Protective Effect Of Astragaloside Ⅳ On Methylglyoxal Induced Perineural Cell Injury

BackgroundDiabetic retinopathy(DR)is one of the common microvascular complications in the patients with type 1 and type 2 diabetes mellitus,being the leading cause of vision loss globally.It has been proved that DR is associated closely with the oxidative stress injury mediated by free radical.Oxidative stress plays an important role in proliferation and differentiation of cells,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.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 Ⅳ(AS-Ⅳ)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.So we supposed that AS-Ⅳ could have protective effects on oxidative damage of RPE induced by MGO.AimTo explore the protective effect of AS-Ⅳ on MGO-induced diabetic pericytic cell injury and explore its molecular mechanism.MethodsDifferent concentrations of MGO were used to induce retinal pericyte injury,CCK-8 was used to detect cell viability,and the optimal concentration and time of cell injury model were determined.However,AS-Ⅳ was pre-administered with various concentrations of AS-Ⅳ 2 hours prior to the addition of MGO,there by confirming the optimal AS-Ⅳ concentration for protecting the retinal pericytes from MGO-induced damage.The DCFH-DA was used as fluorescent probe to measure the level of intracellular ROS,and the detection of the activity of SOD was followed by the manufacturer’s instructions to explor the relation between oxidative stress and the protective effect of AS-Ⅳ on retinal pericyte.Western Blot was used to analyze the expression of Bcl-2 and Bax in mitochondria-mediated apoptotic signaling pathway.The activation levels of Caspase-9 and Caspase-3 were determined by fluorescent enzyme labeling method to explore related cellular pathways..Results1.The cell viability of retinal pericyte presented gradual decrease trend with the increasing concentration of MGO.After incubating for 6h with 800μM MGO,the survival rate of retinal pericyte was about 60%,so 800μM MGO with 6 hours’ incubation was regarded as the optimial condition.AS-Ⅳ preconditioning could increase the cell viability and reduced the cell injury induced by MGO.10 μM AS-Ⅳ could improve the survival rate of retinal pericyte 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-Ⅳ could reduce the production of ROS and the green fluorescence intensity weakened after the AS-Ⅳ pre-administration.Moreover,AS-Ⅳ can increase intracellular SOD activity.3.The morphology of cell nucleus in control was oval with homogeneous blue fluorescence,while in MGO group,some of the cell nucleus were wizened and broken with bright blue fluorescence.AS-Ⅳ could improve the morphology of cell nucleus.By the flow cytometry,we observed that AS-Ⅳ could significantly inhibit the increasing apoptosis rate of retinal pericyte induced by MGO.All the results indicated that AS-Ⅳ had remarkable anti-apoptosis effects.4.We found that AS-Ⅳ could improve the expression of Bcl-2 and raise the ratio of Bcl-2/Bax.AS-Ⅳ could lower the activation of Caspase-9 and Caspase-3,regulating cell apoptosis.ConclusionsAS-Ⅳ can play an anti-apoptotic role by regulating Bcl-2 and Bax in the body,increasing Bcl-2 expression and decreasing Bax expression.It can also regulate the Caspase-3 and Caspase-9.The activation level of inhibits apoptosis of retinal pericyte and has the effect of protecting pericytes.