Nrf2-ARE信号通路对肠缺血再灌注损伤的保护作用研究
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摘要
肠缺血再灌注(intestinal ischemia reperfusion, IIR)损伤是临床外科常见的危急重症之一,常发生于急性肠系膜缺血、严重烧伤、失血、创伤和感染性休克,以及腹主动脉瘤和小肠移植等复杂的外科手术治疗过程中。肠缺血再灌注损伤是系统性炎症反应综合征(systemic inflammatory response syndrome, SIRS)和多器官功能障碍综合征(multiple organ dysfunction syndrome, MODS)的始动因素,具有极高的发病率和死亡率。继发于小肠移植术后的肠缺血再灌注损伤还会进一步加重免疫排斥反应,并最终导致小肠移植的失败。肠缺血再灌注损伤的预防和治疗是外科临床面临的重要问题。
肠缺血再灌注损伤的危害不仅局限于肠道本身,同时它还会引起严重的远隔脏器损伤,其机制复杂。其中,肠道屏障功能被破坏,菌群移位,毒性物质如炎症介质、细菌毒素等释放入血,活性氧自由基(reactive oxygen species, ROS)被激活,形成氧化应激,是损伤机制中极为关键的环节。我们和其他学者的研究表明,这种氧化应激和炎症介质所引起的远隔器官损伤的病理生理过程是通过多种分子机制和细胞因子的参与完成的,多种信号通路参与其中,包括：1.核因子κB (nuclear factor kappa B, NFκB)的活化；2.泛素-蛋白酶体系统(ubiquitin proteasome system,UPS)的作用；3.髓样细胞表达的触发受体-1(Triggering Receptor Expressed on Myeloid Cells 1, TREM-1)信号通路的作用；4.聚腺苷二磷酸核糖聚合酶-1(Poly(ADP-ribose)polymerase 1, PARP-1)信号通路的作用等。研究发现,在各种信号通路被ROS激活并产生炎症放大的同时,另外一些基因蛋白也可能被活化并产生保护作用。
转录因子NF-E2相关因子2(transcription factor NF-E2-related factor-2, Nrf2)是一种重要的内源性抗氧化应激的调节因子,生理状态下定位于细胞浆,与胞质接头蛋白(Kelch-like ECH associating protein 1, Keap-1)结合形成复合物。当受到氧化应激、亲电子试剂等的刺激后,迅速解离出来并活化转位进入细胞核,与抗氧化反应元件(antioxidant response element,ARE)结合,进而诱导其调控的靶基因表达血红素加氧酶1(heme oxygenase 1,HO-1).谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)和NAD(P)H：醌氧化还原酶1(NAD(P)H:quinone oxidoreductase 1,NQO1)等抗氧化和解毒酶,从而对抗ROS引发的氧化应激。因此,调控Nrf2-ARE信号通路可能是预防和治疗氧化应激所致疾病的重要手段和方法。
随着Nrf2-ARE信号通路能够减轻ROS损害作用的发现,有学者对其在脏器缺血再灌注损伤中的保护作用进行了相关研究,研究显示,Nrf2-ARE信号通路对于心、脑和肾脏缺血再灌注损伤具有重要的保护作用,但其在肠缺血再灌注损伤中的保护作用尚不清楚。
莱菔硫烷(Sulforaphane,SFN)是一种异硫氰酸盐,主要来源于绿花椰菜一类的十字花科蔬菜,具有抗氧化、抗肿瘤等生物学特性,作为化学预防药物已引起广大研究者的关注。该化合物是已知的Nrf2重要的激活剂,能够诱发Nrf2活化转位入核,与ARE结合,正向调控Ⅱ相解毒酶的表达,清除ROS。近年来研究开发的中药活性成分表没食子儿茶素没食子酸酯(Epigallocatechin gallate,EGCG)是从绿茶中提取的主要的活性和水溶性成分,是儿茶素中含量最高的组分,具有抗氧化作用。
本研究采用大鼠肠缺血再灌注模型,应用已知药物诱导激活Nrf2-ARE信号通路,探讨Nrf2-ARE信号通路对于肠缺血再灌注肠道及远隔脏器损伤的保护作用,再进一步验证中药活性成分EGCG预处理对肠缺血再灌注多脏器损伤的保护作用,以期为肠缺血再灌注多脏器损伤的预防和治疗提供一种新的靶点,同时也为EGCG的开发利用提供更有利的理论依据。
第一部分：Nrf2-ARE信号通路在大鼠肠缺血再灌注损伤中的作用
目的：通过分析Nrf2-ARE信号通路诱导药物预处理对肠道Nrf2和HO-1的表达和对氧化应激指标的影响,研究Nrf2-ARE信号通路在大鼠肠缺血再灌注(IIR)损伤中的保护作用及机制,为IIR损伤提供一种新的预防和治疗靶点。
方法：32只雄性SD大鼠随机分为control组、SFN control组、IIR组和SFN+IIR组,通过夹闭肠系膜上动脉(superior mesenteric artery,SMA)60 min、再灌注120 min,建立IIR损伤模型。预处理组于缺血前30分钟分别以5 mg／kg SFN行腹腔注射,对照组仅行剖腹术及SMA分离术。再灌注结束后,取小肠组织。观察大鼠肠组织形态学和病理学,测定小肠组织匀浆超氧化物岐化酶(SOD)、髓过氧化物酶(MPO)谷胱甘肽(GSH)、GSH-Px,采用免疫组化法观察小肠组织Nrf2和HO-1表达并用Western-blot法进行检测验证。
结果：1.与control相比,IIR组：(1)小肠粘膜及粘膜下出现水肿、出血,小肠粘膜缺损、断裂(P<0.01)；(2)小肠组织SOD活性降低(P<0.01),MPO活性增强(P<0.01)；(3)小肠组织GSH水平均显著降低(P<0.01)；(4)小肠组织Nrf2、HO-1表达增强(P<0.01,P<0.01)。2.与control相比,SFN control组：(1)小肠组织GSH-Px水平明显增加(P<0.01)；(2)小肠组织中Nrf2和HO-1的蛋白表达增多(P<0.01,P<0.01)。3.与IIR组相比,SFN+IIR组：(1)小肠组织的病理表现及损伤程度明显减轻(P<0.05)；(2)小肠组织匀浆SOD活性升高(P<0.05),MPO活性降低(P<0.05)；(3)小肠组织GSH和GSH-Px水平明显升高(P<0.05,P<0.01)；(4)小肠组织Nrf2、HO-1表达增强(P<0.01,P<0.05)。
结论：肠缺血1小时再灌注2小时可导致明显的小肠组织损伤,表现为小肠扩张,肠粘膜及粘膜下水肿、出血,粘膜缺损、断裂,氧化应激指标增加和抗氧化应激指标的降低。通过Nrf2-ARE信号通路诱导药物预处理后,增加了小肠组织Nrf2和HO-1的表达,同时伴有小肠组织损伤的减轻,氧化应激指标降低和抗氧化应激指标的升高,说明Nrf2信号通路参与了肠缺血再灌注损伤的保护过程,Nrf2-ARE信号通路在大鼠肠缺血再灌注(IIR)肠道损伤中起保护作用。
第二部分：SFN在大鼠肠缺血再灌注肝、肺损伤中的保护作用
目的：通过分析已知药物SFN预处理诱导Nrf2-ARE信号通路对IIR动物模型肝、肺组织Nrf2和HO-1的表达和对氧化应激指标的影响,研究SFN通过诱导Nrf2-ARE信号通路产生对大鼠IIR引发的远隔脏器肝、肺损伤的保护作用及机制。
方法：32只雄性SD大鼠随机分为control组、SFN control组、IIR组、SFN+IIR组,通过夹闭SMA 60 min、再灌注120 min,建立IIR损伤模型。预处理组于缺血前30分钟分别以5 mg／kg SFN行腹腔注射,对照组仅行剖腹术及SMA分离术。再灌注结束后,取肝、肺组织。观察大鼠肝、肺组织形态学和病理学,检测肝功能：血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)；测定肺组织支气管肺泡灌洗液蛋白(brochia alveolus lung fluid protein, BALFP);测定肝、肺组织匀浆SOD、MPO、GSH、GSH、Px,采用免疫组化法观察肝、肺组织Nrf2和HO-1表达并用Western-blot法检测验证。
结果：1.肠缺血再灌注诱发了肝、肺损伤,表现为与control相比,IIR组：(1)血清ALT、AST明显升高(P<0.01,P<0.01)；(2)肺组织BALFP增高(P<0.01)；(3)肝、肺组织出现明显的水肿、出血和炎细胞浸润；(4)肝、肺组织SOD活性降低(P<0.01,P<0.05),MPO活性增强(P<0.01,P<0.05)；(5)肝、肺组织GSH水平均显著降低(P<0.01,P<0.05)；(6)肝、肺组织HO-1表达增强(P<0.01,P<0.01)；(7)肝、肺组织Nrf2表达增强(P<0.05,P<0.01)。2.与control相比,SFN control组：(1)肝、肺组织GSH-Px水平明显增加(P<0.05,P<0.01)；(2)肝、肺组织中HO-1表达增多(P<0.05,P<0.01)；(3)肝、肺组织中Nrf2表达增多(P<0.05,P<0.01)。3.与IIR组相比,SFN+IIR组：(1)肝损伤减轻,血清ALT、AST明显减低(P<0.01,P<0.05)；(2)肺损伤减轻,肺组织BALFP降低(P<0.05)；(3)肝、肺组织的病理表现及损伤程度明显减轻；(4)肝、肺组织匀浆SOD活性升高(P<0.01,P<0.05),MPO活性降低(P<0.01,P<0.05)；(5)肝、肺组织GSH水平明显升高(P<0.05,P<0.01)；(6)肝、肺组织GSH-Px水平明显升高(P<0.05,P<0.01)；(7)肝、肺组织HO-1表达增强(P<0.01,P<0.01)；(8)肝、肺组织Nrf2表达增强(P<0.05,P<0.05)。
结论：IIR引发远隔脏器肝、肺损伤,表现为肝、肺组织水肿、出血和炎细胞浸润,SFN预处理后,激发活化Nrf2-ARE信号通路,肝、肺组织Nrf2、HO-1表达增加,抑制了组织炎性浸润及氧化应激,肝、肺组织病理损伤的程度减轻,说明SFN通过激发活化Nrf2-ARE信号通路对大鼠IIR引发的远隔脏器肝、肺损伤起保护作用。
第三部分：EGCG在大鼠肠缺血再灌注肝、肺损伤中的保护作用
目的：研究EGCG对大鼠肠缺血再灌注肝、肺损伤的保护作用,探讨其是否具有与SFN相同的Nrf2-ARE信号通路激活作用,从而对肠缺血再灌注肝、肺损伤产生保护作用,为EGCG的开发利用提供更有利的理论依据。
方法：32只雄性SD大鼠随机分为对照组、EGCG control组、IIR组、EGCG+IIR组,通过夹闭肠系膜上动脉60 min、再灌注120 min,建立IIR损伤模型。预处理组于缺血前30分钟分别以50 mg/kg EGCG行腹腔注射,对照组仅行剖腹术及SMA分离术。再灌注结束后,取肺组织。观察大鼠肝、肺组织形态学和病理学,检测肝功能：ALT、AST;测定肺组织含水率；测定肝、肺组织匀浆SOD、MPO、GSH、GSH-Px,采用免疫组化法观察肝、肺组织Nrf2和HO-1表达并用Western-blot法检测验证。
结果：1.肠缺血再灌注诱发了肝、肺损伤,与对照组相比,IIR组：(1)血清ALT、AST明显升高(P<0.01,P<0.05)；(2)肺组织含水率增高(P<0.01)；(3)肝、肺组织出现明显的水肿、出血和炎细胞浸润；(4)肝、肺组织SOD活性降低(P<0.01,P<0.01),MPO活性增强(P<0.01,P<0.01)；(5)肝、肺组织GSH水平均显著降低(P<0.05,P<0.05)；(6)肝、肺组织HO-1表达增强(P<0.01,P<0.01)；(7)肝、肺组织Nrf2表达增强(P<0.05,P<0.05)。2.与对照组相比,EGCG control组：(1)肝、肺组织GSH-Px水平明显增加(P<0.01,P<0.01)；(2)肝、肺组织中HO-1表达增多(P<0.01,P<0.01)；(3)肝、肺组织中Nrf2表达增多(P<0.05,P<0.01)。3.与IIR组相比,EGCG+IIR组：(1)肝损伤减轻,血清ALT、AST明显减低(P<0.01,P<0.05)；(2)肺损伤减轻,肺组织含水率降低(P<0.01)；(3)肝、肺组织的病理表现及损伤程度明显减轻；(4)肝、肺组织匀浆SOD活性升高(P<0.01,P<0.05),MPO活性降低(P<0.01,P<0.01)；(5)肝、肺组织GSH水平明显升高(P<0.01,P<0.01)；(6)肝、肺组织GSH-Px水平明显升高(P<0.01,P<0.01)；(7)肝、肺组织HO-1表达增强(P<0.01,P<0.01)；(8)肝、肺组织Nrf2表达增强(P<0.01,P<0.05)。
结论：IIR引发远隔脏器肝、肺损伤,表现为肝、肺组织水肿、出血和炎细胞浸润,通过EGCG预处理,有效降低了肝、肺损伤的程度,同时肝、肺组织Nrf2、HO-1表达增加。实验证明,天然中草药绿茶的有效活性成分EGCG,具有与已知药物SFN相同的作用效果,通过激发活化Nrf2-ARE信号通路,对大鼠肠缺血再灌注远隔脏器肝、肺损伤具有保护作用。
小结
本研究利用大鼠肠缺血再灌注模型,采用生化自动分析、免疫组化和Western blot分析等分子生物学方法对原发脏器肠道,以及远隔脏器肝、肺功能(AST、ATL、肺含水率和BALFP);肠、肝、肺组织氧化损伤水平(SOD)；白细胞趋化因子(MPO)；Nrf2的激活表达和Ⅱ相抗氧化反应酶系统(GSH、GSH-Px、HO-1)进行了检测,探讨Nrf2-ARE信号通路在肠缺血再灌注损伤保护机制中的作用,以及SFN和EGCG预处理对肠缺血再灌注肠道及远隔脏器损伤的保护作用。
结果表明：肠缺血再灌注不但能够导致原发脏器肠道的损伤,还能够引起远隔脏器肝、肺组织的损伤,病理学表现为肠、肝、肺组织的出血、渗出、中性粒细胞浸润；组织结构破坏；肝功能受损；肺组织含水率和BALFP增加；肠、肝和肺组织中的氧化损伤指标和炎症性指标如：SOD含量下降,MPO的含量增加。采用SFN和EGCG预处理后,肠、肝和肺组织Nrf2的核表达增强,Nrf2-ARE信号通路被激活,Ⅱ相抗氧化反应酶系统的水平增加,肠、肝和肺组织损伤程度明显减轻,说明Nrf2-ARE信号通路对于肠缺血再灌注肠道损伤及远隔脏器肝、肺损伤起保护作用,同时,进一步验证了天然中草药绿茶的有效活性成分EGCG,可以诱导活化Nrf2-ARE信号通路,对大鼠肠缺血再灌注肝、肺损伤具有保护作用。
Intestinal ischemia reperfusion (ⅡR) injury is one of the most severe clinical disorders, which is caused by many factors, such as acute mesenteric ischemia, severe burn, hemorrhagic, traumatic or septic shock, and some surgical procedures, including small bowel transplantation and abdominal aortic surgery. It is known thatⅡR injury is an important initiating factor in the pathogenesis of systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS).ⅡR injury which is associated with intestinal transplantation usually lead to the failure of the operation due to the aggravating of immunological rejection. Efforts to prevent and controlⅡR injury remain the challenging and promising for clinical surgery.
ⅡR injury is not only limited to the intestine itself, but involves severe distant tissue destruction. The mechanism ofⅡR injury is complicated. AfterⅡR, intestinal barrier function is damaged, bacteria translocation, toxic substances release into the blood, such as inflammatory mediators, bacterial toxins and reactive oxygen species (ROS) is activated, contributing to the oxidative stress. This is considered the key factor. Multiple signaling pathways have involved in this pathologic process which demenstrated by our and others studies, such as nuclear factor kappa B (NFκB), ubiquitin proteasome system (UPS), triggering receptor expressed on myeloid cells 1 (TREM-1), poly(ADP-ribose) polymerase 1 (PARP-1), etc. ROS in various signaling pathways are activated and produce inflammatory amplification, while other proteins may also be activated to produce a protective effect.
Transcription factor NF-E2-related factor-2 (Nrf2) regulates a major environmental and oxidative stress response. It is held in the cytoplasm by a cytoskeletal-associated specific inhibitory protein (Kelch-like ECH associating protein 1, Keap1) under circumstances of normal cellular quiescent state. Upon the stimulation of oxidative stress, cysteine residues within the hinge region of Keap1 can be modified and cause a conformational change in KEAP1 with the loss of Nrf2 binding, then Nrf2 translocated into the nucleus, which binds to the antioxidant response element (ARE) in the promoter region of a number of genes, encoding for antioxidative and phase 2 enzymes, such as heme oxygenase 1 (HO-1), glutathione peroxidase (GSH-Px), and NAD(P)H:quinone oxidoreductase 1 (NQO1), which can antagonize oxidative stress induced by ROS. Thus, regulating Nrf2-ARE signaling pathway may be a potent approach against diseases caused by oxidative stress.
With the discovery of the Nrf2-ARE signaling pathway can reduce the tissue damage induced by ROS, more and more studies have shown that modulating Nrf2-ARE signaling pathway play a protective role in splanchnic ischemia reperfusion injuries including heart, brain and renal. However, no report addresses a role of Nrf2-ARE signaling pathway in protectingⅡR injury.
Sulforaphane (SFN) is an isothiocyanate derived from natural product which is present in cruciferous vegetables such as broccoli and has been used as antioxidants and anti-tumor agents, which drawn many researchers attention. The cytoprotective effect of this compound is exerted by modulating Nrf2-ARE pathway. Epigallocatechin gallate (EGCG), the major component including activity and water-solubility of polyphenols in green tea, which is an effective component of traditional Chinese drug, has recently drawn considerable attention for antioxidative properties.
Our aim is to use a known pharmacal inducer of Nrf2 to explore the mechanisms of Nrf2-ARE signaling pathway in IIR by a rat model, then testify the role of the active ingredient of Chinese medicine-EGCG pretreatment on multiple organs dysfunction induced byⅡR, to seek a new target for prevention and treatment onⅡR injury at the molecular level, and provide more favorable theoretical basis on EGCG development and utilization.
PartⅠ:Effect of Nrf2-ARE signaling pathway on the protection of intestinal ischemia reperfusion injury in rats
Objective:To investigate the role of Nrf2-ARE signaling pathway on the protection of intestinal ischemia reperfusion (ⅡR) injury through intervening on the expressions of Nrf2 and HO-1 and the effects on indicators of oxidative stress, and to provide a novel target for prevention and treatment on IIR injury at the molecular level.
Methods:Thirty-two male Sprague Dawley rats were randomly divided into 4 groups:control, SFN control, IIR, and SFN+ⅡR groups (n=8 in each group). TheⅡR model was established by clamping superior mesenteric artery (SMA) for 1 hour and reperfusion for 2 hours. Pretreatment groups at 30 minutes before ischemia,5mg/kg SFN abdominal injection respectively, the control group carried out only laparotomy and isolation of the SMA without occlusion. Intestinal histology was investigated. Intestinal tissue superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH) and GSH-Px activity were assayed. The Intestinal Nrf2 and HO-1 were determined by immunohisto-chemical analysis and western blot analysis.
Results:Firstly, compared with the control group,ⅡR group:(1) Intestine was obvious edema, hemorrhage, bleed, exudation, and lack of small intestinal mucosa (P<0.01); (2) Intestinal SOD activity was decreased (P<0.01). Intestinal MPO activity was increased (P<0.01); (3) Intestinal GSH level was significantly enhanced (P<0.01); (4) Expressions of Nrf2 and HO-1 in the intestinal tissue were increased (P<0.01, P<0.01). Secondly, compared with the control group, SFN control group:(1) Intestinal GSH-Px activity increased significantly (P<0.01); (2) Expressions of Nrf2 and HO-1 in the intestinal tissue were increased (P<0.01,P<0.01). Thirdly, compared with theⅡR group, SFN+ⅡR group:(1) Intestine tissue pathological scores of injury significantly were reduced (P<0.05); (2) Intestinal SOD activity was elevated markedly (P<0.05), MPO activity was conspicuously decreased (P<0.05); (3) Intestinal GSH and GSH-Px levels were significantly enhanced (P<0.05,P<0.01); (4) The expressions of Nrf2 and HO-1 in the intestinal tissue were increased (P<0.01, P<0.05).
Conclusions:ⅡR induced intestinal injury, characterized by the histological edema, hemorrhage, lack of small intestinal mucosa. The injury of intestine was alleviated through upregulating Nrf2-ARE signaling pathway, which is accompanied with the expressions of Nrf2 and HO-1 in the intestinal tissue. The activation of Nrf2-ARE signaling pathway plays an important role in the protection of intestinal ischemia reperfusion.
PartⅡ:SFN protects liver and lung injuries induced by intestinal ischemia reperfusion
Objective:To investigate the role of a known inducer of Nrf2-ARE signaling pathway on the liver and lung injuries induced byⅡR through evaluating the expressions of Nrf2 and HO-1 and the effects on indicators of oxidative stress, then clarify the effect of SFN by inducing Nrf2-ARE signaling pathway on liver and lung injuries triggered byⅡR in rats.
Methods:Thirty-two male Sprague Dawley rats were randomly divided into 4 groups:control, SFN control,ⅡR, and SFN+ⅡR groups (n=8 in each group). TheⅡR model was established by clamping SMA for 1 hour and reperfusion for 2 hours. Pretreatment groups at 30 minutes before ischemia, 5mg/kg SFN abdominal injection respectively, the control group carried out only laparotomy and isolation of the SMA without occlusion. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lung brochia alveolus lung fluid protein (BALFP) was measured. Liver and lung histology was investigated. Liver and lung tissue SOD, MPO, GSH and GSH-Px activity were assayed. The liver and lung Nrf2 and HO-1 were determined by immunohistochemical analysis and western blot analysis.
Results:Firstly, compared with the control group,ⅡR group:(1) Serum ALT and AST were significantly enhanced (P<0.01, P<0.01); (2) Lung BALFP was increased significantly (P<0.01); (3) Liver and lung were obvious edema, hemorrhage, and neutrophil infiltration; (4) Liver and lung SOD activities were decreased (P<0.01, P<0.05), MPO activities were increased (P<0.01, P<0.05); (5) Liver and lung GSH levels were significantly enhanced (P<0.01, P<0.05); (6) Expressions of HO-1 in the liver and lung tissues were increased (P<0.01, P<0.01); (7) Expressions of Nrf2 in the liver and lung tissues were increased (P<0.05, P<0.01). Secondly, compared with the control group, SFN control group:(1) Liver and lung GSH-Px activities increased significantly (P<0.05, P<0.01); (2) Expressions of HO-1 in the liver and lung tissue were increased (P<0.05, P<0.01); (3) Expressions of Nrf2 in the liver and lung tissue were increased (P<0.05, P<0.01). Thirdly, compared with theⅡR group, SFN+IIR group:(1) Serum ALT and AST were significantly decreased (P<0.01, P<0.05); (2) Lung BALFP was decreased significantly (P<0.05); (3) The pathological injuries of liver and lung were improved; (4) Liver and lung SOD activities were elevated significantly (P<0.01, P<0.05), MPO activities were decreased significantly (P<0.01, P<0.05); (5) Liver and lung GSH levels were significantly enhanced (P<0.05, P<0.01); (6) Liver and lung GSH-Px levels were significantly enhanced (P<0.05, P<0.01); (7) Expressions of HO-1 in the liver and lung tissue were increased (P<0.01, P<0.01); (8) Expressions of Nrf2 in the liver and lung tissue were increased (P<0.05, P<0.05).
Conclusions:ⅡR induced liver and lung injuries, characterized by the histological edema, hemorrhage, and infiltration with inflammatory cells. The injuries of liver and lung were alleviated through Nrf2-ARE signaling pathway, which is accompanied with the overexpressions of Nrf2 and HO-1 in the liver and lung tissue. The activation of Nrf2-ARE signaling pathway induced by SFN plays an important role in the protection of liver and lung injuries induced byⅡR.
PartⅢ:EGCG protects liver and lung injuries induced by intestinal ischemia reperfusion in rats
Objective:To investigate EGCG on the protection of liver and lung injuries induced byⅡR, and testify if the role of EGCG pretreatment on multiple organs dysfunction induced by IIR act as SFN through Nrf2-ARE signaling pathway. To provide more favorable theoretical basis on the development and utilization of EGCG.
Methods:Thirty-two male Sprague Dawley rats were randomly divided into 4 groups:control, EGCG control,ⅡR, and EGCG+ⅡR groups (n=8 in each group). TheⅡR model was established by clamping SMA for 1 hour and reperfusion for 2 hours. Pretreatment groups at 30 minutes before ischemia,50mg/kg EGCG abdominal injection respectively, the control group carried out only laparotomy and isolation of the SMA without occlusion. Serum levels of AST, ALT, and lung water content was assayed. Liver and lung histology was investigated. Liver and lung tissue SOD, MPO, GSH and GSH-Px activity were assayed. The liver and lung Nrf2 and HO-1 were determined by immunohistochemical analysis and western blot analysis.
Results:Firstly, compared with the control group,ⅡR group:(1) Serum ALT and AST were significantly enhanced (P<0.01, P<0.05); (2) Lung water content was increased significantly (P<0.01); (3) Liver and lung were obvious edema, hemorrhage, and neutrophil infiltration; (4) Liver and lung SOD activities were decreased (P<0.01, P<0.01), MPO activities were increased (P<0.01, P<0.01); (5) Liver and lung GSH levels were significantly enhanced (P<0.05, P<0.05); (6) Expressions of HO-1 in the liver and lung tissues were increased (P<0.01, P<0.01); (7) Expressions of Nrf2 in the liver and lung tissues were increased (P<0.05, P<0.05). Secondly, compared with the control group, EGCG control group:(1) Liver and lung GSH-Px activities increased significantly (P<0.01, P<0.01); (2) Expressions of HO-1 in the liver and lung tissue were increased (P<0.01, P<0.01); (3) Expressions of Nrf2 in the liver and lung tissue were increased (P<0.05, P<0.01). Thirdly, compared with theⅡR group, EGCG+ⅡR group: (1) Serum ALT and AST were significantly decreased (P<0.01, P<0.05); (2) Lung water content was decreased significantly (P<0.01); (3) The pathological injuries of liver and lung were improved; (4) Liver and lung SOD activities were elevated significantly (P<0.01, P<0.05), MPO activities were, decreased significantly (P<0.01, P<0.01); (5) Liver and lung GSH levels were significantly enhanced (P<0.01, P<0.01); (6) Liver and lung GSH-Px levels were significantly enhanced (P<0.01, P<0.01); (7) Expressions of HO-1 in the liver and lung tissue were increased (P<0.01, P<0.01); (8) Expressions of Nrf2 in the liver and lung tissue were increased (P<0.01,P<0.05).
Conclusions:ⅡR induced liver and lung injuries, characterized by the histological edema, hemorrhage, and infiltration with inflammatory cells. The injuries of liver and lung can be alleviated through EGCG pretreatment, which is accompanied with the overexpressions of Nrf2 and HO-1 in the liver and lung tissue. EGCG, the major component of polyphenols in green tea, can protect liver and lung injuries induced byⅡR through Nrf2-ARE signaling pathway.
We use intestinal ischemia reperfusion (ⅡR) rat model to analyze the intestine, liver and lung injuries marker (histology, serum ALT, AST, lung water content and BLAFP), tissue oxidative stress (SOD, MPO), and the expression of Nrf2-ARE pathway (Nrf2, GSH, GSH-Px and HO-1) using biochemical analysis, immunohistochemical analysis and Western blot analysis to evaluate the role of Nrf2-ARE signaling pathway on the protective effect ofⅡR and the effect of SFN, EGCG pretreatment to liver and lung injuries induced byⅡR.
The result showed as follows:Intestinal ischemia reperfusion (ⅡR) not only leads to intestinal damage itself, but also causes severe destruction of remote organs, characterized by the histological edema, hemorrhage and infiltration with inflammatory cells, significant increasing of ALT, AST, lung water content, and BLAFP. Intestine, liver and lung SOD and MPO levels changed markedly inⅡR group. Pretreatment with SFN or EGCG, the intestine, liver and lung injuries were improved, which is accompanied with the level of tissue Nrf2. At the meantime, phase 2 antioxidant and detoxification enzymes increased markedly. The Nrf2-ARE signaling pathway has been shown to have beneficial effects on the prevention ofⅡR. This also demonstrate that EGCG, which is the major component of polyphenols in green tea, plays an important role in the induction of Nrf2-ARE signaling pathway, and the protection of liver and lung injuries induced byⅡR.
肠缺血再灌注损伤的危害不仅局限于肠道本身,同时它还会引起严重的远隔脏器损伤,其机制复杂。其中,肠道屏障功能被破坏,菌群移位,毒性物质如炎症介质、细菌毒素等释放入血,活性氧自由基(reactive oxygen species, ROS)被激活,形成氧化应激,是损伤机制中极为关键的环节。我们和其他学者的研究表明,这种氧化应激和炎症介质所引起的远隔器官损伤的病理生理过程是通过多种分子机制和细胞因子的参与完成的,多种信号通路参与其中,包括：1.核因子κB (nuclear factor kappa B, NFκB)的活化；2.泛素-蛋白酶体系统(ubiquitin proteasome system,UPS)的作用；3.髓样细胞表达的触发受体-1(Triggering Receptor Expressed on Myeloid Cells 1, TREM-1)信号通路的作用；4.聚腺苷二磷酸核糖聚合酶-1(Poly(ADP-ribose)polymerase 1, PARP-1)信号通路的作用等。研究发现,在各种信号通路被ROS激活并产生炎症放大的同时,另外一些基因蛋白也可能被活化并产生保护作用。
转录因子NF-E2相关因子2(transcription factor NF-E2-related factor-2, Nrf2)是一种重要的内源性抗氧化应激的调节因子,生理状态下定位于细胞浆,与胞质接头蛋白(Kelch-like ECH associating protein 1, Keap-1)结合形成复合物。当受到氧化应激、亲电子试剂等的刺激后,迅速解离出来并活化转位进入细胞核,与抗氧化反应元件(antioxidant response element,ARE)结合,进而诱导其调控的靶基因表达血红素加氧酶1(heme oxygenase 1,HO-1).谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)和NAD(P)H：醌氧化还原酶1(NAD(P)H:quinone oxidoreductase 1,NQO1)等抗氧化和解毒酶,从而对抗ROS引发的氧化应激。因此,调控Nrf2-ARE信号通路可能是预防和治疗氧化应激所致疾病的重要手段和方法。
随着Nrf2-ARE信号通路能够减轻ROS损害作用的发现,有学者对其在脏器缺血再灌注损伤中的保护作用进行了相关研究,研究显示,Nrf2-ARE信号通路对于心、脑和肾脏缺血再灌注损伤具有重要的保护作用,但其在肠缺血再灌注损伤中的保护作用尚不清楚。
莱菔硫烷(Sulforaphane,SFN)是一种异硫氰酸盐,主要来源于绿花椰菜一类的十字花科蔬菜,具有抗氧化、抗肿瘤等生物学特性,作为化学预防药物已引起广大研究者的关注。该化合物是已知的Nrf2重要的激活剂,能够诱发Nrf2活化转位入核,与ARE结合,正向调控Ⅱ相解毒酶的表达,清除ROS。近年来研究开发的中药活性成分表没食子儿茶素没食子酸酯(Epigallocatechin gallate,EGCG)是从绿茶中提取的主要的活性和水溶性成分,是儿茶素中含量最高的组分,具有抗氧化作用。
本研究采用大鼠肠缺血再灌注模型,应用已知药物诱导激活Nrf2-ARE信号通路,探讨Nrf2-ARE信号通路对于肠缺血再灌注肠道及远隔脏器损伤的保护作用,再进一步验证中药活性成分EGCG预处理对肠缺血再灌注多脏器损伤的保护作用,以期为肠缺血再灌注多脏器损伤的预防和治疗提供一种新的靶点,同时也为EGCG的开发利用提供更有利的理论依据。
第一部分：Nrf2-ARE信号通路在大鼠肠缺血再灌注损伤中的作用
目的：通过分析Nrf2-ARE信号通路诱导药物预处理对肠道Nrf2和HO-1的表达和对氧化应激指标的影响,研究Nrf2-ARE信号通路在大鼠肠缺血再灌注(IIR)损伤中的保护作用及机制,为IIR损伤提供一种新的预防和治疗靶点。
方法：32只雄性SD大鼠随机分为control组、SFN control组、IIR组和SFN+IIR组,通过夹闭肠系膜上动脉(superior mesenteric artery,SMA)60 min、再灌注120 min,建立IIR损伤模型。预处理组于缺血前30分钟分别以5 mg／kg SFN行腹腔注射,对照组仅行剖腹术及SMA分离术。再灌注结束后,取小肠组织。观察大鼠肠组织形态学和病理学,测定小肠组织匀浆超氧化物岐化酶(SOD)、髓过氧化物酶(MPO)谷胱甘肽(GSH)、GSH-Px,采用免疫组化法观察小肠组织Nrf2和HO-1表达并用Western-blot法进行检测验证。
结果：1.与control相比,IIR组：(1)小肠粘膜及粘膜下出现水肿、出血,小肠粘膜缺损、断裂(P<0.01)；(2)小肠组织SOD活性降低(P<0.01),MPO活性增强(P<0.01)；(3)小肠组织GSH水平均显著降低(P<0.01)；(4)小肠组织Nrf2、HO-1表达增强(P<0.01,P<0.01)。2.与control相比,SFN control组：(1)小肠组织GSH-Px水平明显增加(P<0.01)；(2)小肠组织中Nrf2和HO-1的蛋白表达增多(P<0.01,P<0.01)。3.与IIR组相比,SFN+IIR组：(1)小肠组织的病理表现及损伤程度明显减轻(P<0.05)；(2)小肠组织匀浆SOD活性升高(P<0.05),MPO活性降低(P<0.05)；(3)小肠组织GSH和GSH-Px水平明显升高(P<0.05,P<0.01)；(4)小肠组织Nrf2、HO-1表达增强(P<0.01,P<0.05)。
结论：肠缺血1小时再灌注2小时可导致明显的小肠组织损伤,表现为小肠扩张,肠粘膜及粘膜下水肿、出血,粘膜缺损、断裂,氧化应激指标增加和抗氧化应激指标的降低。通过Nrf2-ARE信号通路诱导药物预处理后,增加了小肠组织Nrf2和HO-1的表达,同时伴有小肠组织损伤的减轻,氧化应激指标降低和抗氧化应激指标的升高,说明Nrf2信号通路参与了肠缺血再灌注损伤的保护过程,Nrf2-ARE信号通路在大鼠肠缺血再灌注(IIR)肠道损伤中起保护作用。
第二部分：SFN在大鼠肠缺血再灌注肝、肺损伤中的保护作用
目的：通过分析已知药物SFN预处理诱导Nrf2-ARE信号通路对IIR动物模型肝、肺组织Nrf2和HO-1的表达和对氧化应激指标的影响,研究SFN通过诱导Nrf2-ARE信号通路产生对大鼠IIR引发的远隔脏器肝、肺损伤的保护作用及机制。
方法：32只雄性SD大鼠随机分为control组、SFN control组、IIR组、SFN+IIR组,通过夹闭SMA 60 min、再灌注120 min,建立IIR损伤模型。预处理组于缺血前30分钟分别以5 mg／kg SFN行腹腔注射,对照组仅行剖腹术及SMA分离术。再灌注结束后,取肝、肺组织。观察大鼠肝、肺组织形态学和病理学,检测肝功能：血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)；测定肺组织支气管肺泡灌洗液蛋白(brochia alveolus lung fluid protein, BALFP);测定肝、肺组织匀浆SOD、MPO、GSH、GSH、Px,采用免疫组化法观察肝、肺组织Nrf2和HO-1表达并用Western-blot法检测验证。
结果：1.肠缺血再灌注诱发了肝、肺损伤,表现为与control相比,IIR组：(1)血清ALT、AST明显升高(P<0.01,P<0.01)；(2)肺组织BALFP增高(P<0.01)；(3)肝、肺组织出现明显的水肿、出血和炎细胞浸润；(4)肝、肺组织SOD活性降低(P<0.01,P<0.05),MPO活性增强(P<0.01,P<0.05)；(5)肝、肺组织GSH水平均显著降低(P<0.01,P<0.05)；(6)肝、肺组织HO-1表达增强(P<0.01,P<0.01)；(7)肝、肺组织Nrf2表达增强(P<0.05,P<0.01)。2.与control相比,SFN control组：(1)肝、肺组织GSH-Px水平明显增加(P<0.05,P<0.01)；(2)肝、肺组织中HO-1表达增多(P<0.05,P<0.01)；(3)肝、肺组织中Nrf2表达增多(P<0.05,P<0.01)。3.与IIR组相比,SFN+IIR组：(1)肝损伤减轻,血清ALT、AST明显减低(P<0.01,P<0.05)；(2)肺损伤减轻,肺组织BALFP降低(P<0.05)；(3)肝、肺组织的病理表现及损伤程度明显减轻；(4)肝、肺组织匀浆SOD活性升高(P<0.01,P<0.05),MPO活性降低(P<0.01,P<0.05)；(5)肝、肺组织GSH水平明显升高(P<0.05,P<0.01)；(6)肝、肺组织GSH-Px水平明显升高(P<0.05,P<0.01)；(7)肝、肺组织HO-1表达增强(P<0.01,P<0.01)；(8)肝、肺组织Nrf2表达增强(P<0.05,P<0.05)。
结论：IIR引发远隔脏器肝、肺损伤,表现为肝、肺组织水肿、出血和炎细胞浸润,SFN预处理后,激发活化Nrf2-ARE信号通路,肝、肺组织Nrf2、HO-1表达增加,抑制了组织炎性浸润及氧化应激,肝、肺组织病理损伤的程度减轻,说明SFN通过激发活化Nrf2-ARE信号通路对大鼠IIR引发的远隔脏器肝、肺损伤起保护作用。
第三部分：EGCG在大鼠肠缺血再灌注肝、肺损伤中的保护作用
目的：研究EGCG对大鼠肠缺血再灌注肝、肺损伤的保护作用,探讨其是否具有与SFN相同的Nrf2-ARE信号通路激活作用,从而对肠缺血再灌注肝、肺损伤产生保护作用,为EGCG的开发利用提供更有利的理论依据。
方法：32只雄性SD大鼠随机分为对照组、EGCG control组、IIR组、EGCG+IIR组,通过夹闭肠系膜上动脉60 min、再灌注120 min,建立IIR损伤模型。预处理组于缺血前30分钟分别以50 mg/kg EGCG行腹腔注射,对照组仅行剖腹术及SMA分离术。再灌注结束后,取肺组织。观察大鼠肝、肺组织形态学和病理学,检测肝功能：ALT、AST;测定肺组织含水率；测定肝、肺组织匀浆SOD、MPO、GSH、GSH-Px,采用免疫组化法观察肝、肺组织Nrf2和HO-1表达并用Western-blot法检测验证。
结果：1.肠缺血再灌注诱发了肝、肺损伤,与对照组相比,IIR组：(1)血清ALT、AST明显升高(P<0.01,P<0.05)；(2)肺组织含水率增高(P<0.01)；(3)肝、肺组织出现明显的水肿、出血和炎细胞浸润；(4)肝、肺组织SOD活性降低(P<0.01,P<0.01),MPO活性增强(P<0.01,P<0.01)；(5)肝、肺组织GSH水平均显著降低(P<0.05,P<0.05)；(6)肝、肺组织HO-1表达增强(P<0.01,P<0.01)；(7)肝、肺组织Nrf2表达增强(P<0.05,P<0.05)。2.与对照组相比,EGCG control组：(1)肝、肺组织GSH-Px水平明显增加(P<0.01,P<0.01)；(2)肝、肺组织中HO-1表达增多(P<0.01,P<0.01)；(3)肝、肺组织中Nrf2表达增多(P<0.05,P<0.01)。3.与IIR组相比,EGCG+IIR组：(1)肝损伤减轻,血清ALT、AST明显减低(P<0.01,P<0.05)；(2)肺损伤减轻,肺组织含水率降低(P<0.01)；(3)肝、肺组织的病理表现及损伤程度明显减轻；(4)肝、肺组织匀浆SOD活性升高(P<0.01,P<0.05),MPO活性降低(P<0.01,P<0.01)；(5)肝、肺组织GSH水平明显升高(P<0.01,P<0.01)；(6)肝、肺组织GSH-Px水平明显升高(P<0.01,P<0.01)；(7)肝、肺组织HO-1表达增强(P<0.01,P<0.01)；(8)肝、肺组织Nrf2表达增强(P<0.01,P<0.05)。
结论：IIR引发远隔脏器肝、肺损伤,表现为肝、肺组织水肿、出血和炎细胞浸润,通过EGCG预处理,有效降低了肝、肺损伤的程度,同时肝、肺组织Nrf2、HO-1表达增加。实验证明,天然中草药绿茶的有效活性成分EGCG,具有与已知药物SFN相同的作用效果,通过激发活化Nrf2-ARE信号通路,对大鼠肠缺血再灌注远隔脏器肝、肺损伤具有保护作用。
小结
本研究利用大鼠肠缺血再灌注模型,采用生化自动分析、免疫组化和Western blot分析等分子生物学方法对原发脏器肠道,以及远隔脏器肝、肺功能(AST、ATL、肺含水率和BALFP);肠、肝、肺组织氧化损伤水平(SOD)；白细胞趋化因子(MPO)；Nrf2的激活表达和Ⅱ相抗氧化反应酶系统(GSH、GSH-Px、HO-1)进行了检测,探讨Nrf2-ARE信号通路在肠缺血再灌注损伤保护机制中的作用,以及SFN和EGCG预处理对肠缺血再灌注肠道及远隔脏器损伤的保护作用。
结果表明：肠缺血再灌注不但能够导致原发脏器肠道的损伤,还能够引起远隔脏器肝、肺组织的损伤,病理学表现为肠、肝、肺组织的出血、渗出、中性粒细胞浸润；组织结构破坏；肝功能受损；肺组织含水率和BALFP增加；肠、肝和肺组织中的氧化损伤指标和炎症性指标如：SOD含量下降,MPO的含量增加。采用SFN和EGCG预处理后,肠、肝和肺组织Nrf2的核表达增强,Nrf2-ARE信号通路被激活,Ⅱ相抗氧化反应酶系统的水平增加,肠、肝和肺组织损伤程度明显减轻,说明Nrf2-ARE信号通路对于肠缺血再灌注肠道损伤及远隔脏器肝、肺损伤起保护作用,同时,进一步验证了天然中草药绿茶的有效活性成分EGCG,可以诱导活化Nrf2-ARE信号通路,对大鼠肠缺血再灌注肝、肺损伤具有保护作用。
Intestinal ischemia reperfusion (ⅡR) injury is one of the most severe clinical disorders, which is caused by many factors, such as acute mesenteric ischemia, severe burn, hemorrhagic, traumatic or septic shock, and some surgical procedures, including small bowel transplantation and abdominal aortic surgery. It is known thatⅡR injury is an important initiating factor in the pathogenesis of systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS).ⅡR injury which is associated with intestinal transplantation usually lead to the failure of the operation due to the aggravating of immunological rejection. Efforts to prevent and controlⅡR injury remain the challenging and promising for clinical surgery.
ⅡR injury is not only limited to the intestine itself, but involves severe distant tissue destruction. The mechanism ofⅡR injury is complicated. AfterⅡR, intestinal barrier function is damaged, bacteria translocation, toxic substances release into the blood, such as inflammatory mediators, bacterial toxins and reactive oxygen species (ROS) is activated, contributing to the oxidative stress. This is considered the key factor. Multiple signaling pathways have involved in this pathologic process which demenstrated by our and others studies, such as nuclear factor kappa B (NFκB), ubiquitin proteasome system (UPS), triggering receptor expressed on myeloid cells 1 (TREM-1), poly(ADP-ribose) polymerase 1 (PARP-1), etc. ROS in various signaling pathways are activated and produce inflammatory amplification, while other proteins may also be activated to produce a protective effect.
Transcription factor NF-E2-related factor-2 (Nrf2) regulates a major environmental and oxidative stress response. It is held in the cytoplasm by a cytoskeletal-associated specific inhibitory protein (Kelch-like ECH associating protein 1, Keap1) under circumstances of normal cellular quiescent state. Upon the stimulation of oxidative stress, cysteine residues within the hinge region of Keap1 can be modified and cause a conformational change in KEAP1 with the loss of Nrf2 binding, then Nrf2 translocated into the nucleus, which binds to the antioxidant response element (ARE) in the promoter region of a number of genes, encoding for antioxidative and phase 2 enzymes, such as heme oxygenase 1 (HO-1), glutathione peroxidase (GSH-Px), and NAD(P)H:quinone oxidoreductase 1 (NQO1), which can antagonize oxidative stress induced by ROS. Thus, regulating Nrf2-ARE signaling pathway may be a potent approach against diseases caused by oxidative stress.
With the discovery of the Nrf2-ARE signaling pathway can reduce the tissue damage induced by ROS, more and more studies have shown that modulating Nrf2-ARE signaling pathway play a protective role in splanchnic ischemia reperfusion injuries including heart, brain and renal. However, no report addresses a role of Nrf2-ARE signaling pathway in protectingⅡR injury.
Sulforaphane (SFN) is an isothiocyanate derived from natural product which is present in cruciferous vegetables such as broccoli and has been used as antioxidants and anti-tumor agents, which drawn many researchers attention. The cytoprotective effect of this compound is exerted by modulating Nrf2-ARE pathway. Epigallocatechin gallate (EGCG), the major component including activity and water-solubility of polyphenols in green tea, which is an effective component of traditional Chinese drug, has recently drawn considerable attention for antioxidative properties.
Our aim is to use a known pharmacal inducer of Nrf2 to explore the mechanisms of Nrf2-ARE signaling pathway in IIR by a rat model, then testify the role of the active ingredient of Chinese medicine-EGCG pretreatment on multiple organs dysfunction induced byⅡR, to seek a new target for prevention and treatment onⅡR injury at the molecular level, and provide more favorable theoretical basis on EGCG development and utilization.
PartⅠ:Effect of Nrf2-ARE signaling pathway on the protection of intestinal ischemia reperfusion injury in rats
Objective:To investigate the role of Nrf2-ARE signaling pathway on the protection of intestinal ischemia reperfusion (ⅡR) injury through intervening on the expressions of Nrf2 and HO-1 and the effects on indicators of oxidative stress, and to provide a novel target for prevention and treatment on IIR injury at the molecular level.
Methods:Thirty-two male Sprague Dawley rats were randomly divided into 4 groups:control, SFN control, IIR, and SFN+ⅡR groups (n=8 in each group). TheⅡR model was established by clamping superior mesenteric artery (SMA) for 1 hour and reperfusion for 2 hours. Pretreatment groups at 30 minutes before ischemia,5mg/kg SFN abdominal injection respectively, the control group carried out only laparotomy and isolation of the SMA without occlusion. Intestinal histology was investigated. Intestinal tissue superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH) and GSH-Px activity were assayed. The Intestinal Nrf2 and HO-1 were determined by immunohisto-chemical analysis and western blot analysis.
Results:Firstly, compared with the control group,ⅡR group:(1) Intestine was obvious edema, hemorrhage, bleed, exudation, and lack of small intestinal mucosa (P<0.01); (2) Intestinal SOD activity was decreased (P<0.01). Intestinal MPO activity was increased (P<0.01); (3) Intestinal GSH level was significantly enhanced (P<0.01); (4) Expressions of Nrf2 and HO-1 in the intestinal tissue were increased (P<0.01, P<0.01). Secondly, compared with the control group, SFN control group:(1) Intestinal GSH-Px activity increased significantly (P<0.01); (2) Expressions of Nrf2 and HO-1 in the intestinal tissue were increased (P<0.01,P<0.01). Thirdly, compared with theⅡR group, SFN+ⅡR group:(1) Intestine tissue pathological scores of injury significantly were reduced (P<0.05); (2) Intestinal SOD activity was elevated markedly (P<0.05), MPO activity was conspicuously decreased (P<0.05); (3) Intestinal GSH and GSH-Px levels were significantly enhanced (P<0.05,P<0.01); (4) The expressions of Nrf2 and HO-1 in the intestinal tissue were increased (P<0.01, P<0.05).
Conclusions:ⅡR induced intestinal injury, characterized by the histological edema, hemorrhage, lack of small intestinal mucosa. The injury of intestine was alleviated through upregulating Nrf2-ARE signaling pathway, which is accompanied with the expressions of Nrf2 and HO-1 in the intestinal tissue. The activation of Nrf2-ARE signaling pathway plays an important role in the protection of intestinal ischemia reperfusion.
PartⅡ:SFN protects liver and lung injuries induced by intestinal ischemia reperfusion
Objective:To investigate the role of a known inducer of Nrf2-ARE signaling pathway on the liver and lung injuries induced byⅡR through evaluating the expressions of Nrf2 and HO-1 and the effects on indicators of oxidative stress, then clarify the effect of SFN by inducing Nrf2-ARE signaling pathway on liver and lung injuries triggered byⅡR in rats.
Methods:Thirty-two male Sprague Dawley rats were randomly divided into 4 groups:control, SFN control,ⅡR, and SFN+ⅡR groups (n=8 in each group). TheⅡR model was established by clamping SMA for 1 hour and reperfusion for 2 hours. Pretreatment groups at 30 minutes before ischemia, 5mg/kg SFN abdominal injection respectively, the control group carried out only laparotomy and isolation of the SMA without occlusion. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lung brochia alveolus lung fluid protein (BALFP) was measured. Liver and lung histology was investigated. Liver and lung tissue SOD, MPO, GSH and GSH-Px activity were assayed. The liver and lung Nrf2 and HO-1 were determined by immunohistochemical analysis and western blot analysis.
Results:Firstly, compared with the control group,ⅡR group:(1) Serum ALT and AST were significantly enhanced (P<0.01, P<0.01); (2) Lung BALFP was increased significantly (P<0.01); (3) Liver and lung were obvious edema, hemorrhage, and neutrophil infiltration; (4) Liver and lung SOD activities were decreased (P<0.01, P<0.05), MPO activities were increased (P<0.01, P<0.05); (5) Liver and lung GSH levels were significantly enhanced (P<0.01, P<0.05); (6) Expressions of HO-1 in the liver and lung tissues were increased (P<0.01, P<0.01); (7) Expressions of Nrf2 in the liver and lung tissues were increased (P<0.05, P<0.01). Secondly, compared with the control group, SFN control group:(1) Liver and lung GSH-Px activities increased significantly (P<0.05, P<0.01); (2) Expressions of HO-1 in the liver and lung tissue were increased (P<0.05, P<0.01); (3) Expressions of Nrf2 in the liver and lung tissue were increased (P<0.05, P<0.01). Thirdly, compared with theⅡR group, SFN+IIR group:(1) Serum ALT and AST were significantly decreased (P<0.01, P<0.05); (2) Lung BALFP was decreased significantly (P<0.05); (3) The pathological injuries of liver and lung were improved; (4) Liver and lung SOD activities were elevated significantly (P<0.01, P<0.05), MPO activities were decreased significantly (P<0.01, P<0.05); (5) Liver and lung GSH levels were significantly enhanced (P<0.05, P<0.01); (6) Liver and lung GSH-Px levels were significantly enhanced (P<0.05, P<0.01); (7) Expressions of HO-1 in the liver and lung tissue were increased (P<0.01, P<0.01); (8) Expressions of Nrf2 in the liver and lung tissue were increased (P<0.05, P<0.05).
Conclusions:ⅡR induced liver and lung injuries, characterized by the histological edema, hemorrhage, and infiltration with inflammatory cells. The injuries of liver and lung were alleviated through Nrf2-ARE signaling pathway, which is accompanied with the overexpressions of Nrf2 and HO-1 in the liver and lung tissue. The activation of Nrf2-ARE signaling pathway induced by SFN plays an important role in the protection of liver and lung injuries induced byⅡR.
PartⅢ:EGCG protects liver and lung injuries induced by intestinal ischemia reperfusion in rats
Objective:To investigate EGCG on the protection of liver and lung injuries induced byⅡR, and testify if the role of EGCG pretreatment on multiple organs dysfunction induced by IIR act as SFN through Nrf2-ARE signaling pathway. To provide more favorable theoretical basis on the development and utilization of EGCG.
Methods:Thirty-two male Sprague Dawley rats were randomly divided into 4 groups:control, EGCG control,ⅡR, and EGCG+ⅡR groups (n=8 in each group). TheⅡR model was established by clamping SMA for 1 hour and reperfusion for 2 hours. Pretreatment groups at 30 minutes before ischemia,50mg/kg EGCG abdominal injection respectively, the control group carried out only laparotomy and isolation of the SMA without occlusion. Serum levels of AST, ALT, and lung water content was assayed. Liver and lung histology was investigated. Liver and lung tissue SOD, MPO, GSH and GSH-Px activity were assayed. The liver and lung Nrf2 and HO-1 were determined by immunohistochemical analysis and western blot analysis.
Results:Firstly, compared with the control group,ⅡR group:(1) Serum ALT and AST were significantly enhanced (P<0.01, P<0.05); (2) Lung water content was increased significantly (P<0.01); (3) Liver and lung were obvious edema, hemorrhage, and neutrophil infiltration; (4) Liver and lung SOD activities were decreased (P<0.01, P<0.01), MPO activities were increased (P<0.01, P<0.01); (5) Liver and lung GSH levels were significantly enhanced (P<0.05, P<0.05); (6) Expressions of HO-1 in the liver and lung tissues were increased (P<0.01, P<0.01); (7) Expressions of Nrf2 in the liver and lung tissues were increased (P<0.05, P<0.05). Secondly, compared with the control group, EGCG control group:(1) Liver and lung GSH-Px activities increased significantly (P<0.01, P<0.01); (2) Expressions of HO-1 in the liver and lung tissue were increased (P<0.01, P<0.01); (3) Expressions of Nrf2 in the liver and lung tissue were increased (P<0.05, P<0.01). Thirdly, compared with theⅡR group, EGCG+ⅡR group: (1) Serum ALT and AST were significantly decreased (P<0.01, P<0.05); (2) Lung water content was decreased significantly (P<0.01); (3) The pathological injuries of liver and lung were improved; (4) Liver and lung SOD activities were elevated significantly (P<0.01, P<0.05), MPO activities were, decreased significantly (P<0.01, P<0.01); (5) Liver and lung GSH levels were significantly enhanced (P<0.01, P<0.01); (6) Liver and lung GSH-Px levels were significantly enhanced (P<0.01, P<0.01); (7) Expressions of HO-1 in the liver and lung tissue were increased (P<0.01, P<0.01); (8) Expressions of Nrf2 in the liver and lung tissue were increased (P<0.01,P<0.05).
Conclusions:ⅡR induced liver and lung injuries, characterized by the histological edema, hemorrhage, and infiltration with inflammatory cells. The injuries of liver and lung can be alleviated through EGCG pretreatment, which is accompanied with the overexpressions of Nrf2 and HO-1 in the liver and lung tissue. EGCG, the major component of polyphenols in green tea, can protect liver and lung injuries induced byⅡR through Nrf2-ARE signaling pathway.
We use intestinal ischemia reperfusion (ⅡR) rat model to analyze the intestine, liver and lung injuries marker (histology, serum ALT, AST, lung water content and BLAFP), tissue oxidative stress (SOD, MPO), and the expression of Nrf2-ARE pathway (Nrf2, GSH, GSH-Px and HO-1) using biochemical analysis, immunohistochemical analysis and Western blot analysis to evaluate the role of Nrf2-ARE signaling pathway on the protective effect ofⅡR and the effect of SFN, EGCG pretreatment to liver and lung injuries induced byⅡR.
The result showed as follows:Intestinal ischemia reperfusion (ⅡR) not only leads to intestinal damage itself, but also causes severe destruction of remote organs, characterized by the histological edema, hemorrhage and infiltration with inflammatory cells, significant increasing of ALT, AST, lung water content, and BLAFP. Intestine, liver and lung SOD and MPO levels changed markedly inⅡR group. Pretreatment with SFN or EGCG, the intestine, liver and lung injuries were improved, which is accompanied with the level of tissue Nrf2. At the meantime, phase 2 antioxidant and detoxification enzymes increased markedly. The Nrf2-ARE signaling pathway has been shown to have beneficial effects on the prevention ofⅡR. This also demonstrate that EGCG, which is the major component of polyphenols in green tea, plays an important role in the induction of Nrf2-ARE signaling pathway, and the protection of liver and lung injuries induced byⅡR.
引文
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