Liver Protection Of Serine/Threonine Kinase Pim-3 Gene Against Lipopolysaccharide/D-galactosamine-induced Fulminant Hepatic Failure

Fulminant hepatic failure(FHF)is a life-threatening clinical syndrome characterized by massive hepatic apoptosis and severe impairment of liver function. Serine/threonine kinase Pim-3 gene may play an important role in protecting liver tissue against FHF because of its inhibition of hepatocyte apoptosis and promotion of cell growth.To probe the hypothesis,we finished an in vivo liver-target transfer of Pim-3 gene.We aimed to investigate the effect and mechanism of Pim-3 gene on FHF.First of all,by reverse transcription polymerase chain reaction(RT-PCR), Pim-3 gene was successfully isolated and cloned from rat myocardium tissues. Pim-3-expressed plasmid was then constructed by genetic recombination technology. After smoothly transfected into eukaryotic cells,the construct was found to have an effect on cell survival and apoptosis in vitro.Secondly,by an injection of green fluorescent protein(GFP)-expressed plasmid DNA into mouse tail vein using hydrodynamics-based or regular procedure,we found that different from regular injection,hydrodynamics-based procedure induced overexpression of GFP in mouse liver,thus was a convenient,efficient method of liver-target transfer of exogenous gene in vivo.Thirdly,FHF was induced by intraperitoneal injections of lipopolysaccharide(LPS)and D-galactosamine(D-GalN)in rats.The optimal dose of drugs was found to establish an animal model.We demonstrated that a challenge with low dose LPS in conjunction with D-GalN could induce nonlethal but marked liver failure,the morphological feature of which is mainly hepatic apoptosis,which may be associated with high expression of iNOS(in the early)and p53 gene(in the mid and late stage).Fourthly,by an intravenous injection of the naked pEGFP-N2/Pim-3 plasmid using hydrodynamics-based procedure,the construct was found to achieve high expression in living cells and had an inhibitory effect on hepatic apoptosis in rats.Finally,through a challenge of lethal doses of LPS/D-GalN, one hundred percent lethality was induced in the model rats within 72h.Treatment with foreign Pim-3 gene by an in vivo transfer protected D-GalN-sensitized rats from LPS-induced liver damage and mortality.Hepatic enzymes showed dramatic increases in vector and Ringer's groups,whereas only a slight increases was observed in Pim-3-treated rats.Liver architecture was preserved,whereas few neutrophil infiltrates and hemorrhagic necrosis were observed in the treated rats. Liver apoptosis was also strikingly inhibited by Pim-3 treatment.Moreover,levels of inflammatory mediator TNF-αand IL-1βwere declined,and expression of iNOS and p53 were dramatically suppressed by exogenous Pim-3 gene.However, overexpression of Bcl-2 protein was observed in Pim-3-treated rats after administrating LPS/D-GalN challenge.These results suggest that exogenous Pim-3 gene can protect rats from LPS/D-GalN-induced FHF by inhibiting liver apoptosis and improving inflammatory response of liver tissues.PartⅠ:Cloning,plasmid construct of Pim-3 gene and its effects on eukaryotic cellsObjective To clone Pim-3 gene and construct its GFP-expressed plasmid,and to further investigate expression and activity of the construct in eukaryotic cells.Methods Pim-3 gene was cloned from myocardium tissues of juvenile Wistar rat by RT-PCR and subcloned into GFP-expressed plasmid vecter pEGFP-N2 by restriction enzyme.The recombinant plasmid pEGFP-N2/Pim-3 was constructed by T4-ligase and then identified by enzyme digestion and sequencing;Hepatoma SMMC7721 cells were cultured and passaged by conventional method,and primary rat hepatocytes were isolated by collegenase perfusion;The plasmid pEGFP-N2/Pim-3 containing Pim-3 and GFP gene was transfected into SMMC7721 cells and primary hepatocytes with cationic liposome;The efficiency of gene transfection was appraised by GFP expression under fluorescence microscope;Cell apoptosis and viability were detected by flow cytometry and MTT assay.Results pEGFP-N2/Pim-3 plasmid was successfully constructed;In SMMC7721 cells,GFP expression was observed at 24h and markedly enhanced at 48h after transfection of the construct;Apoptosis of hepatoma cells was significantly inhibited by exogenous Pim-3 gene.48h after transfection,apoptotic percentage of hepatoma cells in control,liposome,vector and construct groups were(10.2±6.3)%, (11.0±5.9)%,(10.7±4.1)%and(3.5±1.3)%,respectively.Compared with apoptotic percentage of other groups,that of the construct group was markedly lower(P＜0.01);MTT assay showed viability of hepatoma cells was significantly enhanced after transfection of exogenous Pim-3 gene;Primary hepatocytes were successfully obtained and showed GFP expression at 48h after plasmid transfection.By flow cytometry assay,survival rates of primary hepatocytes in control,liposome,vector and construct groups were respectively 74.12±11.20,69.87±13.45,72.03±12.47 and 90.11±9.27.Survival rates of the construct group were significantly higher than those of other groups(P＜0.01).Conclusions The constructed pEGFP-N2/Pim-3 plasmid can efficiently express in eukaryotic cells and have substantial effects of apoptotic inhibition or growth promotion on hepatoma cells and primary hepatocytes.PartⅡ:Organ-specific transfer of naked green fluorescent protein (GFP)-expressed plasmid by tail vein injection in miceObjective To investigate the target organ for gene transfer by an intravenous injection of GFP-expressed plasmid DNA using hydrodynamics-based or regular procedure.Methods Forty mice were randomly divided into three groups:normal control (n=8),hydrodynamics-based procedure(n=16)and regular procedure group(n=16). The mice in the latter two groups were further divided into transfer(n=8)and control group(n=8),respectively.24 h after tail vein injection,the tissue and blood samples were collected.The contents of serum alanine aminotransferase(ALT)and aspartate transaminase(AST)were detected by blood biochemistry meter.The expression levels of GFP in liver,spleen,heart,lung,kidney and brain were appraised under fluorescent microscope by frozen sections.Results The levels of serum transaminase between tail vein injection and normal control mice have no difference statistically;The hydrodynamics-based injection of naked GFP plasmid DNA into mice induces high expression level of GFP gene in liver,and approximately 45%of expression rate is achieved.However, the regular injection results in low expression level only in kidney,and no trace in liver,brain,lung,heart and spleen.Conclusions Hepatic delivery of foreign gene can be accomplished by hydrodynamics-based injection with simplicity and high efficiency,and GFP is a reliable and convenient tracer agent in the hydrodynamics-based procedure in mice.PartⅢ:Rat model of acute liver injury or hepatic failure induced by lipopolysaccharide in D-galactosamine-sensitized ratsObjective LPS is implicated in the pathology of acute liver injury and can induce lethal liver failure when simultaneously administered with D-GalN.Up to now,nonlethal liver failure,the liver injury of clinical implication,is incompletely understood after challenged by low dose LPS/D-GalN.Therefore,the aim of this article is to investigate effects of liver injury of nonlethal dose LPS/D-GalN and a role of apoptosis in this disorder.Methods Forty eight rats were randomly divided into three groups(16 for each).The rats in each group were further divided into treatment(n=8)and control (n=8).The rats in the treatment group received intraperitoneal injections of LPS (50μg/kg body wt)and D-GalN(300mg/kg body wt)in 1 ml stroke-physiological saline solution(SPSS),while the rats in control were treated with SPSS only.The blood and tissue samples were collected at 6,24 and 48h,respectively.The contents of serum ALT,AST and total bilirubin(TBIL)were detected by autoassay meter of blood biochemistry.The morphological changes were observed under light microscope and electromicroscope.The apoptosis of liver cells was dectected by TUNEL assay.The expression of TNF-α,IL-1β,iNOS and p53 gene were dectected by RT-PCR,and the activities of caspase-3,-8,-9 and-12 were measured.Results Blood biochemistry indexes,including ALT,AST and TBIL rose at 6h,reached the peak at 24h and sustained high levels at 48h after LPS/D-GalN injection.Abnormal liver appearance was found at 24h and 48h.Histopathological changes of hepatic injuries accompanied by hepatocellular death,inflammatory infiltration and hemorrhage began to appear at 6h,markedly aggravated at 24h and 48h.Cell apoptosis was significantly induced by nonlethal dose LPS/D-GalN challenge,and the apoptotic indexes(AIs)in 24h-and 48h-treated rats were approximately 70%by TUNEL assay.The inflammatory cytokine IL-1βmRNA levels rose markedly at 6h,and maintained high degrees at 24h and 48h,however, TNF-αlevels were normal in the liver tissues of 6h-,24h-and 48h-treated rats. mRNA expressions of damage gene iNOS were also induced early by LPS/D-GalN challenge and reached the peak at 6h and then gradually stepped down;contrarily, high expression levels of apoptosis-inducing gene p53 mRNA were not found in the early(6h)but emerged in the crest-time of liver apoptosis(24h)and maintained the level till the late stage(48h).In addition,we found that,in 24h-treated rats, caspase-3,-8,-9 and-12 were markedly activated by LPS/D-GalN challenge.Conclusion These results suggest that a challenge with low dose LPS in conjunction with D-GalN can induce nonlethal but marked liver failure,the morphological feature of which is mainly hepatic apoptosis,which may be associated with high expression of iNOS(in the early)and p53 gene(in the mid and late stage);and at least three apoptosis pathways participate in the pathogenesis.PartⅣ:In vivo expression and inhibitory role of hepatic apoptosis of GFP-expressed plasmid of rat serine/threonine kinase Pim-3 geneObjective To investigate the in vivo expression of the construct pEGFP-N2/Pim-3 and its effect on cell apoptosis in rat liver.Methods In vivo gene transfer to rat liver cells was achieved by an intravenous injection of the naked construct using a hydrodynamics-based procedure; The hepatic apoptosis was induced by intraperitoneal injections of LPS and D-GalN and detected by TUNEL assay and caspase-3 activity;The expression levels of reporter gene GFP and target gene Pim-3 were respectively appraised under fluorescent microscope and by RT-PCR. Results High expression of target gene Pim-3 and reporter gene GFP was achieved in rat liver after transfer of the recombinant plasmid;Acute liver apoptosis induced by LPS/D-GalN was reversed by the efficient in vivo expression of exogenous Pim-3 gene.Conclusions The construct pEGFP-N2/Pim-3 plasmid can achieve high expression in living cells and have an inhibitory effect on hepatic apoptosis in rat.PartⅤ:In vivo transfer of Pim-3-expressed plasmid protects against lipopolysaccharide/D-galactosamine-induced fulminant hepatic failureObjective To investigate the effects and mechanisms of serine/threonine kinase Pim-3 gene on fulminant hepatic failure.Methods Thirty-two rats were randomly divided into four groups(eight for each group).Group A was normal control.Group B,C and D were pretreated with Ringer's solution,vector plasmid and recombinant plasmid,respectively,and received intraperitoneal injections of LPS and D-GalN after 1 day.8h after injections of LPS/D-GalN,liver tissues and blood samples were collected;The contents of serum transaminase was tested by automatic blood biochemistry meter;The morphological changes were observed by light microscopy using hematoxylin and eosin(HE)staining;GFP expression levels were appraised under fluorescent microscope by frozen sections;Cell apoptosis of liver tissues was detected by the assay of TUNEL and Caspase-3 activity.Gene expression was detected by RT-PCR and Western blot,and protein secretion in serum was determined by enzyme linked immunosorbent assay(ELISA). Results 24h after LPS/D-GalN challenge,87.5%lethality was induced in group B and C,but only 12.5%in group D;Hepatic enzymes showed dramatic increases in group B and C,whereas only a slight increases was observed in Pim-3-treated rats;Overexpression of reporter gene GFP was induced by single rapid injection of a large amount of naked plasmid solution via rat tail vein (hydrodynamics-based gene transfer technique)and found in group C and D under fluorescence microscope.But group A and B had no trace of GFP;Histopathologic analysis performed on liver sections taken from group B and C after LPS/D-GalN challenge showed widespread destruction of liver architecture,erythrocyte agglutination and neutrophil infiltration.Numerous apoptotic cells were evidenced by the TUNEL assay.Strikingly,rats with Pim-3 gene before LPS/D-GalN injection were resistant to the lethal effect of the drugs' challenge.By marked contrast,liver apoptosis in Pim-3-treated rats was markedly inhibited,evidenced in isolated foci and Caspase-3 activity.Liver architecture was completely preserved,whereas few nentrophil or lymphoid infiltrates were observed;Expression of Pim-3 is constructively found in rat normal liver and inhibited by LPS/D-GalN challenge,but enhanced by in vivo transfer of exogenous Pim-3 gene;Inflammatory cytokines TNF-αand IL-1βsignificantly rised in liver tissues and systemic levels after injections of LPS and D-GalN,but this response was suppressed by in vivo transfer of Pim-3 gene;In addition,expressions of iNOS and p53(but not Bax)induced by LPS/D-GalN challenge were dramatically suppressed by exogenous Pim-3 gene. However,overexpression of Bcl-2 protein was observed in Pim-3-treated rats after administrating LPS/D-GalN challenge.Conclusions Exogenous Pim-3 gene can protect rats from LPS/D-GalN-induced fulminant hepatic failure by inhibiting liver apoptosis and improving inflammatory response of liver tissues,which can be associated with many mechanisms including inhibiting expression of inflammatory mediators such as TNF-αand IL-1β,injury gene iNOS and apoptosis-induced gene p53,and promoting expression of antiapoptosis gene Bcl-2.