罗格列酮对D-氨基半乳糖联合脂多糖诱导小鼠急性肝衰竭的保护作用

目的 观察罗格列酮对D-氨基半乳糖(D-GalN)联合脂多糖(LPS)诱导的小鼠急性肝衰竭的保护作用,并研究其可能的作用机制.方法 雄性昆明小鼠随机分为3组:正常组、对照组、治疗组.对照组和治疗组以D-GalN/LPS腹腔注射构建小鼠急性肝衰竭模型,正常组则相应予以生理盐水腹腔注射;治疗组于造模前2 h予以罗格列酮灌胃,正常组和对照组则相应予以生理盐水灌胃.比较各组小鼠24 h存活率、血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)水平、肝组织病变程度及肝组织中肿瘤坏死因子-α(TNF-α)和天冬氨酸特异半胱氨酸蛋白酶-3(Caspase-3)mRNA表达水平.结果 治疗组小鼠24 h存活率明显高十对照组(P<0.05),血清ALT、AST水平明显低于对照组(P<0.05);治疗组与对照组比较,肝组织炎性细胞浸润明显减少,肝细胞以变性为主,未见明显坏死;治疗组小鼠肝组织TNF-α、Caspase-3 mRNA表达水平明显低于对照组(P<0.05).结论 罗格列酮对D-GalN/LPS小鼠急性肝衰竭有保护作用,可改善肝细胞炎症和坏死.降低急性肝衰竭死亡率,其机制可能与罗格列酮下调TNF-α、Caspasc-3表达有关.     

罗格列酮对D-氨基半乳糖联合脂多糖诱导的小鼠急性肝衰竭的保护作用

目的观察罗格列酮对D-氨基半乳糖(D-GalN)联合脂多糖(LPS)诱导的小鼠急性肝衰竭的保护作用,并研究其可能的作用机制。方法雄性昆明小鼠随机分为三组:正常组、对照组和治疗组。对照组和治疗组以D-GalN/LPS腹腔注射构建小鼠急性肝衰竭模型,正常组则相应予以生理盐水腹腔注射;治疗组于造模前2 h予以罗格列酮灌胃,正常组和对照组则相应予以生理盐水灌胃。比较各组小鼠24 h存活率,检测血清丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)水平,肝组织病变程度及肝组织中肿瘤坏死因子-α(TNF-α)和天冬氨酸特异半胱氨酸蛋白酶-3(Caspase-3)mRNA的表达水平。结果治疗组小鼠24 h存活率明显高于对照组(P0.05),血清ALT、AST水平明显低于对照组(P0.05);治疗组与对照组比较,肝组织炎性细胞浸润明显减少,肝细胞以变性为主,未见明显坏死;治疗组小鼠肝组织中TNF-α、Caspase-3 mRNA表达水平明显低于对照组(P0.05)。结论罗格列酮对D-GalN/LPS小鼠急性肝衰竭有保护作用,可改善肝细胞炎症和坏死,降低急性肝衰竭的死亡率,其机制可能与罗格列酮下调TNF-α、Caspase-3的表达有关。     

IL-18、TNFα在脂多糖介导的急性肝坏死发生中的作用研究

目的探讨IL-18、TNFα在急性肝损伤中的作用.方法采用D-氨基半乳糖(D-Gal)900mg/kg与脂多糖(LPS)10μg/kg诱导BALB/C小鼠急性肝坏死动物模型,腹腔内注入D-Gal/LPS后0～9h分别检测血清转氨酶(ALT、AST)、肝组织病理、DNA梯形条带,评估肝损伤情况;用半定量RT-PCR和相应的分析软件分析不同时间点肝组织中IL-18 mRNA、TNFαmRNA表达及ELISA试剂盒检测血浆IL-18、TNFα的蛋白表达.结果D-Gal/LPS给予后4h血清转氨酶明显升高,7h小鼠开始死亡,10h死亡率达80%.肝组织病理学检查发现,5h肝窦扩张、炎性细胞浸润、库普弗细胞增生;7h肝细胞大量凋亡、坏死或肝组织出现大量出血性坏死;5h电镜示肝细胞核仁碎裂、线粒体肿胀或空泡变性;7h核仁边聚,呈半月型,表现为典型的凋亡形态学变化,线粒体大部分空泡变性.DNA电泳显示5h始出现梯形条带.正常情况下肝组织IL-18 mRNA少量表达,经D-Gal/LPS诱导后小鼠肝组织IL-18 mRNA表达量明显增加,表达高峰在LPS刺激后1～2h(与0h比较,P＜0.01);正常情况下肝组织TNFα mRNA不表达或呈微弱表达,但在该模型诱导过程中肝组织TNFα mRNA有不同程度的表达,且其表达的时间模式与IL-18 mRNA不同,TNFα mRNA表达高峰见于LPS刺激后2h和5h(与0h比较,P＜0.01).血浆IL-18、TNFα蛋白表达也与mRNA表达一致.结论本实验诱导的急性肝坏死模型中,肝细胞凋亡和坏死同时存在,在肝细胞坏死的发生过程中细胞因子IL-18 mRNA、TNFα mRNA及其蛋白在肝内的表达量明显增多,其变化早于转氨酶指标和组织学变化,而且在LPS刺激后IL-18 mRNA的表达先于TNFαmRNA,提示IL-18、TNFα均参与了此型肝损伤,且IL-18可能是TNFα的上游细胞因子.     

暴发性肝衰竭中Toll样受体2表达的实验研究

目的 分析D-氨基半乳糖(D-Gal)/脂多糖(LPS)诱导的暴发性肝衰竭模型中肝组织Toll样受体2(TLR2)的表达变化及与细胞因子白细胞介素-18(IL-18)、肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)表达的关系,探讨TLR2在启动炎性应答而致肝损伤中的作用。 方法 BALB/C小鼠腹内联合注射D-Gal 900 mg/kg与LPS 10μg/kg后观察其存活率,并检测不同时间点血清转氨酶和血浆IL-18、TNF-α和IFN-γ含量。用半定量逆转录-聚合酶链反应和Tanon Gis2.0软件分析各时间点肝组织中TLR2 mRNA表达,并与血浆IL-18、TNF-α、IFN-γ含量进行相关分析;免疫组织化学观察肝组织TLR2蛋白的表达。 结果 给药后4 h,血清转氨酶明显升高(与0 h比较,P<0.05);10 h小鼠死亡率达80％。血浆IL-1 8、TNF一α和IFN-γ含量逐步上升,IL-18在1 h即显著升高,之后持续高表达;TNF-α在2 h、5 h有两个分泌高峰;IFN-γ在2 h前增加不明显(F=2.5 7,P=0.1 3),但3 h及以后则显著升高(与0 h比较,P<0.01)。正常小鼠肝组织少量表达TLR2 mRNA,给药后1 h表达即显著增强(与0 h比较,P<0.05);免疫组织化学也显示TLR2蛋白有类似的变化,尤其肝窦内皮细胞、库普弗细胞表达更为显著;且部分肝细胞凋亡、坏死后,残存肝组织仍有较高TLR2表达。相关分析表明,肝组织TLR2 mRNA表达与     

肝纤维化能够保护小鼠抵抗D-GalN/LPS诱导的致死性损伤

目的:证明四氯化碳(carbon tetrachloride,CCl4)诱导的肝纤维化小鼠对致死性D-氨基半乳糖/脂多糖(D-galactosamine and lipopolysaccharide,D-GalN/LPS)攻击的耐受性.方法:建立CCl4诱导的肝纤维化小鼠模型,于纤维化6 wk时以致死剂量的D-GalN(700 mg/kg)/LPS(50μg/kg)进行攻击,以同样处理的正常小鼠作为对照,即实验共分为4组:正常对照组(Nor)、急性损伤组(Nor+D-GalN/LPS)、肝纤维化组(Fib)、肝纤维化+急性攻击组(Fib+D-GalN/LPS).根据攻击前后小鼠生存率、转氨酶水平及肝组织学的变化来评估正常和纤维化小鼠对致死性D-GalN/LPS损伤的耐受性.结果:生存分析显示,Fib+D-GalN/LPS组的生存率显著高于Nor+D-GalN/LPS组(100%vs20%).血清转氨酶结果表明,Fib+D-GalN/LPS组肝损伤程度明显轻于Nor+D-GalN/LPS组,其sALT水平分别为(6630 U/L±1675 U/L)和(22429 U/L±5446 U/L)(P<0.01).接受攻击的纤维化和正常小鼠的sALT分别升高了14.3倍和455.9倍.肝组织学检查结果也证明,接受致死性D-GalN/LPS攻击的纤维化小鼠的肝损伤较同样处理的正常小鼠明显减轻.结论:CCl4诱导的肝纤维化可保护小鼠抵抗致死性D-GalN/LPS损伤的攻击.     

HGF及其受体c-met在肝衰竭与部分肝切除模型中表达差异性研究

目的研究HGF及其受体c-met在肝衰竭和部分肝切除两种动物模型肝组织的表达差异。方法分别采用D-GalN/LPS腹腔注射和70%肝脏外科切除术建立肝衰竭和部分肝切除模型,同时以健康大鼠作为对照组。采用ELISA法检测血清HGF水平,采用Real-time PCR和Western-blot法分别检测肝组织c-met基因和蛋白的表达水平。结果与对照组比,部分肝切除模型大鼠血清HGF及其受体c-met表达水平均有明显升高（P〈0.01）,而在肝衰竭模型大鼠血清HGF水平虽有升高,但c-met蛋白表达水平却明显降低（P〈0.01）。结论肝细胞c-met表达水平的降低是D-GalN/LPS诱导肝衰竭大鼠肝细胞再生障碍的主要原因。     

Urotensin Ⅱ在急性肝衰竭小鼠肝组织中的表达及损伤作用

目的:探讨Urotensin Ⅱ(UⅡ)在急性肝衰竭(acute liver failure,ALF)小鼠肝组织中的表达及损伤作用.方法:♂Balb/c小鼠随机分成4组(每组6只):正常对照组(A组)、预处理对照组(B组)、模型组(C组)和预处理模型组(D组).模型动物以脂多糖(lipopolysaccharide,LPS)/D-半乳糖胺(D-galactosamine,D-GalN)腹腔注射,预处理动物在造模前30min,用UⅡ受体拮抗剂Urantide0.6mg/kg尾静脉注射.LPS/D-GalN攻击12h后,采集血清和肝组织标本,并观察24h小鼠存活情况;采用Reitman-Frankel法检测血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)和天冬氨酸氨基转移酶(aspartate amino-transferase,AST)活性水平;采用HE染色显微镜观察肝组织损伤程度;RT-PCR法检测UⅡ及其受体UTmRNA的表达;ELISA法检测血清UⅡ多肽分泌水平;免疫组织化学方法检测肝组织UⅡ多肽及其UT受体蛋白质表达.结果:C组小鼠死亡率为66.7%,A、B和D组所有动物均存活;LPS/D-GalN攻击引起C和D组小鼠血清ALT和AST水平显著升高(P<0.01),而D组较C组显著降低(2271.09U/L±102.24U/Lvs1160.67U/L±258.32U/L,1569.42U/L±204.04U/Lvs1030.31U/L±108.09U/L,P<0.01);C组小鼠肝组织结构破坏明显,见大片出血性坏死及炎症表现,D组肝组织结构保持完整,仅有局灶性出血坏死,炎症明显减轻;C和D组小鼠血清UⅡ多肽水平较A和B组高(P<0.01),但D组较C组明显降低(3.73g/L±0.52g/Lvs1.90g/L±0.27g/L,P<0.01);LPS/D-GalN诱导了C和D组小鼠肝组织UⅡ和UT的mRNA及蛋白质高水平表达,而D组的表达水平较C组显著降低(P<0.01).结论:LPS/D-GalN可诱导ALF小鼠肝组织表达和分泌UⅡ,并促进肝组织UT受体的表达;UⅡ的表达与分泌可能存在正反馈调控机制;UⅡ/UT受体介导了LPS/D-GalN诱导的ALF的发生.     

急性肝衰竭TNF-α、Caspase-3及TGF-β1 mRNA的表达及罗格列酮的干预

目的: 观察D-氨基半乳糖(D-Ga lN)联合脂多糖(LPS)诱导的小鼠急性肝衰竭TNF-α、Caspase-3、TGF-β1 mRNA的表达,并探讨罗格列酮的干预作用.方法: ♂昆明小鼠随机分为3组: 正常组、对照组、干预组.对照组和干预组以D-Ga lN/LPS腹腔注射构建小鼠急性肝衰竭模型,正常组则相应予以生理盐水腹腔注射;干预组于造模前2 h予以罗格列酮灌胃,正常组和对照组则相应予以生理盐水灌胃.比较各组小鼠血清丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)水平,肝组织病变程度及肝组织TNF-α、Caspase-3,TGF-β1 mRNA的表达水平.结果: D-GalN联合LPS腹腔注射成功构建了小鼠急性肝衰竭模型,对照组肝组织中TNF-α、Caspase-3、TGF-β1 mRNA的表达较正常组明显增高( P＜0.001);干预组小鼠血清ALT,AST水平明显低于对照组(403.6±76.1 U/L vs 3664.8±646.1 U/L,464.6±63.0 U/L vs 3514.0±468.9 U/L,均P＜0.001),肝组织中TNF-α、Caspase-3、TGF-β1 mRNA表达水平明显低于对照组(0.270±0.042 vs 0.459±0.072,0.388±0.033 vs 0.553±0.033,0.261±0.031 vs 0.403±0.042,均P＜0.001);与对照组比较,干预组肝组织炎性细胞浸润明显减少,肝细胞以变性为主,未见明显坏死.结论: 罗格列酮可能通过下调T N F - α、Caspase-3、TGF-β1的表达对D-GalN/LPS小鼠急性肝衰竭起保护作用.     

miR-122在D-氨基半乳糖联合脂多糖诱导急性肝衰竭小鼠体内的表达及其意义

目的 通过监测急性肝功能衰竭小鼠体内miR-122的表达,探讨miR-122与小鼠急性肝衰竭疾病程度和进展之间的关系,为肝功能衰竭的早期诊断提供新的生物学标志物. 方法将BALB/C小鼠随机分为4组,实验组用D-氨基半乳糖(D-GalN,900 mg/kg)联合脂多糖(LPS,10 μg/kg)腹腔注射建立肝衰竭模型,对照组3组,分别予以D-GalN(900 mg/kg),LPS(10 μg/kg)和等渗盐水腹腔注射,在不同时间点观察小鼠病死率、肝脏组织学变化,给药后0、1、3、5、7、9 h分别留取血清、肝脏组织标本,实时定量逆转录多聚酶链反应检测小鼠体内miRNA-122和炎症因子的表达,LNA(锁核酸)-Northern blot验证miRNA-122的表达,生化分析仪检测血清中ALT、AST水平,酶联免疫吸附法检测血清中炎症因子水平.组间均数比较用two-WayANOVA方差分析,相关性分析采用Pearson和Spearman相关分析.结果 D-GalN/LPS给药24 h,小鼠病死率率达80%以上,而3个对照组则无一只小鼠死亡;肝脏特异性miR-122在正常小鼠肝脏内含量丰富(ct≈14),D-GalN/LPS诱导后1 h,miR-122即发生了明显的变化(P=0.013),表现为上调,之后随疾病的进展,miR-122表达进行性下降,9 h下调最为明显(ct≈15,P=0.002);ALT/AST于给药1 h无明显变化,3 h后呈明显上升趋势,7 h达高峰,之后ALT/AST急剧下降;对miR-122和ALT的表达对比,发现在该模型中miR-122比ALT变化快,且持久;肝衰竭相关炎症因子肿瘤坏死因子(TNF)α和白细胞介素(IL)-6在肝组织和血清中的变化一致,均上调(P<0.05); miR-122和ALT、TNFα和IL-6的相关性分析显示miR-122与以上三项指标均呈良好的相关性(相关系数分别为-0.505、0.493和0.674、).结论 肝衰竭小鼠体内肝脏特异性miR-122和ALT呈负相关关系,但又较ALT更敏感,更持久地反映肝细胞损伤程度,且miR-122表达变化与肝脏炎症损伤相关因素TNF α、IL-6均具良好的相关性,推测miR-122有望成为判断急性肝衰竭肝细胞损伤程度的一个新的分子生物学标志物.     

Caspase-12在D-氨基半乳糖联合脂多糖诱导小鼠急性肝功能衰竭中的表达及作用

目的探讨Caspase-12在D-氨基半乳糖(D-Gal)/脂多糖(LPS)诱导小鼠急性肝功能衰竭发生发展过程中表达水平的变化及Caspase-12介导的内质网应激肝细胞凋亡途径在急性肝功能衰竭中的作用。方法以D-Gal/LPS联合腹腔注射诱导小鼠急性肝功能衰竭建立实验模型,在不同时间点动态检测血清氨基转移酶水平和观察肝组织病理变化,评估肝细胞凋亡和肝坏死演变过程;应用琼脂糖凝胶电泳检测肝细胞DNA凋亡条带;用半定量逆转录聚合酶链反应检测肝组织中Caspase-12 mRNA表达水平;west- ern blot检测Caspase-12、Bip/GRP78蛋白表达。结果药物诱导5h时肝组织中典型凋亡细胞增多,血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)开始升高,Caspase-12 mRNA表达明显增加, Caspase-12蛋白量表达反而减少;7h镜下出现大量肝细胞凋亡和坏死,ALT、AST水平达高峰,分别为(2564±1384)U/L和(1198±497)U/L,正常对照组为(59±17)U/L和(135±12)U/L,Caspase- 12 mRNA表达仍增加,Caspase-12蛋白表达量继续降低;9h 肝细胞坏死明显,伴散在凋亡细胞,ALT、AST水平明显下降,Caspase-12 mRNA表达较7 h下降。Bip/GRP78蛋自表达从5 h开始,至7 h逐渐增加。结论D-Gal/LPS诱导小鼠急性肝功能衰竭早期Caspase-12 mRNA表达水平逐渐升高,后期(7-9 h)降低,与肝细胞凋亡发生的时相一致;Caspase-12蛋白酶因内质网应激而被大量活化,提示Caspase-12介导的内质网应激肝细胞凋亡参与炎症性急性肝功能衰竭的发生发展,是急性肝功能衰竭中肝细胞损伤的重要机制之一,提示早期干预Caspase-12表达及活化对急性肝功能衰竭可能具有保护作用。     

雷帕霉素对急性肝功能衰竭大鼠Toll样受体-4表达的影响

目的 探讨雷帕霉素抑制Janus激酶/信号转导和转录激活子(JAK/STAT)通路对急性肝功能衰竭大鼠Toll样受体(TLR)-4基因表达的影响.方法采用腹腔注射D-氨基半乳糖(D-GalN)800 mg/kg和脂多糖(LPS)8 μg/只,建立急性肝功能衰竭大鼠模型,分别在注射D-GalN和LPS后2、6、12、24、48 h 5个时间点留取大鼠血及肝脏标本.SD大鼠分为对照组(n=6)、急性肝功能衰竭模型组(n=30)、STAT抑制剂雷帕霉素(RPM)干预组(n=30),在各不同时间点检测ALT、AST.ELISA法检测血清TNF-α、IL-6水平,RT-PCR法检测大鼠肝组织TLR-4 mRNA表达.数据行t检验.结果急性肝功能衰竭组大鼠在造模后2 h TNF-α、IL-6水平均显著升高,6 h达峰值,RPM可明显抑制TNF-α、IL-6水平.急性肝功能衰竭组大鼠6、12、24、48 h肝组织中TLR-4 mRNA分别为0.745±0.135、1.092±0.175、1.115±0.152和0.812±0.130,RPM干预后分别为0.545±0.118、0.798±0.124、0.857±0.109和0.595±0.152,各时间点两组比较,差异均有统计学意义(t值分别为2.726、3.349、3.382和2.567,均P＜0.05).TLR-4 mRNA表达与ALT、AST均呈正相关(r值分别为0.722、0.712,均P＜0.01).结论抑制JAK/STAT通路可明显下调急性肝功能衰竭大鼠肝组织TLR-4表达,JAK/STAT通路可能参与急性肝功能衰竭过程中TLR-4mRNA表达的调控.     

The therapeutic effect of CORM-3 on acute liver failure induced by lipopolysaccharide/D-galactosamine in mice

BACKGROUND: Acute liver failure(ALF) is a severe and lifethreatening clinical syndrome resulting in a high mortality and extremely poor prognosis. Recently, a water-soluble CO-releasing molecule(CORM-3) has been shown to have anti-inflammatory effect. The present study was to investigate the effect of CORM-3 on ALF and elucidate its underlying mechanism.METHODS: ALF was induced by a combination of LPS/D-Gal N in mice which were treated with CORM-3 or inactive CORM-3(i CORM-3). The efficacy of CORM-3 was evaluated based on survival, liver histopathology, serum aminotransferase activities(ALT and AST) and total bilirubin(TBi L). Serum levels of inflammatory cytokines(TNF-α, IL-6, IL-1β and IL-10) and liver immunohistochemistry of NF-κB-p65 were determined; the expression of inflammatory mediators such as i NOS, COX-2 and TLR4 was measured using Western blotting.RESULTS: The pretreatment with CORM-3 significantly improved the liver histology and the survival rate of mice compared with the controls; CORM-3 also decreased the levels of ALT, AST and TBi L. Furthermore, CORM-3 significantly inhibited the increased concentration of pro-inflammatory cytokines(TNF-α, IL-6 and IL-1β) and increased the anti-inflammatory cytokine(IL-10) productions in ALF mice. Moreover, CORM-3 significantly reduced the increased expression of i NOS and TLR4 in liver tissues and inhibited the nuclear expression of NF-κB-p65. CORM-3 had no effect on the increased expression of COX-2 in the ALF mice. An i CORM-3 failed to prevent acute liver damage induced by LPS/D-Gal N. CONCLUSION: These findings provided evidence that CORM-3 may offer a novel alternative approach for the management of ALF through anti-inflammatory functions.     

Sinapic acid ameliorates D-galactosamine/lipopolysaccharide-induced fulminant hepatitis in rats: Role of nuclear factor erythroid-related factor 2/heme oxygenase-1 pathways

BACKGROUND Sinapic acid(SA)has been shown to have various pharmacological properties such as antioxidant,antifibrotic,anti-inflammatory,and anticancer activities.Its mechanism of action is dependent upon its ability to curb free radical production and protect against oxidative stress-induced tissue injuries.AIM To study the hepatoprotective effects of SA against lipopolysaccharide(LPS)/Dgalactosamine(D-GalN)-induced acute liver failure(ALF)in rats.METHODS Experimental ALF was induced with an intraperitoneal(i.p.)administration of 8μg LPS and 800 mg/kg D-GalN in normal saline.SA was administered orally once daily starting 7 d before LPS/D-GalN treatment.RESULTS Data showed that SA ameliorates acute liver dysfunction,decreases serum levels of alanine transaminase(ALT),and aspartate aminotransferase(AST),as well as malondialdehyde(MDA)and NO levels in ALF model rats.However,pretreatment with SA(20 mg/kg and 40 mg/kg)reduced nuclear factor kappalight-chain-enhancer of activated B cells(NF-κB)activation and levels of inflammatory cytokines(tumor necrosis factor-αand interleukin 6).Also,SA increased the activity of the nuclear factor erythroid-related factor 2/heme oxygenase-1(Nrf2/HO-1)signaling pathway.CONCLUSION In conclusion,SA offers significant protection against LPS/D-GalN-induced ALF in rats by upregulating Nrf2/HO-1 and downregulating NF-κB.     

Sphingosine kinase 1 dependent protein kinase C-δ activation plays an important role in acute liver failure in mice

AIM: To investigate the role of protein kinase C (PKC)-δ activation in the pathogenesis of acute liver failure (ALF) in a well-characterized mouse model of D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced ALF.METHODS: BALB/c mice were randomly assigned to five groups, and ALF was induced in mice by intraperitoneal injection of D-GaIN (600 mg/kg) and LPS (10 μg/kg). Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at different time points within one week were determined using a multiparameteric analyzer. Serum levels of high-mobility group box 1 (HMGB1), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 as well as nuclear factor (NF)-κB activity were determined by enzyme-linked immunosorbent assay. Hepatic morphological changes at 36 h after ALF induction were assessed by hematoxylin and eosin staining. Expression of PKC-δ in liver tissue and peripheral blood mononuclear cells (PBMCs) was analyzed by Western blot.RESULTS: The expression and activation of PKC-δ were up-regulated in liver tissue and PBMCs of mice with D-GalN/LPS-induced ALF. Inhibition of PKC-δ activation with rottlerin significantly increased the survival rates and decreased serum ALT/AST levels at 6, 12 and 24 h compared with the control group (P < 0.001). Rottlerin treatment also significantly decreased serum levels of HMGB1 at 6, 12, and 24 h, TNF-α, IL-6 and IL-1 β at 12 h compared with the control group (P < 0.01). The inflammatory cell infiltration and necrosis in liver tissue were also decreased in the rottlerin treatment group. Furthermore, sphingosine kinase 1 (SphK1) dependent PKC-δ activation played an important role in promoting NF-κB activation and inflammatory cytokine production in ALF.CONCLUSION: SphK1 dependent PKC-δ activation plays an important role in promoting NF-κB activation and inflammatory response in ALF, and inhibition of PKC-δ activation might be a potential therapeutic strategy for this disease.     

Inhibition of sphingosine kinase 1 ameliorates acute liver failure by reducing high-mobility group box 1 cytoplasmic translocation in liver cells

AIM: To determine the therapeutic potential of sphingosine kinase 1(Sphk1) inhibition and its underlying mechanism in a well-characterized mouse model of D-galactosamine(D-Gal N)/lipopolysaccharide(LPS)-induced acute liver failure(ALF).METHODS: Balb/c mice were randomly assigned to different groups,with ALF induced by intraperitoneal injection of D-Ga IN(600 mg/kg) and LPS(10 μg/kg). The Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) levels at different time points within one week were determined using a multi-parametric analyzer. Serum high-mobility group box 1(HMGB1),tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6,IL-10,and sphingosine-1-phosphate were detected by enzyme-linked immunosorbent assay. Hepatic morphological changes at 36 h after acute liver injury induction were assessed by hematoxylin and eosin staining. HMGB1 expression in hepatocytes and cytoplasmic translocation were detected by immunohistochemistry. Expression of Sphk1 in liver tissue and peripheral blood mononuclear cells(PBMCs) was analyzed by Western blot.RESULTS: The expression of Sphk1 in liver tissue and PBMCs was upregulated in Gal N/LPS-induced ALF. Upregulated Sphk1 expression in liver tissue was mainly caused by Kupffer cells,the resident macrophages of the liver. The survival rates of mice in the N,Ndimethylsphingosine(DMS,a specific inhibitor of Sph K1) treatment group were significantly higher than that of the control group(P 0.001). DMS treatment significantly decreased the levels of serum ALT and AST at 6,12,and 24 h compared with that of the control group(P 0.01 for all). Serum HMGB1 levels at 6,12,and 24 h,as well as serum TNF-α,IL-6,and IL-1β levels at 12 h,were significantly lower in the DMS treatment group than in the control group(P 0.01 for all). Furthermore,hepatic inflammation,necrosis,and HMGB1 cytoplasm translocation in liver cells were significantly decreased in the DMS treatment group compared to the control group(43.72% ± 5.51% vs 3.57% ± 0.83%,χ2 = 12.81,P 0.01).CONCLUSION: Inhibition of Sph K1 ameliorates ALF by reducing HMGB1 cytoplasmic translocation in liver cells,and so might be a potential therapeutic strategy for this disease.