High Glucose, S Effects On HRPE Cells For Oxidative Stress And The Expression Of MMP-2,MMP-9and The Intervention Role Of α-lipoic Acid

Objective: Diabetic retinopathy (DR), one of the most common chronicmicrovascular complications of diabetes, has been the leading cause ofacquired vision down and even blindness with young adults inworldwide.Macular degeneration and proliferative retinopathy are the mostcommon diabetic retinopathy. Almost all type1diabetes and more than half ofpatients with type2diabetes have different levels of retinopathy withprogresse, which seriously destroy the patient's quality for life. The mainpathogenesis of DR contain activated protein kinase (PKC), the accumulationof advanced glycation end products, polyol pathway abnormalities, thedoctrine of oxidative stress, inflammatory response and a variety involvementof cytokines. The oxidative stress theory is currently recognized as thepathogenesis, and that fact oxidative stress could play a center role of theabove-mentioned mechanisms also is accredited.High concentration ofglucose could make the ability to clear oxygen free radica decreased, resultingin the accumulation of oxidation products, especially in the performance of theretinal tissue. Oxidative stress may cause the activation of PKC, accumulationof glycation products,abnormal polyol and other metabolic pathways.Atrecent,the role of matrix metalloproteinases(MMPs)in the progress retinopathyalso become a hot topic.MMPs, a family of proteolytic enzymes, pendent onZn²⁺ andthen promote extracellular matrix(ECM) degradation biologically.The premise for DR fundamental pathological mechanisms of newangiogenesis is the degradation of the retinal basement membrane and ECM,the migration of pericytes. The major collagen within Vascular basementmembrane is type Ⅳcollagen.MMP-2and MMP-9,the most critical gelatinase,result to degradation of collagen type Ⅳ. Studies in domestic or foreign, nomatter the clinical trials of patients with DR, diabetic animal model, or cultured retinal cells under high glucose conditions, were confirmed MMP-2and MMP-9expression increased. In conclusion, MMPs play an importantrole in the pathogenesis of DR. However, the authentic mechanisms of activityof MMPs increased by high glucose is not yet clear. Lipoic acid is a widelystrong antioxidants, in retinal cells without exception. Research in domestic,that50μ l of acrolein cause human retinal pigment epithelial (HRPE) celloxidative damage simultaneous for the intervention of different concentrationsof lipoic acid,has been reported.The results prove that certain concentration oflipoic acid could lead its protective effect to HRPE cells. In this study,different concentrations of lipoic acid effect cultured HRPE cells48hours inhigh glucose, in order to detect the level of MMP-2,MMP-9expression andoxidative stress. It is possible to find new pathway to search for the preventionand treatment of DR, by the mechanism of changes in expression of MMPscaused by High glucose.Methods: HRPE cells were cultured, and the logarithmic phase-growthcells were randomly divided into eight groups:normal control group(glucose5.56mmol/L, and DMEM solution)；high glucose group (glucose30mmol/L,and DMEM solution)；the five intervention groups:(alpha-lipoic acidconcentrations10μmol/L,50μmol/L,100μmol/L,200μmol/L,300μmol L,added glucose30mmol/L, DMEM solution)；mannitol group (mannitol30mmol/L). For48hours, observe the HRPE cell morphology. Viability ofHRPEcells is evaluated by MTT assay, the activity of the superoxide (SOD)and the levels of malondialdehyde (MDA), glutathione (GSH) is measured,MMP-2and MMP-9protein expression level is detected by Western Blot.Results:1The expression of SOD,GSH activity was significantly reduced and theMDA content increased in high glucose group(P<0.05), compared withnormal control group, while those indexes in mannitol group did not changesignificantly (P>0.05).In the intervention of lipoic acid, the expression activity of SOD, GSHgrew higher while the content of MDA lower than ones in high glucose group. The changes in rest of the four groups are obviously different from high sugargroup in dose-dependent manner(P <0.05), in addition to LA10μmol/L group.In addition to lipoic acid100,200μmol/L group,the expression of MDA forthe rest of any two groups were significantly different (P <0.05)；comparedwith lipoic acid50,100,200μmol/L group, the SOD activity in lipoic acid300μmol/L group was statistically (P <0.05)；while GSH had no significantdifference with groups (P>0.05).2Compared with normal control group, the protein expression ofMMP-2,MMP-9were significantly increased in high glucose group (P <0.05),while mannitol group did not change significantly (P>0.05).with theintervention LA, the protein expression MMP-2,MMP-9were declined. Theothers were a significant difference in dose-dependent manner (P <0.05), inaddition to10μmol/L group. And MMP-2expression in any two sets ofdifferent concentration of LA was statistically significant (P <0.05). while theprotein expression of MMP-9had no significant difference with groups (P>0.05).Conclusion:1Human retinal pigment epithelial cells are apt to get attack from oxidativestress and the protein expression of MMP-2,MMP-9always increas up in thehigh-sugar environment, which may play an important role in the process ofdiabetic retinopathy.2Oxidative stress, as a result of high-sugar environment, may stimulate retinalpigment epithelial cells to rise up MMP-2, MMP-9expression. It may be anintermediary factor between the higher expression of MMP-2, MMP-9and thehigh-sugar environment.3Alpha-lipoic acid can significantly inhibit the oxidative stress level of theretinal pigment epithelial cells, as well as effectively reduce MMP-2,MMP-9high expression. As a powerful antioxidant, it is very likely to play anactive role in the prevention and treatment of diabetic retinopathy.