Agmatine Protects Muller Cells From High-concentration Glucose-induced Cell Damage Via Inhibition Of N-methyl-D-aspartic Acid Receptor And Its Signal Pathway In Diabetic Rat

Diabetic retinopathy (DR) is the most serious and common microvascularcomplications of diabetic (DM).With the continuous improvement of people’s livingstandards in recent years,diabetes has been increasing in the country,the incidence ofDR increased significantly.Early DR primarily non-proliferative.As the duration ofthe extension,DR progression to proliferative lesions characterized by theproliferation of new blood vessels.Patients with concurrent symptoms includegradual emergence of severe intraocular hemorrhage,neovascular glaucoma, andretinal detachment.DR cause severe visual dysfunction, and have a very disruptiveto the normal life of the patient.Past studies suggest that DR is a microvascular disease, but recent studies havefound that the presence of retinal damage neurons in the DR lesions.Moreover,retinal degeneration earlier than retinal vascular changes.Nervous system damageplays an important role in the pathogenesis of DR.As important retinal glialcells,Müller cells of retinal neurons to provide structural support and nutrition.Inretinal ganglion cells (RGC) growth,damage,repair and regeneration have played animportant role. Therefore,the search can be suppressed apoptosis diabetes drugMüller cells and to study the mechanism of the prevention and treatment of DRsignificance.Agmatine is an endogenous polyamine formed in the mammalian brain,hasanalgesic,anti-tumor and neuroprotective effects.In vitro studies have shown thatagmatine can suppresse oxidative stress,hypoxia or TNF-α-induced RGC apoptosis.But whether the protective effect on agmatine and related mechanisms exist in the DR process has not been reported.The study found significantly elevated inSTZ-induced diabetic rat model of cerebral cortex glutamate and NMDA receptorexpression levels, NMDA receptor expression levels and may play an important rolein the excessive activation of diabetes,and agmatine by blocking NMDA receptoractivity can inhibit glutamate-induced cerebellar granule cell death,we hypothesizedthat agmatine by the action of the NMDA receptor and thus may play a protectiverole in diabetic retinopathy injury.In this experiment, we have successfullyestablished animal models and cell models of early DR and conducting agmatineinterventions to study the protective effect of agmatine on DR and Müller cells, andanalyze the molecular mechanisms.This study provides new drug candidates for theclinical treatment of DR.1. Effect of agmatine on the growth and apoptosis of Müller cell in highglucoseMüller cells is one of the main glial cells in retinal tissue, involved in neuronssignal transduction,protect neurons,the balance of the environment,and maintainBRB stable.To investigate the protective effect of agmatine on diabetes Müller cellsin an in vitro model,we choose the rat Müller cells cultured in high glucose for thestudy.Some studies mentioned that in the DR process ganglion cell apoptosis is themain in retinal tissue.In this experiment, Müller cells cultured in high glucosesignificantly reduced viability,and the presence of agmatine can improve cellviability. Hoechst staining and Annexin V/PI flow cytometry showed that apoptosisalso exists Müller cells in high glucose conditions, and the apoptosis wassignificantly reduced in the agmatine intervention group, indicating that agmatineplay a protective role on Müller cells under high glucose, but this effect wasinhibited by NMDA,suggesting that agmatine probably by inhibiting NMDAR playa protective role of Müller cells.2. Effect of agmatine on Müller cell inflammation in high glucoseMüller cells play an important role in retinal diseases,changes in Müller cells in the retina have a significant impact on disease progression and neuronal function.Inflammation is a major pathological response of glial cells.In the early animalmodels of diabetes,the researchers found that the presence of inflammation in retinaltissue,and further found that the presence of inflammation in DR retinaltissue.TNF-α is an major marker of early inflammation associated,and activatephospholipase A2, which can cause hydrolysis of the structure and function ofendothelial cell damage, promote platelet,neutrophil and monocyte aggregation,increased vascular permeability,resulting in increased BRB damage.Inaddition,TNF-α can mediate VEGF,IL-1induced expression of other leukocytedeposition in retinal vascular injury and BRB.In this experiment, meanwhile ELISA and Real-time PCR were used to detectthe expression and secretion of TNF-α,and found that the Müller cells cultured inhigh glucose significantly increased the expression and secretion of TNF-α,andagmatine intervention can significantly reduce TNF-α release and mRNA expressionlevels,this effect was inhibited by NMDA,suggesting that agmatine may reduce theinflammation in genetic level by inhibiting NMDAR in high glucose.3. Effect of agmatine on the retina of diabetic ratsThe retinal structure and function in STZ-induced diabetic rats are very similarwith the retinal lesions in early DR patients,so it is selected as the commonly usedexperimental animal models.The STZ-induced type1diabetes modeling shorter,DRappeared earlier, in favor of early lesions observed,therefore, choose the model ofthe experimental study.Transmission electron microscopy and TUNEL staining showed that retinaltissue of diabetic rats microstructural damage nerve cells, and the cells begin toapoptosis.AGMATINE intervention can reduce the extent of DR rat retinal tissuelesions.4. The research on molecular mechanism of AGMATINE on protection ofMüller cells in high glucoseLDH activity was used to detect the fluorescence labeled probes to detect calcium intracellular free calcium concentration in cells, we found in high glucose,cell activity decreased calcium influx,intracellular free calcium levels rise,increasedapoptosis. The intervention by agmatine can significantly inhibit the release of LDH,cell activity increased, reducing the intracellular calcium ion concentration on cellinjury has a protective effect, which may be related to NMDAR inhibition.To explore the anti-apoptotic mechanism of agmatine, we investigated theBcl-2family proteins,which plays an important role in the regulation of themitochondrial apoptotic pathway.Western blot analysis showed that,Bcl-2expressionwas reduced and the expression of Bax was increased in Müller cells with highglucose.Agmatine significantly increased Bcl-2protein levels while reducing Baxprotein levels,and reduce the expression of apoptotic pathway effector moleculeCaspase-3,and inhibited apoptosis.Therefore,agmatine may play a role inanti-apoptosis through regulation of Bcl-2family proteins.Mitogen-activated protein kinases (MAPKs) signaling pathway is closelyrelated to cell stress and apoptosis.MAPKs signal can be activated byNMDA-induced Ca2+influx and promote neurotoxicity produced by NMDA in ratretina.In this study, Western blot test results showed that high glucose in thepresence of MAPK protein hyperphosphorylation,and agmatine can reduce theiracidification levels.This effect was blocked by NMDA.This observation indicatesthat agmatine intervention may reduced the level of MAPK phosphorylation byinhibiting NMDAR,and thus play a cytoprotective mechanism.In this experiment,we found that agmatine can reduce lesions in diabetic ratretina,improve the viability of Müller cells in high glucose,reduce cell inflammation,regulate the apoptosis-related protein expression,inhibite the MAPK signalingpathway.Experimental results demonstrate that agmatine can inhibit Müller cellsapoptosis in high glucose through anti-inflammatory and anti-apoptotic pathways.Moreover,all these protective effects are blocked NMDA,indicating that agmatineprotection of Müller cells may be through inhibition of NMDA receptors to exert itseffects. Although we do not know whether there are other regulatory mechanismsinvolved in the protective effect of agmatine,but it can be sure that: agmatine canplay a role in reducing diabetic retinal damage,and should be considered as a newtherapeutic drug candidate DR.