束缚应激加重对乙酰氨基酚肝毒性作用及其机制研究
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摘要
研究背景和目的
对乙酰氨基酚(商品名为扑热息痛,Acetaminophen,简称APAP)是世界上应用最广泛的非处方类解热镇痛药。在美国销售的含有APAP的制剂超过300种,平均每年的销量是十亿片。尽管在常规治疗剂量下APAP非常安全,但是该药的治疗窗较窄。因为过量使用APAP每年有大于100,000例的患者需要到中毒治疗中心接受治疗,其中包括超过56,000的门诊患者和2600例的住院病人和估计458例死于急性肝衰竭的病人。根据美国急性肝衰竭研究委员会报告,美国50%的急性肝衰竭病人都是由于APAP中毒,每年的人数超过1100人。有人报道,在APAP导致急性肝衰竭的发生率每年呈递增趋势,1998年是21%,而2003年是51%。由于对乙酰氨基酚的肝毒性,在2011年1月美国FDA发布公告要求在处方药中APAP的含量每片不得超过325mg。在2014年2月FDA又发布报告,建议医生和医疗人员禁用含APAP超过325mg的药品。虽然过量使用是导致APAP肝脏毒性的重要原因,但也有很多导致APAP中毒的风险因素,包括营养不良、饮酒和联合用药。迄今为止未见有研究报道外界应激对APAP肝毒性的影响。
应激是一种机体状态,指机体在内部或外界刺激或应激原的影响下后,机体产生的一系列非特异性应答反应。应激原可以是物理因素,也可以是精神因素或二者的综合作用。当机体受到强烈刺激时,机体就会兴奋交感-肾上腺髓质系统(sympathoadrenomedullary system,SAM)和下丘脑-垂体-肾上腺皮质轴(hypothalamic-pituitary-adrenal,HPA),表现为释放儿茶酚胺和糖皮质激素等外周递质。很早之前,大家已经认识到应激能够影响许多疾病的发生发展过程和结果。目前人们主要关注的是应激与心血管系统疾病、神经系统疾病、消化系统疾病、免疫系统疾病、HIV/AIDS以及癌症等之间的关系。大量的研究结果表明应激可以抑制特异性免疫反应,诱导产生或加重抑郁症、心血管系统疾病、肝脏疾病,并促进HIV/AIDS和癌症的病情发展。
目前,有越来越多的证据表明应激对肝脏疾病和功能存在影响。根据早期的临床报道,社会心理应激可以影响肝脏疾病的发生、发展及结果。例如,Hirose等发现情绪应激反应(如受到催眠暗示的恐惧或焦虑)可以明显增加肝脏血流量(HBF)。Fukudo等研究发现,酒精性肝炎中社会心理应激的严重程度与肝炎恶化及肝脏纤维化变化之间存在紧密的联系。在Beck Depression Inventory杂志中,也有学者发现在慢性乙型病毒性肝炎患者,其血清丙氨酸氨基转移酶(alanine transaminase,ALT)水平与其精神抑郁程度之间存在明显的正相关关系。而且,即使剔除了包括年龄、性别、教育水平、抽烟、饮酒与否以及病程等混杂因素,在Grossarth-Maticek分型中慢性丙型病毒性肝炎的严重性与I型人格人群具有相关性。在原位肝脏移植中,发生急性排斥反应的患者手术前的人格品质更倾向于是那些“顺从的”“相对易于受人控制的”以及“淡漠心境”者。在动物试验研究中,人们同样证明了应激与肝脏疾病之间的紧密相互联系。例如,电击应激可以加重四氯化碳处理过的小鼠肝脏损害,也可以使α-半乳糖胺诱导的肝炎加重,这种肝炎和疟疾、沙门氏菌感染诱导的肝损害以及乙型、丙型病毒性肝炎有关。有人在给大鼠施加多种不同的急性应激后,电镜检查发现肝脏粗面内质网破裂并膨胀,糖原耗竭,线粒体肿胀,其中最显著的变化是细胞膜的自噬泡数量增多和体积变大。有意思的是,即使在正常的啮齿类动物中,束缚以及电击应激可以触发温和的肝脏损害,这种肝损害以轻度的血清丙氨酸转氨酶(ALT)水平升高为特点。此外,社会隔绝应激(social isolation stress)可以增加转化生长因子-α转基因小鼠自发性肝细胞肝癌的发生率,同时还可以加速小鼠结肠肿瘤肝转移进程。
动物束缚应激模型是一种直接、非损伤性和简单的心理与生理应激模型。束缚应激可产生心理性的逃避反应和限制机体行动。束缚应激模型不但可准确且可靠地控制应激实验条件,而且与给药造模、手术造模以及外界强烈的温度改变或噪音相比,束缚应激模型可以有效地检测促肾上腺皮质素、皮质酮、fos蛋白、HPA轴的脱敏和细胞因子的表达。同时束缚应激可以协同多种因素进行研究,例如学习记忆、摄食情况和声音刺激等。在最近15年间,束缚应激模型一直是使用最多的应激动物模型。
本课题研究目的是：一、考察束缚应激对APAP给药后肝毒性的影响及可能的发生机制；二、探讨儿茶酚胺递质和肾上腺受体在束缚应激诱导肝损伤过程中作用。
方法
实验选用Balb/c小鼠,雄性,SPF级,8-10周龄,购自军事医学科学院实验动物中心。动物饲养于解放军疾病预防控制所毒理学评价研究中心屏障设施内,温度控制在19-26℃,12小时照明,12小时黑暗,动物自由进食饮水。正式实验前动物检疫观察3天。实验前一天禁食过夜,实验当天同一时间(上午8：00)将动物放入束缚筒内,束缚后保证小鼠自由呼吸,身体无法扭动同时不受挤压。束缚应激后动物或者被立刻麻醉处死或者放回笼内继续饲养。动物处死时,均用戊巴比妥钠麻醉,从腹主动脉采血。动物采血后,立刻取下肝脏,生理盐水冲洗,取左肝页用中性福尔马林固定,石蜡包埋用于病理组织学检查。剩余的肝脏用液氮速冻并保存于-80℃。
在考察束缚应激对对乙酰氨基酚肝毒性影响的实验中,在束缚应激后的各时间点腹腔注射150mg/kg的APAP或生理盐水。APAP给药后4小时,动物麻醉处死采样。在考察儿茶酚胺递质和肾上腺素受体对束缚应激诱导肝损伤影响的实验中,束缚前30分钟腹腔注射1mg/kg的哌唑嗪用于阻断α-1受体；束缚前30分钟腹腔注射2mg/kg的育亨宾用于阻断a-2受体；束缚前30分钟腹腔注射20mg/kg的倍他洛尔用于阻断β-1受体；束缚前30分钟腹腔注射5mg/kg的ICI118,551用于阻断β-2受体；束缚前24小时皮下注射2mg/kg的利血平用于耗竭儿茶酚胺递质。
用全自动生化分析仪检测血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST),用用酶循环法测定肝脏中谷胱甘肽的含量；用酶联免疫法(ELISA)检测血清IL-6和TNF-a含量。对肝组织进行病理学检测,HE常规染色观察病变程度。用TUNEL法原位检测细胞凋亡情况。用免疫组化法检测检钡Kupffer细胞活性情况。用蛋白免疫印迹(Western Blot)法检测肝组织caspse-3、Caspase-9、Bcl-2,和Bax的蛋白表达量。
统计学处理
实验数据采用SPSS13.0统计软件分析处理,用均数士标准差(Meand士SD)表示；单因素试验的多组比较采用单因素方差分析(One. way ANOVA),方差齐时采用多重比较LSD法,方差不齐时采用Dunnett' ST3法。在考察束缚和药物相互干预的试验中采用双因素方差分析比较各处理方式的主效应及交互效应。当P<0.05时,差异被认为有统计学意义。
结果
第一部分：小鼠束缚应激模型的建立
1.动物在束缚应激0、0.5、1.5、3、6小时后,组间血清丙氨酸氨基转移酶(ALT)和门冬氨酸氨基转移酶(AST)的差异有统计学意义(F=5.114,24.666；P均小于0.01),表现为ALT和AST在束缚前3小时持续性的升高。与正常对照组(束缚0小时)相比,束缚应激1.5和3小时后ALT和AST的差异均有统计学意义(P<0.05或P<0.01)。其中在束缚应激3小时后,ALT的水平达到正常值的3倍,根据海氏法则可认为小鼠束缚应激3小时肝细胞达到了损伤的标准。但是,当束缚应激6小时后,血清ALT和AST的水平开始降低,此时AST与正常对照组相比差异仍有统计学意义(P<0.05)。
2.当小鼠束缚1.5、3小时及束缚3小时放松6小时后,各组间血清ALT和AST水平的差异有统计学意义(F=16.000,27.268；P均小于0.01)。当小鼠束缚应激3小时后,血清ALT和AST的水平达到最高值,然后将动物放回笼中自由活动,放松后血清ALT和AST水平开始恢复,放松6小时时,血清ALT和AST水平与正常对照组相比差异仍有统计学意义(P<0.05)；在放松24小时后,ALT值基本达到正常值水平。但是AST与正常对照组相比差异仍有统计学意义(P<0.01)。
3.束缚不同时间后肝脏总谷胱甘肽(GSH+GSSG)含量、氧化型谷胱甘肽(GSSG)含量和GSSG/GSH比值的组间差异有有统计学意义(F=7.199,13.522,22.817;P均小于0.01)。与血清ALT和AST水平的变化情况相同,束缚应激3小时后(GSH+GSSG)含量降低、GSSG含量和GSSG/GSH比值和GSSG/GSH比值逐步升高,与正常对照组相比,这些指标的差异均有统计学意义(P<0.01),其中束缚1.5小时后氧化型谷胱甘肽(GSSG)含量和GSSG/GSH比值与正常对照组相比亦显著性升高,差异有统计学意义(P<0.05或P<0.01)。束缚3小时后将动物放回笼中自由活动,放松后总谷胱甘肽(GSH+GSSG)、GSSG和GSSG/GSH开始恢复,至放松24小时后肝脏中的谷胱甘肽含量恢复至正常值水平。
4.正常对照组和束缚应激0.5、1.5小时的动物肝脏病理组织学检查未见异常,小鼠束缚应激3小时后仅可见有轻度的肝细胞胞浆疏松淡染,未发现明显的病理学改变。
第二部分：束缚应激对对乙酰氨基酚肝毒性的影响
1.在小鼠束缚应激0.5、1.5、3小时以及束缚3小时放松6、24小时后分别给予150mg/kg的APAP,结果发现,组间ALT和AST的差异有统计学意义。(F=14.555,12.584；P值均小于0.01)。小鼠束缚3小时可使APAP引起的ALT和AST升高增强,束缚小3时放6小时后亦可使APAP引起的ALT升高增强,与单纯APAP给药组相比,差异均有统计学意义(P<0.01),其中束缚3小时后立刻给予APAP,血清ALT和AST的升高幅度最大,达到单纯给药水平的4倍。但是束缚应激0.5、1.5小时以及束缚3小时放松24小时后给药,对APAP引起的ALT和AST变化影响不明显,提示束缚应激加重APAP肝毒性与束缚时间有关。
2.显微镜下观察HE染色后的小鼠肝组织病理切片可见,正常对照组小鼠肝小叶轮廓清楚完整,肝细胞索排列整齐,肝窦清晰可见；APAP组动物肝脏中央静脉周围散在肝细胞坏死,伴随空泡变性；束缚3小时+APAP组动物肝脏病变更加严重,中央静脉周围片状肝细胞坏死,肝细胞排列紊乱,伴随空泡变性及肝窦扩张充血；束缚3小时组小鼠未发现明显的病理学改变,仅可见有轻度的肝细胞胞浆疏松淡染。
3.束缚应激对正常对照组和APAP组动物肝脏的总谷胱甘肽(GSH+GSSG)含量、氧化型谷胱甘肽(GSSG)含量和GSSG/GSH比值均有显著性影响(F=24.113,3.079,1.239,P<0.000,P=0.098,P=0.282;F=48.763,8.412,0.088,P<0.000, P=0.010,P=0.770;F=33.764,13.909,9.282, P<0.000,P=0.002,P=0.008)。束缚应激后正常对照组和APAP组总谷胱甘肽(GSH+GSSG)含量均显著降低,氧化型谷胱甘肽(GSSG)含量和GSSG/GSH比值均显著升高,与非束缚应激组相比,这些差异均有统计学意义(P<0.05或P<0.01)。表明束缚应激不但可破坏正常动物的氧化还原平衡状态,还可加重APAP引起的肝脏谷胱甘肽耗竭。另外,APAP给药后束缚应激组动物(GSH+GSSG)含量显著降低,GSSG/GSH比值显著升高,与单纯束缚应激组相比这些差异有统计学意义(P<0.05或P<0.01)。APAP单独给药后肝脏GSSG含量升高,与正常对照组相比差异有统计学意义(P<0.05)。
4.束缚3小时十APAP组与APAP组相比,血清IL-6水平、TNF-a水平以及肝脏Kupffer细胞的数量均无显著性差异。与空白对照组相比,束缚3小时+APAP组和APAP组动物肝脏Kupffer细胞数量增多。
5.束缚应激对正常对照组的IL-6水平有显著影响,对APAP组动物IL-6水平无明显影响(F=14.902,14.553,0.094,P=0.001,P=0.001,P=0.762)。束缚3小时组和正常对照组相比,血清IL-6和TNF-a水平呈现不同的变化趋势,束缚应激后IL-6水平显著升高(P<0.01),但TNF-a水平无显著变化,均在很低的水平。束缚3小时组和正常对照组动物肝脏Kupffer细胞数量也无明显差别。
6.用Western blot方法检测肝脏caspse-3蛋白表达量,正常对照组动物肝脏caspse-3蛋白表达量很低,几乎检测不到。束缚3小时组动物肝脏caspse-3蛋白表达量较正常对照组上升,但是束缚3小时-APAP组和APAP组动物肝脏caspse-3蛋白含量亦是很低或几乎检测不到。
第三部分：束缚应激损伤肝脏的发生机制
1.受体阻断剂或耗竭剂、束缚应激对动物血清ALT和AST水平均有显著影响(F=127.803,6.392,6.515;F=240.972,7.713,9.062;P值均小于0.01),且受体阻断剂或耗竭剂与束缚应激之间存在交互效应。束缚应激后正常对照组和各受体阻断剂或耗竭剂组ALT和AST水平均显著升高,与非束缚应激组相比,这些差异均有统计学意义(P<0.05或P<0.01)。组间进行两两比较显示,当用利血平完全耗竭儿茶酚胺或阻断α-1或阻断a-2受体后,与单纯束缚应激组相比,这三组血清ALT和AST水平均明显降低,其差异有统计学意义(P<0.05或P<0.01)；当阻断β-1或β-2受体后,与单纯束缚应激组相比,给药组血清ALT和AST水平没有显著性差异。此外α-1受体阻断剂哌唑嗪、α-2受体阻断剂育亨宾、β-1受体阻断剂倍他洛尔、β-2受体阻断剂ICI118,551和儿茶酚胺递质耗竭剂利血平对正常对照组动物血清ALT和AST水平均无影响。
2.受体阻断剂或耗竭剂、束缚应激对动物肝脏中总谷胱甘肽(GSH+GSSG)、GSSG和GSSG/GSH比值均有显著影响(F=75.340,4.130,2.211,P<0.000,P=0.003,P=0.068；F=107.928,4.685,6.951,P值均小于0.01；F=95.870,9.456,10.282,P值均小于0.01),且受体阻断剂或耗竭剂与束缚应激之间存在交互效应影响GSSG和GSSG/GSH比)。组间进行两两比较显示,当用利血平完全耗竭儿茶酚胺或阻断α-1或阻断α-2受体后,与单纯束缚应激组相比,这三组肝脏总谷胱甘肽含量显著升高,GSSG和GSSG/GSH比值显著降低,其差异有高度统计学意义(P<0.01)；哌唑嗪组、育亨宾组、倍他洛尔组、ICI118组以及正常对照组在束缚应激后肝脏总谷胱甘肽含量显著减少,与相应未束缚组相比,其差异有统计学意义(P<0.05或P<0.01)；育亨宾组、倍他洛尔组、ICI118组以及正常对照组在束缚应激后肝脏GSSG含量显著升高,,与相应未束缚组相比,其差异有统计学意义(P<0.05或P<0.01)；倍他洛尔组、ICI118组以及正常对照组在束缚应激后肝脏GSSG/GSH比值显著升高,与相应未束缚组相比,其差异有统计学意义(P<0.01)；另外,与交感神经阻断剂对束缚应激后血清转氨酶的影响相同,给予各种阻断剂后,对正常对照组动物肝脏中总谷胱甘肽(GSH+GSSG)、GSSG和GSSG/GSH比值的影响无影响。
3.正常对照组小鼠肝脏中几乎观察不到凋亡的肝细胞,束缚应激3小时后,出现凋亡的肝细胞数增多,其凋亡指数显著增加,与正常对照组相比,差异有高度统计学意义(P<0.01)。当用哌唑嗪阻断α-1受体或用育亨宾阻断a-2受体后出现凋亡的肝细胞数减少,与正常对照组相比无显著性差异,且这两个给药组的肝细胞凋亡数相近。
4.束缚应激3小时,Caspase-3和Caspase-9活化蛋白表达明显增多,Bax/Bcl-2比值亦明显升高；当用哌唑嗪阻断α-1受体或用育亨宾阻断a-2受体后,束缚后Caspase-3活化蛋白表达与正常对照组接近,而Caspase-9活化蛋白表达和Bax/Bcl-2比值仍有所增加,但与单纯束缚应激组相比增加幅度降低。
结论
1.束缚应激可明显加重APAP的肝毒性,表现为ALT和AST的升高,肝细胞坏死明显。其发生机制是束缚应激破坏了肝脏的氧化还原平衡状态,降低了肝脏内的谷胱甘肽储备量,而不是通过炎症反应所介导。同时证明了束缚应激加重APAP诱导肝细胞死亡的主要方式是坏死而不是凋亡。
2.束缚应激可通过减少肝脏谷胱甘肽的储备量而导致可逆性的肝损伤,并通过caspase-9和Bcl-2家族蛋白诱导肝细胞凋亡。
3.α-1和α-2受体参与了束缚应激损伤肝脏的过程。
Background and Objectives:
Acetaminophen (called paracetamol outside the United States) is a popular and widely used analgesic and antipyretic agent. Over300different preparations are now available in the United States with more than one billion pills sold annually. Although it is remarkably safe when used at usual therapeutic doses, it has a relatively narrow therapeutic window. Acetaminophen overdose is the leading cause for calls to Poison Control Centers (over100,000/yr), and accounts for more than56,000emergency room visits,2600hospitalizations and estimated458deaths due to acute liver failure (ALF) each year. Data from the U.S. Acute Liver Failure Study Group registry of more than1100patients with acute liver failure (ALF) from across the United States, suggests that acetaminophen poisoning alone currently constitutes nearly50%of all ALF. In January2011, the U.S. Food and Drug Administration (FDA) was asking manufacturers of prescription combination products that contain acetaminophen to limit the amount of acetaminophen to no more than325milligrams (mg) in each tablet or capsule. In February2014, FDA urged doctors to discontinue prescribing drugs that contain more than325milligrams of acetaminophen per tablet or capsule, to reduce the risk of liver injury. While the dose of acetaminophen ingested is clearly important in the development of hepatotoxicity, a number of other risk factors can severely aggravate liver injury induced by APAP, for example, chronic alcohol use, malnutrition and the concurrent intake of some medicinal agents. The effects of stress on APAP remain unexplored by now.
Stress is an ever-present part of modern life. The general concept of "stress" describes the state of a living organism of the non-specific response of the body to any demand for change when, under the influence of internal or external stimuli or "stressors". Stressors can include physical or mental forces or combinations of both. The adaptive response to stressors comprises the activation of the hypothalamic-pituitary-adrenal (HPA) axis and components of the sympathoadreno-medullary (SAM) system, releasing the key peripheral mediators, i.e. glucocorticoids and catecholamines. For a long time, stress had been implicated as a cofactor in the severity and progression of a number of diseases. The current focus on the stress and disease phenomenon is directed towards the interactions of the immune system, the CNS, cardiovascular disease, liver injury, HIV/AIDS and carcinomas. Studies reveal that stress plays a role in suppressing functions of specific immunity, in triggering or worsening depression, cardiovascular disease, and liver injury and in speeding the progression of HIV/AIDS and carcinomas.
Today, growing evidence has shown that stress can have an effect on liver disease and liver function, both in human and animal studies. Some early clinical reports suggested that psychosocial stress might affect the initiation, course and outcome of liver diseases. For example, Hirose et al revealed that emotional stress, such as that induced by hypnotic suggestion of'fear' and 'anxiety', significantly decreased hepatic blood flow (HBF). Fukudo et al demonstrated a significant positive correlation between the severity of psychosocial stress and the exaggeration of inflammatory and fibrosing changes in alcoholic hepatitis. A significant positive correlation between the alanine aminotransferase (ALT) level and the degree of depression, measured by the short form of the Beck Depression Inventory, was found in patients suffering from chronic hepatitis B. Moreover, the type I personality scales of Grossarth-Maticek have been associated with the severity of chronic hepatitis C, even after adjustment for the confounding factors of age, sex, education level, smoking, drinking, and duration of illness. In orthotropic liver transplantation, patients with acute rejection were significantly more likely to have the preoperative personality traits'submissive','relatively controlled', and 'of indifferent mood' than patients without acute rejection.
Such a close interaction between stress and liver diseases is also suggested by basic research using several animal models. For example, electric foot-shock stress exacerbated liver injury in rats treated with carbon tetrachloride (CCl4), an animal model of drug induced liver injury. More recently, this stress paradigm has been reported to aggravate a-galactosylceramide-induced hepatitis, which is related to malaria and Salmonella infection induced liver injury and viral hepatitis B and C. After exposure to stress, electron microscopy of the liver revealed rough endoplasmic reticulum fragmentation and dilatation, glycogen depletion, and mitochondrial enlargement. The most striking change, however, was an increase in the number and size of autophagic vacuoles which were limited by single or multiple membranes. Interestingly, even in normal rodents, restraint and electric foot-shock stress triggered mild liver injury, which is defined by slightly elevated ALT levels. Moreover, social isolation stress elevated the incidence of spontaneous hepatocellular carcinoma in transforming growth factor-a transgenic mice and accelerated the development of liver metastasis in the colon of carcinoma cell-injected mice.
Animal immobilization or restraint is known to be a straightforward, painless and convenient model to induce both psychological and physical stress. Immobilization or restraint can induce psychological escape reaction and physical muscle work. Compared to the stress induction achieved via drug administration, surgical interventions, extremes of temperature or acoustic stimulation, restraint allows for efficiently examining changes in levels of adrenocorticotropic hormones, corticosterone, fos-protein, desensitization of HPA response and expression of cytokines. A number of frank behavioral responses such as learning and conditioning, food intake or responses to acoustic startle and similar environmental challenges can readily be incorporated along with studies. In the recent15years,"restraint stress" continues to dominate stress-induced methodology.
The aim of the present study was:①To assess the effects and the potential mechanisms of restraint stress on the progression of liver injury in mice treated with APAP.②To investigate the potential involvement of catecholamines and adrenoceptors in the regulation of acute restraint stress-induced liver injury.
Methods and materials
Adult male BALB/c mice (8-10weeks old) were obtained from the Academy of Military Medical Science (Beijing, China). All animals were housed in an environmentally controlled room with a12h light/dark cycle and allowed free access to food and water. The temperature in the colony room was maintained at19-26℃. The mice were allowed to acclimatize themselves to the colony for3days before the experiments began. Animals were fasted overnight before the experiments. On the day of the experiment (starting at8:00AM), mice were introduced into a restraint tube for varying periods. The restraint tube was well ventilated and prevented animals from turning or ambulation, but it did not squeeze the mice. After restraint, mice were immediately sacrificed (see later) or returned to individual cages at room temperature for recovery. During the recovery period, food and water were restored for mice. Both control and restrained mice were euthanized with sodium pentobarbital either immediately after restraint or at specific time points after cessation of stress. Blood samples were collected from the abdominal aorta. The liver was removed and rinsed in saline. The left lateral Lobe of liver was fixed in10%phosphate-buffered formalin and embedded in paraffin for histological analyses. The remaining liver was snap-frozen in liquid nitrogen and stored at-80℃.
For APAP administration experiments, animals were given intraperitoneally (i.p.)150mg/kg APAP or vehicle at specific time points after restraint stress. Four hours after drug administration, animals were euthanized for blood sampling. For pharmacological manipulations of catecholamines and adrenoceptors experiments, Prazosin (1mg/kg) was administered intraperitoneally30min before restraint stress to block α-1receptors. Yohimbine (2mg/kg) was administered intraperitoneally30min before restraint stress to block a-2receptors. Betaxolol (20mg/kg) was administered intraperitoneally30min before restraint stress to block β-1receptors. ICI118,551(5mg/kg) was administered intraperitoneally30min before restraint stress to block β-2receptors. Reserpine (2mg/kg) was subcutaneously injected24h prior to restraint stress in order to achieve a permanent depletion of catecholamines.
Serum alanine transaminase (ALT) and aspartate transaminase (AST) were then determined using commercial test kits. Total soluble GSH and GSSG were measured in the liver homogenate using the enzymatic recycling method. Serum IL-6and TNF-a levels were measured by using an enzyme linked immunoassay kit. Sections of liver were stained with hematoxylin and eosin (H&E) for routine histological examinations and morphometric analyses. Apoptosis detection was performed using a TUNEL peroxidase apoptosis detection kit. Immunohistochemical detection of F4/80protein was performed in sections of formalin-fixed, paraffin embedded livers for the detection of Kupffer cells. The expression of caspse-3、Caspase-9、Bcl-2and Bax in the liver were detected by Western blot analysis.
Statistics
The data were analyzed with SPSS13.0. All results were expressed as mean±SD. Comparisons between multiple groups in single-factor experiment were performed with one-way ANOVA followed by a post hoc LSD test. If the data were not normally distributed, we used the Dunnett'S T3test for the comparison by nonparametric ANOVA. Two-way ANOVA was used to analyze two-factor interaction effects. Results were considered significant when p-values were less than0.05.
Results:
Experiment I:Establishment of restraint stress model in mice
1.When mice were subjected to restrain for0.5,1.5or3h, serum ALT and AST activities continued to increase. However, extending restraint to6h started to decrease serum enzyme activities. In different restraint periods, both ALT and AST activities reached or exceeded3times the upper limit of normal after3h of restraint. According to "Hy's Law" adopted by the U.S. Food and Drug Administration (FDA), severe hepatocellular injury has been defined as being cases where the serum ALT or AST is>3times the upper limit of normal.
2. Serum ALT and AST reached maximum after3h of restraint. When the animals subjected to restrain3h were liberated, their ALT and AST activity progressively decreased. Significant differences between ALT and AST levels in the restraint group and those of the control group were also observed at6h after cessation of stress. ALT activity in the restraint3h group returned to control values24hours later, but AST activity remained elevated.
3. Similar to plasma ALT and AST activities, restrained animals exhibited progressive changes in hepatic GSH (GSH+GSSG), GSSG and GSH/GSSG ratio. Hepatic GSH and GSH/GSSG content were significantly higher after1.5and3h of restraint in the restrained group than that of unrestrained animals (p<0.05or p<0.01). However, restrained animals exhibited significantly lower GSSG content after1.5and3h of restraint compared with unrestrained animals (p<0.01).
4.Histopathological examinations revealed that there was no obvious liver lesion in the restraint mice compared to the control. Only mice exposure to3h restraint showed mild hepatic degeneration with the presence of slight cytoplasmic relaxation.
ExperimentⅡ:Effect of restraint stress on APAP induced liver injury
1.A dose of150mg/kg APAP was administered to mice subjected to restraint0.5,1.5or3h and recovery6or24h after restraint3h. Recovery6h after restraint3h significantly potentiated the APAP induced elevation of ALT and AST levels (P<0.05), and3h of restraint exhibited a higher ALT and AST levels, which was4times of that seen in mice receiving the same dose but without restraint. Animals restrained for other time points had a minimal effect on APAP induced change of ALT and AST levels, revealed that the enhancement of APAP induced liver injury was dependent on the duration of restraint.
2.Histopathological examinations revealed that livers were normal in appearance with no change in the lobular architecture in the control. In the APAP group, limited hepatic necrosis was seen in the centrilobular regions with cytoplasmic vacuolization, but hepatocytes in the centrilobular regions showed significant and extensive centrilobular necrosis with vacuolization and congestion following restraint3h plus acetaminophen treatment.
3. The mice restrained3h showed significantly higher hepatic GSSG content and GSH/GSSG than that of unrestrained animals, and GSH+GSSG content were significantly lower than that of unrestrained animals. The mice exposed to restraint3h exhibited a higher hepatic GSSG content and GSH/GSSG and lower GSH+GSSG content than that seen in mice receiving the same dose but without restraint (P<0.05or P<0.01)
4. In restraint3h plus APAP group, there were no significant differences in serum IL-6and TNF-α levels compared to APAP group without restraint. Similarly, no significant differences were noted in the density of F4/80-positive Kupffer cells between them.
5.IL-6and TNF-a activity showed different profiles after restraint3h. IL-6activity was statistically significantly increased after exposure to3h restraint stress, while no increase in TNF-a activity was found following restraint. Similarly, no significant differences were noted in the density of F4/80-positive Kupffer cells between control and restrained mice.
6. As to both restraint3h plus APAP group and APAP group, none of them showed any processing of the procaspase-3to its active fragment. The level of active caspase-3was elevated in the liver tissue of mice subjected to3h of restraint compared to unrestraint.
ExperimentⅢ:Potential mechanisms of restraint induced liver injury
1. Treatment with reserpine, prazosin, yohimbine, betaxolol and ICI118551did not influence ALT and AST activities of unrestrained control mice compared with vehicle control group. In a state of a generalized catecholamine depletion produced by reserpine, the elevation of ALT or AST activities induced by restraint were significantly less than control animals (p<0.05). After blockade of α-1or a-2adrenoceptor by prazosin or yohimbine, the magnitude of the increase in ALT and AST activities induced by restraint had a similarly predominant drop compared with control animals (p<0.05), which were similar to the effects of reserpine. However, no changes were observed in ALT and AST activities induced by restraint stress after β-1or β-2adrenergic receptor antagonist treatment (betaxolol or ICI118,551). Regardless of drug treatment, ALT and AST activities of restrained groups were significantly higher than those of corresponding control groups (p<0.05or p<0.01).
2. Treatment with reserpine, prazosin, yohimbine, betaxolol and ICI118,551did not affect hepatic GSH (GSH+GSSG), GSSG and GSH/GSSG ratio of unrestrained control mice compared with vehicle control group. After catecholamine depletion by reserpine or blockade of a-lor a-2adrenoceptor by prazosin or yohimbine, the magnitude of the increase in GSSG content and GSH/GSSG ratio and the decrease in GSH content induced by restraint were statistically attenuated compared with that of control animals (p<0.01), and these drugs had the same attenuating effect. However, hepatic total GSH, GSSG and GSH/GSSG ratio of restrained animals treated with drugs (reserpine, prazosin, or yohimbine) still significantly different from corresponding control animals (p<0.05or p<0.01). Changes of hepatic glutathione content induced by restraint were not affected by beta-1or beta-2adrenergic receptor antagonists (betaxolol or ICI118,551).
3.Liver sections were stained with the TUNEL assay to evaluate the effects of prazosin and yohimbine on hepatocellular apoptosis. After restraint stress for3h, apoptotic hepatocytes were frequently observed, while no apparent apoptotic cell was found in the liver tissue of unrestrained mice. Quantitative analysis of TUNEL positive hepatocytes showed that the number of apoptotic hepatocyte in mice exposed to3h restraint was significantly higher than that in unrestrained control mice (p<0.01). Treatment with prazosin and yohimbine reduced the number of apoptotic cells in the liver and the two agents exhibited a similar inhibiting effect on the hepatocellular apoptosis.
4. Restraint stress obviously increased the expression of cleaved forms of caspase-3, the proapoptotic factor Bax and the activated form of caspase-9, and decreased the expression of the antiapoptotic factors Bcl-2when compared with control group, leading to a high Bax/Bcl-2ratio. In the prazosin and yohimbine treatment group, the above factors induced by restraint were attenuated, but these factors were still unable to return to normal level of the unrestraint group.
Conclusion
1. Restraint stress can exacerbate acetaminophen-induced liver injury as indicated by an increase in necrotic hepatic tissue and serum aminotransferase activity. Reduction in hepatic GSH content and disruption of the balance between oxidants and antioxidants in live, but not releasing of inflammatory cytokines, are responsible for such stress induced aggravation of APAP induced liver injury. In addition, necrosis but not apoptosis is the principal mechanism of hepatocytes death after exacerbation of APAP induced liver injury by restraint stress.
2. Restraint stress can induce reversible liver injury by reducing hepatic GSH content and promote hepatocytes apoptosis through caspase-9and Bcl-2family of apoptotic regulatory proteins.
3.α-1and α-2adrenoceptors mediate restraint-induced liver.
对乙酰氨基酚(商品名为扑热息痛,Acetaminophen,简称APAP)是世界上应用最广泛的非处方类解热镇痛药。在美国销售的含有APAP的制剂超过300种,平均每年的销量是十亿片。尽管在常规治疗剂量下APAP非常安全,但是该药的治疗窗较窄。因为过量使用APAP每年有大于100,000例的患者需要到中毒治疗中心接受治疗,其中包括超过56,000的门诊患者和2600例的住院病人和估计458例死于急性肝衰竭的病人。根据美国急性肝衰竭研究委员会报告,美国50%的急性肝衰竭病人都是由于APAP中毒,每年的人数超过1100人。有人报道,在APAP导致急性肝衰竭的发生率每年呈递增趋势,1998年是21%,而2003年是51%。由于对乙酰氨基酚的肝毒性,在2011年1月美国FDA发布公告要求在处方药中APAP的含量每片不得超过325mg。在2014年2月FDA又发布报告,建议医生和医疗人员禁用含APAP超过325mg的药品。虽然过量使用是导致APAP肝脏毒性的重要原因,但也有很多导致APAP中毒的风险因素,包括营养不良、饮酒和联合用药。迄今为止未见有研究报道外界应激对APAP肝毒性的影响。
应激是一种机体状态,指机体在内部或外界刺激或应激原的影响下后,机体产生的一系列非特异性应答反应。应激原可以是物理因素,也可以是精神因素或二者的综合作用。当机体受到强烈刺激时,机体就会兴奋交感-肾上腺髓质系统(sympathoadrenomedullary system,SAM)和下丘脑-垂体-肾上腺皮质轴(hypothalamic-pituitary-adrenal,HPA),表现为释放儿茶酚胺和糖皮质激素等外周递质。很早之前,大家已经认识到应激能够影响许多疾病的发生发展过程和结果。目前人们主要关注的是应激与心血管系统疾病、神经系统疾病、消化系统疾病、免疫系统疾病、HIV/AIDS以及癌症等之间的关系。大量的研究结果表明应激可以抑制特异性免疫反应,诱导产生或加重抑郁症、心血管系统疾病、肝脏疾病,并促进HIV/AIDS和癌症的病情发展。
目前,有越来越多的证据表明应激对肝脏疾病和功能存在影响。根据早期的临床报道,社会心理应激可以影响肝脏疾病的发生、发展及结果。例如,Hirose等发现情绪应激反应(如受到催眠暗示的恐惧或焦虑)可以明显增加肝脏血流量(HBF)。Fukudo等研究发现,酒精性肝炎中社会心理应激的严重程度与肝炎恶化及肝脏纤维化变化之间存在紧密的联系。在Beck Depression Inventory杂志中,也有学者发现在慢性乙型病毒性肝炎患者,其血清丙氨酸氨基转移酶(alanine transaminase,ALT)水平与其精神抑郁程度之间存在明显的正相关关系。而且,即使剔除了包括年龄、性别、教育水平、抽烟、饮酒与否以及病程等混杂因素,在Grossarth-Maticek分型中慢性丙型病毒性肝炎的严重性与I型人格人群具有相关性。在原位肝脏移植中,发生急性排斥反应的患者手术前的人格品质更倾向于是那些“顺从的”“相对易于受人控制的”以及“淡漠心境”者。在动物试验研究中,人们同样证明了应激与肝脏疾病之间的紧密相互联系。例如,电击应激可以加重四氯化碳处理过的小鼠肝脏损害,也可以使α-半乳糖胺诱导的肝炎加重,这种肝炎和疟疾、沙门氏菌感染诱导的肝损害以及乙型、丙型病毒性肝炎有关。有人在给大鼠施加多种不同的急性应激后,电镜检查发现肝脏粗面内质网破裂并膨胀,糖原耗竭,线粒体肿胀,其中最显著的变化是细胞膜的自噬泡数量增多和体积变大。有意思的是,即使在正常的啮齿类动物中,束缚以及电击应激可以触发温和的肝脏损害,这种肝损害以轻度的血清丙氨酸转氨酶(ALT)水平升高为特点。此外,社会隔绝应激(social isolation stress)可以增加转化生长因子-α转基因小鼠自发性肝细胞肝癌的发生率,同时还可以加速小鼠结肠肿瘤肝转移进程。
动物束缚应激模型是一种直接、非损伤性和简单的心理与生理应激模型。束缚应激可产生心理性的逃避反应和限制机体行动。束缚应激模型不但可准确且可靠地控制应激实验条件,而且与给药造模、手术造模以及外界强烈的温度改变或噪音相比,束缚应激模型可以有效地检测促肾上腺皮质素、皮质酮、fos蛋白、HPA轴的脱敏和细胞因子的表达。同时束缚应激可以协同多种因素进行研究,例如学习记忆、摄食情况和声音刺激等。在最近15年间,束缚应激模型一直是使用最多的应激动物模型。
本课题研究目的是：一、考察束缚应激对APAP给药后肝毒性的影响及可能的发生机制；二、探讨儿茶酚胺递质和肾上腺受体在束缚应激诱导肝损伤过程中作用。
方法
实验选用Balb/c小鼠,雄性,SPF级,8-10周龄,购自军事医学科学院实验动物中心。动物饲养于解放军疾病预防控制所毒理学评价研究中心屏障设施内,温度控制在19-26℃,12小时照明,12小时黑暗,动物自由进食饮水。正式实验前动物检疫观察3天。实验前一天禁食过夜,实验当天同一时间(上午8：00)将动物放入束缚筒内,束缚后保证小鼠自由呼吸,身体无法扭动同时不受挤压。束缚应激后动物或者被立刻麻醉处死或者放回笼内继续饲养。动物处死时,均用戊巴比妥钠麻醉,从腹主动脉采血。动物采血后,立刻取下肝脏,生理盐水冲洗,取左肝页用中性福尔马林固定,石蜡包埋用于病理组织学检查。剩余的肝脏用液氮速冻并保存于-80℃。
在考察束缚应激对对乙酰氨基酚肝毒性影响的实验中,在束缚应激后的各时间点腹腔注射150mg/kg的APAP或生理盐水。APAP给药后4小时,动物麻醉处死采样。在考察儿茶酚胺递质和肾上腺素受体对束缚应激诱导肝损伤影响的实验中,束缚前30分钟腹腔注射1mg/kg的哌唑嗪用于阻断α-1受体；束缚前30分钟腹腔注射2mg/kg的育亨宾用于阻断a-2受体；束缚前30分钟腹腔注射20mg/kg的倍他洛尔用于阻断β-1受体；束缚前30分钟腹腔注射5mg/kg的ICI118,551用于阻断β-2受体；束缚前24小时皮下注射2mg/kg的利血平用于耗竭儿茶酚胺递质。
用全自动生化分析仪检测血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST),用用酶循环法测定肝脏中谷胱甘肽的含量；用酶联免疫法(ELISA)检测血清IL-6和TNF-a含量。对肝组织进行病理学检测,HE常规染色观察病变程度。用TUNEL法原位检测细胞凋亡情况。用免疫组化法检测检钡Kupffer细胞活性情况。用蛋白免疫印迹(Western Blot)法检测肝组织caspse-3、Caspase-9、Bcl-2,和Bax的蛋白表达量。
统计学处理
实验数据采用SPSS13.0统计软件分析处理,用均数士标准差(Meand士SD)表示；单因素试验的多组比较采用单因素方差分析(One. way ANOVA),方差齐时采用多重比较LSD法,方差不齐时采用Dunnett' ST3法。在考察束缚和药物相互干预的试验中采用双因素方差分析比较各处理方式的主效应及交互效应。当P<0.05时,差异被认为有统计学意义。
结果
第一部分：小鼠束缚应激模型的建立
1.动物在束缚应激0、0.5、1.5、3、6小时后,组间血清丙氨酸氨基转移酶(ALT)和门冬氨酸氨基转移酶(AST)的差异有统计学意义(F=5.114,24.666；P均小于0.01),表现为ALT和AST在束缚前3小时持续性的升高。与正常对照组(束缚0小时)相比,束缚应激1.5和3小时后ALT和AST的差异均有统计学意义(P<0.05或P<0.01)。其中在束缚应激3小时后,ALT的水平达到正常值的3倍,根据海氏法则可认为小鼠束缚应激3小时肝细胞达到了损伤的标准。但是,当束缚应激6小时后,血清ALT和AST的水平开始降低,此时AST与正常对照组相比差异仍有统计学意义(P<0.05)。
2.当小鼠束缚1.5、3小时及束缚3小时放松6小时后,各组间血清ALT和AST水平的差异有统计学意义(F=16.000,27.268；P均小于0.01)。当小鼠束缚应激3小时后,血清ALT和AST的水平达到最高值,然后将动物放回笼中自由活动,放松后血清ALT和AST水平开始恢复,放松6小时时,血清ALT和AST水平与正常对照组相比差异仍有统计学意义(P<0.05)；在放松24小时后,ALT值基本达到正常值水平。但是AST与正常对照组相比差异仍有统计学意义(P<0.01)。
3.束缚不同时间后肝脏总谷胱甘肽(GSH+GSSG)含量、氧化型谷胱甘肽(GSSG)含量和GSSG/GSH比值的组间差异有有统计学意义(F=7.199,13.522,22.817;P均小于0.01)。与血清ALT和AST水平的变化情况相同,束缚应激3小时后(GSH+GSSG)含量降低、GSSG含量和GSSG/GSH比值和GSSG/GSH比值逐步升高,与正常对照组相比,这些指标的差异均有统计学意义(P<0.01),其中束缚1.5小时后氧化型谷胱甘肽(GSSG)含量和GSSG/GSH比值与正常对照组相比亦显著性升高,差异有统计学意义(P<0.05或P<0.01)。束缚3小时后将动物放回笼中自由活动,放松后总谷胱甘肽(GSH+GSSG)、GSSG和GSSG/GSH开始恢复,至放松24小时后肝脏中的谷胱甘肽含量恢复至正常值水平。
4.正常对照组和束缚应激0.5、1.5小时的动物肝脏病理组织学检查未见异常,小鼠束缚应激3小时后仅可见有轻度的肝细胞胞浆疏松淡染,未发现明显的病理学改变。
第二部分：束缚应激对对乙酰氨基酚肝毒性的影响
1.在小鼠束缚应激0.5、1.5、3小时以及束缚3小时放松6、24小时后分别给予150mg/kg的APAP,结果发现,组间ALT和AST的差异有统计学意义。(F=14.555,12.584；P值均小于0.01)。小鼠束缚3小时可使APAP引起的ALT和AST升高增强,束缚小3时放6小时后亦可使APAP引起的ALT升高增强,与单纯APAP给药组相比,差异均有统计学意义(P<0.01),其中束缚3小时后立刻给予APAP,血清ALT和AST的升高幅度最大,达到单纯给药水平的4倍。但是束缚应激0.5、1.5小时以及束缚3小时放松24小时后给药,对APAP引起的ALT和AST变化影响不明显,提示束缚应激加重APAP肝毒性与束缚时间有关。
2.显微镜下观察HE染色后的小鼠肝组织病理切片可见,正常对照组小鼠肝小叶轮廓清楚完整,肝细胞索排列整齐,肝窦清晰可见；APAP组动物肝脏中央静脉周围散在肝细胞坏死,伴随空泡变性；束缚3小时+APAP组动物肝脏病变更加严重,中央静脉周围片状肝细胞坏死,肝细胞排列紊乱,伴随空泡变性及肝窦扩张充血；束缚3小时组小鼠未发现明显的病理学改变,仅可见有轻度的肝细胞胞浆疏松淡染。
3.束缚应激对正常对照组和APAP组动物肝脏的总谷胱甘肽(GSH+GSSG)含量、氧化型谷胱甘肽(GSSG)含量和GSSG/GSH比值均有显著性影响(F=24.113,3.079,1.239,P<0.000,P=0.098,P=0.282;F=48.763,8.412,0.088,P<0.000, P=0.010,P=0.770;F=33.764,13.909,9.282, P<0.000,P=0.002,P=0.008)。束缚应激后正常对照组和APAP组总谷胱甘肽(GSH+GSSG)含量均显著降低,氧化型谷胱甘肽(GSSG)含量和GSSG/GSH比值均显著升高,与非束缚应激组相比,这些差异均有统计学意义(P<0.05或P<0.01)。表明束缚应激不但可破坏正常动物的氧化还原平衡状态,还可加重APAP引起的肝脏谷胱甘肽耗竭。另外,APAP给药后束缚应激组动物(GSH+GSSG)含量显著降低,GSSG/GSH比值显著升高,与单纯束缚应激组相比这些差异有统计学意义(P<0.05或P<0.01)。APAP单独给药后肝脏GSSG含量升高,与正常对照组相比差异有统计学意义(P<0.05)。
4.束缚3小时十APAP组与APAP组相比,血清IL-6水平、TNF-a水平以及肝脏Kupffer细胞的数量均无显著性差异。与空白对照组相比,束缚3小时+APAP组和APAP组动物肝脏Kupffer细胞数量增多。
5.束缚应激对正常对照组的IL-6水平有显著影响,对APAP组动物IL-6水平无明显影响(F=14.902,14.553,0.094,P=0.001,P=0.001,P=0.762)。束缚3小时组和正常对照组相比,血清IL-6和TNF-a水平呈现不同的变化趋势,束缚应激后IL-6水平显著升高(P<0.01),但TNF-a水平无显著变化,均在很低的水平。束缚3小时组和正常对照组动物肝脏Kupffer细胞数量也无明显差别。
6.用Western blot方法检测肝脏caspse-3蛋白表达量,正常对照组动物肝脏caspse-3蛋白表达量很低,几乎检测不到。束缚3小时组动物肝脏caspse-3蛋白表达量较正常对照组上升,但是束缚3小时-APAP组和APAP组动物肝脏caspse-3蛋白含量亦是很低或几乎检测不到。
第三部分：束缚应激损伤肝脏的发生机制
1.受体阻断剂或耗竭剂、束缚应激对动物血清ALT和AST水平均有显著影响(F=127.803,6.392,6.515;F=240.972,7.713,9.062;P值均小于0.01),且受体阻断剂或耗竭剂与束缚应激之间存在交互效应。束缚应激后正常对照组和各受体阻断剂或耗竭剂组ALT和AST水平均显著升高,与非束缚应激组相比,这些差异均有统计学意义(P<0.05或P<0.01)。组间进行两两比较显示,当用利血平完全耗竭儿茶酚胺或阻断α-1或阻断a-2受体后,与单纯束缚应激组相比,这三组血清ALT和AST水平均明显降低,其差异有统计学意义(P<0.05或P<0.01)；当阻断β-1或β-2受体后,与单纯束缚应激组相比,给药组血清ALT和AST水平没有显著性差异。此外α-1受体阻断剂哌唑嗪、α-2受体阻断剂育亨宾、β-1受体阻断剂倍他洛尔、β-2受体阻断剂ICI118,551和儿茶酚胺递质耗竭剂利血平对正常对照组动物血清ALT和AST水平均无影响。
2.受体阻断剂或耗竭剂、束缚应激对动物肝脏中总谷胱甘肽(GSH+GSSG)、GSSG和GSSG/GSH比值均有显著影响(F=75.340,4.130,2.211,P<0.000,P=0.003,P=0.068；F=107.928,4.685,6.951,P值均小于0.01；F=95.870,9.456,10.282,P值均小于0.01),且受体阻断剂或耗竭剂与束缚应激之间存在交互效应影响GSSG和GSSG/GSH比)。组间进行两两比较显示,当用利血平完全耗竭儿茶酚胺或阻断α-1或阻断α-2受体后,与单纯束缚应激组相比,这三组肝脏总谷胱甘肽含量显著升高,GSSG和GSSG/GSH比值显著降低,其差异有高度统计学意义(P<0.01)；哌唑嗪组、育亨宾组、倍他洛尔组、ICI118组以及正常对照组在束缚应激后肝脏总谷胱甘肽含量显著减少,与相应未束缚组相比,其差异有统计学意义(P<0.05或P<0.01)；育亨宾组、倍他洛尔组、ICI118组以及正常对照组在束缚应激后肝脏GSSG含量显著升高,,与相应未束缚组相比,其差异有统计学意义(P<0.05或P<0.01)；倍他洛尔组、ICI118组以及正常对照组在束缚应激后肝脏GSSG/GSH比值显著升高,与相应未束缚组相比,其差异有统计学意义(P<0.01)；另外,与交感神经阻断剂对束缚应激后血清转氨酶的影响相同,给予各种阻断剂后,对正常对照组动物肝脏中总谷胱甘肽(GSH+GSSG)、GSSG和GSSG/GSH比值的影响无影响。
3.正常对照组小鼠肝脏中几乎观察不到凋亡的肝细胞,束缚应激3小时后,出现凋亡的肝细胞数增多,其凋亡指数显著增加,与正常对照组相比,差异有高度统计学意义(P<0.01)。当用哌唑嗪阻断α-1受体或用育亨宾阻断a-2受体后出现凋亡的肝细胞数减少,与正常对照组相比无显著性差异,且这两个给药组的肝细胞凋亡数相近。
4.束缚应激3小时,Caspase-3和Caspase-9活化蛋白表达明显增多,Bax/Bcl-2比值亦明显升高；当用哌唑嗪阻断α-1受体或用育亨宾阻断a-2受体后,束缚后Caspase-3活化蛋白表达与正常对照组接近,而Caspase-9活化蛋白表达和Bax/Bcl-2比值仍有所增加,但与单纯束缚应激组相比增加幅度降低。
结论
1.束缚应激可明显加重APAP的肝毒性,表现为ALT和AST的升高,肝细胞坏死明显。其发生机制是束缚应激破坏了肝脏的氧化还原平衡状态,降低了肝脏内的谷胱甘肽储备量,而不是通过炎症反应所介导。同时证明了束缚应激加重APAP诱导肝细胞死亡的主要方式是坏死而不是凋亡。
2.束缚应激可通过减少肝脏谷胱甘肽的储备量而导致可逆性的肝损伤,并通过caspase-9和Bcl-2家族蛋白诱导肝细胞凋亡。
3.α-1和α-2受体参与了束缚应激损伤肝脏的过程。
Background and Objectives:
Acetaminophen (called paracetamol outside the United States) is a popular and widely used analgesic and antipyretic agent. Over300different preparations are now available in the United States with more than one billion pills sold annually. Although it is remarkably safe when used at usual therapeutic doses, it has a relatively narrow therapeutic window. Acetaminophen overdose is the leading cause for calls to Poison Control Centers (over100,000/yr), and accounts for more than56,000emergency room visits,2600hospitalizations and estimated458deaths due to acute liver failure (ALF) each year. Data from the U.S. Acute Liver Failure Study Group registry of more than1100patients with acute liver failure (ALF) from across the United States, suggests that acetaminophen poisoning alone currently constitutes nearly50%of all ALF. In January2011, the U.S. Food and Drug Administration (FDA) was asking manufacturers of prescription combination products that contain acetaminophen to limit the amount of acetaminophen to no more than325milligrams (mg) in each tablet or capsule. In February2014, FDA urged doctors to discontinue prescribing drugs that contain more than325milligrams of acetaminophen per tablet or capsule, to reduce the risk of liver injury. While the dose of acetaminophen ingested is clearly important in the development of hepatotoxicity, a number of other risk factors can severely aggravate liver injury induced by APAP, for example, chronic alcohol use, malnutrition and the concurrent intake of some medicinal agents. The effects of stress on APAP remain unexplored by now.
Stress is an ever-present part of modern life. The general concept of "stress" describes the state of a living organism of the non-specific response of the body to any demand for change when, under the influence of internal or external stimuli or "stressors". Stressors can include physical or mental forces or combinations of both. The adaptive response to stressors comprises the activation of the hypothalamic-pituitary-adrenal (HPA) axis and components of the sympathoadreno-medullary (SAM) system, releasing the key peripheral mediators, i.e. glucocorticoids and catecholamines. For a long time, stress had been implicated as a cofactor in the severity and progression of a number of diseases. The current focus on the stress and disease phenomenon is directed towards the interactions of the immune system, the CNS, cardiovascular disease, liver injury, HIV/AIDS and carcinomas. Studies reveal that stress plays a role in suppressing functions of specific immunity, in triggering or worsening depression, cardiovascular disease, and liver injury and in speeding the progression of HIV/AIDS and carcinomas.
Today, growing evidence has shown that stress can have an effect on liver disease and liver function, both in human and animal studies. Some early clinical reports suggested that psychosocial stress might affect the initiation, course and outcome of liver diseases. For example, Hirose et al revealed that emotional stress, such as that induced by hypnotic suggestion of'fear' and 'anxiety', significantly decreased hepatic blood flow (HBF). Fukudo et al demonstrated a significant positive correlation between the severity of psychosocial stress and the exaggeration of inflammatory and fibrosing changes in alcoholic hepatitis. A significant positive correlation between the alanine aminotransferase (ALT) level and the degree of depression, measured by the short form of the Beck Depression Inventory, was found in patients suffering from chronic hepatitis B. Moreover, the type I personality scales of Grossarth-Maticek have been associated with the severity of chronic hepatitis C, even after adjustment for the confounding factors of age, sex, education level, smoking, drinking, and duration of illness. In orthotropic liver transplantation, patients with acute rejection were significantly more likely to have the preoperative personality traits'submissive','relatively controlled', and 'of indifferent mood' than patients without acute rejection.
Such a close interaction between stress and liver diseases is also suggested by basic research using several animal models. For example, electric foot-shock stress exacerbated liver injury in rats treated with carbon tetrachloride (CCl4), an animal model of drug induced liver injury. More recently, this stress paradigm has been reported to aggravate a-galactosylceramide-induced hepatitis, which is related to malaria and Salmonella infection induced liver injury and viral hepatitis B and C. After exposure to stress, electron microscopy of the liver revealed rough endoplasmic reticulum fragmentation and dilatation, glycogen depletion, and mitochondrial enlargement. The most striking change, however, was an increase in the number and size of autophagic vacuoles which were limited by single or multiple membranes. Interestingly, even in normal rodents, restraint and electric foot-shock stress triggered mild liver injury, which is defined by slightly elevated ALT levels. Moreover, social isolation stress elevated the incidence of spontaneous hepatocellular carcinoma in transforming growth factor-a transgenic mice and accelerated the development of liver metastasis in the colon of carcinoma cell-injected mice.
Animal immobilization or restraint is known to be a straightforward, painless and convenient model to induce both psychological and physical stress. Immobilization or restraint can induce psychological escape reaction and physical muscle work. Compared to the stress induction achieved via drug administration, surgical interventions, extremes of temperature or acoustic stimulation, restraint allows for efficiently examining changes in levels of adrenocorticotropic hormones, corticosterone, fos-protein, desensitization of HPA response and expression of cytokines. A number of frank behavioral responses such as learning and conditioning, food intake or responses to acoustic startle and similar environmental challenges can readily be incorporated along with studies. In the recent15years,"restraint stress" continues to dominate stress-induced methodology.
The aim of the present study was:①To assess the effects and the potential mechanisms of restraint stress on the progression of liver injury in mice treated with APAP.②To investigate the potential involvement of catecholamines and adrenoceptors in the regulation of acute restraint stress-induced liver injury.
Methods and materials
Adult male BALB/c mice (8-10weeks old) were obtained from the Academy of Military Medical Science (Beijing, China). All animals were housed in an environmentally controlled room with a12h light/dark cycle and allowed free access to food and water. The temperature in the colony room was maintained at19-26℃. The mice were allowed to acclimatize themselves to the colony for3days before the experiments began. Animals were fasted overnight before the experiments. On the day of the experiment (starting at8:00AM), mice were introduced into a restraint tube for varying periods. The restraint tube was well ventilated and prevented animals from turning or ambulation, but it did not squeeze the mice. After restraint, mice were immediately sacrificed (see later) or returned to individual cages at room temperature for recovery. During the recovery period, food and water were restored for mice. Both control and restrained mice were euthanized with sodium pentobarbital either immediately after restraint or at specific time points after cessation of stress. Blood samples were collected from the abdominal aorta. The liver was removed and rinsed in saline. The left lateral Lobe of liver was fixed in10%phosphate-buffered formalin and embedded in paraffin for histological analyses. The remaining liver was snap-frozen in liquid nitrogen and stored at-80℃.
For APAP administration experiments, animals were given intraperitoneally (i.p.)150mg/kg APAP or vehicle at specific time points after restraint stress. Four hours after drug administration, animals were euthanized for blood sampling. For pharmacological manipulations of catecholamines and adrenoceptors experiments, Prazosin (1mg/kg) was administered intraperitoneally30min before restraint stress to block α-1receptors. Yohimbine (2mg/kg) was administered intraperitoneally30min before restraint stress to block a-2receptors. Betaxolol (20mg/kg) was administered intraperitoneally30min before restraint stress to block β-1receptors. ICI118,551(5mg/kg) was administered intraperitoneally30min before restraint stress to block β-2receptors. Reserpine (2mg/kg) was subcutaneously injected24h prior to restraint stress in order to achieve a permanent depletion of catecholamines.
Serum alanine transaminase (ALT) and aspartate transaminase (AST) were then determined using commercial test kits. Total soluble GSH and GSSG were measured in the liver homogenate using the enzymatic recycling method. Serum IL-6and TNF-a levels were measured by using an enzyme linked immunoassay kit. Sections of liver were stained with hematoxylin and eosin (H&E) for routine histological examinations and morphometric analyses. Apoptosis detection was performed using a TUNEL peroxidase apoptosis detection kit. Immunohistochemical detection of F4/80protein was performed in sections of formalin-fixed, paraffin embedded livers for the detection of Kupffer cells. The expression of caspse-3、Caspase-9、Bcl-2and Bax in the liver were detected by Western blot analysis.
Statistics
The data were analyzed with SPSS13.0. All results were expressed as mean±SD. Comparisons between multiple groups in single-factor experiment were performed with one-way ANOVA followed by a post hoc LSD test. If the data were not normally distributed, we used the Dunnett'S T3test for the comparison by nonparametric ANOVA. Two-way ANOVA was used to analyze two-factor interaction effects. Results were considered significant when p-values were less than0.05.
Results:
Experiment I:Establishment of restraint stress model in mice
1.When mice were subjected to restrain for0.5,1.5or3h, serum ALT and AST activities continued to increase. However, extending restraint to6h started to decrease serum enzyme activities. In different restraint periods, both ALT and AST activities reached or exceeded3times the upper limit of normal after3h of restraint. According to "Hy's Law" adopted by the U.S. Food and Drug Administration (FDA), severe hepatocellular injury has been defined as being cases where the serum ALT or AST is>3times the upper limit of normal.
2. Serum ALT and AST reached maximum after3h of restraint. When the animals subjected to restrain3h were liberated, their ALT and AST activity progressively decreased. Significant differences between ALT and AST levels in the restraint group and those of the control group were also observed at6h after cessation of stress. ALT activity in the restraint3h group returned to control values24hours later, but AST activity remained elevated.
3. Similar to plasma ALT and AST activities, restrained animals exhibited progressive changes in hepatic GSH (GSH+GSSG), GSSG and GSH/GSSG ratio. Hepatic GSH and GSH/GSSG content were significantly higher after1.5and3h of restraint in the restrained group than that of unrestrained animals (p<0.05or p<0.01). However, restrained animals exhibited significantly lower GSSG content after1.5and3h of restraint compared with unrestrained animals (p<0.01).
4.Histopathological examinations revealed that there was no obvious liver lesion in the restraint mice compared to the control. Only mice exposure to3h restraint showed mild hepatic degeneration with the presence of slight cytoplasmic relaxation.
ExperimentⅡ:Effect of restraint stress on APAP induced liver injury
1.A dose of150mg/kg APAP was administered to mice subjected to restraint0.5,1.5or3h and recovery6or24h after restraint3h. Recovery6h after restraint3h significantly potentiated the APAP induced elevation of ALT and AST levels (P<0.05), and3h of restraint exhibited a higher ALT and AST levels, which was4times of that seen in mice receiving the same dose but without restraint. Animals restrained for other time points had a minimal effect on APAP induced change of ALT and AST levels, revealed that the enhancement of APAP induced liver injury was dependent on the duration of restraint.
2.Histopathological examinations revealed that livers were normal in appearance with no change in the lobular architecture in the control. In the APAP group, limited hepatic necrosis was seen in the centrilobular regions with cytoplasmic vacuolization, but hepatocytes in the centrilobular regions showed significant and extensive centrilobular necrosis with vacuolization and congestion following restraint3h plus acetaminophen treatment.
3. The mice restrained3h showed significantly higher hepatic GSSG content and GSH/GSSG than that of unrestrained animals, and GSH+GSSG content were significantly lower than that of unrestrained animals. The mice exposed to restraint3h exhibited a higher hepatic GSSG content and GSH/GSSG and lower GSH+GSSG content than that seen in mice receiving the same dose but without restraint (P<0.05or P<0.01)
4. In restraint3h plus APAP group, there were no significant differences in serum IL-6and TNF-α levels compared to APAP group without restraint. Similarly, no significant differences were noted in the density of F4/80-positive Kupffer cells between them.
5.IL-6and TNF-a activity showed different profiles after restraint3h. IL-6activity was statistically significantly increased after exposure to3h restraint stress, while no increase in TNF-a activity was found following restraint. Similarly, no significant differences were noted in the density of F4/80-positive Kupffer cells between control and restrained mice.
6. As to both restraint3h plus APAP group and APAP group, none of them showed any processing of the procaspase-3to its active fragment. The level of active caspase-3was elevated in the liver tissue of mice subjected to3h of restraint compared to unrestraint.
ExperimentⅢ:Potential mechanisms of restraint induced liver injury
1. Treatment with reserpine, prazosin, yohimbine, betaxolol and ICI118551did not influence ALT and AST activities of unrestrained control mice compared with vehicle control group. In a state of a generalized catecholamine depletion produced by reserpine, the elevation of ALT or AST activities induced by restraint were significantly less than control animals (p<0.05). After blockade of α-1or a-2adrenoceptor by prazosin or yohimbine, the magnitude of the increase in ALT and AST activities induced by restraint had a similarly predominant drop compared with control animals (p<0.05), which were similar to the effects of reserpine. However, no changes were observed in ALT and AST activities induced by restraint stress after β-1or β-2adrenergic receptor antagonist treatment (betaxolol or ICI118,551). Regardless of drug treatment, ALT and AST activities of restrained groups were significantly higher than those of corresponding control groups (p<0.05or p<0.01).
2. Treatment with reserpine, prazosin, yohimbine, betaxolol and ICI118,551did not affect hepatic GSH (GSH+GSSG), GSSG and GSH/GSSG ratio of unrestrained control mice compared with vehicle control group. After catecholamine depletion by reserpine or blockade of a-lor a-2adrenoceptor by prazosin or yohimbine, the magnitude of the increase in GSSG content and GSH/GSSG ratio and the decrease in GSH content induced by restraint were statistically attenuated compared with that of control animals (p<0.01), and these drugs had the same attenuating effect. However, hepatic total GSH, GSSG and GSH/GSSG ratio of restrained animals treated with drugs (reserpine, prazosin, or yohimbine) still significantly different from corresponding control animals (p<0.05or p<0.01). Changes of hepatic glutathione content induced by restraint were not affected by beta-1or beta-2adrenergic receptor antagonists (betaxolol or ICI118,551).
3.Liver sections were stained with the TUNEL assay to evaluate the effects of prazosin and yohimbine on hepatocellular apoptosis. After restraint stress for3h, apoptotic hepatocytes were frequently observed, while no apparent apoptotic cell was found in the liver tissue of unrestrained mice. Quantitative analysis of TUNEL positive hepatocytes showed that the number of apoptotic hepatocyte in mice exposed to3h restraint was significantly higher than that in unrestrained control mice (p<0.01). Treatment with prazosin and yohimbine reduced the number of apoptotic cells in the liver and the two agents exhibited a similar inhibiting effect on the hepatocellular apoptosis.
4. Restraint stress obviously increased the expression of cleaved forms of caspase-3, the proapoptotic factor Bax and the activated form of caspase-9, and decreased the expression of the antiapoptotic factors Bcl-2when compared with control group, leading to a high Bax/Bcl-2ratio. In the prazosin and yohimbine treatment group, the above factors induced by restraint were attenuated, but these factors were still unable to return to normal level of the unrestraint group.
Conclusion
1. Restraint stress can exacerbate acetaminophen-induced liver injury as indicated by an increase in necrotic hepatic tissue and serum aminotransferase activity. Reduction in hepatic GSH content and disruption of the balance between oxidants and antioxidants in live, but not releasing of inflammatory cytokines, are responsible for such stress induced aggravation of APAP induced liver injury. In addition, necrosis but not apoptosis is the principal mechanism of hepatocytes death after exacerbation of APAP induced liver injury by restraint stress.
2. Restraint stress can induce reversible liver injury by reducing hepatic GSH content and promote hepatocytes apoptosis through caspase-9and Bcl-2family of apoptotic regulatory proteins.
3.α-1and α-2adrenoceptors mediate restraint-induced liver.
引文
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