骨髓间充质干细胞旁分泌胰岛素样生长因子1促受损肝L02细胞增殖的效应及其分子机制

目的 探讨骨髓间充质干细胞(BM-MSCs)旁分泌胰岛素样生长因子1(IGF-1)促受损肝L02细胞增殖作用的分子机制. 方法 利用Transwell小室建立MSCs与D-氨基半乳糖(D-GalN)损伤L02肝细胞的非接触式共培养模型,四甲基偶氮唑盐法检测BM-MSCs对D-GalN损伤组L02细胞增殖的影响,酶联免疫吸附法检测共培养体系中IGF-1的表达水平及其主要来源细胞,Western blot 检测损伤L02细胞的IGF-1受体(IGF-IR)表达水平及外源性和BM-MSCs源性IGF-1对D-GalN损伤L02细胞增殖的影响.两组间数据比较采用t检验,两组以上数据的比较采用方差分析. 结果 共培养条件下的BM-MSCs能非接触依赖性地促进D-GalN损伤组L02细胞增殖,其在24、48、72 h的吸光度值分别为0.60±0.09、0.82±0.05、0.90±0.06,明显高于D-GalN损伤组在相应时间点的0.36±0.08、0.52±0.06、0.68±0.06(t值分别为2.493、3.116、2.285,P值均＜0.05).经D-GalN损伤组L02细胞分泌上清液处理48 h的BM-MSCs的IGF-1分泌量为(156±24) ng/ml,与D-GalN损伤组L02细胞共培养48 h的BM-MSC IGF-1分泌量为(185±36) ng/ml,均较正常BM-MSCs分泌量(19±9)ng/ml明显上升(t值分别为5.345和4.473,P值均＜0.01).外源性rh-IGF-1能明显促进L02细胞增殖,且在160 ng/ml时达到最大效应,处理D-GalN损伤L02细胞96h的吸光度值为1.70±0.09,明显高于rh-IGF-1浓度0 ng/ml组的0.79±0.09(t=9.460,P＜0.05).BM-MSCs与D-GalN损伤L02细胞共培养组在加入IGF-1R单克隆抗体处理前后,24、48、72 h时的吸光度值分别为1.80±0.11比1.30±0.16、1.70±0.12比1.30±0.10、1.69±0.11比1.25±0.12,IGF-1R单克隆抗体处理后的L02细胞增殖明显被抑制(t值分别为2.909、2.328、2.560,P值均＜0.05).结论 BM-MSCs源性IGF-1通过结合损伤L02细胞的IGF-1R,在BM-MSCs的旁分泌促受损肝L02细胞增殖中发挥主要作用.     

复方甘草酸单铵和异甘草酸镁对化学损伤人肝细胞的保护作用

目的:研究2 类甘草甜素药物对半乳糖胺( D-GalN)和四氯化碳(CCl4)损伤培养人肝细胞(L-02)的保护作用及差异.方法:培养L-02, 用复方甘草酸单铵(compoundammonium glycyrrhizin, CAG)和异甘草酸镁(magnesium isoglycyrrhizinate, MI)分别进行保护后, 再经D-GalN或CCl4处理. 观察肝细胞生长状态、测定AST、LDH酶活力、及细胞内谷胱甘肽(GSH)含量. 从而评价CAG和MI对D-GalN和CCl4损伤L-02的保护作用差异.结果:浓度为1 g/L的CAG和MI均能提高细胞的存活率, 显著抑制D-GalN和CCl4所致的AST及LDH释放. CAG抑制D-GalN和CCl4所致的AST与LDH效果显著好于MI(均P<0.05);浓度为1 g/L的CAG和MI均能显著抑制2种化学损伤细胞内的GSH降低(CCl4损伤:7.59±1.27, 5.23±0.70 vs 3.33±0.40; D-GalN损伤:7.93±0.36, 5.40±0.52 vs 3.77±0.45, P<0.01或0.05), CAG效果明显好于MI.结论:1 g/L的CAG和MI 2种药物对D-GalN和CCl4致人肝细胞损伤均有保护作用, 其机制与抑制GSH降低相关. 2种甘草甜素药物相比较,CAG保护肝细胞效果好于MI.     

大黄对急性肝衰竭大鼠肝细胞凋亡的影响

目的 探讨大黄对急性肝衰竭大鼠肝细胞凋亡的影响及可能机制.方法 Wistar大鼠随机分为4组:正常对照组、模型组、大黄组和促肝细胞生长素(PHGF)组.大黄组和PHGF组于造模前3d分别每天灌服大黄水煎液和皮下注射PHGF 1 ml/100 mg,正常组和模型组每天灌服0.9％氯化钠注射液1 ml/100 mg.采用D-氨基半乳糖(D-GalN)/内毒素脂多糖(LPS)腹腔注射制备大鼠急性肝衰竭模型,造模8h后开腹取大鼠肝组织作病理检查,HE染色光镜下观察大鼠肝组织病理学变化,应用TUNEL法检测肝细胞凋亡情况,并应用免疫组化法分别检测肝组织中Fas、FasL和caspase-3的表达.结果 D-GαlN和LPS可引起严重的急性肝坏死并有广泛的肝细胞凋亡,伴Fas、FasL和caspaSe-3在肝细胞中强烈表达,大黄能减轻肝组织损伤,并可降低肝细胞凋亡及Fas、FasL和caspase-3表达(P均＜0.05).结论 大黄可抑制急性肝衰竭大鼠肝细胞凋亡,其机制可能与减轻肝细胞Fas、FasL和caspase-3表达有关.     

大片形吸虫排泄分泌产物对体外培养LO2人肝细胞的影响

目的初步探讨大片形吸虫排泄分泌产物(FgESP)对体外培养LO2人肝细胞的增殖及对肝细胞损伤相关的生化指标的影响。方法将LO2人肝细胞分为5个密度组(1 000、 3 000、 5 000、 7 000、 10 000个/孔),铺于96孔板中,空白对照组只加入培养基,根据LO2细胞生长曲线选择最适细胞铺板密度。在96孔板中,以最适细胞密度铺板,分为0.02、 0.10、 0.20、 0.40、 1.00 mg/ml FgESP等5组,每组设4个重复,以PBS作为阴性对照组,在培养箱中孵育4、 8、 12、 24、 48和72 h后进行MTT细胞活性检测,测吸光度(A_(490)值)。在24孔板中,分为0.02、 0.10、 0.20、 0.40、 1.00 mg/ml FgESP等5组,每组设3个重复,以PBS作为阴性对照组,分别在培养4、 8、 12、 24、 48和72 h后,收集LO2细胞上清,测定碱性磷酸酶(ALP)、天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转氨酶(ALT)、白蛋白(ALB)的含量。采用GraphPad Prism 6.0软件进行统计学分析。结果根据LO2人肝细胞生长曲线,筛选出5 000个/孔为96孔板最佳铺板密度。FgESP作用72 h后, MTT细胞活性检测结果显示, 0.02、 0.10、 0.20、 0.40、 1.00 mg/ml FgESP等5组的A_(490)值分别为1.29±0.01、 1.28±0.06、1.13±0.08、 0.97±0.06、 0.25±0.01,均低于对照组(1.45±0.05)(P 0.01)。生化指标检测结果显示, 0.40、1.00 mg/ml FgESP作用细胞LO2人肝72 h后, ALT含量分别为2.00±0.00、 3.67±0.58, AST含量分别为5.33±0.58、7.67±0.58,均高于对照组的(P 0.01);作用48 h后, 1.00 mg/ml FgESP组ALT、 AST含量分别为2.00±0.00、7.00±0.00,高于对照组的0.33±0.58、 3.67±0.58 (P 0.01);在12～72 h内, 0.10、 0.20、 0.40、 1.00 mg/ml FgESP作用后ALP含量升高(P 0.01),而0.02 mg/ml FgESP仅在作用72 h后ALP含量升高(P 0.01);各实验组ALB含量与阴性对照组差异无统计学意义(P 0.05)。结论 FgESP体外作用可抑制LO2人肝细胞增殖能力,且细胞增殖的抑制及损伤程度与FgESP的浓度相关。     

大黄素改善D氨基半乳糖胺造成的L02细胞损伤

目的:探讨大黄素对急性肝细胞损伤的防护作用。方法:运用网络药理学筛选大黄素防护急性肝损伤的潜在靶点和通路。将L02细胞分为对照组、模型组、治疗组，对照组细胞予以普通培养液培养，模型组细胞培养液中加入浓度为40 mmol/L的D氨基半乳糖胺(D-GalN)干预12 h诱导急性肝细胞损伤模型，治疗组细胞在模型组的基础上预先加入10μmol/L的大黄素干预12 h。采用实时荧光定量PCR检测各组细胞SLC2A1、SLC2A4和Caspase-3基因表达情况，流式细胞术检测细胞凋亡，蛋白印迹法法检测Cleaved-Caspase-3和BCL2蛋白的表达。结果:网络药理学提示大黄素可以通过调节细胞凋亡信号通路，代谢相关信号通路干预急性肝衰竭。实验结果表明模型组肝细胞凋亡率均显著高于对照组(P<0.001);治疗组与模型组相比，肝细胞凋亡率显著降低(P<0.001);模型组细胞的SLC2A1、Caspase-3基因表达量上调(P<0.01),Cleaved-Caspase-3蛋白表达明显高于对照组；治疗组细胞的SLC2A1和Caspase-3基因表达量较模型组显著下调(P<...     

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.     

粒细胞集落刺激因子对大鼠骨髓间充质干细胞增殖的影响

目的探讨粒细胞集落刺激因子（G-CSF）对骨髓间充质干细胞（BMSC）增殖的影响及其在BMSC向神经元样细胞分化中的作用。方法取16只SD大鼠的骨髓,体外培养BMSC,采用差速贴壁法分离细胞,并进行鉴定。按不同浓度给予G-CSF,分为四组（G0-3组）,G0组不含G-CSF,设为阴性对照;G1～3组分别含G-CSF5、10、20ng/ml,按1×10^5/ml密度接种于平底96孔培养板中（每孔100μl）。MTT法测定各组加入G-CSF第1、3、5、7天吸光度（A值）的变化,观察G-CSF对BMSC增殖的影响。以碱性成纤维细胞生长因子（bFGF）预诱导24h后,按神经胶质细胞衍化营养因子（GDNF）和不同浓度G-CSF组合分组（B1-5）进一步诱导,B1不含GDNF、G-CSF,镜下观察细胞形态变化。反转录-聚合酶链反应（RT-PCR）检测神经细胞标记物GFR-α1的表达。结果①在给予G-CSF第1、3、5、7天,G0组A值呈下降趋势（F=23.5083,P〈0.05）,G1～3组内A值呈上升趋势（F1=202.5940,F2=1301.8815,F3=1222.0091,均P〈0.05）;同一时间点组间A值比较,G2、G3组较G1组增高（P〈0.05）,G2与G3组比较,差异无统计学意义（P〉0.05）。②B2～5组细胞逐渐变成圆形、多角形,伸出树突,并且均表达GFR-α1,而B1组无类似改变,也不表达GFR-α1。比较各诱导组在同一时间点（诱导第1、3、7天）组间灰度比值,B2-5组比较差异有统计学意义（P〈0.05）。结论G-CSF能促进大鼠BMSC的增殖,其适宜浓度为10ng/ml。联用GDNF和适宜浓度的G-CSF,比单用GDNF能更有效地诱导BMSC分化为神经元样细胞。     

腺病毒介导人Rb94基因对食管癌细胞的生长作用观察

目的 观察含有视网膜母细胞瘤(Rb)94基因的重组腺病毒表达载体(Ad-Rb94)对人食管癌细胞生长的抑制作用. 方法 将Ad-Rb94于体外转染食管癌细胞EC-9706(Ad-Rb94组),设对照腺病毒组(Ad-lacZ组)和空白对照组,用MTT比色法和流式细胞仪观察EC-9706细胞的生长曲线和细胞周期. 结果 Ad-Rb94组EC-9706细胞在转染后第3天开始生长缓慢,在转染后第5天本组细胞的生长与Ad-lacZ组和空白对照组相比明显受抑制(P<0.05).Ad-Rb94组G1期细胞比空白对照组增加14%～19%,G2期和S期细胞分别减少4%～6%和10%～13%. 结论 腺病毒介导的外源性Rb94基因可有效转染人食管癌细胞,并对其生长有抑制作用.     

沙棘总黄酮对人脐静脉血管内皮损伤细胞增殖及细胞周期的影响

目的探讨沙棘总黄酮对损伤人脐静脉血管内皮细胞株（CRL-1730）的保护作用。方法采用H2O2诱导CRL-1730细胞损伤模型,并将细胞随机分为四组,正常组不予任何干预;模型组加入250μmol/L H2O2;沙棘组加入沙棘总黄酮终浓度分别为50、100、200μg/m l,继续培养24 h后加入H2O2（剂量同上）;丹参组加入丹参注射液终浓度2.5 mg/m l,继续培养24 h后加入H2O2（剂量同上）。细胞干预4 h后用MTT比色法检测各组细胞活性,并用流式细胞仪测定各组细胞周期。结果与正常组比较,模型组细胞增殖活性下降,S期细胞比率降低,G2/M期细胞比率增加。与模型组比较,沙棘组及丹参组细胞增殖活性升高,G0/G1期细胞比率降低,S期及G2/M期细胞比率均增加。结论沙棘总黄酮对受损血管内皮细胞有保护作用,可提高细胞增殖活性,增加S期细胞比率。     

中药抗癌方诱导人结肠癌细胞株细胞凋亡的实验研究

目的研究中药抗癌方(KAF)对人结肠癌细胞凋亡的影响。方法选择人结肠癌细胞株HB8693作为靶细胞,用MTT掺入试验测定细胞毒作用,用流式细胞仪检测计算HC8693细胞株的亚G1峰细胞(凋亡)的百分率,并与空白对照组比较。结果KAF对HC8693的LDso为25.4％;在5％及10％浓度作用于HC8693后的亚G1峰细胞百分数分别为(22.0±2.6)％和(29.5±3.1)％,与空白对照组(6.3±1.0)％相比,皆有极显著性差异(P＜0.01)。结论KAF能诱导人结肠癌细胞凋亡。     

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。     

Toll样受体-4小干扰RNA预处理防治小鼠急性肝损伤

目的 观察Toll样受体(TLR)-4小干扰RNA(siRNA)对D-氨基半乳糖盐酸盐/月旨多糖(D-GalN/LPS)诱导的肝损伤小鼠的保护作用.方法 150只雄性C57BL/6小鼠均分为PBS预处理组、阴性对照质粒预处理组、TS4预处理组、TS6预处理组和TS7预处理组.腹腔内联合注射LPS(10 ng/g)及D-GalN(1 mg/g)诱导小鼠急性肝损伤.在D-GalN/LPS联合注射前24及48 h通过尾静脉高压水注射法导人siRNA质粒50 mg/L.末端脱氧核苷酸转移酶介导的dUTP缺口标记术(TUNEL)检测细胞凋亡水平,免疫组织化学法检测肝组织中TLR-4表达,RT-PCR检测TLR-4、TNF-α及巨噬细胞炎性蛋白(MIP)-2 mRNA水平,ELISA检测小鼠血清中MIP-2及TNF-α水平,标准自动分析仪检测血清中ALT及AST水平,苏木精-伊红染色观察肝脏组织学变化,Fisher确切概率法比较各组间小鼠存活率.结果 TLR-4 siRNA可减轻肝细胞坏死、减轻炎性反应,并可显著降低血清转氨酶水平.TS4预处理组(0.065±0.015)比PBS预处理组(0.346±0.062)的TUNEL标记指数(LI)明显降低(t=9.796,P<0.05).TLR-4 siRNA预处理下调TLR-4 mRNA及蛋白表达水平,显著降低TNF-α及MIP-2 mRNA表达及细胞因子水平,显著降低D-GalN/LPS联合诱导的急性肝损伤C57BL/6小鼠的死亡率和肝损伤.结论 TLR-4 siRNA抑制TLR-4表达在防治肝损伤方面可能具有潜在应用价值.     

Protection against liver injury by PGE1 or anti-TNF-alpha is associated with a reduction of TNF-R1 expression in hepatocytes

BACKGROUND: Tumour necrosis factor-alpha (TNF-5) has been shown to exacerbate or protect against liver injury in different experimental models. In a previous study, we observed that enhancement of TNF-alpha expression in hepatocytes by prostaglandin E1 (PGE1) pre-administration induced iNOS expression and cytoprotection against experimental liver injury in rats. Nevertheless, the reduction of TNF-alpha bioactivity by anti-TNF-alpha antibodies also reduced liver injury by D-GalN. The purpose of the present study was to evaluate whether protection by PGE1 or anti-TNF-alpha was related to a common effect on the membrane-bound TNF-alpha receptor expression. METHODS: Liver injury was induced in male Wistar rats by intraperitoneal injection of D-galactosamine (D-GalN) (1 g/kg). PGE1 or anti-TNF-alpha was administered at 30 or 60 min before D-GalN, respectively. Liver injury was evaluated by alanine aminotransferase (ALT) activity in serum and histological examination in liver sections. TNF-alpha was determined by ELISA in serum. The expression of TNF-alpha receptor type 1 (TNF-R1) and TNF-alpha receptor type 2 (TNF-R2) in hepatocytes was assessed by immunohistochemistry and immunoprecipitation + Western-blot analysis. RESULTS: PGE1 or anti-TNF-alpha reduced liver injury induced by D-GalN. Although PGE1 enhanced and anti-TNF-alpha reduced TNF-alpha concentration in serum, both protective treatments reduced the expression of TNF-R1 in hepatocytes. TNF-R2 was not detected in our experimental conditions. CONCLUSIONS: Our study showed that reduction of liver injury by PGE1 or anti-TNF-alpha antibodies was related to a reduction of TNF-R1 expression in hepatocytes.     

Effect of PGE1 on TNF-alpha status and hepatic D-galactosamine-induced apoptosis in rats

Prostaglandin E1 has hepatoprotective properties in several clinical and experimental models of liver dysfunction. Hepatotoxicity induced by D-galactosamine (D-GalN) is a suitable animal model of human acute hepatic failure. The aim of the study was to investigate if prostaglandin E1 (PGE1) protection against hepatic D-GalN-induced apoptosis was related to tumour necrosis factor-alpha (TNF-alpha) content in serum. This cytokine is associated with in vitro apoptosis and general inflammatory disorders. In this study, PGE1 was administered 30 min before D-GalN to rats. In other experiments, several doses of TNF-alpha were administered 15min after PGE1 to D-Ga1N-treated rats. Several parameters related to apoptosis and necrosis were measured by flow cytometry, gel electrophoresis, biochemical analysis, and optical and electron microscopy. Tumour necrosis factor-alpha was quantified by competitive enzyme-linked immunosorbent assay (ELISA). PGE1 by itself did not modify the cell cycle of hepatocytes and liver toxicity, but increased TNF-alpha in serum in comparison with the control group. D-Galactosamine increased the percentage of hepatocytes in apoptosis and in the S phase of the cell cycle, and decreased those in G0/G1. Such an increase of hepatocytes in apoptosis was correlated with a higher number of apoptotic bodies and DNA fragmentation in liver than control samples. Also, D-GalN increased alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase and TNF-alpha in serum compared with the control group. Pre-administration of PGE1 to D-GalN-treated rats reduced all the parameters of apoptosis and necrosis in liver, and increased additionallyTNF-alpha content in serum. In those experiments where low doses of TNF-alpha were administered to PGE1 and D-GalN-treated rats an inverse relationship appeared between TNF-alpha and ALT content in serum. In conclusion, the protective effects of PGE1 on D-GalN-induced apoptosis may be linked to its capacity to modulate cell division and/or its immunomodulatory activity. In this sense, our experimental results suggest that TNF-alpha could be involved in protection or exacerbation of liver damage in relation to the pathophysiological status of the liver.     

Cyclosporin A protects Balb/c mice from liver damage induced by superan tigen SEB and D-GalN

AIM To investigate the pathogenic effect of SEB and D-GalN on liver and the protection of cyclosporin A, the relationship between hepatic apoptosis and necrosis and the possible mechanism of acute hepatic necrosis.METHODS　After staphylococcal enterotoxin B (SEB) mixed with D-galactosamine (D-GalN) were injected intraperitoneally into Balb/cmiceand those previously treated with cyclosporin A, blood samples were collected and livers were isolated at 2, 6, 12 and 24h. Patterns of hepatocellular death were studied morphologically and biochemically, circulating cytokines (TNF-α, IFN-γ) and mice mortality within 24h was assessed.RESULTSTheSEBcouldinducethetypicalapoptoticchangesofhepatocytes, the D-GalN could induce hepatocytes apoptosis and degeneration at the same time, and the mice having received the SEB+D-GalN injections developed apoptosis at 2 and 6h, but after 12h hepatocytes were characterized by severe injury, whereas all the examinations in the cyclosporin A treated mice were normal.CONCLUSION Hepaticcellapoptosis might be related to necrosis, and massive hepatocyte apoptosis is likely the initiating step of acute hepatic necrosis in mice. The effects induced by SEB and D-GalN on hepatocytes might be mediated by T cells, and could be prevented by cyclosporin A.     

TNF-alpha dependent production of inducible nitric oxide is involved in PGE(1) protection against acute liver injury

BACKGROUND: Tumour necrosis factor alpha (TNF-alpha) and nitric oxide modulate damage in several experimental models of liver injury. We have previously shown that protection against D-galactosamine (D-GalN) induced liver injury by prostaglandin E(1) (PGE(1)) was accompanied by an increase in TNF-alpha and nitrite/nitrate in serum. AIMS: The aim of the present study was to evaluate the role of TNF-alpha and nitric oxide during protection by PGE(1) of liver damage induced by D-GalN. METHODS: Liver injury was induced in male Wistar rats by intraperitoneal injection of 1 g/kg of D-GalN. PGE(1) was administered 30 minutes before D-GalN. Inducible nitric oxide synthase (iNOS) was inhibited by methylisothiourea (MT), and TNF-alpha concentration in serum was lowered by administration of anti-TNF-alpha antibodies. Liver injury was evaluated by alanine aminotransferase activity in serum, and histological examination and DNA fragmentation in liver. TNF-alpha and nitrite/nitrate concentrations were determined in serum. Expression of TNF-alpha and iNOS was also assessed in liver sections. RESULTS: PGE(1) decreased liver injury and increased TNF-alpha and nitrite/nitrate concentrations in serum of rats treated with D-GalN. PGE(1) protection was related to enhanced expression of TNF-alpha and iNOS in hepatocytes. Administration of anti-TNF-alpha antibodies or MT blocked the protection by PGE(1) of liver injury induced by D-GalN. CONCLUSIONS: This study suggests that prior administration of PGE(1) to D-GalN treated animals enhanced expression of TNF-alpha and iNOS in hepatocytes, and that this was causally related to protection by PGE(1) against D-GalN induced liver injury.     

G-CSF Enhanced SDF-1 Gradient Between Bone Marrow and Liver Associated with Mobilization of Peripheral Blood CD34+ Cells in Rats with Acute Liver Failure

The role of stromal cell-derived factor-1 (SDF-1) in modulating massive liver damage is not well known. In this study, expression of SDF-1 in bone marrow and liver was investigated in rats with acute liver failure (ALF) when mobilized using granulocyte colony-stimulating factor (G-CSF). ALF was induced in rats by D-galactosamine (D-GalN). Starting after 2 hours following D-GalN induction, the animals were injected with G-CSF 50 μg/kg daily or saline as placebo for 5 days. The percentages of CD34+ cells in peripheral blood and the expression of SDF-1 in bone marrow and liver were then determined. The percentages of peripheral CD34+ cells demonstrated a transient increase in placebo rats following D-GalN induction and a significant increase in rats after G-CSF administration. SDF-1 expression showed a transient decrease in bone marrow and a transient increase in liver tissue from placebo rats. However, a significant decrease of SDF-1 expression in bone marrow and a remarkable increase in liver tissue were observed in animals from the G-CSF group. It was concluded that G-CSF can enhance the reduced expression of SDF-1 in bone marrow and increased expression in liver in ALF rats, forming a greater SDF-1 gradient, and chemoattracting CD34+ cells’ migration from bone marrow to an injured liver.     

赤芍承气汤对急性肝衰竭大鼠肝细胞凋亡的影响

目的：观察赤芍承气汤对内毒素脂多糖（LPS）致急性肝衰竭大鼠肝细胞凋亡的影响。方法：以内毒素（LPS）＋D－氨基半乳糖（D－GalN)）联合制备大鼠急性肝衰竭模型,模型组用生理盐水、治疗组用复方中药赤芍 承气汤分别灌胃,4天后开腹取鼠肝组织作病理检查,以HE染色观察大鼠肝组织病理学变化;以免疫组化法（SP法）检测Fas,FasL蛋白在大鼠肝 的表达情况,并观察赤芍承气汤对肝细胞凋亡的影响。结果：LPS＋D－GalN可引起严重的急性肝坏死并有广阔物肝细胞凋亡,伴Fas,FasL蛋白在肝细胞中强烈表达;肝细胞Fas／FasL阳性率,模型组为83％：92％,中药组为53％：50％,经统计学分析,P＜0．01;其阳性表达率随肝细胞坏死程度加重而增加,结论：赤芍承气汤可减轻肝细胞Fas／FasL的表达,抵抗内毒素诱发的肝细胞凋亡。     

大黄酚对大鼠肾上腺髓质嗜铬细胞瘤分化株细胞缺氧损伤的保护作用

目的观察大黄酚对缺氧引起的大鼠肾上腺髓质嗜铬细胞瘤分化株（PC12）细胞损伤的保护作用。方法培养PC12细胞,采用自制的密闭三气培养箱建立缺氧所致的PC12细胞损伤模型,根据不同条件,分为对照组、模型组、大黄酚组（包括1μg/ml组和5μg/ml组）。于缺氧处理前及处理后1、2、6、12、24h,每个时间点取6个复孔。采用四甲基偶氮唑盐法（MTT）检测PC12细胞增殖活性,高倍显微镜下观察PC12细胞核形态,测定各组上清液中超氧化物歧化酶（SOD）含量,免疫组化法测定各组线虫抗凋亡基因的人类同源基因表达蛋白（BCL-2）的表达,实时定量PCR测定p38丝裂原活化蛋白激酶（MAPK）和含半胱氨酸天冬氨酸蛋白酶3（Caspase-3）mRNA的表达。结果①缺氧处理后12 h,MTT法测定大黄酚1μg/ml组的细胞活力高于模型组;缺氧处理24 h,大黄酚1μg/ml组和5μg/ml组的细胞活力均高于模型组,差异均有统计学意义（P〈0.05）。②从缺氧处理后1 h开始,大黄酚1μg/ml组和5μg/ml组的SOD值高于模型组,差异有统计学意义（P〈0.05）。③从缺氧后2 h开始,大黄酚1μg/ml组和5μg/ml的BCL-2阳性率均高于模型组。④从缺氧处理2h开始,模型组p38MAPK mRNA表达量高于大黄酚1μg/ml组和5μg/ml组,差异有统计学意义（P〈0.05）。从缺氧处理6 h开始,模型组Caspase-3mRNA表达量高于大黄酚1μg/ml组和5μg/ml组,差异有统计学意义（P〈0.05）。⑤缺氧处理后12、24 h,模型组细胞的细胞核皱缩、形态不清,内见颗粒样物质形成增多;大黄酚组的细胞核形态较清楚,少见颗粒样物质形成。结论大黄酚可以减轻缺氧所致PC12细胞的损伤,对PC12细胞有保护作用。     

TNF-alpha but not IL-1alpha is correlated with PGE1-dependent protection against acute D-galactosamine-induced liver injury.

BACKGROUND: Prostaglandin E1 (PGE1) treatment of humans and rodents during acute hepatic failure ameliorates different parameters of hepatic dysfunction. PURPOSE: To investigate whether prevention of acute liver injury induced by D-galactosamine (D-GalN) with preadministration of PGE1 is correlated with a change in the concentration of two proinflammatory cytokines, as tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1alpha, and/or nitrite+nitrate (NOx), as nitric oxide-related end products in serum. RESULTS: D-GalN significantly increased alanine aminotransferase (ALT) and TNF-alpha concentration in serum 5 and 10 mins, respectively, after treatment compared with the control group (P< or =0.05). D-GalN did not change the IL-1alpha concentration at any time during the study. Preadministration of PGE1 to D-GalN-treated rats significantly reduced the ALT content and increased significantly the TNF-alpha concentration in serum 1, 2.5, 5 and 10 mins after D-GalN treatment compared with the D-GalN group (P< or =0.05). Nitric oxide was not involved in either the toxic effect due to D-GalN or the protection observed with PGE1 against D-GalN toxicity. CONCLUSIONS: Acute liver injury induced by D-GalN is correlated with an increased TNF-alpha release. Preadministration of PGE1 to D-GalN-treated rats exerted a priming effect on inflammatory cells to release enhanced levels of TNF-alpha but not IL-1alpha. These findings indicate that stimulation of TNF-alpha release may be involved in the acute D-GalN-induced liver injury and also in PGE1 protection from hepatotoxicity in clinical and experimental studies.