| Multiple sclerosis(MS) is a common autoimmune disease affecting the central nervous system. It is the most widespread disabling neurological condition of young adults around the world with a high recurrence and disability rate. Recent studies have found that the prevalence of the disease is increasing in china. The cost of medication to treat MS has risen during the past several decades. Along with the advancement of immunology, the discovery of new auxiliary T cell subsets,, and the clarified relationship between T cells and the incidence of MS, there is a hope to bring forth a curative treatment for MS.Recent studies have confirmed that the pathogenic role of IL-23 is due to the production of IL-17 induced CD4+T cells in IL-23 cells. IL-17 is the most critical proinflammatory cytokine in MS. But these studies are in experimental autoimmune encephalomyelitis, to carry out, few researches have been done on the MS, and to its closely related to IL-17, IL-23, IL-21, IL-22, IL-6, TGF beta and other cytokines study less.The IL-17, IL-23, IL-6 and other cytokines in the serum of patients with MS content determination, comparative analysis the change of cytokines in serum and cerebrospinal fluid(CSF) nor MS relapse in acute period and ease the comparison of the change of cytokines level period. The cell factor of the study is less, and the design is not perfect. We also study between acute RRMS patients with large dose of methylprednisolone impact treatment 1 week after treatment and 1 month after cytokine content in serum and cerebrospinal fluid changes, cell factor correlation, cytokines and EDSS correlation and recovery period and acute relapses in serum cytokine levels of comparative study on. Our research content is comprehensive, reasonable design, to explore the pathogenesis of MS is of great significance.Inflammatory cytokines can not only lead to MS patients with intracranial inflammatory lesions, but also may be closely related with non- inflammatory lesions. When MS patients with axonal and neuronal damage occurs, the cytoskeleton and axis in the membrane proteins(tau protein, S100 protein, 14-3-3 protein, neurofilament protein) will release to the cerebrospinal fluid, causing a corresponding secondary immune response. The study has confirmed that the S100 protein is located in the axonal cytoskeleton, neurons and glial cells, and the 14-3-3 protein is located in the neurons of the axonal cytoskeleton, neurons and glial cells. When the non demyelination in MS, these non demyelinated nerve damage associated protein expression increased, through the detection kit, to a certain extent can reflect the damage degree, suggesting the efficacy of immune therapy and prognosis of the patients.At present, the part of the MS CSF tau protein, S100 protein, 14-3-3 protein and neurofilament protein research at home and abroad. It has been proved that the increase of tau protein in cerebrospinal fluid of patients with acute MS, S100 protein and neurofilament protein, but the conclusion of the study is not consistent and not to alleviate the dynamic observation of tau protein, S100 protein, 14-3-3 protein and neurofilament protein on the MS patients serum. This study through detection of acute recurrent stage and remission stage RRMS patients, the serum levels of tau protein, S100 protein, 14-3-3 protein, neurofilament protein and explore methylprednisolone on acute relapsing MS in the serum of patients with tau protein, S100 protein, 14-3-3 protein, nerve silk protein level of influence and correlation with EDSS, explore inflammatory cytokines may also lead to MS patients with demyelinating lesion basis.In the treatment, the adrenal cortex hormone is still a number of acute phase of the treatment of acute treatment of conventional drugs, can inhibit a variety of inflammatory cells and cytokines secretion. Although clinical practice proves that the treatment of hormone therapy is effective, it can not prevent the recurrence and progression of the disease. Therefore, it is very important to further explore the relationship between pro inflammatory cytokines and MS and to clarify the therapeutic target.Objective 1. To investigate the changes of level of serum IL-17 A, IL-23 1 week before and after in patients with RRMS the after methylprednisolone pulse therapy and with EDSS correlation;2. To investigate the variation of Th17 cell cytokines, including IL-23, IL-17 A, IL-21, IL-22, TGF-β and IL-6 in serum and CSF of RRMS patients after methylprednisolone pulse therapy and the correlation;3. To explore the changes of serum IL-23, IL-17 A, IL-21, IL-22, TGF- beta, and IL-6 levels in the serum of patients with acute and remission stage of RRMS, and their relationship with the recurrence of RRMS;4. To investigate the effects of methylprednisolone on RRMS patients in acute stage and recovery effect of tau protein, S100 protein, 14-3-3 protein, neurofilament protein levels in the serum, and EDSS in the serum of patients with acute stage and recovery stage, and their relationship with the recurrence of RRMS;5. To explore the evidence of IL-23, IL-17 A, IL-21, IL-22, TGF- beta, IL-6 in RRMS patients induce non inflammatory disease.Methods:All patients were diagnosed as RRMS by Mc Donald standard 2010. They were referred to the Neurology Department of the First Affiliated Hospital at Zhengzhou University after 48 h diagnosis from September 2011 to September 2014. The final patient group included 35 males and 69 females with a mean age of 31.2 ± 11.5. The patients had clinical history ranging from 2 months to 9.5 years with a mean duration of 38.2 months. The patients exhibited 2-8 times relapse with an average relapsing time of 3.9. From all the patients, 52.88% had positive oligoclonal bands(OCBs). There were 104 patients had developed other symptoms and displayed new brain lesions in MRI diagnosis.Study design Part 1: The study population consisted of two groups of MS patients(MS-A and MS-B) and one group of healthy control persons. The serum and CSF IL-17 A levels were analysed by ELISA kit to investigate the correlation between IL-17 A and the severity of RRMS disease by Pearson analysis. Base on the neurological assessment of EDSS scales, the RRMS patients before treatment were divided into two clinical groups: MS-A: EDSS <5, 7 men and 13 women with a mean age of 34.20 ± 7.4; MS-B: EDSS > 5, 9 men and 11 women aged 33.8 ± 9.2. All patients gave informed consent according to a protocol approved by the local ethics committee of Zhengzhou University. After one month treatment, 5 m L peripheral blood was collected by venous puncture and 3 m L of CSF were obtained by lumbar puncture from the patients. The samples were centrifuged at 4,000?rpm at 4°C for 5 minutes followed by immediate frozen and stored at-80°C pending to biochemical analyses, without being thawed or refrozen. The final recruited RRMS patients were treated with intravenous methylprednisolone at the third day of hospital admission. They were injected with methylprednisolone 20 mg/kg/d for 3 days followed by 10 mg/kg/d for 4 days. After that, they orally took prednisone for 3 weeks with a progressive withdrawal of 10 mg/kg/w; namely 30 mg/kg/d for the first week followed by 20 mg/kg/d for the second and 10 mg/kg/d for the third week. Meanwhile, medication to protect the gastric mucosa, calcium supplementation as well as potassium supplementation were administered. After three weeks treatment, 5 m L peripheral blood was collected by venous puncture and 3 m L of CSF were obtained by lumbar puncture from the patients. The samples were processed as above mentioned and kept for future test. Additionally, the control subjects were recruited from 20 non-inflammatory neurological disease(NIND) patients.Part 2: Overall, 38 RRMS patients, 13 male and 25 female aged 31.4±9.5 were recruited to investigate the variation of Th17 cell cytokines of IL-23, IL-17 A, IL-21, IL-22, TGF-β and IL-6 in serum and CSF after methylprednisolone pulse therapy. After one month treatment, 5 m L peripheral blood was collected by venous puncture and 3 m L of CSF were obtained by lumbar puncture from the patients. The samples were centrifuged at 4,000?rpm at 4 °C for 5 minutes followed by immediate frozen and stored at-80°C pending to biochemical analyses. 20 NIND patients were recruited in the present stage for control.Part 3: The design program: participate in the first or second part of the study in the treatment of RRMS patients in the remission period after the treatment of half a year. ELISA method for detection of NIND group and RRMS patients before and after treatment and remission of serum Th17 cell related cytokines and Tau protein, S100 protein, 14-3-3 protein, the level of the silk protein and its correlation with EDSS. Patients were included: included 41 cases of RRMS patients were followed up, 14 males, 27 females, aged 33.6±10.1, 41 patients had no new onset of symptoms and signs, and review the cranial MRI showed no lesions. Enrolled patients into three groups according to the EDSS: MS(a) group(EDSS:<4), MS(b) group(EDSS of more than or equal to 4-<6), MS(c) group(EDSS: is equal to or more than nine points), where MS(a) group(n = 17), 5 men and 20 women with a mean age of 33.12±10.28; MS(B group) and 18 cases, 7 men and 11 women with a mean age of 36.2±11.29; MS group(c) in 6 cases, 2 men and 4 women with a mean age of 35.44±15.70.Results: 1. The serum levels of IL-23 and IL-17 A decreased after 1 week and 1 month after methylprednisolone pulse therapy in RRMS patients; after 1 month of therapy in cerebrospinal fluid of IL-23 and 17 A levels compared with those before treatment also decreased, but still higher than NIND patients; IL-17 A levels in CSF of RRMS patients were higher than those in serum;2. EDSS in the MS group(A) and MS(B) were decreased, MS(C) group did not decrease significantly after methylprednisolone pulse therapy in RRMS patients;3. Clinical EDSS showed improvement in the patients with mild symptoms and confirmed a significant correlation between EDSS and IL-17 A level in the CSF of RRMS patients.4. The serum and CSF levels of IL-17 A, IL-23, IL-21 and IL-22 decreased after methylprednisolone therapy but still higher than NIND patients. TGF-β level was increased after methylprednisolone therapy.5. Before methylprednisolone therapy, RRMS patients had lower serum and CSF levels of TGF-β than NIND patients; after treatment, the CSF level of TGF-β in RRMS patients was obviously higher than NIND patients.6. Before methylprednisolone therapy, in serum, IL-23 strongly correlated with IL-17A; in CSF, IL-23, IL-17 A and IL-21 are closely related to each other.7. After methylprednisolone treatment, the serum levels of IL-23, IL-17 A, IL-21 and IL-22 in remission stage decreased significantly but still higher than NIND patients; the level of TGF-β increased and higher than NIND group value.8. The positive correlation between IL-17 A and IL-23 in serum and cerebrospinal fluid of RRMS patients was positively correlated with IL-23, IL-22 and IL-17 A, suggesting that the 3 inflammatory cytokines have the same direction in the pathogenesis of RRMS;9. Serum levels of IL-23, IL-17 A, IL-21, IL-22, and RRMS in patients with remission stage were significantly higher than those in the patients with acute relapse and after treatment, but still higher than that in the NIND group;10. in the treatment of serum S100 protein levels were decreased in RRMS patients with methylprednisolone pulse therapy, but the recovery period is still higher than NIND group; serum tau and 14-3-3 protein and neurofilament protein levels without significant changes;11. The level of S100 protein in the serum of RRMS patients was positively correlated with EDSS.Conclusions: 1. The levels of IL-23, IL-17 A and EDSS decreased continuously after 1 week ang 1 month of methylprednisolone pulse therapy, which suggests that IL-23 and IL-17 A are associated with the onset of MS;2. Methylprednisolone may by reducing IL-23 and IL-17 A levels in serum and cerebrospinal fluid of patients with RRMS achieve anti-inflammatory effect;3. The level of IL-17 A was correlated with the severity of RRMS;4. IL-17 A, IL-23, IL-21, IL-22 and TGF-β associated with RRMS;5. Methylprednisolone may inhibit inflammation cytokine IL-23 and IL-17 A, IL-21, IL-22, up regulation of TGF- beta play anti-inflammatory effect in RRMS;6. IL-23, IL-17 A, IL-21, IL-22 was still high in serum of patients with RRMS remission stage.these high levels of inflammatory cytokines may be related to the recurrence of RRMS;7. It was positively associated with IL-17 A, IL-23 and IL-22 in RRMS, indicating that the blocking IL-17 A, IL-23 or IL-22 inflammatory pathway, there may block the progress of the disease;8. The levels of serum IL-23, IL-17 A, IL-21, IL-22, and RRMS in remission phase were more acute than those in patients with acute relapse and after treatment, that may be related to the recurrence in RRMS;9. The S100 protein levels is still higher in recovery, suggesting the glial cell proliferation in response to persistent in RRMS;10. The level of S100 protein can predict the clinical severity of RRMS patients;11. IL-23, IL-17 A, IL-21, IL-22 and other cytokines in patients with RRMS was related to the inflammatory and non- inflammatory disease. |
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