Protective Effects Of Pentazocine On Retinal Müller Cells Under High-glucose Conditions

Objective: Diabetic Retinopathy is one of the most common and seriousmicrovascular complication of diabetes, which causes of blindness inworking-age individuals in developed countries. DR become the hots anddifficult spot both in clinical and in basic research. To date the pathogenesis ofdiabetic retinopathy has not been fully elucidated. And the basic principle ofDR treatment is to control blood glucose; correct hyperglycemia causedmetabolic disorder, and improves microcirculation and blood flow of retina.The mainstay of therapy for DR is panretinal photocoagulation so far, and thusnew therapies able to prevent the development of DR are greatly needed.The retinal Müller cells are contributed to the structural stabilization ofthe retina, modulation of immune and inflammatory responses. Besidesproviding neurons with trophic substances and remove metabolic wastes,Müller cells also play a critical role in regulating the extracellular spacevolume, ion and water homeostasis, and the inner blood-retinal barriermaintenance. Studies show that diabetes has complicated internalhyperglucose environment, and the change of the retinal Müller cells reflectedclosely to the early pathological and physiological process of DR, secreting avariety of inflammatory factors and participating in a large number of stressreactions and so on. It was reported that Müller cells plays an important role inthe nerve cells apoptosis and vascular leakage as well. Upon the important rolein the development of DR, retinal Müller cells have been noticed as a researchfocus in recent years.Pentazocine, a specific ligand of sigma receptor1(σR1), has high affinityand specificity in combination with σR1to produce nerve protective effect.The σR1has stable expression in the Müller cells cultured in high glucose invitro or the cells within the rat retina with diabetic retinopathy. This proposal is by means of culture retinal Müller cells in high glucose as the model in vitroto investigate the changes of the Müller cells in diabetic retinopathy.Comparison the cell activity， VEGF and ATF4expression with or withoutthe pentazocine treatment. This article will explore the molecular mechanismof protective effect of pentazocine on retinal Müller cells and providespotential therapeutic approach for treatment of diabetic retinopathy.Methods:1Müller Cell Culture and Immunocytochemistry:The enzyme digestion method is adopted to cultivate and purify theprimary retinal cells of SD rats. Cell immunochemical method was applied toidentify the retinal Müller cells.2Groups of Experiments:Experiment was designed to be three groups, normal control with normallevel of glucose (LG，5.5mmol/L), high glucose group (HG,30mmol/L),pentazocine treatment group (HG in the presence of PTZ3umol/L, HG+PTZ).The method of MTT is used to detect cellular activity, with different timecourse and different dose.3Real-time PCRVEGF and ATF4mRNA expression by the Müller cells in threeexperimental groups were examined by RT-PCR, production of VEGF andATF4was measured by Western blot.4Statiscal analysis.Experimental data were expressed as the mean±SD. Group means werecompared by one-way ANOVA followed by the Dunnett post hoc test usingSPSS (Statistical Package for the Social Sciences)13.0for Windows. A valueof P<0.05was considered significant in all cases.Results:1Isolation and identification of the retinal Müller cells: Primary cultureof retinal Müller cells from SD rats was performed by us enzyme digestionmethod. After8-14d, the Müller cells became confluent and continued toculture for use passages. M ü ller cells were confirmed by immunocytochemistry using antibody against the Müller cell markers GS.Almost all cells were positive for GS, indicating that the cultured cells wereMüller cells.2Cell activity assay: After6h,12h,24h with high glucose cultured, thecells vitality of HG group were decreased compared with LG group, but theOD value was no significant difference between groups;72h post highglucose culture, compared with LG, the HG group cells viabilities decreasedsignificantly (P <0.05), and the cells vitality of HG group were decreased by(23.3±0.1)%compared with LG group; HG+PTZ group cells activities washigher than the HG group (P <0.05). And the determined high glucose actiontime was72h and the level of drug concentration was3ummol/L.3VEGF and ATF4mRNA expression: After the retinal Müller cells wereexposed to HG (30mmol/L) for24h, the expressions of VEGF and ATF4mRNA were up regulated (P <0.05). The results showed that HG significantlyincreased mRNA level of VEGF by almost1-fold and ATF4by0.67fold inMüller cells. Pentazocine reduced HG-induced VEGF and ATF4expression.Moreover, compared with LH group there was no significant difference (P>0.05)4Western blot results: After72h high glucose culture, the expressions ofVEGF in LG, HG, HG+PTZ groups were0.597±0.015,0.735±0.076,0.622±0.006; the expressions of ATF4in three groups were0.183±0.024,0.249±0.008,0.192±0.006, respectively. In addition, compared with LH, theexpression of VEGF and ATF4in Müller cells significantly increased (P <0.05), and the expression of VEGF and ATF4in HG+PTZ group decreased(P<0.05), there was no statistical difference between LG and HG+PTZgroups (P>0.05).Conclusions:1The expression of VEGF and ATF4was up regulated in high glucosecondition in cultured rat retinal Müller cells.2Pentazocine significantly inhibited HG-induced VEGF and ATF4 mRNA and protein expression in retinal Müller cells.