Effects And Mechanisms Of Hesperitin Derivative-11 On CCl₄-induced Rat Hepatic Fibrosis

Hepatic fibrosis?HF?is a continuous pathological injury and repair process occurred in all cases of chronic injury,which can lead to the deposition of extracellular matrix?ECM?that caused the abnormal deposition of liver structure and function change,eventually lead to cirrhosis or even liver cancer.There is no effective treatment for liver fibrosis.The study shows that the main source of the abnormal deposition of ECM is the activation of hepatic stellate cells?HSCs?.The activation and proliferation of HSCs play a central role in the pathogenesis of hepatic fibrosis,which can be regulated by many cytokines and signaling pathways.Therefore,inhibiting the proliferation and activation of HSCs may effectively inhibit the development of liver fibrosis.Our previous study showed that hesperetin derivative 7?HDND-7?could inhibit the proliferation and activation of HSCs which played an important role in anti hepatic fibrosis.Hesperitin derivative?HD-11?is a monomeric compound derived from Hesperidin,which is a naturally occurring flavanone glycoside that exerts extensive clinical effects such as anti-inflammatory,anti-oxidant and anti-angiogenic.Our laboratory synthesized a series of hesperidin derivatives?HDs?through structural modification.In our studies,we evaluated the role of HDs in the activation and proliferation of TGF-?1-induced HSC-T6 cells and found that HD-11 may inhibit the activation and the proliferation of HSCs which suggested a further research and development prospects.However,the role of HD-11 in liver fibrosis is not clear.It was also found that HD-11 could promote the expression of PTEN.Phosphatase and tension homolog deleted on chromosome 10?PTEN?has been reported to have a variety of cellular physiological functions,such as cell growth,proliferation and apoptosis.PTEN is known as a tumor suppressor gene that involved in a wide variety of human cancer,such as breast cancer,colorectal cancer,liver cancer and so on.In addition,the role of PTEN in other diseases has been discovered,such as liver fibrosis,obesity,autoimmune diseases and so on.Previous studies also found that the expression of PTEN was inhibited in the process of liver fibrosis,and elevated PTEN could inhibit the expression of ?-SMA and col1?1.It has been reported that AKT is a direct downstream target of PTEN through a variety of signal pathways affecting cell proliferation and apoptosis.Given the important role in PTEN in cells activation and proliferation,we conceived of the anti-fibrotic effect of HD-11 may via suppressing PTEN/AKT signaling pathway.The above content has not been reported in the literature,so this study focuses on the therapeutic effect of hesperidin derivatives on CCl₄ induced liver fibrosis in rats and its potential mechanism.In order to verify this conjecture,we conducted the following research: 1.The anti-fibrotic effect of HD-11 in vivoForty normal Sprague-Dawlay?SD?rats?male,180-220g?were randomly divided into five groups?eight rats per group?including control group,model group,low,medium and high dose groups.Hepatic fibrosis was generated by biweekly intraperitoneal injection of CCl₄?40% in olive oil?at a dose of 2 ml/kg for 12 weeks.Control rats were treated intraperitoneally with the same volume of olive oil at the same time intervals.Dosage group rats were treated with lavage of low,medium and high dose of HD-11?2 times a week?for 4 weeks at the time of 8 weeks CCl₄ administration in the base of model group.In order to determine the tissue injury and the degree of fibrosis of each group,HE and Masson staining and immunohistochemical examination were used to observe the histopathological changes of livers under a light microscopy;serum ALT and AST levels were detected to observe the degree of the hepatotoxicity damage;Western blot and RT-PCR were used to detect the expression of ?-SMA and collagen I?col1?1?in liver tissue with liver fibrosis and primary hepatic stellate cells in protein and mRNA level.The results suggested that the treatment of different concentrations of HD-11?1g/kg,2g/kg,4g/kg?decreased the expression of ?-SMA and col1?1 and attenuated the CCl₄–induced liver fibrosis.2.The effect of HD-11 on the proliferation and activation of HSC-T6 cells in vitro The MTT method was used to detect different concentrations?0,1.25,2.5,5,10?M?of HD-11 on HSC-T6 cell activity.In order to determine the cytotoxicity of HD-11 on HSC-T6 cells,TGF-?1?10ng/ml?and different concentrations of HD-11?0,1.25,2.5,5,10?M?were used to treat HSC-T6 cells for 48 h.The MTT method selected the minimum concentration free of toxicity on HSC-T6 cells.The expression of ?-SMA and col1?1 were measured by Western blot and RT-PCR.The results showed that HD-11 can obviously inhibit the proliferation of HSC-T6 by arresting in the S phase,and had no effect on cell apoptosis.3.The effect of HD-11 on PTEN/AKT pathway.In vivo,immunohistochemistry technique was used to detect PTEN expression in liver tissue of rats.Western blot and RT-PCR were used to detect the expression of PTEN in rat liver tissue and primary hepatic stellate cells in protein and mRNA level.In vitro,HSC-T6 cells were treated with TGF-?1?10ng/ml?and different concentrations of HD-11 for 48 h.PTEN expression was detected by immunofluorescence,Western blot and RT-PCR,respectively.After the specific inhibitor of PTEN?bpv?,PTEN-siRNA or GV141-PTEN was transfected,HSC-T6 cells were stimulated with TGF-?1?10ng/ml?for 48 h.Western blot and RT-PCR were used to detect PTEN,?-SMA and col1?1 in protein and mRNA levels.The results showed that HD-11 could raise the expression of PTEN;when PTEN was silenced or overexpressioned,the expression of ?-SMA and col1?1 were up-regulated or down regulated,at this time,treatment with HD-11 had no effect on the expression of ?-SMA and col1?1.In vivo,immunohistochemical technique was used to detect the expression of p-AKT in rat liver tissue.Western blot technique was used to detect p-AKT,cyclinD1 and c-myc expression in rat liver tissue and primary hepatic stellate cells.In vitro,first of all,HSC-T6 cells were given the TGF-?1?10ng/ml?and different concentrations of HD-11 for 48 h,Western blot was used to detect expression of p-AKT,cyclinD1 and c-myc.Then,using the same method of silencing or overexpression of PTEN,Western blot was used to detect the expression of p-AKT,cyclinD1 and c-myc.The results showed that the expression of p-AKT in tissues and cells was inhibited by HD-11;when PTEN was knockdown or overexpression,the expression of p-AKT,cyclinD1 and c-myc was up-regulated or down regulated,at this time,treatment with HD-11 had no effect on the expression of p-AKT,cyclinD and c-myc.To sum up,HD-11 inhibits the activation of the downstream PI3K/AKT signaling pathway by up regulating the expression of PTEN,thereby inhibiting the proliferation of hepatic stellate cells and eventually blocking the progression of hepatic fibrosis...