内毒素对大鼠肝细胞线粒体膜电位的影响及黄芪注射液的干预作用

目的:观察内毒素血症对大鼠肝细胞线粒体膜电位(△Ψm)的影响及黄芪注射液的干预作用.方法:将40只大鼠随机分为正常对照组及内毒素注射后6h、12h组和黄芪处理组.分离肝细胞并检测其线粒体△Ψm水平,同时提取大鼠肝细胞线粒体测定其丙二醛(MDA)的含量及超氧化物岐化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)的活性.结果:在注射内毒素6h后,线粒体△Ψm减少,MDA的含量升高,SOD、GSH-PX活性减少,12h后,线粒体△Ψm明显减少,MDA升高更加明显,SOD、GSH-PX活性明显减少.与对应的内毒素组比较,经黄芪处理后,肝细胞线粒体△Ψm升高,MDA的含量明显减少,SOD、GSH-PX活性升高.结论:内毒素血症能造成肝细胞线粒体△Ψm下降和线粒体的氧化损伤,黄芪注射液可有效抑制内毒素血症肝细胞线粒体△Ψm的下降,减少内源性自由基的生成,提高机体的抗氧化损伤能力,具有保护肝细胞的作用.     

丹参素和肝细胞生长因子对药物性肝细胞损伤的影响

目的:观察丹参素、猪再生肝细胞生长因子(PRHGF)和猪促肝细胞生长素(PHGF)对药物性肝损伤的影响。方法:以硫代乙酰胺(TAA)复制体外培养大鼠肝细胞损伤模型,通过检测培养液中LDH选出量和四甲基偶氮唑盐(MTT)显色情况了解肝细胞的存活和增殖情况,并同时检测细胞内、外SOD含量。结果:培养介质中加入TAA后,致LDH逸出明显增加,肝细胞增生指数下降,上清液和细胞内SOD活性显著降低。TAA和丹参素或PRHGF合用,则肝细胞逸出的LDH明显减少,增生指数上升并超过正常对照组,上清液和细胞内SOD活性显著升高。结论:丹参素、PRHGF和PHGF均能阻止肝细胞TAA损伤和促进肝细胞增生的作用。     

解毒化瘀方对内毒素血症大鼠肝细胞线粒体氧化损伤的影响

目的:观察解毒化瘀方对内毒素血症( endotoxemia,ETM)大鼠肝细胞线粒体氧化损伤的影响.方法:将56只大鼠随机分为正常对照组及内毒素注射后6小时、12小时、24小时组和解毒化瘀方6小时、12小时、24小时组.眼眶取血,检测大鼠血清肿瘤坏死因子(TNF)-α水平,同时取肝组织分离线粒体,测定其丙二醛(MDA)的含量及超氧化物岐化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性.结果:在注射内毒素6小时后,大鼠血清TNF-α水平及线粒体MDA的含量升高,线粒体SOD、GSH-Px活性下降,随着时间的延长,TNF-α水平及MDA的含量进一步升高,SOD、GSH-Px活性明显下降.与对应的内毒素组比较,经解毒化瘀方处理后,TNF-α水平及MDA的含量均降低,SOD、GSH-Px活性均升高.结论:解毒化瘀方对内毒素刺激Kupffer细胞释放TNF-α有抑制作用,同时减少内源性自由基的生成,提高机体的抗氧化损伤能力,具有保护肝细胞的作用.     

内毒素亲和吸附剂对失血性休克大鼠NO、SOD及MDA水平的影响

目的探讨内毒素亲和吸附剂(SPV)对失血性休克大鼠模型一氧化氮(NO)、超氧化物歧化酶(SOD)及丙二醛(MDA)水平的影响。方法选取成年的SD大鼠60只,随机分为正常对照组10只,模型组和药物组各25只。模型组和药物组建立失血性休克大鼠模型,药物组造模前采用SPV进行灌胃。复苏后1、4、8、16 h,对所有大鼠的血清NO、SOD、MDA、内毒素及肿瘤坏死因子(TNF)-α水平进行检测。结果复苏后1、4、8、16 h,与模型组比较,药物组大鼠血清NO水平较低,血清SOD水平较高,血清MDA、内毒素、TNF-α水平较低(均P0.05);结论 SPV能够明显降低失血性休克大鼠血清NO、MDA、内毒素及TNF-α水平,提高SOD活性,对失血性休克引起的氧化应激损伤有保护作用。     

丹参素对实验性肝细胞损伤的防护作用

在原代培养小鼠肝细胞上用氰化钾缺氧损伤模型，观察不同剂量的氰化钾肝细胞的损伤及丹参素对损伤肝细胞的保护作用，结果表明，氰化钾对肝细胞具有毒性，丹参素能使含２．５ｍｍｏｌ／Ｌ氰化钾肝细胞培养在ＬＤＨ活性明显降低，显著增加肝细胞ＳＯＤ活力，降低ＬＰＯ含量。提示氰化钾所致肝细胞损伤与自由基毒性作用密切相关，丹参素具有稳定细胞膜，清除自由基的作用。     

芍药甙防治大鼠肝细胞体外损伤形态学及生物化学研究

目的:研究芍药甙对大鼠肝细胞体外损伤的防治作用。方法:采用D-氨基半乳糖和内毒素损伤大鼠原代培养肝细胞的方法造模,通过检测细胞形态变化和生化指标LDH、GOT、SOD等,观察芍药甙对其预防和治疗作用。结果:与各损伤组相对应的预防、治疗组,细胞表面结构、细胞核、内质网、高尔基复合体及线粒体、溶酶体等结构恢复正常。LDH、GOT、SOD也明显降低(P<0.05～0.01)。结论:芍药甙可通过增强肝细胞膜及溶酶体膜的稳定性,对肝细胞损伤起防治作用。     

心痛贴对急性心肌梗死犬氧化应激、心肌代谢和超微结构的影响

目的 观察心痛贴(XTT)对犬急性心肌梗死的保护作用机制.方法 30只犬随机分成5组:假手术组、模型组、XTT小剂量组、大剂量组、阳性药对照组.结扎犬冠状动脉左前降支(LAD)复制急性心肌梗死模型,将XTT贴于犬左前胸近腋窝部位,测定给药后6 h血清超氧化物歧化酶(SOD)、脂质过氧化物(LPO)、游离脂肪酸(FFA)、氧化氮(NO)含量变化,透射电镜观察心肌细胞超微结构改变.结果 XTT能降低血清FFA及LPO含量,提高NO含量和SOD活性(P<0.05、0.01),可改善心肌梗死后心肌细胞超微结构.结论 XTT可改善心肌细胞超微结构,对缺血心肌具有明显保护作用,与其增强抗氧化酶活性,减少脂质过氧化反应,纠正心肌FFA代谢紊乱,提高NO水平有关.     

粉防己碱对小鼠脑肝组织的保护作用

目的 :观察粉防己碱对小鼠脑肝组织的保护作用。方法 :采用小鼠脑肝组织进行体外培养 ,加入粉防己碱 ,测其一氧化氮(NO )、丙二醛 (MDA)、超氧化物歧化酶 (SOD)含量。结果 :粉防己碱能增加组织SOD活性及NO含量 ,降低MDA水平。结论 :粉防已对脑肝组织有明显的保护作用     

内毒素血症对大鼠肝细胞线粒体能量代谢的影响及黄芪注射液的干预作用

[目的]观察内毒素血症(Endotoxemia,ETM)对大鼠肝细胞线粒体能量代谢的影响及黄芪注射液的干预作用.[方法]将40只大鼠随机分为正常对照组及内毒素(LPS)注射后6、12 h组和黄芪处理6、12 h组.取肝组织分离线粒体,测定其琥珀酸脱氢酶(SDH)及细胞色素氧化酶(CCO)活性,同时行高压液相色谱检测线粒体内腺苷酸能荷(EC)水平.[结果]在注射LPS 6h后,线粒体SDH、CCO活性降低,EC值减少;12h后,线粒体SDH、CCO活性明显降低,EC值进一步减少,与对应的LPS组比较,经黄芪处理后,肝细胞线粒体SDH、CCO活性及EC值均升高.[结论]ETM可造成肝细胞线粒体能量储备的下降,黄芪注射液能提升其肝细胞线粒体氧化磷酸化水平,增加线粒体腺苷酸EC值,促进肝细胞线粒体的能量合成和代谢,具有保护肝细胞的作用.     

苦楝素诱导小鼠肝细胞DNA损伤

目的:观察苦楝素对DNA损伤及损伤修复的影响。方法:体外采用苦楝素与小鼠胚胎肝细胞BNL CL2细胞株共培养,CCK-8检测肝细胞活力,吸光度检测肝细胞内ROS含量,Western blot检测γ-H2AX、Parp-1、poly ADP-ribose蛋白表达,观察不同浓度苦楝素以及苦楝素不同作用时间对DNA损伤及损伤修复的影响。结果:随着苦楝素浓度升高,肝细胞BNL.CL2细胞活力逐渐下降(P0.0001),ROS含量升高(P0.05),同时γ-H2AX蛋白质含量随着苦楝素作用时间的延长而升高(P0.0001)。苦楝素作用0.5～1.0 h Parp-1自身PAR化量达最高(P0.05);而苦楝素作用时间自1～24 h Parp-1的PAR逐渐减少(P0.05)。结论:苦楝素可通过增加细胞内ROS诱导DNA损伤,同时抑制Parp-1的活性从而抑制DNA损伤的修复。     

Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes.

Nitric oxide (NO.) is a short-lived mediator which can be induced in a variety of cell types and produces many physiologic and metabolic changes in target cells. The inducible or high-output NO. synthase (NOS) pathway was first characterized in macrophages activated by lipopolysaccharide (LPS) and interferon gamma (IFN-gamma). Hepatocytes also express an inducible NOS following exposure to the combination of endotoxin (LPS) and tumor necrosis factor (TNF), interleukin 1 (IL-1), and IFN-gamma. In this study, to identify which of these cytokines, if any, was acting to induce the gene expression for hepatocyte NOS, we measured the levels of rat hepatocyte NOS mRNA by Northern blot analysis after stimulation by various combinations of endotoxin and cytokines in vitro. We found the mRNA for hepatocyte NOS to be a single band at approximately 4.5 kilobases which was maximally up-regulated (approximately 70-fold) by the combination of TNF, IL-1, IFN-gamma, and LPS. Abundance of NOS mRNA peaked 6-8 hr after stimulation and then declined by 25% at 24 hr. Unstimulated hepatocytes in vitro showed only a trace mRNA band after prolonged autoradiographic exposure. As single agents, TNF and IL-1 were the most effective inducers of hepatocyte NOS mRNA. Combinations of two or three stimuli revealed strong synergy between TNF, IL-1, and IFN-gamma. The increased mRNA levels correlated with elevated nitrogen oxide release and cGMP levels in the culture supernatants. Dexamethasone and cycloheximide inhibited induction of mRNA for hepatocyte NOS in a dose-dependent fashion. The addition of NG-monomethyl-L-arginine had no effect on mRNA levels but effectively blocked NO. formation. The inducible hepatocyte NOS mRNA was also detected in rat hepatocytes following chronic hepatic inflammation triggered by Corynebacterium parvum injection in vivo. These data demonstrate that the inducible NOS is functional in rat hepatocytes both in vitro and in vivo and that this pathway is under complex control. Endotoxin and inflammatory cytokines act synergistically to up-regulate gene expression for hepatocyte NOS, whereas glucocorticoids down-regulate the mRNA.     

内毒素对正常大鼠和肝硬化大鼠肝脏一氧化氮合酶活性及局部一氧化氮水平的影响

观察内毒素血症时肝硬化大鼠和正常大鼠肝脏一氧化氨台酶(NOS)活性以及NO水平的变化．探讨两者在肝硬化大鼠对内毒素高敏感性中的作用。方法：肝硬化大鼠模型由四氯化碳台并乙醇诱导．内毒素4mg／kg体重经尾静脉注射。分别于注射后2、6、12小时测定血清咎丙转氨酶(ALT)、谷草转氨酸(AST）肝脏组织中NOS活性以及N0代谢产物NO^-／NO^-水平。结果：肝硬化大鼠对内毒素损伤敏感性增高。正常大鼠在内毒素作用2小时后NOS活性和NO水平均显著增高．并持续至12d,时,但肝硬化大鼠肝脏NOS活性则未见显著变化,NO水平在12小时才开始增高。结论：肝硬化时肝脏NOS对内毒素刺激的敏感性降低,NO产生减少,可能是导致肝硬化肝脏对内毒素损伤高敏感性的原因之一。     

Marrow culture in diffusion chambers in rabbits: III. Effect of endotoxin and leukocyte products on cell production

Soluble leukocyte products harvested from incubated peritoneal exudate leukocytes, injected intravenously or intramuscularly, increased production of granulocytes and macrophages in marrow in diffusion chambers implanted into the peritoneal cavity of rabbits. Red cell production in the chambers was not consistently affected. Endotoxin increased production of all cell types. Endotoxin tolerance induced by daily injection of endotoxin to host rabbits abolished granulopoietic stimulation by endotoxin given during the culture period but did not diminish the granulopoietic stimulation produced by injected leukocyte products. Attempts to induce tolerance to leukocyte products by daily injections did not reduce the granulopoietic stimulation produced by either endotoxin or leukocyte products injected during the culture period. Intraperitoneally administered leukocytes products markedly inhibited production of all cell types. Endotoxin or leukocyte products given to normal rabbits increased plasma colony stimulating activity (CSA); the increase occurred sooner after leukocyte products than after endotoxin. Endotoxin-tolerant animals showed no rise in CSA after endotoxin, but their response to leukocyte products was normal. Leukocyte products added to agar cultures neither supported nor inhibited colony growth but augmented CSA-stimulated colony production. Endotoxin, leukocyte products, and CSA are different and may interact in regulating granulopoiesis.     

Nitric oxide production by hepatocytes contributes to the inhibitory effect of endotoxin on insulin-like growth factor I gene expression

We tested whether endotoxin (lipopolysaccharide, LPS) inhibits IGF-I gene expression in hepatocytes and the possible role of Kupffer cells and nitric oxide (NO) in this effect. LPS decreased IGF-I mRNA in hepatocyte cultures and increased the nitrite + nitrate levels in the culture medium. Furthermore, there was a negative correlation between the IGF-I mRNA and the nitrite+nitrate levels. When hepatocytes were cocultured with Kupffer cells, the inhibitory effect of LPS on IGF-I mRNA was higher than in hepatocyte cultures, but the stimulatory effect on nitrite+nitrate was similar in both conditions. The exogenous NO donated by S-nitroso-n-acetyl-d,l-penicillamide also decreased the IGF-I gene expression in hepatocyte cultures. In addition, two specific inducible NO synthase (iNOS) inhibitors, l-N6-(1-iminoethyl)lysine (l-NIL) and aminoguanidine, prevented the effect of LPS on nitrite+nitrate levels and on IGF-I gene expression in hepatocyte cultures. These data indicate that iNOS-derived NO may cause downregulation of IGF-I gene expression in hepatocytes. However, in cocultures, the iNOS inhibitor l-NIL prevented the effect of LPS on nitrite+nitrate levels, but only attenuated the LPS-induced decrease in IGF-I gene expression. We conclude that in hepatocytes, LPS-induced decrease in IGF-I is mainly due to induction of iNOS, whereas in the presence of Kupffer cells LPS inhibits IGF-I through NO release and through other inhibitory pathways.     

Role of Endotoxin in NF-κB Activation by Ethanol in Rat Hepatocytes

Background Endotoxin plays an important role in the progression of alcoholic liver injury. However, the role of endotoxin in acute activation of NF-κB by ethanol remains unclear.
Methods In primary rat hepatocyte cultures treated with ethanol and/or endotoxin, the DNA-binding activity of NF-κB in the nuclear extract was estimated by electrophoretic mobility shift assay. After pretreatment of a CYP2E1 inhibitor, 4-methyl pyrazole or diallyl sulfide, NF-κB activity was also measured in the same manner.
Results Ethanol or endotoxin caused activation of NF-κB in primary rat hepatocytes. Taking 50 mM of ethanol and endotoxin together raised the rapid increase in NF-κB activation, but both 100 mM of ethanol and endotoxin treatment reduced the increase. Addition of diallyl sulfide decreased the activity at rapid phase but increased it after 60 min, whereas addition of 4-methyl pyrazole caused no change of the activity at rapid phase but raised it after 60 min. Pretreatment with both endotoxin and a CYP2E1 inhibitor raised the activation of NF-κB constantly after ethanol addition. These findings suggest that endotoxin plays a critical role in the metabolism-independent activation of NF-κB by ethanol.
Conclusions Endotoxin raised metabolism-independent activation of NF-κB by high ethanol in rat hepatocytes.     

The effects of age and inflammation on antioxidant enzyme activity in the eye

The effects of age and inflammation on antioxidant enzyme activity were examined in the rabbit eye. Iridic, choroidal and retinal superoxide dismutase activity (SOD) was measured in adult (6 mo) and aged (4+ yr) animals before and after intravitreal endotoxin administration. No age-related decrease in SOD was noted in control animals. In fact, an age-related increase in SOD activity was noted in each tissue. Endotoxin administration was associated with significant SOD induction in irides, choroids and retinas of adult animals but not of aged animals. Iridic catalase activity, however, exhibited a 22% age-related decrease in control animals and a further 45% decrease after the inflammatory stimulus. Malondialdehyde (MDA), quantified by measurement of thiobarbituric acid (TBA) reaction as an index of lipid peroxidation, increased at each age exhibiting a linear relationship in tissues from young (6 wk), adult and aged animals in endotoxin-treated eyes (r=0.999) while not exhibiting an age-related difference in control eyes. The present data indicate that the propensity of ocular tissues from aged animals to undergo increased oxidative damage subsequent to inflammation may be due to both the age-related decrease in catalase activity and the inability of tissues from aged animals to induce SOD in response to an inflammatory stimulus.     

The Effect of Endotoxin on Circulating Lymphocytes in Normal Man

S ummary . The effect of endotoxin administration on peripheral blood lymphocytes was studied in normal man. A partially purified bacterial endotoxin from Pseudomonas aeruginosa was given intravenously to 10 healthy volunteers. Endotoxin administration resulted in a substantial lymphocytopenia with reduction of both thymus-dependent T and bone marrow-derived B lymphocytes. Absolute numbers of circulating B lymphocytes decreased to a greater extent than T lymphocytes. T-lymphocyte functional characteristics, including mitogenic response to phyto-haernagglutinin and responder cell activity in mixed lymphocyte culture, were unaffected by endotoxin administration. Response to concanavalin A was consistently reduced. Stimulation of allogeneic lymphocytes in mixed lymphocyte culture, a function of B lymphocytes and monocytes, was also decreased. When endotoxin was used as an in vitro mitogen, a significant increase in DNA synthesis was observed only in lymphocytes from individuals previously given endotoxin and not in normal control lymphocytes. These studies indicate that experimental endotoxaemia is associated with a reduction in absolute numbers of circulating T and B lymphocytes, a decrease in B-lymphocyte bur not T-lymphocyte functional activity, and redistribution of a concanavalin A-responsive subpopulation of T lymphocytes. The data suggest that endotoxin has important effects on lymphocyte distribution and function in man.     

抗生素杀菌过程致内毒素释放的比较性研究

目的了解不同杀菌性抗生素杀菌过程致细菌内毒素释放的不同特点。方法采用鲎基质偶氮法检测常用抗生素在２倍最低抑菌浓度下对大肠杆菌杀菌过程中细菌内毒素的释放水平，分别计算活菌数持续下降阶段平均每减少一个对数数量级（ｌｇ）菌落形成单位所致内毒素的释放量（减数期释放率）和杀菌后期存活菌数基本稳定阶段内毒素水平的增加比率（稳定期递增率）。结果各种抗生素依其致内毒素释放能力的大小大致可分为高、中、低三群。β内酰胺类抗生素内部各品种间差别显著，氨曲南、头孢他定、头孢唑啉、头孢呋辛、氨苄西林属高释放品种，阿莫西林、克拉维酸、亚胺培南属低释放品种。氨基糖苷类各品种间差别无显著性，均属中等释放品种。喹诺酮类的环丙沙星属高释放品种，氧氟沙星属中等释放品种。而多粘菌素Ｂ则低于所有品种。各种抗生素的稳定期递增率除氨基糖苷类只有低递增率外，其余和减数期释放率的趋势基本一致。结论临床常用各种抗生素杀菌过程致内毒素释放的能力有着很大差异，增强此方面的认识有助于选择使用抗生素抗感染时对全身炎性反应综合征的控制。