α7nAChR在挤压综合征中的作用研究

发布时间：2018-03-01 10:46

本文关键词： α7nAChR 挤压综合征山莨菪碱高K+血症雌二醇胰岛素敏感性　出处：《第二军医大学》2017年博士论文　论文类型：学位论文

【摘要】：研究目的挤压伤指的是躯干或四肢受到挤压,导致被挤压部位肌肉肿胀和/或神经功能失常。挤压伤多发生于地震、车祸等。挤压综合症则是指当挤压时间过长,被挤压部位肌肉大量坏死,当解除挤压后,大量的细胞内物质从坏死肌肉释放入血,导致代谢性酸中毒、高血K~+症、以及其他代谢性紊乱。同时坏死的肌肉组织吸收大量的水分,导致水钠在局部潴留,从而引发低血容量性休克。挤压综合症患者如果不及时治疗,后期会出现肾衰竭。大地震时,挤压综合征的发生率约为2~15%,是除了外伤以外导致死亡最主要的原因。高达20%的挤压综合症患者死因是高血K~+症引起的心律失常或者低血容量性休克。但是因为院前挤压综合征的诊断比较难,临床缺乏降低挤压综合征患者早期死亡安全有效的药物。山莨菪碱是一种传统的抗休克药物,可以提高血液的微循环,临床上多用于治疗伴有血流障碍性的疾病,比如脓毒性休克和弥散性血管内凝血障碍。我们既往的研究发现山莨菪碱的抗休克作用主要是通过作用于M受体,使乙酰胆碱更多的和α7nAChR相结合,提高血压而不改变心率,从而发挥抗炎抗休克作用。胰岛素在临床上和葡萄糖合用,可以通过激活Na+/K~+-ATPase促进血K~+进入细胞而降低血K~+浓度。有研究报道N受体激动剂尼古丁可以激活α7nAChR进而提高胰岛素的敏感性,那么山莨菪碱能否通过α7nAChR的激活用于挤压综合征的预防给药或者治疗呢?另外文献报道挤压伤发生时,高K~+血症主要见于成年男性,而E_2可以降低血清K~+浓度。E_2可以通过作用于胰岛素信号通路提高心肌细胞Na~+/K~+-ATPase的表达和活性。但是,挤压综合征中E_2和高K~+血症,E_2和胰岛素敏感性的关系未见报道。本实验的目的在于通过一系列的实验探索:1、α7nAChR激活对挤压综合征死亡率的影响及其机制研究;2、E_2在α7nAChR激活对挤压综合征的预防作用中扮演的角色。研究方法1、山莨菪碱对挤压综合征大鼠死亡率及血生化和电解质指标的影响用SD大鼠制做挤压综合征模型。正常组:生理盐水;对照组:挤压+生理盐水;预防给药组:解除挤压前30min给予山莨菪碱;治疗给药组:解除挤压后1h给予山莨菪碱。解除挤压后观察SD大鼠24h死亡率。另外造模,解除挤压后6h眶静脉丛取血,静置、离心得到血清,检测血清K~+、Na~+、Cl~-、Scr、CK、CK-MB和BUN水平。2、山莨菪碱对挤压综合征大鼠胰岛素敏感性的影响用SD大鼠制做挤压综合征模型。正常组:生理盐水;对照组:挤压+生理盐水;预防给药组:解除挤压前30min给予山莨菪碱。解除挤压后6h眶静脉丛取血,静置、离心得到血清,检测血清胰岛素和葡萄糖水平。3、α7nAChR参与了山莨菪碱对挤压综合征死亡率、血清K~+和胰岛素敏感性的作用工具药:α7nAChR激动剂PUN和抑制剂MLA。用C57BL/6小鼠制做挤压综合征模型。对照组:造挤压综合征模型并给予等量的生理盐水;预防给药组:解除挤压前30min给予山莨菪碱。MLA组:解除挤压前30min给予MLA;山莨菪碱+MLA:解除挤压前1h给予MLA,解除挤压前30min给予山莨菪碱;PUN组:解除挤压前30min给予PUN。解除挤压后观察C57BL/624h死亡情况。另外同样的五组,解除挤压后6h取血,静置、离心得到血清,检测血清K~+,胰岛素和葡萄糖水平。同时检测正常小鼠血清K~+,胰岛素和葡萄糖水平。4、用α7nAChR-/-小鼠验证α7nAChR参与了山莨菪碱对挤压综合征死亡率、血清K~+和胰岛素敏感性的作用用α7nAChR+/+和α7nAChR-/-小鼠制做挤压综合征模型。野生型和纯合子各分为两组,对照组:造挤压综合征模型并给予等量的生理盐水;预防给药组:解除挤压前30min给予山莨菪碱。解除挤压后6h取血,静置、离心得到血清,检测血清K~+,胰岛素和葡萄糖水平。另外同样的四组,解除挤压后观察24h死亡情况。5、山莨菪碱对高K~+血症大鼠死亡率和血清K~+的作用对照组:生理盐水,30min后给予KCl;实验组:给予山莨菪碱,30min后给予同样的KCl。30min后取血,检测血清K~+浓度。同样的另外两组观察24h死亡情况。6、α7nAChR参与了山莨菪碱对细胞外高K~+的作用成肌细胞分成五组:山莨菪碱组,乙酰胆碱组,山莨菪碱+乙酰胆碱组,尼古丁组和PUN组。在细胞培养基中加入外源性K~+造高K~+模型。给药时间在加入K~+之前30min。K~+加入6h后取培养基检测K~+和葡萄糖浓度。同样的用n受体抑制剂Mec和除α7nAChR以外的N受体阻断剂hex探究α7nAChR的作用。7、胰岛素信号通路介导山莨菪碱降低细胞外高K~+的作用HNMPA-(AM)3:胰岛素受体抑制剂。C2C12骨骼肌成纤维细胞分成五组:正常组,对照组,山莨菪碱+乙酰胆碱,HNMPA-(AM)3和山莨菪碱+乙酰胆碱+HNMPA-(AM)3组。免疫荧光检测HNMPA-(AM)3对Na~+/K~+-ATPase在细胞膜表面分布的影响。使用胰岛素受体抑制剂HNMPA-(AM)3、mTOR抑制剂雷帕霉素、pi3k抑制剂LY294002、转录活化因子Stat3抑制剂Stattic和Na~+/K~+-ATPase抑制剂哇巴因探究胰岛素/Na~+/K~+-ATPase通路对细胞外高K~+的作用。8、挤压综合征死亡率、雌激素、胰岛素敏感性和血清K~+的性别差异雌雄小鼠各半造挤压综合征模型,观察24h小鼠死亡情况。另外造模,解除挤压后6h取血,测量雌雄小鼠E_2、血清K~+、胰岛素和葡萄糖水平。9、α7nAChR的激活对挤压综合征小鼠血清K~+和E_2的影响工具药:α7nAChR抑制剂MLA。用C57BL/6小鼠制做挤压综合征模型。对照组:造挤压综合征模型并给予等量的生理盐水;预防给药组:解除挤压前30min给予山莨菪碱。MLA组:解除挤压前30min给予MLA;山莨菪碱+MLA:解除挤压前1h给予MLA,解除挤压前30min给予山莨菪碱。解除挤压后6h取血,静置、离心得到血清,检测血清K~+浓度、胰岛素和葡萄糖水平。同时检测正常小鼠血清K~+浓度、胰岛素和葡萄糖水平。10、卵巢摘除术对挤压综合征小鼠血清K~+、E_2和胰岛素敏感性的影响假手术组和手术组各分成四组:正常组、挤压组、山莨菪碱组和E_2组。后三组均挤压。取血,检测血清K~+和E_2水平。另外假手术组和手术组各再造模分组:挤压组、山莨菪碱组和E_2组。11、E_2对挤压综合征小鼠死亡率的影响假手术组和手术组各再造模分组:挤压组、山莨菪碱组和E_2组。监测解除挤压后24h死亡情况。12、探究山莨菪碱对挤压综合征大鼠血压的影响以及α7nAChR在其中发挥的作用SD大鼠分成五组:正常组、对照组、MLA组、山莨菪碱组和MLA+山莨菪碱组。后四组接受挤压。解除挤压前,各组先测量一段大鼠血压和心率,解除挤压后持续监测3.5h再停止测量。记录分析。实验结果1、和不给药组相比,解除挤压前30min和解除挤压后1h给予山莨菪碱可以显著降低挤压综合征大鼠24h死亡率以及挤压综合征大鼠血清K~+、scr、ck、ck-mb和bun水平2、和不给药组相比,给予山莨菪碱可以提高挤压综合征大鼠胰岛素敏感性。3、山莨菪碱降低挤压综合征小鼠24 h死亡率的作用可以被α7nAChR抑制剂MLA阻断。而α7nAChR激动剂PNU可以显著降低挤压综合征小鼠24 h死亡率。PNU提高挤压综合征小鼠胰岛素敏感性,MLA降低挤压综合征小鼠胰岛素敏感性。4、山莨菪碱可以降低野生型挤压综合征小鼠24 h死亡率,但是对纯合子挤压综合征小鼠24 h死亡率没有显著作用。5、山莨菪碱预防给药可以通过降低血K~+降低高血K~+症大鼠死亡率。6山莨菪碱单用、山莨菪碱和乙酰胆碱合用均可降低细胞外高K~+和葡萄糖浓度。山莨菪碱和乙酰胆碱合用比山莨菪碱单用效果更明显。Mec和MLA可以阻断这一作用。尼古丁和PNU单用均可以降低细胞外高K~+和葡萄糖浓度。7、山莨菪碱+乙酰胆碱使更多的Na+/K~+-ATPase磷酸化,而胰岛素受体抑制剂HNMPA-(AM)3可以阻断这一作用。HNMPA-(AM)3、雷帕霉素、LY294002、Stattic和哇巴因可阻断山莨菪碱和乙酰胆碱降低细胞外高K~+的作用。8、挤压综合征雌性小鼠和雄性小鼠死亡率差异无统计学意义,但是挤压以后雌性小鼠血清K~+低于雄性小鼠,E_2水平高于雄性小鼠,胰岛素敏感性高于雄性小鼠。9、α7nACh R的激活可以降低挤压综合征小鼠血清K~+浓度及提高血清E_2的浓度。10、卵巢摘除术可以提高挤压综合征小鼠血清K~+浓度,降低胰岛素敏感性,降低血清E_2浓度。11、E_2可以降低挤压综合征小鼠死亡率。12、山莨菪碱升高挤压综合征大鼠血压,MLA可以阻断山莨菪山莨菪碱的这一作用。山莨菪碱对挤压综合征大鼠心率没有影响。研究结论:α7nAChR的激活可以降低挤压综合症早期死亡率,这种作用部分是通过胰岛素/Na+/K~+-ATPase信号通路介导的血K~+降低来实现的,并且雌激素在其中发挥重要作用;部分作用可能是通过升高血压来实现的。激活α7nAChR的药物(如山莨菪碱)以及E_2,有望成为降低挤压综合症早期死亡率的有效药物。
[Abstract]:The purpose of the study is to crush injury refers to the trunk or limbs caused by extrusion, extrusion parts of muscle swelling and / or nerve dysfunction. Crush injuries occurred in earthquake accident. Crush syndrome refers to when the extrusion time is too long, crushed muscle necrosis, when lifting pressure, a lot of material within the cell release from necrotic muscle into the blood, leading to metabolic acidosis, high blood K~+ disease and other metabolic disorders. At the same time, necrosis of muscle tissue to absorb a large amount of water, resulting in the local water and sodium retention, causing hypovolemic shock. Crush syndrome patients without treatment, the latter will appear kidney failure. Earthquake, crush syndrome incidence is about 2~15%, in addition to outside trauma resulting in the death of the main reasons. The causes of death in patients with crush syndrome is high up to 20% K~+ blood diseases caused by arrhythmia or low blood volume Shock. But because the pre hospital diagnosis of crush syndrome is difficult, lack of clinical lower crush syndrome early death in patients with safe and effective drugs. Anisodamine is a traditional anti shock drug can improve the blood microcirculation, clinical for the treatment of many diseases associated with blood disorders, such as septic shock and diffusion intravascular coagulation disorders. Our previous studies found that anisodamine anti shock function is mainly mediated by M receptors, make more acetylcholine and alpha 7nAChR combined with the increase in blood pressure without changing heart rate, so as to exert anti-inflammatory anti shock effects. Insulin in clinical and can reduce blood glucose, the concentration of K~+ by the activation of Na+/K~+-ATPase promotes blood K~+ into cells. Studies have reported that N receptor agonist nicotine can activate alpha 7nAChR and improve insulin sensitivity, so anisodustanguticus Alkali can through the activation of alpha 7nAChR for prevention of crush syndrome to medicine or treatment? Also reported crush injury occurs, high K~+ hyperlipidemia mainly in adult males, while E_2 can reduce the concentration of serum K~+.E_2 can act on the insulin signaling pathway to improve the expression and activity of Na~+/K~+-ATPase in myocardial cell. However, E_2 and high K~+ hyperlipidemia crush syndrome, E_2 and insulin sensitivity is not reported. The purpose of this experiment is that through a series of experiments: 1, alpha 7nAChR activation effect on mortality of crush syndrome and its mechanism study; 2, E_2 in alpha 7nAChR activation on play preventive effect of crush syndrome in the role. Study on the 1 methods, the model effect of anisodamine on crush syndrome rat mortality and blood biochemical and electrolyte index for SD rats made of crush syndrome. Normal group: saline control group: Extrusion + saline; preventive treatment group: treated with Anisodamine before pressure release 30min; treatment group: treated with Anisodamine 1h after pressure release. Release of SD rats was observed. In addition, the mortality of 24h after extrusion molding, extrusion after release 6h orbital venous plexus blood, static, centrifuged to obtain serum detection serum K~+, Na~+, Cl~-, Scr, CK, CK-MB and BUN level of.2, the protective effects of anisodamine on insulin sensitivity in rats with crush syndrome with SD rat model made of crush syndrome. Normal group: normal saline; control group: extrusion + saline; preventive treatment group: in addition to 30min prior to extrusion solution anisodamine. Lift after extrusion 6h orbital venous plexus blood, static, centrifuged to obtain serum, serum glucose and insulin levels of.3 alpha, 7nAChR in anisodamine on mortality of crush syndrome, serum K~+ and function of insulin sensitivity: 7nAChR alpha agonist and inhibitor PUN Model C57BL/6 mice made MLA. crush syndrome. Control group: saline made crush syndrome model and given the same amount; drug prevention group: 30min before pressure release anisodamine group.MLA: remove extrusion 30min prior to MLA; anisodamine +MLA: pressure release 1H prior to MLA, 30min treated with Anisodamine before pressure release; group PUN: pressure release given before 30min PUN. released C57BL/624h observation of death after extrusion. In the same five groups, 6h after blood pressure release, static, centrifuged to obtain serum, serum K~+, insulin and glucose levels. At the same time detection of normal mice serum K~+, insulin and glucose levels of.4 alpha 7nAChR-/- mice alpha 7nAChR is involved in the verification of anisodamine on mortality of crush syndrome, model with a 7nAChR+/+ and a 7nAChR-/- mouse made serum K~+ and insulin sensitivity in the role of crush syndrome in wild type and pure. Zygotes were divided into two groups, control group: saline made crush syndrome model and given the same amount; drug prevention group: 30min treated with Anisodamine before pressure release. After 6h blood pressure release, static, centrifuged to obtain serum, serum K~+, insulin and glucose levels. The other four groups of the same lift, after extrusion to observe 24h death.5. The effect of anisodamine on high K~+ hyperlipidemia rat mortality and serum K~+ in control group: normal saline, 30min after administration of KCl; experimental group: treated with anisodamine, 30min after giving the same KCl.30min blood serum K~+ concentrations were detected. The same in two groups 24h the death of.6, alpha 7nAChR in anisodamine on high extracellular K~+ function into muscle cells were divided into five groups: anisodamine group, acetylcholine group, anisodamine + group acetylcholine, nicotine group and PUN group. In the cell culture medium with exogenous K~+ high K + model. Time of administration before joining K~+ 30min.K~+ after joining 6h based detection of K~+ and glucose concentration in culture. The same with the N receptor inhibitor Mec and besides alpha 7nAChR N receptor blocking agent hex.7 explore alpha 7nAChR, insulin signaling pathway mediated by anisodamine reduce the effects of extracellular K~+ (HNMPA- high AM) 3: insulin receptor inhibitor.C2C12 in skeletal muscle fibroblasts were divided into five groups: normal group, control group, anisodamine + acetylcholine, HNMPA- (AM) 3 and anisodamine + +HNMPA- acetylcholine (AM) 3 groups. Immunofluorescence detection of HNMPA- (AM) 3 to Na~+ /K~+-ATPase in influencing the distribution of cell membrane. Insulin receptor inhibitor HNMPA- (AM) 3, mTOR inhibitor rapamycin, PI3K inhibitor LY294002, Stat3 inhibitor and activator of transcription factor Stattic inhibitor Na~+/K~+-ATPase /Na~+/K~+-ATPase on insulin ouabain on high extracellular K~+ The role of.8, crush syndrome mortality, estrogen, male and female mice were sex differences in insulin sensitivity and serum K~+ of crush syndrome model, to observe the 24h model mice death. In addition, 6h after blood pressure release, measurement of male and female mice E_2, serum K~+, insulin and glucose levels.9, activation of alpha 7nAChR drug effects on serum K~+ and E_2 mice of crush syndrome: model C57BL/6 mice produced alpha 7nAChR inhibitor MLA. crush syndrome. Control group: saline made crush syndrome model and given the same amount; drug prevention group: 30min before pressure release anisodamine group.MLA: remove extrusion 30min prior to MLA; anisodamine +MLA: pressure release 1H prior to MLA, 30min treated with Anisodamine before pressure release. After 6h blood pressure release, static, centrifuged to obtain serum, serum K~+ concentration, insulin and glucose levels at the same time. Detection of normal mice serum K~+ concentrations, insulin and glucose levels.10, enucleation on serum K~+ crush syndrome ovary, E_2 and insulin sensitivity of the sham operation group and operation group were divided into four groups: normal group, crush group, anisodamine group and E_2 group. The three groups after extrusion. Blood serum the K~+ and E_2 level detection. In sham operation group and operation group, model group: extrusion reconstruction group, anisodamine group and E_2 group of.11, the effect of E_2 on the mortality of mice with crush syndrome in sham operation group and operation group the model group: reconstruction crush group, anisodamine group and E_2 group. The monitoring release after extrusion 24h the death of.12, SD rats to explore the influence of anisodamine on blood pressure in rats of crush syndrome and alpha 7nAChR in the play is divided into five groups: normal group, control group, MLA group, MLA+ group and anisodamine anisodamine group. The four groups after extrusion. Lift the squeeze The pressure before the first measurement of a rat heart rate and blood pressure, remove stop measuring continuous monitoring of 3.5H extrusion. The experimental results were recorded and analyzed. 1, compared with no drug group, pressure release 30min before and after removal of crush 1H treated with Anisodamine can significantly reduce the mortality of 24h rats with crush syndrome and serum K~+. Crush syndrome rat SCR, CK, CK-MB and BUN levels of 2, compared with no drug group, treated with Anisodamine can improve the insulin sensitivity of.3 rats with crush syndrome, anisodamine reduced mice crush syndrome 24 h mortality effect can be a 7nAChR inhibitor MLA and 7nAChR alpha agonist PNU could significantly to reduce the mortality rate of 24 h mice of crush syndrome.PNU improves insulin sensitivity in mice crush syndrome, MLA syndrome mice reduce extrusion.4 insulin sensitivity, anisodamine can reduce the wild type crush syndrome mice 24 h death Rate, but significant.5 has no effect on mice homozygous crush syndrome 24 h mortality, anisodamine prophylactically can reduce blood K~+ reduce the level of blood K~+ in the rat mortality.6? Anisodamine alone, anisodamine and acetylcholine in combination can reduce the extracellular K~+ and high glucose concentration combined with Anisodamine and acetylcholine. Anisodamine than single effect more obvious.Mec and MLA can block the effect of nicotine. And PNU could both reduce the extracellular K~+ and high glucose concentration.7, anisodamine + acetylcholine phosphorylates Na+/K~+-ATPase more, and insulin receptor (AM) inhibitor HNMPA- 3 can inhibit the effect of.HNMPA- (AM) 3 rapamycin, LY294002, Stattic, and the resulting block wow anisodamine and reduce the effects of acetylcholine.8 high K~+ cells, crush syndrome and mortality in female mice and male mice had no statistical difference Learn the meaning, but after extrusion female mice serum K~+ was lower than that of male mice, E_2 level is higher than that of male mice, insulin sensitivity is higher than that of male mice.9 alpha 7nACh activation of R can reduce the crush syndrome in mice serum K~+ concentration and higher.10 concentration of serum E_2, ovariectomy can increase the serum K~+ concentration of crush syndrome, reduce insulin the sensitivity of serum concentration of E_2.11, E_2 can reduce the mortality of mice.12 crush syndrome, anisodamine increased blood pressure in rats of crush syndrome, MLA can inhibit the action of anisodamine anisodamine. Anisodamine has no effect on heart rate in rats with crush syndrome. Conclusion: alpha activation of 7nAChR can reduce the early mortality of extrusion this syndrome, partly through insulin /Na+/K~+-ATPase signaling mediated by decreased serum K~+ to achieve, and estrogen plays Some important effects may be achieved by increasing the blood pressure. Activation of alpha 7nAChR drugs (such as anisodamine) and E_2 is expected to become an effective drug to reduce the early mortality of crush syndrome.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R96

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