Effects And Mechanisms Of HDND-7 On CCl₄-induced Mice Hepatic Fibrosis

Liver fibrosis is essentially a continuous wound-healing response of the liver to repeated injury, which caused by a variety of etiology such as viruses, drugs, schistosomiasis and alcohol. It’s mainly characterized by the excessive deposition of extracellular matrix(ECM) components and caused by the unbalanced fiber hyperplasia and decomposition. An increased number of experimental studies have confirmed that the main source of excessive deposition of ECM is the activation of hepatic stellate cell and HSCs activation and proliferation are the central event in the process of the development of liver fibrosis, which regulated by a large number of signaling pathways and cytokines. Therefore, inhibiting HSC activation may slow the development of liver fibrosis.HDN, a flavanone found abundantly in fruit peels of the genus citrus, is one of the effects of the main active ingredient. Literature showed that, hesperidin exhibit several key biological and pharmacological properties, such as anti-inflammation, anti-oxidant and anti-tumor and anti-hyperlipidemia. However, it’s water solubility and bioavailability limits the medicinal. In the previous studies, our laboratory had synthesis of a series of hesperidin derivatives. In our studies,we evaluated the role of HDNDs in the activation and proliferation of PDGF-BB-induced hepatic stellate cells. We found that HDND-7 may inhibit the activation and the proliferation of HSCs through suppressing the Wnt/β-catenin pathway.The Wnt signaling pathway has been reported to play a variety of cellular physiological functions, such as cell development, proliferation, and apoptosis.Some elements of Wnt/β-catenin pathway involved in wound healing and fibrosis and up-regulating in fibroblasts which derived from HSC. Among them Wnt/β-catenin(canonical) signaling was proved to play important role in maintaining the quiescent state of HSC. In addition, the Wnt/β-catenin signaling pathway is widely involved in the proliferation of various tumor, such as liver cancer, lung cancer, bladder cancer and so on. Given the important role of Wnt/β-catenin in the cell activation and proliferation, we conceived of the anti-fibrotic of HDND-7 may via suppressing Wnt/β-catenin pathway. In order to confirming this idea, we conducted the following research. 1. The anti-fibrotic of HDND-7 in liver fibrosis tissues of mice.Liver fibrosis model was established by lavage of CCl4 in mouse and HE, Masson and immunohistochemistry were used to test the injury of different groups of liver tissues. The RT-PCR and Western blot were used to measure the levels of α-SMA, collagen I, β-catenin, cyclind1 and c-myc in the mouse liver fibrosis tissues. The results suggested that the treatment of different concentration of HDND-7(50mg/kg, 100mg/kg, 150mg/kg) attenuate the CCl4–treated liver fibrosis and decreased the expression of α-SMA, collagen I, β-catenin, cyclind1 and c-myc. 2. The effect of HDND-7 on the proliferation and activation of HSC-T6 cellsHSC-T6 cells were treated with HDND-7(10, 50, 100, 200 μM), then we used MTT assay to evaluated the role of HDND-7 on the viability of PDGF-BB-activated HSC-T6 cells. HSC-T6 cells were pretreated with PDGF(10 ng/ml) for 2 h, then incubated with HDND-7(10, 50, 100 μM) for another 24 h or 48 h. MTT and FCM were used to detect the role of HDND-7 on HSC-T6 cell proliferation and cycle distribution. The expression of α-SMA and Collagen I were measured by RT-PCR and western blot. The results showed that HDND-7 might mainly function by negatively regulating PDGF-BB-induced HSC-T6 cells activation and proliferation. 3. The effect of HDND-7 on canonical Wnt/β-catenin pathway.HSC-T6 cells were pretreated with PDGF(10 ng/ml) for 2 h,then incubated with the various concentrations of HDND-7 10,50,100 μM) for another 24 h or 48 h. RT-PCR and western blot were used to determine the expression of β-catenin, cyclind1 and c-myc in HSC-T6 cells. HSC-T6 cells were pretreated with PDGF(10 ng/ml) and icRT3(60ng/ml) for 2 h,then incubated with HDND-7(10,50,100 μM) for another 24 h or 48 h. Then, the expression of α-SMA, β-catenin, cyclind1 and c-myc were determined in HSC-T6 cells. The result that HDND-7 might inhibit the activation and proliferation of PDGF-BB-induced HSC-T6 cells may be partly through targeting Wnt/β-catenin signaling pathway.