【作者】 李明；

【导师】 李兴福；

【作者基本信息】 山东大学 ， 内科学（风湿病学）（专业学位）， 2016， 博士

【摘要】 研究背景：系统性红斑狼疮(Systemic lupus erythematosus, SLE)是典型的自身免疫性疾病,其主要特征是血清中出现以抗核抗体为主的多种自身抗体和多系统损害。近年来,关于氧化应激失衡在SLE发病中作用的多项研究证实：SLE的发病及器官损害与机体氧化应激失衡有关。正常情况下,体内氧化抗氧化系统是个平衡过程,在某些病理情况下,氧化过程超过抗氧化系统的抗衡能力,平衡打破以后,活性氧化物(ROS)增多,后者通过一系列化学反应损伤细胞膜、DNA及脂质和蛋白质,导致相应的功能障碍,此过程称为氧化应激。活性氧化物(ROS)是导致组织病理损伤的主要活跃性物质,清除机制相当复杂,直接检测非常困难,血清一氧化氮(NO)、丙二醛(MDA)水平是传统的反应氧化过度的指标。近年的研究结果显示8-表-前列腺素F2 a(8-epi-PGF2α)是氧化应激的敏感指标,它是体内脂质过氧化时的产物,性质稳定,易被检测,能很好的反映体内自由基损害程度。体内抗氧化系统分酶类和非酶类两部分,这些抗氧化系统物质可以对抗ROS,通过一系列反应,把过氧化物还原为羟基化合物,促进过氧化氢(H2O2)的分解为水,从而把有毒性的化合物转化为无毒性的代谢产物,避免了组织细胞受氧化应激的损害。糖皮质激素作为临床应用广泛的传统的免疫抑制剂,是目前治疗SLE的主要药物,可以使患者生存率明显提高,但是,其不良反应如无菌性骨坏死、严重感染增加、严重骨质疏松、心脑血管病变等让无数患者付出了沉痛的代价。因此,近年来关于SLE非激素或少激素治疗的研究日益增多,经典免疫抑制剂如环磷酰胺治疗SLE的方案日趋合理多样,新型免疫抑制剂如来氟米特、吗替麦考酚酯、他克莫司等在缓解及控制维持SLE特别是狼疮性肾炎病情方面,已经取得了可喜的进展。以单克隆抗体药物为代表的靶向治疗更是在小规模的临床试验中取得了突破性进展。此外,在非免疫抑制剂药物的尝试中,出现了诸多有前景的选择,如羟氯喹的临床资料已经使之成为SLE治疗的基础用药,其他药物的尝试也开始有陆续的临床观察如他汀类药物、维生素D及抗氧化剂的临床应用,其中,近年来已有多种抗氧化剂用于治疗SLE,部分药物的基础实验结果展示出了良好的临床应用前景。基础病理学证实氧化应激参与了机体内炎症的产生,作为自身免疫性炎症性疾病,SLE的发病及组织损害关系与氧化应激关系也已经被多项研究证实。因此,有多项研究开始探讨SLE抗氧化剂治疗的可能前景。N-乙酰半胱氨酸(NAC)是经典的抗氧化剂,它是体内非必需氨基酸L-半光氨酸乙酰化的衍生物,能直接干扰各种自由基生成,且能清除已生成的自由基,同时能提高机体内的谷胱甘肽的含量,抑制炎症反应核因子-kB,拮抗细胞凋亡,调节细胞代谢活性,所以具有广泛的生物学活性,在机体的炎症反应、免疫应答等多个生理活动调控过程中发挥重要作用。2012年,首个随机双盲病例对照的N-乙酰半胱氨酸(NAC)治疗SLE研究在Arthritis Rheum杂志发表,随后美国学者也进行了NAC的治疗SLE的临床观察,标志着NAC可能会成为SLE的治疗药物之一。但目前尚没有国内相关研究资料。本课题旨在探讨NAC对国内汉族人群SLE的疗效,但因伦理学因素,未设安慰剂对照组,而是选择轻症SLE予以两种不同治疗方案,通过含NAC联合方案的协同增益作用来观察NAC的疗效。以期探讨SLE非糖皮质激素治疗的更多临床选择。为较全面地观察NAC在SLE中抗氧化治疗的效果,我们选用MRL/lpr小鼠作为狼疮小鼠模型,应用N-乙酰半胱氨酸干预MRL/lpr小鼠,观察小鼠血清中NO、 MDA、SOD、GSH-PX水平的变化情况,同时检测小鼠血清抗ds-DNA水平及尿蛋白变化,以初步探讨NAC对SLE氧化应激的影响及治疗效应。同时对轻症SLE患者予以含NAC的非糖皮质激素方案治疗,分析NAC在轻度SLE治疗中的临床协同作用。研究目的第一部分动物实验部分1.通过观察自发性狼疮小鼠模型MRL/lpr小鼠血清氧化应激指标的变化情况,了解系统性红斑狼疮初期机体氧化应激特征。2.初步了解抗氧化剂N-乙酰半胱氨酸对狼疮小鼠模型MRL/lpr小鼠氧化应激指标的影响及对狼疮小鼠的治疗作用。第二部分临床研究部分1.观察N-乙酰半胱氨酸(NAC)对轻症系统性红斑狼疮患者治疗前后血清一氧化氮(N0)、谷胱甘肽过氧化物酶(GSH-PX).人8异前列腺素F2 Q(8-iso-PGF2 α)的影响,同时观察患者血沉(ESR)、抗双链DNA(Ds-DNA).免疫球蛋白G(IgG).补体3(C3)及系统性红斑狼疮病情活动评分(SLEDAI)的变化。2.探讨N-乙酰半胱氨酸联合合非糖皮质激素方案(硫酸羟氯喹)治疗轻症系统性红斑狼疮能否增加近期疗效。研究方法第一部分动物实验部分实验分为3组：正常对照组(BALB/c小鼠)6只；狼疮模型鼠(MRL/1pr鼠)组20只；随机分为非干预组10只,NAC观察组10只,观察组各鼠给予NAC150mg/d腹腔注射,共30天。正常对照组和非干预组各鼠每天给予等量0.9%氯化钠注射液腹腔注射。试验30天后留取足量尿液,处死小鼠获取血清标本,分别检测小鼠尿蛋白、抗ds-DNA抗体变化。同时分别采用硝酸根还原酶法、硫代巴比妥酸法、黄嘌呤氧化酶-羟胺法、化学比色法检测各组小鼠血清一氧化氮(N0)、丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-PX)的表达水平,观察分析各指标的变化情况,分析NAC的治疗作用。第二部分临床研究部分65例轻度SLE患者分对照组30例,应用硫酸羟氯喹治疗,观察组35例,除硫酸羟氯喹治疗外加用NAC(1200mg/日),在治疗前、治疗6月检测血清一氧化氮(N0)、谷胱甘肽(GSH)、人8异前列腺素F2 α(8-iso-PGF2α)、抗双链DNA(Ds-DNA).免疫球蛋白G(IgG).补体3(C3)。同时记录患者血沉(ESR)、系统性红斑狼疮病情活动评分(SLEDAI)的变化。研究结果第一部分动物实验部分1.狼疮模型鼠组的MDA.NO水平分别为(12.35±2.56)nmol/ml和(51.23 ±22.15)umol/1,明显高于对照组的(5.26±1.89)nmol/ml和(32.36±18.56) umol/1(P均<0.05),狼疮模型鼠组的SOD.GSH-PX水平分别为(51.05±12.35)U/m1和(155.19±32.48)U/ml,明显低于对照组的(75.41±20.54)U/ml和(195.34±23.02)U/ml(P均<0.05)。2.狼疮鼠模型组血清MDA、NO水平和抗dsD-NA抗体呈现正相关(r=0.5329,0.6675,P<O.05)而GSH-PX、SOD则与抗dsDNA抗体呈现负相关(r=-0.6476,-0.5746,P<O.05)。3.应用N-乙酰半胱氨酸干预4周后,观察组狼疮模型鼠的MDA.NO水平分别为(11.23±1.64)nmol/ml和(46.28±19.35)umol/l,明显低于非干预组组的(14.12±3.20)nmol/ml和(32.36±18.56)umol/l(P均<0.05),狼疮模型鼠的SOD.GSH-PX水平分别为(83.25±21.46)U/ml和(221.30±23.85)U/ml,明显高于非干预组的(60.12±22.41)U/m1和(183.62±22.14)U/ml(P＜均0.05)。4.应用N-乙酰半胱氨酸干预4周后,观察组狼疮模型鼠的尿蛋白(102.57±18.21)ng/ml,明显低于非干预组(126.24±21.32)ng/ml(P<0.05):观察组狼疮模型鼠的血清ds-DNA(15.40±2.10)ng/ml,明显低于非干预组(18.02 ±2.32)ng/ml(P<0.05).第二部分临床研究部分1.治疗6月后,观察组SLE患者血清NO(36.45±8.4)umol/1.8-iso-PGF2 α(41.26±8.36)ug/l与治疗前NO(56.53±8.9)umol/1.8-iso-PGF2 α(65.32 ±10.23)ug/l比较明显降低(P<0.05),而GSH明显升高(1.01±3.26 mg/gVS0.45 ±0.23 mg/g)(P<0.05).2.对照组SLE治疗6月比较,各指标(N050.37±8.9umol/1,8-iso-PGF2 α 55.65±8.45ug/l,GSHO.62±0.34mg/g)与治疗前(NO57.05±7.8umol/l, 8-iso-PGF2 α 63.59±11.36 ug/l,GSHO.49±0.3lmg/g)均无显著差异(P>O.05)。3.观察组SLE治疗6月SLEDAI(3.78±1.20)与治疗前(7.33±1.41)比较明显降低(P<0.05)；对照组SLE治疗6月SLEDAI(5.50±0.92)与治疗前(7.50±1.41)比较亦明显降低(P<0.05)。治疗6月后,观察组SLEDAI(3.78±1.20)与对照组SLEDAI(5.50±0.92)明显降低(P<0.05)。观察组及对照组治疗6月与治疗前比较IgG无明显差异(19.64±6.83g/1VS23.22±7.23g/1:20.11±5.69 g/1 VS24.95±8.21g/1)(P均>0.05)。观察组及对照组治疗6月与治疗前比较C3变化无明显差异(0.82±0.24g/1VSO.85±0.32g/1:0.73±0.21 g/1 VSO.72±0.26g/1)(P＞0.05).治疗6月观察组ESR(38.65±10.36)mm/h,抗ds-DNA(45.36±16.32)IU/1与治疗前ESR(55.11±13.56)mm/h,抗ds-DNA(78.56±15.36) IU/l比较明显降低(P＜0.05)；对照组SLE治疗6月比较各指标(ESR48.67± 10.54mm/h,抗ds-DNA 60.38±11.85IU/l)与治疗前(ESR 56.39±14.20mm/h,抗ds-DNA 75.63±14.35IU/l)比较均无显著差异(P>0.05)。研究结论1.狼疮模型鼠体内氧化抗氧化失衡,氧化程度较正常对照组高,氧化应激程度与SLE的病情相关。2.NAC治疗能够改善减轻SLE导致的氧化应激状态。3.NAC对早期轻症SLE患者的病情有一定治疗协同作用,能降低SLE早期病情活动性。更多还原

【Abstract】 BackgroundSystemic Lupus Erythematosus (SLE) is an autoimmune disease typically with multi-system involvement, mainly characterized with antinuclear antibodies and other auto-antibodies appear in serum and multi-system damage and multi-system damage. The relationship between oxidative stress imbalance and SLE has been proved by many studies recent years. As an autoimmune disease with high incidence, the pathogenesis of SLE is related to damage of free radicals generated by oxidative stress.Oxidative stress is a balance, which would be destroyed if oxidation factors surpass the scavenging ability of antioxidant system under pathological conditions. And this is also called oxidative stress, causing damage to lipids, proteins, cell membranes and DNA. Reactive oxygen species (ROS) are major active substances that cause pathological damage with rather complicated scavenging mechanism. It is quite difficult to directly detect ROS, while nitric oxide (NO) and malondialdehyde (MDA) levels in serum are conventional indicators of excessive oxidation.8-epi-PGF2α is a recently discovered sensitive indicator, which can reflect oxidative stress and damage degree of free radicals, and is a stable chemical representing the extent of lipid peroxidation. Antioxidant systems in the body of human beings mainly include Superoxide Dismutase (SOD) and Glutathione Peroxidase (GSH-PX). These antioxidants can antagonize ROS, reduce toxic peroxides to nontoxic hydroxyl compounds, promote the decomposition of H2O2, and thus protect the structure and function of cell membranes from interference and damage caused by peroxides.Considering that oxidative stress is closely related to autoimmune diseases like SLE, many research organizations focus on the study of SLE anti-oxidant treatments. N-acetylcysteine (NAC), which is derivative from an unnecessary amino acid L-cysteine acetylation, is a classic antioxidant. It has direct interference on various free radicals generation and ability to scavenge generated free radicals, and can regulate the activity of cell metabolism. Meanwhile, NAC increases the amount of glutathione in the body, is also anti-apoptotic, inhibits inflammatory nuclear factor-kappa B, has a wide range of biological activity, and plays an important role in processes including inflammation, immune response and cell growth regulation.The first randomized double blind case-control stiduy of N-acetyl cysteine (NAC) in the treatment of SLE was published in Arhtritis Rheum magazine published in 2012.Then the American scholar also has carried on the NAC treatment of SLE clinical observation, marked the NAC may become one of the treatment of SLE. But there are no domestic related research materials.The purpose of this subject is to investigate NAC treatment the curative effect of SLE domestic Han population. But because of ethical factors, not the placebo group, but the coordination of NAC and hydroxychloroquine sulfate in the treatment of SLE was observed, Then explore the advantages of joint treatment and the curative effect of NAC for the treatment of SLE.The research goal is to find more clinical therapy choice without iglucocorticoids in treatment of SLE.In order to observe the efficacy of NAC in SLE early-stage anti-oxidative treatment, mouse model was used to stimulate the pathophysiology of SLE. Antioxidant NAC was applied to MRL/lpr mice. In order to explore the impact and effects of NAC treatment on SLE oxidative stress, changes in serum levels of NO, MDA, SOD and GSH-PX in MRL/lpr mice were observed, and changes in serum ds-DNA level and urinary protein were also tested. Meanwhile, in order to analyze the effect of NAC on SLE acute phase, patients with mild SLE were treated with non-glucocorticoid containing NAC. Research ObjectivesPart 1. Animal Experiments1.Understand the characters of lupus early-stage oxidative stress by observing the changes of serum oxidative stress indicators in spontaneous lupus MRL/lpr mice.2.Preliminary explore the effect and efficacy of antioxidant NAC on oxidative stress indicators in spontameous lupus MRL/lpr mice.Part 2. Clinical Experiments1.Observe the short-term changes of NO, GSH,8-iso-PGF2a, ESR, Ds-DNA, IgG and systemic lupus erythematosus disease assessment indices (SLEDAI) after using NAC for SLE treatment.2.Investigate whether short-term efficacy can be increased by treating SLE patients with NAC in combination with non-glucocorticoid. Research MethodsPart 1. Animal ExperimentsThe study had 3 groups:The normal control group (BALB/c mice) had six mice. MRL/lpr group had 20 mice, which were randomly divided into non-intervention and intervention groups with 10 mice each. Mice in intervention group were given NAC 150 mg intra-peritoneal injection daily for 30 days. Mice in normal control group and observation group were given 0.9% NaCl intra-peritoneal injection daily for 30 days. Sufficient amounts of urine and serum samples were obtained by killing the mice after 30 days to test changes of urinary protein and anti-dsDNA antibody respectively. Serum levels of NO, MDA, SOD and GSH-PX were measured respectively by itrate reductase method, thiobarbituric acid method, xanthine oxidase-hydroxylamine method, chemical colorimetric method for mice in every group. The changes in the levels of these indicators were observed and analyzed in order to explore the NAC efficacy on SLE.Part 2:Clinical Experiments65 patients with mild SLE were divided into control group with 28cases and observation group with 35 cases. All the patients were treated with hydroxychloroquine sulfate while patients in observation group were treated with NAC (1200mg/daily) additionally. Changes of serum NO, GSH,8-iso-PGF2a, ESR, Ds-DNA, IgG and SLEDAI were tested before and 6 months after treatments.ResultsPart 1. Animal ExperimentThe MDA (12.35±2.56 nmol/mL) and NO (51.23±22.15 umol/L) levels in lupus mice were significantly higher than the MDA (5.26 ±1.89 nmol/ml) and NO (32.36 ±18.56 umol/L) in control group (P<0.05). While the SOD (51.05 ±12.35 U/mL) and GSH-PX (155.19±32.48 U/mL) were significantly lower than the SOD (75.41 ±20.54 U/mL) and GSH-PX (195.34 ±23.02 U/mL) in control group (P<0.05).Serum MDA and NO levels in lupus mice were positively correlated with anti-dsDNA antibody (r=0.53290.66752, P<0.05) while GSH-PX and SOD were negatively correlated (r=-0.6476,-0.5746, P<0.05).After 4-week intervention with NAC, MDA and NO levels in lupus mice in intervention group were 11.23±1.64 nmol/mL and 46.28±19.35 umol/L respectively, significantly lower than MDA (14.12±3.20 nmol/mL) and NO (32.36 ±18.56 umol/L) in non-intervention group (P<0.05). SOD and GSH-PX levels in intervention group were 83.25±21.46 U/mL and 221.30±23.85 U/mL respectively, significantly higher than SOD (60.12±22.41 U/mL) and GSH-PX (183.62±22.14 U/mL) in non-intervention group (P<0.05).After 4-week intervention with NAC, urinary protein level of 102.57±18.21ng/mL in lupus mice intervention group was significantly lower than that (126.24±21.32 ng/mL) in non-intervention group (P<0.05). However, serum ds-DNA level (15.40±2.10 ng/mL) in intervention group was significantly lower than that (18.02 ±2.32ng/ml) in non-intervention group (P<0.05).Part 2. Clinical ExperimentsAfter 6-month treatment, NO (36.45±8.4 umol/L) and 8-iso-PGF2a (41.26±8.36 ug/L) of SLE patients in observation group significantly decreased compared with NO (56.53±8.9 umol/L) and 8-iso-PGF2a (65.32±10.23 ug/L) levels before treatment (P<0.05), while GSH level increased obviously (1.01±3.26 mg/g vs.0.45±0.23 mg/g) (P<0.05).After 6-month SLE treatment for control group, there was no significant difference for various indicators (NO 50.37±8.9 umol/L,8-iso-PGF2a 55.65±8.45 ug/L, GSH 0.62±0.34 mg/g) compared with those (NO 57.05±7.8 umol/L, 8-iso-PGF2a63.59± 11.36 ug/L, GSH 0.49+0.31 mg/g) before treatment (P>0.05).SLEDAI (3.78± 1.20) in observation group 6 months after SLE treatment decreased obviously compared with that (7.33±1.41) before treatment (P<0.05); SLEDAl (5.50±0.92) in control group 6 months after SLE treatment also decreased obviously compared with that (7.50±.41) before treatment (P<0.05).IgG levels in observation group and control group before and 6 months after treatment had no significant difference (19.64±6.83 g/L vs.23.22±7.23 g/L; 20.11±5.69 g/L vs.24.95±8.21 g/L) (P>0.05).and C3 levels in observation group and control group before and 6 months after treatment had no significant difference too (0.82±0.24g/lVS0.85±0.32g/l;0.73±0.21 g/1 VS0.72±0.26g/l) (P>0.05).ESR (38.65±0.36 mm/h) and ds-DNA (45.36±16.32 IU/L) in observation group 6 months after treatment decreased obviously as compared with ESR (55.11±13.56 mm/h) and ds-DNA (78.56±15.36 IU/L) before treatment (P<0.05). For control group, however, various indicators had no significant change before (ESR 56.39±14.20 mm/h, ds-DNA 75.63±14.35 IU/L) and 6 months after (ESR 48.67±10.54 mm/h, ds-DNA 60.38±11.85 IU/L) treatment (P>0.05).ConclusionThere was imbalance between oxidant and antioxidant in the bodies of lupus model mice. The degree of oxidation was higher than normal control group, and the degree of oxidative stress was related to lupus disease.NAC is able to relieve oxidative stress caused by lupus.NAC has a certain role in controlling the patients’ conditions with early-stage mild SLE, and has efficacy in decreasing the early activity of SLE.更多还原