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ObjectiveTo investigate the effects of Yishen Daluo prescription （YSDL） on Ras homolog（Rho）/Rho-associated coiled-coil containing protein kinase（ROCK）signaling pathway in mice with experimental autoimmune encephalomyelitis （EAE） based on the silencing of β-arrestin1 gene. MethodSixty C57BL/6 female mice were randomly divided into a blank group， a model group， a virus group， a YSDL group， a virus + YSDL group， and a prednisone acetate group （hormone group）. The EAE model was induced in mice except for those in the normal group. Adeno-associated virus（AAV）solution （150 μL， 1×1011 vg·mL-1） was injected into the tail vein of each mouse in the virus group and the virus + YSDL group on the 4th day of immunization. Drugs were administered on the 8th day of modeling. Specifically， normal saline was given to the mice in the normal group，the model group，and the virus group at 10 mL∙kg-1， prednisone acetate suspension to those in the hormone group at 3.9 g∙kg-1，and YSDL to those in other groups at 20 g∙kg-1 for 14 consecutive days. The mice were weighed and scored every day. The neurological function scores of mice in each group were recorded every day after immunization. Hematoxylin-eosin （HE） staining was used to determine the inflammatory response and lesion location in the brain tissues and spinal cord tissues of mice. The protein expression of β-arrestin1，Ras homolog gene family member A（RhoA）， and Rho-associated coiled-coil forming protein kinase Ⅰ（ROCK Ⅰ） in spinal cord and brain tissues of EAE mice was determined by Western blot. ResultCompared with the model group， the virus group and the virus + YSDL group showed decreased neurological function scores （P<0.01），and the YSDL group also showed decreased neurological function scores（P<0.05）. HE results showed that there was obvious inflammatory reaction in the central nervous system （CNS） of the model group， which was alleviated to varying degrees in other groups compared with the model group. Western blot results showed that compared with the blank group， the model group showed increased protein expression levels of β-arrestin1， RhoA， and ROCK Ⅰ in the spinal cord tissues （P<0.01）. Compared with the model group， the virus group， the YSDL group， the virus + YSDL group， and the hormone group showed decreased protein expression levels of β-arrestin1， RhoA， and ROCKⅠ in the spinal cord tissues （P<0.01）. Compared with the blank group， the model group showed increased protein expression levels of β-arrestin1， RhoA， and ROCK Ⅰ in the brain tissues （P<0.01）. Compared with the model group， the virus group， the YSDL group， the virus + YSDL group， and the hormone group showed decreased protein expression level of β-arrestin1 in the brain tissues （P<0.01）， and the virus group and the YSDL group showed decreased protein expression levels of RhoA， and ROCKⅠ in the brain tissues （P<0.05）. Additionally， the virus + YSDL group and the hormone group showed decreased protein expression levels of RhoA and ROCKⅠ in the brain tissues （P<0.01）. ConclusionYSDL can improve the clinical symptoms of EAE mice and improve the inflammatory response of CNS. The mechanism is presumably attributed to the fact that YSDL inhibits the expression of β-arrestin1 in CNS，thereby reducing the expression of Rho/ROCK signaling pathway. Furthermore， YSDL may have a synergistic effect with the inhibition of β-arrestin1 gene expression.
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@#【Objective】 To study the mechanisms and therapeutic effects of human olfactory mucosa-derived mesenchymal stem cells（OMSC）on experimental autoimmune encephalomyelitis（EAE）in mice.【Methods】Under local anesthesia by using nasal endoscopy，olfactory epithelia of healthy donors were obtained，digested and cultured up to the 5th passage. OMSC were identified，differentiated and stained. EAE models were induced in C57 female mice by myelin oligodendrocyte glycoprotein（MOG35- 55）and pertussis toxin（PT）. Neurological function was documented daily. On day 16 after immunization（peak of incidence），the mice were divided randomly into two groups and treated with OMSC and PBS via tail vein injection respectively. On day 24 after immunization ，blood was collected from angular vein and levels of IL-10，IL-17，IFN-γ and IL-6 were determined by cytometric beads array（CBA）. The size of the spinal cord lesion in mice was observed and measured by using HE and LFB staining. Peripheral blood lymphocytes（PBL）of healthy donors were obtained and then co-cultured with OMSC. The proportions of CD4+ T cells secreting IFN- γ（Th1 cells）in lymphocyte group and co-culture group were compared after 2 days of cultivation. Adding IDO or COX pathway inhibitor to co- culture group and cultivating for 2 days，we observed and compared the proportions of Th1 cells in lymphocyte group，co-culture group and inhibitor treatment group respectively.【Results】OMSC exhibited certain mesenchymal stem cell-like characteristics with respect to expression of stem cell surface markers and multilineage differentiation potentials. After induced by MOG35- 55 and PT，EAE models showed different levels of neurological damage. Compared with those in PBS treatment group，in OMSC treatment group，the severity of neural dysfunction in mice was significantly reduced（P =0.002），the level of IFN-γ in serum was lower（P = 0.032），but no significant differences in the levels of IL-10，IL-17 and IL-6 were found between two groups. HE and LFB staining revealed that the inflammatory infiltration and demyelinating areas in OMSC treatment group were less than those in PBS treatment group. The proportion of Th1 cells was lower in co-culture group than that in lymphocyte group（P = 0.001），higher in IDO inhibitor group than that in co-culture group（P = 0.01），but no significant difference was found between IDO inhibitor group and lymphocyte group or between COX inhibitor group and co-culture group.【Conclusions】OMSC may regulate the proportion of Th1 lymphocytes through IDO pathway so as to inhibit the demyelinating injuries of EAE in mice. This study provides a new idea for the clinical treatment of multiple sclerosis.
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Objective:To investigate the effect of miR-322-5p which targets Akt3 on Th17 differentiation in experimental autoimmune encephalomyelitis (EAE) interfered by interferon-β (IFN-β). Methods:The effect of IFN-β on EAE mice which were randomly divided into IFN-β group and PBS group was examine. The percents of Th17 in the two groups were compared by fluorescence activated cell sorting. The miRNA array was made to find different miRNAs between those two groups. MiR-322-5p was screened for further research. The target gene of miR-322-5p was predicted using softwares and the common predicted target gene Akt3 was got. The expression of Akt3 was detected after IFN-β intervention and miR-322-5p overexpression. The target relationship between Akt3 and miR-322-5p was verified by luciferase reporter assay. At last, the effect of Akt3 on Th17 differentiation was explored in vitro. Results:Compared to PBS group, the percent of Th17 was significantly downregulated, the expression of miR-322-5p was significantly upregulated and Akt3 was significantly downregulated in IFN-β group. The expression of Akt3 was obviously decreased after overexpressing miR-322-5p. Luciferase reporter assay showed that Akt3 was directly targeted by miR-322-5p. The percent of Th17 differentiation was greatly promoted by Akt3 in vitro. Conclusion:IFN-β significantly ameliorates the severity of EAE by affecting miR-322-5p which can inhibit Th17 differentiation by targeting Akt3.
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In recent years,it has been found that there are lymphoid follicle structures outside the lymphoid tissues and organs, which are called ectopic lymphoid tissues,also known as tertiary lymphoid tissues(TLTs).TLTs currently have been found in a number of diseases such as multiple sclerosis,diabetes,cancer,atherosclerosis and rheumatoid arthritis and the like.Here we focus on the molecular mechanism of TLTs and the relationship between TLTs and multiple sclerosis (MS)/experimental autoimmune encephalomyelitis (EAE) disease,and briefly describe the role of TLTs in the development of MS/EAE,the relevant mechanisms and research progress,make people have more understanding and insights on the formation of TLTs and the mechanism that it involved in disease,to provid theoretical knowledge for clinical treatment.
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Objective:To investigate the therapeutic effects of astragalus polysaccharide (APS) on experimental autoimmune encephalomyelitis(EAE) and to explore the regulating effects on microglia activation that is associated with the pathogenesis of EAE and its possible mechanisms.Methods:Animal experiments:EAE model was induced by MOG35-55in C57BL/6 mice.APS was given by gavage.EAE was scored according to a 0-5 scale to observe the therapeutic effects of APS.Cell experiments:The effects of lipopolysac-charide (LPS) on cell viability of BV-2 microglial cell line were investigated by MTT assay and then the appropriate concentration of LPS to activate the BV-2 microglial cell line was selected.The microglia activation model was established.The changes in BV-2 microglial cell line morphology were observed with an inverted microscope.The cytokines of TNF-α and IFN-γ in the cell culture supernatant of BV-2 microglial cell line were detected by ELISA.The activated BV-2 microglial cells were treated with APS in different concentrations.The regulatory roles of the APS on the BV-2 microglial cell activation were observed.Western blot and Real-time PCR method were used to measure the protein and mRNA level of the PD-L1 on the cell surface of BV-2 microglial cells treated with APS.Results:APS could effectively ameliorate the symptoms in EAE mice and could suppress neuroinflammation of EAE significantly.The microglia activation model in vitro induced by LPS was successful.APS in certain concentration could inhibit the activation of microglia,increase the viabilily of the active microglia.Meanwhile,it could downregulated the level of the cytokines including IFN-γ and TNF-α and upregulated the expression of protein and mRNA of PD-L1 on activated microglia.Conclusion:APS can effectively inhibit the autoimmune reaction of EAE and effectively suppress the microglia activation induced by LPS,reduce the pro-duction of IFN-γ and TNF-α.APS plays a crucial role in reducing the inflammation induced by microglia activation.The potential mech-anisms might be related to the upregualtion of the PD-1/PD-L1 pathway.
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Objective To explore the effects of vasoactive intestinal peptide (VIP)on the ratio of CD4+CD25+Treg/CD4+T cell and the expression of TGF-β1 in experimental autoimmune encephalomyelitis (EAE)rats. Methods We randomly divided 60 healthy female Wistar rats into normal control group,EAE control group,VIP low-dose group and VIP high-dose group.We used myelin basic protein (MBP)+ complete adjuvant (CFA)to establish EAE model. Since the day of model construction, the low- and high-dose VIP groups received intraperitoneal injection of 4 nmol/kg (0.2 mL)and 16 nmol/kg (0.8 mL)of VIP every other day,respectively;normal control group and EAE group received injection of saline of 0.8 mL for 10 days in a row.We recorded the peak of neurological dysfunction score (NDS)changes in the rats,observed the pathological changes and GFAP+astrocyte activation in the brain at the morbidity peak of rats with HE staining,and detected the ratio of CD4+CD25+Treg/CD4+T in the spleen with FACS and TGF-β1 cytokine level in brain tissue with ELISA.Results The peak nerve dysfunction score was decreased in each VIP dose group.In normal control group,there were decreased inflammatory cell infiltration and decreased number of active astrocytes in the brain tissue.The degree of infiltration of inflammatory cells and astrocyte activation in VIP control groups were significantly lower than those in EAE group.The CD4+CD25+Treg/CD4+T cell ratio of the spleen tissue in each dose VIP treated group rats was higher than that in EAE control group.The cytokine level of TGF-β1 in the brain tissue increased in each VIP dose group in the dose-dependent manner.Conclusion Through up-regulating the ratio of CD4+CD25+Treg/CD4+T cell in the spleen tissue,increasing TGF-β1 content in brain tissue,and inhibiting the infiltration of inflammatory cells and the astrocyte activation,VIP plays an important role in prevention and control of EAE.
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Objective: To evaluate the mechanism of T follicular helper cells ( Tfh ) in experimental autoimmune encephalomyelitis (EAE) via in vivo experiments. Methods:C57BL/6 mice were randomly divided into four groups,CFA group,EAE group,anti-ICOSL group and control group. Lymphocytes of different time points isolated from draining lymph nodes and spleen were stained for T follicular helper cells surface marker and T cells activation surface marker and analyzed by FACS. Observed parameters include inflammatory infiltration,demyelination in spinal cord and germinal center in spleen. ELISA was used to measure the level of antigen specific antibodies. Results: Mice in anti-ICOSL treated group developed mild disease was with lower clinical scores when compared with the EAE group. HE staining results turned out with alleviated inflammation and Luxol Fast Blue staining( LFB) showed no demyelization in anti-ICOSL treated mice compared with non-treated EAE models. Flow cytometry results revealed that percentages of T follicular helper cells decreased though the whole activated degree T cells was not influenced in anti-ICOSL treated group. Fewer ger minal center was found in both anti-ICOSL group and CFA group with reduced secretion of MOG-specific Ab. Conclusion:T follicular helper cells supported the development of cognate B cells,promoted the formation of germinal center,facilitate pathogenic MOG-specific Ab secretion,thus enhance EAE.
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Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease model mediated by specific allergenic CD4+T cell and accompanied by mononuclear cells invasion in central nervous system (CNS) and demyelination. Similar to the histopathology and immune characters of multiple sclerosis(MS), EAE has been used as a classical animal model for the study of MS. In recent years, as an immune regulator for the treatment of relapsing-remitting MS, study of estrogen has entered the pre-clinical phaseⅡtrials. Larger numbers of studies have verified that high concentration of estrogen could improve EAE. However, the specific mechanisms are still in controversy. For these considerations, this review summarizes the protective mechanisms of estrogen in EAE.
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@# ObjectiveTo explore the mechanism causing the sex differences in experimental autoimmune encephalomyelitis (EAE).MethodsEAE model was established with PLP 139-151 in SJL/J mice. The sex differences of illness state were evaluated by neurologic score, and that of response of peripheral lymphocytes to autologous antigens were detected by methods of MTT and ELISA.ResultsThe onset time of female SJL mice was 15±2.1 days less than that of male ones (22±4.3 days), and the feature of diseases course of female mice was relapse after recovery, but that of male mice was transient. No significant differences were observed in T cell responses and interferon-γ productions between male and female mice (P>0.05). Male mice secreted more interleukin-4 than female mice (P<0.01).ConclusionThe sex differences of EAE in mice are due to peripheral lymphocytes secreting more interleukin-4 in male mice.
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OBJECTIVE: We studied effects of dexamethasone on neurogenic bladder and paralysis in experimental autoimmune encephalomyelitis (EAE) rat model for multiple sclerosis. METHOD: Thirty-five female Lewis rats were used in the study. Thirteen rats used as normal cystometrogram controls. Twenty-two rats induced EAE were divided into two groups: ten rats as control and twelve rats as dexamethasone injection group. Bladder dysfunction by cystometrogram, severity of weakness, and duration of paralysis were evaluated every other day after the onset of paralysis. RESULTS: Dexamethasone injection group compared to control group presented short duration of bladder dysfunction (2.5 vs. 4.2 day, p0.05), but it was not statistically significant. CONCLUSION: Dexamethasone ameliorates the course of paralysis and bladder dysfunction in EAE. We suggest that dexamethasone treatment is an effective method in treating neurogenic bladder and paralysis in multiple sclerosis.
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Animals , Female , Humans , Rats , Dexamethasone , Encephalomyelitis, Autoimmune, Experimental , Models, Animal , Multiple Sclerosis , Paralysis , Urinary Bladder , Urinary Bladder, NeurogenicABSTRACT
Objective:To explore the effects of peroxisome proliferator-actived receptors-?(PPAR-?)agonists rosiglitazone and non-steroidal anti-inflammatory drugs(NSAIDs) ibuprofen on tumor necrosis factor-?(TNF-?)mRNA expression in the brain of rats with acute experimental autoimmune encephalomyelitis(EAE).Methods:EAE models on rat were established by inoculating the homogenate containing spinal cord of guinea pig and complete Freund's adjuvant,and then treated by rosiglitazone and ibuprofen respectively.The improvement of clinical manifestations was assessed to determine the curative effect of the two medicines and TNF-? mRNA expression level was detected by RT-PCR.Results:Compared with EAE group,the groups with rosiglitazone and ibuprofen showed improvement in clinical manifestations ,and the expression of TNF-? mRNA reduced markedly(P0.05).Conclusions:PPAR-? agonists rosiglitazone and NSAIDs ibuprofen could obviusely improve the clinical manifestations of EAE rats,and decrease the level of the expression of TNF-? mRNA,which suggested PPAR-? agonists may have the anti-inflammation neural protective function on EAE or multiple sclerosis(MS),and the anti-inflammation of ibuprofen may be related to the activation of PPAR-?.
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Objective:To explore the efficacy and therapeutic mechanism of immune inhibitor tacrolimus(FK506) for experimental autoimmune encephalomyelitis rats and compare the efficacy of FK506 and confirmed traditional methyllprednisolone.Methods:the guinea pig spinal cord homogenate in complete freund's adjuvant was used to induce the acute EAE in SD rats FK506 at the dosage of 1 mg/kg and 0.5 mg/kg were intraperitoneally injected to EAE rats respetively for daily for ten days.Clinical features were observed,their peripheral blood lymphocytes CD3,CD4,CD8 and NK subsets were counted,pathology changes of brain and spinal cord were observed,and the evidence of nerve regeneration was shown by immunohistochemistry stain.Result:FK506 could relieve remits the clinical phenonoue of EAE promote the climical and release and recoveny of EAE,decrease the inflammatory infiltration in the CNS,decrease demyelination and axonal damage in CNS,promote for axons regeneration.Furthermore,no replase after stopping drug was observed.Conclusion:FK506 as a immune inhibitor can be used the Autoimmune disease,such as EAE.
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Objective To explore the surface molecule changes of the experimental autoimmune encephalomyelitis (EAE) rat brain vascular endothelial cells. Methods The expressions of vascular cell adhesion molecule\|1(VCAM\|1) and major histocompatibility complex (MHC) class Ⅱ molecules in the EAE rat brain vascular endothelial cells were tested by immunohistochemistry. The level of tumor necrosis factor (TNF) and interferon\|?(IFN\|?) in defferent clinic grade EAE were tested by MTT [3\|(4 5\|dimethythiazol\|zyl)\|2^5\|diphenyl) tetrazolium bromide] method. Results There were inducible expressions of VCAM\|1 and MHC class Ⅱ molecules in the EAE rat brain vascular endothelial cells compared with control. The sera of EAE rats possessed the activities of TNF and IFN\|?.Conclusion\ In the CNS inflammation, the change surface molecules in the brain vascular endothelial cells may suggest that it plays important roles in EAE pathogenesis.\;[