Expression And Role Of TGF-β1/smad3 In The Acute Liver Injury In Mice Induced By Carbon Tetrachloride

INTRODUCTION Liver is a multi-functional metabolic organ. Various factors, such as viral infection, drugs, trauma and chemical agents, might contribute to liver injury, which could lead to health disorder, even fatality. Therefore, exploring the mechanism of hepatic injury would exert an important impact on setting up effective therapeutic strategy. TGF-β1, a member of transforming growth factor-beta（TGF-β） family, plays critical roles in the development of liver diseases, including inhibiting proliferation of hepatocytes and promoting production of extracellular matrix（ECM）. Therefore, TGF-β1 has been considered to be a key molecule in chronic liver diseases and hepatic fibrosis. Mammalian homolog of the Drosophila Madgene3（Smad3） is an essential molecule in TGF-β signaling. The abnormal expression of Smad3 can activate hepatic stellate cells（HSC） and promote the production of ECM, which results in chronic liver disease and hepatic fibrosis. Carbon Tetrachloride（CCl₄）, a typical liver toxicant, is to induce classic hepatic injury in mice. Previous studie and our data have shown that TGF-β1/Smad3 was involved in the process of the chronic hepatic diseases. However, the changes of TGF-β1 and Smad3 protein expression as well as their involvement in the process of CCl₄-induced acute liver injury in mice remain unclear. In the present study, we confirmed the roles of TGF-β1 in the CCl₄-induced acute liver injury by establishing the acute liver injury model of mice induced by CCl₄ and detecting the level of TGF-β1 and Smad3 protein expression. In addition, the effect of Samd3 in the acute liver injury in mice induced by CCl₄ was investigated by means of Smad3-overexpression and Smad3 knock-down experiment.1. METHODS 1.1 The Establishment of Model of CCl₄-Induced Acute liver Injury in Mice 36 Balb/c mice were randomly divided into two groups. In experimental group, acute liver injury of mice was induced by intraperitoneal injection of 0.5 ml of CCl₄ in 9.5 ml of olive oil（1:19 v/v）/kg body weight, while in olive oil control group mice were intraperitoneally injected with 10 ml of olive oil/kg body weight. Mice were executed on day 1, 3, 5 after injection. 1.2 The Overexpression of Smad3 in vivo in Mice Balb/c mice were randomly divided into four groups as follows,（1）Olive + PC,（2）C Cl4 + PC,（3）Olive + Smad3,（4）CCl₄ + Smad3. Mice were treated in vivo with Smad3-expressing plasmid pc DNA-Smad3 and control negative plasmid pc DNA3, respectively. Following the instruction of previous literature, the pc DNA-Smad3 plasmids（3μg / mouse） and control pc DNA3 plasmids（3μg / mouse） were enfolded in Lipofectamine 2000, and then administered into mice via tail intravenous injection. 12 h after the injection of plasmids, the mice were intraperitoneally injected with CCl₄ or olive oil, mice were executed on day 1, 3, 5 after treated with CCl₄ or olive oil. 1.3 The Knock-down of Smad3 in vivo in Mice Balb/c male mice were randomly divided into four groups as follows: Olive oil+p GCsi-U6/Ne（NC）, CCl₄+ NC, Olive oil+ p GCsi-U6/Neo-Smad3 sh RNA（Smad3 sh RNA） and CCl₄+ Smad3 sh RNA. Mice were transfected with p GCsi-U6/Neo plasmid（3μg / mouse） or p GCsi-U6/Neo-Smad3 sh RNA（3μg / mouse） enfolded by Lipofectamine 2000 via tail intravenous injection. 12 h after the transfection, the CCl₄-treated or olive oil-treated mice were intraperitoneally injected with CCl₄ or olive oil as mentioned above. The mice were executed on day 1, 3, 5 after treated with CCl₄ or olive oil.2. RESULTS 2.1 Levels of Serum ALT and AST and Pathologic Changes of Livers in CCl₄-treated Mice Change of serum level of transaminases ALT and AST is relationship with liver injury. The results showed that, the serum levels of ALT and AST were significantly elevated on days 1 and 3 after the administration of CCl₄, comparing to those in control mice treated with olive oil. The peak of elevation was on the 1st day and gradually declined. H&E staining was performed to detect pathologic findings in mice livers. The results showed that lobular structure in the control mouse with olive oil treatment was clear and the hepatic cells arranged in neat rows. In contrast, livers treated by CCl₄ for 1 day presented large areas of annular necrotic lesions around the hepatic lobule portal area while 3 days after CCl₄ treatment, more inflammatory cells infiltration were exhibited in the hepatic lobule portal area in spite of the reduced area of nectosis. The injury liver repair and inflammatory cells infiltration could be observed 5 days after CCl₄ treatment. These results indicated that CCl₄ successfully induced acute liver injury in mice. 2.2 Levels of TGF-β1 and Smad3 in Liver Injury of Mice Induced by CCl₄ To observe the change of TGF-β1 expression in liver injury induced by CCl₄, the levels of TGF-β1 in both serum and hepatic tissue homogenate were assayed by ELISA. Furthermore, the TGF-β1 and Smads m RNA expression levels were detected by RT-PCR, Smad3 protein expression levels was detected with Western blotting. The results revealed that TGF-β1 levels were also elevated significantly in sera of CCl₄-treated mice, comparing to those in control mice treated with olive oil. The peak levels was detected on the 3rd day after injection; RT-PCR results showed that TGF-β1 and TβRII, Smad2, Smad3 and Smad4 m RNA expression was also up-regulated remarkably in livers of CCl₄-treated mice, comparing to those of control; Western blotting results revealed that Smad3 and phospho-Smad3（p-Smad3） protein levels were elevated significantly in CCl₄-treated mice comparing to those in control. These data above generally indicated that the m RNA transcription and protein levels of TGF-β1 and Smad3 were elevated in CCl₄-induced acute liver injury of mice. 2.3 Effects of Smad3-overexpression on CCl₄-Induced Acute Liver Injury 2.3.1 Evaluation of the Transfection Efficiency of in vivo Smad3-overexpression To determine thansfection effects in the of Smad3 overexpression mice in vivo,the Smad3 m RNA and protein expression levels were detected in liver tissues of the smad3 overexpression mice. RT-PCR and western blotting results showed significant overexpression of both Smad3 m RNA and protein comparing to those in control, indicating that the mice model of the smad3 overexpression was established successfully in vivo. 2.3.2 Effect of Smad3-overexpression on Liver Injury of Mice Treated With CCl₄ The effect of Smad3-Overexpression was evaluated by the assay of ALT and AST. The results showed that even though both treated by CCl₄, higher levels of ALT and AST were exhibited in the group of Smad3-overexpression, with the peak occuring on the 1st and 3rd day comparing to those in the control group of negative plasmid. The results above indicated that Smad3-Overexpression may lead to a worse acute liver injury. H&E staining showed no significant difference in area of annular necrotic lesions around the hepatic lobule portal between the Smad3-overexpressing mice and the negative plasmid control mice on the 1st day after CCl₄ treatment. However, on the 3rd and 5th day, larger area of annular necrotic lesions with massive inflammatory cells infiltration was observed in the Smad3-overexpressing mice comparing to the control mice. These results indicated that Smsd3-overexpression in vivo aggravated CCl₄-induced acute liver injury in mice and prevented the repair of the injured liver. 2.3.3 Effects of Smad3-overexpression on Amount of Macrophages and Neutrophils in Livers and Apoptosis of Hepatocytes In this study, the amount of of macrophages and neutrophils was detected by flow cytometry. Levels of inflammatory cytokines IL-1β and IL-6 in sera of mice were determined by ELISA. The results showed that the amount of macrophages and neutrophils in livers of Smad3-overexpressing mice treated with CCl₄ were remarkably higher than those in control of negative plasmid mice treated with CCl₄. ELISA results showed that IL-1β and IL-6 expression levels were increased apparently in sera of Smad3-overexpressing mice treated with CCl₄ comparing to those in control mice of negative plasmid treated with CCl₄, and the peak was observed on the 3rd day and subsequently declined on 5th day, but still higher than the control group. TUNEL staining was used to examine apoptosis of hepatocytes. The results showed that significantly more TUNEL-positive hepatocytes were observed in the Smad3-overexpressing mice on the 1st and 3rd day after CCl₄ treatment comparing to those in the negative plasmid control mice treated with CCl₄. These results indicated that the Smad3-overexpressing mice might aggravate liver injury and prevent the repair of the injured liver by promoting inflammatory cells infiltration, inflammatory cytokines release, and apoptosis. 2.4 Effect of Smad3 Knock-down on Liver Injury of CCl₄-Induced Mice 2.4.1 Evaluation of the Transfection Efficiency of in vivo Smad3 Knock-down To determine the transfection effects of Smad3 knock down mice in vivo, the Smad3 m RNA and protein expression levels were detected in the liver of smad3 knock down mice by RT-PCR and Western blotting. RT-PCR and western blotting results showed significant decrease of both Smad3 m RNA and protein comparing to those in control mice, indicating that the mice model of the smad3 sh RNA was established in vivo. 2.4.2 Effect of Smad3 sh RNA on Acute Liver Injury in Mice Induced by CCl₄ The effect of Smad3 sh RNA was evaluated by the assay of ALT and AST. The results showed that even though both treated by CCl₄, lower levels of ALT and AST were exhibited in the group of Smad3 sh RNA, comparing to those in the control group of negative plasmid. The results above indicated that Smad3 sh RNA may lead to a mitigation acute liver injury. H&E staining showed smaller area of annular necrotic lesions around the hepatic lobule portal with less inflammatory cells infiltration in the Smad3 sh RNA mice comparing to those in the control group of negative plasmid. These results indicated that Smsd3 sh RNA in vivo mitigated CCl₄-induced acute liver injury in mice and promoted the repair of the injured liver. 2.4.3 Effects of Smad3 sh RNA on Amount of Macrophages and Neutrophils in Livers and Apoptosis of Hepatocytes In our study, we detected the quantity of Macrophages, Neutrophils and NK cells by flow cytometry. Levels of inflammatory cytokines IL-1β and IL-6 in sera of mice were determined by ELISA. The results showed that the amount of macrophages and neutrophils in livers of Smad3 sh RNA mice treated with CCl₄ were remarkably lower than those in control of negative plasmid mice treated with CCl₄. ELISA results showed that IL-1β and IL-6 expression levels were decreased apparently in sera of Smad3 sh RNA mice treated with CCl₄ comparing to those in control mice of negative plasmid treated with CCl₄, TUNEL staining was used to examine apoptosis of hepatocytes. The results showed that significantly less TUNEL-positive hepatocytes were observed in the Smad3 sh RNA mice on the 1st and 3rd day after CCl₄ treatment comparing to those in the negative plasmid control mice treated with CCl₄. These results indicated that the Smad3 sh RNA mice might mitigated liver injury and promoted the repair of the injured liver by decreased inflammatory cells infiltration, inflammatory cytokines release, and apoptosis.3. CONCLUSION The overall results indicated that high level expression of TGF-β1 and Smad3 presented in the CCl₄-induced liver injury. The overexpression of Smad3 aggravated the CCl₄-induced Acute Liver Injury, while Smad3 sh RNA can alleviated the CCl₄-induced acute liver injury. The destructive or protective effect of Smad3 might be conducted by promoting inflammatory cells infiltration, inflammatory cytokines release such as Macrophages, Neutrophils and NK cells, and apoptosis of hepatocytes through the mitochondrial pathway. All the result above confirmed that TGF-β1/Smad3 take part in the liver injury and liver repair process. Smad3 functions to promote inflammatory cells infiltration, inflammatory cytokines release and apoptosis of hepatocyte. Moreover, the protective effect of Smad3 sh RNA enabled its clinical applicability in the treatment of acute liver injury.