The Study Of Vasoactive Intestinal Peptide Preventive Effect In EAE Rats

Objective:To observe the preventive effect of vasoactive intestinal peptide (VIP) in experimental autoimmune encephalomyelitis(EAE), and to explore the immunological mechanism for the VIP treatment of multiple sclerosis (MS) finding adequate theoretical basis. Method:Randomized double-blind method, 60 female Wistar rats (200g-250g/rats) were divided into 4 groups,15 in each group:normal control group, EAE control group, VIP low-dose treatment group and VIP high-dose treatment group.MBP+CFA was injected into the hind paws subcutaneous of EAE control group,VIP each-dose treatment groups rats(0.2ml /100g) for active immunization, The control group received normal saline+ CFA. At 0 hours and 48 hours, immuned to the rats by intraperitoneal injection of pertussis hormone (PTX) (0.2ml/only) to establish EAE model. Since modeling the date, VIP low and high dose treatment group were given intraperitoneal injection every other day VIP 4nmol/kg (0.2ml),16nmol/kg (0.8ml), EAE control group and the control group given every other day intraperitoneal injection of 0.8ml of saline for 10 days. Such situation below could be considered as the peak of EAE disease:the symptom did not aggravate or quadriplegia for 3 consecutive days, or the rats being dead; Killed the rats at the peak time. And to the normal group, we abserved for 8 weeks and then killed them.1. Record the onset rats constituent ratio, incubation period, progression and the peak of neurological dysfunction score changes.2. Light microscopic observation of the brains and pinal pathological changes in each group rats.3. By immunohistochemistry, using anti-glial fibrillary acidic protein (GFAP) antibody to detect the active astrocytes in each group and calculated anti-GFAP average optical density values (IOD)for each rats; using anti-myelin myelin protein (MBP) antibody to stain demyelination of brain tissue, and calculated the IOD of anti-MBP.4 The ratio of CD4+CD25+Tregs in spleens was detected using FACS analysis.5. The cytokine levels of IL-17A, TGF-β1 in brain tissue were analyzed by radioimmunoassay.6. The cytokine levels of IFN-y,IL-4 in serum were analyzed by ELISA kit. Results:1. The incidence: (1)Latency change:compared with EAE control group, The latency of different dosage of VIP prolonged, with significant difference (P<0.01); the latency of VIP high dose control group was longer than that of VIP low dose group(P<0.01). (2)The progress of change:compared with EAE control group: the progression of different dosage of VIP was shortened (P<0.05, P<0.01), the progression period of VIP high dose group of VIP was shorter than that of low dose group significantly (P<0.05). (3) The peak incidence of neurological dysfunction score:compared with EAE control group the neurological dysfunction score of VIP low dose group was lower, no statistical significance (P>0.05); the neurological dysfunction score of VIP high dose group was lower than EAE and VIP low dose group (P<0.01).2. Pathological changes in the brain and spinal cord:with no pathological changes in brain and spinal cord of normal control group; there were varying degrees of pathological changes of typical EAE in brain and spinal cord of EAE control group and VIP treated rats, small perivascular inflammatory cell infiltration was "cuff" shape change, demyelinating around the venous vascular white matter was obvious, the infiltration of inflammatory cells and the demyelination in the brain and spinal cord of different dosage of VIP treated group is lower than that of EAE control group, high dose of VIP treated group was lower than that of low dose VIP treated group. The inflammatory cell infiltration scores in the brain and spinal cord of EAE control group and different dosage of VIP treated group were significantly higher than those in control group (all P<0.01); the inflammatory cell infiltration scores in the brain and spinal cord of each dosage VIP treated group were significantly lower than those in EAE control group (all P<0.01); the inflammatory cell infiltration scores in the brain and spinal cord of VIP high -dose treated group were significantly lower than those in VIP low-dose treated group(P<0.05).3. The immunohistochemiscal results in brain tissue of each group rats at the peak incidence, astrocytes activation:normal control group has no activated astrocytes; the activation of astrocytes in EAE control group showed diffuse distribution, perivascular reaction stronger; the number of GFAP positive cells was significantly reduced in VIP treated groups, but still with perivascular obvious reaction. The anti GFAP positive average optical density (IOD) in brain tissue of different doses of VIP treated group were lower than those in EAE group (P<0.01, P<0.05); compared with VIP low-dose treated group, the mean optical density value of anti-GFAP positive (IOD) significantly reduced in brain tissue of VIP high-dose treated group(P<0.01) Demyelination changes:Normal control group, myelin intact; In the brain tissue of the EAE control group, there widely exist punctuated demyelination, and the positive expression of MBP average optical density (IOD) significantly decreased compared with different dosage of VIP treated groups(P<0.01, P<0.05). Compared with VIP low-dose treated group, the average optical density of MBP positive expression in VIP high-dose group increased significantly (P<0.01).4 In the incidence peak the changes of CD4+CD25+Treg/CD4+.T cell ratio in splenic tissue of each group rats: the CD4+CD25+Treg/CD4+T ratio of spleen tissue in EAE control group was lower than that in nomal control group (P<0.01); the CD4+CD25+Treg/CD4+T ratio of spleen tissue in each dose VIP treated group rats were higher than that in EAE. control group (P＜O.01, P<0.05); the CD4+CD25+Treg/CD4+T ratio of spleen tissue in VIP high dose treated group rats was higher than that in VIP-low dose group (all P<0.01).5. In the peak incidence, the content changes of IL-17A, TGF-β1 in brain tissue homogenate of each group rats:the IL-17A of the brain tissue homogenate in EAE control group and each dose VIP treated groups were markedly elevated compared with normal control group (all P<0.01); the content of IL-17A in different dosage VIP treated groups were significantly lower than that in the EAE control group (all P<0.01); the content of IL-17A in brain tissue of VIP high-dose treated group was lower than that in VIP low-dose treated group (P＜O.01). Compared to normal control group, the content of TGF-p 1 in the EAE control group and each dose VIP treated groups markedly decreased (P<0.01, P,<0.05).the content of TGF-β1 in the brain tissue of different dosage VIP treated groups were significantly higher than those in EAE group (all P<0.01); VIP high-dose group of brain tissue homogenate TGF-(31 content was significantly higher than that that of VIP low-dose group (P<0.01).6. The content changes of IFN-y, IL-4 in the serum of each group rats:Compared with normal control group, the serum content of IFN-y in the serum of EAE control group and different dosage VIP treated markedly elevated (P<0.01, P<0.05); the content of IFN-y in serum of each does VIP treated groups were significantly lower than that of EAE control group (P<0.01, P< 0.05); the content of IFN-y in serum of VIP high-dose group was lower than that in VIP low-dose treated group (P<0.05). Compared with normal control group, the serum content of IL-4 in serum of EAE control group and different dosage VIP treated groups markedly descended (all P<0.01); the content of IL-4 in serum of each does VIP treated groups were higher than that of EAE control group(all P<0.01); the content of IL-4 in serum of VIP high-dose group was higher than that in VIP low-dose treated group(P<0.01).7. Correlation analysis:①the contents of IL-17A in brain and IFN-y in serum in the EAE control group,VIP low-dose group and VIP high-dose group were significantly positive correlated with the peak neural dysfunction score (all P<0.01); the content TGF-(31 in brain、IL-4 in serum and the CD4+CD25+Treg/CD4+T ratio of spleen tissue, were significantly negative correlated with the peak neural dysfunction score (P<0.01); ②the content of IL-17A in brain and IFN-γ in serum in the EAE control group,VIP low-dose group and VIP high-dose group were significantly positive correlated with the HE stain pathology score(P<0.01); the CD4+CD25+Treg/CD4+T ratio of spleen tissue, the content of TGF-β1in brain and IL-4 in serum were significantly negative correlated with the HE stain pathology score(P<0.01).③the content of IL-17A in brain and IFN-y in serum in the EAE control group, the VIP low-dose group and the VIP high-dose group were significantly positive correlated with the anti-GFAP astroeytes average IOD(P<0.01); the CD4+CD25+Treg/CD4+T ratio of spleen tissue, the content of TGF-β1in brain and IL-4 in serum were significantly negative correlated with the anti-GFAP astroeytes average IOD(P<0.01).④the content of IL-17A in brain and IFN-y in serum in the EAE control group, the VIP low-dose group and the VIP high-dose group were significantly negative correlated with the anti-MBP average IOD(P<0.01), the CD4+CD25+Treg/CD4+T ratio of spleen tissue, the content of TGF-β1in brain and IL-4 in serum were significantly positive correlated with the anti-MBP average IOD(P<0.01). Conclusion: 1.There was an imbalance of Thl/Th2 cells, Thl7/CD4+CD25+Treg cells in EAE rats, in the peak incidence, the level of Th1 type cytokines IFN-yand Th17 type cytokines IL-17 in the body increased, the level of Th2 type cytokine IL-4, CD4+CD25+Treg cytokine TGF-β1 reduced, The pattern of migration tended to Th1 type immune and destructed the peripheral and central tolerance mediated by CD4+CD25+Treg cells. The immune dysfunction closely related to incubation period, progression and the peak of neurological dysfunction of EAE rats.2.By the treatment of VIP,the incubation period of EAE rats can be extended, progression shortened, neurological dysfunction scores reduced, brain tissue infiltration of inflammatory cells reduced, astrocyte activation and demyelination of pathological damage inhibited, thus it played a role in prevention and treatment of EAE.3.Through activisting Th2 cells, increasing CD4+CD25+Treg cell number and secretion, inhibiting Th1 cells, Th17 cell activity, keeping the Thl/Th2 cells, Th17/CD4+CD25+Treg cells in balance, and promoting inflammatory cytokines to suppress IL-4, TGF-β1 production, inhibiting of proinflammatory cytokines IFN-y, IL-17 production, maintaining the central and peripheral immune tolerance, VIP reduced the clinical manifestations and pathological changes, and thus played a protective role in EAE rats.