【作者】 王华；

【导师】 徐德祥；

【作者基本信息】 安徽医科大学 ， 卫生毒理学， 2009， 硕士

【摘要】 细菌脂多糖(LPS)和氨基半乳糖(GalN)共处理诱导以肝脏细胞凋亡为主要特征的急性肝损伤,肿瘤坏死因子α(TNF-α)在GalN/LPS引起小鼠急性凋亡性肝损伤过程中起关键作用。体外研究发现,核因子κB(NF-κB)激活对抗TNF-α介导的肝细胞凋亡,而外源性活性氧(ROS)或内源性氧化应激均可加重TNF-α介导的肝细胞凋亡。本课题通过深入研究NF-κB抑制剂二硫代氨基甲酸吡咯烷(PDTC)及抗氧化剂褪黑素(MT)和N-乙酰半胱氨酸(NAC)分别对GalN/LPS诱导小鼠急性凋亡性肝损伤的不同效应,继而阐明NF-κB激活和ROS生成在GalN/LPS诱导小鼠急性凋亡性肝损伤中的不同作用。1.PDTC对GalN/LPS诱导小鼠急性凋亡性肝损伤的效应目的通过研究PDTC对GalN/LPS诱导小鼠急性凋亡性肝损伤的效应,以阐明NF-κB在LPS诱导小鼠急性凋亡性肝损伤中的作用。方法本研究设置生理盐水(NS)组、LPS组、GalN组、GalN/LPS组、PDTC+GalN/LPS组和PDTC组。GalN/LPS组同时给予小鼠LPS(20μg/kg,i.p.)和GalN(600 mg/kg,i.p.),PDTC+GalN/LPS组小鼠于LPS(20μg/kg,i.p.)处理前24 h和2 h分别经腹腔注射PDTC(100+100 mg/kg),LPS组、GalN组和PDTC组分别给予小鼠LPS(20μg/kg,i.p.)、GalN(600 mg/kg,i.p.)和PDTC(100+100 mg/kg),NS组小鼠给予等容积生理盐水。每组10只小鼠被用于观察LPS处理后72 h内的动物死亡情况;每组6只小鼠经LPS处理后1.5 h被取血、处死并留取肝脏,用RT-PCR检测肝脏组织TNF-α、IL-1β和IL-6 mRNA表达水平,用EMSA分析肝脏NF-κB结合活性,用ELISA测定血清TNF-α含量;每组12只小鼠于LPS处理后8 h取血、处死并留取肝脏,测定血清丙氨酸转氨酶(ALT)活力、一氧化氮(NO)水平和肝组织还原性谷胱甘肽(GSH)含量,用比色法检测肝脏caspase-3活性,用TUNEL技术和DNA断裂分析方法检测肝脏细胞凋亡,并对肝组织切片行常规HE染色。结果GalN/LPS共处理显著升高小鼠血清ALT活力;肝脏组织病理学检查发现,GalN/LPS组小鼠肝脏严重充血、坏死并伴有大量炎性细胞浸润,肝脏组织TUNEL阳性细胞显著增多;GalN/LPS处理的小鼠肝脏组织caspase-3活性明显升高;在GalN/LPS共处理72 h内有90%小鼠发生死亡,所有死亡小鼠均伴有肝脏严重充血。PDTC预处理抑制GalN/LPS诱导的肝脏NF-κB激活和TNF-α表达,但PDTC预处理反而加重GalN/LPS引起的小鼠肝脏细胞凋亡、进一步升高血清ALT活力、加重肝脏充血和坏死并加速小鼠死亡。结论NF-κB抑制剂PDTC通过抑制肝脏实质细胞NF-κB介导的抗凋亡机制加重GalN/LPS诱导的小鼠急性凋亡性肝损伤。2.MT对GalN/LPS诱导小鼠急性凋亡性肝损伤的效应目的通过探讨MT对GalN/LPS诱导小鼠急性凋亡性肝损伤的效应,以阐明ROS在LPS诱导小鼠急性凋亡性肝损伤中的作用。方法实验小鼠被随机分为3组。GalN/LPS组小鼠被同时给予GalN(600 mg/kg,i.p.)和LPS(20μg/kg,i.p.);MT+GalN/LPS于GalN/LPS处理前0.5 h给予MT(5.0 mg/kg,i.p.),且在GalN/LPS处理后1 h、2 h再分别腹腔给予MT(2.5 mg/kg)处理;NS组小鼠经腹腔注射等容积生理盐水。经GalN/LPS处理8 h后,摘眼球取血、处死并取肝脏,检测血清ALT活力、TNF-α和NO水平,测定肝脏组织caspase-3活性、还原性GSH含量、GSH-Px活性和GSH-Rd活性,采用DNA梯度分析方法检测肝脏细胞凋亡,并对小鼠部分肝脏行病理组织学检查。结果GalN/LPS处理显著升高小鼠血清ALT活力,引起小鼠肝脏组织严重充血、坏死并伴有大量炎性细胞浸润,而且GalN/LPS明显升高小鼠肝脏组织caspase-3活性和增多肝脏凋亡细胞数。进一步研究结果显示,MT处理明显降低GalN/LPS升高的小鼠血清ALT活力。同时,MT处理也明显减轻肝脏充血和坏死。另一项实验结果表明,MT明显减轻GalN/LPS引起小鼠肝脏细胞凋亡,表现为肝脏caspase-3活性下降和DNA断裂的减少。而且,MT处理明显减轻GalN/LPS引起的肝脏组织GSH损耗,显著升高肝脏组织GSH-Rd和GSH-Px活性。结论抗氧化剂MT减弱GalN/LPS引起的小鼠急性凋亡性肝损伤。3.NAC对GalN/LPS诱导小鼠急性凋亡性肝损伤的效应目的通过探讨N-乙酰半胱氨酸(NAC)对GalN/LPS诱导小鼠急性凋亡性肝损伤的效应,以阐明ROS在LPS诱导小鼠急性凋亡性肝损伤中的作用。方法雌性ICR小鼠被随机分成4组。除NS组外,所有小鼠均被同时给予GalN(600 mg/kg,i.p.)和LPS(20μg/kg,i.p.);NAC/GalN/LPS组小鼠于GalN/LPS共处理前30 min给予NAC(150 mg/kg,i.p.);BSO/NAC/GalN/LPS组小鼠于GalN/LPS共处理前12 h和2 h被分别注射BSO(100 mg/kg,i.p.),并于GalN/LPS共处理前30 min给予NAC(150 mg/kg,i.p.);NS组小鼠经腹腔注射给予等容量生理盐水。GalN/LPS处理1.5 h后,部分动物被剖杀、取血,并测定血清TNF-α含量;于GalN/LPS处理8 h后,剩余动物被剖杀、取血和肝脏,测定血清ALT活力和NO水平,检测肝脏组织caspase-3活性与GSH含量,采用DNA断裂分析方法检测肝脏凋亡,并对部分小鼠肝脏行病理组织学检查。结果GalN/LPS处理显著升高小鼠血清ALT活力,引起小鼠肝脏组织严重充血、坏死并伴有大量炎性细胞浸润,而且GalN/LPS明显升高小鼠肝脏组织caspase-3活性和增多肝脏凋亡细胞数。进一步研究结果显示,NAC处理明显降低GalN/LPS升高的小鼠血清ALT活力。同时,NAC处理也明显减轻肝脏充血和坏死。另一项实验结果表明,NAC明显减轻GalN/LPS引起小鼠肝脏细胞凋亡,表现为肝脏caspase-3活性下降和DNA断裂的减少。而且,NAC处理明显减轻GalN/LPS引起的肝脏组织还原型GSH损耗。结论抗氧化剂NAC保护GalN/LPS引起的小鼠急性凋亡性肝损伤。综上所述,本研究可得出如下结论:NF-κB激活在LPS诱导小鼠急性凋亡性肝脏损伤中起保护作用,而ROS生成在LPS诱导小鼠急性凋亡性肝脏损伤中起促进作用。更多还原

【Abstract】 Mice were co-injected with lipopolysaccharide (LPS) and D-galactosamine (GalN) to induce acute apoptotic liver injury. Tumor necrosis factor alpha (TNF-α) plays an important role in GalN/LPS-evoked acute apoptotic liver damage. In vitro studies have showed that nuclear factor kappa B (NF-κB) activation protected against TNF-α-mediated hepatocellular apoptosis, whereas exogenous reactive oxygen species (ROS) and endogenous oxidative stress aggravated TNF-α-triggered hepatocellular apoptosis. Accordingly, the present study aimed to investigate the different effects of pyrrolidine dithiocarbamate (PDTC), a hibitor of NF-κB activity, and two kinds of antioxidants, melatonin (MT) and N-acetylcysteine (NAC),on LPS-induced acute apoptotic liver damage, and to clarify different roles of NF-κB activation and ROS production in GalN/LPS-induced acute apoptotic liver injury.1. Effects of PDTC on GalN/LPS-induced acute apoptotic liver injury in miceObjective The present study was to investigate the effects of PDTC on LPS-induced acute apoptotic liver injury in mice and to clarify the role of NF-κB activation in GalN/LPS-induced acute apoptotic liver injury. Methods All mice were randomly divided into six groups. Mice in GalN/LPS group were co-injected with GalN (600 mg/kg, i.p.) and LPS (20μg/kg, i.p.). Mice in PDTC+GalN/LPS group were injected with two doses of PDTC, one (100 mg/kg, i.p.) at 24 h before LPS and the other at 2 h before LPS (20μg/kg, i.p.). Mice in control groups were treated with LPS (20μg/kg, i.p.), GalN (600 mg/kg, i.p.), PDTC (100 mg/kg, i.p.) or saline. Ten mice each group were observed for animal survival within 72 h after LPS treatment. Six mice in each group were sacrificed 1.5 h after LPS for collecting blood and isolating livers. The expression of hepatic TNF-αmRNA was determined by reverse transcription and polymerase chain reaction (RT-PCR). Hepatic NF-κB binding activity was measured using electrophoretic mobility shift assay (EMSA). Serum TNF-αlevel was analyzed by enzyme-linked immunosorbent assay (ELISA). Twelve mice in each group were sacrificed 8 h after LPS treatment. Serum was collected for measurement of alanine aminotransferase (ALT) and nitrate plus nitrite. Livers were dissected for measurements of glutathione (GSH) content, caspase-3 activity and hepatocellular apoptosis and histological examination. Results Co-injection of GalN and LPS markedly increased serum ALT activity. Histopathological examination of liver sections revealed that GalN/LPS induced hepatic congestion and necrosis, and massive macrophages infiltration, increased the number of TUNEL-positive cells in mouse liver. GalN/LPS treatments significantly increased hepatic caspase-3 activity, led to 90% mortality within 72 h and with severe congestion and necrosis in the liver of all the dead mice. PDTC pretreatment significantly inhibited GalN/LPS-induced hepatic NF-κB activation and TNF-αexpression. In contrast, PDTC aggravated GalN/LPS-triggered hepatocellular apoptosis, increased serum ALT activity, exacerbated hepatic hemorrhage and necrosis, and accelerated death. Conclusion PDTC aggravates GalN/LPS-induced acute apoptotic liver injury via inhibiting NF-κB-mediated anti-apoptotic effects.2. Effects of MT on GalN/LPS-induced acute apoptotic liver injury in miceObjective The present study was to investigate the effects of MT on GalN/LPS-induced acute apoptotic liver injury and to elucidate the roles of ROS in GalN/LPS-induced acute apoptotic liver injury. Methods Mice were randomly divided into three groups. Mice in GalN/LPS group were co-injected with GalN (600 mg/kg, i.p.) and LPS (20μg/kg, i.p.). Mice in MT+GalN/LPS group were administered with three doses of MT, one (5.0 mg/kg, i.p.) at 0.5 h before GalN/LPS, another (2.5 mg/kg, i.p.) at 1.0 h after GalN/LPS, and the other (2.5 mg/kg, i.p.) at 2.0 h after GalN/LPS treatments. The control mice were given with normal saline. Mice were sacrificed at 8 h after GalN/LPS. Blood serum was collected for measurements of ALT, TNF-α, and nitrate plus nitrite. Livers were excised to determine hepatic GSH content and antioxidant enzyme activity. Hepatocellular apoptosis were analyzed by measurement of DNA fragmentation and caspase-3 activity. Results Serum ALT activities were significantly increased 8 h after GalN/LPS, massive hemorrhage being observed in histological sections of liver from GalN/LPS-treated mice. MT markedly attenuated GalN/LPS-induced elevation of serum ALT. In parallel, MT significantly improved GalN/LPS-evoked hepatic congestion. Additional experiment showed that MT distinctly attenuated GalN/LPS-triggered hepatic apoptosis, measured by inhibition of caspase-3 activities and reduction of DNA fragment. Moreover, MT significantly increased hepatic glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) activities and attenuated hepatic GSH depletion in GalN/LPS-treated mice. Conclusion MT protects mice against GalN/LPS-evoked acute apoptotic liver injury.3. Effects of NAC on GalN/LPS-induced acute apoptotic liver injury in miceObjective The present study was to investigate the effects of NAC on GalN/LPS-induced acute apoptotic liver injury and to elucidate the roles of ROS in GalN/LPS-induced acute apoptotic liver injury. Methods All female ICR mice were randomly divided into four groups. Except for NS group, all mice were treated with GalN (600 mg/kg, i.p.) and LPS (20μg/kg, i.p.). Mice in NAC/GalN/LPS group were injected with NAC(150 mg/kg,i.p.)at 0.5 h before GalN/LPS administrations. Mice in buthionine sulfoximine (BSO)/NAC/GalN/LPS group were challenged with BSO (100+100 mg/kg,i.p.) plus NAC (150 mg/kg,i.p.) before GalN/LPS treatments. The control mice were given with saline. Some mice were killed at 1.5 h after GalN/LPS administrations. Serum was collected for determinations of TNF-αlevel by ELISA. The remaining mice were sacrificed at 8 h after GalN/LPS. Blood was collected by drawing the eyeball. Serum ALT acitivity and NO production were measured. Livers were dissected for measurements of caspase-3 activity and GSH content. Hepatocellular apoptosis were analyzed by DNA laddering. Liver sections were examined by HE stain. Results There was a significant increase in serum ALT activity of GalN/LPS-treated mice as compared with the controls. Massive hemorrhage was also observed in histological sections of liver from GalN/LPS-treated mice. NAC markedly attenuated GalN/LPS-induced elevation of serum ALT. In parallel, NAC significantly improved GalN/LPS-evoked hepatic congestion. Another experiment indicated that NAC distinctly attenuated GalN/LPS-triggered hepatic apoptosis, measured by inhibition of caspase-3 activities and attenuation of DNA fragmentation. Moreover, NAC significantly attenuated hepatic GSH depletion in GalN/LPS-treated mice. Conclusion NAC prevents mice from GalN/LPS-evoked acute apoptotic liver injury.Taken together, these results indicate that NF-κB activation plays a protective role in LPS-induced acute apoptotic liver injury, whereas ROS release exerts its detrimental effects in GalN/LPS-induced acute apoptotic liver damage.更多还原