二甲基甲酰胺通过活性氧介导线粒体通路诱导H9c2心肌细胞凋亡研究

目的 初步探讨二甲基甲酰胺（dimethylformamide,DMF）是否通过线粒体通路致H9c2心肌细胞凋亡及可能机制。方法 H9c2心肌细胞体外培养,分组给予不同处理：正常对照组,N-乙酰半胱氨酸（N-acetyl-L-cysteine,NAC）（4 mmol/L）组,DMF（200 mmol/L）组,NAC（4 mmol/L）+DMF（200 mmol/L）组,24 h后使用免疫荧光细胞化学法（immune-fluorescence cytochemistry,IFC）检测细胞内细胞色素C（cytochrome C,Cyt-c）定位,蛋白免疫印迹法检测胞浆Cyt-c、Bcl-2、Cleaved caspse-3水平。结果 IFC观察发现,正常对照及NAC组Cyt-c呈点状或斑块状分布于胞浆,DMF作用24 h导致Cyt-c弥散分布于胞浆,而NAC可抑制DMF的这种作用;蛋白免疫印迹结果表明,Cyt-c蛋白水平组间差异具有统计学意义（F=26.14,P<0.001）,与对照组相比,DMF组Cyt-c水平升高,NAC+DMF组较DMF组Cyt-c水平降低,差异均有统计学意义（均有P<0.001）;Bcl-2蛋白水平组间差异具有统计学意义（F=28.92,P<0.001）,与对照组相比,DMF组Bcl-2蛋白水平降低,与DMF组相比,NAC+DMF组Bcl-2蛋白水平升高,差异均有统计学意义（均有P<0.001）;Cleaved caspase-3蛋白水平组间差异具有统计学意义（F=28.98,P<0.001））,与对照组相比,DMF组Cleaved caspase-3水平上升,NAC+DMF组较DMF组Cleaved caspase-3水平降低,差异均具有统计学意义（均有P<0.001）。结论 DMF可以通过线粒体通路诱导H9c2心肌细胞凋亡,氧化应激可能参与该通路的发生。     

连接介导PCR分析线粒体DNA的氧化损伤频率及损伤热点

目的 论证线粒体ＤＮＡ（ｍｉｔｏｃｈｏｎｄｒｉａａｌ,ｍｔＤＮＡ）“常见缺失”与ＤＮＡ氧化损伤的关系。方法 采用连接介导ＰＣＲ（ｌｉｇａｔｉｏｎ ｍｅｄｉａｔｅｄ ＰＣｔｒ,ＬＭＰＣＲ）,在大鼠肝细胞标（ＢＲＩ,３Ａ）暴露于５０ｍｍｏｌ／Ｌ过氧化氢（Ｈ２Ｏ２）形成氧化损僵后,分析该缺失的断裂区段（８０７１～８１９０,约１２０ｂｐ）内,各核苷酸氧化损伤的频率及热点。结果 在８０７１～８１９０区段,存     

Enhanced induction of apoptosis by combined treatment of human carcinoma cells with X rays and death receptor agonists

The death receptors Fas and DR5 are known to be expressed not only in immune cells but also in various tumor cells. The aim of the present study was to determine whether X irradiation enhanced induction of apoptosis in Tp53 wild type and Tp53-mutated tumor cell lines treated with agonists against these death receptors. We showed that 5 Gy of X irradiation significantly up-regulated the expression of death receptors Fas and DR5 on the plasma membrane in gastric cancer cell lines MKN45 and MKN28, lung cancer cell line A549, and prostate cancer cell line DU145, and that subsequent treatments with agonistic molecules for these death receptors, Fas antibody CH11 and TRAIL, increased the formation of active fragment p20 of caspase 3 followed by the induction of apoptosis. This death-receptor-mediated apoptosis was independent of Tp53 status since MKN28 and DU145 were Tp53-mutated. The post-irradiation treatment of the cells with N-acetyl-L-cysteine (NAC) abolished the up-regulation of the expression of Fas and DR5 on the plasma membrane. NAC also attenuated the increase in the formation of p20 and the induction of apoptosis by agonistic molecules. These results suggested that the increase in the induction of apoptosis by combined treatment with X irradiation and CH11 or TRAIL occurred through a change of the intracellular redox state independent of Tp53 status in human carcinoma cell lines.     

美金刚胺对甲基汞致大鼠谷氨酸代谢障碍及氧化损伤的影响

目的探讨盐酸美金刚胺(memantine hydrochloride,MEM)对甲基汞所致谷氨酸代谢障碍以及氧化损伤的影响。方法实验用Wistar大鼠30只,按体重随机分成3组。第1组为对照组,第2组为单纯染汞组,第3组为MEM预处理组。对照组和单纯染汞组皮下注射生理盐水,MEM预处理组皮下注射5μmol/kg MEM;2 h后,对照组腹腔注射生理盐水,单纯染汞组和MEM预处理组腹腔注射12μmol/kg氯化甲基汞,注射容量为5 ml/kg。干预隔日一次,每周3次,染毒每日1次,每周5次,连续干预与染毒4周。于最后一次染毒24 h后每组取6只大鼠,麻醉后处死,测定大脑皮质汞含量,谷氨酸(Glu)、谷氨酰胺(Gln)含量,谷氨酰胺合成酶(GS)、磷酸活化的谷氨酰胺酶(PAG)活力,还原型谷胱甘肽(GSH)、丙二醛(MDA)含量,超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活力。每组其余4只大鼠,制备大脑皮质单细胞悬液,测定活性氧簇(ROS)含量以及细胞凋亡率。结果与对照组比较,单纯染汞组大鼠大脑皮质汞含量显著升高,Glu含量及PAG活力显著升高,Gln含量及GS活力显著降低;GSH含量、SOD及...     

丹参对全胃肠外营养致肝损伤的防治疗效观察

目的通过临床病例观察和分析，探讨注射用丹参在全胃肠外营养（totalparenteralnutrition；TPN）治疗中肝损伤的疗效。方法将2007～2008年临床收集的各种需要短期TPN治疗的住院病例24例，随机分为对照组和治疗组，两组均予TPN治疗，治疗组加用注射用丹参静脉滴注，检测病例肝功能的变化。结果对照组7例伴有肝损伤，其中2例伴中度ALT升高；治疗组4例伴有轻度肝损；相对治疗组，对照组ALT明显升高，具有明显的差异。短期TPN引起的肝损伤以ALT和ALP的升高为主。结论丹参对TPN引起的肝损伤具有一定的防治作用。     

扶正解毒颗粒改善镍暴露者肝酶活性的研究

目的 观察中药扶正解毒颗粒(FJK)对镍暴露者肝损伤的临床疗效及改善酶活性的作用。方法 将60例患者随机分为两组；A组31例，采用中药FJK合用保肝西药治疗；B组29例，仅用西药保肝疗法。疗程均为4周。观察治疗前后两组临床症状、体征的疗效，血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、白蛋白(ALB)、总胆红素(TBIL)、血清胆碱酯酶(SChE)、血清镍、红细胞膜Na^ —K^ —ATP酶及Ca^2 —ATP酶变化。结果 与B组比较，A组临床症状、体征的改善，ALT、AST、血镍水平及SChE、NNa^ —K^ —ATP酶及Ca^2 —AT酶活性的改善均优于B组(P＜0．05—0．01).结论 FJK具有降镍，恢复ATP酶活性，改善肝功能作用，临床疗效肯定。     

线粒体分裂融合基因在氧化应激诱导宫颈癌Hela细胞损伤中的作用

目的:利用维生素K3(VitaminK3,Vk3)复制子宫颈癌Hela细胞损伤模型,观察线粒体融合分裂基因的变化,探讨线粒体融合分裂基因在Vk3诱导Hela细胞凋亡过程中的作用。方法:MTT法检测各组Hela细胞存活率,Hoechst33258染色观察细胞凋亡,RT-PCR方法检测各组Hela细胞中线粒体融合基因Mfn1、Mfn2和Opa1及线粒体分裂基因Drp1、Fist1和MTP18mRNA表达。结果:Vk3能够降低Hela细胞存活率,并且Hoechst33258染色结果显示细胞呈现凋亡形态变化,凋亡率增加,Mfn1、Opa1、和MTP18mRNA表达量明显下降(P<0.05);而与Vk3单独作用组比较,Vk3+NAC联合作用组Hela细胞存活率增加(P<0.05),凋亡率降低,Mfn1、Opa1、和MTP18mRNA表达量明显增加(P<0.05)。结论:线粒体融合和分裂基因表达的降低都有可能参与了氧化应激诱导的细胞损伤作用。     

金属硫蛋白对急性化学性肝损伤的保护作用研究

[目的]研究含金属硫蛋白(metallothionein,MT)蛋奶粉对CCl4致大鼠急性肝损伤的保护作用。[方法]70只Wistar大鼠随机分为正常对照组10只、模型组20只、低和高剂量MT蛋奶粉治疗组各20只。低和高剂量MT蛋奶粉治疗组大鼠以含MT蛋奶粉灌胃1周,然后对除正常对照组以外的大鼠复制CCl4肝损伤模型,于造模当日,正常对照组和模型组大鼠用蒸馏水,治疗组用含MT蛋奶粉连续灌胃2周后,断头处死大鼠,观察各组大鼠血清酶类含量的变化和肝脏过氧化物质和抗氧化酶类(SOD、CAT)和GSH含量的变化。[结果]与正常对照组相比,模型组大鼠血清中ALT、AST、GGT和ALP含量显著升高(P<0.01),而含MT蛋奶粉治疗组大鼠血清中ALT、AST、GGT和ALP含量均显著低于模型组,尤其以高剂量组为显著(P<0.01)。含MT蛋奶粉组大鼠肝脏MDA含量显著低于模型组(P<0.01),抗氧化酶类SOD活性和抗氧化物质GSH含量显著高于模型组(P<0.05或P<0.01),对CAT活性并无显著性影响。[结论]含MT蛋奶粉对CCl4造成的大鼠急性肝损伤具有一定程度的保护作用。     

丹酚酸对四氯化碳致大鼠肝损伤保护作用

目的 探讨具有特定组成的丹酚酸(SA)对四氯化碳(CCl₄)所致大鼠肝损伤的预防性保护作用及其机制。方法 将健康SD成年大鼠随机分为5组,对照组和模型组给予蒸馏水,各实验组分别用3种不同剂量SA灌胃,2周后以花生油(对照组)或CCl₄(其余各组)腹腔注射,24 h后测定大鼠血清天门冬氨酸氨基转移酶(AST)及丙氨酸氨基转移酶(ALT)、血清和肝组织丙二醛(MDA)、谷胱甘肽(GSH)含量以及超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性,并观察肝组织病理变化。结果 模型组大鼠血清AST、ALT值分别为615 U/L和243 U/L,而SA组大鼠AST、ALT分别为348~452和134~181 U/L,活性显著下降(P<0.05)。与模型组比较,当SA剂量为300mg/(kg·bw)时,大鼠血清和肝匀浆中的SOD活性分别上升22.7%和16.7%,MDA含量降低19.3%和30.5%,GSH含量升高9.2%和9.1%,差异均有统计学意义(P<0.05)。实验组和对照组间GSH-Px活性没有显著变化(P>0.05)。模型组大鼠肝脏小叶中心部出现明显点状和灶状坏死,而SA不同剂量组大鼠肝脏病变明显减轻。结论 前期给予适量的SA处理可有效抵抗CCl₄所致大鼠急性肝损伤,其保护作用机制可能与提高大鼠机体抗氧化能力有关。     

Increased hepatic de novo lipogenesis and mitochondrial efficiency in a model of obesity induced by diets rich in fructose

Purpose

To assess hepatic de novo lipogenesis and mitochondrial energetics as well as whole-body energy homeostasis in sedentary rats fed a fructose-rich diet.

Methods

Male rats of 90 days of age were fed a high-fructose or control diet for 8 weeks. Body composition, energy balance, oxygen consumption, carbon dioxide production, non-protein respiratory quotient, de novo lipogenesis and insulin resistance were measured. Determination of specific activity of hepatic enzymes of de novo lipogenesis, mitochondrial mass, oxidative capacity and degree of coupling, together with parameters of oxidative stress and antioxidant defence, was also carried out.

Results

Body energy and lipid content as well as plasma insulin and non-esterified fatty acids were significantly higher in fructose-fed than in control rats. Significantly higher rates of net de novo lipogenesis and activities of hepatic lipogenic enzymes fatty acid synthase and stearoyl CoA desaturase-1 were found in fructose-fed rats compared to controls. Mitochondrial protein mass and degree of coupling were significantly higher in fructose-fed rats compared to controls. Hepatic mitochondria showed oxidative damage, both in the lipid and in the protein component, together with decreased activity of antioxidant defence.

Conclusion

Liver mitochondrial compartment is highly affected by fructose feeding. The increased mitochondrial efficiency allows liver cells to burn less substrates to produce ATP for de novo lipogenesis and gluconeogenesis. In addition, increased lipogenesis gives rise to whole body and ectopic lipid deposition, and higher mitochondrial coupling causes mitochondrial oxidative stress.     

运动联合大蒜素降低高脂血症大鼠血脂的实验研究

目的 通过建立高脂血症大鼠模型,探讨中等强度运动联合大蒜素对高脂血症大鼠血脂代谢、肝组织氧化损伤的影响。方法:大鼠50只,分为普通饲料组（A组）、高脂饲料组（B组）、高脂饲料＋大蒜素组（C组）、高脂饲料＋运动组（D组）、高脂饲料＋运动大＋蒜素组（E组）。A组普通饲料喂养,B组、C组、D组、E组高脂饲料喂养,C组、E组灌服剂量为30mg/kg·d大蒜素,D组、E组进行10周运动。测定各组大鼠血TC、TG、LDL-C、HDL-C、AST、ALT,肝组织MDA、SOD、GSH-Px。结果:与A组相比,B组、C组、D组、E组TC、TG、LDL-C、AST、ALT、MDA升高,HDL-C、GSH-Px降低（P＜0.05）,B组、C组、D组SOD降低（P＜0.05）;与B组相比,C组、D组、E组TC、TG、LDL-C、AST、ALT、MDA降低,GSH-Px、SOD、HDL-C升高（P＜0.05;）;与C组相比,D组TC、TG、LDL降低（P＜0.05）,E组TC、TG、LDL降低,HDL-C升高（P＜0.05）;与C组、D组相比,E组AST、ALT、MDA降低,GSH-Px、SOD升高（P＜0.05）。结论:中等强度运动和大蒜素均可降低高脂血症大鼠血脂,抑制肝组织脂质过氧化反应,减少肝细胞损伤,且两者具有协同效应。大蒜素对高脂血症大鼠血脂的调节效果不如中等强度运动。     

Plant polyphenols attenuate hepatic injury after hemorrhage/resuscitation by inhibition of apoptosis, oxidative stress, and inflammation via NF-kappaB in rats

Purpose  

Oxidative stress and inflammation contribute to hepatic injury after hemorrhage/resuscitation (H/R). Natural plant polyphenols, i.e., green tea extract (GTE) possess high anti-oxidant and anti-inflammatory activities in various models of acute inflammation. However, possible protective effects and feasible mechanisms by which plant polyphenols modulate pro-inflammatory, apoptotic, and oxidant signaling after H/R in the liver remain unknown. Therefore, we investigated the effects of GTE and its impact on the activation of NF-kappaB in the pathogenesis of hepatic injury induced by H/R.     

复合营养素对PM_(2.5)所致大鼠急性肺部氧化应激和炎症损伤的影响

目的探讨某复合营养素对大气PM_(2.5)致大鼠急性肺部氧化应激和炎症损伤的预防作用。方法将40只SD大鼠随机分为4组:空白组、PM_(2.5)模型组、低剂量营养素(150 mg/kg)+PM_(2.5)组、高剂量营养素(750 mg/kg)+PM_(2.5)组。营养素组连续预灌胃14 d营养素溶液,空白组、模型组灌胃生理盐水。第15天灌胃后除空白组外的各组大鼠气管滴注PM_(2.5)(9.0 mg/kg)混悬液染毒,每隔24 h 1次,共3次,空白组气管滴注生理盐水。末次染毒24 h后,收集血样、支气管肺泡灌洗液、肺组织匀浆和病理切片。分析测定血清丙二醛(MDA)、超氧化物歧化酶(SOD)水平,肺泡灌洗液中乳酸脱氢酶(LDH)、酸性磷酸酶(ACP)水平;肺匀浆液中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)水平。结果与空白组比较,PM_(2.5)模型组的大鼠MDA、ACP、LDH、TNF-α、IL-1β水平增加、SOD活力降低(P<0.05或P<0.01);与PM_(2.5)模型组比较,高、低剂量营养素+PM_(2.5)组能降低MDA、TNF-α、IL-1β水平及升高SOD活力(P<0.05或P<0.01),高剂量营养素+PM_(2.5)组能降低ACP、LDH水平(P<0.05或P<0.01),并改善大鼠肺组织病理变化。结论该复合营养素对PM_(2.5)致大鼠急性肺部氧化应激及炎症损伤具有保护作用。     

Stimulatory effect of glutamine on human monocyte activation as measured by interleukin-6 and soluble interleukin-6 receptor release

The activation state of murine peritoneal macrophages is shown to depend on extracellular glutamine concentration. However, there are no studies on the effect of glutamine concentration on the activation state of human monocytes. We studied the effect of extracellular glutamine concentration on interleukin (IL-6) and soluble IL-6 receptor (sIL-6R) production by activated monocytes. Monocytes separated by density gradient centrifugation and adherence to glass from buffy coats obtained from healthy donors were activated by 1 pg/ml or 1 ng/ml lipopolysaccharide (LIPS) and incubated for 48 h in 0, 0.1, 0.2, 0.6 and 2.0 mM glutamine. Levels of IL-6 and sIL-6R in culture medium were measured by immunoassay. The extracellular glutamine concentration had a significant effect on IL-6 secretion by activated human monocytes. The mean levels of IL-6 in 0.1 mM glutamine were only marginally higher (P = 0.54) compared to those in the absence of glutamine. A minimum of 0.2 mM glutamine was required to reach the maximal production of IL-6. At all glutamine concentrations the higher concentration of LPS (1 ng/ml) induced higher mean levels of IL-6 than the lower one (1 pg/ml). Glutamine concentration did not affect sIL-6R production. Our results indicate that very low levels of glutamine in plasma may impair the activation of human monocytes as measured by IL-6 secretion.     

镉中毒诱发的自由基对红细胞损伤及带3蛋白表达的影响

目的研究镉离子诱发的过氧化氢与红细胞损伤及带３蛋白（ｂａｎｄ３）表达的关系。方法应用免疫扫描电镜示踪带３蛋白及铈细胞化学法显示过氧化氢。应用Ｘ射线能量失散谱对Ｃｄ的细胞分布进行了分析。结果红细胞膜表面不但有大量过氧化氢反应标志,而且带３蛋白表达明显减少且细胞畸变。结论结果提示,镉离子能诱发自由基的大量产生,导致红细胞膜及其骨架的自由基损伤,从而影响红细胞的稳定性和变形性及带３蛋白的表达。     

胆碱酯酶在老年肺部感染致多器官功能障碍中变化及与预后关系

目的 探讨老年人肺部感染致多器官功能障碍血清胆碱酯酶(CHE)水平变化及与预后的关系,以期为该病诊治及病情监测提供依据.方法 以2009年1月-2011年12月住院老年肺部感染导致多脏器功能障碍综合征患者100例为研究对象,随机抽取同期健康体检老年患者40例为对照组,应用丁酰硫代胆碱法分别测定对照组、多器官功能障碍组、肝功能不全组CHE水平.结果 对照组血清CHE水平为(7800.03±1632.78)IU/L,肺部感染致多器官功能障碍组CHE水平为(4072.98±2396.36) IU/L,其中肝功能不全组为(4734.79±2445.64)IU/L,较对照组均明显降低(P＜0.05);对照组CHE异常比例为0.00％,老年多器官功能障碍组、肝功能不全组分别为:71.00％和54.16％,较对照组均明显升高(P＜0.05);按照预后将老年肺部感染致多器官功能障碍患者分为生存组与死亡组,生存组CHE水平为(4771.06±2431.42) IU/L,而死亡组则为(2387.96±1259.58)IU/L,死亡组较生存组明显降低(P＜0.05);生存组CHE异常比例为59.20％,而死亡组达96.60％,死亡组较生存组明显升高(P＜0.05).结论 老年肺部感染致多器官功能障碍患者血清CHE水平显著降低,其降低程度可能与预后相关,可作为患者病情监测、治疗与预后判断的综合指标.     

BDNF及高亲和力受体TrKB在胆红素神经损伤豚鼠皮层及海马表达变化的实验研究

目的:探讨新生豚鼠胆红素脑神经损伤元时脑源性神经营养因子(BDNF)及受体酪氨酸激酶B(TrKB)在大脑皮质及海马表达特点。方法:取生后2～5天内的豚鼠60只,随机分为3组,C组、T1组、T2组。C组为对照组;T1组:腹腔注射晶体胆红素100μg/g;T2组腹腔注射胆红素200μg/g,分别在4h、8h处死。做脑组织切片,电镜,光镜观察病理改变,并采用免疫组化和图像分析,观察不同时间点BDNF、TrKB在皮层及海马表达变化。结果:①胆红素脑神经元损伤模型成功建立。②T14h、T24h在早期皮层及海马是降低的。随时间延长,BDNF表达明显上升,T14h、T24h与C4h比较,P<0.05,T14h、T24h与T18h、T28h比较,P<0.05。③TrKB在皮层和海马表达是随时间延长和损伤加重表达增加,T14h、T18h、T24h、T28h与C4h、C8h比较,P<0.05。结论:胆红素脑神经元损伤中,皮层和海马BDNF、TrKB升高可能减轻神经元损伤,在抑制神经元凋亡和神经元修复中发挥重要作用,有利神经元修复和再生,可能是胆红素脑神经元损伤时机体的自我保护机制之一。     

凋亡因子在缺氧缺水缺食致心脏损伤中作用

目的 探讨凋亡相关因子Bax、Bcl-2、细胞色素C和caspase-3在缺氧缺水缺食条件下大鼠心肌细胞损伤中变化及其意义.方法将80只Wistar大鼠置于常压低氧舱(10％氧浓度)并禁食水,于实验1、3、5、7d,采用原位末端标记、免疫组织化学和原位杂交技术等方法,检测缺氧缺水缺食后心脏凋亡指数变化及凋亡相关因子Bax、Bcl-2、细胞色素C和caspase-3在其中的作用.结果 10％缺氧复合缺水缺食后1d和7d大鼠心肌细胞凋亡指数(62.63±14.97)、(20.19±3.26)增加;Bax、细胞色素C和caspase-3蛋白在单纯缺氧和缺氧缺水缺食后均可见1～7d表达增加,缺水缺食后在3～7d表达增加.Bcl-2在单纯缺氧和缺水缺食后1～7 d表达减少,缺氧缺水缺食后1～3d表达减少;Caspase-3 mRNA在单纯缺氧组和缺氧缺水缺食后1 d(4.19±1.42)、(3.87±0.86)和7 d(10.43±2.79)、(12.12 +2.27)表达增加,缺水缺食后1 d(3.85±0.88)表达增加.结论10％缺氧复合缺水缺食可引起心肌细胞凋亡,与Bax表达增加、Bcl-2表达减少有关;可能进一步通过线粒体途径,释放细胞色素C,激活caspase-3,引发心肌细胞凋亡.