Therapeutic Effect Of LhRNA Targeting HBx/PD-L1 And Edaravone In Chronic Liver Diseases And Related Mechanisms

Part I:Therapeutic effect of long-hairpin RNA(lhRNA)targeting HBx and PD-L1 in chronic HBV infection and related mechanismsBackgroundHepatocellular carcinoma(HCC)is one of a commonmalignant tumors with poor prognosis,which is the fifth most prevalent fatal cancer and the third leading cause of cancer deaths in the word.And the development of HCC is a pathologic processes with multi-step involved in,charactered as hepatitis-cirrhosis-liver cancer "trilogy".Chronic hepatitis B virus(HBV)infection is the most important factor inducing the development of HCC,occupying more than half of the cases worldwide.Unfortunately,the therapeutic options of HBV related HCC are still hampered by many obstacles.Therefore,seeking for new therapeutic approaches to improving survival rate and life quality of the patient is still a burning questionHBV-encoded X protein(HBx)is a multifunctional regulator with a crucial role in the pathogenesis of HBV-related HCC.As a transactivator,growing evidence has proved that it has mutiple important biological functions in cell proliferation,cell cycle control,anti-apoptosis,tumor invasion and metastasis.Moreover,it can modulate epithelial-mesenchymal transition,regulatory non-coding RNAs(ncRNA)and various signal-transduction pathways to accelerate the development of HCC.HBx is highly conserved among the different genome of the virus,with a low mutation rate,and is an ideal target for therapy of chronic HBV infection and HBV-related hepatoma.Recently,PD-L1 expression is reported to be elevated on hepatocytes in CHB patients,which plays an important role in HBV induced immune escape.It can suppress antiviral function and the proliferative ability of T cells,and contributes to T cell apoptosis by interacting with its receptor,PD-1.In this study,PD-L1 and HBx were silenced simultaneously by using the RNA interference(RNAi)technology to explore the feasibility of the therapeutic schemes in HBV-related hepatoma.Methods1.We designed and synthesized three PD-L1 si RNA,and then selected one sequence with a better silencing efficacy by means of PCR technology;Meanwhile,we synthesized HBx siRNA,and confirmed its efficiency of silence by means of PCR and Western Blot methods;2.We constructed long-hairpin RNA(lhRNA)vector targeting HBx and PD-L1 genes(lhRNA-HBx/PD-L1)simultaneously;3.HepG2.2.15 cells were used as the research cellular model,and the silencing efficacy of lhRNA-HBx/PD-Ll was verified by using PCR and Western Blot assays;4.CCK-8 assay was performed to test the proliferation of HepG2.2.15 cells transfected with lhRNA-HBx/PD-L1;5.FACS-based AnnexinV-FITC/PI staining and PI staining was performed to test the apoptosis and cycle of HepG2.2.15 cells transfected with lhRNA-HBx/PD-L1 respectively;6.Western blot analysis was performed to analyze the expression levels of BCL-2,BCL-XL and proteins related with mTOR signlingin of HepG2.2.15 cells transfected with lhRNA-HBx/PD-L1;7.PCR analysis was performed to determine the mRNA expression of BCL-2,BCL-XL and genes related with mTOR signlingin of HepG2.2.15 cells transfected with 1hRNA-HBx/PD-L1;8.CCK-8 assay was performed to test the sensitivity of HepG2.2.15 cells transfected with lhRNA-HBx/PD-Ll to CHB hPBMCs-mediated cytotoxicity;9.FACS-based analysiswas performed todetect the protein levels of molecules related to cytolysis of CHB hPBMCs incubated with HepG2.2.15 cells transfected lhRNA-HBx/PD-L1;10.PCR and ELISA assays were performed to detect the cytokine expression profile of HepG2.2.15 cells transfected with lhRNA-HBx/PD-L1.Results1.Selection of PD-L1 siRNA and silencing efficacy of HBx siRNAQPCR was performed to test the gene expression of PD-L1 in U251 cells transfected with desighed three PD-L1 siRNA using lipofectamineTM 2000,and selected the PD-L1 siRNA-1 as a better siRNA to be used in the following assays;Meanwhile,we confirmed the silencing efficacy of HBx siRNA both in the gene and protein level successfully;2.LhRNA-HBx/PD-L1 was constructed successfullyWe constructed lhRNA-HBx/PD-L1 vector silencing PD-L1 and HBx successfully,and confirmed the silencing efficacy of constructed vectors both in the gene and protein level in HepG2.2.15 cells;3.LhRNA-HBx/PD-L1 inhibited the growth of HepG2.2.15 cellsConstructed vectors can suppress the ability to proliferation and cell cycle progression,and contribute to cell apoptosis of HepG2.2.15 cells;Moreover they can Inhibit the Bcl-2 and Bcl-xl expresson,as well as the activation of mTOR signling both in gene and protein level;4.LhRNA-HBx/PD-L1 enhanced the cytotoxicity sensibility of HepG2.2.15 to CHB hPBMCCytotoxicity sensibility of HepG2.2.15 cells transfected with constructed vector to CHB hPBMC was enhanced;5.LhRNA-HBx/PD-Ll reversred the anti-virus function of CD8+T and NK cells in CHB hPBMCThe cytotoxicity and IFN-y secretion of CD8+T and NK cells in CHB hPBMC were enhanced,when incubated with HepG2.2.1 5 cells transfected with constructed vectors;Conclusions1.We constructrd the long-hairpin RNA(lhRNA)targeting HBx and PD-L1 vector successfully;2.Constructed vectors can suppress the ability to proliferate and cell cycle progression,and contribute to cell apoptosis in HepG2.2.15 cells;3.Cytotoxicity sensibility of HepG2.2.15 cells transfected with constructed vector to CHB hPBMC was enhanced;And on the other hand,the cytotoxicity and IFN-? secretion of CD8+T and NK cells in CHB hPBMC were enhanced,when incubated with HepG2.2.15 cells transfected with constructed vectors;4.The study would bring forward a brand-new idea for therapeutic strategy to curing the HBV-related liver disease.Part ?:Underlying roles of EDA in the progression of liver fibrosis and related mechanisms.BackgroundHepatic fibrosis is the wound-healing response of the liver undergoing the chronic injury,which can be induced by various factors.Initially,this can lead to the liver failure to a low degree,termed as early fibrosis,having the potential to regress by terminating the injury,and can progress into cirrhosis,charactered asabnormal nodules with following distorpted liver architecture with the persistent presence of injury.Since neurosurgical patients are usually followed by a state of comatose and deep sedationand need to be treatmented for a long time,which prone to result in the liver injury,and even induce liver fibrosis.In addition,Edaravone(3-methyl-1-phenyl-2-pyrazolin-5-one,EDA),a novel free radical scavenger,clinically serves as a novel neuroprotective agent to treat the acute ischemic stroke in patients,effectively decreasing the infarct size,increasing the neurological scores.It makes sense to illuminate the impact of EDA in the development of liver fibrosis.Thus,the aim of the study was to determine the role of EDA in the pathogenesis of hepatic fibrosis,as well as its potential molecular mechanism,via carrying out molecular mechanistic studies both in vivo and in vitro.Methods1.Liver fibrosis mouse models were established by repeatedly intraperitonea linjection of thioacetamide(TAA);2.Immunohistochemistry,Sirus Red Staining,H&E,Western Blot,qPCR,ALT and AST assays were performed to detect hepatic collagen deposition,HSCs activation and state of hapetic inflammation and injury;3.FACS-based analysis was performed to test the changes of immune cell subsets in the liver of model mice;4.Immunohistochemistry,qPCR and FACS-based analysis were performed to determine the changes of macrophages in the liver of model mice;5.qPCR and Western blot assays were performed to determine the changes of inflammatory factor in the liver of model mice;6.QPCR,ELISA and Western blot assays were performed to test the expression of I1-1?.FACS-based analysis was performed to test the level of ROS,and Western blot assay was performed to test the activaty of NF-kB and AP-1 in LPS-stimulated Abdominal macrophages;7.Mouse livers were perfused in situ with collagenase type I to isolate primary hepatic stellate cells.Cell immunofluorescence staining and qPCR assays were performed to test the level of activation of HSCs co-cultured with conditioned medium collected from LPS-stimulated Abdominal macrophages;8.Western blot and qPCR were performed to test the expression of IL-1? of THP-1 treated with EDA;Western blot was performed to test the level of HSCs activation co-cultured with conditioned medium collected from LPS-stimulated THP-1 in vitro.Results1.EDA ameliorates TAA-induced experimental liver fibrosis in vivoEDA was administered intraAbdominally to Thioacetamide(TAA)induced liver fibrosis mouse model,and the results of Sirius Red??-SMA staining?qPCR and Western blot showed that EDA-treatment group had less collagen deposition in ECM and HSC activation,compared to TAA group.These dates indicate that EDA can alleviate the TAA-induced experimental liver fibrosis.2.EDA has a hepatic protective role to TAA-induced chronic liver injuryEDA have a role of hepatic protection to some extent,as demonstrated by H&E staining and ALT/AST assays;In addition,the decreased liver injury could be reflected by decreased hepatic mRNA and protein level of the caspase3 and FADD in the EDA-treatment mice,compared to TAA group.Taken together,these findings support the hepatic protective role of EDA in TAA-induced chronic liver injury.?3.Hepatic inflammation are decreased in EDA-treatment mice after TAA-induced liver fibrosisFACS-based analysis?F4/80 immunostaining and qPCR showed that,there was a decreased mononuclear macrophage infiltration in the EDA-treatment liver,compared to TAA group;Quantification of the mRNA level of IL-1? in the liver of EDA-treatment mice show a remarkable decrease,whereas the hepatic mRNA levels of IL-1?,TNF-? and IL-6 were not further decreased in EDA-treated mice,compared to the TAA littermates These data indicate that EDA alleviates monocyte-derived macrophages recruitment and hepatic inflammation.4.EDA decrease IL-? production of macrophages via inhibiting the ROS generation and activation of NF-?BQPCR?EL ASA and Western blot assays showed that EDA reduced the expression of IL-1? in LPS-exposed macrophages in vitro;FACS-based analysis and Western blot assays confirmed that EDA cleared ROS produced by the macrophage,restraining the activation of NF-?B and AP-1,and ultimately leading to decreased production of IL-1?,and acquire a more attenuated pro-inflammatory phenotype.5.EDA-treated macrophages decrease the fibrogenic properties of hepatic myofibroblasts via IL-1?Mouse hepatic myofibroblasts isolated from mice were cultured in the presence of conditioned medium(CM)collected from LPS-exposed or EDA-treated Abdominal macrophages.The expression of molecules related with HSC activation were significantly reduced in myofibroblasts exposed to CM collected from EDA-treated Abdominal macrophages.And qPCR and Western blot assays showed that EDA reduced the expression of IL-1? in LPS-exposed THP-1-induced macrophages;And LX-2 cells were cultured in the presence of conditioned medium(CM)collected from LPS-exposed or EDA-treated THP-1-induced macrophages.The expression of molecules related with a-SMA were significantly reduced in LX-2 cells exposed to CM collected from EDA-treated THP-1-induced macrophages.And the inhibition was alleviated when extro IL-1? cytokine in conditioned medium was added.Conclusions1.EDA protects against liver fibrosis via decreasing the IL-1? secretion of macrophage in vivo;2.EDA cleared ROS produced by the macrophage,restraining the activation of NF-?B and AP-1,and ultimately leading to decreased production of IL-1?,and acquire a more attenuated pro-inflammatory phenotype;3.EDA can also decrease production of IL-1? of human macrophages and supress the fibrogenic properties of LX-2 cells;4.These datas indicate us that using EDA for a long time don't have harm to the liver of neurosurgical patient,instead they mey exert a beneficial role to them.