Effects of Ganfukang on expression of connective tissue growth factor and focal adhesion kinase/protein kinase B signal pathway in hepatic fibrosis rats.

OBJECTIVE: To investigate the effect of Ganfukang (GFK) on connective tissue growth factor (CTGF) and focal adhesion kinase (FAK)/protein kinase B (PKB or Akt) signal pathway in a hepatic fibrosis rat model and to explore the underlying therapeutic molecular mechanisms of GFK. METHODS: Fifty SD rats were randomly divided into five groups as follows: the control group, the model group (repeated subcutaneous injection of CCl4), and the three GFK treatment groups (31.25, 312.5, and 3125 mg/kg, intragastric administration). Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry were used to examine the expression of CTGF, integrin α5, integrin ß1, FAK/Akt signal pathway, cyclinD1, and collagen in the different-treated rats. RESULTS: GFK attenuated the up-regulation of CTGF, integrin α5, and integrin ß1 in hepatic fibrosis rats and suppressed both the phosphorylation of FAK and the phosphorylation of Akt simultaneously (P<0.01). At the same time, the expression of cyclinD1, collagen I, and collagen III was decreased by GFK significantly (P<0.01). CONCLUSIONS: CTGF and FAK/Akt signal pathway were activated in the CCl4-induced hepatic fibrosis rats, which contribute to increased expression of cyclinD1 and collagen genes. The mechanisms of the anti-fibrosis activity of GFK may be due to its effects against CTGF and FAk/Akt signal pathway.

Herb medicine Gan-fu-kang attenuates liver injury in a rat fibrotic model.

AIM OF THE STUDY: To verify therapeutic effects of Gan-fu-kang (GFK), a traditional Chinese medicine compound, in a rat model and to investigate the underlying mechanisms. MATERIALS AND METHODS: Liver fibrosis was established by 12 weeks of carbon tetrachloride (CCl(4)) treatment (0.5mg/kg, twice per week) followed by 8 weeks of "recovery" in rats. Rats randomly received GFK (31.25, 312.5 and 3125 mg/kg/day, p.o.) or vehicle from weeks 9 to 20, and were sacrificed at the end of week 20 for histological, biochemical, and molecular biological examinations. In a separate set of experiments, rats received 12 weeks of CCl(4) treatment, concomitant with GFK (312.5mg/kg/day, p.o.) during the same period in some subjects, but were then sacrificed immediately. An additional group of rats receiving no CCl(4) treatment served as normal controls. RESULTS AND CONCLUSIONS: (1) CCl(4) treatment resulted in severe liver damage and fibrosis. (2) In the main block of the 20-week study, GFK attenuated liver damage and fibrosis. (3) In the 12-week study, GFK produced prevention effect against hepatic injury. (4) GFK suppressed the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), type I collagen, platelet-derived growth factor-BB (PDGF-BB)/PDGF receptor-beta chains (PDGFRbeta) and mitogen-activated protein kinases (MAPKs)/active protein-1 (AP-1) signal pathways. Taken together, these results indicated that GFK could attenuate liver injuries in both settings. Our findings also suggest that the AP-1 pathway is the likely molecular substrate for the observed GFK effects.

[Changes and significance of peripheral blood platelet count in tumor shrinkage induced by a low dose of CTX in T739 mice].

AIM: To establish a mouse model for BTT739 tumor-bearing mice cured by a low dose of cyclophosphamide (CTX). And then to observe the dynamic changes and significance of peripheral blood counts especially blood platelet count during tumor shrinkage induced by a low dose of CTX in T739 mice. METHODS: Mouse bladder carcinoma tissues were inoculated subcutaneously into T739 mice. Seven days later, different doses of CTX or the same volume of NS were administered intraperitoneally to treat these tumor-bearing T739 mice. Tumor sizes were observed and recorded subsequently to find out the minimal dose of CTX that could cure most of these tumor-bearing mice. Then another 12 tumor-bearing mice were randomly divided into 15 mg/kg CTX treatment group and control group. Blood samples were obtained from orbital venous sinus on different times after CTX treatment. Complete blood counts were performed and the relationship between peripheral blood platelet counts and tumor shrinkage was analyzed. RESULTS: Within 2 weeks after CTX treatment, the speed of tumor shrinkage had a positive relationship with the dose of CTX used; but the survival rate of the tumor-bearing mice had a negative relationship with the dose of CTX used in 2 months after CTX treatment. 15 mg/kg CTX could cure most of the tumor bearing mice, while it had no remarkably inhibitive effects on peripheral blood cells. The perpherial platelet count increased to (1483.4+/-184.4)x10(9)/L in mice 6 h after CTX treatment. There was significant difference compared with that in mice of control group (1086.6+/-81.0)x10(9)/L (P<0.01). During the 2nd to 14th day after CTX treatment, there was no obvious difference in the platelet count between treatment group and control group (P>0.05). CONCLUSION: CTX 15 mg/kg could cure most of bladder tumor-bearing T739 mice. The transient increase of the peripheral platelet count in 6 h after CTX treatment may relate to the antitumor effects of CTX.

Effects of traditional chinese medicine on endotoxin and its receptors in rats with non-alcoholic steatohepatitis.

AIMS: The aim of this research is to study the effects of traditional Chinese medicine on endotoxin and its receptors in rats with nonalcoholic steatohepatitis (NASH). METHODS: Fifty-six SD rats were divided into seven groups. All the animals were fed high fatty diet for 12 weeks. Rats with non-alcoholic steatohepatitis (NASH) were treated with traditional Chinese medicine according to low-dose, middle-dose, high-dose and Lipitor from fifth week. All rats were killed at the end of 12th week. The liver pathology changes were observed under light microscope. The levels of serum lipoid, alanine aminotransferase (ALT), endotoxin (ET), tumor necrosis factor-alpha (TNF-alpha) and interleukine-1beta (IL-1beta) were determined. The expressions of CD14 and nuclear transcriptional factor kappaB (NF-kappaB) were observed by immunohistochemistry. The expressions of lipopolysaccharide binding protein (LBP), toll-like receptor-4 (TLR-4), myeloid differentiation-2 (MD-2) and induced nitric oxide synthase (iNOS) mRNA were detected by the reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The levels of serum endotoxin in the middle dose group (0.0225 +/- 0.0112 EU/l) were lower than those in high fatty diet model group (0.2249 +/- 0.0982 EU/l) at 12th week, the difference was significant (P < 0.01). In the middle dose group, mean values of serum TNF-alpha and IL-1beta levels decreased dramatically (1.604 +/- 0.302 ng/ml and 0.052 +/- 0.024 ng/ml) compared with those in the high fatty diet model group (4.029 +/- 1.180 ng/ml and 14.944 +/- 0.491 ng/ml; P < 0.01 and P < 0.01). The expressions of CD14 and NF-kappaB in the middle dose group decreased compared with those in the high fatty diet model group. The expressions of LBP mRNA (0.284 +/- 0.105) and TLR-4 mRNA (0.290 +/- 0.123) in the middle dose group down regulated compared with those in the high fatty diet model group (1.060 +/- 0.158 and 1.261 +/- 0.368; P < 0.01 and P < 0.01). In the middle dose group MD-2 and iNOS gene expressions were 0.132 +/- 0.058 and 0.164 +/- 0.061, respectively, which were significantly lower compared with the high fatty diet model group (0.795 +/- 0.294 and 1.029 +/- 0.388; P < 0.01 and P < 0.01). CONCLUSIONS: The mechanism of non-alcoholic steatohepatitis (NASH) maybe related to increasing the levels of serum endotoxin, upregulating endotoxin receptors of hepatic tissue and enhancing liver inflammatory injury. Traditional Chinese medicine is a good treatment for non-alcoholic steatohepatitis (NASH). It can produce a marked effect via relieving LPS-induced liver injury.

[Relationship between TNFalpha and tumor rejection induced by a single dose of melphalan in C57BL/6 mice].

AIM: To investigate the relationship between TNFalpha and tumor rejection induced by a single dose of melphalan in C57BL/6 mice. METHODS: Different gene type mice (TNFR1(+/+), TNFR1(+/-) and TNFR1(-/-)) with the same genetic background of C57BL/6 were used in this experiment. Murine lymphoma EL4 cells were inoculated subcutaneously into the different gene type mice simultaneously. Twelve days later, 7.5 mg/kg melphalan was used intraperitoneally to treat the tumor-bearing mice with TNFR1(+/+), TNFR1(+/-) and TNFR1(-/-). The tumors in the different gene type mice were observed and recorded every one to three day. RESULTS: After the treatment of 7.5 mg/kg melphalan during the first week, the tumors in the different gene type mice shrank at a similar rate. In the following 2 months, the tumors in the TNFR1(+/+) and TNFR1(+/-) C57BL/6 mice gradually shrank and were cured but most tumors in the TNFR1(-/-) C57BL/6 mice relapsed after melphalan treatment. CONCLUSION: TNFalpha plays an important role in melphalan-induced tumor rejection. The anti-tumor effect of melphalan has no relationship with the expression of tumor necrosis factor 1 in tumor-bearing mice. TNFR1 is required to prevent or avoid the relapse of tumors in mice instead of tumor cells.

Effect of Chinese traditional compound, Gan-fu-kang, on CCl(4)-induced liver fibrosis in rats and its probable molecular mechanisms.

AIM: To explore the antifibrotic effect of traditional Chinese medicine compound Gan-fu-kang (GFK) on CCl(4)-induced liver fibrosis in rats and its probable mechanisms. METHODS: The effects of GFK on CCl(4)-induced liver fibrosis were tested in rats. The liver histopathology was examined by light microscope, polaring microscope and electron microscope. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assayed and the content of albumin (ALB) and hydroxyproline in the liver was measured. The expression of transforming growth factor-beta(1) (TGF-beta(1)) and laminin (LN) was determined by immunohistochemistry. Semi-quantitive computation of collagen types I and III and laminin was done. The expression of MMP-2 and TIMP-1 was assayed by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Upon pathological examination, GFK treatment had significantly reversed liver fibrosis. Hepatic extracellular matrix (ECM) deposition was significantly reduced, as evidenced by the reduction of the content of hydroxyproline, collagen types I and III, and laminin. Hepatic function was improved by GFK treatment, as evidenced by the increase of plasma ALB and A/G, and by the decrease of serum ALT and AST. TGF-beta(1) in liver was significantly reduced. A significant expression of MMP-2 and TIMP-1 mRNA in liver were downregulated after GFK treatment. CONCLUSION: The traditional Chinese medicine compound recipe GFK has an antifibrotic effect on CCl(4)-induced liver fibrosis in rats, which improves hepatic function and lessens the deposition of collagen in the liver. The probable antifibrotic mechanisms were: inhibiting the expression of TGF-beta(1) and decreasing expressions of MMP-2 and TIMP-1.

[Effect of cardiotrophin-1 on cardiac transcription factor GATA4 expression in rat cardiomyocytes].

OBJECTIVE: To investigate the effect of cardiotrophin-1 (CT-1) on the GATA4 expression and related signaling pathways (JAK-STAT3, ERK1/2 and PI3-K) in rat cardiomyocytes. METHODS: Using semi-quantitative RT-PCR and EMSA, we measured the dose and time dependent effects of CT-1 on GATA4 mRNA and binding activity in cultured rat cardiomyocytes. Parthenolide (a STAT inhibitor), U-0126 (an ERK inhibitor) and LY-294002 (a PI3-K inhibitor) alone or in combination were added to the culture medium to assess the role of above signaling pathways in CT-1 mediated effects. RESULTS: GATA4 mRNA expression significantly increased at 3 h post 0.1 nmol/L CT-1 exposure, peaked at 6 h and remained high till 24 h post exposure. The GATA4 binding activity began to increase at 10 min and peaked at 60 min and returned to baseline level 180 min. Six hours post CT-1 (0.01 nmol/L, 0.1 nmol/L, 1 nmol/L) exposure, the GATA4 mRNA expression increased in a dose-dependent manner. The GATA4 binding activity peaked with 0.1 nmol/L CT-1 and higher dose did not further increase the binding activity. U-0126 increased the GATA4 mRNA expression and enhanced the GATA4 binding activity and these effects could be partially attenuated with addition of Parthenolide. Parthenolide also prevented the increase of GATA4 mRNA and binding activity induced by CT-1. LY-294002 had no effects GATA4 mRNA and binding activity. CONCLUSION: CT-1 increases the GATA4 mRNA expression and binding activity in rat cardiomyocytes via STAT3/ERK1/2 pathways and these effects are independent of PI3-K pathway.