| The protective effect of zeaxanthin on human limbal and conjunctival epithelial cells against UV-induced cell death and oxidative stressPart IAIM:Zeaxanthin is an important macular pigment with photon absorbing and anti-oxidative stress capacities.The aim of this study was to understand if it has protective effect on human limbal and conjunctival epithelial cells against UV-radiation and excessive oxidative stress.METHODS:Human limbal and conjunctival epithelial cells were isolated from cadaver corneal tissues and cultured in vitro.They were challenged with UVB radiation and H2O2 with and without zeaxanthin pretreatment.Cell viability,p38 and c-JUN NH(2)-terminal kinase(JNK)phosphorylation,IL-6,IL-8 and MCP-1 secretion and malondialdehyde(MDA)content were measured.Results:Zeaxanthin had no measurable cytotoxicity on limbal or conjunctival epithelial cells when used at concentrations of 5μg/mL and below.At 30 mJ/cm2 UVB,the pretreatment of zeaxanthin increased the percentage of live cells from 50%to 69%(p=0.01)and from 66%to 75%(p=0.05)for limbal and conjunctival epithelial cells,respectively.At the meantime,the concentrations of IL-6,IL-8 and MCP-1 in the culture medium reduced to 66%(for IL-6 and MCP-1)and 56%(for IL-8)of the levels without zeaxanthin.This was accompanied by reduced p38 and JNK protein phosphorylation.Pretreatment of zeaxanthin also reduced intracellular MDA content caused by H2O2 stimulation from 0.86 μM to 0.52 μM(p=0.02)in limbal epithelial cells and from 0.96 μM to 0.56 μM in conjunctival epithelial cells(p=0.03).However,zeaxanthin did not have significant effect on H2O2-induced cell death in limbal or conjunctival epithelial cellsConclusions:Our results showed that zeaxanthin is an effective reagent in reducing the detrimental effect of UV-radiation and oxidative stress on ocular surface epithelial cellsPart Ⅱ(1) The effect of blood glucose on the oxidative stress of aqueous humorObjective:The purpose of this study was to compare aqueous oxidative stress levels in cataract patients with different blood glucose.Methods:Seventy-one cataract patients who underwent phacoemulsification and intraocular lens implantation in Xinhua hospital from July 2016 to October 2016 were divided into four groups according to the preoperative fasting blood glucose(FBG)levels:Group 1,FBG<5.6mmol/L,30 patients;Group 2,5.6 ≤ FBG<7.0 mmol/,20 patients;Group 3,FBG≥7.0 mmol/L but deny the history of diabetes,10 patients;G roup 4,positive history of diabetes,11 patients.Patients(regardless of blood glucose)were also divided into four age-groups:Group A,age<70,9 patients;Group B,70≤age<80,20 patients;Group C,80≤age<90,34 patients;Group D,age≥90,8 patients.Furthermore,patients in Group 1 were divided into four age-groups:Group 1A,age<70,3 patients;Group 1B,70≤age<80,8 patients;Group 1C,80≤age<90,15 patients;Group 1D,age≥90,4 patients.Patients in Group 1 and Group 2 were also divided into four age-groups:Group(1+2)A,age<70,7 patients;Group(1+2)B,70≤age<80,14 patients;Group(1+2)C,80≤age<90,23 patients;Group(1+20)D,age≥90,6 patientsThe concentrations of MDA and TAC in aqueous humor of each patient were measured respectively.The concentration of MDA in plasma was also measuredResults:Among the patients grouped according to blood glucose,there were statistically significant differences in MDA levels in the aqueous humor but not plasma,between the group 1(normal glucose group),group 2(critical glucose group),and group 3(hyperglycemia group)and the group 4(diabetes group)(P<0.001)Pearson correlation analysis showed that there was a positive correlation between aqueous humor MDA and FBG level(r=0.593,P<0.001).However,MDA levels showed no significant differences among patients at different ages.We found no significant differences in TAC among patients with different FBG status or ages However,among patients with normal FBG levels and without diabetes,we found positive correlation between aqueous MDA and TAC(r=0.402,p=0.034)Conclusion:The concentration of MDA in aqueous humor is strongly has correlated with blood glucose level.However,TAC remained constant despite of glucose and age changesPart Ⅱ(2) Effect of intravitreal injection of anti-vascular endothelial growth factor on aqueous oxidative stress level and inflammatory cytokine concentrations in patients with proliferative diabetic retinopathyObjectives:The purpose of this study was to determine the effect of intravitreal injection of anti-vascular endothelial growth factor(VEGF)on aqueous oxidative stress level and inflammatory cytokine concentrations in patients with proliferative diabetic retinopathy(PDR).Methods:A total of 74 patients with proliferative diabetic retinopathy underwent pars plana vitrectomy(PPV)were enrolled in this study,including 35 males and 39 females.ALL the patients received intravitreal Conbercept theraphy 7 days before PPV.50-100μl of undiluted aqueous humor samples were collected at the time of intravitreal Conbercept and PPV treatment.Exclude patients with other serious history of heart,liver,kidney and other systemic diseases;glaucoma,uveitis and other history of eye diseases;previously had a history of retinal laser,vitreous injection,history of eye surgery.All patients were tested for IL-6,MCP-1,and MDA concentrations in the aqueous humor before and after intravitreal injection of anti-VEGF drugs.Results:7 days after intravitreal Conbercept,the levels of IL-6,MCP-1(3889.86±6583.57 pg/ml、1501.43±1012.96 pg/ml)in aqueous humor were significantly higher than the levels of pre-operation(317.16±1763.89 pg/ml,1089.38±679.09 pg/ml),the differences were statistically significant(P<0.001).The levels of MDA(22.21±8.05 pg/ml))in aqueous humor were significantly lower than the levels of pre-operation(32.03±16.80 pg/ml),the difference was statistically significant(P<0.05).Spearman’s correlation analysis showed that there were always strongly correlations between the concentrations of IL-6 and MCP-1 in the aqueous humor of the patients before and after the intravitreal Conbercept,including r=0.681 before injection,P<0.001,and r=0.614 after intravitreal injection,P<0.001.Ⅱ-6 and MCP-1 were also strongly correlated before and after intravitreal injection themselves,with r=0.376 before and after injection(IL-6),P<0.05,and r=0.638 before and after injection(MCP-1),P<0.001.We also find before the intravitreal Conbercept,there was a strong correlation between MDA and MCP-1 in the aqueous humor of patients,r=0.651,P<0.001.There was also a strong correlation between MDA and IL-6,r=0.530,P<0.05.No correlation between MDA,MCP-1,and IL-6 was found after intravitreal Conbercept.Conclusion:Intravitreal injection of anti-VEGF drugs may not reduce the concentration of inflammatory factors in aqueous humor of PDR in the short term,but it may reduce the oxidative stress. |
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