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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.

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.

Aim: To study the effect of IL-10 on the expression of growth factors--transforming growth factor-beta1 (TGF-beta1), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) of hepatic stellate cells (HSCs) of hepatic fibrosis rat and the anti-fibrogenic role of exogenous IL-10., Methods: Hepatic fibrosis was induced by CCl(4) administration intra-peritoneally. Sixty clean male Sprague-Dawley (SD) rats were randomly divided into three groups: normal control group (GN, 8 rats), hepatic fibrosis model group (GC, 28 rats) and IL-10 treated group (GI, 24 rats). At the beginning of the 7th and 11th wk, rats in each group were routinely perfused with pronase E and type IV collagenase through a portal vein catheter and the suspension obtained from the liver was spun by centrifugation with 11% Nycodenz density gradient to isolate HSCs. Histological examination was used to determine the degree of hepatic fibrosis. RT-PCR was employed to analyze mRNA expression from freshly isolated cells. Immunocytochemistry was performed to detect protein expression in primary cultured HSCs., Results: Rat hepatic fibrosis was developed with the increase of injection frequency of CCl(4), and HSCs were successfully isolated. At the 7th and 11th wk, TGF-beta1, EGF, and HGF mRNA in GC increased obviously compared with GN (P = 0.001/0.042, 0.001/0.001, 0.001/0.001) and GI (P = 0.001/0.007, 0.002/0.001, 0.001/0.001). For TGF-beta1, no difference was observed between GI and GN. For EGF, mRNA level in GI increased compared with GN during the 7th wk (P = 0.005) and 11th wk (P = 0.049). For HGF, mRNA level in GI decreased compared with GN at the 7th wk (P = 0.001) and 11th wk (P = 0.021). Between these two time points, TGF-beta1 expression at the 7th wk was higher than that of the 11th wk (P = 0.049), but for EGF, the former was lower than the latter (P = 0.022). As for PDGF mRNA, there was no significant difference between these groups, but difference seemed to exist in protein levels. Results by immunocytochemistry of TGF-beta1 and EGF were paralleled with the above findings., Conclusion: The expression of TGF-beta1, EGF and HGF increased in HSC of hepatic fibrosis rat and decreased after treatment with IL-10. IL-10 plays an anti-fibrogenic role by suppressing growth factors expression.

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.

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.

Aim: To study the expression and serum level of HBxAg, Fas and FasL in tissues of HCC patients, and to assess the relationship between HBxAg and Fas/FasL system., Methods: Tissues from 50 patients with HCC were tested for the expression of HBxAg, Fas and FasL by S-P immunohistochemistry. Serum levels of sFas/sFasL and HBsAg/HBeAg were measured by ELISA assay. HBV X gene was detected by PCR in serum and confirmed by automatic sequencing. Fifty cases of liver cirrhosis and 30 normal controls were involved in serum analysis., Results: The expression of HBxAg, Fas and FasL in carcinoma tissues was 96 %, 84 % and 98 %, respectively. Staining of HBxAg, Fas and FasL was observed predominately in cytoplasms, no significant difference was found in intensity between HBxAg, Fas and FasL (P>0.05). HBxAg, Fas and FasL might express in the same area of carcinoma tissues and this co-expression could be found in most patients with HCC. The mean levels of sFas in serum from HCC, cirrhosis and normal controls were 762.29 +/- 391.56 microg.L(-1), 835.36 +/- 407.33 microg.L(-1) and 238.27 +/- 135.29 microg.L(-1). The mean levels of sFasL in serum from HCC, cirrhosis and normal controls were 156.36 +/- 9.61 microg.L(-1), 173.63 +/- 18.74 microg.L(-1) and 121.96 +/- 7.83 microg.L(-1). Statistical analysis showed that both sFas and sFasL in HCC and cirrhosis patients were significantly higher than those in normal controls (P<0.01). Serum HBV X gene was found in 32% of HCC patients and 46% of cirrhotic patients. There was no significant relationship between serum level of sFas/sFasL and serum X gene detection (P>0.05). Eight percent of HCC patients with negative HBsAg and HBeAg in serum might have X gene in serum and HBxAg expression in carcinoma tissues., Conclusion: Our data suggest that HBxAg and Fas/FasL system plays an important role in the development of human HCC. Expression of HBxAg can leads to expression of Fas/FasL system which and reverse apoptosis of hepatocellular carcinoma induced by FasL.

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.

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.