Effect Of Ribosomal Protein S5on Hepatic Fibrosis

【Background and aims】Hepatic fibrosis (HF), a pathological response to various chronic liver injuries, is recognized as a dynamic and reversal process. It is characterized by deposition of extracellular matrix (ECM) in the Disse’s space. Therefore, inhibition or even reversal of hepatic fibrosis is a major strategy for the treatment of chronic liver disease.Activated hepaticstellate cells (HSCs) are proliferative and fibrogenic, with accumulation of ECM. They are considerated as the essencial factor of hepatic fibrosis. Additionally, during liver injuries, HSCs are stimulated by various factors to obain the charactoristcs of myofibroblast (MFb).Epithelial-to-mesenchymal transition (EMT) is a process that epithelial cells gradually lose their epithelial signatures while acquiring the characteristics of mesenchymal cells in cell morphology, structure, biological function, adhesion and migration ability. Some researches revealed that EMT was a predominant mediator to participate in the process of organ fibrosis. Therefore, inhibition of EMT process in the hepatic fibrogenesis will provide a new therapeutic target for hepatic fibrosis.Our preverious studies suggested that the pharmacological effect of matrine derivative M19was largely increased after modifition to matrine. By serial affinity and competition affinity chromatography, we isolated one of the specific binding proteins of matrine, referring to ribosomal protein S5(RPS5). Furthermore, we found that the mRNA and protein expressions of RPS5were greatly decreased in the model group livers. These results suggested that RPS5may play a role in hepatic fibrosis.RPS5, belonging to a family of ribosomal proteins, participates in the complex task of coordinating protein biosynthesis to maintain cell homeostasis and survival. However, recent researches reveal that several RPS5possesses distinct extra-ribosomal functions in apoptosis, DNA repair and transcription, and are critical to the development and progressions of many kinds of diseases. In this study, we examined the effect of RPS5on hepatic fibrosis and EMT progress by injection of AdshRPS5or AdRPS5in vitro or in vivo. Furthermore, in order to research the anti-fibrosis mechanisms of RPS5, we investigated the related signaling pathways and interactivated proteins.【Methods】1. The role of RPS5in activation and EMT progress of HSC-T6cells.HSC-T6cells were infected with AdshRPS5or AdRPS5for48h. Realtime RT-PCR and western blot were carried out to determine mRNA and protein expression level of HSC activation makers and EMT makers. MTT based assay was used to detect cell proliferation after infection for8h,24h,48h and72h.2. The role of RPS5in hepatic fibrosis models in ratsTo investigate the effect of RPS5on liver fibrosis, hepatic fibrosis was induced in rats by DMN injection intraperitoneally with1%DMN (10μg/kg) for three consecutive days per week for up to4weeks. After DMN injection for6times, rats were infused with4x109pfu of AdshNC or AdshRPS5via the tail vein, respectively. Two weeks after gene delivery, the animals were killed. All paraffin-embedded liver tissues were stained with H&E, Masson’s tri chrome, and Sirius red staining. For the semi quantitative analysis, five image fields were selected randomly from each section and the percentages were calculated. Immunohistochemistry were performed on paraffin-embedded liver sections to investigate levels of proteins.Total hepatic hydroxyproline levels of liver samples were determined according to the protocol of the Hydroxyproline Testing Kit. Realtime RT-PCR and immunohistochemistry were performed to determine the mRNA and protein levels of hapetic fibrosis and EMT makers.To research the relationship between RPS5and hepatic fibrosis Bile duct ligation model, BDL model was induced in rats2d after infection with4x109pfu of AdshRPS5or AdshNC. Two weeks after BDL, the animals were sacrificed for examinations of liver function and immunochemistry as previous described.To study whether endogenous RPS5could regulate liver fibrogenesis,4x109pfu of AdRPS5or AdNC was injected twice at2days before DMN injection and6times after DMN injection, and the animals were killed at the end of3weeks. Liver tissues were analysised as previous described.3. Mechanism for anti-fibrosis of RPS5in vitroHSC-T6cells were infected with AdshRPS5or AdshNC for48h, and then western blot was carried out to determine protein expression level of Akt signaling pathway; immunoprecipitation was used to assess the interaction between RPS5and p-Akt; Immunohistochemistry was performed to detect the p-Akt protein level in liver sections of hepatic fibrosis models.[Results]1. The role of RPS5in activation and EMT progress of HSC-T6cells.MTT analysis revealed that AdshRPS5significantly induced HSC-T6cell proliferation after infection. The suppression rate was up to25%(P<0.05). Realtime RT-PCR showed the significant upregulation of mesenchymal maker vimentin and downregulation of epithelial maker E-cadherin after infection AdshRPS5for48h in HSC-T6. Western blot verified the effect on protein levels.Realtime PCR showed that AdRPS5inhibited HSC-T6cell activation and EMT progress.2. The role of RPS5in hepatic fibrosis models in ratsWe then tested the effect of AdRPS5in hepatic fibrosis rat model. As expected, DMN injection induced prominent hepatic fibrosis in rats as shown by Masson’s trichrome and Sirius red staining and decreased RPS5expression. AdshRPS5promoted hepatic fibrosis proceeding in rats, with the increasing170%ECM,36%hydroxyproline content, and upregulation of collagen III mRNA level (P <0.01).For BDL models, AdshRPS5treatment induced the decrease of RPS5and induced ECM (Masson’s trichrome staining) by157%(P <0.05). The hydroxyproline content in AdshRPS5-treated group (271.6±106.8μg/g) also increased compared with that in AdshNC group (228.3±74.0μg/g, P <0.01). In addition, the increased protein levels of a-SMA, Vimentin were also detected by immunohistochemistry.In contrast, AdRPS5treatment blocked the decrease of RPS5and reduced ECM (Masson’s trichrome staining) by11%(P<0.05). The hydroxyproline content in AdRPS5-treated group (400.8±57.2μg/g) also decreased compared with that in AdNC group (471.3±74.5μg/g), with the percentage of15%. In addition, AdRPS5significantly suppressed mRNA levels of collagen III and collagen I. The increased protein levels of RPS5, a-SMA and vimentin were also detected by immunohistochemistry.3. Mechanism for anti-fibrosis of RPS5in vitroWestern blot showed that AdshRPS5promoted phosphorylation of Akt in HSC-T6cells after infection with AdshRPS5for48h; Immunohistochemistry revealed the upregulation of p-Akt protein level in liver sections of hepatic fibrosis models; immunoprecipitation showed the interaction between RPS5and p-Akt; dephosphorylate experiment revealed that RPS5dephosphorylates phospho-Thr-308of Akt in vitro.【Conclusion】All of our results revealed that:1. RPS5knockdown promoted HSC-T6activation and EMT progress.2. Knockdown of RPS5in vivo aggravated experimental hepatic fibrosis induced by DMN and BDL; Over-expression of RPS5in vivo attenuated experimental hepatic fibrosis induced by DMN.3. RPS5exerts anti-fibrosis effect via regulation Akt phosphorylation in HSC-T6; RPS5interactivated with p-Akt.4. The protein level was increased during hepatic fibrosis progress.