Neuroprotective And Immunoregulation Effects Of Gypenosides In Experimental Autoimmune Optic Neuritis

Optic neuritis(ON)is a common neuro-ophthalmologic inflammatory disease that results in persistent vision impairment in young adults.Idiopathic demyelinating optic neuritis(IDON),the most common clinical type,is strongly associated with multiple sclerosis(MS).However the pathogenesis of MS or ON remains largely obscure.A widespread theory is that:MS is an autoimmune disease characterized by the CD4+ T cell-mediated demylination in the central nervous system.CD4+T cell cause the inflammatory cascade reaction and oxidative stress which produced a large number of reactive oxygen species and consuming endogenous antioxidant enzyme.In addition to a wide range of demyelinating lesions in acute or chronic MS and ON,axonal loss and neuronal apoptosis occur and are closely related to irreversible loss of vision.Corticosteroids are commonly used in the treatment of ON.The evidences of evidence-based medicineare are provided by random controlled trial from ONTT(Optic Neuritis Treatment Trial).The pharmacological effects of corticosteroids are based on decreasing inflammation,but not neuroprotection.Therefore,they have limited effects on improving the prognosis of visual acuity.One study even indicates that methylprednisolone accelerate neuron apoptosis in the central nervous system.The primary treatments for MS or ON are the corticosteroids and immuno-suppressive agents.With the serious side effects of corticosteroids and their lack of neuroprotective effect,patients with ON urgently need a novel medication with anti-inflammatory properties,immune regulation and neuroprotective effects to improve the visual prognosis.Gynostemma pentaphyllum makino,a popular herb used in traditional Chinese medicine,has been used in food and tea at length owing to its ability to prevent chronic diseases such as hepatitis,hypertension,and gastritis.Gypenosides(Gp),the saponin extracts from Gynostemma pentaphyllum,has been shown to provide various bioactivities such as antioxidation,hepatoprotection,antilipidemia and inflammation reduction.The neuroprotective effect of Gp has been proven and current research is mainly focused on ischemia-reperfusion injury,Alzheimer’s Disease,Parkinson’s disease.However,whether Gp has a protective effect on ON has not been established.So we focus on the immunoregulation and neuroprotection effect of Gp and generated new hypotheses:The therapeutic action of Gp on ON,a neurodegeneration diseases cause by autoimmune demyelinating.In present study,an retinal ganglion cells oxidative damage model and experimental autoimmune optic neuritis(EAON)vivo model were employed to evaluate the potential therapy effect of Gp on antioxidant,anti-apoptosis and neuroprotection in cell and EAON animal,Part 1 Protective Effects of Gypenosides against hydrogen peroxide-induced oxidative stress in retinal ganglion cellsObjective:To investigate the protective effect and the possible mechanism of gypenosides(Gp)on oxidative stress of retinal ganglion cells.Methods:To cultivate and identify the primary culure RGC and establishe the oxidative damage model by H2O2.Purified RGCs were exposed to H2O2 at the concentrations of 100 μMol/L and treat with or without 50/100/200 μg/ml Gp,meanwhile,the control group was set as well.To the study of signaling pathways,we set up the control,oxidative stress,oxidative stress and inhibitor,Gp treatment,Gp and inhibitor groups.The cell survival rates were measured by MTT and the apoptosis was observed by Hoechst 33342/Tunel staining.The intracellular reactive oxygen species(ROS)levels were detected by DCFH-DA staining and flow cytometry.The spectrophotometer and enzyme standard instrument were applied to test the intracellular level of ATP and SOD.The expression levels of iNOS,COX-2,Nrf-2,HO-l,Gpx1/2,Bcl-2,Bax,Caspase-3 were analyzed by Real-Time PCR and Western blot.The signaling pathway of ERK and PI3k/Akt were also assessed by Western blot too.Results:The expression of specific antibody Thy 1.1 was detected by immunohistochemical staining in primary cultured cell and morphlolgy of cell consistent with neurons.Concentrations of Gp in 50,100,200 μg/ml were selected as optimal concentration in the study by MTT assay.Hoechst 33342 and Tunel staining showed that all-concentrations of Gp can reduce the RGC apoptosis induced by H2O2.Gypenosides could reduce the intracellular ROS level at all of concentration.Medium and high concentrations of Gp could effectively increased the intracellular level of ATP concentrations and increased SOD activity.Real Time-PCR result indicated all the concentration of Gp downregulated iNOS,COX-2 and Gpxl/2 mRNA expression.Only high concentrations of Gp upregulated mRNA expression levels of Nrf-2 and HO-1.In terms of apoptosis index,the low and high concentration of Gp upregulated gene expression levels of Bcl-2 but high concentration could downregulated Bax mRNA.All concentration of Gp could downregulated Caspase-3 expression.Western blot demonstrated that medium and high concentration of Gp downregulated iNOS expression and high concentration of Gp downregulated COX-2 expression.All the concentration of Gp upregulated Nrf-2 expression but medium or high concentration of Gp upregulated HO-1 and Gpx1/2 expression.In the aspect of apoptosis protein detection,all the concentration of Gp could downregulated the cleaved caspase-3 expression and increase the ratio of bcl-2 to bax.In the study of signaling pathways we found Akt pathway could be activated significantly by medium concentrations Gp treatment but there was no activation in ERK passway.Conclusions:Our findings suggest that Gp treatment can improve activity and reduce the apoptosis of RGC.Gypenosides helps RGC to resistant to oxidation damage by reduce the amount of ROS in cells directly and raise the level of ATP or enhance the SOD activation.Gp treatment also reduce the generation of iNOS,COX-2 and increase the Nrf-2 levels so as to increase the content of HO-land Gpx1/2 which can enhance the abiliy of antioxidation in RGC.Gp protects RGC cells from H2O2 insults via alleviate apoptosis through mitochondrial apoptotic pathway and activation of Akt signaling pathway.Part 2 Neuroprotective Effects of Gypenosides in Treating Experimental Autoimmune Optic Neuritis in C57BL/6 MiceObjective:To determine whether Gypenosides(Gp)have protective effects in the treatment of experimental autoimmune optic neuritis(EAON).Methods:Mice were randomly divided into seven groups:control group,model group,three different density Gp monotherapy,methylprednisolone monotherapy,combination of Gp and methylprednisolone group.The control group was subcutaneously injected with oil emulsion adjuvant and all sother groups were subcutaneously immunized with an emulsified mixture of myelin oligodendrocyte glycoprotein(MOG)35-55 peptide to induce EAON.Mice in the Gp groups were administered injections daily with three concentrations(15 mg/kg,30 mg/kg,45 mg/kg)of Gp respectively.Mice in the methylprednisolone group and the combination treatment group were injected daily with methylprednisolone(20 mg/kg)or injected methylprednisolone(20 mg/kg)+Gp(30 mg/kg),respectively.After MOG immunization,visual evoked potential(VEP)and histopathologic staining(Lfb and Bielschowsky’s staining)were performed at 14,20,30and 40d post-inoculation(p.i.).Demyelination and axonal damage were observed by transmission electron microscope.Results:Compared with the control group,p2 latency was prolonged in the model group.Combination treatment can alleviated the change in VEP at 20d p.i.The pathomorphological results show a decrease in demyelination in the combination group compared with the model group(20d p.i.).Gp treatment also alleviated the degree of axonal loss(40d p.i.)by medium concentration of Gp or combined therapy.There was obviously change in the ultrastructural alteration:a significant demyelination and axonal damage were detected in EAON group from the onset to the later period of the disease.Gp treatment could alleviate axon damage in some degree and combination treatment prevent demyelination and axonal loss effectively.Conclusion:Treatment with Gp exerted protective effects on the optic nerve axons in cases of experimental optic neuritis.When combined with Gp,methylprednisolone reduces demyelination and axon damage in the acute stage of EAON.These results indicate that Gp might be a useful supplement for optic neuritis(ON)treatment.Part 3 Neuroprotective and Immunoregulation Effects of Gypenosides in Treating Experimental Autoimmune Optic Neuritis in Lewis RatObjective:To determine whether Gypenosides have immunoregulation effects and neuroprotective effects in the treatment of experimental autoimmune optic neuritis(EAON).Methods:Rat were randomly divided into 4 groups:control group,model group,Gp monotherapy,methylprednisolone monotherapy.EAON was induced in Lewis rat by footpad subcutaneous application of an emulsion containing 25μgMBP.The control group was injected with oil emulsion adjuvant.Mice in the Gp groups were oral administered daily with concentrations(100mg/kg)of gypenosides.Mice in the methylprednisolone group were daily injected with methylprednisolone(20 mg/kg)into the tail vein for 3 days.At 14d post-inoculation(p.i.),CD4+/CD8+ T cell ratio and CD4+T cells subset were examined by flow cytometry.The secretion of cytokines(IL-4,IL-10,IFN-γ,TNF-α)were detected by ELISA Kit.The MDA concentration in the optic nerve tissue were measured by spectrophotometer.Optical coherence tomography(OCT)and Tunel staning examination were performed at 14 and 42 d post-inoculation(p.i.).The expression levels of caspase-3,Akt,p-Akt were analyzed by Western blot method at 14 day p.i.Results:In acute EAON rats,treatment with Gp significantly increase the population of CD4+ T cell and Th2.Treg cell in CD4+T cell subset.Gp also reduce Tbl or Thl7 T cells in the optic nerve,corresponding to the upregulation expression of TL-4,IL-10 and downregulation expression of IFN-γ,TNF-α.Gypenosides can protect the rat optic nerve tissue from lipid peroxidation reaction demonstrat a antioxidant effect.Gypenoside attenuates optic injury by means of antioxidation in experimental autoimmune optic neuritis.OCT studies revealed a decline in thickness of the retinal nerve fiber layer(RNFL)in EAON rat,it is Gp but not Mp could reduced the RNFL deteriorate in the late stage of EAON.Treat with Gypenosides or methylprednisolone could both alleviate apoptosis in EAON rat at 14 and 42 p.i.Gp treatment decrease the expression of the cleaved caspase-3 and activate Akt signaling pathway.Conclusions:These results strongly indicate that gpenosides has a immunoregulation effect on EAON by suppressing T helper 1(Th1)and Th17 T cells and upregulating regulatory T cells and Th2 cell.Additionally,Gp alleviated neurological oxidation impairment of EAON and prevent RGC from apoptosis and activated of the PI3K/Akt pathway simultaneously.These neuro-protective effects represents a novel neuroprotective strategy in treating with demyelination disease.