Research On Protective Effect And Molecular Mechanism Of Astragalosides ? On Retinal Pigment Epithelium Injury Induced By Methylglyoxal

BackgroundDiabetic retinopathy(DR)is one of the common microvascular complications in the patients with type 1 or type 2 diabetes mellitus,being the leading cause of vision loss globally.It has been proved that DR is closely associated with the oxidative stress injury mediated by free radical.Oxidative stress plays an important role in proliferation and differentiation of cells.It can regulate the physiological function and biochemical reaction of cells and can also induce the apoptosis.Retinal pigment epithelium(RPE)is the mainly component of the outer blood-retinal barrier(oBRB),as a monolayer of pigmented cells situated between the neuroretina and the choroids.The tight junction between neighbouring RPE and adjacent endothelial cells is essential for the integrity of the retina.It has been confirmed that the RPE dysfunction was observed in early DR.Methylglyoxal(MGO),one of the active dicarbonyl compounds formed by glycolysis,is the precursor of advanced glycation ends products(AGEs).Large amount of MGO,produced by the high level of glycolysis in diabetes with persistently increased blood glucose,leads to the abnormal accumulation of AGEs in RPE basement membrance,resulting in cell injury and DR.Astragaloside ?(AS-?)is the major bioactive substance in astragali radix,with various pharmacological activities,including anti-inflammation,anti-aging,scavenging reactive oxide species,ameliorating the state of insulin resistance,accelerating cell proliferation and anti-apoptosis.Moreover,AS-IV can reduce the blood glucose in diabetes,inhibit the oxidative stress and decrease the formation of AGEs.Therefore,we supposed that AS-IV could have protective effects on oxidative damage of RPE induced by MGO.AimTo investigate the protective effects of AS-? on human retinal pigment epithelial cell(ARPE-19)injury induced by MGO,and to explore its molecular mechanism.MethodsMGO with different concentrations was performed to induce the damage of ARPE-19,and cell viability was detected by CCK-8 to determine the optimal concentration and time for the model of cell injury.Aftrer that,AS-IV pretreatment of various concentrations was preformed 6 h before the addition of MGO,by which the optimal concentration of AS-IV to protect ARPE-19 against MGO-induced injury could be confirmed.The DCFH-DA was used as fluorescent probe to measure the level of intracellular ROS,and the detection of the activity of SOD and the concentration of MDA was followed by the manufacturer's instructions to explore the relation between oxidative stress and the protective effect of AS-? on ARPE-19.Furthermore,the morphology of cell nucleus was observed by high-content imaging system after Hoechst 33342 staining.ARPE-19 cells were labelled by Annexin V-FITC/PI and the apoptosis was detected by flow cytometry.JC-1 staining was employed to evaluate the change of mitochondrial membrane potential.Western Blot was used to analyse the expression of Bcl-2,Bax,PARP,Akt and p-Akt in apoptosis signal pathway mediated by mitochondrion.And the level of Caspase-9 and Caspase-3 activation was measured by fluorescence enzyme-labelled method,to explore the related molecular mechanism.Results1.The cell viability of ARPE-19 gradually decreased with the increasing concentration of MGO.After incubating for 16h with 1 mmol·L-1 MGO,the survival rate of ARPE-19 was about 60%,so 1 mmol·L-1 MGO with 16-hours' incubation was regarded as the optimial condition.AS-IV pretreatmentcould increase the cell viability and reduce the cell injury induced by MGO.10 ?mol·L-1 AS-? could improve the cell viability of ARPE-19 to the maximum,compared with MGO group.2.By the analysis of fluorescent images,we found the level of ROS in MGO group significantly increased,leading to the marked increase of green fluorescence intensity,while AS-? could reduce the production of ROS and the green fluorescence intensity weakened after the AS-? pretreatment.What's more,AS-? could reverse the activity reduction of SOD and the augment of MDA induced by MGO.3.The morphology of cell nucleus in control group was oval with homogeneous blue fluorescence,while in MGO group,some of the cell nucleus were wizened and broken with bright blue fluorescence.AS-? could improve the morphology of apoptotic cell nucleus.By the flow cytometry,we observed that AS-? could significantly inhibit the increasing apoptosis rate of ARPE-19 induced by MGO.All the results indicated that AS-?exhibited remarkable anti-apoptosis effects.4.The fluorescence ratio of red/green in MGO group significantly decreased compared with control group;AS-? could increase the ratio of red/green,which suggested AS-? could prevent the decrement of mitochondrial membrane potiential of ARPE-19 caused by MGO.Also,we found that AS-? could improve the expression of Bcl-2 and raise the ratio of Bcl-2/Bax.AS-? could inhibit the cleavage of PARP which plays a key role in repairing the damaged DNA,and accelerate the phosphorylation of Akt to promote the survival of ARPE-19.In addition,AS-? could lower the activation of Caspase-9 and Caspase-3,exerting the ability to inhibit cell apoptosis.ConclusionsAS-? presented excellent protective effect against the damage of ARPE-19 induced by MGO.AS-? could resist the oxidative stress injury of ARPE-19 by reducing the ROS and MDA and increasing SOD.Furthermore,AS-? could regulate the protein expression in Bcl-2 family and Caspase family to exert the anti-apoptosis effect and enhance the expression of p-Akt to promote the survival of ARPE-19.The whole data implied AS-? had the potential to be developed a potent drug in the treatment of DR,although the therapeutic effect of AS-? on DR remains to be confirmed by further experiments in vivo.Other molecular mechanisms underlying in the protection effect of AS-? on ARPE-19 need more researches.