The Therapeutic Efficiency And Mechanism Of Bone Marrow Mesenchymal Stem Cell Combined With Resveratrol In EAE

Multiple sclerosis?MS?is a heterogeneous inflammatory and neurodegenerative disease,in which perivascular inflammation,demyelination and axonal loss happen in the central nervous system.Experimental autoimmune encephalitis?EAE?is an inflammatory demyelinating disease,which served as a useful model MS.Resveratrol is a small polyphenolic compound and possess therapeutic activity in various immune-mediated diseases.Mouse bone marrow mesenchymal stem cells?mBM-MSC?were shown to have neuroprotection capabilities in EAE.We study the therapeutic efficiency and mechanism of mBM-MSC combined with Resveratrol in EAE to provides a novel potential experimental protocol for alleviating EAE symptoms.Part? Resveratrol Defends Blood-brain Barrier Integrity in Experime-ntal Autoimmune Encephalomyelitis MiceObjective: To study the machenism underlying the therapeutic efficiency of resveratrol in EAE,we investigated whether resveratrol play a role in the protectin of Blood-brain Barrier Integrity.Methods: C57BL/6 mice at 8-10 weeks of age was induced by immunization with 250?g myelin oligodendrocyte glycoprotein?MOG?p35-55 dissolved in complete Freund's adjuvant?CFA?,which contained 4-mg/ml heat-killed Mycobacterium tuberculosis H37 Ra.After emusfication,as per 0.1 ml was administered subcutaneously into mice by dividing each side of the spine into four points.At time 0 and at 48 h post-immunization,mice were injected intraperitoneally with 500 ng pertussis toxin.Mice were examined daily after induction for clinical signs of EAE and scored according to Knoz.The effect of different doses of resveratrol was evaluated through the score.The Evans blue?EB?dye was injected?2% in PBS?into the mouse tail vein and was allowed in circulation.The brains were removed and snap-frozen.Sections?20 ?m?were then photographed for qualitative analysis.The expression of ZO-1,occluding and claudin-5,as well as the ICAM1 and VCAM-1 were determined by Western blot.The mRNA level of iNOS,IL-1?,arginase 1 and IL-10 of the brain tissue from EAE with or without treatment was determined by real time PCR.Brain local oxidation status was evaluated by NADPH oxidase?NOX?expression and NADPH activity.Results:1 Resveratrol treatment attenuated severity of clinical EAE.Treatment with 10 mg/kg resveratrol failed to suppress EAE,whereas both 25 and 50 mg/kg resveratrol significantly attenuated the paralysis of EAE during 50 days observation period.The average and maximum clinical scores of 25 and 50 mg/kg resveratrol-treated mice were significantly lower than the PBS treatment group.2 Quantitative results showed lower EB leakage detected in brains of resveratrol-treated EAE mice at the doses of 25 and 50 mg/kg compared to vehicle control,while there was no significant difference between 10 mg/kg resveratrol treatment group and the untreated vehicle control group.3 Resveratrol treatment ameliorated EAE-induced loss of tight junction proteins.The expressions of ZO-1,occludin and claudin-5 significantly declined in EAE mice compared with the sham control mice.Treatment with 25 and 50 mg/kg resveratrol produced a marked,dose-related reduction in the loss of ZO-1,occludin and claudin-5.4 Resveratrol inhibited the upregulation of ICAM-1 and VCAM-1 in the brain tissues of EAE mice.Treatment with 10 mg/kg resveratrol did not change the levels of these two adhesion factors,mice treated with 25 and 50 mg/kg resveratrol showed a dose-dependent repression in the EAE-induced expressions of ICAM-1 and VCAM-1 in the brain,as compared with the vehicle control group.5 Markedly elevated iNOS and IL-1? were detected in brain samples of EAE mice,which were then significantly suppressed by 25 and 50 mg/kg of resveratrol treatment.Local anti-inflammatory factors arginase 1 and IL-10 were slightly upregulated in untreated vehicle EAE mice,whereas greatly upregulated in EAE mice after 25 and 50 mg/kg resveratrol treatment.Consistently,we found that 10 mg/kg resveratrol treatment didn't affect these two factors.6 Resveratrol inhibited NADPH oxidase?NOX?expression and activity in the brain.Resveratrol did not affect NOX1 expression compared to vehicle control.The upregulated NOX2 and NOX4 were significantly suppressed by resveratrol treatment in a dose-dependent manner.The increased NADPH activity in EAE mice was also significantly suppressed by resveratrol at the doses of 25 and 50 mg/kg.Part ? The therapeutic efficiency of bone marrow mesenchymal stem cell combined with resveratrol in EAEObjective: Bone marrow mesenchymal stem cell?BM-MSC?has been demonstrated to have neuroprotection on Experimental autoimmune encephalitis?EAE?,but the environment with oxide and inflammation influences the activity of BM-MSC.We studied the protection of EAE-related pathological environments of resveratrol on BM-MSC.Methods: The bone marrow cells were isolated from 6-8 week C57BL/ 6 mice and cultured for 6 weeks.After the purification,they were labeled with anti-Sca-1,CD44,CD14,CD33 and CD45 antibody and analyzed with a FACS Calibur flow cytometer.We investigated the resveratrol protection of EAE-related pathological environments using MTT assay with H₂O₂ and TNF-?.Cultured cells were pretreated with resveratrol in different concentrations?0.1,1,10 M?for 24 h,either accompanied with 20 ng/mL TNF-? exposure for the entire duration of culture,or followed by 4 h H₂O₂?500 mM?treatment,respectively.We assayed cell viability and LDH release of MSCs in vitro 1,3,5.Results:1 Flow cytometry analysis of the immunoflurescently labeled mMASs demonstrated the expressiosn of the Sca-1 and CD44,but not CD14,CD33 or CD45.Expression profile of these cell surface markers fitted the phenotypic features of MSCs.2 Resveratrol treatment could protect cells from EAE-related pathological environments.H₂O₂ and TNF-? both caused significant decrease in cell survival and increase in LDH release.The cytotoxic effects of H₂O₂ and TNF-? were attenuated by resveratrol.Part ? The effect of bone marrow mesenchymal stem cell combined with resveratrol on immune inflammation in EAEObjective: To study in depth the machenism underlying the therapeutic efficiency of bone marrow mesenchymal stem cell combined with resveratrol in EAE,we investigated the effect of bone marrow mesenchymal stem cell combined with resveratrol on immune inflammation in EAE mice.Methods: We investigated the therapeutic efficiency of bone marrow mesenchymal stem cell combined with resveratrol in EAE by clinical score.The infliteration of immune cells such as monocytes and lymphocytes into brain was observed using H&E and Immunohistochemistry with anti-CD4 antibody.ELISA was conducted to detect the secretion of IFN-?/TNF-?and IL-4/IL-10 inserum.Results:1 Treatment with mBM-MSCs and resveratrol significantly decreased the clinical severity of EAE.The combination treatment significantly postponed clinical symptom onset.Treatment with mBM-MSCs or resveratrol significantly decreased the average clinical scores?average clinical score in the PBS treatment,2.63 ± 0.42;mBM-MSCs treatment,1.34 ± 0.31;resveratrol treatment,1.44 ± 0.38;P<0.01,PBS versus mBM-MSCs treatment;P<0.01,PBS versus resveratrol treatment?and the maximum clinical scores?PBS treatment,3.76 ± 0.32;mBM-MSCs treatment,1.83±0.27;resveratrol treatment,1.74 ± 0.31;P< 0.01;PBS versus mBM-MSCs treatment;P<0.01,PBS versus resveratrol treatment?.The effect of combination-treatment was much more prominent than either single treatment?average clinical scores of the combinationtreatment group,0.64 ± 0.27,P<0.01;mBM-MSCs versus combination treatment,P<0.01;resveratrol versus combination treatment;maximal clinical score of the combination-treatment group: 0.91 ± 0.22,P<0.01 mBM-MSCs versus combination treatment;P<0.01,resveratrol versus combination treatment?.2 Combined treatment reduced the number of inflammatory cells in EAE mouse spinal cord.mBM-MSCs or resveratrol treatment alone decreased the numbers of infiltrating cells compared with the PBS treatment?P<0.05,PBS versus mBM-MSCs treatment;P<0.01,PBS versus resveratrol treatment?,and a further significant decrease occurred in the combination treatment group?P<0.01,mBM-MSCs versus combination treatment;P < 0.01,resveratrol versus combination treatment?.3 Combination treatment promoted a shift in balance from T helper type 1?Th1?to Th2 cytokine in EAE mice.A significant decrease in IFN-? and TNF-? Th1 cytokines,and increase in IL-4 and IL-10 Th2 cytokines in the mBM-MSCs or resveratrol treatment groups were observed?IFN-?: P<0.01,PBS versus mBM-MSCs treatment;P<0.01,PBS versus resveratrol treatment;TNF-?: P<0.05,PBS versus mBM-MSCs treatment;P<0.01,PBS versus resveratrol treatment;IL-4: P<0.01,PBS versus mBM-MSCs treatment;P< 0.01,PBS versus resveratrol treatment;IL-10: P<0.01,PBS versus mBMMSCs treatment;P<0.01,PBS versus resveratrol treatment?.In addition,a more prominent decrease in IFN-?/TNF-? Th1 cytokines and increase in IL-4/IL-10 Th2 cytokines in the combination treatment group were observed?IFN-?: P<0.01,mBM-MSCs versus combination treatment;P<0.05,resveratrol versus combination treatment;TNF-?: P<0.05,mBM-MSCs versus combination treatment;P<0.05,resveratrol versus combination treatment;IL-4: P< 0.05,mBM-MSCs versus combination treatment;P<0.05,resveratrol versus combination treatment;IL-10: P<0.01,mBM-MSCs versus combination treatment;P<0.01,resveratrol versus combination treatment?.Conclusions:1 Resveratrol treatment protected Blood-brain Barrier integrity in EAE mice.Resveratrol treatment ameliorated EAE-induced loss of tight junction proteins,ZO-1,occludin and claudin-5.Resveratrol inhibited the upregulation of ICAM-1 and VCAM-1 in the brain tissues of EAE mice.Resveratrol inhibited NADPH oxidase?NOX?expression and activity in the brain.2 H₂O₂ and TNF-? both caused significant decrease in cell survival and increase in LDH release.Resveratrol treatment could protect cells from EAE-related pathological environments with oxide and inflammation in vitro.3 Combination treatment with mBM-MSCs and resveratrol could ameli-orate clinical severity.Combined treatment reduces the number of inflammatory cells in EAE mouse spinal cord and promotes a shift in balance from T helper type 1?Th1?to Th2 cytokine in EAE mice.