| AIM AND BACKGROUNDTransforming growth factor β1 (TGF-β1) was known as the key cytokine in liver fibrogensis. Previously, we found that TGF-pi was also an important factor in liver fibrosis resulted from Schistosomiasis Japonica although there is some difference in mechanism of fibrogensis between Schistosomiasis Japonica-induced liver injury and other facotors-induced liver damage, such as virus, alcohol, carbon tetrachloride (CCL4), dimethyl nitrosamine (DMN), bile ducts ligation (BDL). In our previous experiments, we observed that the liver fibrosis of rabbits infected by Schistosomiasis Japonica could be reversed to some extent after treatment with praziqutel. After interferon-γ (IFN-γ) was given followed, liver fibrosis significantly improved. Smad was identified as the signal transduction protein involved in TGF- P 1 signal transduction process about ten years ago. In fact, little was known about roles of the downstream protein of TGF-pi in the development of Schistosomiasis Japonica-induced liver fibrosis. Therefore, we established the mice model of liver fibrosis resulted from Schistosomiasis Japonica to observe two transmembrane receptors of TGF-pi and Smads in the development of fibrogensis and investigate the effects of preziquantel and IFN-γ on TGF-β1 receptors (TpR I and TβRⅡ) and Smads to explore whether the antifibrotic roles of praziqutel and IFN-y was through influencing the downstream protein of TGF—β1.MATERIALS AND METHODSInduction of liver fibrosis in mice BALB/c mice aged 68wk weighing 18g to 20g were obtained from Chinese Science Academy Animal Experiment Center, and were maintained with Rodent Diet (Animal Experiment Center of medical school of ZheJiang University) and water ad lib. Mice were cared for and used in accordance with the Declaration of Helsinki. After maintenance for a week, mice were infected subcutaneously with 20±1 cercariae of Schistosoma japonicum at Medicine and Science Academy of ZheJiang Province. These animals survived at least 24 weeks post-infection allowing comparison among various stages of infection. All infected mice were divided into two groups: untreated group and praziqutel -treated group. In the untreated group, eight, eight, eight, and twelve mice were killed for liver sample at 8 weeks, 12 week, 16 week, and 24 weeks post-infection, respectively. The praziqutel -treated group was divided into two subgroups: praziquatel group and praziquatel combined with IFN-γ group. In praziquatel group, animals 16 weeks post-infection were given the drug by gavage once at a concentrate of 500mg/kg body weight and then injedted with saline via muscles per day. In praziquatel combined with IFN-γ group, animals were injected via the muscle, according to 50,000u per day per animal for 8 weeks after praziquatel treatment. All animals were killed after treatment for liver specimen. Some were snap- frozen in fluid nitrogen immediately, and then conserved in the -80 °C refrigerator for reverse transcription polymerase chain reaction (RT-PCR). Other liver pieces were fixed in 10% buffered formalin for histology assay to detect the size of egg granuloma and determine the liver fibrosis degree and immunohistochemistry assay.RNA Extraction and RT-PCR Total RNA was extracted from the snap-frozen liver tissue of individual mice. Samples were homogenized inTrizol and total RNA was isolated according to the manufacturer's suggested protocol. For RT experiments, 1μg of RNA was used for cDNA synthesis, which was performed in a sample volume of 20 μl using random primers and Mu-MLV reverse transciptase in the reaction for one hour at 42 °C, then at 70°C for 10 min. 1μl cDNA was added to 24 μl PCR mixture, which included 18.25μl dH2O, 2.5μl 10×PCR buffer, 2μl2.5nMdNTP,20pmol/ul upstream and down stream primer 0.5μl respectively, 2U/ul Taq DNA polymerase 0.25μl. Amplification conditions were at 95 °C for 3 min, 38 cycles of denaturation at 94°C for 30s, annealing at 57°Cfor 30s and extension at 72°C for 30s (TGF-βl, TPR I and TβRⅡ ) ,30 cycles of denaturation... |
