Experimental Study On The Mechanism Of Gynostemma Glycosides In Treating Optic Nerve Injury And Retinitis In Rats

Background and purposeOptic neuritis is a common optic neuropathy that results in the loss of retinal ganglion cells(RGC)and visual impairment.The main pathological changes are optic nerve inflammation,demyelination and optic nerve axon damage.The etiology of optic neuritis is complex,which may be caused by primary optic neuritis or autoimmune disease or infection.Optic neuritis can cause sharp decline in vision within 1-2 weeks,and some patients can recover spontaneously within 1-3 months.However,about half of patients still have different degrees of permanent visual impairment.At present,the treatment of optic neuritis mainly uses glucocorticoids.However,these drugs do not prevent optic nerve axonal injury and thus do not improve the impairment of RGC and visual function in the course of optic neuritis.Therefore,exploring drugs with protective functions for optic ganglion cells has important significance for the treatment of optic neuritis.Gypenosides(GPs)are the total saponins of Gynostemma pentaphyllum in traditional Chinese medicinal materials.Studies have shown that GPs have anti-inflammatory effects and protective effects on neurodegenerative diseases,suggesting that gypenosides may also exert protective effects on neurons of optic neuritis.In this study,the rat model of optic retinitis and NMDA-induced excitotoxicity model of RGC-5 cells were studied to evaluate the therapeutic effect of GPs on optic neuritis and protection of optic ganglion cells,and possible molecular mechanisms were studied.Part 1 Protective Effects of Gypenosides on the Optic Nerve and Retina in Optic Neuritis Model RatsObjectiveOptic neuritis is a common optic neuropathy,which is divided into primary optic nerve inflammatory reaction,demyelination and optic nerve axon damage,which can eventually lead to the loss of retinal ganglion cells(RGCs)and visual impairment.In this study,the optic neuritis model was established by intraperitoneally injecting lipopolysaccharide(LPS)into the optic nerve,and observed the protective effect of gypenosides(GPs)on optic nerve tissue and RGCs in the retina.MethodsSprague Dawley rats were injected with LPS to induce optic neuritis,and made the injury model of optic nerve and retinitis in rats.The experimental animals were randomly divided into control group,optic neuritis group(ON)and ON + GPs group..The histopathological changes of each group were observed by HE staining.The expression of ED1/CD68 and GFAP was detected by immunofluorescence.Luxol fast blue(LFB)staining was used to evaluate the myelin sheath Degree of loss,retinal apoptosis detected by TUNEL.Results1.GPs reduce optic nerve injury in optic neuritis rats.2.GPs inhibit LPS-induced increases in macrophages and astrocytes in the optic nerve.3.GPs relieve LPS-induced retinal RGC injury.ConclusionsThe current research shows that GPs significantly attenuates LPS-induced neuroinflammation-mediated optic nerve tissue and retinal damage,inhibits microglial activation,astrocyte proliferation,and plays a protective role in demyelination and RGC.GPs can be used as a candidate drug for optic neuritis.Part 2 Gypenosides on Optic Nerve Inflammation Expression of Inflammatory Cytokines and Anti-inflammatory MechanismObjectiveDamage to the optic nerve caused by trauma or inflammation can be manifested as activation of the inflammatory response,release of a large number of inflammatory factors,and exacerbate nerves damage.Studies have shown that the pro-inflammatory cytokines TNF-a and IL-6 and the inflammation-related enzymes COX2 and iNOS are involved in the optic nerve inflammation,NF-κB and STAT are two important inflammatory signaling pathway.In this study,we detected the expression of the pro-inflammatory cytokines TNF-α and IL-6,the inflammation-related enzymes COX2 and iNOS and inflammatory signals molecular of NF-κB and STAT to further explore the mechanism of GPs on treatment of Optic neuritis.MethodsThe optic nerve of Sprague Dawley rats was injected with LPS to establish the model of optic neuritis.The animals were randomly divided into control group,optic neuritis group(ON)and ON+GPs group.The optic neuritis(ON)group and the ON+GPs group,fresh tissue was used to detect the related gene expression of each group by Real-time PCR assays,the expression related proteins differences by Western blotting.Results1.GPs inhibit the expression of pro-inflammatory cytokines TNF-a,IL-6 and the inflammation-related enzymes COX2,iNOS in the optic nerve injury model of Optic neuritis rats induced by LPS.2.GPs inhibit the activation of STAT1,3 and NF-κB in the optic nerve injury model of Optic neuritis rats induced by LPS.ConclusionsGPs significantly attenuated LPS-induced expression of pro-inflammatory cytokines TNF-α,IL-6 and inflammation-related enzymes COX2,iNOS,which may be partly by inhibition of NF-κB and STAT signaling pathways.Part 3 Protective Effect of Gypenosides on Excitotoxicity Induced by NMDA in RGC-5 CellsObjectiveThe pathological basis of optic nerve damage is the progressive death of retinal ganglion cells(RGCs)and the loss of optic nerve fibers,leading to irreversible changes in visual function.Many N-methyl-D-aspartate(NMDA)receptor antagonists have been used to treat neurodegenerative diseases caused by glutamate excitotoxicity in the clinic.To investigate the mechanism of action of gypenosides on NMDA-induced RGC-5 cells in vitro.MethodsNMDA(400 μM)treatment of RGC-5 cells to establish a model of neuronal excitotoxicity,and then cell culture,passage,counting,cryopreservation,administration,stand-by staining of apoptosis was detected by Annexin V-FITC-PI staining.Relative gene expression was detected by Real-time PCR and expression of related inflammatory factors was detected by ELISA.Results1.GPs inhibit NMDA-induced excitotoxicity of RGC-5 cells.2.GPs inhibit NMDA-induced expression of inflammatory cytokines in RGC-5 cells.3.GPs may inhibit the expression of inflammatory factors through gene expression level and play a protective role in RGC-5 cellsConclusionsIn this study,GPs were used to effectively inhibit the NMDA-induced neurotoxicity of RGCs,and the expression of inflammatory cytokines TNF-a,IL-6 and INF-γ and pro-inflammatory enzymes COX2 and iNOS.These suggest that it is possible to GPs antagonize NMDA-induced neurotoxicity of RGCs cells by inhibiting the expression of inflammatory cytokines.