慢性氟中毒大鼠肾组织细胞凋亡的实验研究

目的：观察细胞凋亡在氟中毒肾脏损害发生机制中的作用。方法：经饮水投氟复制氟中毒模型。采用原位末端标记法（TUNEL）测定了慢性氟中毒大鼠肾脏组织细胞凋亡的情况，应用全自动生化分析仪检测大鼠尿液乳酸脱氢酶（LDH）、碱性磷酸酶（ALP）、γ－谷胺酰转移酶（γ－GT）、N－乙酰氨基葡萄糖苷酶（NAG）的活性。结果：TUNEL检测显示，在慢性氟中毒大鼠肾脏中，可见到明显的NBT／BCIP着染阳性的凋亡细胞，而且这种表达具有选择性，多出现在肾皮髓质交界处。氟中毒大鼠尿液LDH、ALP、γ－GT、NAG的活性变化不明显。投氟组与对照组比较无明显差异。结论：在慢性氟中毒大鼠肾脏细胞凋亡明显，凋亡发生部位与病理改变明显的部位吻合，慢性氟中毒大鼠肾损害过程中很可能有细胞凋亡机制参与。     

不同氟中毒动物模型肾脏功能与离子代谢的变化

目的：观察不同氟中毒动物模型肾脏功能与离子代谢的变化。方法;经饮水投氟复制氟中毒模型。采用全自动生化分析仪检测血清内反映肾功能指标和相关离子代谢,应用放射免疫技术测定血,尿中的β2－MG含量。结果：氟中毒家兔和大鼠的BUN,Cr,UA变化不明显,β2－MG投氟组与对照组比较无明显差异。氟中毒家兔和大鼠离子代谢紊乱,并且以Ca^2 代谢紊乱明显。结论：高氟剂量或在伴有钙缺乏的情况下,血清内离子代谢紊乱明显,尤以Ca^2 表现显,但氟中毒时肾功能损害不明显。     

过量氟对大鼠肝细胞内钙水平和肝细胞凋亡的影响

目的 研究在大鼠过量摄氟后体内对肝细胞中游离钙([Ca~(2+)]i)水平和肝细胞凋亡的影响。方法 应用饮水加入氟化钠进行大鼠染毒实验,采用Fura-2/AM荧光指示剂测定慢性氟中毒大鼠肝细胞内[Ca~(2+)]i浓度的变化,同时利用流式细胞术测定肝细胞凋亡率。结果 过量氟可刺激肝细胞内[Ca~(2+)]i浓度增高,常食(高钙饮食)投氟组与常食对照组相比差异显著(P<0.05),偏食低钙投氟组高于低钙对照组,差异显著(P<0.05);肝细胞凋亡率在正常饮食加氟组与对照组相比差异无显著性(P>0.05),低钙饮食加氟组肝细胞凋亡率明显增高,与低钙对照组相比,差异显著(P<0.05);相关分析显示,低钙饮食投氟组肝细胞内[Ca~(2+)]i浓度增高与细胞凋亡率有相关性趋势(r=0.576)。结论(1)过量氟所致的大鼠肝细胞内[Ca~(2+)]i持续增高对肝细胞凋亡有不同程度的影响,可能在氟骨症病理过程中起重要作用;(2)投氟伴随低钙可加重细胞内[Ca~(2+)]i超负荷和细胞凋亡,提示钙营养与氟中毒发病有着重要联系;(3)肝细胞内[Ca~(2+)]i增高与肝细胞凋亡率之间有无相关性有待于进一步研究证实。     

过量氟对大鼠脑肝肾细胞周期与细胞凋亡的影响

目的 研究氟对大鼠脑、肝、肾组织细胞凋亡和细胞周期的作用特点与 氧化侵袭之间的关系,进一步探讨氟中毒发生机理。方法 在常规食、偏食条件下,经饮水投不同剂量氟,应用流式细胞仪方法检测脑肝、肾组织细胞凋亡,细胞周期与活性氧产生状态。结果 常规食投氟100－150mg/L、偏食投氟50－100mg/L对脑、肝、肾组织活性氧产生无明显影响。氟对脑组织神经细胞周期的影响主要在S期,可明显减少S期神经细胞数量;对肝细胞周期的作用主要在G1期和G2期,偏食投氟各组G1期细胞数目普遍低于常食投氟组,G2期细胞数目则高于常规投氟组。过量氟对肾组织的作用,主要为偏食投氟100mg/L情况下,可明显诱导细胞凋亡的变化。结论 过量氟对细胞周期的影响,对不同组织的作用时相似有一定程度的差别,对脑神经细胞主要作用于S期,肝细胞的影响主要是G1、G2期;对肾细胞主要表现为诱导细胞凋亡过程。     

氟致离体人肝细胞凋亡及硒的保护作用研究

目的 研究硒、氟对离体人肝细胞周期和凋亡的影响。方法 体外培养的人肝细胞接触氟和（或）硒12h后,用流式细胞仪检测肝细胞凋亡百分率和细胞周期构成比。结果 氟可使肝细胞凋亡百分率明显升高,S期细胞数增加,但G0／G1期和G2／M期细胞数无明显改变;加硒可明显拮抗氟的这种影响。结论 氟对人肝细胞的损害与氟诱导细胞凋亡和改变细胞周期分布有关,一定 量的硒可明显拮抗氟诱导的肝细胞凋亡及其对细胞周期的影响。     

氟中毒大鼠肝细胞凋亡研究

目的研究氟化物对肝细胞凋亡的影响。方法用偏食饲料、饮水加氟化钠１００ｍｇ／Ｌ或同时加碳酸钙３２００ｍｇ／ｋｇ饲料喂饲大鼠２个月,用流式细胞术检测肝细胞凋亡小体百分率,同时检测了谷胱苷肽过氧化物酶活性和脂质过氧化物含量。结果发现氟中毒组大鼠肝细胞凋亡小体百分率较对照组明显升高（Ｐ＝０．０１～０．０５）,加钙可使这种改变更为明显（Ｐ＜０．０１）;而ＧＳＨ－ｐｘ活性则明显降低。结论氟化物的毒性可导致肝细胞凋亡,对机体抗氧化能力的降低可能起一定作用。     

氟中毒大鼠脑组织自由基代谢的变化

目的 探讨氧化侵袭在氟中毒大鼠脑组织损害中所发挥的作用。方法 大鼠经饮水投氟１０周,采用生化方法检测氟中毒大鼠脑组织脂质过氧化物含量和抗氧化酶系活性水平。结果 常食对照组和投氟组脂质过氧化物含量和抗氧化酶系活性改变不明显;偏食投氟１００ｍｇ／Ｌ氟组脑组织ＭＤＡ较其对照组增高,红细胞ＣＡＴ较其对照组活性下降,其他生化反应的改变不明显。结论 投氟伴随低钙可能是氟中毒脑组织损害的重要环节之一,钙营养与氟     

自由基与氧化应激在氟中毒骨病变发生中的作用

本实验用常规兔饲料饲养家兔,经饮水投氟复制家兔氟中毒模型,检测了自由基代谢指标,表明红细胞超氧化物歧化酶（Ｃu-Zn-SOD)、全血谷胱甘肽过氧化物酶（ＧＳＨ－Ｐx)等抗氧化酶类测定结果,投氟组与对照组间无显差异,血清脂质过氧化物（ＬＰＯ）水平高剂量投氟组与对照组未见明显差异,剂量投氟组较对照组反而降低,用ＥＳＲ测骨组织的自由基信号,高、中剂量投氟组均显高于对照组,表明氟中毒时可发生自由基增我,自由基和氧化应激改变与过量氟所致骨相或非骨相损害之间缺乏明确的相关性,目前尚得不出氧化应激参与氟中毒骨病变发生机理的结论。     

氟中毒大鼠肾组织形态改变与氧化应激

目的：定量分析氟中毒大鼠肾组织形态改变，并探讨氧化应激与肾损伤的关系。方法：给大鼠饮水投氟10周。采用图象分析对肾组织进行体视学定量分析，应用流式细胞术检测肾细胞活性氧含量，通过生化技术检测红细胞抗氧化酶活性改变。结果：氟中毒大鼠肾小囊囊腔和皮髓质交界处肾小管均扩张明显，红细胞过氧化氢酶在投与同等剂量氟的情况下，偏食组较常食组的酶活性下降明显。结论：氟中毒引起肾损害的靶部位以皮髓质交界处肾小管扩张为主，并且随着投氟剂量的增加或低钙的情况下损害更为明显。     

氟中毒大鼠肝肾细胞凋亡与p53的关系

目的 研究氟中毒大鼠肝、肾细胞凋亡与相关基因p53的关系。方法 用流式细胞术检测氟中毒大鼠及氟加硒大鼠肝、肾细胞凋亡百分率及相关基因p53。结果 氟中毒大鼠组及氟加硒组肝、肾细胞凋亡率明显高于对照组（P＜0.01）且相关基因p53的表达也明显高于对照组（P＜0.01)。并且加硒组肝、肾细胞凋亡率明显低于氟中毒组。结论 氟可以导致细胞凋亡，加硒可保护细胞减少细胞凋亡。p53参与并介导细胞凋亡。     

肿瘤坏死因子α及caspase-3表达与暴发性肝衰竭细胞凋亡

目的　研究肿瘤坏死因子α(TNFα)与caspase 3表达在实验性暴发性肝衰竭 (FHF)中对肝细胞凋亡的作用。方法　用脂多糖和D 氨基半乳糖制备FHF小鼠模型 ;ELISA和逆转录PCR法检测血清TNFα水平及肝组织TNFαmRNA表达 ;原位杂交法检测肝组织内caspase 3表达 ;DNA琼脂糖凝胶电泳和末端转移酶介导的dUTP缺口末端标记 (TUNEL)检测肝细胞凋亡。结果　用药后 2h开始 ,TNFαmRNA表达增加 ( 0 91± 0 75 ,正常值 0 32± 0 10 ) ,血清TNFα水平升高 [( 32 0 5 0± 86 5 7)ng/L ,正常值 ( 16 6 6± 7 0 1)ng/L],caspase 3少量表达 ;8h后 ,血清ALT和总胆红素 (TBil)水平显著增加 [分别为 ( 5 6 0 6 6± 6 0 2 0 )U/L和 ( 16 3 6 6± 34 5 1) μmol/L ,正常值为 ( 2 3 5 6± 8 0 3)U/L和 ( 14 90± 4 80 ) μmol/L],caspase 3表达至最高峰 ,并出现典型的肝细胞凋亡改变 ;12h后 ,血清ALT和TBil水平达最高峰 ,caspase 3表达较 8h减少 ,肝细胞坏死和凋亡同时存在。抗TNFα单抗阻断试验后 ,肝细胞凋亡和坏死明显减轻 ,TUNEL和caspase 3表达显著减少。 结论　TNFα有促进肝细胞凋亡与坏死作用 ,肝细胞凋亡与caspase 3的激活有关 ,在时间上肝细胞凋亡先于坏死。     

PERK/eIF2α通路在酒精性肝损伤大鼠肝细胞凋亡中的作用

目的 探讨内质网类似激酶(PERK)/真核生物翻译起始因子(eIF)2α信号通路在酒精性肝损伤大鼠肝细胞凋亡中的作用.方法 建立大鼠酒精性肝损伤模型.设4、6、10周和12周4个时间点,动态观察肝组织病理变化;流式细胞术检测肝细胞凋亡率;酶联免疫吸附法检测血清同型半胱氨酸(tHCY)水平;实时荧光定量聚合酶链反应和Western blot检测肝组织PERK/eIF2α通路信号分子mRNA和蛋白的表达水平.多组样本均数的两两比较采用One-Way ANOVA分析.结果 4周时造模大鼠发生急性肝损伤改变,12周时则出现慢性肝损伤改变;6周时造模大鼠肝细胞凋亡率较正常组显著增加(P＜0.05),随着造模时间的延长,肝细胞凋亡程度逐渐加剧,12周时早期和总凋亡率分别达到26%和29%;自6周起,造模大鼠血清tHCY水平明显高于正常大鼠(P＜0.01);自4周起,造模大鼠肝组织eIF-2α蛋白发生明显磷酸化,12周时peIF-2α蛋白表达量上升了2.81倍(P＜0.01),葡萄糖调节蛋白(GRP) 78/Bip、GRP94、caspase 12和caspase-3则表现为过度活化,12周时基因和蛋白表达量分别为正常大鼠的4.70、12.95、3.83、4.05倍和3.93、6.93、9.88、3.31倍(P＜0.01).结论 PERK/eIF2α通路的活化与酒精性肝损伤大鼠肝细胞凋亡的发生和持续发展密切相关.     

Bcl—2腺病毒载体抗肿瘤坏死因子α及氨基半乳糖诱导小鼠肝细胞凋亡

目的　探讨Bcl-2家族蛋白在TNFα致肝损伤及肝细胞凋亡中的作用。方法　以TNFα联合D-氨基半乳糖诱发小鼠肝损伤,以免疫组织化学法检测Bax、Bak蛋白在鼠肝组织中的表达情况;并以Bcl-2腺病毒载体感染肝细胞,观察其抗肝细胞凋亡作用。结果　TNFα可引起严重的肝损伤并有广泛的肝细胞凋亡,伴Bax、Bak蛋白在肝细胞中表达增强;Bcl-2腺病毒感染可使肝损伤小鼠ALT水平由（1372.9±251.4）U/L下降至（796.5±78.7）U/L,统计分析差异有显著意义（P＜0.0005）。结论　TNFα诱导肝细胞凋亡可能与其诱导Bax、Bak蛋白在肝细胞中表达增强有关;Bcl-2腺病毒载体可在小鼠肝细胞中持续表达至少一个月并可部分抵抗TNFα诱发的肝细胞凋亡。     

Involvement of platelet-activating factor in hepatic apoptosis and necrosis in chronic ethanol-fed rats given endotoxin

BACKGROUND/AIMS: Platelet-activating factor (PAF)-a potent activator of neutrophils-plays an important role in the pathogenesis of endotoxin-induced tissue injury. However, the role of PAF in hepatic damage during alcoholic hepatitis remains unclear. The aims of the present study were to test whether PAF contributes to hepatic injury in an animal model of alcoholic hepatitis and to investigate the involvement of the Fas-receptor/Fas-ligand system in this process. METHODS: Male Sprague-Dawley rats were pair-fed with Lieber-DeCarli ethanol liquid diet or isocaloric control diet for 6 weeks. Liver injury was induced by the intravenous (i.v.) injection of lipopolysaccharide (LPS) (1 mg/kg). Rats were pretreated with a specific PAF receptor antagonist (TCV-309; 100 mg/kg i.v.) or vehicle 1 h before LPS treatment. RESULTS: Chronic ethanol administration remarkably sensitized the rats to the effects of LPS, with resultant severe hepatocellular injury, accompanied by significant increases in serum levels of alanine aminotransferase (ALT), tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 (CINC/gro). Histological examination of the damaged livers showed hepatocyte apoptosis and necrosis with extensive infiltration by neutrophils, whereas immunohistochemical studies revealed enhanced Fas-receptor expression on hepatocytes and hepatic accumulation of neutrophils expressing Fas-ligand. Pretreatment with the PAF receptor antagonist protected against hepatic injury, suppressing hepatocyte apoptosis and necrosis, infiltration of neutrophils, expression of Fas-receptor and Fas-ligand, and serum TNF-alpha levels. CONCLUSIONS: Our study suggests that PAF is an important mediator of hepatic injury in the ethanol/endotoxin model of alcoholic hepatitis.     

Apoptosis as a mechanism for liver disease progression

Hepatocyte injury is ubiquitous in clinical practice, and the mode of cell death associated with this injury is often apoptosis, especially by death receptors. Information from experimental systems demonstrates that hepatocyte apoptosis is sufficient to cause liver hepatic fibrogenesis. The mechanisms linking hepatocyte apoptosis to hepatic fibrosis remain incompletely understood, but likely relate to engulfment of apoptotic bodies by professional phagocytic cells and stellate cells, and release of mediators by cells undergoing apoptosis. Inhibition of apoptosis with caspase inhibitors has demonstrated beneficial effects in murine models of hepatic fibrosis. Recent studies implicating Toll-like receptor 9 in liver injury and fibrosis are also of particular interest. Engulfment of apoptotic bodies is one mechanism by which the TLR9 ligand (CpG DNA motifs) could be delivered to this intracellular receptor. These concepts suggest therapy focused on interrupting the cellular mechanisms linking apoptosis to fibrosis would be useful in human liver diseases.     

Interleukin 1beta protects mice from Fas-mediated hepatocyte apoptosis and death.

BACKGROUND & AIMS: Fas-mediated apoptosis is one of the major death processes of hepatocytes in liver diseases. The aim of this study was to determine whether interleukin (IL)-1beta regulates the Fas-mediated apoptotic process of differentiated hepatocytes in vivo. METHODS: IL-1beta was injected into Balb/cA mice 5 hours before lethal challenge with agonistic anti-Fas administration. Survival and hepatocyte apoptotic process of these mice were examined. RESULTS: IL-1beta pretreatment prolonged animal survival in a dose-dependent manner, and 500 ng of IL-1beta completely protected mice from lethality. Both serum alanine aminotransferase value and hepatic DNA fragmentation were significantly suppressed by IL-1beta pretreatment. IL-1beta affected neither hepatic distribution of anti-Fas antibody nor Fas expression levels on hepatocytes but significantly suppressed Fas-induced activation of hepatic caspase 3-like protease. Suppression of Fas-induced activation of the caspase by IL-1beta was diminished by coadministration with D-galactosamine and reversed by coinjection with an excess amount of uridine. CONCLUSIONS: These results suggest that IL-1beta suppresses Fas-mediated hepatocyte apoptosis by inducing molecule(s) that suppress the apoptosis control machinery upstream of caspase 3. This observation raises the possibility that IL-1beta acts as a negative regulator of Fas-mediated hepatocyte apoptosis during liver injury.     

酒精中毒患者血清中肝细胞再生因子水平的测定及其意义

探讨酒精中毒患者血清中肝细胞再生因子水平的变化及其意义。应用组织细胞培养法及同位素标记技术对 30例酒精中毒患者和 30例健康对照组血清中肝细胞再生因子水平进行测定。急性酒精中毒患者伴有肝功能异常者血清中肝细胞再生因子水平高于慢性酒精中毒 (P <0 0 5 ) ,并显著高于正常对照组 (P <0 0 1)。为估计酒精中毒患者的肝损伤程度提供参考价值。     

Fibronectin is essential for survival but is dispensable for proliferation of hepatocytes in acute liver injury in mice

Acute liver injury causes massive hepatocyte apoptosis and/or fatal liver damage. Fibronectin, an extracellular matrix glycoprotein, is prominently expressed during adult tissue repair. However, the extent of fibronectin dependence on hepatocyte response to acute liver damage remains to be defined. Because identification of hepatic survival factors is critical for successful therapeutic intervention in liver failure, this relationship has been investigated using a fibronectin-deficient mouse model of acute liver injury. Here, we show that lack of fibronectin induces significantly increased hepatocyte apoptosis, which is accompanied by significant down-regulation of the antiapoptotic protein, B-cell lymphoma-extra large (Bcl-xL). Furthermore, fibronectin deficiency leads to a significantly elevated production of hepatocyte growth factor in hepatic stellate cells postinjury, which, in turn, results in an earlier onset and acceleration of hepatocyte regeneration. Primary hepatocytes on fibronectin are protected from reactive oxygen species-induced cellular damage, retaining the expression of Bcl-xL, whereas those on type I collagen are not. This retained expression of Bcl-xL is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. CONCLUSION: We provide evidence that fibronectin-mediated matrix survival signals for hepatocytes are transduced through the PI3K/Bcl-xL-signaling axis in response to injury. This work defines fibronectin as a novel antiapoptotic factor for hepatocytes after acute liver injury, but demonstrates that fibronectin is not essential for subsequent hepatocyte proliferation.     

囊型肝包虫囊周肝细胞"消失"机制的初步研究

目的 观察囊型肝包虫周围肝细胞的病理形态学变化（肝细胞萎缩、坏死、凋亡）,初步探讨囊型肝包虫病肝细胞“消失”机制。方法 对30例肝包虫囊肿周围肝组织通过光镜观察肝组织的病理形态学变化,运用TUNEL法测定肝细胞凋亡,采用免疫组化技术检测肝包虫囊肿周围肝组织及正常肝组织中Bcl—2及Bax蛋白的表达。结果 TUNEL检测囊型肝包虫病患者肝细胞凋亡指数（TI）为0．12％,与正常肝组织（TI=O．16％）比较差异无显著性（P〉0．05）;Bc卜2及Bax蛋白的囊周肝组织呈低表达,分别为6．67％和13．33％,正常肝组织Bcl一2和Bax均为10．00％,差异无显著性（P〉O＋05）。病理组织学观察示肝细胞萎缩,肝细胞坏死明显。结论 肝细胞萎缩、坏死可能是引起肝细胞“消失”的主要机制,肝细胞压迫性和营养不良性萎缩、肝细胞坏死、肝细胞凋亡共同参与囊型肝包虫病肝细胞“消失”过程。     

Yinchenhao decoction attenuates obstructive jaundice-induced liver injury and hepatocyte apoptosis by suppressing protein kinase RNA-like endoplasmic reticulum kinase-induced pathway

BACKGROUND Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes, and leads to apoptosis. Apoptosis is very important in regulating the homeostasis of the hepatobiliary system. Endoplasmic reticulum(ER) stress is one of the signaling pathways that induce apoptosis. Moreover, the protein kinase RNA-like endoplasmic reticulum kinase(PERK)-induced apoptotic pathway is the main way; but its role in liver injury remains unclear. Yinchenhao decoction(YCHD) is a traditional Chinese medicine formula that alleviates liver injury and apoptosis,yet its mechanism is unknown. We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice(OJ).AIM To investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein(CHOP)-growth arrest and DNA damage-inducible protein34(GADD34) pathway and B cell lymphoma/leukemia-2 related X protein(Bax)/B cell lymphoma/leukemia-2(Bcl-2) ratio.METHODS For in vivo experiments, 30 rats were divided into three groups: control group, OJ model group, and YCHD-treated group. Blood was collected to detect the indicators of liver function, and liver tissues were used for histological analysis.For in vitro experiments, 30 rats were divided into three groups: G1, G2, and G3.The rats in group G1 had their bile duct exposed without ligation, the rats in group G2 underwent total bile duct ligation, and the rats in group G3 were given a gavage of YCHD. According to the serum pharmacology, serum was extracted and centrifuged from the rat blood to cultivate the BRL-3 A cells. Terminal deoxynucleotidyl transferase mediated d UTP nick end-labelling(TUNEL) assay was used to detect BRL-3 A hepatocyte apoptosis. Alanine aminotransferase(ALT) and aspartate transaminase(AST) levels in the medium were detected.Western blot and quantitative real-time polymerase chain reaction(q RT-PCR)analyses were used to detect protein and gene expression levels of PERK, CHOP,GADD34, Bax, and Bcl-2 in the liver tissues and BRL-3 A cells.RESULTS Biochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group. The TUNEL assay showed that massive BRL-3 A rat hepatocyte apoptosis was induced by OJ. Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ. Western blot or q RT-PCR analyses showed that the protein and m RNA expression levels of PERK, CHOP, and GADD34 were significantly increased both in the rat liver tissue and BRL-3 A rat hepatocytes by OJ. The Bax and Bcl-2 levels were increased, and the Bax/Bcl-2 ratio was also increased. When YCHD was used, the PERK, CHOP, GADD34,and Bax levels quickly decreased, while the Bcl-2 levels increased, and the Bax/Bcl-2 ratio decreased.CONCLUSION OJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio.YCHD can attenuate these changes.