右美托咪定在脓毒症大鼠发挥肝脏保护作用的机制研究

发布时间：2018-09-01 17:07

【摘要】：右美托嘧啶(dexmedetomidine,Dex),一种高选择性的α2肾上腺素能受体激动剂,因其具有镇静、镇痛、抑制交感系统活性且不伴呼吸抑制等优势,从而在ICU患者应用中广受青睐。前期有研究证实:右美托嘧啶可明显提高脓毒症患者及大鼠的生存率,同时可通过抗炎,抑制氧化应激等机制发挥器官保护作用,但关于其在脓毒症时是否可发挥肝脏保护作用与相关具体机制,尚无结论性证据研究。且有大量的基础及临床研究发现,肝脏的功能状态可直接影响脓毒症患者的生存率及预后,而脓毒症时以坏死为主要类型的肝细胞的大量死亡是导致肝脏功能损伤的主要因素之一。另外,一种新的可调控的细胞死亡类型--necroptosis被证实参与到了众多炎性疾病过程中来,另有实验证实特异性的敲除RIP3-necrptosis的重要通路蛋白-可在脓毒症时减少肝脏中炎症细胞的浸润,从而发挥肝脏保护作用。因此,本研究旨在探讨右美托嘧啶是否可在脓毒症大鼠中发挥肝脏保护作用,以及这种保护作用与抑制necroptosis的发生是否相关。第一部分:不同剂量右美托嘧啶对脓毒症大鼠肝脏功能的不同作用目的:通过观察不同剂量右美托嘧啶对脓毒症大鼠肝脏细胞死亡的不同影响,初步探讨其最佳剂量和作用机制。方法:SPF级健康雄性SD大鼠80只,体重200~220g,6~8周龄,于尾静脉置管后按照随机数字表法将其随机分为5组(n=16):假手术组(Sh组),脓毒症组(S组),高剂量Dex组(H组),中剂量Dex组(M组)和低剂量Dex组(L组)。其中每组抽取10只观察24h生存率。S、H、M及L组采用CLP方法制备大鼠脓毒症模型。Sh组仅暴露盲肠,而不进行结扎或穿孔。H,M和D组分别于术前经尾静脉置管以5.0μg/kg/h、2.5μg/kg/h、1.0μg/kg/h的剂量持续输入Dex1h,Sh和S组持续输入等体积生理盐水。分别于术后6,12和24h经股动脉和尾静脉取动脉血和静脉血,检测动脉血气(ABG),血清AST,ALT水平。于术后24h处死大鼠取肝脏,进行HE及TUNEL染色观察肝脏形态学及细胞死亡情况,同时检测肝脏中IL-6和TNF-α含量。结果:与Sh组相比,凡是接受了CLP手术的大鼠,肝酶水平、乳酸含量以及IL-6和TNF-α均增加(P均0.05),PaO_2明显降低(P均0.05),肝脏形态学也有不同的病理改变,细胞死亡数目也有所增加。而与S组相比,5μg/kg/h Dex预处理可明显降低肝酶以及乳酸水平,减少肝脏中IL-6和TNF-α的产生,并且可以改善肝脏病理学改变及减少肝脏细胞的死亡。结论:不同剂量右美托嘧啶对脓毒症大鼠肝脏将产生不同作用,2.5μg/kg/h和5μg/kg/h Dex均可通过抗炎和抑制细胞死亡作用而发挥肝脏保护作用,而5μg/kg/h效果更好。第二部分:脓毒症时肝细胞的死亡类型目的:通过检测使用necroptosis特异性抑制剂necrostatin-1(Nec-1)后,肝脏组织中necroptosis相关通路蛋白表达情况的改变,从而探讨necroptosis是否参与到了脓毒症时肝脏细胞的死亡中来。方法:SPF级别健康雄性SD大鼠24只(220~220g,6~8周),大鼠尾静脉置管置入后采用随机数字表法被分入4组(n=6):假手术组(Sh组)、脓毒症组(S组),溶剂DMSO组(D组)和Nec-1组(N组)。S、D和N组大鼠采用CLP方法制备大鼠脓毒症模型。Sh组大鼠处理同第一部分操作。N组和D组分别与术前1h经尾静脉注入1.0mg/kg Nec-1和等容量的DMSO;Sh组和S组分别于术前以同样方式注入等容量生理盐水。所有大鼠于术后6h处死并分离肝脏,采用western blot法测定肝脏组织中RIP1、RIP3及MLKL的表达,同时应用化学荧光法检测组织中ROS的含量。结果:与Sh组相比,S组,N组和D组肝组织ROS水平升高,RIP1、RIP3及MLKL表达上调(P0.05);与S组相比,N组肝中ROS浓度降低,RIP1、RIP3和MLKL表达下调(P0.05);Sh组与D组各种指标表达无统计学差异(P0.05)。结论:necroptosis参与到了脓毒症大鼠肝细胞的死亡中来。第三部分:右美托嘧啶可抑制necroptosis从而发挥肝脏保护作用目的:通过观察使用Dex预处理是否能够引起necroptosis相关通路蛋白的表达,进而探究Dex在脓毒症时的肝脏保护作用的发生与抑制程序性坏死是否相关。方法:本部分实验由三个部分组成:(1)32只SPF级别成年大鼠成功置管后,随机分为3组(n=8):假手术组(Sh组),脓毒症组(S组)和5μg/kg/h Dex组(D组)。(2)40只大鼠随机分为5组(n=8):假手术组(Sh组),脓毒症组(S组),1.0mg/kg Nec-1组(N1组),0.5mg/kg Nec-1组(N2组)和0.25mg/kg Nec-1组(N3组)。(3)24只大鼠分为3(n=8):假手术组(Sh组),脓毒症组(S组),Dex联合Nec-1组(ND组)。结果:(1)与Sh组相比,S组和D组的RIP1、RIP3、MLKL和HMGB1表达及ROS水平均上调(P均0.05);与S组相比,接受了Dex预处理的大鼠三种蛋白表达及ROS水平均下调(P均0.05);(2)与Sh组相比,其余四组大鼠四种蛋白表达均上调(P均0.05);与S组相比,N1和N2组四种蛋白表达均下降(P均0.05),而N3组无明显差异(P0.05)(3)与Sh相比,S组和ND组四中蛋白表达均升高(P0.05),而与S组相比,ND组四种蛋白表达下调(P均0.05)。结论:右美托嘧啶可通过抑制脓毒症时肝脏细胞necroptosis的发生,进而发挥肝脏保护作用。
[Abstract]:Dexmedetomidine (Dex), a highly selective alpha 2 adrenergic receptor agonist, is widely used in ICU patients because of its sedative, analgesic, sympathetic suppression and no respiratory depression. Previous studies have confirmed that dexmedetomidine can significantly improve the survival of sepsis patients and rats. However, there is no conclusive evidence to prove whether it can play a protective role in sepsis or not. And a large number of basic and clinical studies have found that the functional status of the liver can directly affect the survival rate and survival rate of patients with sepsis. In addition, a new regulated type of cell death, necroptosis, has been shown to be involved in many inflammatory diseases, and specific knockout of RIP3-necrptosis has been demonstrated to be an important link. The aim of this study was to investigate whether dexmedetopyrimidine could play a hepatoprotective role in septic rats and whether this protective effect was related to the inhibition of necroptosis. OBJECTIVE: To observe the effects of different doses of dexmedetopyrimidine on hepatic cell death in septic rats, and to explore the optimal dosage and mechanism of action. Methods: 80 healthy male SD rats of SPF grade, weighing 200-220 g, aged 6-8 weeks, were treated with caudal vein catheterization according to random number table method. They were randomly divided into five groups (n=16): sham operation group (Sh group), sepsis group (S group), high dose Dex group (H group), middle dose Dex group (M group) and low dose Dex group (L group). Ten rats in each group were selected to observe the 24-hour survival rate. Dex1h, Sh and S groups were continuously infused with normal saline of the same volume at the doses of 5.0ug/kg/h, 2.5ug/kg/h, 1.0ug/kg/h. Arterial blood gas (ABG), serum AST and ALT levels were measured at 6, 12 and 24 hours after operation, respectively. Results: Compared with Sh group, the levels of liver enzymes, lactate, IL-6 and TNF-alpha increased (P 0.05), and the levels of PaO_2 decreased (P 0.05). The morphological changes of liver were also different. Cell death was observed. Compared with S group, pretreatment with 5 ug/kg/h Dex significantly decreased the levels of liver enzymes and lactic acid, decreased the production of IL-6 and TNF-a, and improved the pathological changes of liver and reduced the death of liver cells. Both h and 5ug/kg/h Dex can protect the liver from inflammation and inhibit cell death, but 5ug/kg/h is more effective. Part 2: Types of liver cell death in sepsis Objective: To detect the expression of necroptosis-related pathway protein in liver tissue after the use of necroptosis-specific inhibitor Necrostatin-1 (Nec-1). Methods: Twenty-four healthy male SD rats of SPF grade (220-220 g, 6-8 weeks) were divided into four groups (n=6) by random number table method: sham operation group (Sh group), sepsis group (S group), solvent DMSO group (D group) and Nec-1 group (N group). Rats in groups S, D and N were treated with the same procedure as those in the first part. Group N and D were injected with 1.0mg/kg Nec-1 and D MSO via caudal vein one hour before operation, and group Sh and group S were injected with normal saline in the same way before operation. All rats were sacrificed and their livers were separated 6 hours after operation. Western blot was used to detect the expression of RIP1, RIP3 and MLKL in liver tissues, and chemical fluorescence was used to detect the content of ROS in liver tissues. Conclusion: Neroptosis is involved in the death of hepatocytes in septic rats. Part III: Dextropyrimidine can inhibit the expression of necroptosis-related pathway proteins by observing whether the use of Dex preconditioning can induce the expression of necroptosis-related pathway proteins. Methods: The experiment consisted of three parts: (1) 32 adult rats of SPF grade were randomly divided into three groups (n = 8): sham operation group (S h group), sepsis group (S group) and 5 UG / kg / h Dex group (D group). (2) Forty rats were randomly divided into five groups (n = 8). 8: Sham operation group (Sh group), sepsis group (S group), 1.0mg/kg Nec-1 group (N1 group), 0.5mg/kg Nec-1 group (N2 group) and 0.25mg/kg Nec-1 group (N3 group). (3) Twenty-four rats were divided into 3 (n=8): sham operation group (Sh group), sepsis group (S group), Dex combined with Nec-1 group (ND group). Results: (1) Compared with Sh group, RIML, RIP3, KL and ROHB1 levels in S and D groups were up-regulated. Compared with S group, the expression of three proteins and the level of ROS in Dex pretreated rats were all down-regulated (P 0.05); (2) Compared with Sh group, the expression of four proteins in other four groups were up-regulated (P 0.05); compared with S group, the expression of four proteins in N1 and N2 groups were down-regulated (P 0.05), but there was no significant difference in N3 group (P 0.05) (3) compared with Sh group and ND group. Compared with S group, the expression of four proteins in ND group was down-regulated (P 0.05). CONCLUSION: dexmedetopyrimidine can protect the liver by inhibiting the occurrence of necroptosis in liver cells in sepsis.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7

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