利鲁唑在脂多糖诱导眼内炎症中的干预研究

发布时间：2018-07-11 18:45

本文选题：脂多糖 + 眼内炎　；参考：《郑州大学》2015年博士论文

【摘要】：眼外伤、内眼手术是感染性眼内炎的主要原因,多为细菌进入眼内导致感染所致,如凝固酶阴性葡萄球菌、金黄色葡萄球菌、链球菌、革兰氏阴性细菌等。细菌性眼内炎是严重威胁视力的眼科急症,细菌性眼内炎预后受细菌数量、毒力、患者年龄及免疫状态等多种因素影响。虽然及时采取抗生素药物治疗、玻璃体切除术等治疗,但是视力预后仍不理想,常导致严重的视功能损害。细菌在增殖、破裂的过程中释放的毒性物质(内毒素、外毒素)可诱发严重的炎症反应及细胞坏死[1,2,3]。其中细菌的裂解产物是诱发眼内炎症的重要因素之一。脂多糖是革兰阴性杆菌细胞壁的主要成分,也是其主要毒性致病因子,可用于玻璃体腔注射,诱导感染性眼内炎症反应[4]。炎症反应导致血眼屏障破坏,炎性细胞向组织间隙迁移浸润,蛋白渗漏,精密的视网膜结构被破坏,最终导致功能不同程度的丧失。如何消除眼内炎中眼内组织破坏、改善最终视力预后一直是我们面临的难题和挑战。在积极抗菌治疗的同时,能否有效并及时地控制眼内炎症反应是治疗的关键,也是预后的决定性因素。利鲁唑(Riluzole)属于苯并噻唑类衍生物,1996年开始作为一种新型谷氨酸释放抑制剂被批准应用于肌萎缩性脊髓侧索硬化症(amyotrophic lateral sclerosis,ALS)的治疗,其稳定细胞内外离子浓度的作用使其在神经保护、抗惊厥、抗抑郁等方面具有广泛的作用,提示其在更多疾病中的临床应用价值。根据其药理机制,我们推测其可能在眼内炎症控制中起到积极作用。本研究拟观察利鲁唑对脂多糖诱导大鼠眼内炎症的干预作用,并对其机制进行初步研究。目的通过玻璃体腔注射大肠杆菌脂多糖(LPS,lipopolysaccharide from Escherichia coli)建立大鼠眼内炎模型,观察其炎症表现、组织病理学特征以及利鲁唑的干预作用,然后对其作用机制进行研究,为眼内炎的治疗提供新线索。材料与方法将Sprague-Dawley(SD)大鼠随机分为3组,1组:生理盐水对照组;2组:眼内炎组;3组:眼内炎利鲁唑干预组。生理盐水组大鼠右眼注入5μl无菌生理盐水。2、3组大鼠右眼玻璃体腔内注入LPS(2μg,5μl),建立LPS诱导的眼内炎模型。左眼为正常对照眼。利鲁唑干预组在LPS注射前12 h和术中及术后连续3 d腹腔内注射利鲁唑注射液(0.1%,10mg/kg)。第一部分:造模后6h、12h、24h、48h、3d、5d、7d进行眼部炎症评分,病理组织学检查观察炎症细胞浸润,行免疫组化测定谷氨酰胺合成酶(glutamine synthetase,GS)在视网膜组织的表达变化及分布,采用荧光定量聚合酶链反应(RT-PCR)技术检测视网膜组织中GS的表达情况,观察利鲁唑的干预作用。第二部分:对利鲁唑的干预机制进行初步探讨,分光光度计法测定玻璃体中谷氨酸(glutamate,Glu)含量,采用TUNEL法检测视网膜组织细胞凋亡。所有数据均用(x±s)表示,应用SPSS21.0统计软件包进行统计学处理,采用单因素方差分析进行组间比较,采用LSD检验进行两两比较,P0.05为差异有统计学意义。结果1.临床炎症评分玻璃体腔内注射LPS诱导出典型眼内炎临床表现,造模后干预组炎症反应明显较2组轻,造模后24 h、48h、3d时间点两组临床炎症评分间差异均有统计学意义(P0.05)。1组未见明显炎症反应。2.病理组织学检查常规HE染色后显微镜下观察白细胞浸润,对切片中以锯齿缘为前界的玻璃体腔中的中性粒细胞、淋巴细胞、单核-巨噬细胞等炎症细胞进行计数分析。2组、3组在造模后均可观察到大量外周血白细胞眼内的浸润。HE染色显示各时间点3组玻璃体内炎症细胞计数低于2组,差异有统计学意义(P0.05)。而Ⅰ组造模后玻璃体腔、视网膜大部分未见、或散在少量的炎症细胞浸润,视网膜形态基本正常,结构清晰。与1组相比,GS在眼内炎组的表达升高,主要表达于内颗粒层、神经节细胞层Müller细胞胞浆中。2组、3组在6h、12h、24h、48h、3d GS的表达具有显著性差异(P0.05),3组GS的表达明显低于2组,但仍高于对照组。3.实时荧光定量PCR方法检测视网膜组织GS的表达48h时,对照组视网膜中检测到低水平表达,眼内炎组GSm RNA表达较对照组明显增强(P0.05),利鲁唑干预组GSm RNA表达较眼内炎组降低(P0.05),但仍高于对照组。4.谷氨酸浓度2组和3组大鼠玻璃体中谷氨酸含量在炎症初期就开始升高,48h时达到高峰,分别为324.01±2.47μmol/l、253.95±10.76μmol/l。2组在5d时谷氨酸浓度较之前出现小幅回升(298.04±4.83μmol/l),然后逐渐下降。6h后各时间点,3组造模眼玻璃体谷氨酸浓度明显低于2组,差异均有统计学意义(P0.05),但仍明显高于1组。5.LPS诱导眼内炎后,TUNEL染色阳性细胞散在分布于视网膜神经节细胞层、内颗粒层,3d时出现凋亡细胞数量明显增多。2组细胞凋亡数目明显多于3组。生理盐水对照组视网膜未见明显细胞凋亡,6h,12h,24h时眼内炎组与干预组相比细胞凋亡程度无明显差异,其后各时间点3组凋亡细胞指数均低于同期眼内炎组,差异有统计学意义(P0.05)。结论利鲁唑可以抑制内毒素诱导的眼内炎中炎症细胞的眼内浸润,减少组织损害,其机制可能是通过减少视网膜组织细胞凋亡,使玻璃体谷氨酸含量降低,抑制Müller细胞活化。该研究结果提示利鲁唑在细菌性眼内炎中的保护作用,为眼内炎的辅助治疗提供依据。
[Abstract]:Ocular trauma and internal eye surgery are the main causes of infective endophthalmitis, most of which are caused by bacterial entry into the eye, such as coagulase negative staphylococcus, Staphylococcus aureus, Streptococcus, Gram-negative bacteria, etc. bacterial endophthalmitis is a serious threat to visual acuity, and the prognosis of bacterial endophthalmitis is affected by the number of bacteria, virulence, and patients. A variety of factors such as age and immune status. Although timely use of antibiotics, vitrectomy, and other treatments, the visual prognosis is still not ideal, which often leads to severe visual impairment. The toxic substances released in the process of proliferation and rupture (endotoxin, exotoxin) can induce severe inflammatory reactions and cell necrosis [1 2,3]. is one of the important factors to induce intraocular inflammation. Lipopolysaccharide is the main component of the cell wall of gram-negative bacilli, and it is also the main toxic factor of the gram negative bacilli. It can be used in the intravitreal injection to induce the infection of the intraocular inflammation and the [4]. inflammatory reaction leads to the destruction of the blood barrier and the inflammatory cells migrate to the interstitial space. It is a difficult problem and challenge to eliminate the destruction of the intraocular tissue in the ophthalmitis and improve the prognosis of the final eyesight. In the meantime, the effect and timely control of the intraocular inflammation is the key to the treatment. Key, also a decisive factor in prognosis. Riluzole is a benzothiazole derivative. It began as a new type of glutamate release inhibitor in 1996 and was approved to be applied to the treatment of amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS), which stabilizes the role of intracellular and extracellular ion concentration to protect it from neuroprotection. The anticonvulsant, antidepressant, and other aspects have a wide range of functions, suggesting its clinical application in more diseases. According to its pharmacological mechanism, we speculate that it may play an active role in the control of intraocular inflammation. This study intends to observe the intervention effect of Lulu on lipopolysaccharide induced endophthalmitis in rats and to make a preliminary study of its mechanism. Objective to establish an endophthalmitis model by injecting LPS (lipopolysaccharide from Escherichia coli) into the cavity of the vitreous body to observe the inflammation, histopathological features and the intervention of lelozole, and then study its mechanism of action to provide new clues for the treatment of endophthalmitis. Materials and methods will apply Sprague- to the treatment of endophthalmitis. Dawley (SD) rats were randomly divided into 3 groups, 1 groups: normal saline control group, 2 group: Endophthalmitis group, 3 group of endophthalmitis leuzole intervention group. The right eye of the normal saline group was injected into the right eye of the right eye of the normal saline group, the right eye of group.2,3 rats were injected LPS (2 mu, 5 mu L), and the LPS induced endophthalmitis model was established. The left eye was the normal eye to the eye. Li Lu azole intervention group. 12 h before LPS injection and intraoperative and postoperative 3 D intraperitoneal injection of ralu injection (0.1%, 10mg/kg). First part: 6h, 12h, 24h, 48h, 3D, 5D, 7d, followed by inflammation scores, histopathological examination of inflammatory cells, and immunohistochemical determination of glutamine synthetase in retina tissue The expression of GS in retinal tissue was detected by fluorescence quantitative polymerase chain reaction (RT-PCR), and the intervention of lurazole was observed. The second part: preliminary discussion on the intervention mechanism of lurazole, spectrophotometric determination of the content of glutamate (Glu) in vitreous body, and the detection of optic network by TUNEL All data were expressed by (x + s), using SPSS21.0 statistical software package for statistical treatment, using single factor analysis of variance to compare groups and using LSD test for 22 comparison, P0.05 was statistically significant. Results 1. clinical inflammation score in vitreous cavity injected LPS to induce typical endophthalmitis The inflammatory response in the intervention group was significantly lighter than that of the 2 groups after the model, 24 h, 48h, and 3D time points in the two groups. There was a significant difference between the two groups of clinical inflammation scores (P0.05) there was no obvious inflammatory reaction in the.1 group and.2. histopathological examination was observed under the routine HE staining, and the leukocyte infiltration was observed under the microscope and the serrations in the anterior boundary of the glass body were in the section. Inflammatory cells such as neutrophils, lymphocytes, mononuclear macrophages and other inflammatory cells were counted and analyzed in.2 group. The 3 groups could observe the infiltration of.HE in the eyes of a large number of peripheral blood leucocytes in the 3 groups. The count of inflammatory cells in the vitreous body of 3 groups was lower than that of the 2 groups, and the difference was statistically significant (P0.05). As compared with the 1 groups, the expression of GS in the endophthalmitis group increased, mainly in the inner granular layer, the.2 group in the ganglion cell layer M u ller cell cytoplasm, and the 3 groups in 6h, 12h, 24h, 48h, 3D GS (P0.05), 3 groups of GS tables. It was significantly lower than the 2 groups, but still higher than the control group.3. real-time fluorescence quantitative PCR method to detect the expression of GS in the retinal tissue 48h, the control group detected a low level of expression in the retina, the expression of GSm RNA in the group of endophthalmitis was significantly enhanced (P0.05), and the RNA table of the rizole intervention group was lower than that of the endophthalmitis group (P0.05), but still higher than the control group.4.. The glutamic acid content in the vitreous body of the 2 group and the 3 group of rats began to rise in the early stage of the inflammation, and reached the peak at 48h, 324.01 + 2.47 mu mol/l respectively. The glutamic acid concentration in the 253.95 + 10.76 mol/l.2 group appeared slightly higher than before (298.04 + 4.83 Mu mol/l), and then gradually decreased at each time point after.6h, and the vitreous valleys of the 3 groups were made. The concentration of ammonia acid was significantly lower than that of the 2 group (P0.05), but it was still significantly higher than the 1 groups of.5.LPS induced endophthalmitis. The TUNEL staining positive cells were scattered in the retinal ganglion cell layer, the inner granular layer, and the number of apoptotic cells in the 3D group increased obviously in.2 group more than in the 3 groups. No obvious apoptosis was found in the membrane. There was no significant difference in the degree of apoptosis between the endophthalmitis group and the intervention group at 6h, 12h and 24h. The 3 groups of apoptotic cells in each time point were lower than those in the same period of endophthalmitis. The difference was statistically significant (P0.05). Conclusion raluzole could inhibit the intraocular infiltration of inflammatory cells in endotoxin induced endophthalmitis and the reduction group. The mechanism may be to reduce the content of vitreous glutamic acid and inhibit the activation of M u ller cells by reducing the apoptosis of retinal tissue cells. The results suggest that the protective effect of raluzole in bacterial endophthalmitis provides a basis for the adjuvant treatment of endophthalmitis.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R77

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