| 1.Objectives and significanceLupus 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 importantIn 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 project2.1 Subjects18 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 situationRecord weight of the mice Weekly and observe diet, activities generally.2.3 Urinary protein and serum antibodies testingTo measure the ANA, and dsDNA and urinary protein concentration.2.4 Weight determination of immune organAfter 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 examinationThe kidneys of mice are fixed in 10% neutral formalin, embedded in paraffin and then sliced and detected by HE staining.2.6 MetabonomicsSerum 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 mechanisms2.7 Data processing and statisticsData and figures are analyzed by using SPSS 13.0, MestReNova5.3.1 and software package of SIMCA-P 12.0.1.3 Results3.1 The general condition of miceBefore 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 proteinuria3.2.1 Urine protein concentrationBefore 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 antibodiesBefore 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 organsThe 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 pathologyModel 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 metabolomicsThe 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 Conclusion4.1 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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