【作者】 张鑫；

【导师】 蔡国君；

【作者基本信息】 第二军医大学 ， 药理学， 2012， 硕士

【摘要】 研究目的：感染性休克是由微生物及其代谢产物在机体内引起微循环障碍，导致细胞与器官代谢和功能损害的全身反应性综合征。当机体遭受微生物毒素侵入时，机体会分泌大量的炎症介质，如肿瘤坏死因子-α (TNF-α)、白介素-1(IL-1)、白介素-6(IL-6)以及一氧化氮(Nitric oxide, NO)等，导致机体血液循环遭到破坏，产生低血压，激活凝血途径等，最后致使器官损害甚至衰竭。目前，针对感染性休克的治疗方法有很多，包括积极治疗原发病，补充血容量，给予血管活性物质以及抗生素，拮抗细胞因子防治细胞损伤，甚至血液透析以保护重要器官功能防治衰竭等。尽管其病理过程以及其治疗措施已经取得了很大的进展，但是未能有效的应用于临床，如细胞因子拮抗剂或单克隆抗体在动物实验都能取得一定的效果，但其临床试验结果却令人失望；以及其高昂的治疗费用，愈后病人的生活质量等问题的存在，致使感染性休克成为非心血管重症监护病房的主要死因，死亡率高达40%-75%，并且每年都不断上升。因此，寻找新的行之有效的治疗方法以及其具体的作用机制就变得非常重要。酮色林(Ketanserin)，作为一种5-羟色胺2A (5-hydroxytryptamine2A,5-HT2A)受体拮抗剂，大剂量时，对α1受体有阻断作用。因其具有良好的降压作用，在临床上一直被用来治疗高血压。近几年，研究者发现了5-HT2A在炎症反应和免疫反应中的作用，已在动物模型上研究了其拮抗剂对糖尿病肾病、关节炎等治疗作用。本课题组在教研室前期的研究基础上，已经证实了酮色林对内毒素休克有着良好的治疗作用，但是其具体的分子机制还未见报道。一氧化氮具有许多生物活性，一度成为研究者关注的焦点，许多研究已经证实了NO在感染性休克中的病理意义，是导致感染性休克病人低血压的关键分子。体内NO合成的基本底物是L-精氨酸(L-arginine)与O2，在一氧化氮合酶(nitric oxide synthase,NOS)的催化作用下生成NO与L-瓜氨酸(L-citrulline)。NOS又三种同工酶：主要存在于内皮细胞的eNOS (endothelial nitric oxide synthase)，存在于神经细胞的nNOS (neuronalnitric oxide synthase)，以及存在于巨噬细胞、角质细胞中等的可诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)。eNOS和nNOS统称为cNOS (constitutive nitricoxide synthase)。在感染性休克中，研究发现细菌毒素可诱导iNOS表达，进而产生千倍于生理基础的NO。这些过量的NO可激活平滑肌细胞内的可溶性鸟苷酸(sGC)，持续产生大量的cGMP，舒张血管平滑肌，增加细胞通透性，降低血管对收缩因子的反应性，使血压更加降低，也可抑制心肌收缩力，加重休克。所以，降低NO含量应可成为治疗感染性休克的一种有效的方法。关于感染性休克病理过程的研究非常多也很深入，其中涉及其中的有核因子kappa B (nuclear factor kappa B, NF-κB)和丝裂原活化蛋白激酶(mitogen-activatedprotein kinases, MAPKs)等。因此，我们将酮色林内毒素休克的分子机制作为研究重点，提出假设：酮色林通过抑制iNOS表达，达到治疗内毒素休克的目的，其中涉及的信号转导分子有NF-κB和MAPKs。研究方法：本研究首先以内毒素诱导的休克小鼠为模型，利用整体动物实验方法研究酮色林对内毒素诱导的iNOS蛋白表达的作用，使用Western blotting和免疫组织化学的方法对iNOS表达进行检测；以及用Griess reagent法检测体内NO水平的变化。其次，利用小鼠巨噬细胞系(RAW264.7细胞)，检测酮色林对内毒素刺激诱导其iNOS蛋白质和mRNA水平的影响，分别使用Western blotting法和RT-PCR法；并用ELISA法检测酮色林对其炎症因子(TNF-α、IL-6)水平的影响。最后，我们在RAW264.7细胞上研究酮色林抑制内毒素诱导iNOS表达的具体分子机制，观察5-HT2A、NF-κB以及MAPKs在整个过程发挥的作用，分别使用工具药研究方法和分子生物学检测方法。结果：1.以内毒素诱导的休克小鼠为模型，分别给予不同剂量(3mg/kg、10mg/kg)酮色林，检测其对iNOS表达以及NO水平的影响。结果显示酮色林在不同剂量时，可不同程度iNOS表达的抑制，降低NO水平，其中10mg/kg剂量组作用更为明显。2.在RAW264.7细胞上，给予内毒素(100ng/ml)刺激，共同孵育酮色林(10μM)，不同时间(0-12h)检测其对iNOS蛋白表的作用，实验结果显示在内毒素诱导iNOS表达的初期可以明显抑制其表达，其中8小时作用最为明显；进一步，我们观察了不同剂量(0-10μM)酮色林对iNOS蛋白质和mRNA水平的影响，实验结果显示酮色林以剂量依赖性的抑制iNOS的表达。3.在RAW264.7细胞上，直接观察酮色林对内毒素刺激产生NO水平的作用。不同剂量(1μM、10μM)酮色林与内毒素(100ng/ml)共同作用8小时，采用流式细胞仪检测NO探针荧光的方法，发现酮色林可不同程度降低NO产生水平。4.在RAW264.7细胞上，发现酮色林可剂量依赖性和时间了依赖性地降低内毒素刺激后炎症因子分泌的水平。5.在RAW264.7细胞上，发现酮色林抑制内毒素刺激iNOS表达是通过拮抗5-HT2A受体实现的。分别使用5-HT2A受体和α1受体特异性拮抗剂，观察其对内毒素刺激iNOS表达的作用，实验结果显示，利坦色林对iNOS有抑制作用，但是哌唑嗪无抑制作用。6.在RAW264.7细胞上，使用内毒素刺激，我们检测了酮色林对MAPKs磷酸化程度的影响，实验结果显示酮色林可以抑制ERK1/2的磷酸化程度，对p38、JNK无明显进一步使用ERK1/2抑制剂(PD98059)，发现酮色林抑制iNOS表达的作用取消了。因此，酮色林通过抑制ERK1/2的磷酸化抑制的iNOS表达水平。7.在RAW264.7细胞上，使用内毒素刺激，我们还检测了酮色林对NF-κB的影响，不同剂量(10μM、100μM)酮色林都对NF-κB活性有抑制作用。结论：酮色林通过拮抗5-HT2A受体，抑制ERK1/2的磷酸化，从未抑制iNOS表达，降低体内NO水平，达到治疗内毒素休克的目的。更多还原

【Abstract】 Backgrounds and Objectives: Sepsis is a systemic inflammatory response to thepresence of infection, mediated via the production of many cytokines, including tumournecrosis factor α (TNF-α), interleukin (IL)-6, and IL-1, as well as nitric oxide (NO) whichcauses changes in the circulation, increases endothelial permeability and actives thecoagulation pathways. These mechanisms play roles during septic shock, but on a systemicscale, leading to diffuse endothelial disruptior vascular permeability, vasodilation, andthromnosis of end-organ capillaries. Endothelial damage itself can further activateinflammatory and coagulation cascades, creating a positive feedback loop, and leading tofurther endothelial and end-organ damage. Treatment of septic shock is based on support oforgan perfusion with fluids, administering vasoactive agents and antibiotics. Although, thepathophysiology of septic shock and the process of inflammation are understand moredeeply, the mortality rates are16%with sepsis,20%with severe sepsis, and46%withseptic shock. Therefore, it is important to find new strategies for septic shock treatment andaccurate mechanism.Ketanserin, an antihypertensive drug with a central sympathoinhitory action, is aselective5-HT2Areceptor antagonist with additional α1-adrenoceptor-blocking prooerties.Studies have demonstrated that ketanserin effectively lowers blood pressure (BP),decreases blood pressure variability (BPV) and enhances BRS in SHR. However, clinicalapplication of ketanserin was not limited to anti-hypertension. Research demonstrated thatintracoronary administration of ketanserin augments coronary collateral flow and decreasesmyocardial ischemia during balloon angioplasty. This could be of clinical significance inthe management of acute ischemic syndromes. Another clinical research showed thattreated with2%ketanserin ointment for8weeks, observed a significant decrease inrelative wound area compared with the placebo group in chronic leg ulcers patients. Theseresults indicate that ketanserin is a valuable therapy for difficult-to-treat leg ulcers. Thiseffect of ketanserin is through blocked of5-HT2Areceptor and improved microcirculation.Our previous study demonstrated that ketanserin could protect the organ damage in hypertension by restoring arterial baroreflex function. Furthermore, we had demonstratedthat ketanserin has a beneficial effect in septic shock through restoration of baroreflexfunction.Nitric oxide (NO) is one of many vasoactive substances released, from a variety ofcells, under conditions of endotoxaemia and sepsis. Under physiological conditions it isproduced by two constitutive calcium-dependent enzymes (nitric oxide synthase; NOS) inneurons (nNOS) and endothelial cells (eNOS) and has functions ranging fromneurotransmission and vasodilatation to inhibition of platelet adhesion and aggregation.Following bacterial infection, especially with Gram-negative organisms, the formation ofNO from L-arginine is enhanced due to the induction of a NOS enzyme (iNOS) in cardiacmyocytes and vascular smooth muscle cells. Studies ascertained that the excess NO formedby iNOS resulted in the pathophysiology of sepsis shock, including haemorrhage,vasodilation, intravascular coagulation, and hypotension.When infection occurs, many signaling molecules are activated, such as nuclearfactor-κB (NF-κB), cyclic AMP responsive element-binding protein (CREB) andmitogen-activated protein kinases (MAPKs) family, which are classified into at least threecomponents: extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK),and p38MAPK. Recaent studies have implicated that NF-κB and MAPKs induce therelease of immune-related cytotoxic factors such as iNOS, COX-2, and proinflammatorycytokines.Considering the critical role of iNOS in the sepsis shock, we hypothesized thatketanserin cures the sepsis shock via inhibiting the expression of iNOS. Furthermore, weexplained the molecular mechanism.Methods: The study was based on the model of endotoxin-induced shock mice andRAW264.7stimulated by LPS. Kunming mice were injected with LPS (30mg/kg, i.p.) toinduce septic shock. Ketanserin was administered (3mg/kg,10mg/kg, i.p.) immediatelyafter injuected with LPS for12hours. The expression of iNOS was detected by Westernblot and immunohistochemistry. The level of NO was measured by Griess reagent. RAW264.7cells were pre-treated with indicated ketanserin for10minutes, then stimulatedwith LPS (100ng/ml) for indicated time. Then, the expression of iNOS protein andMAPKs were detected using Western blot. The level of iNOS mRNA was measured byreal-time PCR. The cells were treated with different dosage of ritanserin or prazosin for10minutes and stimulated with LPS (100ng/ml) for8hours in order to measure the role onthe expression of iNOS. Moreover, the activity of NF-κB was measured using EMSA.Results：1. Kunming mice were injected with LPS (30mg/kg, i.p.) to induce septic shock,then injected ketanserin (3mg/kg,10mg/kg, i.p.) immediately after injected withLPS for12hours. Results showed that ketanserin could both inhibited theexpression of iNOS protein and decreased the level of NO, especially the dosge of10mg/kg.2. RAW264.7cells were pre-treated with ketanserin (10μM) for10min andstimulated with LPS (100ng/ml) for indicated time (0-12h).The expression ofiNOS was inhibited at the early time period of the iNOS induced, especially at8hours after stimulated with LPS. Furthermore, RAW264.7cells were pre-treatedwith ketanserin (0.1-10μM) and stimulated with LPS (100ng/ml) for8hours.Results show that ketanserin could inhibit the expression of iNOS and the level ofNO dose-inpendently3. RAW264.7cells were pre-treated with ketanserin (1μM,10μM) for10min andstimulated with LPS (100ng/ml) for5hours. The level of iNOS mRNA wasdecreased by ketanserin significantly.4. RAW264.7cells were pre-treated with ketanserin (0.1-10μM) for10minutes andstimulated with LPS (100ng/ml) for indicated time. The level TNF-α and IL-6were significantly decreased by ketanserin.5. RAW264.7cells were pre-incubated with ritanserin (0-10μM) or prazosin(0-1μM) for10minutes, then stimulated with LPS (100ng/ml) for8hours. Theexpression of iNOS was examined by western blotting. Ritanserin, a specific antagonist of5-HT2A receptor, could significantly inhibit the expression of iNOSin RAW264.7cells stimulated with LPS. However, prazosin which is a specificantagonist of α1receptor, failed to inhibit the expression of iNOS.6. RAW264.7cells were pre-treated with ketanserin (10μM) for10min andstimulated with LPS (100ng/ml) for indicated time (0-2hours). The effect ofketanserin on the phosphorylation of MAPKs was detected by Western blot. Theresults showed ketanserin could inhibit the phosphorylation of ERK1/2, while hadno effect on that of p38and JNK. Moreover, RAW264.7cells were pre-treatedwith ketanserin (10μM) or PD98059(40μM) for10min and stimulated withLPS (100ng/ml) for8h. The effect of ketanserin on iNOS was canceled.7. RAW264.7cells were pre-treated with ketanserin (10μM,100μM) for10minutes and stimulated with LPS (100ng/ml) for8hours. Result showed that theactivity of NF-κB was inhibited by ketanserin.Conclusion: Ketanserin could bind with5-HT2Areceptor and inhibit iNOS pathwaythrough ERK1/2signaling pathway.更多还原