硫化氢对脑缺血再灌注后炎症的影响

发布时间：2018-05-25 10:42

本文选题：脑缺血 + 硫化氢　；参考：《河北医科大学》2014年硕士论文

【摘要】：目的：硫化氢(hydrogen sulfide, H2S)是有着“一种臭鸡蛋气味的气体”,长期接触这种有害气体,将导致神经系统、呼吸系统以及消化系统等许多组织器官的损害。但是有大量资料证明,H2S不只有毒性作用,而且还广泛参与机体多种生理及病理的过程,被认为是继一氧化氮(NO)和一氧化碳(CO)之后的第三类气体信号分子。正常动物体内可以合成H2S,参与内源性H2S合成的两种重要的酶是胱硫醚β-合酶(cystathionine beta-synthase,CBS)和胱硫醚Y-裂解酶(cystathionine γ lyase, CSE)。其中CBS主要分布于中枢神经系统,而CSE则主要分布于神经系统以外的组织。近几年的研究表明,H2S可以通过抗氧化、抑制凋亡等发挥抗脑、心肌、肝及肾等多器官组织缺血再灌注损伤作用。炎症在脑缺血再灌注损伤发生发展中发挥重要作用。本实验采用血管性痴呆(Vascular Dementia, VD)大鼠模型,通过腹腔给药的方式,研究不同剂量硫化氢供体NaHHS及H2S合成酶抑制剂羟氨(hydroxylamine, HA)和炔丙基甘氨酸(propargylglycine, PAG),对建立大鼠脑缺血再灌注模型后的海马CA1区锥体神经元损伤和脑组织炎症变化的影响,并探讨其炎症反应机制。采用病理切片和HE染色的方法观察各组大鼠海马CA1区细胞形态的变化；免疫组化和Westen blot方法检测大鼠脑缺血再灌注中脑组织H2S生成酶CBS表达变化；ELISA方法检测H2S对脑缺血再灌注中炎症介质的影响。为了证明H2S通过抗炎发挥抗脑缺血再灌注损伤作用。因此,本实验旨在从神经元、炎性因子、内源性H2S等方面,证明H2S的抗脑缺血再灌注损伤作用,并为其提供理论依据。方法：选用纯系健康雄性Wistar大鼠,体重310g左右,将动物随机分为9组,正常组(Normal group),假手术组(sham),脑缺血再灌注组(ischemia-reperfusion injury:IR),硫化氢治疗组(IR+NaHS)根据注射剂量进一步分为：2mg/kg,5mg/kg,10mg/kg,20mg/kg4个亚组,硫化氢羟氨抑制组(IR+NaHS+HA),硫化氢炔丙基甘氨酸抑制组(IR+NaHS+PAG)。每组5只动物,采用四血管阻断法(4-VO)制作VD大鼠模型,假手术组麻醉其手术过程与VD模型组相同,但不电凝双侧椎动脉,不阻断双侧颈总动脉。实验结束后取大鼠脑组织,制作大脑切片；HE染色,采用光镜观察各组大鼠海马CA1区的形态学变化；采用免疫组化的方法检测CBS表达情况；取双侧海马组织并匀浆,酶联免疫吸附法(ELISA)测定IL-1β, TNF-α;采用Western blot方法检测各组大鼠海马组织CBS表达情况。结果：1.形态学：Normal组和Sham组大鼠海马CA1区锥体细胞排列整齐、致密,细胞形态完整,胞核饱满,核仁清晰,无明显的细胞损伤。IR组大鼠出现了明显的细胞损伤,海马CA1区锥体细胞稀疏,排列紊乱；核仁消失；硫化氢治疗组中,2,5 mg/kg剂量组海马CA1区组织形态与IR组基本一致；10 mg/kg组与IR组相比,神经元数量有所增加,可见不完整的锥体细胞层；20 mg/kg组神经元存活状态明显改善,组织学特征与Normal组和Sham组类似,神经元排列比较整齐,细胞形态也比较完整。IR+NaHS+HA组和IR+NaHS+PAG组与硫化氢治疗组20mg/kg相比出现了明显的细胞损伤。2蛋白CBS的表达：免疫组化结果显示,Normal组、Sham组大鼠海马CA1区可见到一定量CBS阳性免疫颗粒,呈棕黄色,染色较浅,均匀分布。与Sham组相比,IR组大鼠海马CA1的CBS阳性免疫颗粒显著减少,甚至在锥体细胞层及其周围出现大片CBS表达缺失区域。与IR组相比,低剂量硫化氢(2、5mg/kg)海马CA1区CBS的免疫阳性染色强度均无明显变化,随着硫化氢的剂量加大免疫染色阳性随之增强。硫化氢大剂量(20mg/kg)组大鼠可见大量棕褐色CBS阳性免疫颗粒分布于海马CA1区,尤其在海马锥体细胞层可见大量、深染的CBS阳性颗粒包绕锥体神经元细胞。IR+NaHS+HA组和 IR+NaHS+PAG组与硫化氢治疗组20mg/kg相比,CBS阳性免疫颗粒明显减少。3.westen blot结果：在Normal组和Sham组动物海马CA1区组织内有CBS蛋白的基础生成。与sham组相比,全脑缺血组的CBS的蛋白表达全脑缺血后显著降低。大剂量硫化氢可明显上调全脑缺血大鼠CBS蛋白的表达；而低剂量硫化氢对全脑缺血大鼠CBS蛋白表达下调无明显影响。同时注射HA和PAG后硫化氢诱导的CBS的蛋白表达上调作用消失。4. ELISA结果：在Normal组和Sham组动物海马CA1区组织内有IL-1p和TNF-a蛋白的基础生成。与Normal组和Sham组相比,脑缺血组动物的IL-1β和TNF-a蛋白浓度于脑缺血后显著上调。治疗性给予硫化氢后,明显抑制了脑缺血引起的IL-1β和TNF-a蛋白生成的上调,表现为其上升的高峰显著降低。并且,大剂量组(20mg/kg)的抑制作用最为明显。IR+NaHS+HA组和IR+NaHS+PAG组与硫化氢治疗组20mg/kg相比IL-1β和TNF-a蛋白显著上调。结论：脑缺血前给予一定剂量的硫化氢可有效地减轻全脑缺血再灌注大鼠海马CA1区细胞损伤,且大剂量组的保护作用更明显。而H2S合成酶的抑制剂HA和PAG可以抑制上述作用；HA和PAG可以抑制硫化氢上调CBS表达和下调脑缺血过程中炎症因子IL-1β和TNF-a的表达,可能是硫化氢抗缺血性脑损伤的机制之一,并进一步表明硫化氢的抗脑缺血作用。
[Abstract]:Objective: hydrogen sulfide (H2S) is a "Stinky egg odor". Long-term exposure to this harmful gas will cause damage to many tissues, such as the nervous system, the respiratory system and the digestive system. But there is a lot of information that H2S is not only toxic, but also widely participates in many physiological and diseases of the body. The process is considered to be the third class of gas signal molecules following nitric oxide (NO) and carbon monoxide (CO). The normal animals can synthesize H2S. The two important enzymes involved in endogenous H2S synthesis are cystetyl beta synthase (cystathionine beta-synthase, CBS) and cystthioether Y- lyase (cystathionine gamma lyase, CSE). The central nervous system is distributed in the central nervous system, while CSE is mainly distributed outside the nervous system. Recent studies have shown that H2S can play an important role in the ischemic reperfusion injury of multi organ tissues, such as brain, myocardium, liver and kidney, through antioxidation and inhibition of apoptosis. The inflammation plays an important role in the development of cerebral ischemia-reperfusion injury. The rat model of Vascular Dementia (VD) was used to study the NaHHS and H2S synthetase inhibitors (hydroxylamine, HA) and propargyl glycine (propargylglycine, PAG) of different doses of hydrogen sulfide donor NaHHS and H2S synthase inhibitors by intraperitoneal administration. The damage of pyramidal neurons in the hippocampus CA1 region after the rat model of cerebral ischemia reperfusion was established. The effects of inflammatory changes in brain tissue and the mechanism of inflammatory reaction were investigated. The morphological changes of hippocampal CA1 area were observed by pathological section and HE staining. Immunohistochemical and Westen blot methods were used to detect the expression of H2S producing enzyme CBS in brain tissue of rat cerebral ischemia reperfusion, and ELISA method was used to detect the cerebral ischemia reperfusion. The effect of medium inflammatory mediators. In order to prove that H2S plays the role of anti cerebral ischemia and reperfusion injury through anti-inflammatory. Therefore, this experiment aims to prove the anti cerebral ischemia reperfusion injury effect of H2S from the aspects of neurons, inflammatory factors and endogenous H2S, and provide a theoretical basis for it. Methods: a healthy male Wistar rat was selected, and the weight of the rat was about 310G, The animals were randomly divided into 9 groups, the normal group (Normal group), the sham operation group (sham), the cerebral ischemia reperfusion group (ischemia-reperfusion injury:IR), and the hydrogen sulfide treatment group (IR+NaHS) were further divided into 2mg/kg, 5mg/kg, 10mg/kg, 20mg/kg4 subgroups, hydrogen sulfide hydroxyl ammonia inhibition group (IR+NaHS+HA), and hydrogen sulfide propargyl glycine inhibition group. (IR+NaHS+PAG) 5 animals in each group were used to make the VD rat model by the four vascular blocking method (4-VO). The sham operation group was anesthetized with the same procedure as the VD model group, but did not electrocoagulation the bilateral vertebral arteries and did not block the bilateral common carotid artery. After the experiment, the rat brain tissue was taken to make the large brain slices; HE staining was used to observe the CA1 area of the hippocampus in each group by light microscope. The expression of CBS expression was detected by immunohistochemical method; IL-1 beta and TNF- a were measured by bilateral hippocampal tissue and homogenate and enzyme linked immunosorbent assay (ELISA). The expression of CBS in hippocampus of each group was detected by Western blot. Results: 1. morphology: Normal and Sham group rat hippocampal pyramidal cell lines. Neat, dense, complete cell morphology, full nucleolus, clear nucleolus, no obvious cell damage in the.IR group, there were obvious cell damage, the pyramidal cells in the hippocampal CA1 region were sparse, and the nucleolus disappeared; in the hydrogen sulfide treatment group, the CA1 area of the hippocampus in the 2,5 mg/kg dose group was basically the same as that in the IR group; 10 mg/kg group and IR group phase. The number of neurons increased, and the incomplete pyramidal cell layer was seen. The survival state of the 20 mg/kg group was obviously improved. The histological features were similar to that of the Normal group and the Sham group, the neurons were arranged neatly, and the cell morphology was also more complete in the.IR+NaHS+HA group and in the IR+NaHS+PAG group than in the hydrogen sulfide treatment group 20mg/kg. The expression of cell damage.2 protein CBS: the immunohistochemical results showed that a certain amount of CBS positive immune granules in the hippocampus CA1 area of group Sham rats were found to be brown, and the staining was shallow and evenly distributed. Compared with the Sham group, the CBS positive immune particles of the CA1 in the hippocampus of the IR group were significantly reduced, even in the pyramidal cells and around the cell layer and around a large scale CBS table. Compared with the IR group, the immune positive staining intensity of the CBS in the hippocampus CA1 area of the low dose hydrogen sulfide (2,5mg/kg) was not significantly changed, and the positive staining of the immunostaining increased with the increase of the dose of hydrogen sulfide. The large dose of hydrogen sulfide (20mg/kg) group showed that a large number of brown CBS positive immune particles were distributed in the hippocampal CA1 region, especially in the hippocampal cones. In the body cell layer, a large number of CBS positive particles wrapped around the pyramidal neurons were found in the.IR+NaHS+HA group and the IR+NaHS+PAG group, compared with the hydrogen sulfide treatment group 20mg/kg, the CBS positive immune particles significantly reduced the.3.westen blot results: the base formation of CBS protein in the Normal and Sham group CA1 region tissues. Compared with the sham group, the whole brain was compared with the sham group. The protein expression of CBS in the ischemic group decreased significantly after the whole brain ischemia. The high dose of hydrogen sulfide could obviously increase the expression of CBS protein in the rats with whole brain ischemia, while the low dose of hydrogen sulfide had no obvious effect on the down regulation of CBS protein expression in the whole brain ischemia rats. At the same time, the up regulation of the protein expression of CBS induced by hydrogen sulfide induced by injection of HA and PAG disappeared in the.4. ELISA junction. Fruit: the basal formation of IL-1p and TNF-a protein in the hippocampal CA1 region of the Normal and Sham groups. Compared with the Normal and Sham groups, the concentration of IL-1 beta and TNF-a protein in the cerebral ischemia group was significantly up-regulated after cerebral ischemia. After the treatment of hydrogen sulfide, the up regulation of IL-1 beta and TNF-a protein production caused by cerebral ischemia was obviously inhibited. The maximum inhibitory effect of the high dose group (20mg/kg) was most obvious in the.IR+NaHS+HA group and the IR+NaHS+PAG group, and the IL-1 beta and TNF-a protein increased significantly compared with the hydrogen sulfide treatment group 20mg/kg. Conclusion: a certain dose of hydrogen sulfide before cerebral ischemia can effectively reduce the CA1 region of the hippocampus of the rats with whole cerebral ischemia reperfusion. H2S synthase inhibitors, HA and PAG, can inhibit the above action. HA and PAG can inhibit the up-regulated CBS expression of hydrogen sulfide and down regulation of the expression of IL-1 beta and TNF-a in the process of cerebral ischemia. It may be one of the mechanisms of hydrogen sulfide against ischemic brain damage and further indicates sulfur. The effect of hydrogen on cerebral ischemia.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

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