| Background and objective:With the incidence of environmental pollution,changes of living habits,and the popularization of video devices,the incidence of dry eye has increased to affect over 15 to 35% of the world's population.Common DED symptoms include but are not limited to: eye irritation,blurred vision,feelings of eye dryness,and overall discomfort.Though pathogenesis of dry eye is not clear yet,inflammation and oxidative stress play in important role in its development.At present,the main treatment of dry eye is the use of tear substitutes such as artificial tears or gels,and anti-inflammatory drugs such as glucocorticoids and cyclosporine A.However,various drugs have either their limitations in treatment or significant side effects.As evidenced by numerous research studies highlighting the importance of oxidative stress and inflammatory mediators in the progression of dry eye,development of therapeutic strategies that effectively inhibit key inflammatory pathways may provide more effective treatment for patients with dry eye.Recently,multiple papers have highlighted the anti-inflammatory properties of antiviral drugs.One class of antiviral drugs,termed Nucleoside Reverse Transcriptase Inhibitors(NRTIs),are a class of drugs which are widely used in combination as the mainstay of treatment for HIV.In 2014,the anti-inflammatory effects of NRTIs in age-related macular degeneration(AMD)were first reported.Studies have shown that NRTIs can protect AMD animal models by inhibiting the activation of inflammsome.The mechanism of inhibition of caspase-1 activation is specific and independent from their reverse transcriptase inhibitor functions.In ocular surface diseases,studies have also shown that dry eye and diabetic-related ocular surface disease are also involved in the activation of NLRP3 inflammasome.Although previous studies clearly illustrate the anti-inflammatory properties of NRTIs,there are few reports on the protective effects of Zidovudine(AZT),one of the most commonly used NRTIs,in ocular surface diseases—particularly dry eye.In this experiment,we established two different oxidative stress cell models in human corneal epithelial cells.We would like to observe the relationship between oxidative stress and inflammatory response,to investigate whether AZT can regulate the balance between cellular oxidative system and antioxidant system,as well as to dertermine which pathway it works by.As a widely used,inexpensive,and readily available drug,novel repurposing of AZT for the treatment of dry eye may prove to be useful.Subjects and methods1.HCECs were cultured and induced two different ways to establish oxidative stress models in vitro.(1)4-HNE induced oxidative stress model: 10?M,20?M,30?M 4-HNE(2)Hyperosmolar cell stress model: 70 mM,90 mM Nacl2.The following measurements were performed to evaluate the activation of oxidative stress and expression of inflammatory cytokines.Cell death measured by a Pierce LDH Cytotoxicity Assay Kit,total ROS level measured by a DCFDA kit,NF?B p65 expression in cell lysates examined by a NF-?B p65 Total SimpleStep ELISA Kit,profile cytokine analysed by a Proteome Profiler Human Cytokine Array Kit,NLRP3 inflammasome activation markers caspase1 p10/p20 and ROS-related enzymes were measured by western blot.3.Elaluation of anti-inflammatory and antioxidative stress effects of AZT on different oxidative stress models.HCE cells were pretreated with 50 ?M and 100 ?M AZT for 1.5 hours.Then HCE cells were stimulated with 4-HNE or NaCl,the following methods were used for detection:1)Cell cytotoxicity of different HCE cell stress models after AZT pretreatment were meseased by LDH method.2)ROS levels in different HCE cell stress models after pretreatment of AZT can be continuously observed by DCFDA-Cell Reactive Oxygen Assay3)Sandwich ELISA for IL-6 and IL-8 were performed to determine concentration of pro-inflammatory cytokines in media treated with various concentrations of AZT4)NF?B p65 level were detected and profile proteins involved in NF?B pathway were further analysed by a Proteome Profiler Human NFk B Pathway Array Kit.5)ROS-related enzymes expression were measured by Western Blot on protein levels Results:1.Cell survival were improved by AZT in different HCE cell stress modelsAfter 100?M AZT treatment,the cell death in the 20?MHNE group decreased by 7.729%.Cell death in the 70 mM NaCl group also decreased from 8.47±0.70% to 3.60±0.44% with AZT treatment,which was very close to control group(4.147 ±.8772%)(p<0.05).2.ROS level were downregulated by AZT in different cell modelsThe ROS level of control group was 14760±572 RFU at 150 minutes.In the 20 ?M 4-HNE induced group,ROS production increased to 18189.5±525.5 RFU(P<0.05),while in 100 ?M AZT pretreated group,ROS level could be decreased to 14332±3611.5 RFU which was very close to control(P<0.05).Similarly,the 70 mM NaCl-induced ROS level could be increased to 6590.33±821.62 RFU at 150 min,which was significantly higher than that of the control group 4175.50±129.04 RFU(P<0.01).After pretreatment with 100 ?M AZT,the ROS levels in HCECs also decreased to 5016.67±327.06 RFU(P<0.05).3.NF?B p65 expression decreased after AZT pretreatment in different HCE Cell Oxidative Stress ModelsAfter induced by 20?M 4-HNE or 70 mM NaCl for 24 hours,the expression of NF?B p65 increased to 138.952±6.633% or 126.83%±3.79%,respectively,compared to the control group.After pretreatment with 100?M AZT,NF?B p65 expression both in 20?M 4-HNE group and 70 mM NaCl group decreased to 81.9 ± 2.637%(P <0.01)and 87.99 ± 1.53%(P <0.05),compared to control.4.Possible AZT target proteins in NF?B PathwayNF?B pathway protein profile analysis were performed by Proteome Profiler Human NFk B Pathway Array Kit.After pretreatment with 100 ?M AZT,20 ?M 4-HNE was used induce HCE cells and 41 Proteins were detected.From the results,CD40/TNFRSF5,KK?/NEMO,p53,c-Rel,TRAF2,TRAIL R1/DR4,SOCS6,STAT1(pY701)expression levels may increase in 4-HNE treatment group,however,in the AZT100 ?M group TRAIL R1/DR4,SOCS6,and I?B? decreased almost to the level of control.5.AZT could balance the oxidative and antioxidative response system in HCECsSOD1 and NQO1 protein levels were determined by Western Blot.In this study,we observed decreased SOD1 levels and increased NOQ1 levels in HCECs exposed to 4-HNE induced model.However,pretreatment of HCECs with 50?M or 100?M of AZT showed restored levels of antioxidant enzyme SOD1 back to near-control levels.SOD1 expression in HCEC treated with 20 ?M 4-HNE was reduced to 62.06% compared to control group,while 50 ?M and 100 ?M AZT treatment increased to 95.79% and 78.43%(P<0.05).In 20?M 4-HNE induced cell model,NQO1 levels increased to 146.3% compared to control,which could be decreased to 136.6% and 113.11% with 50?M and 100?M AZT treatment(P<0.05).Similarly,in the 70 mM NaCl-induced cell model,SOD1 protein levels in the 70 mM Nacl group were only 39.10% compared to the control group.After pretreated with 50 ?M or 100 ?M AZT,SOD1 expression level could be increased to 49.5% and 65.1% relative to the control(p<0.05).6.AZT can decrease IL-6 expression in different HCEC stress modelsAfter induced by 20 ?M HNE for 24 hours,IL-6 expression was increased to 66.247±3.433 pg/mL pg/mL compared with 34.227±4.702 pg/mL in the control group.If treated by 100 ?M AZT,IL-6 expression could be decreased to only 11.536±1.866 pg/mL(P<0.01).Similarly,in 70 mM NaCl induced group,the IL-6 expression level increased to 48.43±1.153(P<0.01)compared to the control group at 7.406±1.153 pg/mL;100 ?M AZT pretreatment could decrease IL-6 level to 6.37±1.547 pg/mL(P<0.01).Conclusion:1.This study indicated that AZT conferred significant anti-oxidative stress and anti-inflammatory effects on 4-HNE induced or hyperosmolar induced HCECs model.These effects included: an increased expression of SOD1,reduced ROS production,decreased activation of NF? ?B and production of proinflammatory cytokine IL-6,and improved cell viability.2.This study couldn't provide any evidence of AZT playing a role in inhibiting the activation of NLRP3 inflammasome.However,further experiments are needed.3.Through the profiler NF-?B pathway proteins analysis,CD40-TRAF2-NF KappaB-IL-6 pathway may be possible targets by which AZT interferes in the oxidative stress pathway.However,this conclusion needs to be confirmed by further testing of relevant proteins at DNA,RNA and protein levels. |
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