 7 days. Then cells were stimulated with 10ng/ml transforming growth factor-beta1 (TGF-beta1) after incubated with 10-6 M/L SA-B. The effects of SA-B on Extracellular-regulated kinase (ERK) expression and its phosphorylation. Transforming growth factor beta1 receptor I (TbetaR I) and transforming growth factor beta1 receptor II (TbetaR II) on HSCs, type I collagen expression in HSC Induced by TGF-beta1 were detected with western blot assay. Quantity of Type I collagen in the medium of HSCs was detected by ELISA. Matrix metalloproteinase 2, 9, 13 (MMP-2, MMP-9 and MMP-13) in the medium of HSCs was tested by Zymography.</p><p><b>RESULTS</b>The phosphorylation of ERK1/2 in HSCs with or without TGF-beta1 was inhibited by SA-B. The expression of TbetaR I and TbetaR II on HSCs can not be affected by SA-B. The synthesization of Type I collagen in HSCs was decreased by SA-B; The synthesization and secretion of type I collagen in HSCs with TGF-beta1 were reduced by SA-B too. SA-B had no effect on the activity of MMP-2 and MMP-13, but induced the activity of MMP-13.</p><p><b>CONCLUSION</b>SA-B inhibits ERK signaling induced by TGF-b1 in HSC. This inhibition has no association with the expression of TbetaR I and TbetaR II on HSCs. SA-B reduces the synthesization and secretion of Type I collagen in HSC by means of inhibiting TGF-beta1 signaling, which might be not related to the degrading activities of MMPs.</p>

The role of changes of MMP-2, 9 activity in the development of liver fibrosis in rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the role of changes of matrix metalloproteinase-2, 9 (MMP-2, 9) activity in the development of dimethylnitrosamine (DMN)-induced liver fibrosis in rats.</p><p><b>METHODS</b>The rat liver fibrosis model was established by peritoneal injection of DMN (at a dose of 10 mg/kg, 3 times a week, for 4 weeks). The dynamic changes of liver fibrosis were observed at different time points (1d, 2d, 3d, 1 week, 2 weeks, 4 weeks, 6 weeks and 8 weeks). The MMP-2, 9 activity was measured by zymogram method. Liver ultrastructure was observed by electron microscope. The expressions of type IV collagen (CIV), laminin (LN), type I collagen (CI) and alpha-smooth muscle actin (alpha-SMA) were examined by immunohistochemistry. The tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) content was measured by Western blot method.</p><p><b>RESULTS</b>The MMP-2, 9 activity (gray value) significantly increased in the 2d and 3d DMN model rats (2d: normal/model group, MMP-2: 54.72+/-4.56/70.76+/-7.63; F = 16.27, P < 0.05; MMP-9: 25.72+/-4.29/51.76+/-15.33, F=13.38, P < 0.05). The positive staining area percentage of CIV in the sinusoidal walls decreased in the 2d, 3d and 1 weeks model rats (2d: normal/model group, 6.06+/-1.35/2.86+/-0.63, F=69.12, P < 0.05), but significantly increased in the 4w model rats (normal/model group, 6.06+/-1.35/8.04+/-1.50, F=14.42, P < 0.05). There was a remarkable negative correlation between the MMP-9 activity and expression of CIV in the sinusoidal walls (r = -0.729, P < 0.05). Positive expressions of LN and CI increased, and the strongest positive staining of them displayed in the 4w model rats. The formation of basement membrane was also observed in the 4 weeks model rats. Expression of TIMP-2 significantly increased in the late stage of fibrosis.</p><p><b>CONCLUSIONS</b>The increase of MMPs activity, especially MMP-9 which degrades the CIV normally distributed under the sinusoidal endothelium is the important factor in the formation of sinusoidal capillarization. The deposition and reconstitution of LN and new synthetic CIV, adding the deposition of CI constitute the high density basement membrane. The increase of TIMP-2 expression in the late stage of the fibrosis may be one of reasons why natural resolution of DMN-induced liver fibrosis is difficult.</p>