Effect of capsaicin on liver fibrogenesis in rats

AIM：To investigate the effects of capsaicin on rat hepatic stellate cells (HSCs) in vitro and on the liver fibrogenesis in vivo. METHODS：HSCs were cultured with different concentrations of capsaicin. The levels of reactive oxygen species (ROS) were tested with a DCFH-DA kit. The proliferation of HSCs was detected by CCK-8 assay. The expression of α-smooth muscle actin in HSCs was evaluated by Western blotting. The expression of fibrosis-related genes was detected by RT-PCR. The apoptosis of HSCs was measured by flow cytometry. The rat model of liver fibrosis was established by intraperitoneal injection of carbon tetrachloride. Capsaicin at different concentrations was given by gavage. The pathologic changes of the liver sections were observed under microscope with HE staining. Hydroxyproline content in the liver tissues and the levels of collagen Ⅲ and hyaluronic acid in the serum were also measured. RESULTS：Capsaicin inhibited the generation of ROS in a concentration-dependent manner. Compared with control, the proliferation and activation of HSCs were inhibited (P<0.05) and the apoptosis of HSCs was promoted by capsaicin (P<0.05). Capsaicin down-regulated the expression of tissue inhibitor of metalloproteinase 1 and transforming growth factor β 1 in activated HSCs (P<0.05). Capsaicin decreased the levels of hydroxyproline, collagen III and hyaluronic acid in the rats (P<0.05). CONCLUSION：Capsaicin inhibits the proliferation and activation, and promotes the apoptosis of hepatic stellate cells, thus down-regulating the fibrogenesis level of the liver in rats．     

Roles of four liver cells in regulation of PA-plasmin system during liver fibrogenesis in rats

AIM: To investigate the roles of hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs) and endothelial cells (ECs) in the regulation of PA-plasmin system during liver fibrogenesis in rats. METHODS: Experimental liver fibrosis was induced in rats by injection of carbon tetrachloride (CCl₄) twice a week for 12 weeks. Four kinds of liver cells were separated from the normal and fibrotic livers of the rats. The expression levels of urokinase plasminogen activator (uPA), uPA receptor (uPAR) and PA inhibitor-1 (PAI-1) in liver cells were determined by Northern and Western blotting. RESULTS: The expression of PAI-1 and uPAR was markedly increased in HSCs during liver fibrosis in rats as compared to those in the ECs. CONCLUSION: HSCs and ECs may play very important roles in the regulation of PA-plasmin system during liver fibrogenesis in rats. The activated HSCs are main cells to secrete PAI-1 and uPAR.     

Effect of fat-specific protein 27 on activation of rat hepatic stellate cells in vitro

AIM：To investigate the effects of fat-specific protein 27 (Fsp27) on the proliferation and activation of hepatic stellate cells (HSCs) in vitro. METHODS：HSCs were isolated from the liver of SD rats. The mRNA and protein expression of Fsp27 in primary HSCs and activated HSCs was detected by real-time fluorescence quantitative PCR, immunofluorescence staining and Western blotting. After 72 h of transfection with Fsp27-carrying lentivirus (pLV-Fsp27), the proliferation of HSCs was tested by CCK-8 assay, the protein expression of α-smooth muscle actin (α-SMA) in HSCs was detected by Western blotting, and the mRNA expression of fibrosis-related proteins, including matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinase 1 (TIMP-1) and transforming growth factor beta 1 (TGF-β1), was determined by real-time fluorescence quantitative PCR. RESULTS：Rat HSCs were successfully isolated and cultured. The difference of Fsp27 expression between primary HSCs and activated HSCs was significant (P<0.01). The proliferation and activation of HSCs was inhibited 72 h after pLV-Fsp27 transfection (P<0.05). Fsp27 enhanced the mRNA expression of MMP-2 and down-regulate the mRNA expression of TIMP-1 and TGF-β1 in activated HSCs (P<0.05). CONCLUSION：Fsp27 inhibits the proliferation and activation of HSCs and regulates the expression of fibrosis-related proteins. Fsp27 may play an important role in maintenance of the quiescent phenotype of HSCs.     

Chronic alcohol intake-induced epithelial-mesenchymal transition contri-butes to hepatic fibrogenesis in mice

AIM：To evaluate the effect of chronic alcohol intake on the histopathological changes of the liver and to determine the contribution of epithelial-mesenchymal transition (EMT) to hepatic fibrogenesis. METHODS：Thirty male C57BL/6 mice were randomly divided into 3 groups as following: the mice in control group was given (ig) water; the mice in low-dose alcohol group (2.0 g·kg -1·d -1) and high-dose alcohol group (4.0 g·kg -1·d -1) were given (ig) alcohol for 5 months. Alcohol-induced histopathological changes of the liver or development of hepatic fibrosis were evaluated using the histological methods with HE and Masson trichrome staining. The apoptosis of the liver was detected by TUNEL fluorometric staining (counterstained with DAPI). The activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was measured by an automated biochemical analyzer. The expression of fibroblast-specific protein 1 (FSP-1), α-smooth muscle actin (α-SMA) and E-cadherin in the hepatic tissues was detected by immunofluorescence examination. The protein levels of E-cadherin, α-SMA, FSP-1, transforming growth factor β 1 (TGF-β 1) and hypoxia-inducible factor 1α (HIF-1α) were analyzed by Western blotting. RESULTS：Compared with control, the activity of serum ALT and AST, and apoptotic index of liver tissues were increased in the mice treated with alcohol for 5 months. The histopathological changes of the livers in the mice of low-dose alcohol group included steatosis and mild liver fibrosis, while severe liver fibrosis was observed in the high-dose alcohol-treated mice. Chronic alcohol consumption induced the increase in malondialdehyde (MDA) level, and the decreases in the activity of superoxide dismutase (SOD) and catalase (CAT) in the livers. It also reduced E-cadherin expression and increased α-SMA expression. FSP-1 immunostaining and albumn immunostaining positive cells were co-localized in the hepatocytes of low-dose alcohol group, but only FSP-1 positive hepatocytes were observed in high-dose alcohol group. Chronic alcohol consumption decreased E-cadherin expression and increased α-SMA, FSP-1, TGF-β 1 and HIF-1α expression in a dose-dependent manner, but the HIF-1α expression was not altered between the 2 alcohol-treated groups. CONCLUSION：Chronic alcohol intake induces the progression of hepatic fibrosis. Some fibroblasts derive from hepatocytes in liver fibrosis via EMT. The underlying mechanism is associated with the changes of the redox state, and increased TGF-β 1 generation and HIF-1α expression.     

Relationship between expression of MKK-4 and MMP-9 genes in primary hepatic carcinoma with or without invasion and metastasis

AIM：To investigate the expression of mitogen activated protein kinase kinase 4 (MKK-4) and MMP-9 mRNA in primary hepatic carcinoma (PHC), and to analyze its relationship with invasion and metastasis. METHODS：The expression of MKK-4 and MMP-9 mRNA in 34 cases of hepatic carcinoma tissues and adjacent tissues,and in 12 cases of normal liver tissues were detected with RT-PCR. RESULTS：(1) The expression level of MMP-9 mRNA was higher in metastatic cancer tissues than that in other tissuses (P<0.01). (2) There was significant statistical difference among the expression level of MKK-4 mRNA, but the level in metastatic cancer was low (P<0.01). (3) There was no statistical difference among the expression level of MKK-4 or MMP-9 mRNA among the adjacent tissues and normal tissues (P>0.05). (4) MMP-9 mRNA had a tendency to rise as PHC became invasive and metastatic.The expression level of MKK-4 had a tendency to decline in PHC became invasive and metastatic. (5) The expression level of MMP-9 or MKK-4 mRNA had no correlations with tumor volume,or cell differentiation (P>0.05). (6) There were correlations between expressions of MKK-4 and MMP-9 mRNA in PHC (Pearson Correlation, r=-0.925, P<0.01). CONCLUSION：There are high MMP-9 mRNA expression and low MKK-4 mRNA expression in PHC.The expression level of MKK-4 or MMP-9 mRNA is correlated with tumor metastasis.     

Protein expression of EMMPRIN,MMP-2 and P53 detected by technique of quantum dots immunofluorescence in human lung cancer

AIM: To investigate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN/CD147), matrix metalloproteinase2 (MMP-2) and P53 proteins in human lung cancer tissues and to explore the relationship between EMMPRIN protein and malignant biological behaviour of lung cancer.METHODS: Fluorescent semiconductor nanocrystals were applied, which were nanometer-sized light-emitting particles and were emerging as a new class of fluorescent probes for cancer detection due to the unique optical and electronic properties. The technique of QDs immunofluorescence histochemistry (QDs-IHC) was used to detect the protein expression of EMMPRIN, P53 and MMP-2 in the human lung cancer microarray, and co-expression of EMMPRIN/P53 proteins was also simultaneously detected by double-labeling immunofluorescence.RESULTS: Compared with non-cancerous lung tissues, the positive rates of EMMPRIN, P53 and MMP-2 proteins in the lung cancer tissues were 70.00%, 77.14% and 72.86%, respectively, and the differences were all significant (P<0.05). The positive rates of EMMPRIN, P53 and MMP-2 proteins were all significantly related with tumor staging(TNM stage) and lymph node metastasis (P<0.05). The positive correlation between EMMPRIN expression and protein levels of MMP-2 and P53 was observed (P<0.01).CONCLUSION: The protein expression of EMMPRIN, P53 and MMP-2 is correlated with the development of lung cancer. Malignant progression of lung cancer promoted by EMMPRIN may be closely related with the expression of MMP-2 and P53.     

Inhibitory effects of Angelicae sinensis and sodium ferulate on acute inflammatory liver injury and expression of ICAM-1 and E-selectin in mice

AIM: To explore the effects of Angelicae sinensis preparation and sodium ferulate on inflammatory liver injury induced by lipopolysaccharide (LPS) and their molecule mechanism. METHODS: ICR mice were divided into five groups: Angelicae sinensis group, sodium ferulate group, dexamethasone group, inflammation control group and normal group. The model of inflammatory injury in mice was set up by tail vein injection with the bacillus calmette-guerin (BCG) and LPS respectively prior and posterior to administration of those tested drugs. The tested drugs (the preparations of Angelica sinensis, sodium ferulate and dexamethasone) and normal saline were given respectively to the corresponding group. The pathological observation of the liver tissues in mice was made for the intensity of inflammatory liver injuries. The immunohistochemical detection and comparison were performed for the expression of ICAM-1 and E-selectin proteins in the liver tissues in those mice. RESULTS: The intensities of liver inflammatory injuries in mice from drug-treated groups were obviously lighter than that from the inflammation control group (P<0.01). The expressions of ICAM-1 and E-selectin proteins in the liver tissues of mice from the inflammation control group were not only significantly higher than that in normal control, but also obviously higher than that from drug-treated groups (P<0.01). CONCLUSIONS: Angelica sinensis and sodium ferulate alleviate inflammatory liver injury induced by injection of LPS. Their suppressive effects on inflammatory liver injury may be associated with the decrease in the expression of ICAM-1 and E-selectin proteins.     

Dynamic expression of PTEN in liver tissue of rats with hepatic fibrosis

AIM: To investigate the dynamic expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) in liver tissue in the process of hepatic fibrosis in rats. METHODS: The rat model of hepatic fibrosis used in this study was induced by common bile duct ligation (BDL). Hematoxylin and eosin (HE) and Masson’s trichrome staining were used for observing the histological changes in hepatic fibrosis tissue. At 4 time points, the expressions of PTEN protein and mRNA in hepatic tissues of rats were detected by immunohistochemical staining, Western blotting and real-time fluorescent quantitative PCR assay, respectively. RESULTS: The rat model of hepatic fibrosis was established successfully. With each consecutive week after BDL, increased fibrosis, degeneration and necrosis were found in rat liver cells. Not surprisingly, a disruption of normal architecture and a decrease in normal hepatic cells was concomitantly observed. The immunohistochemical staining indicated that there was extensive expression of PTEN in liver tissues of normal rats, it expressed mainly in the cytoplasm, and with the aggravation of hepatic fibrosis, the expression of PTEN in liver tissues decreased gradually (P<0.01). Western blotting and real-time fluorescent quantitative PCR at weekly time points (1, 2, 3, and 4 weeks) after BDL showed that, the expression of PTEN protein and mRNA in fibrotic rat liver tissue decreased gradually with increasing severity of hepatic fibrosis (P<0.01). Furthermore, all values from BDL rats were significantly lower than those from the sham operation group (P<0.01). CONCLUSION: The expressions of PTEN protein and mRNA in fibrotic liver tissue of rat decrease gradually with the progression of hepatic fibrosis, and increasing severity of fibrosis correlated well with decreasing PTEN expression.     

Baicalin attenuates pancreatic fibrosis by inhibiting TGF-β1/TAK-NF-κB signaling pathway in chronic pancreatitis mice

AIM: To explore the role of transforming growth factor-β1 (TGF-β1)/TGF-β-activated kinase (TAK)-nuclear factor-κB (NF-κB) signaling pathway in chronic pancreatitis (CP) mice and the effect of baicalin on pancreatic fibrosis in the mice. METHODS: Kunming mice (n=58) were randomly divided into 3 groups, including control group, CP group and baicalin group. The mice in CP group and baicalin group were intraperitoneally injected with 20% L-arginine. After 2 weeks of CP, the mice in baicalin group were intraperitoneally injected with baicalin (100 mg/kg, once a day). At 2 weeks, 4 weeks and 6 weeks after modeling, the mice were anesthetized and sacrificed. The morphological changes of the pancreas were observed by HE and Masson staining. The serum level of TGF-β1 was analyzed by ELISA. The expression of fibronectin (FN) and NF-κB in the pancreas was observed by immunohistochemistry staining. The protein levels of transforming growth factor-β receptor type Ⅰ (TGF-βRⅠ), phosphorylated TAK1 (p-TAK1) and NF-κB in the pancreas were determined by Western blot. The mRNA expression of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloprotease-1 (TIMP-1) was detected by real-time PCR. RESULTS: At 2 weeks, 4 weeks and 6 weeks after intraperitoneal injection of L-arginine, the pancreatic tissues were obviously injured and exhibited different degrees of fibrosis, and FN expression was significantly increased. After treatment with baicalin, the degrees of pancreatic injury and fibrosis were significantly attenuated and the expression of FN was reduced (P<0.01). Compared with control group, the protein levels of TGF-β1, TGF-βRⅠ, p-TAK1, NF-κB and TIMP-1 in the pancreas of CP group were significantly increased, and the expression of MMP-1 was decreased at each time point. In baicalin group, the protein levels of TGF-β1, TGF-βRⅠ, p-TAK1, NF-κB and TIMP-1 were significantly decreased, and the expression of MMP-1 was markedly increased at the corresponding time points compared with CP group (P<0.01). CONCLUSION: Baicalin effectively atte-nuates pancreatic fibrosis by inhibiting the activation of TGF-β1/TAK-NF-κB signaling pathway and regulating the balance of MMP-1/TIMP-1.     

Expression of calpain 2 and Bax in rat fibrotic liver tissues

AIM：To observe the expression of calpain 2 and Bcl-2-associated X protein (Bax) in rat fibrotic liver tissues and to explore their effects on hepatic fibrosis.
METHODS：Forty male Wistar rats were randomly divided into four groups (each n=10): 4-week control group, 8-week control group, 4-week liver fibrosis group and 8-week liver fibrosis group. Liver fibrosis model was induced by subcutaneous injection of 40% CCl4 (3 mL/kg) every 3 days for 4 or 8 weeks. The apoptosis of hepatocytes was detected by TUNEL. Additionally, the mRNA expression of calpain 2 and bax was determined by real-time PCR, and the protein expression of calpain 2 and Bax was determined by immunohistochemistry and Western blotting. RESULTS：Real-time PCR showed that the mRNA expression of calpain 2 and bax in liver tissues was elevated in 4-week and 8-week liver fibrosis groups. The results of immunohistochemistry and Western blotting revealed that there was no difference of calpain 2 protein expression in liver tissues between 4-week liver fibrosis group and control group, but that in 8-week liver fibrosis group was obviously increased. The protein expression of Bax in 4-week and 8-week liver fibrosis groups was higher than that in control groups. Additionally, the numbers of apoptotic hepatocytes in 4-week and 8-week liver fibrosis groups were obviously increased compared with control groups.CONCLUSION：Calpain 2 and Bax may play important roles in the process of liver fibrosis.     

Preventive effects of anti-IGFBPrP1 antibody on hepatic fibrosis in mice

AIM: To investigate the preventive effect and mechanism of anti-insulin-like growth factor binding protein related protein 1(IGFBPrP1) antibody on hepatic fibrosis induced by thioacetamide (TAA) in mice.METHODS: Twenty-four male C57BL/6 wild-type mice were randomly divided into 3 groups (n= 8 in each group): normal control group, TAA group (4 weeks) and TAA+anti-IGFBPrP1 antibody group (4 weeks). The morphological changes of liver tissues were observed. The expression levels of α-smooth muscle actin (α-SMA), transforming growth factor beta 1 (TGF-β₁), Smad3, phosphorylated Smad2/3 (p-Smad2/3), fibronectin (FN), collagen I, collagen Ⅲ and IGFBPrP1 were detected by the methods of immunohistochemistry and Western blotting.RESULTS: In TAA group (4 weeks), obvious injury of liver was observed, and the expression levels of α-SMA, TGF-β₁, Smad3, p-Smad2/3, FN, collagen Ⅰ, collagen Ⅲ and IGFBPrP1 were significantly increased as compared with normal control group (P<0.01). Compared with TAA group (4 weeks), the injury of the liver was alleviated and the expression levels of the proteins above were decreased in TAA+anti-IGFBPrP1 antibody group (4 weeks, P<0.01). IGFBPrP1 was positively correlated with TGF-β₁, Smad3, p-Smad2/3, FN and collagen I (P<0.01). CONCLUSION: Anti-IGFBPrP1 antibody prevents TAA-induced hepatic fibrosis in mice by inhibiting the activation of hepatic stellate cells, reducing the expression of p-Smad2/3 and inhibiting the TGF-β₁/ Smad3 signal transduction, thereby depressing the deposition of extracellular matrix in liver tissues.     

Cardiac collagen metabolism in murine viral heart diseases

AIM：To investigate the dynamic alteration of cardiac collagen metabolism in mice with acute,chronic myocarditis and dilated cardiomyopathy (DCM).METHODS：BALB/c mice infected with coxsackievirus B3 were used to establish animal models of acute,chronic myocarditis and dilated cardiomyopathy,while uninfected animals were also prepared and served as controls.After verification of models by histopathological methods and echocardiography,serum concentration of aminoterminal propeptide of type Ⅲ procollagen (PIIINP),aminoterminal propeptide of type Ⅰ procollagen (PINP) and carboxyterminal propeptide of type I procollagen (PICP) in each group of mice were detected by enzyme linked immunosorbent assay (ELISA).The expression of matrix metalloproteinase 1 (MMP-1) and its tissue inhibitor (TIMP-1) were determined by Western blotting analysis.The MMP-1 activity was also detected.RESULTS：Marked myocardial fibrosis was observed in all groups of CVB3-infected mice.Reparative fibrosis,promotion of synthesis and degradation of cardiac collagens were presented in heart tissue of acute myocarditis mice.Both reparative and reactive fibrosis,enhanced synthesis and lightened degradation of collagen were present in chronic myocarditis,while reactive fibrosis and excess collagen synthesis were confirmed in DCM.Expression and activity of MMP-1 was progressively decreased.TIMP-1 showed unchanged.The ratio of MMP-1/TIMP-1 was progressively descended.CONCLUSION：Collagen metabolism was special in different phase of viral heart diseases,which may play different roles in the progression and prognosis of these kinds of disease.     

Effects of NGF on collagen secretion and morphology of hepatic stallete cells

AIM: To investigate the effects of nerve growth factor (NGF) on the changes of collagen secretion and morphology of hepatic stallete cells(HSCs). METHODS: Rat HSCs were incubated with different concentrations of NGF for 24 h. Collagen I and III in the supernatants of culture medium secreted by HSCs were detected by enzyme-linked immunosorbent assay. The morphological changes of HSCs were observed under inverted microscope with acridine orange staining and under transmission electronic microscope. RESULTS: When HSCs was incubated with NGF at concentrations of 100, 200 or 400 μg/L for 24 h, the content of collagen I and collagen III in the culture supernatants were significantly reduced compared with control group (P<0.05). After stimulated with NGF at the concentration of 100 μg/L for 24 h, the growth of the HSCs was inhibited and the morphous of the cells became round or oval gradually. The morphological changes of apoptotic cells were also observed by acridine orange staining and transmission electronic microscopy. CONCLUSION: NGF inhibits HSCs to synthesize collagen I and collagen III. Inhibition of collagen production and promotion of apoptosis in HSCs may be the possible mechanisms of NGF to reverse liver fibrosis.     

Liraglutide ameliorates liver injury of type 2 diabetic mice via micro-RNA-33-AMPK pathway

AIM: To observe the effects of liraglutide on the level of microRNA-33 (miR-33) and the expression of AMP-activated protein kinase (AMPK) and apoptosis-related proteins in mice with type 2 diabetes mellitus (T2DM), and to explore its possible mechanism. METHODS: High-fat diet and intraperitoneal injection of streptozocin were used to establish the type 2 diabetic model in C57BL/6 mice. The mice were randomly divided into 4 groups (n=15):in control group, the normal mice were subcutaneously injected with equivalent volume of saline; in model group, the T2DM mice were subcutaneously injected with equivalent volume of saline; in low-and high-dose liraglutide treatment groups, the T2DM mice were subcutaneously injected with 100 and 200 μg·kg^-1·d^-1, respectively. After 4 weeks of administration, the levels of FBG, TG, TC, HDL-C, LDL-C, ALT and AST were determined. HE staining was used to observe the pathological changes of the liver tissues. The protein level of cleaved caspase-3 in the liver tissue was detected by the technique of immunofluorescence. The protein levels of p-AMPK/AMPK and apoptosis-related proteins were detected by Western blot. The expression of miR-33 in the liver tissues was detected by real-time PCR. RESULTS: Compared with model group, the contents of FBG, TG, TC, LDL-C, ALT and AST were decreased significantly, while the content of HDL-C was increased significantly in low-dose liraglutide group and high-dose liraglutide group (P<0.05). The protein levels of phosphorylated AMPK and Bcl-2 were up-regulated significantly, and the expression of cleaved caspase-3 was down-regulated significantly (P<0.05). The level of miR-33 was decreased significantly (P<0.01). CONCLUSION: Liraglutide alleviates liver injury in type 2 diabetic mice, and the mechanism may be associated with reducing the level of miR-33 and increasing the phosphorylation of AMPK in the liver tissues, thereby inhibiting hepatocyte apoptosis.     

Effects of c-myb antisense RNA on the proliferation and collagen Ⅰ gene expression in cultured hepatic stellate cells

AIM:To investigate the effects of c-myb antisense RNA on the proriferation and collagen Ⅰ gene expression in cultured hepatic stellate cells(HSC) in rats.METHODS:The c-myb antisense gene recombinant retroviral vector(pDOR-myb) was constructed, and then was transfected into retroviral package cell line PA 317 by means of N-[1-(2,3-Dioleoyloxy) propyl]-N, N, N-trimethylammonium methyl-sulfate(DOTAP) liposomal transfection reagent. The pseudoviruses produced from the resistant PA317 cells selected with G418 were collected, with which HSCs isolated from rat liver were infected. The cell proliferation was measured by MTT method, c-myb, α₁-Ⅰ collagen mRNA expression and c-myb protein in HSCs were detected with semi-quantitative RT-PCR and western-blot, respectively.RESULTS:HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, c-myb expression levels, the cell proliferation, and α₁-Ⅰ collagen mRNA expression were repressed significantly.CONCLUSIONS:c-myb plays a key role in the activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation and α₁-Ⅰ collagen mRNA expression in the infected HSC. These data suggest that inhibition of c-myb gene expression would be a potential way for the treatment of liver fibrosis.     

Effects of tumor necrosis factor alpha on proliferation and apoptosis of HSCs

AIM:To explore the role of tumor necrosis factor alpha(TNF-α) in the pathogenesis of liver fibrosis.METHODS:The proliferation and apoptosis of hepatic stellate cells (HSCs) in vitro were detected with flow cytometry, electron microscopy and TUNEL.RESULTS:The flow cytometry analysis showed that the cell proliferation index (PI) in the TNF-α(0.5 μg/L, 2.0 μg/L, 8.0 μg/L) groups was evidently lower than that in the control group (P＜0.05). In the cell cycle distribution, the portion of G₀/G₁ phase in the TNF-α groups was significantly higher than that in the control group(P＜0.05), but the portion of S phase in the TNF-α groups was evidently lower than that in the control group(P＜0.05). These indicated that TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. The apoptotic rate in the TNF-α groups was evidently higher than that in the control group(P＜0.05). The gene expression of bcl-2 and bax was also detected with flow cytometry. The expression of bcl-2 in the TNF-α groups was evidently lower than that in the control group(P＜0.05), but the expression of bax in the TNF-α groups was significantly higher than that in the control group(P＜0.05). TUNEL analysis showed the apoptotic rate of HSCs in the TNF-α(2.0 μg/L) group was 18.7%±2.5% compared with 5.3%±1.2% in the control group(P＜0.05).CONCLUSIONS:TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. TNF-α down-regulated bcl-2 gene expression and up-regulated bax gene expression whereupon the apoptosis of HSCs was induced.     

Effect of chelerythrine on TGF-β/Smads signaling pathway in mouse livers with hepatic fibrosis

AIM:To investigate the anti-hepatic fibrosis effect of chelerythrine on mice and the regulation of transforming growth factor-β (TGF-β)/Smads signaling pathway. METHODS:C57BL/6N mice (n=50) were randomly divided into control group, model group and chelerythrine groups (10 mg·kg^-1·d^-1, 20 mg·kg^-1·d^-1 and 40 mg·kg^-1·d^-1, ig). The mouse model of hepatic fibrosis was established by intraperitoneal injection of carbon tetrachloride (CCl₄) in combination with the olive oil for 8 weeks. At the 5th week, different doses of chelerythrine was used to treat hepatic fibrosis in the mice. At the 14th week, hepatic index was detected. Histopathological changes and the degree of hepatic fibrosis were observed by hematoxylin-eosin staining and Van Gieson staining. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hyaluronic acid (HA), and hepatic hydroxyproline (Hyp) content were assayed by spectrophotometry and ELISA. The mRNA expression of TGF-β₁, Smad3, Smad4 and Smad7 in the liver was detected by RT-qPCR, and the protein expression of TGF-β₁, Smad4 and Smad7 was determined by Western blot. RESULTS:The degree of hepatic fibrosis changed markedly in model group compared with control group. The hepatic index, the serum levels of ALT and AST, and the contents of HA and Hyp were significantly increased (P<0.05). The mRNA expression of TGF-β₁, Smad3 and Smad4 was significantly up-regulated, while the mRNA expression of Smad7 was significantly down-regulated (P<0.05). The protein expression of TGF-β₁ and Smad4 was significantly up-regulated, while the protein expression of Smad7 was significantly down-regulated (P<0.05). Compared with model group, the changes of the above indexes in chelerythrine groups were inhibited. CONCLUSION:Chelerythrine protects the mouse liver from CCl₄-induced fibrogenesis injury by regulating TGF-β/Smads signaling pathway.     

Gelatinases expression and their activity in rat alveolar macrophages induced by cigarette smoke medium

AIM: To study the effect of cigarette smoke medium (CSM) on the gene expression and activity of gelatinases from alveolar macrophages (AMs) in the rat, and then to explore their role in the pathogenesis of chronic obstructive pulmonary disease (COPD). METHODS: AMs were obtained from BALF of the rats that had smoked for 12 weeks. CSM was produced following the method of Wirtz and colleagues, and the cultured AMs were respectively stimulated for 24 h by 0%, 1%, 3%, 5%, 10%, 15% CSM. The mRNA levels of MMP-9 and MMP-2 were detected by semi-quantitative RT-PCR, and the enzyme activity was measured by Zymography. RESULTS: When the concentration of CSM was below 5%, the expression and activity of MMP-9 and MMP-2 signficantly increased with the concentration of CSM in a dose-depended manner (P<0.05). While the concentration of CSM exceeded 5%, the expression and activity of MMP-9 and MMP-2 correspondingly decreased with the increase in CSM concentrations (P<0.05), which possibly were related with the cytotoxicity of CSM. CONCLUSION: The expression and activity of MMP-9 and MMP-2 are induced by CSM. The gelatinases induced by smoking from AM may play an important role in the pathogenesis of COPD.