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1、Int J Clin Exp Pathol 2015;8(8):9062- ArticleEndotoxin tolerance alleviates experimental acute liver failure via inhibition of high mobility group box 1Nai-Bin Yang1,2,3,Shun-Lan Ni1,2,3,Shan-Shan Li1,2,3,Sai-Nan Zhang1,2,3,Dan-Ping Hu4,Ming-Qin Lu1,2,31Department of Infection Diseases,The First Aff
2、iliated Hospital of Wenzhou Medical University,Wenzhou 325000,Zhejiang,P.R.China;2Wenzhou Key Laboratory of Hepatology,Wenzhou 325000,Zhejiang,P.R.China;3Hepatology Institute of Wenzhou Medical University,Wenzhou 325000,Zhejiang,P.R.China;4Department of Infectious Diseases,The Third Affiliated Hospi
3、tal of Wenzhou Medical University,108 Wansong Road,Ruian 325200,Zhejiang,ChinaReceived June 11,2015;Accepted July 23,2015;Epub August 1,2015;Published August 15,2015Abstract:High mobility group box 1(HMGB1)has been widely reported to mediate damage caused by inflammatory responses.The aim of our stu
4、dy is to investigate the role of HMGB1 in endotoxin tolerance(ET)alleviating inflamma-tion of acute liver failure(ALF)rats and its possible signaling mechanism.To mimic ET,male Sprague-Dawley rats were pretreated with low dose of lipopolysaccharide(LPS)(0.1 mg/kg once a day intraperitoneally for con
5、secutive five days)before subsequent ALF induction.ALF was induced by intraperitoneal administration of D-GalN/LPS.ET induced by LPS pretreatment significantly improved the survival rate of ALF rats.Moreover,after ALF induction,ET+ALF rats exhibited lower serum enzyme(ALT,AST and TBiL)levels,lower p
6、roduction of inflammatory cytokines(IL-6,TNF-a and HMGB1)and more minor liver histopathological damage than ALF rats.ET+ALF rats showed en-hanced expression levels of HMGB1,decreased levels of STAT1 and p-STAT1,augmented expression of SOCS1 in liver tissues than ALF rats.These results indicated that
7、 ET induced by low-dose LPS pretreatment may alleviate inflammation and liver injury in experimental acute liver failure rats mainly through inhibition of hepatic HMGB1 translocation and release.Keywords:Endotoxin tolerance,high mobility group box 1,acute liver failure,JAK/STAT1 signaling,lipopolysa
8、ccha-ride(LPS)IntroductionAcute liver failure(ALF)is a clinical syndrome in which acute loss of metabolic and synthetic liver function leads to coagulopathy,hepatic encephalopathy and multiorgan failure within a short time in patients.Pathology of ALF is char-acterized by massive hepatocellular necr
9、osis and large scales of inflammation in liver 1.Despite considerable advances have been made in the understanding of its pathophysiol-ogy,ALF is still a serious clinical condition with an unacceptably high mortality rate.Currently,liver transplantation is the most effective treat-ment for ALF,altho
10、ugh shortage of donor organs limits this option.In addition,bioartifi-cial and artificial liver support systems are safe,beneficial but need to be evaluated in more detail 2.Therefore,further investigation is required so as to dissect the exact pathogene-sis of ALF.Disorder of immune balance is con-
11、sidered to contribute to formation and develop-ment of ALF,with massive release of various inflammatory cytokines from necrotic and apop-tosis hepatocytes 3.Endotoxin tolerance(ET)is defined as a reduced inflammatory response of the host(in vivo)or of cultured macrophage/monocyte(in vitro)to LPS fol
12、lowing a previous exposure to this stimu-lus.Although the phenomenon of ET was first described by Beeson 4 more than 60 years ago,its mechanism has still not been fully clari-fied,and thus is far from being applied in clini-cal practice.Recently,illumination of the relationship betw-een ET and some
13、inflammatory mediators sug-gests a new direction for its promising clinical application in diseases,such as sepsis 5.ET Endotoxin tolerance alleviates experimental acute liver failure9063 Int J Clin Exp Pathol 2015;8(8):9062-9071has been demonstrated to ameliorate experi-mental ALF 6,however,molecul
14、ar mechanism of protective effect of ET on ALF remains unclear.HMGB1,a protein generally found in the nucle-us that promotes DNA bending by binding to nucleosomes,has been reported to mediate damage caused by inflammatory responses 7.Aneja et al.demonstrated that preconditioning with low-dose recomb
15、inant HMGB1 induced endotoxin tolerance in mice 8.Moreover,endogenous HMGB1 has been confirmed as a major mediator in ET alleviating sepsis induced by lethal-dose LPS 7.We hypothesized that the protective effect of ET on ALF might rely on regulation of serum HMGB1.SOCS1 9,10,the negative regulator o
16、f JAK/STAT1 signaling path-way,is involved in the regulation of ET and JAK/STAT1 signaling promotes HMGB1 hyperacety-lation and nuclear translocation 11.In pres-ent study,we further confirmed the molecular mechanism of ET regulating translocation and release of HMGB1.Materials and methodsAnimalsMale
17、 Sprague-Dawley rats weighing 180-200 g were purchased from the Shanghai Laboratory Animal Center(Shanghai,China).All rats were housed at the Experimental Animal Laboratory of Wenzhou Medical University(Wenzhou,Zhejiang,China)under standard laboratory conditions(laminar-flow,temperature-controll-ed
18、by 21 2C,12 h light/dark cycle).The rats were fed with a standard chow diet and free water.They were conducted under the stan-dard procedure set by the Committee for the Purpose of Control and Supervision of Experi-ments on Animals and the National Institutes of Health for the specification use of t
19、he experi-mental animals.The research protocol was approved by the Animal Ethics Committee of Wenzhou Medical University.RegentsD-GalN(G0500-1 g)and LPS(L-2880-10 mg)were purchased from Sigma-Aldrich(St.Louis,MO,USA).Enzyme-linked immunosorbent assay(ELISA)kits for tumor necrosis factor-alpha(TNF-)a
20、nd interleukin-6(IL-6),BCA pro-tein assay kits were acquired from the Beyotime institute of biotechnology(Nanjing,China).Enzyme-linked immunosorbent assay(ELISA)kits for High Mobility Group Box 1(HMGB1)were from Shanghai Westang Bio-Tech Co,Ltd.(Shanghai,China).Antibodies against HMGB1 were purchase
21、d from Cell Signaling Technology(Danvers,MA,USA)and Antibodies against p-STAT1,STAT1,SOCS1 and GAPDH were obtained from Santa Cruz Biotechnology(CA,USA).Inhibitors of protease cocktail were acquired from Roche(Summerville,NJ,USA).Automatic biochemistry analyzer was pur-chased from Abbott Laboratorie
22、s,USA.Study design and groupingD-GalN and LPS were dissolved in sterile 0.9%sodium chloride according to the product description.The rats were randomly divided into three groups:control group(n=6),ALF group(n=40),ET+ALF group(n=20).For induc-tion of ET,rats in ET+ALF model were given 0.1 mg/kg LPS i
23、ntraperitoneally once every day for consecutive five days.Rats in the ALF group and control group were injected with the same volume of sterile 0.9%sodium chloride instead of LPS for the same treatment.For induction of ALF,rats in ET+ALF group and ALF group were given one intra-peritoneal injection
24、of 500 L saline containing 800 mg/kg D-GalN and 8 g LPS on the sixth day.Meanwhile,rats in control group were injected with the same volume of sterile 0.9%sodium chloride.In a separate experiment,survival rate was monitored for 3 days after injection of D-GalN/LPS in both ALF group(n=20)and ET+ALF g
25、roup(n=20).Except rats for the survival rate,all rats were finally sacrificed with chloral hydrate(1.0 g/kg,intra-peritoneally(i.p.)at 2,6,12,24,48 hours.Serum cytokine and biochemical markers as-saysAfter the experimental animals were sacrificed,blood of 2 ml was collected from the portal vein,30 m
26、inutes standing,centrifuged at 3000 r/min for 10 min,the upper serum was kept and stored in-80C deep-freezer.Serum concentrations of tumor necrosis factor-(TNF-a),interleukin-6(IL-6)and HMGB1 were deter-mined by using antibody enzyme-linked immu-nosorbent assay(ELISA)kits.All procedures were perform
27、ed in accordance with manufac-turer instructions.The levels of AST,ALT and total bilirubin(TBiL)in the serum were tested by an automatic biochemistry analyzer.Endotoxin tolerance alleviates experimental acute liver failure9064 Int J Clin Exp Pathol 2015;8(8):9062-9071Liver histological examinationAf
28、ter the blood collection,liver samples were dissected and fixed in 10%neutral formalin solution and embedded in paraffin.The embed-ded liver tissues were cut into 4 m sections,stained in hematoxylin and eosin(H&E)and viewed under a video microscope for histolo-pathological changes.Western blot analy
29、sisAfter the blood collection and livers dissect-ion for histological examination,livers were removed immediately,cut into pieces and stored at-80C for further research.The isola-tion of total protein was used lyses buffer(1 ml Radiation immune precipitation(RIPA)paraly-sis liquid,10 l PMSF,10 l sod
30、ium orthovana-date activating agent)accompanied by a inhibi-tor of protease cocktail.Samples were placed on ice bath for 30 minutes and centrifugated at 13000 r/min for 10 min,the supernatant was collected and stored at-80C for preservation,and the protein concentration was measured with BCA protein
31、 assay kit.The samples(15 g protein each)were suffered from polyacryl-amide gel electrophoresis(SDS-PAGE)after heat denaturation at 95C for 5 min.Sub-sequently,the target proteins in gel were shift-ed onto polyvinylidene fluoride membrane(PVDF)membranes.Then,the membranes were blocked by 5%bovine al
32、bumin(5%BSA)for 1.5 h at room temperature.The primary antibodies of HMGB1,p-STAT1,STAT1,SOCS1 and GAPDH were used to incubate the mem-branes at 4C overnight,respectively.The membranes were washed with TBST for 7 min-utes four times,incubated with the secondary antibody at room temperature for 1 h,wa
33、shed again with TBST for 7 minutes four times.Then the film exposure was performed after covered with chemiluminescent reagents.Statistical analysisAll data were analyzed with SPSS version 19.0(IBM,Chicago,USA)and expressed as means standard deviation(SD).Statistical significance was evaluated by on
34、e-way analysis of variance(ANOVA)or the least significant difference(LSD)test.P values less than 0.05 were considered statistically significant(P0.05).ResultsET induced by LPS pretreatment improved the survival rate of ALF ratsWe randomly adopted twenty rats for each group to exclusively investigate
35、 the effect of endotoxin tolerance on the survival rate of rats.The mortalities in 72 hours after injection of D-GalN/LPS or saline for each group were monitored.The mortality rate of rats in ALF group was 60%in 72 hours.Twelve in twenty rats of the ALF group died totally and majority of them died i
36、n the first 12 h.However,the mortality rate in ET+ALF group was 10%.Only two in twenty rats of the ET+ALF group died,at 5 h and 10 h,respectively.None of the twenty rats in control group died in 72 hours(Figure 1).Comparison of the mortality rates of 72 hours after injection of D-GalN/LPS or saline
37、in differ-ent groups clearly revealed that pre-existed endotoxin tolerance condition induced by LPS pretreatment significantly improved the surviv-al of ALF rats.Effect of ET induced by LPS pretreatment on histopathology characteristics of liver tissuesLiver sections from each group were stained wit
38、h hematoxylin and eosin(HE).In ALF group,liver tissues represented serious injuries with damaged hepatic lobules,ruined hepatic cords,and large numbers of infiltrated inflammatory cells,including mainly monocytes and lympho-Figure 1.Significant difference in survival rates was found between ALF grou
39、p and ET+ALF group by Kaplan-Meier survival analysis(P0.01 by Log-rank Test).Endotoxin tolerance alleviates experimental acute liver failure9065 Int J Clin Exp Pathol 2015;8(8):9062-9071cytes.Necrosis and apoptosis can also be found.However,liver injuries in ET+ALF group were less severe with mild n
40、ecrosis of hepato-cytes and less infiltrated inflammatory cells.Normal hepatic cells,central veins and lobules were shown in control group(Figure 2).We concluded pre-existed endotoxin tolerance condition induced by LPS pretreatment amelio-rated inflammation in liver of experimental ALF rats.ET induc
41、ed by low-dose LPS pretreatment al-leviated liver injuries in ALFTo study the effect of ET on liver function of experimental ALT rats,the levels of serum ALT,AST and TBiL in 48 h after ALF induction for each group were examined.Levels of ALT,AST and TBiL were elevated in both ALF group and ET+ALF gr
42、oup than that in control group.However,the increases in ALF group were sig-nificantly higher than that in ET+ALF group at 6 h,12 h,24 h,48 h(P0.05).Moreover,levels of ALT and AST in both groups reached the peak at 12 h and levels of TBiL continuously rise in 48 hours(Figure 3A-C).No obvious variatio
43、n occurred in control group.After comparing levels of serum markers for liver function in 48 hours,we concluded that pre-existed endotoxin tolerance condition induced by low-dose LPS pretreatment signifi-cantly alleviated liver injuries in ALF rats.ET induced by low-dose LPS pretreatment decreased s
44、erum levels of HMGB1 and pro-inflammatory cytokines in ALFPrevious studies have shown the release of cytoplasmic HMGB1 in activated immune cells and hepatocytes can activate macrophages to produce pro-inflammatory cytokines 12-14.To investigate the role of ET on alleviating inflam-mation of ALF rats
45、,we detected serum levels of TNF-,IL-6 and HMGB1 in 48 hours after ALF induction.Data indicated that TNF-,IL-6 levels started to increase immediately after injection of D-GalN/LPS and peaked at 6 hour in both ALF group and ET+ALF group when compared to the control group(P0.05).In addition,levels of
46、both TNF-a and IL-6 in ET+ALF group were lower than that in ALF group at each time point(P0.05).However,HMGB1 levels increased slowly during the first 6 hours and peaked at 12 hour in both groups.Also,levels of HMGB1 in ET+ALF group were lower than their counter-parts in ALF group at each time point
47、(P0.05)(Figure 3D-F).ET induced by low-dose LPS pretreatment en-hanced liver tissues levels of HMGB1 in ALFTo clarify whether alleviated inflammation and liver damage in ET+ALF rats were related to lev-els of HMGB1 in liver tissues,the protein levels of hepatic HMGB1 were measured by western blottin
48、g.As shown in Figure 4E,protein levels of hepatic HMGB1 gradually decreased after ALF induction and reached the nadir at 12 hour postinduction in both ALF group and ET+ALF group.Also,the levels of hepatic HMGB1 in both groups were decreased at every time point after ALF induction,as compared to the
49、control group(P0.05).However,relatively higher pro-tein levels of HMGB1 in ET+ALF group indicated that ET induced by low-dose LPS pretreatment could partly reverse the decline of hepatic HMGB1 in ALF group(P0.05).ET induced by low-dose LPS pretreatment re-duced expression of p-STAT1 and STAT1 in ALF
50、Researchers previously reported that activa-tion of JAK/STAT1 signaling promotes hyper-Figure 2.Representative photographs of liver sections using H&E staining(original magnification:200).A.Liver sections from the control group;B.Liver sections from the ALF group;C.Liver sections from the ET+ALF gro