N-乙酰-L-半胱氨酸改善丙酮醛诱导的皮肤角质形成细胞损伤及机制

发布时间：2018-04-24 02:08

本文选题：糖尿病 + 丙酮醛　；参考：《广州医科大学》2017年硕士论文

【摘要】：背景晚期糖基化终末产物(Advanced glycation end products,AGEs)堆积是糖尿病(Diabetes mellitus,DM)皮肤并发症的重要原因。丙酮醛(Methyglyoxal,MGO)是一种活性二羰基化合物,是AGEs产生的重要中间体,在糖尿病的病理过程中能生成AGEs。N-乙酰-L-半胱氨酸(N-acetyl-cysteine,NAC)是某些临床常用药的主要活性成分,参与内源性还原型谷胱甘肽(reduced glutathione,GSH)和硫化氢(hydrogen sulfide,H2S)的生成。然而,NAC可否通过抑制AGEs的产生或其毒性,减轻DM诱导的皮肤损伤仍鲜有报道。因而,本研究旨在观察NAC对MGO诱导的糖尿病性皮肤细胞损伤的影响及分子机制。方法应用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)法检测糖尿病患者和健康志愿者血浆中AGEs的含量。用牛血清白蛋白(bovine serum albumin,BSA)与MGO于37℃孵育3 d制备AGEs。根据AGEs具有自发荧光和形成棕色溶液的特点,分别用荧光酶标仪检测340/465 nm反应体系中荧光强度以及观察BSA与MGO混合溶液的颜色;进一步观察NAC对MGO诱导的AGEs生成的影响。人永生化角质形成细胞(Human adult low calcuim kertinocytes,Ha Ca T)给予MGO处理,模拟糖尿病血液或组织中高MGO状态,建立糖尿病皮肤伤口愈合障碍的体外模型。从细胞活力、线粒体膜电位、细胞的粘附和迁移功能、炎症因子的分泌、核因子κB(nuclear factor kappa B,NF-κB)亚基p65的核转位、基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)的表达以及细胞外基质的含量进行评价。在MGO处理Ha Ca T细胞前,用NAC预处理1 h,观察NAC对MGO诱导的上述指标改变的影响,以明确其皮肤细胞保护效应。最后,通过检测AGEs的受体(receptor for advanced end products,RAGE)的表达,以及其中和抗体(Neutralizing antibody against RAGE,N-ABR)预处理对MGO诱导的Ha Ca T细胞损伤的影响,最终揭示MGO诱导的细胞损伤以及NAC的细胞保护效应是否依赖于RAGE的激活。结果1.糖尿病患者血浆中AGEs的含量高于正常人,差异具有统计学意义(P0.05)。2.MGO和BSA反应后,溶液颜色加深,荧光增强,提示MGO可诱导AGEs的生成。NAC处理可以降低溶液的颜色和荧光强度(P0.01),提示其可以抑制MGO诱导的AGEs生成。另外,用MGO处理Ha Ca T细胞,可使细胞培养基中AGEs含量增加,NAC预处理能明显抑制MGO诱导的AGEs生成,差异具有统计学意义(P0.01)。3.用MGO处理Ha Ca T细胞,可以剂量依赖性地降低细胞活力、损害线粒体膜电位和细胞的粘附和迁移等行为学功能,这种损害效应可以被NAC预处理所减弱。提示NAC可减弱MGO诱导细胞损伤。4.Ha Ca T细胞经MGO处理后,炎症因子IL-6和IL-8的分泌增加、NF-κB亚基p65核转位增强,NAC预处理能抑制MGO诱导的炎症因子分泌和NF-κB亚基p65核转位。提示NAC能抑制MGO诱导的炎症反应。5.MGO处理Ha Ca T细胞可导致MMP-9的表达上调以及I型胶原的生成减少,这种效应能部分被NAC预处理逆转。提示NAC改善MGO诱导的细胞迁移障碍可能与改善细胞外基质的水平有关。6.MGO处理可上调Ha Ca T细胞内RAGE受体的表达,NAC预处理可以抑制MGO诱导的RAGE受体过表达。重要的是,用RAGE中和抗体阻碍其作用后,MGO引起的细胞受损,炎症因子释放,MMP-9过度表达以及NF-κB亚基p65核转位均被部分改善。提示MGO诱导的皮肤细胞炎症损伤与RAGE受体的激活有关,而NAC拮抗MGO的损伤作用是通过抑制RAGE受体实现的。结论本文研究证实,NAC可减弱MGO诱导的皮肤细胞损伤,其机制与抑制AGEs的生成和AGEs/RAGE/NF-κB信号通路的活化有关。本文的研究为临床应用NAC相关的药物防治糖尿病皮肤并发症提供了基础资料与理论支持。
[Abstract]:The accumulation of advanced glycosylation end products (Advanced glycation end products, AGEs) is an important reason for the skin complications of diabetes (Diabetes mellitus, DM). The acetaldehyde (Methyglyoxal, MGO) is a kind of active two carbonyl compound, an important intermediate in AGEs production, and can produce cysteine acetyl acetate in the pathological process of diabetes. N-acetyl-cysteine (NAC) is the main active component of some common clinical drugs, and participates in the production of endogenous reduced glutathione (GSH) and hydrogen sulfide (hydrogen sulfide, H2S). However, it is still rarely reported that NAC can reduce DM induced skin damage by inhibiting the production of AGEs or its toxicity. The effects of NAC on MGO induced diabetic skin cell damage and its molecular mechanism were observed. Methods the content of AGEs in plasma of diabetic patients and healthy volunteers was detected by enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) and incubated with bovine serum albumin (bovine serum albumin, BSA) and MGO at 37. Es. based on the characteristics of AGEs with spontaneous fluorescence and the formation of brown solution, the fluorescence intensity of 340/465 nm reaction system and the color of BSA and MGO mixed solution were observed by fluorescent enzyme labeling, and the effect of NAC on MGO induced AGEs formation was further observed. T) give MGO treatment, simulate the high MGO state of diabetic blood or tissue, establish an in vitro model of diabetic skin wound healing disorder. From cell viability, mitochondrial membrane potential, cell adhesion and migration, secretion of inflammatory factors, nuclear factor kappa B (nuclear factor kappa B, NF- kappa B) subunit p65, matrix metalloproteinase 9 (matr) The expression of IX metalloproteinase 9, MMP-9) and the content of extracellular matrix were evaluated. Before MGO Ha Ca T cells were treated with NAC, 1 h was pretreated with NAC to observe the effect of NAC on MGO induced changes of the above index, in order to clarify the protective effect of its skin cells. As well as the effects of Neutralizing antibody against RAGE (N-ABR) pretreatment on MGO induced Ha Ca T cell damage, and finally revealed whether MGO induced cell damage and NAC cell protection effect depended on RAGE activation. Results 1. diabetic patients were higher than normal people in plasma, and the difference was statistically significant. After the reaction of P0.05.2.MGO and BSA, the color of the solution was deepened and the fluorescence enhanced, suggesting that MGO could induce the formation of AGEs by.NAC treatment to reduce the color and fluorescence intensity of the solution (P0.01), suggesting that it could inhibit the AGEs generation induced by MGO. Furthermore, the MGO processing of Ha Ca cells could increase the content of the cell culture medium, and the pretreatment could be obvious. Inhibition of MGO induced AGEs generation, the difference has statistical significance (P0.01),.3. using MGO to treat Ha Ca T cells, can reduce cell viability in a dose-dependent manner, damage mitochondrial membrane potential and cell adhesion and migration and other behavioral functions, this damage effect can be reduced by NAC preconditioning. NAC can weaken MGO induced cell damage.4.Ha. The secretion of inflammatory factors IL-6 and IL-8 increased after MGO treatment, and the nuclear transposition of NF- kappa B subunit was enhanced. NAC preprocessing could inhibit the secretion of inflammatory factors induced by MGO and NF- kappa B subunit nuclear transposition. Less, this effect can be partially reversed by NAC pretreatment. Suggesting that NAC improves MGO induced cell migration disorders may be associated with the improvement of the level of extracellular matrix,.6.MGO treatment can up regulate the expression of RAGE receptors in Ha Ca T cells. NAC preconditioning can inhibit RAGE receptor overexpression induced by MGO. It is important that the action of RAGE neutralize antibodies to obstruct its action. Later, MGO induced cell damage, inflammatory factors release, MMP-9 overexpression and NF- kappa B subunit p65 nuclear transposition were partially improved. It suggests that MGO induced skin cell inflammation damage is related to the activation of RAGE receptor, and NAC's antagonistic effect on MGO is achieved by inhibiting RAGE receptor. Conclusion this study confirms that NAC can weaken MGO induced. The mechanism of skin cell damage is related to the inhibition of the formation of AGEs and the activation of the AGEs/RAGE/NF- kappa B signaling pathway. This study provides basic information and theoretical support for the clinical application of NAC related drugs in the prevention and treatment of diabetic skin complications.

【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

【参考文献】