Neuroprotective Effect Of Paeoniflorin On Myelin Oligodendrocyte Glucoprotein 35-55 Induced Experimental Autoimmune Encephalomyelitis In C57BL/6N Mice

Paeoniflorin (PF) is a characteristic monoterpene glucoside isolated from the rootof paeonia lactiflora. It is one of the bioactive components in paeoniae radix. Paeoniaeradix, the dried root of Paeonia lactiflora Pall., is one of the Chinese traditional crudedrugs. It has long been used as a component of traditional Chinese prescription totreat certain types of dementia. It has been reported that PF exhibits manypharmacological effects such as anti-inflammatory, anti-allergic effects,anti-hyperglycemic effects, anti-thrombosis effects, neuromuscular blocking,stimulating the release of noradrenaline, and enhancing glucose uptake. Recently, itwas reported that PF had neuroprotective effects on cerebral ischemic rat and theMPTP mouse model of Parkinson's disease by activating adenosine A1 receptor(A1AR). Experimental autoimmune encephalomyelitis (EAE) is a classic model ofmultiple sclerosis (MS). The neuroprotective effect of PF on myelin oligodendrocyteglucoprotein (MOG) 35-55 induced EAE in C57BL/6N mice were examined. Inaddition, the possible mechanisms underlying its action were studied.PartⅠThe neuroprotective effect of PF on MOG 35-55induced EAE in C57BL/6N miceAim: To induce an EAE model by MOG35-55 in C57BL/6N mice. To detectwhether PF has a neuroprotective effect on this model.Methods: A EAE model in C57BL/6N mice was induced by MOG35-55 withComplete Freund's Adjuvant (CFA) and Pertussis toxin (PTX). Animals were assessed daily for EAE severity, including body weight, clinical score and histologicalanalysis. The neuroprotective effect was observed after subcutaneous administrationof PF (2.5 and 5 mg/kg) for 14 days from 7 days before immunization.Result: 1. Clinical manifestation: The body weight of EAE drops on day-1 postimmunization, but rise from the next day. It drops again on about day-12 postimmunization with motor dysfunction and rise of clinical score. This model exhibits achronic progressive pattern. 2. Histological analysis: Haematoxylin & Eosin (HE)staining of the spinal cord of EAE shows an infiltration of inflammation lymphocytesmainly around the veinlet. Luxol Fast Blue stain shows demyelination in EAE's spinalcore, and Bielschowsky's silver stain reveals the axonal injury. 3. Neuroprotectiveeffect of PF: PF at the dose of 5 mg/kg ameliorated the clinical manifestation and thehistological change of EAE significantly.PartⅡThe depressant effect of PF on neuroinflammation inEAEAim: To study whether PF has a depressant effect on neuroinflammation in EAEat the dose of 5 mg/kg.Method: Spleen lymphocyte proliferation assay was performed. Flow cytometry(FCM) was used to detect the CD4+CD25+ T-regulatory (T_R) of spleen lymphocyte.We also determined the concentration of nitric oxide (NO) in the supernatant oflymphocyte by Greiss's assay. Real-time PCR was performed and the relative foldchange (RFC) of mRNA in spinal core were observed, including INF-γ,IL-1β,iNOS and IL-10. IκBαwas also detected by Western-blot assay to evaluate theactivity of NF-κB.Result: 1. PF has a depressant effect on the specific reaction of lymphocytetowards MOG35-55 of EAE. CD4+CD25+ T-regulatory (T_R) was up-regulated by PF,and the generation of NO in EAE was suppressed. 2. The mRNA expression ofpro-inflammatory cytokines, like INF-γ, IL-1βand iNOS were down-regulated afterthe administration of PF. Meanwhile, IL-10, an anti-inflammatory cytokine, has anincrease in mRNA level. 3. PF has a depressant effect on the degeneration of IκBα,which indicated the activity of NF-κB was suppressed. PartⅢThe neuroprotective effect of PF is related to theactivation of A1ARAim: To investigate whether A1AR is related to the neuroprotective effect of PFon MOG EAE.Method: 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a selective A1ARantagonist was administrated intraperitoneally 15 min before each PF injection.Clinical manifestation, histological change, lymphocyte proliferation, CD4+CD25+T-regulatory, NO, mRNA of cytokines and degeneration of IκBαwere thenevaluated.Result: The neuroprotective effect of PF on MOG EAE was diminished whenDPCPX was administrated intraperitoneally 15 min before PF injection at the dose of0.3mg/kg.Conclusions1. EAE was successfully induced in C57BL/6N mice by MOG35-55 with CFAand PTX.2. PF (5mg/kg) has a neuroprotective effect on MOG EAE mice.3. PF (5mg/kg) has a depressant effect on neuroinflammation in MOG EAEmice.4. The neuroprotective and anti-neuroinflammatory effect of PF is related to theactivation of A1AR.