Hepatitis B Virus X protein elevates Parkin-mediated mitophagy through Lon Peptidase in starvation.

Hepatocellular Carcinoma (HCC) is the fifth most prevalent cancer worldwide. Specially, Hepatitis B viurs X protein (HBx) is a leading factor in the progression of Hepatitis B viurs-related HCC. Nutrient-deprived tumor microenvironment also contributes to tumor development. However, the role of HBx in nutrient-deprived HCC has received little investigation. Here, we show that HBx elevates PINK1-Parkin mediating mitophagy in starvation. HBx not only increases the PINK1/Parkin gene expression but also accelerates Parkin recruitment to partial mitochondria. Further analysis indicates that, HBx either promotes mitochondrial unfolded protein response, with remarkable mitochondrial LONP1 increases, or reduces LONP1 expression in cytosol inducing LONP1-Parkin pathway, both consequently enhancing mitophagy. Moreover, the enhanced mitophagy lowers mitochondrial apoptosis in starved hepatoma cells, and Bax is implied in the machinery. In addition, we define differential centrifuge, 3000 g or 12,000 g to pellet mitochondria, as an effective method to obtain distinct mitochondria. In collect, HBx regulates diverse aspects of LONP1 and Parkin, enhancing mitophagy in starvation. This study may shed new insights into the machinery development of hepatocellular carcinoma.

Lactobacillus plantarum improves LPS-induced Caco2 cell line intestinal barrier damage via cyclic AMP-PKA signaling.

Lactobacillus plantarum (LP) has been shown to exhibit protective effects on intestinal barrier function in septic rats, although the regulatory mechanism has not been established. We determined whether LP imparts such protective effects in a lipopolysaccharide (LPS)-induced Caco2 cell monolayer model and whether cAMP-PKA signaling is the underlying mechanism of action. The cyclic adenosine monophosphate (cAMP) agonist, forskolin (FSK), and the protein kinase A (PKA) inhibitor, HT89, were used to study the protective effect of LP on the destruction of the tight junction (TJ) structure of cells treated with LPS and the corresponding changes in cAMP-PKA signaling. Our experimental results demonstrated that LP promoted the expression of TJ proteins between Caco2 cells after LPS treatment, and increased the electrical barrier detection (TEER) between Caco2 cells. Moreover, transmission electron microscopy (TEM) revealed that the TJ structural integrity of cells treated with LPS + LP was improved compared to cells treated with LPS alone. In addition, our findings were consistent between the FSK and LP intervention group, while HT89 inhibited LP influence. Taken together, our results indicate that LP has an improved protective effect on LPS-induced damage to the monolayer membrane barrier function of Caco2 cells and is regulated by the cAMP-PKA pathway.

Interleukin-10 induces senescence of activated hepatic stellate cells via STAT3-p53 pathway to attenuate liver fibrosis.

Hepatic fibrosis is a wound healing process which results in deposition of excessive abnormal extracellular matrix (ECM) in response to various liver injuries. Activated hepatic stellate cells (HSCs) are the major sources of ECM and induction of senescence of activated HSCs is an attractive therapeutic strategy for liver fibrosis. Our previous studies have shown that interleukin-10 (IL-10) attenuates the carbon tetrachloride (CCL4) - and porcine serum-induced liver fibrosis in rats. However, little is known about the mechanisms of IL-10 regulating the senescence of activated HSCs. The aim of this study is to uncover the underlying pathway by which IL-10 mediates activated HSCs senescence to attenuate liver fibrosis. In vivo, we found that IL-10 gene by hydrodynamics-based transfection attenuated CCL4-induced liver fibrosis associated with senescence of activated HSCs in rats. In vitro experiment confirmed that IL-10 could induce senescence of activated HSCs via inhibiting cell proliferation, inducing cell cycle arrest, increasing the SA-ß-Gal activity and enhancing expression of senescence marker protein p53 and p21. Treatment with Pifithrin-α, a specific inhibitor of p53, could abrogate IL-10-increased SA-ß-Gal activity and expression of P53 and P21in activated HSCs. Lastly, IL-10 also increased the expression of total and phosphorylated signal transducers and activators of transcription 3(STAT3) and promoted phosphorylated STAT3 translocation from cytoplasm to nucleus. Treatment with cryptotanshinone, a specific inhibitor of STAT3, could inhibit the phosphorylation of STAT3 and its downstream proteins p53 and p21 expression and decrease the activity of SA-ß-Gal in activated HSCs induced by IL-10. Taken together, IL-10 induced senescence of activated HSCs via STAT3-p53 pathway to attenuate liver fibrosis in rats and present study will provide a new mechanism of antifibrotic effects of IL-10.

Effect of HBx on inflammation and mitochondrial oxidative stress in mouse hepatocytes.

Hepatitis B virus × protein (HBx) serves an important role in the pathogenesis of the hepatitis B virus infection. Previous studies have reported that the interaction between HBx and hepatocyte mitochondria is an important factor leading to liver cell injury and apoptosis, ultimately inducing the formation of liver cancer. In the present study, a mouse model expressing HBx was constructed using hydrodynamic in vivo transfection based on the interaction between HBx and cytochrome c oxidase (COX) subunit III. The specific mechanism of HBx-induced oxidative stress in mouse hepatocytes and the subsequent effect on mitochondrial function and inflammatory injury was assessed. The results demonstrated that HBx reduced the activity of COX and the expression of superoxide dismutase and upregulated the expression of malondialdehyde, NF-κB and phospho-AKT, thus increasing oxidative stress. In addition, HBx induced an increase in interleukin (IL)-6, IL-1ß and IL-18 expression levels, which created an inflammatory microenvironment in the liver, further promoting hepatocyte inflammatory injury. Therefore, it was proposed that HBx may affect hepatocyte mitochondrial respiration by reducing the activity of cytochrome c oxidase, leading to mitochondrial dysfunction and inducing hepatocyte inflammation and injury.

12-Lipoxygenase promotes epithelial-mesenchymal transition via the Wnt/ß-catenin signaling pathway in gastric cancer cells.

BACKGROUND: 12-Lipoxygenase (12-LOX) plays a major role in the progression and metastasis of various types of cancer. In gastric cancer (GC), the expression level of 12-LOX is significantly up-regulated; however, its function, and underlying mechanism of action remain unclear. METHODS: The mRNA and protein expression levels of 12-LOX were assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analyses, respectively, in GC cell lines. 12-LOX expression was stably up-regulated using lentiviral vector in BGC823 and MGC803 cells, and cell-counting kit-8 (CCK8), colony formation, and invasion assays were performed to verify the function of 12-LOX in proliferation and metastasis. In addition, the expression levels of epithelial-mesenchymal transition (EMT) differentiation markers and downstream targets of the Wnt/ß-catenin signaling pathway were examined by Western blotting. A nude mouse model of tumor growth and metastasis was established to investigate the role of 12-LOX in vivo. RESULTS: Our findings demonstrate that 12-LOX mRNA and protein were highly expressed in GC cell lines. 12-LOX overexpression promoted GC cell proliferation, migration, and invasion both in vitro and in vivo. In addition, up-regulation of 12-LOX promoted the EMT in GC cells, as reflected by a decrease in E-cadherin expression and an increase in N-cadherin and Snail expression. 12-LOX overexpression in GC cells also increased the expression of multiple downstream targets of the Wnt/ß-catenin signaling pathway. CONCLUSION: These findings revealed that 12-LOX functions as an oncogene in promoting GC cell proliferation and metastasis in vitro and in vivo. In addition, 12-LOX might regulate the EMT via the Wnt/ß-catenin signaling pathway, indicating a potential role for 12-LOX as a target in GC treatment.

Interleukin­10 promotes primary rat hepatic stellate cell senescence by upregulating the expression levels of p53 and p21.

Liver fibrosis is characterized by the excessive deposition of extracellular matrix (ECM) components, and activated hepatic stellate cells (HSCs) are a primary source of ECM. Several studies have revealed that the induction of HSC senescence may reduce liver fibrosis. The effect of interleukin­10 (IL­10) on the senescence of activated HSCs is not fully understood. Therefore, the present study examined its effects and potential mechanisms in activated primary rat HSCs. Collagenase perfusion and density gradient centrifugation methods were used to isolate rat HSCs. HSCs were identified by autofluorescence, Oil Red O staining and immunocytochemical analysis. Activated HSCs were treated with 0, 10, 20 or 40 ng/ml IL­10 for 24 h. Senescence­associated ß­galactosidase (SA­ß­Gal) staining, flow cytometry analysis and a cell counting kit­8 assay were performed to detect the senescence, apoptosis and viability of rat HSCs, respectively. Reverse transcription­quantitative polymerase chain reaction, western blot analysis and enzyme linked immunosorbent assays were used to detect the expression of senescence­associated proteins and cytokines. Freshly isolated rat HSCs exhibited a striking blue­green autofluorescence and HSC retinoid droplets were stained bright red by Oil Red O. Immunocytochemical analysis demonstrated the cytoplasmic expression of HSC markers desmin and α­smooth muscle actin. The number of SA­ß­Gal positive HSCs, the apoptotic rate and the expression levels of p53, p21 and tumor necrosis factor­α were significantly increased following IL­10 treatment. HSC viability and IL­6 and IL­8 expression levels were significantly decreased compared with the control group. In summary, primary rat HSCs were successfully isolated and IL­10 was demonstrated to promote the senescence of activated primary rat HSCs through the upregulation of p53 and p21 expression.

Antifibrotic effects of interleukin-10 on experimental hepatic fibrosis.

BACKGROUND/AIMS: To study the effects of interleukin-10 on hepatic stellate cells and liver tissue in experimental rats hepatic fibrosis. METHODOLOGY: Rat hepatic fibrosis model induced by carbon tetrachloride was established. Liver tissues were harvested from the rats administered CCl4 with or without IL-10 treatment and the animals of the control group. The expression of TGF-beta1, MMP-2 and TIMP-1 in the liver tissues was measured by S-P immunohistochemistry. In addition, another model was established; HSCs in rats in each group were isolated. RT-PCR was employed to analyze TGF-beta1, MMP-2 and TIMP-1 mRNA expression in cells and immunocytochemistry was performed to detect protein expression of alpha-SMA, NF-kappaB, TGF-beta1, MMP-2 and TIMP-1 in HSCs. RESULTS: Rat hepatic fibrosis was developed successfully. The fibrosis changes were partially reversed by simultaneous administration of IL-10. The positive signals of TGF-beta1, MMP-2 and TIMP-1 were observed more frequently (P<0.05) in the CCl4-treated group compared to those in the IL-10-treated group and the control group. HSCs were successfully isolated. TGF-beta1, MMP-2 and TIMP-1 mRNA in HSCs increased obviously during the course of hepatic fibrosis, and their levels were decreased after the treatment with IL-10 (P<0.05). The immunocytochemistry positive levels for TGF-beta1, MMP-2, TIMP-1, alpha-SMA and NF-kappaB in the fibrogenesis group were increased significantly compared to the normal group (P<0.01). The positive signals decreased significantly (P<0.05) after the treatment with IL-10. CONCLUSIONS: The expression of TGF-beta1, MMP-2 and TIMP-1 increased in liver or in HSC of hepatic fibrosis rats and decreased after treatment with IL-10. The IL-10 could inhibit the activation of HSCs and make an antifibrogenic process come into effect in this way.

Hepatitis B virus X protein sensitizes HL-7702 cells to oxidative stress-induced apoptosis through modulation of the mitochondrial permeability transition pore.

Chronic hepatitis B virus (HBV) infection is a leading cause of liver cirrhosis and cancer. Among the pathogenic factors of HBV, HBV X protein (HBx) is attracting increased attention. Although it is documented that HBx is a multifunctional regulator that modulates cell inflammation and apoptosis, the exact mechanism remains controversial. In the present study, we explored the effect of HBx on oxidative stress-induced apoptosis in normal liver cell line, HL-7702. Our results showed that the existence of HBx affected mitochon-drial biogenesis by modulating the opening of the mitochondrial permeability transition pore (MPTP). Notably, this phenomenon was associated with a pronounced translocation of Bax from the cytosol to the mitochon-dria during the period of exposure to oxidative stress with a release of cytochrome c and activation of cleaved caspase-3 and PARP. Moreover, MPTP blockage with cyclosporin A prevented the translocation of Bax, and inhibited oxidative stress-induced apoptotic killing in the HBx-expressing HL-7702 cells. Our findings suggest that HBx exhibits pro-apoptotic effects upon normal liver cells following exposure to oxidative stress by modulating the MPTP gateway.

Therapeutic effect of interleukin-10 on CCl4-induced hepatic fibrosis in rats.

AIM: To study the therapeutic effect of exogenous interleukin-10 on CCl4-induced hepatic fibrosis in rats and its possible mechanisms. METHODS: Fourty-seven SD rats were randomly divided into control group (group N) and CCl4-induced hepatic fibrosis model group (group C). After CCl4 was given for 9 wk, the model group was divided into three groups. Rats in group M were put to death immediately,rats in group T were treated with IL-10 for another three wk and then put to death, rats in group R recovered after three weeks and were then killed. The degree of hepatic fibrosis was measured by HE staining and histological activity index (HAI). Histological activity index (HAI), change of collagen types I and III were measured by Picrosirius staining. The expression of TNF-alpha, MMP-2 and TIMP-1 in liver tissue was measured by S-P immunohistochemistry. RESULTS: CCl4- induced experimental rat hepatic fibrosis model was established successfully. The degree of hepatic fibrosis was markedly lower in group T than in groups M and R, and there was no difference between the two groups. The expression of collagen types I and III was significantly suppressed in group T and was slightly suppressed in groups M and R. The positive levels of TNF-alpha, MMP-2 and TIMP-1 in group M increased significantly compared to those in group N (P<0.01). The positive signals decreased significantly in groups T and R (P<0.01),but positive score was significantly lower in group T than in group R (P<0.01). CONCLUSION: Exogenous IL-10 can reverse CCl4-induced hepatic fibrosis in rats. IL-10 may exert its reversible effects on hepatic fibrosis by blocking CCl4-induced inflammation,inhibiting expression of MMP-2 and TIMP-1 and promoting resolution of collagen types I and III.

Relationship between transforming growth factor beta1 and anti-fibrotic effect of interleukin-10.

AIM: To study the effect of interleukin-10 (IL-10) on the expression of transforming growth factor beta1 (TGF-beta1) in hepatic fibrosis rats and the anti-fibrotic role of exogenous IL-10. METHODS: Hepatic fibrosis was induced by carbon tetrachloride administered (CCl(4)) intraperitoneally. The experiment was performed in two stages. In the first stage, 60 SD rats were divided randomly into normal control group 1 (GN(1), n=8), hepatic fibrosis group (GC, n=28)and IL-10 intervened group (GI, n=24). At the beginning of the 7(th) and 11(th) wk, hepatic stellate cells (HSCs) were isolated, reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were performed to detect the expression of TGF-beta1 in HSCs. Histological examination was used to determine the degree of hepatic fibrosis. In the second stage, 47 SD rats were divided randomly into normal control group 2 (GN(2), n=6)and CCl(4) group(GZ, n=41). At the end of the 9(th) wk, rats in GZ group were allocated randomly into model group(GM, n=9), IL-10 treatment group (GT, n=9)and recovered group (GR, n=9). At the end of the 12(th) wk, all rats were sacrificed. RT-PCR and immunohistochemistry were performed to detect the expression of TGF-beta1 in liver tissue. ELISA was used to assay serum TGF-beta1 levels. RESULTS: Hepatic fibrosis developed in rats with the increase of the injection frequency of CCl(4). In the first stage, hepatic fibrosis developed and HSCs were isolated successfully. At the 7(th) and 11(th) wk, TGF-beta1 mRNA in GC group increased significantly compared with that in GN(1) (P=0.001/0.042) and GI groups (P=0.001/0.007), whereas there was no significant difference between the two groups. The levels of TGF-beta1 at the beginning of the 7(th) wk was higher than that of the 11(th) wk (P=0.049). Immunocytochemistry results of TGF-beta1 were consistent with the above findings. In the second stage, TGF-beta1 increased significantly in GM group compared to GN(2). After treatment with IL-10, TGF-beta1 declined obviously. The expression of TGF-beta1 decreased in GR group but was still higher than that in GT group. CONCLUSION: The levels of TGF-beta1 are increased in hepatic fibrosis rats and decreased after treatment with exogenous IL-10. IL-10 may play an anti-fibrotic role by suppressing TGF-beta1 expression.

Expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic stellate cells during rat hepatic fibrosis and its intervention by IL-10.

AIM: To investigate the expression of matrix metallopr-oteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic fibrosis and the antifibrogenic role of exogenous interleukin-10 (IL-10). METHODS: Hepatic fibrosis was induced by CCl(4) administration and 60 male Sprague-Dawley rats were randomly divided into normal control group (group N, 8 rats), CCl(4)-induced group (group C, 28 rats) and IL-10-treated group (group I, 24 rats). At the beginning of the 7(th) and 11(th) wk, rats in each group were routinely perfused with pronase E and type IV collagenase through portal vein catheter and the suspension was centrifuged by 11% Nycodenz density gradient to isolate hepatic stellate cells (HSCs). RT-PCR was used to analyze mRNA of MMP-2 and TIMP-1 from freshly isolated cells. Densitometric data were standardized with beta-actin signals. Immunocytochemistry was performed to detect MMP-2 and TIMP-1 expression in HSC cultured for 72 h. RESULTS: Compared to group N in the 7(th) wk, MMP-2 and TIMP-1 mRNA increased in group C (P = 0.001/0.001) and group I (P = 0.001/0.009). The level of MMP-2 and TIMP-1 mRNA in group I was significantly lower than that in group C (P = 0.001/0.001). In the 11(th) wk, MMP-2 mRNA in group I was still lower than that in group C (P = 0.005), but both dropped compared with that in the 7(th) week (P = 0.001/0.004). TIMP-1 mRNA in group I was still lower than that in group C (P = 0.001), and increased in group C (P = 0.001) while decreased in group I (P = 0.042) compared with that in the 7(th) wk. Same results were found by immunocytochemistry. CONCLUSION: Expression of MMP-2 and TIMP-1 is increased in hepatic fibrosis. IL-10 exhibits an antifibrogenic effect by suppressing MMP-2 and TIMP-1 expression.

HBx co-localizes with COXIII in HL-7702 cells to upregulate mitochondrial function and ROS generation.

Hepatocellular carcinoma (HCC) is one of the most common malignant diseases, and HBx leads to the development of HBV-associated HCC. Mitochondria are key organelles that regulate apoptosis, cellular energetics and signal transduction pathways, and are the source of HBx-induced reactive oxygen species (ROS). Recent findings have shown that HBx interacts with the inner mitochondrial membrane protein, COXIII, via the yeast two-hybrid system, mating experiment and coimmunoprecipitation. The aim of the present study was to examine the co-localizaiton of HBx and COXIII in HL-7702 cells and to investigate ensuing alterations of mitochondrial function. An HL-7702 cell line stably expressing the HBx gene by lentivirus vectors was constructed. Confocal microscopy was utilized to assess the interaction between HBx protein and COXIII. Expression of COXIII, activities of cytochrome c oxidase (COX) and the mitochondrial membrane potential, which were functionally relevant to the HBx protein-COXIII interaction, were investigated in cell cultures. Moreover, the intracellular ROS levels were detected by flow cytometry. The results demonstrated that HBx co-localized with the inner mitochondrial protein, COXIII, in HL-7702 cells, causing the upregulation of COXIII protein expression as well as COX activity. However, HBx did not alter the mitochondrial membrane potential and mitochondria exhibited only slight swelling in HL-7702-HBx cells. Moreover, HBx elevated the generation of mitochondrial ROS in HL-7702-HBx cells. The main finding of the present study was that the co-localization of HBx and COXIII leads to upregulation of the mitochondrial function and ROS generation, which are associated with the oncogenesis of HBV-associated HCC.

Effects of transmitters and interleukin-10 on rat hepatic fibrosis induced by CCl4.

AIM: To study the effects of transmitters ET, AgII, PGI(2), CGRP and GG on experimental rat hepatic fibrosis and the antifibrogenic effects of IL-10. METHODS: One hundred SD rats were randomly divided into 3 groups: control group (N): intraperitoneal injection with saline 2 ml.kg(-1) twice a week; the fibrogenesis group (C): intraperitoneal injection with 50 % CCl(4) 2 ml.kg(-1) twice a week; IL-10 treated group (E): besides same dosage of CCl(4) given, intraperitoneal injection with IL-10 4 ug.kg(-1) from the third week. In the fifth, the seventh and the ninth week, rats in three groups were selected randomly to collect plasma and liver tissues. The levels of ET, AgII, PGI(2), CGRP and GG were assayed by radioimmunoassay (RIA). The liver fibrosis was observed with silver staining. RESULTS: The hepatic fibrosis was developed with the increase of the injection frequency of CCl(4). The ET, AgII, PGI(2), CGRP and GG levels in serum of group N were 71.84+/-60.2 ng.L(-1), 76.21+/-33.3 ng/L, 313.03+/-101.71 ng/L, 61.97+/-21.4 ng/L and 33.62+/-14.37 ng/L, respectively; the levels of them in serum of group C were 523.30+/-129.3 ng/L, 127.24+/-50.0 ng/L, 648.91+/-357.29 ng/L, 127.15+/-62.0 ng/L and 85.26+/-51.83 ng/L, respectively; the levels of them in serum of group E were 452.52+/-99.5 ng/L, 90.60+/-44.7 ng/L, 475.57+/-179.70 ng/L, 102.2+/-29.7 ng/L and 38.05+/-19.94 ng/L, respectively. The histological examination showed that the degrees of the rats liver fibrosis in group E were lower than those in group C. CONCLUSION: The transmitters ET, AgII, PGI(2), CGRP and GG play a significant role in the rat hepatic fibrosis induced by CCl(4). IL-10 has the antagonistic action on these transmitters and can relieve the degree of the liver fibrosis.

Expression of insulin-like growth factor 1 and insulin-like growth factor 1 receptor and its intervention by interleukin-10 in experimental hepatic fibrosis.

AIM: To study the expression of IGF-1 and IGF-1R and its intervention by interleukin-10 in the course of experimental hepatic fibrosis. METHODS: Hepatic fibrosis was induced in rats by carbon tetrachloride intoxication and liver specimens were taken from the rats administered CCl4 with or without IL-10 treatment and the animals of the control group. Immunoreactivities for insulin-like growth factor-1 (IGF-1) and IGF-1 receptor(IGF-1R) were demonstrated by immunohistochemistry, and their intensities were evaluated in different animal groups. RESULTS: The positive levels for IGF-1 and IGF-1R were increased with the development of hepatic fibrosis, with the positive signals localized in cytoplasm and/or at the plasmic membrane of hepatocytes. The positive signals of IGF-1 and IGF-1R were observed more frequently (P<0.01) in the CCl4-treated group (92.0 % and 90.0 %) compared to those in the control group. The positive signals decreased significantly (P<0.05) in IL-10-treated group. The responses in IGF-1 and IGF-1R expression correlated with the time of IL-10 treatment. CONCLUSION: The expression of IGF-1 and IGF-1R immunoreactivities in liver tissue seems to be up-regulated during development of hepatic fibrosis induced by CCl(4), and exogenic IL-10 inhibits the responses.

Effects of cytokines on carbon tetrachloride-induced hepatic fibrogenesis in rats.

AIM: To observe the possible effects of transforming growth factor (TGF) beta(1), interleukin (IL)-6, tumor-necrosis factor (TNF) alpha and IL-10 on experimental rat hepatic fibrosis. METHODS: One hundred SD rats were divided randomly into the three groups. Control group received intraperitoneal injection of saline (2 ml/kg(-1)), twice a week. Fibrogenesis group was injected intraperitoneally with 50% carbon tetrachloride (CCl(4)) (2 ml/kg(-1)) twice a week. Fibrosis-intervention group was given IL-10 at a dose of 4 microg/kg(-1) 20 minutes before CCl(4) administration from the third week. At the fifth, seventh, and ninth weeks, 7 to 10 rats in each group were sacrificed to collect serum. Levels of TGF-beta(1), TNF-alpha, IL-6 and IL-10 were determined by enzyme-linked immunosorbent assay (ELISA). The liver tissues were taken for routine histological examination. RESULTS: Hepatic fibrosis was developed with the injection of CCl(4). Values of the circulating TGFbeta(1), TNFalpha, IL-6 and IL-10 in the control group were 25.49+/-5.56 ng/L(-1), 15.18+/-3.83 ng/L(-1), 63.64+/-13.03 ng/L(-1) and 132.90+/-12.13 ng/L(-1), respectively. Their levels in the CCl(4)-intoxication group were 31.13+/-6.41 ng/L(-1), 18.91+/-5.31 ng/L(-1), 89.08+/-25.39 ng/L(-1) and 57.63+/-18.88 ng/L(-1), respectively, and those in the IL-10-intervention group were 26.11+/-5.32 ng/L(-1), 13.99+/-1.86 ng/L(-1), 74.71+/-21.15 ng/L(-1) and 88.19+/-20.81 ng/L(-1), respectively. A gradual increase was observed in the levels of TGFbeta(1), TNFalpha and IL-6 during hepatic fibrogenesis. These changes were partially reversed by simultaneous administration of IL-10. The histological parameters, characterized by CCl(4)-intoxification, also seemed to be improved with IL-10 treatment, the collagen production was reduced at the ninth week and the histological activity index was decreased from 7.9+/-1.2 to 4.7+/-0.9. CONCLUSION: TGFbeta(1), TNFalpha and IL-6 may play important roles during CCl(4)-induced hepatic fibrogenesis, and IL-10 may counterbalance their effects.

Cytochrome C oxidase III interacts with hepatitis B virus X protein in vivo by yeast two-hybrid system.

AIM: To screen and identify the proteins which interact with hepatitis B virus (HBV) X protein in hepatocytes by yeast two-hybrid system and to explore the effects of X protein in the development of hepatocellular carcinoma (HCC). METHODS: With HBV X gene amplified by polymerase chain reaction (PCR), HBV X bait plasmid, named pAS2-1-X, was constructed by yeast-two hybridization system3 and verified by auto-sequencing assay. pAS2-1-X was transformed into the yeast AH109, and X-BD fusion protein expressed in the yeast cells was detected by Western blotting. The yeast cells cotransformed with pAS2-1-X and normal human liver cDNA library were grown in selective SC/-trp-leu-his-ade medium. The second screen was performed with beta-gal activity detection, and false positive clones were eliminated by segregation analysis, true positive clones were amplified, sequenced and analyzed with bioinformatics. Mating experiment was performed to confirm the binding of putative proteins to X protein in the yeast cells. RESULTS: Bait plasmid pAS2-1-X was successfully constructed and pAS2-1-X correctly expressed BD-X fusion protein in yeast AH109. One hundred and three clones grew in the selective SC/-trp-leu-his-ade medium, and only one clone passed through beta-gal activity detection and segregation analysis. The inserted cDNA fragment showed high homology with Homo sapiens cytochrome C oxidase III (COXIII). Furthermore, mating experiment identified that the binding of COXIII to X protein was specific. CONCLUSION: COXIII protein is a novel protein that can interact with X protein in vivo by yeast two-hybrid system, and may contribute to the development of HCC through the interaction with X protein.

Effect of interleukin-10 and platelet-derived growth factor on expressions of matrix metalloproteinases-2 and tissue inhibitor of metalloproteinases-1 in rat fibrotic liver and cultured hepatic stellate cells.

AIM: To examine the expressions of matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in rat fibrotic liver and in normal rat hepatic stellate cells, and to investigate the changes in their expressions in response to treatment with interleukin-10 (IL-10) and platelet-derived growth factor (PDGF). METHODS: Rat models of CCl4-induced hepatic fibrosis were established and the liver tissues were sampled from the rats with or without IL-10 treatment, and also from the control rats. The expressions of MMP-2 and TIMP-1 in liver tissues were detected by S-P immunohistochemistry, and their expression intensities were evaluated in different groups. Hepatic stellate cells (HSCs) were isolated from normal rat and cultured in vitro prior to exposure to PDGF treatment or co-treatment with IL-10 and PDGF. MMP-2 and TIMP-1 levels were measured by semi-quantitative reverse transcriptional polymerase chain reaction (RT-PCR). RESULTS: CCl4- induced rat hepatic fibrosis models were successfully established. The positive expressions of MMP-2 and TIMP-1 increased obviously with the development of hepatic fibrosis, especially in untreated model group (84.0% and 92.0%, P<0.01). The positive signals decreased significantly following IL-10 treatment (39.3% and 71.4%, P<0.01 and P<0.05) in a time-dependent manner. TIMP-1 mRNA in PDGF-treated group was significantly increased time-dependently in comparison with that of the control group, but PDGF did not obviously affect MMP-2 expression. No difference was noted in TIMP-1 and MMP-2 expressions in HSCs after IL-10 and PDGF treatment (P>0.05). CONCLUSION: MMP-2 and TIMP-1 expressions increase in liver tissues with the development of fibrosis, which can be inhibited by exogenous IL-10 inhibitor. PDGF induces the up-regulation of TIMP-1 but not MMP-2 in the HSCs. IL-10 inhibits TIMP-1 and MMP-2 expressions in HSCs induced by PDGF.

Effects of platelet-derived growth factor and interleukin-10 on Fas/Fas-ligand and Bcl-2/Bax mRNA expression in rat hepatic stellate cells in vitro.

AIM: To investigate the effects of platelet-derived growth factor(PDGF) and interleukin-10 (IL-10) on Fas/Fas-ligand and Bcl-2/Bax mRNA expressions in rat hepatic stellate cells. METHODS: Rat hepatic stellate cells (HSCs) were isolated and purified from rat liver by in situ digestion of collagenase and pronase and single-step density Nycodenz gradient. After activated by culture in vitro, HSCs were divided into 4 groups and treated with nothing (group N), PDGF (group P), IL-10 (group I) and PDGF in combination with IL-10 (group C), respectively. Semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was employed to compare the mRNA expression levels of Fas/FasL and Bcl-2/Bax in HSCs of each group. RESULTS: The expression levels of Fas between the 4 groups had no significant differences (P>0.05). FasL mRNA level in normal culture-activated HSCs (group N) was very low. It increased obviously after HSCs were treated with IL-10 (group I) (0.091+/-0.007 vs 0.385+/-0.051, P<0.01), but remained the low level after treated with PDGF alone (group P) or PDGF in combination with IL-10 (group C). Contrast to the control group, after treated with PDGF and IL-10, either alone or in combination, Bcl-2 mRNA expression was down-regulated and Bax mRNA expression was up-regulated, both following the turn from group P, group I to group C. Expression of Bcl-2 mRNA in group C was significantly lower than that in group P (0.126+/-0.008 vs 0.210+/-0.024, P<0.01). But no significant difference was found between group C and group I, as well as between group I and group P (P>0.05). Similarly, the expression of Bax in group C was higher than that in group P (0.513+/-0.016 vs 0.400+/-0.022, P<0.01). No significant difference was found between group I and group P (P>0.05). But compared with group C, Bax expressions in group I tended to decrease (0.449+/-0.028 vs 0.513+/-0.016, P<0.05). CONCLUSION: PDGF may promote proliferation of HSCs but is neutral with respect to HSC apoptosis. IL-10 may promote the apoptosis of HSCs by up-regulating the expressions of FasL and Bax and down-regulating the expression of Bcl-2, which may be involved in its antifibrosis mechanism.

[Screening the hepatitis B virus PreS1 associated protein by the yeast two-hybrid system].

OBJECTIVE: To screen the hepatitis B virus PreS1 associated protein from normal human liver cDNA library by the yeast two-hybrid system and explore the role of PreS1 protein in the infection of hepatitis B virus (HBV). METHODS: PCR was preformed to amplify the PreS1 gene containing EcoRI and PstI from HBV positive serum, and the production was inserted into plasmid pAS2-1 after digesting with the former two restricted endonuclease, then the bait vector pAS2-1-PreS1 was verified by auto-sequencing assay. The PreS1-BD fusion protein expressed in the yeast cells was confirmed by western blot, after pAS2-1-PreS1 was transfected into the yeast cell AH109. Yeast cells co-transfected with pAS2-1-PreS1 and the normal human liver cDNA library grew in selective SC/-trp-leu-his-ade2 medium, and the second screening was performed with LacZ report gene. Furthermore, segregation analysis and mating experiment were done to eliminate the false positive, then the true positive clones were submitted for PCR and sequencing. The results were submitted to the BLAST notebook of World Wide Wed Site NCBI to seek homologous sequence. RESULTS: Bait vector pAS2-1-PreS1 included the anticipated fragment of PreS1 gene. Western blot showed that pAS2-1-PreS1 could correctly express PreS1-BD fusion protein in the yeast cells. After yeast cells co-transfected with pAS2-1-PreS1 and the normal human liver cDNA library, 97 colonies grew in the selective SC/-trp-leu-his-ade2 medium, only one clone was positive and showed high homology with Homo sapiens nascent-polypeptide-associated complex alpha polypeptide. CONCLUSION: Bait vector pAS2-1-PreS1 is successfully constructed, and nascent-polypeptide-associated complex alpha polypeptide protein expressed in hepatocyte can interact with PreS1 by the yeast two-hybrid system.

Hydrodynamics-based transfection of rat interleukin-10 gene attenuates porcine serum-induced liver fibrosis in rats by inhibiting the activation of hepatic stellate cells.

Liver fibrosis is the common pathological outcome for the majority of chronic liver diseases. Interleukin-10 (IL-10) is a cytokine that downregulates proinflammatory responses and has a modulatory effect on liver fibrogenesis. However, little is known regarding the effect of rat interleukin­10 (rIL­10) gene by hydrodynamics-based transfection (HBT) on liver fibrosis in rats. The aim of this study was to investigate the effect of the rIL-10 gene by HBT on the progression of liver fibrosis induced by porcine serum (PS) in rats and explore its possible mechanism. Plasmid­expressing rIL-10 was transferred into rats by HBT and immunohistochemistry and RT-PCR were used to detect the major organ expressing rIL-10. Liver fibrosis was induced in rats by intraperitoneal administration of PS for 8 weeks. Plasmid pcDNA3-rIL-10 solution was administered weekly by HBT starting at the 5th week. Liver function and hepatic histology were examined. The possible molecular mechanisms of rIL-10 gene therapy were assessed in liver tissue and hepatic stellate cells (HSCs) co-cultured with BRL cells (a hepatocyte line) in vitro. The results showed rIL-10 expression occurred mainly in the liver following rIL-10 gene transfer by HBT. Maintaining a stable expression of rIL-10 in serum was assessed by repeated administration. The rIL-10 gene treatment attenuated liver inflammation and fibrosis in PS-induced fibrotic rats, reduced the deposition of collagen and the expression of α-smooth muscle actin (α-SMA) in fibrotic rats. The in vitro experiment showed that the expression of a-SMA and procollagen type I in HSCs co-cultured with the BRL­transfected rIL-10 gene were significantly decreased. These findings indicate that rIL-10 gene therapy by HBT attenuates PS-induced liver fibrosis in rats and that its mechanism is associated with rIL-10 inhibiting the activation of HSCs and promoting the degeneration of collagen.