昆仙胶囊对MRL/lpr小鼠狼疮性肾炎代谢组学影响的研究
摘要
一、目的与意义
狼疮性肾炎(Lupus nephritis, LN)是系统性红斑狼疮(SLE)累及肾脏所引起的一种免疫复合物肾炎,临床上以发热、关节炎、皮疹及肾脏损害症状为主要表现。约有40%～75%的SLE患者在患病5年内出现临床肾脏受累表现,而肾活检几乎100%有肾脏病理改变。LN是SLE患者最严重的并发症,也是最常见的死亡原因之一,对其进行积极治疗和有效地控制病情进展,至关重要。
到目前为止,有关LN的病因和发病机制尚未完全清楚,但其主要的发病过程已得到深入研究：高度活化的B淋巴细胞产生自身抗体,与自身抗原形成免疫复合物是致病的主要因素,相应的细胞因子在发病过程中起辅助作用。近年来,国内外研究发现,细胞凋亡异常,产生核小体,暴露自身隐蔽抗原,可能是发病的主要始动环节。另外,树突状细胞也参与了LN的发病和发展。
LN治疗的最终目标是防止疾病的复发,保护肾功能,尽可能减少并发症,促进患者的康复。对LN的早期诊断、早期药物治疗是缓解症状和控制疾病进程的关键。现代医学认为,LN的治疗原则应包括免疫抑制治疗和支持治疗,免疫抑制治疗的强度应根据临床表现、血清学检查结果及肾脏病变的组织学活性确定。支持治疗包括严格控制高血压和高脂血症,及其他防治慢性肾脏病(CKD)的治疗手段。虽然西药能在一定程度上控制疾病的活动和发展,但许多药物不良反应明显,停药后病情易复发,且价格昂贵,为此我们有必要结合中医药治疗LN。
中医无系统性红斑狼疮病名,但其临床表现在文献中有类似描述,如“蝴蝶斑”、“阴阳毒”、“日晒疮”等,而狼疮性肾炎则见于“水肿”、“虚损”等病证中。目前,不少医家根据中医理论对LN的病因、病机、辨证论治等方面进行了长期而广泛深入的讨论,形成了较完善的理论体系。多数学者认为,LN起于先天禀赋不足、肝肾阴虚,在情志内伤、劳倦过度、六淫侵袭、阳光暴晒等诱发因素的作用下,导致热毒内盛或瘀血阻络,内侵脏腑而成。在LN病情发展过程中,无论活动期、缓解期和恢复期,中医辨证“肾虚”始终存在。故治疗应以补肾固本贯穿始终。在临床治疗上,发挥中医药多靶点治疗疾病的优势,可以提高本病的整体疗效。因此,如何利用传统中医药的特长,对这一难治性疾病进行有效的治疗,是研究LN的重要课题之一。
代谢组学是一门在新陈代谢的动态进程中,系统研究代谢产物的变化规律,揭示机体生命活动代谢本质的科学。应用核磁共振(NMR)、液相色谱-质谱(LC-MS)、气相色谱—质谱(GC-MS)等高通量、高分辨、高灵敏度的现代仪器分析手段,获得体液中代谢产物的信息。通过代谢组学方法,研究生物体内所有代谢产物(主要是低分子量代谢物)在疾病或外源性物质等因素扰动下的动态变化,可以反映生物体的病理生理变化趋势,进而揭示其变化的机制。因此,代谢组学对研究疾病的发病、诊断、药物治疗有着直接意义。
昆仙胶囊是由昆明山海棠、淫羊藿、枸杞子、菟丝子组成,具有温阳益肾,活血祛风除湿等功效。其主要成分昆明山海棠(THH)是卫矛科雷公藤属药物,与中药雷公藤含有多种相同的活性成分,具有抗炎和免疫抑制作用。近20年来,THH被广泛应用于治疗类风湿关节炎、纤维组织炎、红斑狼疮、慢性肾炎等多种结缔组织病及自身免疫性疾病。昆仙胶囊作为一种新型免疫抑制剂,对慢性肾脏病引起的蛋白尿均有较好的疗效,并且治疗期间无严重的肝、肾功能损害或白细胞降低等情况,服用较安全,已在临床上广泛应用于狼疮性肾炎的治疗。目前,尽管对昆仙胶囊已经有大量的临床疗效报道,但是实验研究相对滞后。所以,应该加强对昆仙胶囊作用机理的研究,为有效利用其药效作用和防治、拮抗不良反应做好基础。
MRL/lpr小鼠是一种先天性免疫缺陷小鼠,是常用的SLE动物模型之一。MRL/lpr小鼠的症状与人类红斑狼疮相似,包括显著的血清自身抗体,免疫复合物肾小球肾炎,血管炎等。通过对其研究阐明了人类LN发病机制中的很多问题,对当前发展新的诊疗方法有重要意义。
为了验证昆仙胶囊对LN的治疗作用,为临床应用提供实验依据,本实验采用MRL/lpr小鼠狼疮肾炎模型,观察昆仙胶囊对狼疮肾炎小鼠血清抗体,蛋白尿,免疫器官的影响及小鼠血清的代谢表型改变,并对其作用机理进行初步探讨。
二、方法和内容
2.1研究对象
将18只12-14周龄MRL/lpr小鼠随机分为三组：模型对照组6只；昆仙胶囊组6只；来氟米特组6只。分别给予生理盐水；昆仙胶囊0.234mg/g/d；来氟米特片0.0026mg/g/d。各组小鼠每天固定时间灌胃,连续给药4周。
2.2一般情况
每周记录小鼠体重并观察各组小鼠精神状态、食量、活动、关节、毛色等一般情况。
2.3尿蛋白及血清抗体检测
给药前后测尿蛋白浓度及血清ANA、抗dsDNA抗体的含量。
2.4小鼠免疫器官重量测定
末次给药24h后称体重,放血处死小鼠,摘胸腺、脾脏、称其湿重,分别计算脾指数和胸腺指数(mg/10g)。脾(胸腺)指数=10×脾(胸腺)重(mg)/体重(g)。
2.5病理组织检查
小鼠双肾置于4%中性甲醛溶液中固定,石蜡包埋、切片,作HE染色,进行光镜检查。
2.6代谢组学研究
用基于1H-NMR技术的代谢组学方法检测用药后各组小鼠血清,观察昆仙胶囊对小鼠血清中代谢产物的影响,并对相应机制进行探讨。
2.7数据整理与统计
采用SPSS13.0、MestReNova5.3.1及SIMCA-P12.0.1软件包对数据和图谱进行分析。
三、结果
3.1各组小鼠的一般情况
实验开始前,各组小鼠均毛色光滑,饮食、活动、体重无差别。在实验过程中,模型对照组小鼠逐渐出现食欲减退,生长缓慢,活动减少,精神萎靡,反应迟钝等表现。
3.2尿蛋白及血清抗体检测
3.2.1尿蛋白检测
给药前,三组小鼠尿蛋白浓度无显著性差异(P>0.05)。给药4周末,昆仙胶囊组和来氟米特组小鼠尿蛋白浓度与模型对照组比较明显降低(P<0.05,P<0.01);并且两组间比较无显著性差异(P>0.05)。比较各组小鼠实验前后尿蛋白浓度发现,两个治疗组小鼠给药后比给药前尿蛋白浓度均降低(P<0.05,P<0.05)；而模型对照组实验前后比较无显著性差异(P>0.05)。
3.2.2血清抗体检测
三组小鼠给药前,血清ANA水平及抗dsDNA抗体水平均无显著性差异(P>0.05)。给药后,两个用药组与模型对照组比较,血清抗体水平均明显降低,差异具有显著性(P<0.01,P<0.01)。与给药前比,模型对照组ANA水平在给药4周末无明显变化(P>0.05),而抗dsDNA抗体；水平显著升高(P<0.01)；昆仙胶囊组与来氟米特组给药后血清ANA水平均明显降低,差异具有显著性(P<0.01,P<0.01),抗dsDNA抗体水平虽比给药前有所升高,但是显著低于同时间模型对照组抗体水平(P<0.05)。
3.3免疫器官重量测定结果
昆仙胶囊组胸腺指数明显升高,与模型对照组比较差异有统计学意义(P<0.05),但脾指数与模型对照组相比无显著差异。来氟米特组脾指数明显上调(P<0.05)。
3.4肾脏病理学检查
光镜下观察到模型对照组小鼠肾小球系膜细胞、内皮细胞呈轻中度增生,而两个给药组小鼠肾脏病变均较模型对照组有所减轻(P<0.01)。
3.5代谢组学改变
两个给药组跟模型对照组比较发现,支链氨基酸、丙氨酸、糖蛋白、酮体、柠檬酸、肌酸、磷酸胆碱、牛磺酸、葡萄糖等代谢组分发生改变。
四、结论
4.1通过观察昆仙胶囊对LN模型小鼠免疫学指标及肾脏病理改变的影响,发现昆仙胶囊可以显著降低自身抗体及尿蛋白水平；并能调节免疫器官,控制肾脏病理损害的进展。提示昆仙胶囊能够控制LN的活动,缓解病情。
4.2用基于1H-NMR技术的代谢组学方法检测用药后各组小鼠血清,研究结果显示,昆仙胶囊组小鼠血清跟模型对照组比较,支链氨基酸、丙氨酸、糖蛋白、柠檬酸、磷酸胆碱、牛磺酸、葡萄糖含量升高；而酮体、肌酸等代谢组分含量则低于模型对照组。推测昆仙胶囊发挥作用的机制包含以下几个方面：一、促进机体正常能量代谢以及增强机体抗氧化能力、免疫力；二、稳定细胞膜,维持细胞内外渗透压平衡；三、改善肾功能；四、控制体内细胞的异常凋亡。
1.Objectives and significance
Lupus nephritis (LN) is an inflammation of the kidney caused by systemic lupus erythematosus (SLE), a disease of the immune system. Clinically, fever, arthritis, skin rashes and kidney damage are mainly symptoms. About 40% to 75% of the SLE patients have the clinical manifestations of kidney damage in 5-year disease. Pathological changes of kidney can be found in almost 100% of the patients by Kidney biopsy. LN is a serious complication of systemic lupus erythematosus and one of the most common cause of death.Its is essential that giving Effective treatment to control the progresses of disease.
Until now, the cause and mechanism of LN is not entirely clear, but the main link has been studyed thoroughly. The immune complexes of autoantibodies produced by Highly activated B lymphocytes and autoantigen are the key elements of disease. Corresponding cytokines play a supporting role in the pathogenesis of LN. in recent years, domestic and foreign research find that abnomal cell apoptosis form nucleosome and exposure their Concealed antigen,which may be the important factor for the onset of lupus. In addition, dendritic cells are involved in the pathogenesis and development of LN.
Ultimate goal of treatment of LN is to prevent the recurrence, protect the renal function, minimize complications and promote the rehabilitation of patients.the early diagnosis and therapy Of LN to relieve symptoms and control the disease process are the key.
Principle of treatment of LN should include immunosuppressive therapy and supportive care. The intensity of immunosuppressive therapy should be based on clinical manifestations, serological findings and renal histological lesions of activity to determine. Supportive care includes strict control of hypertension and hyperlipidemia, and other prevention and treatment of chronic kidney disease (CKD). To a Certain extent, Western medicine can controll activities and development of disease. But there are also many significant adverse drug reactions and relapse after the withdrawal, and these expensive drugs brought a heavy burden to patients. Therefore, we need to combine traditional Chinese medicine to treat LN.
Actually no record concering " systemic lupus erythematosus" in traditional Chinese medicine(TCM)files is found. But it's clinical manifestations described in the literature, such as "Hu Dieban","yin and yang poison","sun sores", etc.LN can be classified as "edema" and "Zhou Bi". Currently, according to TCM theory, many doctors make an intensive and long study of etiology, pathogenesis, diagnosis and treatment and other aspects of LN. Most scholars believe that, LN is due to congential defect and deficiency of yin of both liver and kidney, which lead to heat-toxin, blood stasis and damage of organs And emotional factors, fatigue, sun exposure and six excessive atmosphric factors are the incentives.During development process in the LN condition, "Deficiency of Kidney"is always present in active, remission and recovery stage. Therefore, treatment should attach importance to tonifying Kidney throughout.In clinical treatment, TCM can play the advantages of multi-target in treatment of LN and improve the overall efficacy of the disease. As a result,the issues on how to fully explore the advantages and potential superiorities of TCM to treat refractory diseases is considered as one of the most important issues in LN research.
Metabolism is a subject that researchs the variation of metabolites and reveals the essence of life activities. Because of application of nuclear magnetic resonance (NMR), liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), which are high-throughput, high-resolution, high-sensitivity,people can use these modern instrumental analysis methods to obtain information on metabolites in body fluids. By using metabolomics method to study all the metabolites in vivo (mainly low molecular weight metabolites) in the disease or dynamic change under exogenous factors can reflect changes in trends in the pathophysiology of living organisms, and thus reveal the mechanism. Therefore, metabolomics has a direct meaning to study diseases, diagnosis and drug therapy.
Kunxian capsule are composed of tripterygium hypoglaucum hutch, Epimedium, wolfberry fruit, dodder seed With Warming Kidney, promoting blood circulation, expelling Wind and removing Dampness and other effects. Tripterygium (TH) The main component is the Celastraceae Tripterygium drugs and contains the same active ingredient as Tripterygium wilfordii, and has anti-inflammatory and immunosuppressive effects. In the past 20 years, TH is widely used in the treatment of rheumatoid arthritis, fibrositis, lupus, chronic nephritis, and other connective tissue diseases and autoimmune diseases. Kunxian Capsules as a novel immunosuppressant, have a good effect on proteinuria caused by chronic kidney disease, and have no serious liver and kidney dysfunction or leukopenia during treatment etc. Now it's widely used in clinical treatment of lupus nephritis. At present, although a large number of the clinical efficacy of Kunxian capsule has been reported, experimental research is lagging behind. Therefore, we should strengthen study of the mechanism of Kunxian capsules. And lay the foundation of effective using of its efficacy and controling adverse reactions.
MRL/lpr mice is a congenital immune deficient mice, can occur spontaneously autoimmune disease, manifested as inflammatory cell infiltration, vasculitis, proliferation of mesangial cells and interstitial tubular lesions, and proteinuria and renal function Hypothyroidism, renal pathology and human lupus nephritis are very similar. Through its research to clarify the pathogenesis of human SLE are many problems in the current development of new treatment methods is important
In the present study, the effect of Kunxian capsule on MRL/lpr mice with lupus will be observed by detecting proteinuria and immune organs and analyzing the changes of serum metabolic phenotype.
2.Methods and project
2.1 Subjects
18 MRL/lpr mice,12～14 weeks were selected and divided into the model control group (A group), Kun xian capsule group (B group) and leflunomide group (C group). A Group is given normal saline; B group is given Kunxian Capsules 0.234mg/g/d and C group is given leflunomide tablets 0.0026mg/g/d. Mice are fed in a fixed time every day and the whole administration last 4 weeks.
2.2 General situation
Record weight of the mice Weekly and observe diet, activities generally.
2.3 Urinary protein and serum antibodies testing
To measure the ANA, and dsDNA and urinary protein concentration.
2.4 Weight determination of immune organ
After the last administration, mice are weighed and sacrificed. Then calculate the spleen index and thymus index (mg/10g) respectively. Spleen (thymus) Index=10×spleen (thymus) weight (mg)/body weight (g).
2.5 Histopathologic examination
The kidneys of mice are fixed in 10% neutral formalin, embedded in paraffin and then sliced and detected by HE staining.
2.6 Metabonomics
Serum samples that are collected after administration from mice are researed by 1H NMR spectroscopy. View the differences in metabolites of urine after the treatment with Kunxian capsule, and discuss the corresponding mechanisms
2.7 Data processing and statistics
Data and figures are analyzed by using SPSS 13.0, MestReNova5.3.1 and software package of SIMCA-P 12.0.1.
3 Results
3.1 The general condition of mice
Before the start of the experiment, coat, diet, activity, body weight of the mice have no difference. During the experiment, the model control group were loss of appetite, slow-growing, unresponsive etc.
3.2 Detection of serum antibodies and proteinuria
3.2.1 Urine protein concentration
Before administration, urine protein concentration in three groups of mice had no significant difference (P> 0.05). Administration of 4 weeks, the urine protein concentrations in two treated group was significantly decreased (P<0.05, P<0.01); and between the two groups showed no significant difference (P> 0.05).
3.2.2 Serum antibodies
Before administration, the serum anti-ANA antibody and anti-dsDNA antibody levels were not significantly different (P> 0.05). After administration, serum antibody levels in Kunxian capsule group and Leflunomide group compared with the control group were significantly lower, the difference was significant (P<0.01, P<0.01).
3.3 Results of weight measurement of immune organs
The thymus index in Kun Xian Capsules group were significantly increased compared with the control group (P<0.05). The spleen index in Leflunomide group also significantly increased (P<0.01).
3.4 Renal pathology
Model control group were observed mild or moderate hyperplasia in mesangial cells, endothelial cells. Compared with model control group,two treated group have Minor kidney damage (P<0.01), while Between the two treated groups showed no significant difference (P> 0.05).
3.5 Changes of metabolomics
The difference between treated group and model group were branched-chain amino acids, alanine, sugar, protein, ketones, citric acid, creatine, choline phosphate, taurine, glucose and other metabolic components.
4 Conclusion
4.1 Kunxian Capsules can significantly reduce the level of autoantibodies and proteinuria, can regulate the immune organs, and control the progress of Clinical Pathology. Kunxian Capsules contributes to control the activities of LN and remission of disease.
4.2 After treatment, detect metabolic components in serum by 1H-NMR-based metabonomics technology.The result suggesting that the mechanism of Kunxian Capsules contains the following aspects: 1. It can promote the body's normal energy metabolism and enhanced antioxidant capacity and immunity; 2. It can stable cell membrane to maintain osmotic balance inside and outside cells; 3. It can improve renal function; 4. It can control abnormal cell apoptosis in vivo.
狼疮性肾炎(Lupus nephritis, LN)是系统性红斑狼疮(SLE)累及肾脏所引起的一种免疫复合物肾炎,临床上以发热、关节炎、皮疹及肾脏损害症状为主要表现。约有40%～75%的SLE患者在患病5年内出现临床肾脏受累表现,而肾活检几乎100%有肾脏病理改变。LN是SLE患者最严重的并发症,也是最常见的死亡原因之一,对其进行积极治疗和有效地控制病情进展,至关重要。
到目前为止,有关LN的病因和发病机制尚未完全清楚,但其主要的发病过程已得到深入研究：高度活化的B淋巴细胞产生自身抗体,与自身抗原形成免疫复合物是致病的主要因素,相应的细胞因子在发病过程中起辅助作用。近年来,国内外研究发现,细胞凋亡异常,产生核小体,暴露自身隐蔽抗原,可能是发病的主要始动环节。另外,树突状细胞也参与了LN的发病和发展。
LN治疗的最终目标是防止疾病的复发,保护肾功能,尽可能减少并发症,促进患者的康复。对LN的早期诊断、早期药物治疗是缓解症状和控制疾病进程的关键。现代医学认为,LN的治疗原则应包括免疫抑制治疗和支持治疗,免疫抑制治疗的强度应根据临床表现、血清学检查结果及肾脏病变的组织学活性确定。支持治疗包括严格控制高血压和高脂血症,及其他防治慢性肾脏病(CKD)的治疗手段。虽然西药能在一定程度上控制疾病的活动和发展,但许多药物不良反应明显,停药后病情易复发,且价格昂贵,为此我们有必要结合中医药治疗LN。
中医无系统性红斑狼疮病名,但其临床表现在文献中有类似描述,如“蝴蝶斑”、“阴阳毒”、“日晒疮”等,而狼疮性肾炎则见于“水肿”、“虚损”等病证中。目前,不少医家根据中医理论对LN的病因、病机、辨证论治等方面进行了长期而广泛深入的讨论,形成了较完善的理论体系。多数学者认为,LN起于先天禀赋不足、肝肾阴虚,在情志内伤、劳倦过度、六淫侵袭、阳光暴晒等诱发因素的作用下,导致热毒内盛或瘀血阻络,内侵脏腑而成。在LN病情发展过程中,无论活动期、缓解期和恢复期,中医辨证“肾虚”始终存在。故治疗应以补肾固本贯穿始终。在临床治疗上,发挥中医药多靶点治疗疾病的优势,可以提高本病的整体疗效。因此,如何利用传统中医药的特长,对这一难治性疾病进行有效的治疗,是研究LN的重要课题之一。
代谢组学是一门在新陈代谢的动态进程中,系统研究代谢产物的变化规律,揭示机体生命活动代谢本质的科学。应用核磁共振(NMR)、液相色谱-质谱(LC-MS)、气相色谱—质谱(GC-MS)等高通量、高分辨、高灵敏度的现代仪器分析手段,获得体液中代谢产物的信息。通过代谢组学方法,研究生物体内所有代谢产物(主要是低分子量代谢物)在疾病或外源性物质等因素扰动下的动态变化,可以反映生物体的病理生理变化趋势,进而揭示其变化的机制。因此,代谢组学对研究疾病的发病、诊断、药物治疗有着直接意义。
昆仙胶囊是由昆明山海棠、淫羊藿、枸杞子、菟丝子组成,具有温阳益肾,活血祛风除湿等功效。其主要成分昆明山海棠(THH)是卫矛科雷公藤属药物,与中药雷公藤含有多种相同的活性成分,具有抗炎和免疫抑制作用。近20年来,THH被广泛应用于治疗类风湿关节炎、纤维组织炎、红斑狼疮、慢性肾炎等多种结缔组织病及自身免疫性疾病。昆仙胶囊作为一种新型免疫抑制剂,对慢性肾脏病引起的蛋白尿均有较好的疗效,并且治疗期间无严重的肝、肾功能损害或白细胞降低等情况,服用较安全,已在临床上广泛应用于狼疮性肾炎的治疗。目前,尽管对昆仙胶囊已经有大量的临床疗效报道,但是实验研究相对滞后。所以,应该加强对昆仙胶囊作用机理的研究,为有效利用其药效作用和防治、拮抗不良反应做好基础。
MRL/lpr小鼠是一种先天性免疫缺陷小鼠,是常用的SLE动物模型之一。MRL/lpr小鼠的症状与人类红斑狼疮相似,包括显著的血清自身抗体,免疫复合物肾小球肾炎,血管炎等。通过对其研究阐明了人类LN发病机制中的很多问题,对当前发展新的诊疗方法有重要意义。
为了验证昆仙胶囊对LN的治疗作用,为临床应用提供实验依据,本实验采用MRL/lpr小鼠狼疮肾炎模型,观察昆仙胶囊对狼疮肾炎小鼠血清抗体,蛋白尿,免疫器官的影响及小鼠血清的代谢表型改变,并对其作用机理进行初步探讨。
二、方法和内容
2.1研究对象
将18只12-14周龄MRL/lpr小鼠随机分为三组：模型对照组6只；昆仙胶囊组6只；来氟米特组6只。分别给予生理盐水；昆仙胶囊0.234mg/g/d；来氟米特片0.0026mg/g/d。各组小鼠每天固定时间灌胃,连续给药4周。
2.2一般情况
每周记录小鼠体重并观察各组小鼠精神状态、食量、活动、关节、毛色等一般情况。
2.3尿蛋白及血清抗体检测
给药前后测尿蛋白浓度及血清ANA、抗dsDNA抗体的含量。
2.4小鼠免疫器官重量测定
末次给药24h后称体重,放血处死小鼠,摘胸腺、脾脏、称其湿重,分别计算脾指数和胸腺指数(mg/10g)。脾(胸腺)指数=10×脾(胸腺)重(mg)/体重(g)。
2.5病理组织检查
小鼠双肾置于4%中性甲醛溶液中固定,石蜡包埋、切片,作HE染色,进行光镜检查。
2.6代谢组学研究
用基于1H-NMR技术的代谢组学方法检测用药后各组小鼠血清,观察昆仙胶囊对小鼠血清中代谢产物的影响,并对相应机制进行探讨。
2.7数据整理与统计
采用SPSS13.0、MestReNova5.3.1及SIMCA-P12.0.1软件包对数据和图谱进行分析。
三、结果
3.1各组小鼠的一般情况
实验开始前,各组小鼠均毛色光滑,饮食、活动、体重无差别。在实验过程中,模型对照组小鼠逐渐出现食欲减退,生长缓慢,活动减少,精神萎靡,反应迟钝等表现。
3.2尿蛋白及血清抗体检测
3.2.1尿蛋白检测
给药前,三组小鼠尿蛋白浓度无显著性差异(P>0.05)。给药4周末,昆仙胶囊组和来氟米特组小鼠尿蛋白浓度与模型对照组比较明显降低(P<0.05,P<0.01);并且两组间比较无显著性差异(P>0.05)。比较各组小鼠实验前后尿蛋白浓度发现,两个治疗组小鼠给药后比给药前尿蛋白浓度均降低(P<0.05,P<0.05)；而模型对照组实验前后比较无显著性差异(P>0.05)。
3.2.2血清抗体检测
三组小鼠给药前,血清ANA水平及抗dsDNA抗体水平均无显著性差异(P>0.05)。给药后,两个用药组与模型对照组比较,血清抗体水平均明显降低,差异具有显著性(P<0.01,P<0.01)。与给药前比,模型对照组ANA水平在给药4周末无明显变化(P>0.05),而抗dsDNA抗体；水平显著升高(P<0.01)；昆仙胶囊组与来氟米特组给药后血清ANA水平均明显降低,差异具有显著性(P<0.01,P<0.01),抗dsDNA抗体水平虽比给药前有所升高,但是显著低于同时间模型对照组抗体水平(P<0.05)。
3.3免疫器官重量测定结果
昆仙胶囊组胸腺指数明显升高,与模型对照组比较差异有统计学意义(P<0.05),但脾指数与模型对照组相比无显著差异。来氟米特组脾指数明显上调(P<0.05)。
3.4肾脏病理学检查
光镜下观察到模型对照组小鼠肾小球系膜细胞、内皮细胞呈轻中度增生,而两个给药组小鼠肾脏病变均较模型对照组有所减轻(P<0.01)。
3.5代谢组学改变
两个给药组跟模型对照组比较发现,支链氨基酸、丙氨酸、糖蛋白、酮体、柠檬酸、肌酸、磷酸胆碱、牛磺酸、葡萄糖等代谢组分发生改变。
四、结论
4.1通过观察昆仙胶囊对LN模型小鼠免疫学指标及肾脏病理改变的影响,发现昆仙胶囊可以显著降低自身抗体及尿蛋白水平；并能调节免疫器官,控制肾脏病理损害的进展。提示昆仙胶囊能够控制LN的活动,缓解病情。
4.2用基于1H-NMR技术的代谢组学方法检测用药后各组小鼠血清,研究结果显示,昆仙胶囊组小鼠血清跟模型对照组比较,支链氨基酸、丙氨酸、糖蛋白、柠檬酸、磷酸胆碱、牛磺酸、葡萄糖含量升高；而酮体、肌酸等代谢组分含量则低于模型对照组。推测昆仙胶囊发挥作用的机制包含以下几个方面：一、促进机体正常能量代谢以及增强机体抗氧化能力、免疫力；二、稳定细胞膜,维持细胞内外渗透压平衡；三、改善肾功能；四、控制体内细胞的异常凋亡。
1.Objectives and significance
Lupus nephritis (LN) is an inflammation of the kidney caused by systemic lupus erythematosus (SLE), a disease of the immune system. Clinically, fever, arthritis, skin rashes and kidney damage are mainly symptoms. About 40% to 75% of the SLE patients have the clinical manifestations of kidney damage in 5-year disease. Pathological changes of kidney can be found in almost 100% of the patients by Kidney biopsy. LN is a serious complication of systemic lupus erythematosus and one of the most common cause of death.Its is essential that giving Effective treatment to control the progresses of disease.
Until now, the cause and mechanism of LN is not entirely clear, but the main link has been studyed thoroughly. The immune complexes of autoantibodies produced by Highly activated B lymphocytes and autoantigen are the key elements of disease. Corresponding cytokines play a supporting role in the pathogenesis of LN. in recent years, domestic and foreign research find that abnomal cell apoptosis form nucleosome and exposure their Concealed antigen,which may be the important factor for the onset of lupus. In addition, dendritic cells are involved in the pathogenesis and development of LN.
Ultimate goal of treatment of LN is to prevent the recurrence, protect the renal function, minimize complications and promote the rehabilitation of patients.the early diagnosis and therapy Of LN to relieve symptoms and control the disease process are the key.
Principle of treatment of LN should include immunosuppressive therapy and supportive care. The intensity of immunosuppressive therapy should be based on clinical manifestations, serological findings and renal histological lesions of activity to determine. Supportive care includes strict control of hypertension and hyperlipidemia, and other prevention and treatment of chronic kidney disease (CKD). To a Certain extent, Western medicine can controll activities and development of disease. But there are also many significant adverse drug reactions and relapse after the withdrawal, and these expensive drugs brought a heavy burden to patients. Therefore, we need to combine traditional Chinese medicine to treat LN.
Actually no record concering " systemic lupus erythematosus" in traditional Chinese medicine(TCM)files is found. But it's clinical manifestations described in the literature, such as "Hu Dieban","yin and yang poison","sun sores", etc.LN can be classified as "edema" and "Zhou Bi". Currently, according to TCM theory, many doctors make an intensive and long study of etiology, pathogenesis, diagnosis and treatment and other aspects of LN. Most scholars believe that, LN is due to congential defect and deficiency of yin of both liver and kidney, which lead to heat-toxin, blood stasis and damage of organs And emotional factors, fatigue, sun exposure and six excessive atmosphric factors are the incentives.During development process in the LN condition, "Deficiency of Kidney"is always present in active, remission and recovery stage. Therefore, treatment should attach importance to tonifying Kidney throughout.In clinical treatment, TCM can play the advantages of multi-target in treatment of LN and improve the overall efficacy of the disease. As a result,the issues on how to fully explore the advantages and potential superiorities of TCM to treat refractory diseases is considered as one of the most important issues in LN research.
Metabolism is a subject that researchs the variation of metabolites and reveals the essence of life activities. Because of application of nuclear magnetic resonance (NMR), liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), which are high-throughput, high-resolution, high-sensitivity,people can use these modern instrumental analysis methods to obtain information on metabolites in body fluids. By using metabolomics method to study all the metabolites in vivo (mainly low molecular weight metabolites) in the disease or dynamic change under exogenous factors can reflect changes in trends in the pathophysiology of living organisms, and thus reveal the mechanism. Therefore, metabolomics has a direct meaning to study diseases, diagnosis and drug therapy.
Kunxian capsule are composed of tripterygium hypoglaucum hutch, Epimedium, wolfberry fruit, dodder seed With Warming Kidney, promoting blood circulation, expelling Wind and removing Dampness and other effects. Tripterygium (TH) The main component is the Celastraceae Tripterygium drugs and contains the same active ingredient as Tripterygium wilfordii, and has anti-inflammatory and immunosuppressive effects. In the past 20 years, TH is widely used in the treatment of rheumatoid arthritis, fibrositis, lupus, chronic nephritis, and other connective tissue diseases and autoimmune diseases. Kunxian Capsules as a novel immunosuppressant, have a good effect on proteinuria caused by chronic kidney disease, and have no serious liver and kidney dysfunction or leukopenia during treatment etc. Now it's widely used in clinical treatment of lupus nephritis. At present, although a large number of the clinical efficacy of Kunxian capsule has been reported, experimental research is lagging behind. Therefore, we should strengthen study of the mechanism of Kunxian capsules. And lay the foundation of effective using of its efficacy and controling adverse reactions.
MRL/lpr mice is a congenital immune deficient mice, can occur spontaneously autoimmune disease, manifested as inflammatory cell infiltration, vasculitis, proliferation of mesangial cells and interstitial tubular lesions, and proteinuria and renal function Hypothyroidism, renal pathology and human lupus nephritis are very similar. Through its research to clarify the pathogenesis of human SLE are many problems in the current development of new treatment methods is important
In the present study, the effect of Kunxian capsule on MRL/lpr mice with lupus will be observed by detecting proteinuria and immune organs and analyzing the changes of serum metabolic phenotype.
2.Methods and project
2.1 Subjects
18 MRL/lpr mice,12～14 weeks were selected and divided into the model control group (A group), Kun xian capsule group (B group) and leflunomide group (C group). A Group is given normal saline; B group is given Kunxian Capsules 0.234mg/g/d and C group is given leflunomide tablets 0.0026mg/g/d. Mice are fed in a fixed time every day and the whole administration last 4 weeks.
2.2 General situation
Record weight of the mice Weekly and observe diet, activities generally.
2.3 Urinary protein and serum antibodies testing
To measure the ANA, and dsDNA and urinary protein concentration.
2.4 Weight determination of immune organ
After the last administration, mice are weighed and sacrificed. Then calculate the spleen index and thymus index (mg/10g) respectively. Spleen (thymus) Index=10×spleen (thymus) weight (mg)/body weight (g).
2.5 Histopathologic examination
The kidneys of mice are fixed in 10% neutral formalin, embedded in paraffin and then sliced and detected by HE staining.
2.6 Metabonomics
Serum samples that are collected after administration from mice are researed by 1H NMR spectroscopy. View the differences in metabolites of urine after the treatment with Kunxian capsule, and discuss the corresponding mechanisms
2.7 Data processing and statistics
Data and figures are analyzed by using SPSS 13.0, MestReNova5.3.1 and software package of SIMCA-P 12.0.1.
3 Results
3.1 The general condition of mice
Before the start of the experiment, coat, diet, activity, body weight of the mice have no difference. During the experiment, the model control group were loss of appetite, slow-growing, unresponsive etc.
3.2 Detection of serum antibodies and proteinuria
3.2.1 Urine protein concentration
Before administration, urine protein concentration in three groups of mice had no significant difference (P> 0.05). Administration of 4 weeks, the urine protein concentrations in two treated group was significantly decreased (P<0.05, P<0.01); and between the two groups showed no significant difference (P> 0.05).
3.2.2 Serum antibodies
Before administration, the serum anti-ANA antibody and anti-dsDNA antibody levels were not significantly different (P> 0.05). After administration, serum antibody levels in Kunxian capsule group and Leflunomide group compared with the control group were significantly lower, the difference was significant (P<0.01, P<0.01).
3.3 Results of weight measurement of immune organs
The thymus index in Kun Xian Capsules group were significantly increased compared with the control group (P<0.05). The spleen index in Leflunomide group also significantly increased (P<0.01).
3.4 Renal pathology
Model control group were observed mild or moderate hyperplasia in mesangial cells, endothelial cells. Compared with model control group,two treated group have Minor kidney damage (P<0.01), while Between the two treated groups showed no significant difference (P> 0.05).
3.5 Changes of metabolomics
The difference between treated group and model group were branched-chain amino acids, alanine, sugar, protein, ketones, citric acid, creatine, choline phosphate, taurine, glucose and other metabolic components.
4 Conclusion
4.1 Kunxian Capsules can significantly reduce the level of autoantibodies and proteinuria, can regulate the immune organs, and control the progress of Clinical Pathology. Kunxian Capsules contributes to control the activities of LN and remission of disease.
4.2 After treatment, detect metabolic components in serum by 1H-NMR-based metabonomics technology.The result suggesting that the mechanism of Kunxian Capsules contains the following aspects: 1. It can promote the body's normal energy metabolism and enhanced antioxidant capacity and immunity; 2. It can stable cell membrane to maintain osmotic balance inside and outside cells; 3. It can improve renal function; 4. It can control abnormal cell apoptosis in vivo.
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