HMGB1参与实验性急性肝、肺损伤的作用及其机制
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摘要
肝脏疾病(如病毒性肝炎、自身免疫性肝炎、酒精肝、肝纤维化及肝硬化等)严重危害人类健康。免疫性损伤是导致肝脏疾病的重要因素之一。ConA所致肝损伤模型由于其病理特征类似于人急性肝炎，被认为是研究人类病毒性肝炎和自身免疫性肝病的可靠模型。ConA与肝细胞膜有极强亲和性，注入体内后大量聚集于肝脏，招募并激活多克隆T细胞，并促进其释放多种细胞因子(如TNF-a和IFN-γ)，导致肝细胞损伤。
高迁移率族蛋白B1(high mobility group box，HMGB1)是一种核内蛋白，其分子含与DNA结合的2个结构域A-box和B-box，以及一个高度重复并富含酸性氨基酸的C-末端。HMGB1核内功能主要是使双螺旋极度扭曲以便各种转录因子和染色质相互作用，并可调节类固醇激素受体、NF-κB、p53及RAG1／2重组酶的转录活性。
研究发现，HMGB1可由坏死细胞(而非凋亡细胞)被动释放，或由受刺激的免疫细胞(包括单核／巨噬细胞、DC等)主动分泌至胞外。胞外HMGB1可通过其B盒与相应受体(RAGE、TLR2、TLR4)结合，从而发挥多种生物学效应：①直接介导非特异性炎症反应(包括早期和晚期炎症反应)；②作为一种警报素，激活DC，从而启动、增强适应性免疫应答，参与炎症性疾病如脓毒血症、急性肺损伤、某些自身免疫病如(系统性红斑狼疮、类风湿性关节炎)、同种移植排斥反应及多种病理过程发生和发展。
国内有文献报道，HMGB1参与D-GalN／LPS诱导的肝损伤。另有研究发现，注射Con A后1h，实验动物体内HMGB1 mRNA表达即增加。但HMGB1参与Con A所致肝损伤的确切作用及其机制，国内外均尚未见文献报道。
本课题建立Con A诱导的小鼠免疫性肝损伤模型，探讨HMGB1参与免疫性肝损伤的作用及其机制；在此基础上，制备抗HMGB1多克隆抗体和HMGB1特异性拮抗物HMGB1-A box重组蛋白，观察其对免疫性肝损伤的保护作用，并初步探讨其机制，为设计干预病毒性肝炎和自身免疫性肝炎的策略提供新思路。
一、HMGB1参与Con A所致急性肝损伤
尾静脉注射建立Con A诱导的肝损伤模型，设立PBS对照组。
1．Con A注射后不同时间点HMGB1 mRNA表达
Con A注射后2 h、6 h、12 h、24 h取小鼠肝脏组织，提取总RNA，RT-PCR检测HMGB1 mRNA表达水平。结果显示：与对照组比较，Con A处理后2 h，HMGB1表达即上调，持续至24 h，尤以2 h为明显。
2．肝组织HMGB1表达
Con A注射后12 h取小鼠肝脏，免疫组织化学分析肝组织HMGB1表达。结果发现：正常肝组织中，肝细胞和Kupffer细胞胞核内仅低表达HMGB1；急性肝损伤组Kupffer细胞和肝实质细胞胞核内HMGB1呈阳性表达，肝实质细胞坏死区内呈强阳性表达。
3．外源性HMGB1可加重Con A所致肝损伤
与单纯Con A(10μg／g，i．t．)组比较，非致死剂量的重组HMGB1(20μg／mouse，i．t．)和相同剂量Con A同时注射，其ALT值明显升高，而小剂量重组HMGB1(20μg／mouse)未引起小鼠ALT升高。
以上结果提示：HMGB1参与Con A诱导的急性肝损伤发生。
二、HMGB1特异性拮抗物的制备、纯化和功能鉴定
1．重组HMGB1免疫新西兰兔，制备多克隆抗体，并借助蛋白G柱纯化。
2．用本室黄亚非博士构建的PGEX-4T-2-A box原核表达载体，原核表达获得大量GST-A box融合蛋白；借助亲和纯化及去内毒素处理。
3．生物学实验证实，抗HMGB1多克隆抗体和重组r-Abox可有效拮抗HMGB1的生物学功能。
三、HMGB1特异性拮抗物对Con A所致急性肝损伤的保护作用及机制
1．抗HMGB1多克隆抗体对肝损伤小鼠的影响
与IgG对照组比较，抗HMGB1多克隆抗体处理(0．6mg／mouse，30 min前给药)能显著降低Con A所致肝损伤，表现为：血清谷丙转氨酶(ALT)明显降低；HE染色显示肝细胞水肿变性坏死和炎性浸润均明显较轻。
2．延迟给药对小鼠血清ALT水平的影响
相同剂量Con A(15μg／g)注射2 h后，再给予抗HMGB1多克隆抗体(0．6mg／mouse)处理小鼠，检测Con A注射12 h后小鼠血清ALT水平。结果发现：抗HMGB1多克隆抗体能明显降低肝损伤小鼠血清ALT水平。
3．抗HMGB1多克隆抗体对肝损伤小鼠生存的影响
抗HMGB1抗体预处理小鼠，然后尾静脉注射完全致死剂量Con A(25μg／g)，观察抗HMGB1抗体对小鼠生存的影响。结果发现：与PBS与IgG组比较，抗HMGB1多克隆抗体处理不仅能提高小鼠生存率，并能延长小鼠存活时间。
4．r-A box对肝损伤小鼠血清ALT水平的影响
与对照组比较，r-Abox处理(0．6mg／mouse，30 min前给药)能显著降低Con A所致肝损伤小鼠血清ALT水平。
5．抗HMGB1抗体对炎性细胞因子(TNF-α、IFN-γ)水平的影响
Con A注射后，小鼠血清TNF-α、IFN-γ含量明显增高；抗HMGB1抗体处理能显著降低TNF-α、IFN-γ含量；体外实验证实，抗HMGB1能抑制Con A诱导RAW264．7细胞分泌TNF-α的作用。
6．抗HMGB1抗体对NF-κB活性的影响
注射Con A 3 h后，提取肝组织细胞核蛋白，凝胶迁移率实验(EMSA)检测NF-κB活性。结果发现：急性肝损伤时，胞浆NF-κB被激活并向胞核内转位。经抗HMGB1多克隆抗体预处理的小鼠，其肝组织细胞核内NF-κB活性水平显著低于IgG对照组。
7．抗HMGB1抗体对肝细胞凋亡的影响
注射Con A 12 h后，切取肝组织，经4％多聚甲醛固定，制成石蜡切片，TUNEL法检测肝脏细胞凋亡。结果显示：Con A处理后，肝细胞凋亡增加，而经抗HMGB1抗体预处理的小鼠其细胞凋亡明显低于对照组。
8．抗HMGB1抗体对肝脏单个核细胞T、NKT细胞亚群的影响
注射Con A后0 h、6 h、12 h，分别取各处理组小鼠肝脏，分离肝脏MNC，双色标记检测T、NKT细胞亚群。结果发现，抗HMGB1抗体组与IgG对照组间T、NKT亚群无显著差异。
9．抗HMGB1抗体对肝脏单个核细胞T、NKT细胞活化的影响
注射ConA 12 h后，取小鼠肝脏，分离肝脏MNC，以CD69为标记检测T、NKT细胞活化状态。结果显示：
(1)抗HMGB1抗体处理组的CD69~+T细胞占T细胞比例明显低于IgG对照组。
(2)抗HMGB1抗体处理组的CD69~+NKT细胞占NKT细胞比例明显低于IgG对照组。
10．Con A诱导的肝损伤与TLR4(HMGB1受体)相关
Con A处理后6 h、12 h、24 h，可见TLR4表达，尤其以6 h为明显。注射相同剂量Con A，12 h处死动物，取血清检测ALT。结果发现：TLR4基因缺陷型小鼠(C3H／Hej)ALT水平明显低于TLR4野生型小鼠(C3H／HeOuj)。
四、HO-1通过抑制HMGB1表达减轻LPS所致肺损伤
HO-1诱导剂CoPPⅨ能显著增加肺组织HO-1活性，进而保护LPS所致急性肺损伤，表现为：肺组织中炎性细胞(尤其是中性粒细胞)浸润减少，肺W／D、肺组织EBA含量降低及肺组织损伤减轻等。其机制可能涉及HO-1上调可抑制TNF-a和HMGB1表达。
五、结论
1．HMGB1参与Con A所致急性肝损伤的发生与发展。
2．HMGB1特异性拮抗物能减轻Con A所致肝损伤，其机制可能为：抑制炎性因子释放；抑制NF-κB活性；抑制肝细胞凋亡；抑制T细胞、NKT细胞活化等。
3．HO-1诱导剂CoPPⅨ能显著增强肺组织HO-1活性，进而保护LPS所致急性肺损伤，其机制可能与HO-1抑制TNF-a和HMGB1表达有关。
Study of the Mechanism and Effect of Extracellular HMGB1on Acute Hepatic Injury and Lung Injury in Mice
Ph.D.Candidate:Gong Quan
Superviser:Prof.Gong Feili
Department of Immunology,Tongji Medical College of HuazhongUniversity of Science and Technology
Liver diseases,including viral hepatitis,autoimmune hepatitis,alcoholic liver injury,liver fibrosis and hepatic cirrhosis represent a worldwide health problem in humans.Theimmune-mediated liver injury plays an important role in various liver diseases.The ConA-induced hepatitis in mice,being characterized by an activation of CD4~+T cells and naturalkiller T(NKT) cells and markedly increased production of roinflammatory cytokine,such asTNF-a,IFN-γ,is similar to human immune-mediated hepatitis,Therefore,the Con A-inducedhepatitis in mice is one of model for studying hepatitis,especially viral hepatitis andautoimmune hepatitis.
High mobility group box 1 protein(HMGB1,previously HMG-1,amphoterin) has 215residues in its primary amino acid sequence,it is a highly conserved protein with 99% aminoacid identity between rodents and that of humans.HMGB1 has 2 DNA binding motifs:A boxand B box.Structure-function analyses revealed that the active cytokine domain of HMGB1is localized to the DNA-binding B box,whereas the A box competes with HMGB1 forbinding sites on the surface of activated macrophages and attenuates the biologic function ofthe full length HMGB1,so the recombinant A box(rA-box) acts as a specific antagonist ofHMGB1.Intracellular HMGB1 has been implicated in the regulation of gene transcriptionand in stabilizing nucleosome formation.
Extracellular HMGB1,released from injured or necrotic cells and monocytes/ macrophages stimulated with endotoxin or proinflammatory cytokines,has recently beenproposed as a nonclassical proinflammatory cytokine,and has been found to play a pivotalrole in the pathogenesis of proinflammatory diseases such as sepsis,acute lung injury,autoimmune diseases such as systemic lupus erythematosus(SLE),rheumatoid arthritis(RA),and acute allograft rejection.Anti-HMGB1 treatment using HMGB1 inhibitors has shownbeneficial effects in experimental model of sepsis,hepatic ischemia/reperfusion injury,rheumatoid arthritis and cardiac transplantation.
In current study,we have explored the change of HMGB1 in the Con A induced liverinjury in mice,Data presented here demonstrated that mRNA level of HMGB1 is increased asearly as 2 h after Con A injection,and reaches the peak value 2 h post Con A treatment.Andthe upregulation of HMGB1 is also confirmed by immunohistochemistry analysis.Furthermore,administration of exogenous recombinant HMGB1 worsens Con-A inducedhepatitis.These results suggest that HMGB1 contributes to the pathogenesis of Con Ainduced liver injury.
Based on the above observations,HMGB1 specific antagonists anti-HMGB1 polyclonalantibody and r-A box are produced,and given intraperitoneally 30 min before Con A injectionat the dose of 0.6 mg/mouse.As expected,specific antagonist of HMGB1 treatmentsignificantly reduces the increase of serum level of aminotransferase(ALT),and alsoattenuates the massive necrosis and infiltration of inflammatory cells induced by Con A.Furthermore,anti-HMGB1 antibody pretreatment protected mice from the lethality associatedwith lethal dose of Con A(25μg/g).These results suggest that HMGB1 specific antagonistsanti-HMGB1 polyclonal antibody and r-A box confer protective effect on Con A inducedhepatitis.
We next explored the possible mechanisms by which anti-HMGB1 treatment reducesliver injury induced by Con A.Our results shows that anti-HMGB1 treatment markedlyinhibits the increased production of proinflammatory cytokine TNF-a and IFN-γboth in vivoand in vitro and activation of NF-κB induced by Con A,and inhibits the Con A-inducedhepatocytes apoptosis,anti-HMGB1 treatment also dramatically suppressed the activation ofT cells(CD3~+NK1.1~-),NKT cells(CD3~+NK1.1~+) in liver,but fails to decrease ConA-induced subsets of T cells and NKT cells in liver as determined by flow cytometry. Interestingly,our data shows that,compared with TLR4-intact mice TLR4-defective micewere partly protected from Con A induced liver injury,suggests that HMGBl-mediated ConA induced hepatitis involves TLR4 system.
In addition,we investigate whether administration of CoPPⅨ,an HO-1 inducer,couldsignificantly inhibit TNF-αand Hmgbl expression and thus attenuate the acute lung injury(ALI) induced by lipopolysaccharide(LPS) in mice.Acute lung injury was inducedsuccessfully by intratracheal administration of LPS(0.5 mg/kg) in male BALB/c mice.CoPPⅨor ZnPPⅨ(an HO-1 inhibitor) was administered to mice 24 h prior to LPS exposure.It was found that CoPPⅨ(5,10 mg/kg,i.p.) caused a significant reduction in the total cellsand neutrophils in BALF,a significant reduction in the W/D ratio and EBA leakage at 24 hafter LPS challenge.Furthermore,the histopathologic findings indicated that alveolitis withleukocyte infiltration in the alveolar space was less severe in the CoPPⅨ-treated mice than inthe mice treated with LPS alone.In addition,CoPPⅨwas also believed to havedown-regulated the expression of LPS-induced proinflammatory cytokines,including earlyproinflammatory cytokine TNF-a,and late proinflammatory cytokine Hmgbl.In contrast,noobvious difference was observed between the ZnPPⅨgroup and the LPS group.Thesefindings demonstrate the significant protection of CoPPⅨagainst LPS-induced ALI,and theeffect mechanism of CoPPⅨwas associated with decreasing the expression of TNF-a andHmgbl.
In summary,our results demonstrate that Extracellular HMGB1 plays a pivotal role inCon A-induced hepatic injury.Administration of Neutralizing antibody to HMGB1significantly attenuates Con A-induced liver injury,and this protective effect is associatedwith reduced hepatocytes apoptosis,pro-inflammatory cytokines,and impaired activation of Tand NKT cells.The detrimental effect of HMGB1 is partly dependent on the activation ofTLR4 signaling.And we also find that Heme oxygenase-1 upregulation significantly inhibitsTNF-αand Hmgb1 releasing and attenuates lipopolysaccharide-induced acute lung injury inmice.
高迁移率族蛋白B1(high mobility group box，HMGB1)是一种核内蛋白，其分子含与DNA结合的2个结构域A-box和B-box，以及一个高度重复并富含酸性氨基酸的C-末端。HMGB1核内功能主要是使双螺旋极度扭曲以便各种转录因子和染色质相互作用，并可调节类固醇激素受体、NF-κB、p53及RAG1／2重组酶的转录活性。
研究发现，HMGB1可由坏死细胞(而非凋亡细胞)被动释放，或由受刺激的免疫细胞(包括单核／巨噬细胞、DC等)主动分泌至胞外。胞外HMGB1可通过其B盒与相应受体(RAGE、TLR2、TLR4)结合，从而发挥多种生物学效应：①直接介导非特异性炎症反应(包括早期和晚期炎症反应)；②作为一种警报素，激活DC，从而启动、增强适应性免疫应答，参与炎症性疾病如脓毒血症、急性肺损伤、某些自身免疫病如(系统性红斑狼疮、类风湿性关节炎)、同种移植排斥反应及多种病理过程发生和发展。
国内有文献报道，HMGB1参与D-GalN／LPS诱导的肝损伤。另有研究发现，注射Con A后1h，实验动物体内HMGB1 mRNA表达即增加。但HMGB1参与Con A所致肝损伤的确切作用及其机制，国内外均尚未见文献报道。
本课题建立Con A诱导的小鼠免疫性肝损伤模型，探讨HMGB1参与免疫性肝损伤的作用及其机制；在此基础上，制备抗HMGB1多克隆抗体和HMGB1特异性拮抗物HMGB1-A box重组蛋白，观察其对免疫性肝损伤的保护作用，并初步探讨其机制，为设计干预病毒性肝炎和自身免疫性肝炎的策略提供新思路。
一、HMGB1参与Con A所致急性肝损伤
尾静脉注射建立Con A诱导的肝损伤模型，设立PBS对照组。
1．Con A注射后不同时间点HMGB1 mRNA表达
Con A注射后2 h、6 h、12 h、24 h取小鼠肝脏组织，提取总RNA，RT-PCR检测HMGB1 mRNA表达水平。结果显示：与对照组比较，Con A处理后2 h，HMGB1表达即上调，持续至24 h，尤以2 h为明显。
2．肝组织HMGB1表达
Con A注射后12 h取小鼠肝脏，免疫组织化学分析肝组织HMGB1表达。结果发现：正常肝组织中，肝细胞和Kupffer细胞胞核内仅低表达HMGB1；急性肝损伤组Kupffer细胞和肝实质细胞胞核内HMGB1呈阳性表达，肝实质细胞坏死区内呈强阳性表达。
3．外源性HMGB1可加重Con A所致肝损伤
与单纯Con A(10μg／g，i．t．)组比较，非致死剂量的重组HMGB1(20μg／mouse，i．t．)和相同剂量Con A同时注射，其ALT值明显升高，而小剂量重组HMGB1(20μg／mouse)未引起小鼠ALT升高。
以上结果提示：HMGB1参与Con A诱导的急性肝损伤发生。
二、HMGB1特异性拮抗物的制备、纯化和功能鉴定
1．重组HMGB1免疫新西兰兔，制备多克隆抗体，并借助蛋白G柱纯化。
2．用本室黄亚非博士构建的PGEX-4T-2-A box原核表达载体，原核表达获得大量GST-A box融合蛋白；借助亲和纯化及去内毒素处理。
3．生物学实验证实，抗HMGB1多克隆抗体和重组r-Abox可有效拮抗HMGB1的生物学功能。
三、HMGB1特异性拮抗物对Con A所致急性肝损伤的保护作用及机制
1．抗HMGB1多克隆抗体对肝损伤小鼠的影响
与IgG对照组比较，抗HMGB1多克隆抗体处理(0．6mg／mouse，30 min前给药)能显著降低Con A所致肝损伤，表现为：血清谷丙转氨酶(ALT)明显降低；HE染色显示肝细胞水肿变性坏死和炎性浸润均明显较轻。
2．延迟给药对小鼠血清ALT水平的影响
相同剂量Con A(15μg／g)注射2 h后，再给予抗HMGB1多克隆抗体(0．6mg／mouse)处理小鼠，检测Con A注射12 h后小鼠血清ALT水平。结果发现：抗HMGB1多克隆抗体能明显降低肝损伤小鼠血清ALT水平。
3．抗HMGB1多克隆抗体对肝损伤小鼠生存的影响
抗HMGB1抗体预处理小鼠，然后尾静脉注射完全致死剂量Con A(25μg／g)，观察抗HMGB1抗体对小鼠生存的影响。结果发现：与PBS与IgG组比较，抗HMGB1多克隆抗体处理不仅能提高小鼠生存率，并能延长小鼠存活时间。
4．r-A box对肝损伤小鼠血清ALT水平的影响
与对照组比较，r-Abox处理(0．6mg／mouse，30 min前给药)能显著降低Con A所致肝损伤小鼠血清ALT水平。
5．抗HMGB1抗体对炎性细胞因子(TNF-α、IFN-γ)水平的影响
Con A注射后，小鼠血清TNF-α、IFN-γ含量明显增高；抗HMGB1抗体处理能显著降低TNF-α、IFN-γ含量；体外实验证实，抗HMGB1能抑制Con A诱导RAW264．7细胞分泌TNF-α的作用。
6．抗HMGB1抗体对NF-κB活性的影响
注射Con A 3 h后，提取肝组织细胞核蛋白，凝胶迁移率实验(EMSA)检测NF-κB活性。结果发现：急性肝损伤时，胞浆NF-κB被激活并向胞核内转位。经抗HMGB1多克隆抗体预处理的小鼠，其肝组织细胞核内NF-κB活性水平显著低于IgG对照组。
7．抗HMGB1抗体对肝细胞凋亡的影响
注射Con A 12 h后，切取肝组织，经4％多聚甲醛固定，制成石蜡切片，TUNEL法检测肝脏细胞凋亡。结果显示：Con A处理后，肝细胞凋亡增加，而经抗HMGB1抗体预处理的小鼠其细胞凋亡明显低于对照组。
8．抗HMGB1抗体对肝脏单个核细胞T、NKT细胞亚群的影响
注射Con A后0 h、6 h、12 h，分别取各处理组小鼠肝脏，分离肝脏MNC，双色标记检测T、NKT细胞亚群。结果发现，抗HMGB1抗体组与IgG对照组间T、NKT亚群无显著差异。
9．抗HMGB1抗体对肝脏单个核细胞T、NKT细胞活化的影响
注射ConA 12 h后，取小鼠肝脏，分离肝脏MNC，以CD69为标记检测T、NKT细胞活化状态。结果显示：
(1)抗HMGB1抗体处理组的CD69~+T细胞占T细胞比例明显低于IgG对照组。
(2)抗HMGB1抗体处理组的CD69~+NKT细胞占NKT细胞比例明显低于IgG对照组。
10．Con A诱导的肝损伤与TLR4(HMGB1受体)相关
Con A处理后6 h、12 h、24 h，可见TLR4表达，尤其以6 h为明显。注射相同剂量Con A，12 h处死动物，取血清检测ALT。结果发现：TLR4基因缺陷型小鼠(C3H／Hej)ALT水平明显低于TLR4野生型小鼠(C3H／HeOuj)。
四、HO-1通过抑制HMGB1表达减轻LPS所致肺损伤
HO-1诱导剂CoPPⅨ能显著增加肺组织HO-1活性，进而保护LPS所致急性肺损伤，表现为：肺组织中炎性细胞(尤其是中性粒细胞)浸润减少，肺W／D、肺组织EBA含量降低及肺组织损伤减轻等。其机制可能涉及HO-1上调可抑制TNF-a和HMGB1表达。
五、结论
1．HMGB1参与Con A所致急性肝损伤的发生与发展。
2．HMGB1特异性拮抗物能减轻Con A所致肝损伤，其机制可能为：抑制炎性因子释放；抑制NF-κB活性；抑制肝细胞凋亡；抑制T细胞、NKT细胞活化等。
3．HO-1诱导剂CoPPⅨ能显著增强肺组织HO-1活性，进而保护LPS所致急性肺损伤，其机制可能与HO-1抑制TNF-a和HMGB1表达有关。
Study of the Mechanism and Effect of Extracellular HMGB1on Acute Hepatic Injury and Lung Injury in Mice
Ph.D.Candidate:Gong Quan
Superviser:Prof.Gong Feili
Department of Immunology,Tongji Medical College of HuazhongUniversity of Science and Technology
Liver diseases,including viral hepatitis,autoimmune hepatitis,alcoholic liver injury,liver fibrosis and hepatic cirrhosis represent a worldwide health problem in humans.Theimmune-mediated liver injury plays an important role in various liver diseases.The ConA-induced hepatitis in mice,being characterized by an activation of CD4~+T cells and naturalkiller T(NKT) cells and markedly increased production of roinflammatory cytokine,such asTNF-a,IFN-γ,is similar to human immune-mediated hepatitis,Therefore,the Con A-inducedhepatitis in mice is one of model for studying hepatitis,especially viral hepatitis andautoimmune hepatitis.
High mobility group box 1 protein(HMGB1,previously HMG-1,amphoterin) has 215residues in its primary amino acid sequence,it is a highly conserved protein with 99% aminoacid identity between rodents and that of humans.HMGB1 has 2 DNA binding motifs:A boxand B box.Structure-function analyses revealed that the active cytokine domain of HMGB1is localized to the DNA-binding B box,whereas the A box competes with HMGB1 forbinding sites on the surface of activated macrophages and attenuates the biologic function ofthe full length HMGB1,so the recombinant A box(rA-box) acts as a specific antagonist ofHMGB1.Intracellular HMGB1 has been implicated in the regulation of gene transcriptionand in stabilizing nucleosome formation.
Extracellular HMGB1,released from injured or necrotic cells and monocytes/ macrophages stimulated with endotoxin or proinflammatory cytokines,has recently beenproposed as a nonclassical proinflammatory cytokine,and has been found to play a pivotalrole in the pathogenesis of proinflammatory diseases such as sepsis,acute lung injury,autoimmune diseases such as systemic lupus erythematosus(SLE),rheumatoid arthritis(RA),and acute allograft rejection.Anti-HMGB1 treatment using HMGB1 inhibitors has shownbeneficial effects in experimental model of sepsis,hepatic ischemia/reperfusion injury,rheumatoid arthritis and cardiac transplantation.
In current study,we have explored the change of HMGB1 in the Con A induced liverinjury in mice,Data presented here demonstrated that mRNA level of HMGB1 is increased asearly as 2 h after Con A injection,and reaches the peak value 2 h post Con A treatment.Andthe upregulation of HMGB1 is also confirmed by immunohistochemistry analysis.Furthermore,administration of exogenous recombinant HMGB1 worsens Con-A inducedhepatitis.These results suggest that HMGB1 contributes to the pathogenesis of Con Ainduced liver injury.
Based on the above observations,HMGB1 specific antagonists anti-HMGB1 polyclonalantibody and r-A box are produced,and given intraperitoneally 30 min before Con A injectionat the dose of 0.6 mg/mouse.As expected,specific antagonist of HMGB1 treatmentsignificantly reduces the increase of serum level of aminotransferase(ALT),and alsoattenuates the massive necrosis and infiltration of inflammatory cells induced by Con A.Furthermore,anti-HMGB1 antibody pretreatment protected mice from the lethality associatedwith lethal dose of Con A(25μg/g).These results suggest that HMGB1 specific antagonistsanti-HMGB1 polyclonal antibody and r-A box confer protective effect on Con A inducedhepatitis.
We next explored the possible mechanisms by which anti-HMGB1 treatment reducesliver injury induced by Con A.Our results shows that anti-HMGB1 treatment markedlyinhibits the increased production of proinflammatory cytokine TNF-a and IFN-γboth in vivoand in vitro and activation of NF-κB induced by Con A,and inhibits the Con A-inducedhepatocytes apoptosis,anti-HMGB1 treatment also dramatically suppressed the activation ofT cells(CD3~+NK1.1~-),NKT cells(CD3~+NK1.1~+) in liver,but fails to decrease ConA-induced subsets of T cells and NKT cells in liver as determined by flow cytometry. Interestingly,our data shows that,compared with TLR4-intact mice TLR4-defective micewere partly protected from Con A induced liver injury,suggests that HMGBl-mediated ConA induced hepatitis involves TLR4 system.
In addition,we investigate whether administration of CoPPⅨ,an HO-1 inducer,couldsignificantly inhibit TNF-αand Hmgbl expression and thus attenuate the acute lung injury(ALI) induced by lipopolysaccharide(LPS) in mice.Acute lung injury was inducedsuccessfully by intratracheal administration of LPS(0.5 mg/kg) in male BALB/c mice.CoPPⅨor ZnPPⅨ(an HO-1 inhibitor) was administered to mice 24 h prior to LPS exposure.It was found that CoPPⅨ(5,10 mg/kg,i.p.) caused a significant reduction in the total cellsand neutrophils in BALF,a significant reduction in the W/D ratio and EBA leakage at 24 hafter LPS challenge.Furthermore,the histopathologic findings indicated that alveolitis withleukocyte infiltration in the alveolar space was less severe in the CoPPⅨ-treated mice than inthe mice treated with LPS alone.In addition,CoPPⅨwas also believed to havedown-regulated the expression of LPS-induced proinflammatory cytokines,including earlyproinflammatory cytokine TNF-a,and late proinflammatory cytokine Hmgbl.In contrast,noobvious difference was observed between the ZnPPⅨgroup and the LPS group.Thesefindings demonstrate the significant protection of CoPPⅨagainst LPS-induced ALI,and theeffect mechanism of CoPPⅨwas associated with decreasing the expression of TNF-a andHmgbl.
In summary,our results demonstrate that Extracellular HMGB1 plays a pivotal role inCon A-induced hepatic injury.Administration of Neutralizing antibody to HMGB1significantly attenuates Con A-induced liver injury,and this protective effect is associatedwith reduced hepatocytes apoptosis,pro-inflammatory cytokines,and impaired activation of Tand NKT cells.The detrimental effect of HMGB1 is partly dependent on the activation ofTLR4 signaling.And we also find that Heme oxygenase-1 upregulation significantly inhibitsTNF-αand Hmgb1 releasing and attenuates lipopolysaccharide-induced acute lung injury inmice.
引文
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[1] Matzinger P. The Danger Model: A Renewed Sense of Self [J]. Science, 2002, 296:301-305.
[2] Medzhitov R, Janeway CA. Decoding the patterns of self and nonself by the innate immune system. Science. 2002; 296: 298- 300.
[3] Matzinger P. Tolerance, danger, and the extended family Annu Rev Immunol 1994;12:991-1045.
[4] Goodwin GH, Sanders C, Johns EW. A new group of chromatin-associated proteins with a high content of acidic and basic amino acids. Eur J Biochem. 1973, 38:14-19.
[5] Bianchi ME, Manfredi AA. High-mobility group box 1 (HMGB1) protein at the crossroads between innate and adaptive immunity[J]. Immunol Rev, 2007,220: 35-46.
[6] Thomas JO, Travers AA. HMG1 and 2, and related 'architectural' DNA-binding proteins.Trends Biochem Sci 2001;26:167-174.
[7] Dumitriu IE, Baruah P, Manfredi AA. HMGB1: Guiding immunity from within. Trends Immunol 2005; 26: 381-87.
[8] Calogero S, Grassi F, Aguzzi A, et al. The lack of chromosomal protein Hmgl does not disrupt cell growth, but causes lethal hypoglycaemia in newborn mice. Nat Genet 1999;22:276-80.
[9] M(?)ller S, Ronfani L, Bianchi ME. Regulated expression and subcellular localization of HMGB1, a chromatin protein with a cytokine function. J Intern Med 2004;255:332-343.
[10] Miiller S, Scaffidi P, Degryse B, et al. The double life of HMGB1 chromatin protein:architectural factor and extracellular signal. EMBO J 2001 ;20: 4337-4340.
[11] Bianchi ME, Agresti A. HMG proteins: dynamic players in gene regulation and differentiation. Curr Opin Genet Dev 2005;l5:496-506.
[12] Bianchi, M.E. Significant (re)location: how to use chromatin and/or abundant proteins as messages of life and death. Trends Cell Biol. 2004; 14:287-293.
[13]Muller S, Ronfani L, Bianchi ME. Regulated expression and subcellular localization of HMGB1, a chromatin protein with a cytokine function. J Intern Med. 2004, 255(3):332-43
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[2] Medzhitov R, Janeway CA. Decoding the patterns of self and nonself by the innate immune system. Science. 2002; 296: 298- 300.
[3] Matzinger P. Tolerance, danger, and the extended family Annu Rev Immunol 1994;12:991-1045.
[4] Goodwin GH, Sanders C, Johns EW. A new group of chromatin-associated proteins with a high content of acidic and basic amino acids. Eur J Biochem. 1973, 38:14-19.
[5] Bianchi ME, Manfredi AA. High-mobility group box 1 (HMGB1) protein at the crossroads between innate and adaptive immunity[J]. Immunol Rev, 2007,220: 35-46.
[6] Thomas JO, Travers AA. HMG1 and 2, and related 'architectural' DNA-binding proteins.Trends Biochem Sci 2001;26:167-174.
[7] Dumitriu IE, Baruah P, Manfredi AA. HMGB1: Guiding immunity from within. Trends Immunol 2005; 26: 381-87.
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[9] M(?)ller S, Ronfani L, Bianchi ME. Regulated expression and subcellular localization of HMGB1, a chromatin protein with a cytokine function. J Intern Med 2004;255:332-343.
[10] Miiller S, Scaffidi P, Degryse B, et al. The double life of HMGB1 chromatin protein:architectural factor and extracellular signal. EMBO J 2001 ;20: 4337-4340.
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