肿瘤坏死因子-α诱导肝细胞凋亡在暴发性肝衰竭中的作用

目的 研究肿瘤坏死因子－α（ｔｕｍｏｒ ｎｅｃｒｏｓｉｓ ｆａｃｔｏｒ－α,ＴＮＦ－α）诱导肝细胞凋亡细胞凋亡在暴发性肝衰竭中的作用机制。方法 分别注射脂多糖（ｌｉｐｏｐ ｏｌｙｓａｃｃｈａｒｉｄｅ,ＬＰＳ）和ＴＮＦ－α于Ｄ－氨基半乳糖（Ｄ－ｇａｌａｃｔｏｓａｍｉｎｅ,ＧａｌＮ）致敏的ＢＡＬＢ／ｃ小鼠造成暴发性肝衰竭模型,用脱氧核糖核酸转移酶介导的缺口原位末端标记（ｉｎ ｓｉｔｅ ｅｎｄ ｌａｂｅ     

Preventive effect of cyclosporin A on experimentally induced acute liver injury in rats

Abstract: We examined the effect of cyclosporin A (CsA) on the pathogenesis of acute experimental liver injury in rats induced by injection of heat-killed Propionibacterium acnes (P. acnes) and subsequent injection of lipopolysaccharide (LPS). Pretreatment with CsA significantly reduced serum alanine aminotransferase (ALT), serum tumor necrosis factor-α (TNF-α) production, without changing the TNF-α mRNA level in the liver, and plasma interferon-γ (IFN-γ), following LPS injection in this model. Twenty-four-hour mortality was also markedly improved, from 100% in the P. acnes plus LPS group to 0% in the CsA-pretreated group. Although direct addition of CsA to isolated hepatic macrophages from P. acnes-pretreated rats did not prevent the production of TNF-α and active oxygen species, isolated hepatic macrophages from P. acnes plus CsA-pretreated rats significantly reduced their production in response to the addition of LPS. These results suggest that CsA protects against P. acnes plus LPS-induced acute liver injury, not by direct inhibition of hepatic macrophage activation, but by indirect prevention of hepatic macrophage activation, presumably related to the reduction in plasma IFN-γ levels.     

益赛普对大鼠放射性肺损伤中TNF-α的影响

目的观察注射用重组人肿瘤坏死因子受体-抗体融合蛋白（益赛普）对大鼠放射性肺损伤过程中TNF-α的影响,试图寻找一种预防或治疗放射性肺损伤的有效途径。方法 72只雌性SD大鼠随机分为3组：正常对照组、单纯照射组和益赛普治疗组,每组24只。照射组及治疗组动物麻醉后,行直线加速器全胸部照射一次,剂量为25Gy。照射后第一周内,治疗组大鼠腹腔注射益赛普（5mg·kg^-1）,共计2次。对照组和照射组大鼠注射同等体积的生理盐水。于照射后第1、4、8、和24周处死动物,取部分肺组织行HE染色,观察组织学变化,另提取组织行免疫组化观察组织中TNF-α变化,使用酶联免疫吸附分析法检测血清中TNF-α水平。所有数据采用SPSS统计软件进行方差分析。结果照射组大鼠肺组织血清中TNF-α水平与对照组相比,明显升高,并在第4周时达到顶峰（P〈0.01,q=5.63,q=6.21）;治疗组TNF-α水平与对照组相比,第4周时也增加明显,但是与照射组相比明显下降（P〈0.01,q=4.97q=7.42）。结论大鼠放射性肺损伤时TNF-α水平明显升高,在放射性肺损伤发展中起到重要作用,益赛普能显著降低它们的水平,并抑制炎症反应严重程度,从而能有效地防治放射性肺损伤。     

吡咯烷二硫代氨基甲酸对大鼠急性肺损伤TNF-α的影响

目的 探讨核因子-кB(NF-кB)抑制剂(吡咯烷二硫代氨基甲酸,PDTC)在脂多糖(LPS)所致大鼠急性肺损伤(ALI)中对肿瘤坏死因子-α (TNF-α)的影响.方法 雄性大鼠66只,随机分3组.对照组(N组)6只,腹腔注射生理盐水3 mL/kg; ALI组(L组)30只,腹腔注射LPS3 mg/kg; PDTC干预组(P+L组)30只,先腹腔注射PDTC120 mg/kg,半小时后再腹腔注射LPS3 mg/kg.后两组分别于腹腔注射LPS后1、2、4、8、12 h作为观测时间点.观察肺组织匀浆及血浆中TNF-α的变化.结果 L组各时相肺组织匀浆及血浆中TNF-α较N组升高(P＜0.01),且以2h组升高最为显著,但P+L组TNF-α与L组比较减少(P＜0.05).结论 LPS引起肺组织和血中TNF-α大量释放,NF-кB参与炎症的调控,在ALI中起重要作用.PDTC通过调控炎性介质的表达和释放,可有效地减轻LPS所致大鼠ALI.     

脂多糖预处理对非酒精性脂肪性肝炎的影响

目的 探讨脂多糖(lipopolysaccharide,LPS)预处理对非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)的影响.方法 选取24只成年雄性Wistar大鼠,分为正常对照组、NASH组和LPS预处理组.NASH组饲以高糖高脂饲料;LPS预处理组饲料同肝损伤组,隔日皮下注射LPS 0.5 mg/kg;正常对照组饲以普通饲料;所有大鼠自由进食与饮水.于实验第9周末处死大鼠.制备肝组织切片,计数浸润肝组织的淋巴细胞;测定血浆内毒素水平和丙氨酸转氨酶(ALT)活性、肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)含量.结果 NASH组血浆内毒素水平比正常对照组高;LPS预处理组血浆ALT水平、肝组织淋巴细胞计数与NASH组比较均显著降低;血浆TNF-α水平LPS预处理组与NASH组比较明显降低,而血浆IL-10则相反,差异有统计学意义(P＜0.05).肝组织切片HE染色结果显示,LPS预处理组与NASH组比较,肝细胞内脂肪空泡小、数量少,脂肪变性明显减轻.结论 小剂量LPS预处理与减轻NASH密切相关.其机制可能是LPS预处理影响了细胞因子的释放,导致Th1/Th2细胞因子失衡,Th1向Th2偏离,可能诱导了宿主的免疫耐受,表现为肝组织损伤减轻.

Abstract：

Objective To investigate the changes of Th1/Th2 cytokines and its relationship with lipopolysaccharide (LPS) in pretreatment of relieving nonalcoholic steatohepatitis (NASH).Methods The 24 male Wistar rats were randomly divided into 3 groups: normal control group, liver injury group and LPS pretreatment group. The rats were given normal diet in normal control group,high-sucrose and high-fat diet both in liver injury group and in LPS pretreatment group, and the rats in LPS pretreatment group were given hypodermic injection of LPS 0. 5 mg/kg every other day. The level of plasma endotoxin (ET), activity of alanine aminotransferase (ALT), content of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were determined. At the end of week 9, the rats were executed, and the liver tissue slices were prepared to investigate hepatic pathologic change by hematoxylin and eosin (HE) staining.Results The level of plasma ET was significantly higher in liver injury group than in normal control group. The level of plasma ALT and infiltrating lymphocytes in liver tissue were significantly lower in LPS pretreatment group than in liver injury group. The level of plasma TNF-α was significantly lower in LPS pretreatment group compared with liver injury group.In contrast, the level of plasma IL-10 was higher (P＜0. 05). Histology with HE staining showed that hepatocyte steatosis was obviously relieved with smaller lipid droplet in LPS pretreatment group than in liver injury group. Conclusions LPS pretreatment can alleviate high-sucrose and high-fat induced NASH. The disequilibrium of Th1/Th2 cytokines may be an important part of mechanism.     

全身炎症反应对髂动脉损伤后狭窄的影响

目的用脂多糖(LPS)诱导加重猪全身炎症反应,观察其对髂动脉损伤后狭窄的影响。方法12头小型猪随机分为两组,实验组(n=6)给予静脉注射LPS,对照组(n=6)给予生理盐水。所有动物都行右髂动脉内膜剥脱术,并于术前、术后4h和术后24h抽取静脉血,行酶联免疫吸附反应检查(ELISA)测定炎症细胞因子肿瘤坏死因子-α(TNF-α)血浆浓度。术后28d处死动物,取术侧血管和对侧髂动脉作为对照行病理切片观察管腔变化情况。结果(1)在对照组和实验组中,术后4hTNF-α的血浆浓度均比术前明显升高(P<0.05),并且实验组的升高水平显著高于正常对照组(P<0.05)。(2)病理切片发现,4周后实验组和对照组均有新生内膜形成,导致血管管腔狭窄。(3)在实验组观察到比对照组更大的新生内膜面积和严重的狭窄程度。结论血管损伤后早期的全身炎症反应对后期的新生内膜形成起重要作用。     

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.     

TNF-α在对乙酰氨基酚所致大鼠药物性肝损伤中的作用

目的探讨TNF-α在对乙酰氨基酚所致大鼠急性药物性肝损伤中的作用。方法 40只SD大鼠随机分成空白对照组、益赛普对照组、对乙酰氨基酚组、对乙酰氨基酚+益赛普组,每组10只。单次给药,24 h后采集血液测定生化指标,之后处死大鼠,取肝脏组织做病理检查以及应用ELISA试剂盒检测血清TNF-α。结果对乙酰氨基酚组肝损伤明显,阻断TNF-α后肝损伤减轻。对乙酰氨基酚组肝功生化值及TNF-α水平较空白对照组升高,差异有统计学意义(P0.05),对乙酰氨基酚+益赛普组肝功生化值及TNF-α水平较对乙酰氨基酚组下降,差异有统计学意义(P0.05)。结论 TNF-α的表达在对乙酰氨基酚所致大鼠急性肝损伤肝组织中明显升高,阻断TNF-α后肝损伤减轻,TNF-α可能参与对乙酰氨基酚所致肝损伤。     

肿瘤坏死因子-α、诱导性一氧化氮合酶在扑热息痛肝损害中的表达

目的:探讨炎症反应在扑热息痛(acetaminoph-en,AAP)肝损害中的作用.方法:应用AAP建立SD大鼠肝损害模型;分别于给药后3、6、12、24h处死大鼠,AAP组和对照组分别测定ALT水平,HE染色观察肝脏病理变化,放射免役分析法测定血清TNF-α水平,免疫组织化学和Westernblot方法检测肝组织中TNF-α、iNOS蛋白的表达.结果:AAP组和对照组相比:血清ALT(nKat/L)进行性升高(3、6、12、24h的值分别为:1166.90±151.03vs586.78±89.35,2153.84±254.55vs573.45±75.18,4220.84±928.52vs750.15±81.68,13202.64±1392.78vs780.16±161.37,均P<0.01);肝脏病理损伤进行性加剧,24h达高峰;给药后24h血清TNF-α(g/L)水平显著升高(5.69±0.46vs2.64±0.27,P<0.01),且与血清ALT水平呈显著的正相关(r=0.773,P<0.01);免疫组织化学方法测定肝组织TNF-α、iNOS表达明显增强,分别于6、12h达高峰;Westernblot方法检测肝组织TNF-α、iNOS蛋白的表达显著高于对照组(P<0.01),分别于3、6h达高峰,24h时仍高于正常水平.结论:炎症反应在AAP肝损伤发生、发展的过程中发挥着关键的作用.     

All-trans retinoic acid suppresses liver injury induced by Propionibacterium acnes and lipopolysaccharide in rats

All-trans retinoic acid (ATRA) has been reported to exert major effects on the immune system, including monocytes/macrophages. The present study was designed to determine whether ATRA would modulate macrophage-associated liver injury induced by Propionibacterium acnes and lipopolysaccharide (LPS) in rats. All-trans retinoic acid administration alleviated the liver injury and reduced the incidence of death following hepatic failure. Serum alanine aminotransferase (ALT) levels 5 h after, and survival rates within 12 h after the administration of LPS were significantly lower in the ATRA-treated group (134 ± 119 IU/L and 72.7%) compared with the control group (713 ± 411 IU/L and 18.2%; P < 0.05). Histological findings supported these results. These effects may be due to suppression of tumour necrosis factor-α (TNF-α) and superoxide anions produced by activated macrophages. Serum levels of TNF-α 1 h after LPS administration were significantly lower in the ATRA-treated group (60.5 ± 7.0 ng/mL) as compared with the control group (105.2 ± 39.3 ng/mL; P < 0.05). Formazan deposition that was generated by the perfusion of the liver with nitroblue tetrazolium, also suggested suppression of the release of superoxide anions from hepatic macrophages. These results suggest that ATRA acts as an immunomodulator in liver injury by suppressing the activation of liver macrophages.     

内毒素性肝衰竭大鼠TLR4mRNA表达、血清肿瘤坏死因子-α及肝细胞凋亡的动态变化

目的 观察急性内毒素性肝衰竭大鼠肝组织中TLR4mRNA表达变化规律及其与血清肿瘤坏死因子-α水平、肝细胞凋亡的关系。方法雌性Wistar大鼠给予D氨基半乳糖／脂多糖同时腹腔注射，计算动物死亡率及生存时间，动态观察给药后4、8、12h肝功能、血清肿瘤坏死因子-α、肝组织TLR4mRNA表达及病理变化，以TUNEL法检测原位细胞凋亡，计算凋亡指数。结果80％大鼠死于急性肝衰竭，平均生存时间15．6h±1．8h，病理表现为肝脏大块或亚大块坏死。给药后4、8、12h血清TNF—α增高含量及肝细胞凋亡均增加，血清TNF—α变化早于肝细胞凋亡指数的增加，肝组织TLR4mRNA的表达与血清TNF—α含量呈正相关（r=0．709，P=0．000）。结论 内毒素通过单核吞噬系统TLR4介导TNF—α大量产生，激活炎症级联反应并诱导肝细胞凋亡是内毒素性肝衰竭的重要病理机制之一，阻断肝内外单核吞噬系统TLR4介导的的生理学作用，可能会对内毒素性肝衰竭起到一定防治作用。     

Heat shock response decreases endotoxin-induced acute lung injury in rats

OBJECTIVE: Transient whole-body hyperthermia was reported to reduce lung damage in a rat with intra-abdominal sepsis produced by caecal perforation. METHODOLOGY: In order to determine the effect of heat shock response on acute lung injury induced by endotoxin, which plays a central role in the pathogenesis of sepsis, we instilled either saline or lipopolysaccharide (LPS) intravenously with and without heat pretreatment in rats. The heated rats had their rectal temperature raised to more than 40 degrees C for 13 min 18 h before intravenous administration of saline or LPS. RESULTS: We found that the lung leak was significantly increased among the rats given LPS intravenously with (median, 0.17; range, 0.15-0.22; n = 10) and without heat pretreatment (0.23; 0.17-0.30; n = 10) compared with those of saline-treated rats (0.13; 0.10-0.14; n = 10) (P < 0.05 in each). However, rats given LPS after heat pretreatment had significantly decreased lung leak index compared with those of LPS-treated rats without heat pretreatment (P < 0.05). Rats administered LPS intravenously showed increased myeloperoxidase activity without heat pretreatment (19.01; 9.34-28.00 U/g; n = 10) compared with that of saline-treated rats (7.09; 4.49-10.56 U/g; n = 5) (P < 0.05) (Fig. 2). Myeloperoxidase activity of the rats treated with LPS with heat pretreatment (5.57; 2.87-8.96 U/g; n = 10) was significantly decreased to the level of normal control compared with that of LPS-treated rats without heat pretreatment (P < 0.05). The levels of heat shock proteins (HSP72) in lung tissue, which were examined by western blot analysis, were increased over baseline levels at 23 h after hyperthermic stress. CONCLUSIONS: These observations show that brief heat shock response is associated with the induction of HSP72 protein synthesis and attenuated neutrophil recruitment and acute lung leak is induced by endotoxin in rats.     

Tumor Necrosis Factor-α Plays an Initiating Role in Extracorporeal Circulation-induced Acute Lung Injury

Background

Despite advances in critical care, the mortality rate for patients with acute lung injury (ALI) remains high. The aim of this study was to test the hypothesis that tumor necrosis factor-α (TNF-α) plays an initiating role in the onset of extracorporeal circulation (ECC)-induced ALI.

Methods

Eight New Zealand rabbits subjected to 1 h of ECC and 40 min of observation after termination of ECC were used for monitoring pulmonary nociceptor activity. Fifty Sprague-Dawley (SD) rats that received 2 h of ECC and 4 h of rest were used to measure the pulmonary function and inflammatory cytokines release, including total cells, neutrophils, and TNF-α in bronchoalveolar lavage (BAL) and white blood cell (WBC) and neutrophils in blood. An additional 40 SD rats were randomized to pretreatment with inhalation of phosphate buffer solution (control group), IgG (IgG inh group), or TNF-α antibody (anti-TNF-α inh group) and venous injection of TNF-α antibody (anti-TNF-α iv group). After 2 h of ECC and 4 h of rest, the arterial blood and BAL fluid were collected for measurement of arterial oxygen pressure (PaO₂) and inflammatory cytokines release. The left-lower-lung tissues of animals were stained with hematoxylin & eosin (H&E).

Results

The results demonstrated that the activities of airway nociceptor and TNF-α release were similarly upregulated at the early stage and in a time-related manner in ECC-induced ALI. Pretreatment with TNF-α antibody inhalation, but not venous injection, improved pulmonary function, inhibited pulmonary inflammation, and attenuated pulmonary histopathological changes after ECC.

Conclusion

We concluded that TNF-α played an important role in the pathogenesis of ALI and acted as an initiating cytokine at the early stage of ECC-induced ALI.     

IgA deficiency evidence after anti-TNF-α treatment in a psoriatic arthritis patient: case report

It is known that the use of anti-TNF-α drugs is related to an increased incidence of infective diseases. This therapy can not be administered to patients having active infections and it has to be considered with caution in case of acquired or congenital immunodeficiency diseases. We report the case of a 28-years-old man affected by psoriatic arthritis; he developed some infections during treatment with TNF-α blockers. The infections were caused by a selective IgA deficiency, that was not evident before the anti-TNF-α blockers administration and disappeared after withdrawing the biological therapy. This case-report draws our attention to the possibility of cases of subclinical immunodeficiency, unknown by the patients, but important in the prognosis and in the therapeutic approach to these diseases. Therefore, it is important to evaluate carefully the immunologic status of patients during the pre-therapeutic screening for TNF-α blocking therapy.     

血必净注射液对脓毒症大鼠肺损伤的保护作用

目的：探讨血必净注射液对脓毒症大鼠肺损伤的保护作用及机制。方法：采用盲肠结扎穿孔法（CLP）建立脓毒症模型,雄性Wistar大鼠80只,随机分成4组：正常对照组（N组,8只）;假手术组（S组,8只）;脓毒症组（CLP）（C组,32只）和血必净治疗组（X组,32只）,C组和X组分别于CLP后经大鼠颈静脉注射生理盐水或血必净注射液2ml/kg,分别于CLP后3、6、12和24h各处死8只动物,取肺组织,比色法检测丙二醛（MDA）和髓过氧化物酶（MPO）,酶联免疫吸附法检测血清肿瘤坏死因子α（TNF-α）水平。结果：脓毒症时各时间点肺组织MDA、MPO和血清TNF-α浓度较对照组显著升高,而血必净治疗后则有明显下降（P〈0.01）。结论：血必净注射液可减轻脓毒症时的肺损伤,其机制可能与减少肺组织MDA、MPO和血清TNF-α表达有关。     

枳黄方对急性酒精性肝损伤大鼠内毒素信号转导通路中白细胞分化抗原14的影响

[目的]探讨酒精性肝损伤时，枳黄方对内毒素信号通路中白细胞分化抗原14（CD14）表达的影响。[方法]将75只Wistar大鼠随机分为空白对照（正常）组、酒精攻击（模型）组、枳黄方（治疗）组，10d后，处死大鼠，取大鼠血清和肝脏测定丙氨酸氨基转移酶（ALT）、天冬氨酸转氨酶（AST），苏木精-伊红染色观察肝脏病理变化，基质染色法测定血清内毒素，免疫组化法测定肝脏肿瘤坏死因子α（TNF-α），RT—PCR法测定肝组织CD14mRNA的变化。[结果]治疗组肝脏病理表现较模型组好，其血清内毒素、ALT、AST和肝组织TNF-α、CD14mRNA表达水平均高于正常组（P〈0．05，〈0．01，〈0．01，〈0．01，〈0．05），且均显著低于模型组（均P〈0．01）。[结论]枳黄方能显著降低内毒素信号转导通路上CD14基因水平的表达，减少肝组织TNF-α的表达，这可能是其对大鼠酒精性肝损伤有较明显保护作用的机制之一。