肝再生刺激因子对小鼠实验性急性肝损伤的保护作用

本工作采用雄性初断乳 SD 大鼠肝按 LaBrecque 法提取肝再生刺激因子(HSS),用四氯化碳和半乳糖胺分别损伤小鼠肝来研究 HSS 的保肝作用。结果如后:(1)HSS 可使 CCl_4致肝损伤小鼠的血清 GPT 和 GOT 升高幅度降低,并呈量效关系。(2)肝组织切片表明 HSS 可使 CCl_4损伤肝组织的程度减轻。(3)肝组织化学法表明 HSS 可使 CCl_4损害肝细胞线粒体琥珀酸脱氢酶的活性恢复。(4)胰岛素-胰高糖素可降低半乳糖胺所致的小鼠死亡率,减弱对肝组织的损害和刺激肝细胞增殖,这项实验可作为 HSS 具有保肝作用的证据。     

四氯化碳中毒对大鼠离体再生肝细胞钾离子外漏的影响

本工作用三种剂量四氯化碳(CCl_4,10,15和20mmol/L)损伤正常大鼠离体肝细胞,分别在5,10,15和20min测定细胞内K~+和GPT漏出量。实验观察到细胞内K~+和GPT漏出量与CCl_4染毒的剂量和时间有明显关系,而且K~+漏出量较GPT更能灵敏地反映细胞的损伤程度;用中等剂量CCl_4(15mmol/L)损伤离体再生肝细胞20min后,细胞内K~+漏出的变化百分数明显低于正常肝细胞。这些结果表明,大鼠离体再生肝细胞具有较强的抗CCl_4损伤作用,其机制可能与再生肝细胞膜稳定性较强有关。     

人肝刺激因子对大鼠实验性慢性肝损伤的保护作用

从健康孕妇水囊引产４─６个月龄的胎儿取肝,采用ＬａＢｒｅｃｑｕｅ方法提取人肝刺激因子（ｈＨＳＳ）。经３Ｈ－胸腺嘧啶核苷参入肝ＤＮＡ法测定其生物活性。表明此ｈＨＳＳ可刺激肝细胞ＤＮＡ合成。采用皮下注射ＣＣｌ４和饮用１０％乙醇来制备慢性肝损伤动物模型,观察了ｈＨＳＳ的保护肝脏作用。结果表明：ｈＨＳＳ可使ＣＣｌ４－乙醇所致慢性肝损伤大鼠的死亡率、血清谷丙转氨酶水平、肝组织中羟脯氨酸含量的升高以及肝组织中丙二醛的含量降低。肝组织切片表明：ｈＨＳＳ能减轻肝组织的损伤程度,促进肝细胞再生,并能明显防止肝纤维化的形成和发展。可见,ｈＨＳＳ对ＣＣｌ４－乙醇所致的慢性肝损伤大鼠有明显的保护作用,其机制可能与促进肝细胞再生及抑制肝细胞膜的脂质过氧化有关。     

芍药苷减轻四氯化碳诱导的急性肝损伤

为研究芍药苷对四氯化碳(CCl_4)诱导的急性肝损伤的保护作用及相关机制。将36只健康雄性C57BL/J小鼠随机分成6组:空白组,芍药苷对照组,CCl_4模型组(0.1%,20 mL/kg),高、中、低剂量芍药苷+CCl_4组(10 mg/kg,30 mg/kg,100 mg/kg),每组6只。24 h后眼球取血收集血清,测定丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST)活性。用苏木精-伊红(HE)染色,观察肝脏的组织学改变;试剂盒测定肝组织中SOD、GSH-PX、CAT的活性及MDA和GSH含量;ELISA法检测血清中TNF-α、IL-6含量;试剂盒检测小鼠肝组织中Caspase-3的活性;q RT-PCR检测肝组织中HO-1 mRNA的表达。实验发现PAE降低小鼠血清ALT、AST水平,改善肝脏的病理形态;芍药苷抑制CCl_4诱导氧化应激,升高肝组织HO-1 mRNA水平;降低TNF-α、IL-6含量;芍药苷减低肝组织中Caspase-3活性,减少肝细胞凋亡。由此可知,芍药苷可保护CCl_4诱导的急性肝细胞损伤,该保护作用可能与抑制脂质过氧化,减少促炎细胞因子产生,减少肝细胞凋亡,促进抗氧化蛋白表达有关。     

大鼠再生肝抗CCl4损伤与其线粒体呼吸活性变化的关系

本工作观察了肝部分切除(68%)后96h 大鼠再生肝的抗 CCl_4损伤作用,并用氧电极法测定了再生肝线粒体的呼吸活性。结果如下: (1)CCl_4(50%,10ml/kg)引起的动物死亡率,肝切除组大鼠较假手术组明显降低;(2)CCl_4(50%,5 ml/kg)损伤后,肝切除组大鼠血清胆红素、血清谷丙转氨酶(sGPT)均明显低于假手术组,组织学检查损伤程度也明显减轻;(3)无论是否伴有 CCI_1损伤,肝切除组大鼠肝线粒体的呼吸活性均强于假手术组,且肝线粒体呼吸活性的变化与血清胆红素、sGPT 及肝组织损伤程度的改善是一致的。上述结果提示:再生肝线粒体呼吸活性增高,同时不易受 CCl_4损伤,可能在再生肝抗 CCl_4损伤机制中起一定作用。     

人肝刺激因子对CCl4损伤大鼠离体肝细胞内钙和钾离子稳态的影响

本工作从自愿流产孕妇的胎儿取肝,按照LaBrecque法提取人肝刺激因子(human hepaticstimulator substance,hHSS)。用荧光探针Fura-2/AM测定离体肝细胞内游离钙,用离子分析仪测细胞染毒(四氯化碳CCl_4)前后基质中钾离子含量,观察hHSS对染毒肝细胞内Ca~(2+)和K~+稳态的影响,并测定肝细胞存活率和细胞内转氨酶(ALT)的漏出作为佐证。结果表明,人胎肝中含有hHSS,hHSS能提高离体肝细胞的存活率,维持肝细胞内游离钙的相对恒定,减少细胞内钾离子和ALT的漏出。这些结果提示,hHSS可保护肝细胞内钙,钾离子稳态和肝细胞膜的稳定,从而加强大鼠离体肝细胞抗CCl_4的损伤。     

促肝细胞生长物质(HSS)

促肝细胞生长物质(hepaticstimulatorysubstance,HSS)不仅能够阻止肝损伤、启动肝细胞增殖再生,而且可以部分恢复肝功能以及具有抗氧化作用,因此有较为广阔的应用前景。     

人肝刺激因子对实验性急性肝功能衰竭小鼠的保护作用

取健康孕妇水囊引产的4～6个月龄的胎儿肝脏,按LaBreque法提取肝刺激因子(hHSS)。将hHSS注入肝部分切除的大鼠体内,用~3H-TdR掺入肝DNA测定其生物学活性。给昆明种小鼠腹腔注射D-半乳糖胺(D-Gal)以造成急性肝功能衰竭,观察hHSS对这种急性肝衰的保护效应,结果如下:①hHSS明显降低D-Gal所致的小鼠死亡率(P<0.01);②hHSS显著降低D-Gal所致sGPT和sGOT水平增高(sGPT 208.2±26.1比103.6±21.2 U/100ml;sGOT 502.6±65.4比287.8±39.4 U/100m1);③hHSS明显降低D-Gal所致的小鼠肝组织丙二醛水平增高(330.4±38.9比115.5±43.8nmol/100mg蛋白质);④光镜和电镜的组织学观察表明hHSS明显减轻D-Gal对肝组织的损害。上述实验结果表明,hHSS对D-Gal所致的急性肝衰具有明显的保护作用,其机制可能是保护肝细胞正常代谢和防止膜脂质过氧化。     

肝再生增强因子的cDNA克隆、表达及表达产物的生物活性研究

以肝部分切除后再生肝组织为起始材料,利用RT-PCR扩增出大鼠肝再生增强因子（ALR）,亚克隆于pGEM-T载体,核苷酸序列测定证实为大鼠ALR;将ALRcDNA亚克隆于pBV220质粒,构建了原核表达栽体,并获高效表达菌株,特异表达蛋白占细菌总蛋白的15％,原核表达的ALR在体外缺乏促进大鼠原代培养肝细胞及SMMC-7721肝癌细胞DNA合成的活性,但在体内1／3肝部分切除模型中可刺激肝细胞DNA合成;ALR在生物学活性方面与肝脏刺激物（HSS）存在一定差别,ALR和HSS应是两种不同的活性因子．ALR还具有促肝损伤修复的作用,对其深入研究可能为临床治疗严重肝病提供有效的药物.     

新生牛肝脏再生刺激物质的部分纯化及生物学特性

新生牛肝胞浆液中存在刺激肝细胞DNA合成的物质,经过一定的分离提纯,可使其杂蛋白含量降低770倍,特异活性提高26.7倍。在无血清培养条件下,可使成年原代大鼠肝细胞和肝源性肿瘤细胞DNA合成分别增加5倍及7.2倍左右。HSS为耐热的蛋白质,一定的酸碱及变性剂可使活性丧失,主要活性成分的分子量在43—67KD之间,但小于10KD的物质亦具有一定的活性。该实验结果提示:HSS很可能是正常肝细胞分裂与再生时共同的调节因子。     

肝再生剌激因子对小鼠实验性急性肝损伤的保护作用

A hepatic stimulator substance (HSS) was extracted from the liver of male weanling SD rats according to the method of LaBrecque. The mice were injected with carbon tetrachloride or D-galactosamine to induce hepatic injuries and the protective effect of HSS on thus induced hepatic damage was investigated. The results were as follows: (1) HSS could suppresses the elevation of sGPT and sGOT induced by carbon tetrachloride intoxication in a dose-dependent manner. (2) Hepatic histological findings indicated that the degree of CCl4 or D-galactosamine-induced hepatic lesions could be lessened by HSS. (3) CCl4-induced reduction of hepatic mitochondrial succinic dehydrogenase activity could be restored by HSS. (4) Insulin-glucagon enhanced the survival of D-galactosamine intoxicated mice and stimulated hepatocyte proliferation, thus showing less pronounced hepatic damage.     

乳酸菌源有机硒对肝损伤小鼠脾脏脂质过氧化和NK细胞活性的保护效应研究

本文通过研究乳酸菌源有机硒干预CCl_4致肝损伤小鼠脾脏NK细胞活性和脂质过氧化反应的变化,探讨该有机硒在抗损伤保护过程中的效应及其机制。分别选用60只健康成年小鼠,雌雄对半,随机分成对照组(C组),有机硒组(Se组),CCl_4组、CCl_4-有机硒保护组(CCl_4-Se组),每组15只。通过腹腔注射CCl_4诱发肝损伤后,分别在第2、4周检测脾脏NK细胞活性及其组织匀浆GSH-Px、CAT、SOD活性和MDA含量变化、结果显示,在整个实验期内,C组、Se组和CCl_4-Se组脾组织匀浆GSH—Px、CAT和SOD活性均高于或明显高于CCl_4组,Se和CCl_4-Se组与C组比较除SOD活性在第4周有明显升高外均差异不显著;CCl_4组小鼠脾脏MDA含量均显著高于C组、Se组和CCl_4-Se组,而CCl_4-Se组与C组接近,Se组较CCl_4-Se组和C组低;Se组NK细胞活性最高,第4周明显高于C组,CCl_4组最低且低于或明显低于CCl_4-Se、Se和C组,CCl_4-Se组与C组无明显差异。结果提示,乳酸菌源有机硒能够提高正常机体抗氧化能力,在干预肝损伤过程中,可以通过改善和提高脾组织抗氧化酶活性及NK细胞活性发挥积极有效的作用。     

Hepatoprotective action of schisandrin B against carbon tetrachloride toxicity was mediated by both enhancement of mitochondrial glutathione status and induction of heat shock proteins in mice

In the present study, we investigated the differential role of the mitochondrial glutathione status and induction of heat shock proteins (HSPs) 25/70 in protecting against carbon tetrachloride (CCl_4) hepatotoxicity in schisandrin B (Sch B)-pretreated mice. The time-course of Sch B-induced changes in these hepatic parameters were examined. Dimethyl diphenyl bicarboxylate (DDB), a non-hepatoprotective analog of Sch B, was studied for comparison. Sch B treatment (2 mmol/kg) produced maximal enhancement in hepatic mitochondrial glutathione status as well as increases in hepatic HSP 25/70 levels at 24 h post-dosing. The stimulatory effect of Sch B then gradually subsided, but the activities of hepatic mitochondrial glutathione reductase (GR) and glutathione S-transferases (GST) as well as the level of HSP 25 remained relatively high even at 72 h post-dosing. CCl_4 challenge caused significant impairment in mitochondrial glutathione status and a decrease in HSP 70 level, but the HSP 25 level was significantly elevated. While the extent of hepatoprotection afforded by Sch B pretreatment against CCl_4 was found to inversely correlate with the time elapsed after the dosing, the protective effect was associated with the ability of Sch B to maintain the mitochondrial glutathione status and/or induce further production of HSP 25 in CCl_4-intoxicated condition. On the other hand, DDB treatment (2 mmol/kg), which did not increase mitochondrial GSH level and GST activity or induce further production of HSP 25 after CCl_4 challenge, could not protect against CCl_4 toxicity. The results suggest that the enhancement of mitochondrial glutathione status and induction of HSP 25/70 may contribute independently to the hepatoprotection afforded by Sch B pretreatment.     

大鼠再生肝刺激因子抗四氯化碳损伤的研究

我们以往的研究证明,大鼠再生肝具有抗四氯化碳损伤的能力。本工作进一步研究其机制,首先从部分（６８％）肝切除后不同的再生肝的取肝刺激因子,用＾３Ｈ胸腺嘧喧核苷测定ｒＨＳＳ的生物活性,结果表明部分切除肝后７２ｈ的ｒＨＳＳ活性较对照组约增加７．７倍。然后将ｒＨＳＳ注射给小鼠,观察其抗ＣＣｌ４损伤肝的效应,具体表现如下：ｒＨＳＳ能减少ＣＣｌ４中毒小鼠的死亡率和降低ＣＣｌ４所增高的血清谷丙转氨酶和谷草转氨酶     

大鼠肝再生过程中肝再生刺激物及其mRNA的动态变化

本实验先制备大鼠肝再生模型,在该模型中大鼠成活率达９５％以上,肝再生情况良好,适合于进行下一步的研究。随后,通过耐热性和肝细胞特异性的检测,初步认为从该模型中所提取的活性成分即为肝再生刺激物（ＨＳＳ）。用３Ｈ胸腺嘧啶核苷测定ＨＳＳ及其ｍＲＮＡ体外翻译产物的生物活性,结果表明二者在肝再生过程中均存在动态变化,但前者在肝部分（２／３）切除后７２ｈ活性最高,后者则在２４ｈ达高峰。这一结果为后续的分子克隆工作奠定了基础。     

IL-6 prevents T cell-mediated hepatitis via inhibition of NKT cells in CD4+ T cell- and STAT3-dependent manners

The hepatoprotective effect of IL-6 on various forms of liver injury including T cell-mediated hepatitis has been well documented, and it is believed that induction of antiapoptotic proteins is an important mechanism. In this study, we provide evidence suggesting an additional mechanism involved in the protective role of IL-6 in T cell-mediated hepatitis. In NKT cell-depleted mice, Con A-induced liver injury is diminished; this can be restored by the adoptive transfer of liver mononuclear cells or NKT cells from wild-type mice, but not from IL-6-treated mice. In vitro IL-6 treatment inhibits the ability of mononuclear cells to restore Con A-induced liver injury in NKT-depleted mice, whereas the same treatment does not inhibit purified NKT cells from restoring the injury. The addition of CD3(+) T cells or CD4(+) T cells can restore the inhibitory effect of IL-6 on purified NKT cells, whereas the addition of CD3(+) T cells from CD4-deficient mice fails to restore this inhibitory effect. The expression of IL-6R was detected in 52.6% of hepatic CD3(+) T cells and 32.7% of hepatic CD4(+) T cells, but only in 3.9% of hepatic NK and 1.5% of hepatic NKT cells. Finally, treatment with IL-6 induces STAT3 activation in hepatic lymphocytes and hepatic T cells, and blocking such activation abolishes the inhibitory effect of IL-6 on hepatic lymphocytes to restore liver injury. Taken together, these findings suggest that in addition to its antiapoptotic abilities, as previously well documented, IL-6/STAT3 inhibits NKT cells via targeting CD4(+) T cells and consequently prevents T cell-mediated hepatitis.