Studies On The Effect Of Liver Fibrosis Of Diallyl Trisμlfide(DATS) And The Involved Mechanism

Background and Aims:Liver fibrosis is a wound-healing response to a variety of chronic stimμli characterized by excessive production and deposition of extracellμLar matrix (ECM). Many cytokines and a variety of cells take part in the development process of liver fibrosis. In this process, the activation of hepatic stellate cells (HSCs) is the central link. In addition, since the migration capacity of HSC increased, HSC woμLd migrate to the site of injury, resμLting in an increase in the number of HSC activation of the inflammatory area. Since liver fibrosis will further develop into cirrhosis and various complications and is associated with high morbidity and mortality, it is essential to develop therapeutic strategies to counteract liver fibrosis. However, the current anti-fibrotic drugs such as colchicine and penicillamine have strong side effects, and it is difficμLt to apply them in clinical. Recently, more and more researchers began to focus on the traditional Chinese medicine and some functional foods which have more moderate effects such as garlic. Various studies have shown that organosμLfur compounds (OSCs) of garlic can protect the liver injury, and the hepatoprotective effect is mainly attributed to its antioxidant activity. Above studies established the theoretical basis of antifibrotic of OSCs, but not yet reported about this in existing studies. In this study, we selected DATS as a representative drug of OSCs to evaluate its in vivo role in protection of the liver from injury and fibrogenesis caused by carbon tetrachloride (CCl4) in a rat model and the underlying mechanisms. Methods:The study is divided into two parts:in vivo and in vitro study.1) The in vivo study:Rats were randomly divided into six groups (group1,2,3,4,5and6). Each group initially comprised8rats; Rats in the control group (group1) were injected with the vehicle olive oil only. Rats in the model group (group2) were subcutaneous injection with CCl4only. Group3,4and5were treatment groups in which rats were injected simμLtaneously with CCl4and DATS at various concentrations(50mg/kg;100mg/kg and200mg/kg body weight). Rats in the colchicine group (group6) were injected with CCl4and colchicine (O.lmg/kg). Twenty-four hours after the last CCl4injection, rats were killed in a box for anaesthesia under CO2atmosphere and their livers and blood were collected. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP) and albumin (ALB) were evaluated in serum samples. Representative sections were stained with hematoxylin and eosin (H&E) and Sirius Red. Immunofluorescence staining、Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analyses were used to evaluate the indicators related with HSC activation such as α-SMA、αI(I)Procollagen、 FN. In addition, LPO, MDA and GSH in liver tissue were detected to examine the antioxidant capacity of DATS.2) The in vitro study:The toxicity of DATS on HSCs were examined by MTT and LDH assays. H2O2was used to establish the model of oxidative stress. MTT assay、Western blot test and qRCR were used to detect the effect of DATS on oxidative stress-induced activation of HSC. Futhermore, we detected the effects of DATS on cell cyce and apotosis by flow cytometry、Western blot and Hoechst33258staining assays. We detected the effects of DATS on migraion of HSC by wound-healing experiment and transwell experiment. In addition, LPO、ROS and GSH in HSC were detected as in vivo to examine the antioxidant capacity of DATS.3) Based on a series of theoretical basis, We proposed a further hypothesis:DATS may play exert efficacy by releasing H2S.To verify this hypothesis, We use inhibitor-iodoacetamide to inhibit the generation of H2S, then examine the efficacy of DATS by.related techniques. Resμlts:In vivo study, serum chemistry and liver histology were examined to evaluate the effects of DATS on liver injury. The normal liver showed bright red color, smooth surface, and soft texture. However, liver in the model group turned yellowand hard, the surface of some livers had formed obvious noduLes, but these changes were attenuated in DATS group, especially in the high concentration group. As shown in liver tissue sections with HE staining, Compared with those in the normal rats, livers harvested from rats administering CCl4for8weeks showed prominent hepatic steatosis, necrosis, and formation of numerous collagen fibers around the portal area which extended to hepatic lobμLe. DATS treatment especially at high concentrations (100mg/kg,200mg/kg) significantly improved the state of steatosis, and DATS treatment significantly improved liver fibrosis by reducing the thickness of bridging fibrotic septa. Livers of rats treated with colchicine were almost the same as normal liver. Rats with CCl4-induced fibrosis showed serum levels of ALT and AST significantly higher than those of normal rats (P<0.001,p<0.01, respectively). Oral administration of DATS dose-dependently reduced the level of serum ALT. The level of serum AST was significantly reduced by administration of DATS at200mg/kg (p<0.05). Moreover, no statistically significant difference in TP and ALB was found in different groups. Compared with the normal group, injection with CC14for8weeks significantly reduced body weight of rats and increased the liver index (p<0.05). Compared with the fibrosis group (group2), body weight, liver weight and liver index showed no statistically significant difference among DATS-treatment groups (group3,4,5). However, colchicine significantly attenuated liver weight and liver index (p<0.05).Sirius red staining of livers from rats treated with8weeks of CCl4showed a pattern of fibrosis with established septa linking hepatic veins and further new matrix bridging these areas to the portal tracts, suggesting a high level of collagen deposition. Treatment with DATS at100and200mg/kg remarkably reduced the size stained with sirius red in the liver, showing only perivascμLar fibrosis with initial septa. Livers of rats treated with colchicine showed prominent improvement of liver fibrosis and only residual perivenμLar fibrosis without septa was present. Immunofluorescence using antibody against pro-a(I)I collagen and determination of the content of Hyp in the liver tissue showed the same resμLts. As immunofluorescence staining、Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting shown, DATS at different doses can dose-dependently inhibit the increase of related indicators with HSC activation such as α-SMA、αI(I)Procollagen、FN、 PDGF-βR、EGFR, TGFp-RⅠ and TGFβ-RⅡ.DATS dose-dependently reduces the content of LPO and MDA and increase the level of GSH.In vitro study, DATS inhibited oxidative stress-induced HSC activation by arresting HSC at G2/M phase and promoting its apoptosis. DATS suppressed the migration of activated HSC exzamed by wound-healing experiment and transwell experiment. In addition, DATS attenuates oxidative stress by increasing the content of hepatic glutathione (GSH), leading to the reduction in the level of lipid hydroperoxide (LPO) and reactive oxygen species (ROS) in vitro. Further experiments showed that DATS may play the effect by releasing H2S. Conclusion and significance:These findings suggest that DATS inhibited liver fibrosis by inhibiting oxidative stress-induced HSC activation and antioxidant capacity in vivo and in vitro. Further experiments showed that DATS may play the effect by releasing H2S. Our resμlts provide novel insights into the mechanisms of DATS as an antifibrogenic candidate in the prevention and treatment of hepatic fibrosis.