Glycyrrhizic acid inhibits apoptosis and fibrosis in carbontetrachloride-induced rat liver injury

AIM:To investigate anti-apoptotic effects of glycyrrhizic acid(GA) against fibrosis in carbon tetrachloride(CCl4)-induced liver injury and its contributing factors.METHODS:Liver fibrosis was induced by administration of CCl4 for 8 wk.Pathological changes in the liver of rats were examined by hematoxylin-eosin staining.Collagen fibers were detected by Sirius red staining.Hepatocyte apoptosis was determined by TUNEL assay and the expression levels of cleaved caspase-3,Bax,α-SMA,connective tissue growth factor(CTGF),matrix metalloproteinase(MMP) 2 and MMP9 proteins were evaluated by western blot analysis,and α-SMA m RNA,collagen type Ⅰ and Ⅲ m RNA were estimated by real-time PCR.RESULTS:Treatment with GA significantly improved the pathological changes in the liver and markedly decreased the positive area of Sirius red compared with rats in the CCl4-treated group.TUNEL assay showed that GA significantly reduced the number of TUNEL-positive cells compared with the CCl4-treated group.The expression levels of cleaved caspase-3,Bax,α-SMA,CTGF,MMP2 and MMP9 proteins,and α-SMA m RNA,collagen type Ⅰ and Ⅲ m RNA were also significantly reduced by GA compared with the CCl4-treated group(P < 0.05).CONCLUSION:GA treatment can ameliorate CCl4-induced liver fibrosis by inhibiting hepatocyte apoptosis and hepatic stellate cell activation.

Antifibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline on bile duct ligation induced liver fibrosis in rats

AIM:To investigate the preventive effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) on bile duct ligation (BDL)induced liver fibrosis in rats. METHODS:Liver fibrosis in rats was induced by BDL and AcSDKP was infused subcutaneously for 2 wkvia a osmotic minipump (Alzet 2ML4) immediately after BDL operation. After scarifying, serum and liver specimens were collected. Hematoxylin and eosin staining, Sirius red staining, enzyme linked immunosorbent assay, Western blot or real-time polymerase chain reaction were used to determinate liver functions, histological alterations, collagen deposition, mRNA expression of markers for fibroblasts, transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7). RESULTS:When compared to model rats, chronic exogenous AcSDKP infusion suppressed profibrogenicTGF-β1 signaling, α-smooth muscle actin positivity (α-SMA), fibroblast specific protein-1 (FSP-1) staining and collagen gene expression. Col Ⅰ, Col Ⅲ, matrix metalloproteinase-2, tissue inhibitors of metallopro-teinase-1 and tissue inhibitors of metalloproteinase-2 mRNA expressions were all significantly downregulated by AcSDKP infusion (2.02 ± 1.10vs 14.16 ± 6.50, 2.02 ± 0.45vs 10.00 ± 3.35, 2.91 ± 0.30vs 7.83 ± 1.10, 4.64 ± 1.25 vs 18.52 ± 7.61, 0.46 ± 0.16 vs 0.34 ± 0.12, respectively, P < 0.05). Chronic exogenous AcSDKP infusion attenuated BDL-induced liver injury, inflammation and fibrosis. BDL caused a remarkable increase in alanine transaminase, aspartate transaminase, total bilirubin, and prothrombin time, all of which were reduced by AcSDKP infusion. Mast cells, collagen accumulation, α-SMA, TGF-β1, FSP-1 and BMP-7 increased. The histological appearance of liver specimens was also improved. CONCLUSION:Infusion of exogenous AcSDKP attenu-ated BDL-induced fibrosis in the rat liver. Preservation of AcSDKP may be a useful therapeutic approach in the management of liver fibrosis.

Osteopontin expression is associated with hepatopathologic changes in Schistosoma japonicum infected mice

AIM:To investigate osteopontin expression and its association with hepatopathologic changes in BALB/C mice infected with Schistosoma japonicum.METHODS:The schistosomal hepatopathologic mouse model was established by abdominal infection with schistosomal cercaria.Liver samples were obtained from mice sacrif iced at 6,8,10,14,and 18 wk after in-fection.Liver histopathological changes were observed with hematoxylin-eosin and Masson trichrome staining.The expression of osteopontin was determined with im-munohistochemistry,reverse transcription-polymerase chain reaction,and Western blotting.The expressionof α-smooth muscle actin(α-SMA)and transforming growth factor-β1(TGF-β1)were determined by im-munohistochemistry.Correlations of osteopontin ex-pression with other variables(α-SMA,TGF-β1,hepato-pathologic features including granuloma formation and degree of liver f ibrosis)were analyzed.RESULTS:Typical schistosomal hepatopathologic changes were induced in the animals.Dynamic changes in the expression of osteopontin were observed at week 6.The expression increased,peaked at week 10(P<0.01),and then gradually decreased.Positive correla-tions between osteopontin expression and α-SMA(r=0.720,P<0.01),TGF-β1(r=0.905,P <0.01),granu-loma formation(r=0.875,P<0.01),and degree of liver f ibrosis(r=0.858,P<0.01)were also observed.CONCLUSION:Osteopontin may play an important role in schistosomal hepatopathology and may promote granuloma formation and liver fi brosis through an un-explored mechanism.

Effect of nuclear factor-κB and angiotensin Ⅱ receptor type 1 on the pathogenesis of rat non-alcoholic fatty liver disease

AIM: To investigate the roles of nuclear factor(NF)-κB and angiotensin Ⅱ receptor type 1(AT1R) in the pathogenesis of non-alcoholic fatty liver disease(NAFLD).METHODS: Forty-two healthy adult male SpragueDawley rats were randomly divided into three groups:the control group(normal diet), the model group,and the intervention group(10 wk of a high-fat diet feeding, followed by an intraperitoneal injection of PDTC); 6 rats in each group were sacrificed at 6, 10,and 14 wk. After sacrifice, liver tissue was taken,paraffin sections of liver tissue specimens were prepared, hematoxylin and eosin(HE) staining was performed, and pathological changes in liver tissue(i.e., liver fibrosis) were observed by light microscopy.NF-κB expression in liver tissue was detected by immunohistochemistry, and the expression of AT1 R in the liver tissue was detected by reverse transcriptionpolymerase chain reaction(RT-PCR). The data are expressed as mean ± SD. A two-sample t test was used to compare the control group and the model group at different time points, paired t tests were used to compare the differences between the intervention group and the model group, and analysis of variance was used to compare the model group with the control group. Homogeneity of variance was analyzed with single factor analysis of variance. H variance analysis was used to compare the variance. P < 0.05 wasconsidered statistically significant.RESULTS: The NAFLD model was successful after 6wk and 10 wk. Liver fibrosis was found in four rats in the model group, but in only one rat in the intervention group at 14 wk. Liver steatosis, inflammation, and fibrosis were gradually increased throughout the model. In the intervention group, the body mass,rat liver index, serum lipid, and transaminase levels were not increased compared to the model group.In the model group, the degree of liver steatosis was increased at 6, 10, and 14 wk, and was significantly higher than in the control group(P < 0.01). In the model group, different degrees of liver cell necrosis were visible and small leaves, punctated inflammation,focal necrosis, and obvious ballooning degeneration were observed. Partial necrosis and confluent necrosis were observed. In the model group, liver inflammatory activity scores at 6, 10, and 14 wk were higher than in the control group(P < 0.01). Active inflammation in liver tissue in the intervention group was lower than in the model group(P < 0.05). HE staining showed liver fibrosis only at 14 wk in 4/6 rats in the model group and in 1/6 rats in the intervention group. NF-κB positive cells were stained yellow or ensemble yellow,and NF-κB was localized in the cytoplasm and/or nucleus. The model group showed NF-κB activation at6, 10, and 14 wk in liver cells; at the same time points,there were statistically significant differences in the control group(P < 0.01). Over time, NF-κB expression increased; this was statistically lower(P < 0.05) at14 weeks in the intervention group compared to the model group, but significantly increased(P < 0.05)compared with the control group; RT-PCR showed that AT1 R mRNA expression increased gradually in the model group; at 14 wk, the expression was significantly different compared with expression at 10 weeks as well as at 6 weeks(P < 0.05). In the model group, AT1 R mRNA expression was significantly higher than at the same time point in the control group(P <0.01).CONCLUSION: With increasing severity of NAFLD,NF-κB activity is enhanced, and the inhibition of NF-κB activity may reduce AT1 R mRNA expression in NAFLD.

Decorin accelerates the liver regeneration after partial hepatectomy in fibrotic mice

Background Considering the existence of a large number of liver cell degeneration and necrosis in fibrotic liver, liver function was damaged severely and could not effectively regenerate after partial hepatectomy （PHx）. The aim of this study was to investigate whether decorin （DCN） could promote the liver regeneration after PHx in fibrotic mice. Methods Forty mice （5-week-old, Balb/c） were injected with CCI4 intraperitoneally and liver fibrosis model was established after 5 weeks. The survival mice were randomly divided into two groups： control group and DCN group. Then, we performed 70% PHx on all these mice and injected DCN or phosphate-buffered saline plus normal saline （NS） to each group, respectively, after surgery. Liver body weight ratio （LBR）, quantitative real-time polymerase chain reaction, and immunohistochemistry were used to analyze liver regeneration and fibrosis degree in both groups, and to find out whether exogenous protein DCN could promote the regeneration of fibrosis liver after PHx. Results Expressions of a-smooth muscle actin （SMA） mRNA and LBR had significant increases in the DCN group at postoperative Day 3 （POD 3, P〈0.05）. The protein expressions of CD31, a-SMA, and tumor necrosis factor （TNF）-a were higher in the DCN group than those in the control group by immunohistochemistry at POD 3 （P〈0.05）. Conclusion Exogenous protein DCN could promote liver regeneration after PHx in fibrotic mice. Chin Med J 2014;127 （14）： 2679-2685