Efficacy of human umbilical cord mesenchymal stem cells via different transplantation approaches in treatment of rats with liver cirrhosis / 临床肝胆病杂志

ObjectiveTo investigate the therapeutic effect of the frozen and fresh preparations of human umbilical cord mesenchymal stem cells （hUC-MSC） on a rat model of liver cirrhosis after transplantation via the portal vein or the caudal vein. MethodsA total of 70 specific pathogen-free healthy male Sprague-Dawley rats were randomly divided into normal group （13 rats fed with ordinary tap water and rat food） and liver cirrhosis model group （57 rats given subcutaneous multi-point injection of mixed carbon tetrachloride/olive oil solution）. At week 8， the growth of rats was observed for both groups， and 3 rats were selected from each group for histopathological examination to confirm the formation of liver cirrhosis. A total of 50 rats were selected from the liver cirrhosis model and were divided into model group， portal vein group+fresh cell preparation group， portal vein+frozen cell preparation group， caudal vein+fresh cell preparation group， and caudal vein+frozen cell preparation group using a random number table， with 10 rats in each group. Fresh or frozen hUC-MSC were transplanted via the portal vein or the caudal vein， and after 4 weeks of administration， the different groups were compared in terms of the changes in liver function parameters and liver fibrosis degree. Continuous data were expressed as mean±standard deviation， and the independent-samples t test was used for comparison between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsAt week 8 of modeling， the model group showed the formation of pseudolobules of different sizes in the liver and met the diagnostic criteria for liver cirrhosis， with significant increases in the levels of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， total bilirubin （TBil）， and alkaline phosphatase （ALP） compared with the normal group （all P<0.001）， suggesting that the rat model of liver cirrhosis was established successfully. There were significant differences in the levels of ALT， AST， TBil， and ALP between the five groups （F=232.00， 177.10， 112.30， 121.70， all P<0.001）. Further comparison between two groups showed that the model group had significantly higher levels of ALT， AST， TBil， and ALP than the normal group （all P<0.01）， and the portal vein group+fresh cell preparation group， the portal vein+frozen cell preparation group， the caudal vein+fresh cell preparation group， and the caudal vein+frozen cell preparation group had significantly lower levels of ALT， AST， TBil， and ALP than the model group （all P<0.01）. ConclusionThere are significant improvements in liver function and liver fibrosis degree in a rat model of liver cirrhosis at week 4 after the transplantation of hUC-MSC， and frozen or fresh cell preparation and different transplantation approaches have no significant influence on treatment outcome.

Interventional effect of mangiferin on carbon tetrachloride-induced hepatic fibrosis in mice / 临床肝胆病杂志

ObjectiveTo investigate the interventional effect of mangiferin on carbon tetrachloride (CCl4)-induced hepatic fibrosis in mice and the potential mechanism. MethodsA total of 45 male C57BL/6 mice were randomly divided into three groups: normal control group (NC group), liver fibrosis model group (CCl4 group), and mangiferin pretreatment group (CCl4+M group), with 15 mice in each group. An automatic biochemical analyzer was used to measure the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST); HE staining and Masson staining were performed to observe liver pathological changes; ELISA was used to measure the serum levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα); Western Blot was used to measure the protein expression of α-smooth muscle actin (α-SMA), nuclear factor-kappa B (NF-κB), IL-1β, p62, and microtubule-associated protein 1 light chain 3 (LC3) in the liver; quantitative real-time PCR was used to measure the mRNA expression of type I collagen (Col-I) and α-SMA in the liver. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsHE staining and Masson staining showed a low proportion of mice with hepatocyte degeneration and necrosis and a significant reduction in collagen fibers in the CCl4+M group. Compared with the CCl4 group, the CCl4+M group had significant reductions in the serum levels of ALT and AST (both P＜001). ELISA showed that compared with the CCl4 group, the CCl4+M group had significant reductions in the serum levels of IL-1β, IL-6, and TNF-α (all P＜0.01). Western Blot showed that compared with the CCl4 group, the CCl4+M group had significant reductions in the protein expression of α-SMA, NF-κB, IL-1β, and LC3-II/I in the liver (P＜0.01, P＜0.05, P＜0.01, and P＜0.05) and a significant increase in the protein expression of p62 (P＜0.05). Quantitative real-time PCR showed that compared with the CCl4 group, the CCl4+M group had significant reductions in the mRNA expression of Col-I and α-SMA (P＜0.05 and P＜0.01). ConclusionMangiferin can alleviate CCl4-induced hepatic fibrosis in mice, possibly by reducing inflammation to protect liver function and inhibiting autophagy to reduce the activation of hepatic stellate cells.

Value of acoustic radiation force impulse elastography in evaluation of nonalcoholic fatty liver disease and hepatic fibrosis in rats / 中国医学影像技术

Objective To evaluate the value of acoustic radiation force impulse (ARFI) elastography in assessment of nonalcoholic fatty liver disease (NAFLD) and hepatic fibrosis in rats.Methods Models with various degrees of NAFLD severity were conducted in 110 rats by feeding high fat emulsion.The right liver lobe of rat models were processed and embedded in a fabricated gelatin solution to measure the shear wave velocity (SWV) by ARFI.And the other liver lobes were used for histologic assessment.Based on NAFLD activity score (NAS),the final pathologic NAFLD diagnosis were considered as normal group (NAS=0),simple steatosis (SS) group (1≤NAS≤2),borderline (3≤NAS≤4) group and nonalcoholic steatohepatitis (NASH) group (NAS≥5).The diagnostic accuracy of the SWV parameters in evaluating NAFLD severity and fibrosis stages was studied using ROC curves.Results The difference of SWV values among normal group,SS group,borderline group and NASH group was statistically significant (F=31.53,P＜0.001).Taking SWV≥ 2.54 m/s as the diagnostic standard to differentiate normal rats from rats with SS,and SWV≥2.90 m/s to differentiate SS from NASH in rats,the area under ROC curve (AUC) was 0.922 (95％CI [0.871,0.973],P＜0.001) and 0.882 (95％ CI [0.807,0.956],P＜0.001) respectively.The sensitivity and specificity were 93.5 ％ and 100 ％ for differentiating normal and SS groups,83.3 ％ and 84.2 ％ for differentiating SS and NASH groups.Taking SWV≥3.48 m/s as cutoff to predict fibrosis (≥F2 stage),the AUC was 0.963 (95％CI [0.909,1.000],P＜0.001),the sensitivity was 92.9％ and the specificity was 97.6％.Taking SWV≥3.61 m/s as cutoff to predict severe fibrosis (≥F3 stage),the AUC was 0.997 (95％CI [0.990,1.000],P＜0.001),sensitivity was 100％ and specificity was 98.9％.The same high validity was maintained as in the prediction of cirrhosis (F4 stage) with the cutoff as SWV≥4.50 m/s,and the AUC was 0.993 (95％CI [0.982,1.000],P＜0.001),the sensitivity was 100 ％ and the specificity was 96.8％.Conclusion ARFI elastography is a promising method for differentiating the different severity of NAFLD and staging the degree of hepatic fibrosis with NAFLD in rat models.

Comparison of 70%hepatectomy model established by two different methods in liver fibrosis mice / 天津医药

Objective To explore a reliable method of 70% hepatectomy model in liver fibrosis mice. Methods Sixty-six C57BL6 mice were randomly devided into control group (n=6), the traditional group (n=30, ligation and removal liver lobe) and improved group (n=30, removal of liver lobe after blocking blood flow). Those 60 mice were induced liver fibrosis firstly, then randomly divided into six mice in each group, and were sacrificed at preoperative, 12, 24, 48 and 72 hours after liver resection. Liver tissues and blood samples were collected. The survival rate and incidence of complications were recorded and compared between two groups. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to observe the liver injury after 70%hepatectomy. The ratio of liver weight to body weight and the expression of proliferating cell nuclear antigen (PCNA) were also measured to observe the difference of liver regeneration between the two groups. Results (1) Compared to the pathological control group, liver fibrosis model was established successfully in both traditional group and improved group, which can be used in 70%hepatectomy. So the follow-up experiment can be undertook timely. (2) Compared to traditional group, the survival rate was improved significantly in improved group (96.67%vs. 73.33%), and the incidence of complications was significantly lower (P<0.05). (3) The ALT and AST levels were higher 12 h and 24 h after operation in traditional group than those of improved group (P<0.05), while ALT and AST levels were increased first 12 h after operation and then decreased in both groups (P<0.05). (4) The liver/body weight ratio showed a decreasing trend 12 h after hepatectomy in two groups. The expression of PCNA increased at the beginning of postoperative, and reached its peak at 48 h (P<0.05). However, there was no significant difference at each time point between the two groups. Conclusion By blocking blood flow to establish 70% hepatectomy model in liver fibrosis mice, we can significantly improve the success rate of the model, and reduce the incidence of complications.

The protective effects of different doses of fasudil on hepatic ischemia/reperfusion injury in rats with cirrhosis / 中华普通外科杂志

Objective To investigate the protective effects of different doses of fasudil on hepatic ischemia/reperfussion (I/R) injury in rats with liver cirrhosis.Methods Cirrhosis was induced in rats by subcutaneous injection of 60％ carbon tetrachloride (CC14) corn oil solution (0.4 ml/100 g) twice a week for twelve weeks.Five percent of alcohol was given intermittently in drinking water.Then 40 cirrhotic rats were randomized into 4 groups.In sham group,sham operation was performed.In I/R group A and B,whole rat livers were subjected to warm ischemia by clamping the hepatic artery and portal vein for 30 min.In group A,the selected rats were pretreated with low-dose fasudil 1 mg/kg (intraperitoneal injection) 30 min before the induction of ischemia,and in group B,with high-dose 10 mg/kg.The serum levels of alanine aminotransferase (ALT) and endothelin-1 (ET-1),and the liver tissue superoxide dismutase (SOD)activity,malondialdehyde (MDA) content,the expression of HIF-1a (hypoxia-inducible fador-1a) were measured after reperfusion for 6 hours.Hepatic pathologic changes were observed under microscope.Results Compared with I/R group,the serum ALT,AST,ET-1 levels,MDA content and the expression of HIF-1 a were markedly decreased in group B,while the SOD activity significantly increased (P ＜ 0.05).And the pathologic changes were less severe in group B.Conclusion The high-dose fasudil markedly lessened the expression of HIF-1a,up-regulated the concentration of SOD,and lowered the levels of MDA and ET-1,protecting against heoatic ischemia/reperfusion injury in rats with liver cirrhosis.

Effects of Ruangan granule on transforming growth factor-β1/Smads signaling pathway in liver fibrosis in rats / 国际中医中药杂志

Objective To investigate the effects of Ruangan granule on transforming growth factor-β1(TGF-β1)/Smads signaling pathway in liver fibrosis in rats. Methods A total of 105 Wistar rats were randomly divided into normal control group, model group and colchicine, Dahuang-Zhechong pill group, high-, medium- and low-dose Ruangan granule groups (n=15 in each group). Liver fibrosis was induced by carbon tetrachloride and a high-cholesterol diet. After modeling, the low-, medium- and high-dose Ruangan granule groups were intragastric administrated Ruangan granule mixed suspension 3.6, 7.2, 14.4 g/(kg?d), respectively;Dahuang-Zhechong pill group was administrated with Dahuang-Zhechong pellets mixed suspension of 0.18 g/(kg?d);the colchicine group was intragastric administrated with colchicine mixed suspension of 0.108 mg/(kg?d);and the normal control group and the model group were intragastric administrated with the equal volume of distilled water. All rats were intragastric administrated for 8 weeks. The expressions of TGF-β1, Smad3 and Smad7 proteins in the liver tissue were detected with immunohistochemical staining method. The expressions of TGF-β1, Smad3, Smad7 mRNAs in the liver tussue were detected by RT-PCR. Results The expressions of TGF-β1 (2.59 ± 0.99 vs. 0.43 ± 0.21) and Smad3 (2.56 ± 0.67 vs. 0.41 ± 0.18) proteins and TGF-β1 mRNA (2.25 ± 0.21 vs. 0.71 ± 0.09) and Smad3 (2.34 ± 0.03 vs. 0.78 ± 0.12) mRNAs in the model group were significantly increased than those in the normal control group (all P<0.01). Compared with the model group, the expressions of TGF-β1 (1.12 ± 0.27 vs. 2.59 ± 0.99) and Smad3 (1.05 ± 0.34 vs. 2.56 ± 0.67) proteins in the high-dose Ruangan granule group decreased significantly, the expression of Smad7 increased significantly (2.33 ± 0.62 vs. 0.36 ± 0.18), and the expressions of TGF-β1 (1.09 ± 0.11 vs. 2.25 ± 0.21) and Smad3 (1.10 ± 0.02 vs. 2.34 ± 0.03) mRNAs decreased significantly, the expression of smad7 mRNA (1.18 ± 0.13 vs. 0.38 ± 0.11) increased significantly (P<0.05). Conclusions Ruangan granule can regulate the TGF-β1/Smads signaling pathway via down-regulation of TGF-β1, Smad3 and up-regulation of Smad7 in liver fibrosis in rats.

Effects of Gynostemma Pentaphyllum on serum markers of liver fibrosis in cholesterol-induced non-alcoholic fatty liver disease in rabbits / 国际中医中药杂志

Objective To investigate the effects of Gynostemma Pentaphyllum on serum markers of liver fibrosis in cholesterol-induced non-alcoholic fatty liver disease (NAFLD) in rabbits. Methods Forty adult male white rabbits were randomly divided into a normal control group, a model group, a treatment group and a simvastatin group, with 10 rabbits in each group. NAFLD was induced with a high-cholesterol diet. After modeling, the rabbits in the treatment group were intragastrically administrated with Gynostemma Pentaphyllum 5 mg/(kg?d), the simvastatin group with simvastatin 5 mg/(kg?d), and the model and normal control groups with the equal volume of distilled water for 9 weeks. The serum levels of total cholesterol (TC) and triglyceride (TG), the serum inflammatory maker C-reactive protein (CRP), the serum markers of liver fibrosis such as hyaluronic acid and laminin (LN), and the TG level in the liver tissue were detected. Results Compared with the normal control group, the serum levels of TC (60.50 ± 9.77 mg/L vs.1.30 ± 0.44 mg/L), TG (1.72 ± 0.61 mmol/L vs. 0.85 ± 0.39 mmol/L), CRP (256.79 ± 30.78 mg/L vs. 8.71 ± 1.41 mg/L), HA (798.8 ± 69.4 ng/ml vs. 121.2 ± 6.8 ng/ml),LN (964.8 ± 62.8 ng/ml vs. 142.4 ± 12.2 ng/ml) in the model group were increased significantly (all P0.05). Conclusions Gynostemma Pentaphyllum may regulate lipid metabolism, alleviate inflammation, and decrease serum markers of liver fibrosis, and might protect against liver fibrosis in rabbits with NAFLD.

Lung and liver hhanges due to the induction of cirrhosis in two experimental models / Alteracoes hepaticas e pulmonares decorrentes da inducao de cirrose em dois modelos experimentais.

Context To evaluate lung and liver changes in two experimental models using intraperitoneal carbon tetrachloride (CCl4) and bile duct ligation (BDL). Methods Twenty-four male Wistar rats were divided into a control group (CO) and an experimental group (EX). We evaluated the liver transaminases (AST, ALT, AP), arterial blood gases (PaO2, PCO2 and SpO2) and lipid peroxidation by TBARS (substances that react to thiobarbituric acid) and chemiluminescence. We also evaluated the antioxidant enzyme superoxide dismutase (SOD) and histology of lung tissue and liver. Results There were significant differences in AST, ALT, ALP and PaO2 between CO group and EX group (P<0.05). The levels of TBARS, chemiluminescence and activity of enzyme superoxide dismutase were increased to different degrees in the CCl4 groups: CO and in the BDL -EX (P<0.05, respectively). In the lung histology, an increase in the wall thickness of the pulmonary artery and a diameter reduction in the CCl4 animal model were observed: comparing CO group with EX group, we observed a reduction in thickness and an increase in the diameter of the artery wall lung. Conclusion Both experimental models have caused liver damage and alterations in the artery wall that are associated with major changes in pulmonary gas exchange. .

Objetivo Avaliar as alterações pulmonares e hepáticas em dois modelos experimentais de cirrose hepática pelo uso de tetracloreto de carbono intraperitoneal (CCl4) e ligadura de ducto biliar. Métodos Vinte e quatro ratos machos Wistar foram divididos em grupo controle (CO) e experimental (EX). Foram avaliadas as transaminases hepáticas (AST, ALT, FA), gasometria arterial (PaO2, PCO2 e SatO2) e a lipoperoxidação através de TBARS (substâncias que reagem ao ácido tiobarbitúrico) e por quimiluminescência. Também foi avaliada a atividade antioxidante da enzima superóxido dismutase e a histologia do tecido pulmonar e hepático. Resultados Nas enzimas hepáticas (AST, ALT e FA), bem como na PaO2 foram observadas diferenças significativas (P≤0,05) entre os grupos CO vs EX em ambos modelos. Os níveis de TBARS, quimiluminescência e a atividade da enzima superóxido dismutase encontram-se aumentados nos grupos CCl4 e ligadura de ducto biliar: CO vs EX (P≤0,05). Na análise histológica do pulmão observamos um aumento na espessura da parede da artéria pulmonar e uma redução no diâmetro no modelo CCl4: CO vs EX, e no modelo de ligadura de ducto biliar podemos observar uma redução da espessura e aumento no diâmetro da parede da artéria pulmonar. Conclusão Ambos os modelos experimentais provocaram dano hepático, além de causar alterações na parede da artéria pulmonar contribuindo na redução das trocas gasosas. .

Protective Effects of Extract of Periplaneta Americana on Liver Fibrosis in Rats / 天津医药

Objective To investigate the protective effects of extract of Periplaneta americana (APA) against carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats. Methods Seventy SD rats were divided into five groups:normal con-trol group (C), model control group (M), extract of APA 1 group (APA1), extract of APA 2 group (APA2) and reduced glutathi-one group (R). The liver fibrosis model was induced by injecting 40%CCl4-olive solution subcutaneously for seven weeks in M, APA1, APA 2 and R groups. Drugs were given at the same time. Rats were killed at 7-week (C, M, APA1 and R groups), and were killed at 9-week (APA2 group). The serum values of transaminase (ALT, AST), albumin (ALB), hyaluronic acid (HA) and laminin (LN), and hepatic inflammation and fibrosis were detected respectively. The expressions of apoptotic relat-ed gene Bcl-2 and Bax protein in central veins and portal areas of hepatic lobules were detected by immunohistochemical method. Results Rats showed poor nutritional status, and significantly increased transaminase, HA and LN in M group, but the serum level of ALB was significantly lower than that in C group. There was extensive necrosis of liver cells, obviously fi-brosis or cirrhosis in model rats’liver tissues. The serum contents of ALT, AST, HA and LN were significantly decreased, the serum level of ALB were significantly increased in APA1,APA2 and R groups (P<0.05). There were complete hepatic lobule and no obvious fibrous tissue hyperplasia in liver biopsy of APA1 and R groups. The values of Bcl-2/Bax proteins in central vein of liver tissues were significantly higher in APA1,APA2 and R groups than those of M group (P<0.05), but the ratio of Bcl-2/Bax was lower in periportal area of liver tissues (P<0.05). There were significantly higher body weight and ALB levels in APA1 group than those in R group (P<0.05). The level of transaminase was slightly higher in APA1 group than that of R group. The degeneration of liver cells was found mostly in R group. Conclusion The extract of Periplaneta americana has a role in protecting liver cells, inhibiting the progression of hepatic fibrosis, and improving the nutritional sta-tus of trial rats.

Real-time tissue elastography with tissue dispersion quantitative analysis technique for assessment of rat liver fibrosis / 中华超声影像学杂志

Objective To explore the value of real-time tissue elastography (RTE) with tissue dispersion quantitative analysis technique for assessment of liver fibrosis stage.Methods 51 rats were injected 6％ thioacetamide to induce liver fibrosis model,and 9 rats were injected saline as control group.In modeling 4 weeks,8 weeks,12 weeks respectively,14 rats in group of liver fibrosis model and 3 rats in control group were randomly selected to RTE.All the rats underwent tissue dispersion quantitative analysis,to obtain 12 quantitative parameters of elasticity,which included average relative strain value (MEAN),standard deviation of relative strain value (SD),area ratio of low-strain region (％ AREA),complexity (COMP),kurtosis (KURT),skewness (SKEW),contrast (CONT),entropy (ENT),inverse difference moment (IDM),angular second moment (ASM),correlation (CORR) and liver elasticity index (LF index).Subsequently,rats were sacrificed and their livers were taken for pathology analysis.Liver fibrosis model group was divided into S0,S1,S2,S3,S4 group.The 12 quantitative parameters of elasticity were compared with each group.Results 49 rats were successfully modeled,and 42 rats were analyzed.Except COMP,KURT,CORR,the other quantitative parameters had statistically differences (P ＜ 0.05).The other 9 parameters were correlated with liver fibrosis stage.Among these parameters,MEAN,％ AREA and LF index had higher related coefficient(r =-0.831,0.882,0.866).The ROC curve was made by MEAN,LF index and ％AREA to estimate the fibrosis stage,when S≥S1,S≥S2,S≥S3,S =S4,the areas under the ROCcurve were 0.884,0.925,0.934,0.962 (MEAN);0.917,0.958,0.984,0.962 (％AREA);0.917,0.948,0.966,0.967 (LF index),respectively.Conclusions As a non-invasive examination,RTE dispersion quantitative analysis technology can be used to quantitatively assess liver fibrosis.

Fsp27 gene inhibits the development of CCl4-related fibrosis in rats / 中国医师杂志

Objective To investigate the influence of fat-specific protein 27 (Fsp27) gene on the regulation of liver fibrogenesis in vivo.Methods Hepatic stellate cells (HSCs) were isolated from rat liver.Fsp27 gene was detected in primary HSCs and activated HSCs by real-time quantitative PCR (RTqPCR).Lentiviral vector carrying Fsp27 gene was constructed.The model of liver fibrosis was established by infusing carbon tetrachloride (CC14).The rats with liver fibrogenesis were infected by the virus.Liver sections were made to observe the structure and form of liver histocytes.The content of fibrous protein in liver and serum was detected by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay.Resukts HSCs were isolated and cultured successfully.The difference of Fsp27 gene between primary HSCs and activated HSCs was significant(P ＜ 0.01).The model of liver fibrosis was achieved.After infecting the model rats,we found the fibrosis level in treatment group was lower compared with control group.Conclusions Fsp27 treatment can decrease collagen deposition in the liver and inhibit the formation of fibrosis.

Relationship between serum transforming growth factor ß1 and liver collagen content in rats treated with carbon tetrachloride / Relação entre fator transformador de crescimento β1 e conteúdo hepático de colágeno em ratos tratados com tetracloreto de carbono

CONTEXT: Transforming Growth Factor ß1 (TGFß1) plays a fundamental role in fibrogenesis, although its importance as a biomarker of liver disease is still matter of debate. OBJECTIVE: Quantify serum TGFß1 and its association to liver collagen content in rats exposed to Carbon Tetrachloride (CCl4). METHODS: Rats were submitted to a fibrosis model using CCl4 and sacrificed after 6, 10, 12 and 16 weeks of treatment. Serum levels of TGFß1 were measured by ELISA and collagen content was defined by morphometric analysis. RESULTS: Serum levels of TGFß1 increased between 6 and 10 weeks, whereas collagen density increased between 12 and 16 weeks. A negative correlation was observed between liver collagen deposition and serum levels of TGFß1 (r = -0. 48; P<0. 05). CONCLUSION: Serum levels of TGFß1 were inversely proportional to collagen intensity in cirrhotic livers of rats exposed to CCl4, thus suggesting a limited use as biomarker in advanced liver disease.

CONTEXTO: A citocina TGFß1 (Fator Transformador de Crescimento, TGFß1) desempenha um papel fundamental na fibrogênese, mas sua importância como biomarcador da doença hepática ainda tem sido debatida. OBJETIVO: Quantificar o TGFß1 sérico e estudar a sua associação com o conteúdo de colágeno no tecido hepático em ratos expostos ao Tetracloreto de Carbono (CCl4). MÉTODOS: Ratos foram submetidos ao modelo de fibrose por CCl4 e sacrificados após 6, 10, 12 e 16 semanas de tratamento. Os níveis séricos de TGFß1 foram quantificados por ELISA e a densidade de colágeno foi definida por morfometria. RESULTADOS: Os níveis séricos de TGFß1 aumentaram entre 6 e 10 semanas, enquanto a densidade de colágeno aumentou entre 12 e 16 semanas. Foi detectada uma correlação negativa entre a deposição hepática de colágeno e a concentração sérica de TGFß1 (r = -0,48; P<0,05). CONCLUSÃO: O nível sérico de TGFß1 foi inversamente proporcional à intensidade do colágeno no fígado de ratos com cirrose por CCl4, o que indica que seu uso como biomarcador em estágios avançados da doença pode ter utilidade limitada.

Adipose-derived mesenchymal stem cells inhibit the proliferation and activation of hepatic stellate cell in vitro and inhibit rat liver fibrogenesis in vivo / 中华普通外科杂志

Objective To investigate the effect of adipose derived stem cells (ADSCs) on hepatic stellate cells (HSCs) in vitro and on liver fibrosis in vivo.Methods ADSCs and HSCs were isolated from adipose tissue and liver respectively in SD rats.The coculture system was set up by transwell insert.The 5th passage HSCs were cultured on the 6-well plastic plate,and ADSCs or BRLs seeded on the transwell insert.The proliferation of HSCs was tested by CCK-8 test kit.Smoothmuscle α-actin (α-SMA) expression of HSCs were tested by Western blot.Rat models of liver fibrosis was established.Rats in ADSCs treatment group were infused with ADSCs and those in control group were infused with Buffalo rat liver cells (BRLs).Liver sections were studied by immunocytochemistry.Liver hydroxyproline (Hyp) content,serum laminin (LN)and hyaluronic acid (HA) were tested,the cytokines in the culture medium were assayed.Results HSCs and ADSCs were isolated successfully.After coculture for 72 h,compared with the control group,the proliferation and activation of HSCs was inhibited by ADSCs( absorbance of each group were 2.172 ±0.107,1.424 ± 0.013,1.209 ± 0.117,F =90.605,P ＜ 0.05 ; Gray-scale values of each group were 1.4 ± 0.2,152 ± 14,258 ± 18,F =283.348,P ＜ 0.05 ),ADSCs infusion inhibits liver fibrosis in model rats ( F =77.234,65.164,58.309,all P ＜ 0.05 ).More hepatocyte growth factor(HGF) and less transforming growth factor-β1 (TGF-β1) (F=1.767,P＜0.05)and nerve growth factor (NGF) (F=2.301,P＜0.05) were secreted by ADSCs than by BRLs.Conclusions ADSCs inhibit the proliferation and activation of hepatic stellate cells.Treatment with ADSCs decreases collagen deposition in the liver and inhibits liver fibrosis.

Effect of remifentanil on hepatic ischemia-reperfusion injury in rats with liver cirrhosis / 中华麻醉学杂志

ObjectiveTo investigate the effect of remifentanil on hepatic ischemia-reperfusion (I/R) injury in rats with liver cirrhosis.MethodsThirty male SD rats weighing 260-300 g were randomly divided into 3 groups (n =10 each):group liver cirrhosis (group C); group liver cirrhosis + hepatic I/R (group I/R) and group remifentanil (group R).Liver cirrhosis was produced in all animals in the 3 goups.I/R injury was induced by 20 min occlusion of the hepatic artery and portal vein entering the middle and left lobes of the liver followed by 4 h reperfusion at 1 week after establishment of hepatic cirrhosis in I/R and R groups.In group R remifentanil was infused iv at 1 μg·kg-1 ·min-1 starting from 10 min before ischemia until the end of 4 h reperfusion.Venous blood samples were taken from inferior vena cava at the end of 4 h reperfusion for measurement of serum ALT and AST activities.The animals were then sacrificed and liver specimens were taken from middle lobe for determination of Bcl-2 and Bax expression (by immuno-histochemistry) and hepatocyte apoptosis (by TUNEL) and microscopic examination.Apoptosis index (percentage of apoptotic cells) was calculated.ResultsI/R significantly increased serum ALT and AST activities,Bax expression and apoptosis index and decreased Bcl-2 expression in group I/R as compared with group C.Remifentanil significantly attenuated the I/R-induced changes in serum ALT and AST activities,Bax and Bcl-2 expression and apoptosis in group R as compared with group I/R.Remifentanil also ameliorated I/R-induced liver damage.ConclusionRemifentanil can auenuate hepatic I/R injury in rats with liver cirrhosis by up-regulating Bcl-2 expression and down-regulating Bax expression and inhibiting apoptosis.

Dynamic expressions and it's significance of small heterodimer partner in rat models of liver fibrosis / 中华传染病杂志

Objective To observe the expressions of small heterodimer partner (SHP) in rat model of hepatic fibrosis and its role in fibrosis.Methods A total of 30 healthy male SD rats were divided into two groups: 6 rats in control group and 24 rats in model group.The model group was further divided into four subgroups which were sacrificed at different time points,2,4,6 and 8 weeks after intraperitoneal injection of dimethylnitrosamine (DMN).After establishment of the rat model,blood samples and liver tissue specimens were collected at week 2,4,6 and 8 respectively.The sections of liver tissue were stained with HE and Masson and then were observed under optical microscope.The expressions of SHP mRNA and protein were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot,respectively.The comparison of means among the groups was performed by univariate ANOVA.Results The hepatic fibrosis was most obvious at 4 weeks and 6 weeks after the intraperitoneal injection.In model groups,alanine aminotransferase (ALT) and aspartateaminotransferase (AST) levels gradually increased and reached a peak at week 4,which were (169.2±16.2) and (193.3±31.1) U/L,respectively.Meanwhile,albumin level in the model group decreased gradually,reaching the nadir at week 6,and the differences were statistically significant at all selected time points between the model group and control group (FAST =83.10,FALT =104.63,FAlb =54.24; all P＜0.05).After modeling,the expression of SHP mRNA and protein in model group were significantly increased than those in control group,and reached a peak at week 4 (0.4494±0.0555 and 1.1155 ±0.1546,respectively),then both mRNA and protein levels decreased gradually at week 6 and 8 which obviously lower than the control group.Transforming growth factor (TGF)β1 mRNA expression level also increased gradually,reaching a peak at week 4 (0.9625±0.1196),and the differences between model subgroups and control group were statistically significant (F=25.740,171.383,118.393 and 94.343; all P＜0.05).Linear correlation analysis showed that SHP mRNA was positively correlated with TGFβ1 mRNA (r=0.593,P＜0.01).Conclusion During the progression of hepatic fibrosis,the SHP expression increases at the beginning and then turns to decrease,which suggests that SHP may play an important role in the development of hepatic fibrosis.

Intestinal permeability assessed by 51Cr-EDTA in rats with CCl4 - induced cirrhosis / Permeabilidade intestinal ao 51-Cr-EDTA em ratos com cirrose induzida por CCl4

CONTEXT: The straight relationship between cirrhosis and impaired intestinal barrier has not been elucidated yet. OBJECTIVES: To verify 51Cr-EDTA-intestinal permeability in rats with CCl4-induced cirrhosis and controls. METHOD: Fifty male Wistar rats weighing 150-180 g were separated in three groups: 25 animals received CCl4 0.25 mL/kg with olive oil by gavage with 12 g/rat/day food restriction for 10 weeks (CCl4-induced cirrhosis); 12 received the same food restriction for 10 weeks (CCl4-non exposed). Other 13 rats received indomethacin 15 mg/kg by gavage as positive control of intestinal inflammation. RESULTS: The median (25-75 interquartile range) 51Cr-EDTA-IP values of cirrhotic and CCl4-non exposed rats were 0.90 percent (0.63-1.79) and 0.90 percent (0.60-1.52) respectively, without significant difference (P = 0.65). Animals from indomethacin group showed 51Cr-EDTA-IP, median 7.3 percent (5.1-14.7), significantly higher than cirrhotic and CCl4-non exposed rats (P<0.001). CONCLUSION: This study showed the lack of difference between 51Cr-EDTA-intestinal permeability in rats with and without cirrhosis. Further studies are necessary to better clarify the relationship between intestinal permeability and cirrhosis.

CONTEXTO: A relação direta entre cirrose e alterações na barreira intestinal ainda não foi devidamente esclarecida. OBJETIVO: Verificar a permeabilidade intestinal ao 51Cr-EDTA em ratos com cirrose induzida por tetracloreto de carbono (CCl4) e controles. MÉTODO: Cinquenta ratos Wistar machos pesando 150-180 g foram separados em três grupos: 25 animais receberam CCl4 0,25 mL/kg diluído em óleo de oliva por gavagem com restrição dietética de 12 g/rato/dia por 10 semanas (grupo cirrose induzida por CCl4); 12 receberam a mesma restrição dietética por 10 semanas (grupo não exposto ao CCl4). Outros 13 ratos receberam indometacina 15 mg/kg por gavagem como controle positivo de inflamação intestinal. RESULTADOS: A mediana (intervalo interquartil 25-75) dos valores de permeabilidade intestinal ao 51Cr-EDTA dos grupos cirrose induzida por CCl4 e não exposto ao CCl4 foram 0,90 por cento (0,63-1,79) e 0,90 por cento (0,60-1,52), respectivamente, sem significância estatística (P = 0,65). Os animais do grupo indometacina apresentaram uma mediana de permeabilidade intestinal ao 51Cr-EDTA de 7,3 por cento (5,1-14,7), sendo significativamente maior do que os grupos cirrose induzida por CCl4 e não exposto ao CCl4 (P<0,001). CONCLUSÃO: Este estudo não demonstrou diferenças entre a permeabilidade intestinal ao 51Cr-EDTA em ratos com e sem cirrose. Mais estudos são necessários para melhor esclarecer a relação entre a permeabilidade intestinal e cirrose.

Melatonin protects the liver and erythrocytes against oxidative stress in cirrhotic rats / Melatonina protege o fígado e eritrócitos do estresse oxidativo em ratos cirróticos

CONTEXT: Cirrhosis is a progressive chronic hepatopathy which constitutes an irreversible stage of liver dysfunction. OBJECTIVES: To evaluate the oxidative stress in the blood of cirrhotic rats treated with the antioxidant melatonin. METHODS: Cirrhosis was induced through inhalation of carbon tetrachloride. Liver integrity was evaluated by measuring serum enzymes, oxidative damage measured by lipoperoxidation, and antioxidant enzyme activity in erythrocytes. Lipoperoxidation, total nitrates, collagen, and histology by picrosirius staining were evaluated in the livers of these animals (n = 15), which were divided in three groups: control, carbon tetrachloride, and carbon tetrachloride + melatonin. Melatonin (20 mg/kg) was administered intraperitoneal from week 10 of carbon tetrachloride inhalation. In order to shorten the cirrhosis induction time, phenobarbital (0.3 g/L) was added to the animals' drinking water. RESULTS: A significant impairment in the liver integrity of melatonin-treated animals as compared to cirrhotic animals was observed. In rat erythrocytes and liver, lipoperoxidation was significantly increased in the cirrhotic rats as compared to controls, as measured through thiobarbituric acid reactive substances, and significantly decreased in melatonin-treated animals as compared to cirrhotic ones. In blood, a decrease in superoxide dismutase and glutathione peroxidase enzymes was detected in the cirrhotic group as compared to the control group, with increased superoxide dismutase activity when melatonin was administered. A reduction in the levels of total nitrates was detected in the hepatic tissue of the animals in the carbon tetrachloride group as compared to the control group and an increase of these levels in the carbon tetrachloride + melatonin group. As for hepatic collagen, we found a significant increase in the carbon tetrachloride group as compared to the controls and a regression of these values in the treated group. ...

CONTEXTO: A cirrose é uma hepatopatia crônica e progressiva que constitui estágio irreversível de disfunção hepática. É associada a alterações na circulação sistêmica. OBJETIVOS: Avaliar o estresse oxidativo no sangue de ratos cirróticos e tratados com antioxidante melatonina. MÉTODOS: A cirrose foi induzida através da inalação de tetracloreto de carbono. Foram avaliadas as provas de integridade hepática através das medidas das enzimas séricas, o dano oxidativo medido pela lipoperoxidação e a atividade das enzimas antioxidantes no eritrócito. No fígado desses animais, foram avaliados a lipoperoxidação, os nitratos totais, colágeno e histologia através de picrosíruis. Os animais (n = 15) foram divididos em três grupos experimentais: controle, tetracloreto de carbono e tetracloreto de carbono + melatonina. A melatonina foi administrada por via intraperitonial após a 10ª semana de inalação na concentração de 20 mg/kg. Com o objetivo de abreviar o tempo de indução, foi administrado para todos animais, fenobarbital na água de beber na concentração de 0,3 g/L. RESULTADOS: Observou-se redução significativa nas provas de integridade hepática nos animais tratados com melatonina, em relação aos animais cirróticos. Nos eritrócitos e fígados dos ratos, foi observado aumento significativo da lipoperoxidação nos ratos cirróticos em comparação com os controles, através da medida das substâncias que reagem ao ácido tiobarbitúrico, e redução nos animais tratados com melatonina. No sangue, observou-se diminuição dos valores das enzimas superóxido dismutase e glutationa peroxidase do grupo cirrótico em comparação ao grupo controle, elevando a atividade da superóxido dismutase quando administrada melatonina. Na avaliação dos nitratos totais, no tecido hepático, observou-se redução dos valores nos animais do grupo tetracloreto de carbono em comparação ao grupo CO e um aumento desses valores nos ratos do grupo tratado com melatonina. Na medida do colágeno ...

Hepatopulmonary syndrome: an update / Síndrome hepatopulmonar: atualização

Hepatopulmonary syndrome (HPS) is a clinical threesome composed of liver disease, intrapulmonary vascular dilatation (IPVD) and arterial gas abnormalities. Its occurrence has been described in up to 32 percent of cirrhotic candidates for liver transplantation. It also affects non-cirrhotic patients with portal hypertension. Its pathogenesis is not well defined, but an association of factors such as imbalance in the endothelin receptor response, pulmonary microvascular remodeling and genetic predisposition is thought to lead to IPVD. Diagnosis is based on imaging methods that identify these dilatations, such as contrast echocardiography or perfusion scintigraphy with 99mTc, as well as analysis of arterial gases to identify elevated alveolar-arterial differences in O2 or hypoxemia. There is no effective pharmacological treatment and complete resolution only occurs through liver transplantation. The importance of diagnosing HPS lies in prioritizing transplant candidates, since presence of HPS is associated with worse prognosis. The aim of this paper was to review the pathogenetic theories and current diagnostic criteria regarding HPS, and to critically analyze the prioritization of patients with HPS on the liver transplant waiting list. Searches were carried out in the Medline (Medical Literature Analysis and Retrieval System Online) via PubMed, Cochrane Library and Lilacs (Literatura Latino-Americana e do Caribe em Ciências da Saúde) databases for articles published between January 2002 and December 2007 involving adults and written either in English or in Portuguese, using the term hepatopulmonary syndrome. The studies of greatest relevance were included in the review, along with text books and articles cited in references that were obtained through the review.

A síndrome hepatopulmonar (SHP) é considerada uma tríade clínica composta de doença hepática, dilatações vasculares intrapulmonares (IPVD) e alterações de gases arteriais. Há descrição de sua ocorrência em até 32 por cento dos cirróticos candidatos ao transplante de fígado, acometendo também não cirróticos com hipertensão portal. Sua etiopatogenia não está bem definida, mas se especula que a associação de fatores como o desequilíbrio na resposta dos receptores vasculares de endotelina, o remodelamento microvascular pulmonar e a predisposição genética propiciem as IPVD. O diagnóstico baseia-se em métodos de imagem que identifiquem essas dilatações, como a ecocardiografia com contraste ou a cintilografia de perfusão com 99mTc, além da análise de gases arteriais, para identificar elevação da diferença alvéolo-arterial de O2 ou hipoxemia. Não existe tratamento medicamentoso eficaz e sua resolução completa ocorre apenas com o transplante de fígado. A importância do diagnóstico da SHP está em priorizar os candidatos ao transplante, uma vez que sua presença está associada ao pior prognóstico. O objetivo deste artigo é fazer revisão das teorias de etiopatogenia da SHP, dos seus critérios diagnósticos atuais, além de realizar análise crítica sobre a prioridade dos pacientes com a síndrome na lista de espera pelo transplante de fígado. Buscaram-se na base de dados Medline (Medical Literature Analysis and Retrieval System Online) via PubMed, Cochrane Library e Lilacs (Literatura Latino-Americana e do Caribe em Ciências da Saúde), os artigos publicados no período de janeiro de 2002 a dezembro de 2007, envolvendo adultos, escritos nos idiomas inglês ou português, que apresentassem o termo síndrome hepatopulmonar. Os estudos de maior relevância foram incluídos, além de livros-texto e artigos citados nas referências obtidas na revisão.

Hepatoprotective effect of water soluble extract of Coleus barbatus on cholestasis on young rats / Efeito hepatoprotetor do extrato aquoso de Coleus barbatus na colestase em ratos jovens

PURPOSE: To test the effects of water extract of Coleus barbatus (WEB) on liver damage in biliary obstruction in young rats. METHODS: Forty 21 day-old male Wistar rats were divided into four groups of ten 21 day old (P21) submitted to sham or actual operation (S or L) combined with WEB or Water (B or A). At P48 pentobarbital sleeping time (ST) was measured. At P49 they were submitted to euthanasia to determine of serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), liver wet weight (PFF) and, on hepatic histological slides, the frequency of mitoses (FM), the number of necrotic areas (NN), intensity of fibrosis (IF) and intensity of ductal proliferation (IPD). Two Way ANOVA, the S.N.K. test and the Wilcoxon test for paired multiple comparisons were employed to study the effects of cholestasis and those of EAB and their interactions. The Pearson's coefficient of linear correlation of between paired histological variables separately for the groups LA and LD was determined. The test results were considered statistically significant when the p of alpha error <0.05. RESULTS: Cholestasis increased the TS, ALT, AST, PFF, MI, NN, IF and IPD. The EAB decreased the TS and IM in the animals without cholestasis (sham operated animals). The EAB decreased the TS, ALT, AST, PFF, MI, NN and IF of the cholestatic animals. In the LA group there was a positive correlation between the IPD and the IF, a negative correlation between the IPD and the FM and a negative correlation between the IF and the FM. In the LD group there was a negative correlation between the NN and the IPD. CONCLUSIONS: In the absence of cholestasis the EAB reduces the pentobarbital sleeping time and decreases the frequency of mitoses. The EAB has a hepatoprotective effect on the biliary cirrhosis secondary to extra-hepatic biliary obstruction.

OBJETIVO: Testar os efeitos do extrato aquoso de Coleus barbatus (EAB) na cirrose biliar secundária por obstrução das vias biliares extra-hepáticas em ratos jovens. MÉTODOS: Quarenta ratos Wistar machos com 21 dias de vida (P21), foram distribuídos em quatro grupos de 10 animais, submetidos a operação simulada ou dupla ligadura e ressecção do ducto biliar (S ou L) combinados EAB e a Água (B ou A). No P48, foi medido o tempo de sono com o pentobarbital (TS). No P49, foram submetidos a eutanásia para a determinação das atividades séricas do aspartato aminotransferase (AST) e da alanina aminotransferases (ALT); após a eutanásia foram avaliados o peso fresco do fígado (PFF) e, em cortes histológicos do fígado, a freqüência de mitoses (FM), o número de áreas de necrose (NN), a intensidade da fibrose (IF) e da proliferação ductal (IPD). Os efeitos da colestase, os do EAB e suas interações foram testados pela ANOVA com dois fatores, e as comparações múltiplas pareadas foram realizadas pelo teste de S.N.K ou teste de Wilcoxon. Também foi determinada a correlação linear de Pearson entre as variáveis histológicas duas a duas separadamente para os grupos LA e LD. O nível de significância estatística para os vários testes foi de p do erro alfa <0,05. RESULTADOS: A colestase aumentou significativamente o TS, a ALT, a AST, o PFF, a MI, o NN, a IF e a IPD. O EAB diminuiu o TS e a IM nos animais sem colestase (operação simulada). O EAB diminuiu o TS, a ALT, a AST, o PFF, a MI, o NN e IF na colestase. No grupo LA houve correlação positiva entre a IPD e a IF, correlação negativa entre a IPD e a FM e correlação negativa entre a IF a FM. No grupo LD houve correlação negativa entre o NN e a IPD. CONCLUSÕES: Na ausência de colestase o EAB encurta o tempo de sono e diminui a freqüência de mitoses. O EAB apresenta efeito hepatoprotetor no modelo de cirrose biliar secundária a obstrução biliar extra-hepática.

Role of N-acetylcysteine on fibrosis and oxidative stress in cirrhotic rats / Papel da N-acetilcisteína na fibrose e estresse oxidativo em ratos cirróticos

BACKGROUND: Hepatic cirrhosis is the final stage of liver dysfunction, characterized by diffuse fibrosis which is the main response to the liver injury. The inhalatory carbon tetrachloride is an effective experimental model that triggers cirrhosis and allows to obtain histological and physiological modifications similar to the one seen in humans. AIM: To investigate the effects of N-acetylcysteine (NAC) on the fibrosis and oxidative stress in the liver of cirrhotic rats, analyzing liver function tests, lipoperoxidation, activity of glutathione peroxidase enzyme, collagen quantification, histopathology, as well as the nitric oxide role. METHODS: The animals were randomly in three experimentals groups: control (CO); cirrhotic (CCl4) and CCl4 + NAC. Evaluate the lipid peroxidation, the glutathione peroxidase enzyme, the collagen and the expression of inducible nitric oxide synthase (iNOS). RESULTS: The cirrhotic group treated with N-acetylcysteine showed trough the histological analysis and collagen quantification lower degrees of fibrosis. This group has also shown less damage to the cellular membranes, less decrease on the glutathione peroxidase levels and less expression of inducible nitric oxide synthase when matched with the cirrhotic group without treatment. CONCLUSION: N-acetylcysteine seams to offer protection against hepatic fibrosis and oxidative stress in cirrhotic rat livers.

RACIONAL: A cirrose é o estágio final da disfunção hepática, sendo caracterizada por fibrose difusa, que compõe a resposta principal do organismo ao dano hepático. O tetracloreto de carbono inalatório é um modelo experimental efetivo, que desencadeia a cirrose e permite obter modificações histológicas e fisiológicas similares às vistas em humanos. OBJETIVO: Investigar os efeitos da N-acetilcisteina (NAC) sobre a fibrose e o estresse oxidativo no fígado de ratos cirróticos, analisando as provas hepáticas, a lipoperoxidação, a atividade da enzima glutationa peroxidase, a quantificação do colágeno, a histopatologia, bem como o papel do óxido nítrico. MÉTODOS: Os animais foram divididos em três grupos experimentais: controle (CO); cirrótico (CCl4) e CCl4 + NAC. Foram avaliados a lipoperoxidação, a enzima glutationa peroxidase, a histologia hepática, a quantificação de colágeno e a expressão da óxido nítrico síntase induzível (iNOS). RESULTADOS: O grupo cirrótico tratado com a NAC demonstrou, através da análise histológica e da quantificação de colágeno, menores graus de fibrose. Este grupo demonstrou, ainda, menos dano às membranas celulares, menor decréscimo nos níveis de glutationa peroxidase e menor expressão da iNOS quando comparado com o grupo cirrótico sem tratamento. CONCLUSÃO: A NAC parece oferecer proteção contra a fibrose hepática e o estresse oxidativo no fígado de ratos cirróticos.