The Effect And Pathogenesis Of Blue Light On Corneal Endothelial Cells

Part Ⅰ:The changes of corneal endothelial cell density in young and middleaged people of different agesPurpose:To access the changes of corneal endothelial cell density(ECD)in young and middle-aged people of different ages and its potential causes.Methods:PubMed,EMBASE,Cochrane Library and web of science were used to search the literature related to corneal endothelial cell density in China.As of December 2021,the literature containing the average value,standard deviation of ECD and case number of young and middle-aged people without other eye diseases except ametropia were included for meta-analysis.The year of examination and ECD were analyzed by linear regression Correlation analysis and subgroup analysis,which were used to compare the changes of corneal endothelial cell density in young and middleaged people in different ages.Heterogeneity was accessed.Results:50 studies were identified including 6,432 subjects,ranging in average age from 18.7 to 34 years.The total ECD was 2855.09±183.5(mean±standard deviation)/mm2,95%confidence interval(95%CI):2807.14～2903.03/mm2.According to linear regression analysis and correlation analysis,ECD showed a gradual downward trend with the development of years,and the two showed a significant negative correlation,which was statistically significant(P<0.0001).At the same time,the study was grouped by its examination year.There was no difference in age between the groups.Compared with the past 10 years,the average ECD of young and middleaged people decreased by about 129/mm2(P<0.01),and compared with the past 5 years,the ECD decreased by about 160/mm2(P<0.0001).The difference between the groups was statistically significant.Similar results were obtained after excluding the influence of region and inspection mode.Conclusion:with the development of the times,ECD of healthy young and middle-aged people in China gradually decreases,indicating that the spontaneous loss rate of corneal endothelial cells in modern people is higher than before.The reasons behind this may be related to the revolutionary change of light source,the wide application of LED and the increased time of using eyes.This phenomenon and its related mechanism need to be further clarified and studied.Part Ⅱ:Effects and mechanisms of blue light on corneal endothelial cellsPurpose:Blue light is defined as visible light with wavelengths ranging from 400 to 500 nm,and light-emitting diodes(LEDs),which are widely used today,emit large amounts of blue light.Blue light can reach the endothelial layer through the clear cornea,and corneal endothelial cells(CECs)are the key to maintaining corneal transparency.At present,the research on blue light and corneal endothelial cells is still in the blank.This study aims to explore the effect and mechanism of blue light irradiation on corneal endothelial cellsMethods:In vivo and in vitro LED blue light irradiation models were established.In vivo,male C57BL/6J mice aged 12 weeks were exposed to LED blue light for 4 hours a day.In vitro,primary cultured rabbit corneal endothelial cells were exposed to LED blue light for different times.The thickness and transparency of cornea and the morphology and density of CECs were observed by slit-lamp photography,in vivo laser confocal microscopy(IVCM)and optical coherence tomography(OCT).The ultrastructure of mouse corneal endothelium was detected by scanning electron microscope and transmission electron microscope.qRT-PCR,western blot and/or immunofluorescence staining were used to detect corneal endothelial barrier and pump function indicators ZO-1,N-cadherin,sodium and potassium pump.qRT-PCR,immunofluorescence staining and related kits were used to detect cell senescencerelated indicators including cell proliferation,cell cycle,SA-β-galactosidase(SA-βGal),senescence-associated secretory phenotype(SASP),and senescence-related genes p16 and p21;TUNEL staining was used to detect apoptosis;The ROS levels in corneal endothelial cells were detected by flow cytometry and immunostaining.Immunohistochemical staining(IHC)and/or qRT-PCR were used to detect the expression changes of oxidative stress related indicators 3-NT,NOX4,Nrf2,SOD2,HO-1 and Catalase.The changes of corneal endothelial cell morphology,function and senescence related indexes were observed by adding exogenous ROS scavenger NAC and Nrf2 signaling pathway inhibitor ML385 before blue light irradiation in vitro.Finally,in the in vivo and in vitro models,the changes of corneal endothelial cell morphology,function and oxidative stress related indicators were observed during the normal light was applied for a period of time after the blue light modeling.Results:The damage of CECs morphology and function increased in a dose and time dependent manner after blue light irradiation,and even caused endothelial decompensation after long time irradiation.Blue light irradiation in vivo increased the loss rate of endothelial cells with age and upregulated the expression of p16,p21 and SASP of corneal endothelium.Blue light irradiation accelerated cell senescence of CECs,increased cell apoptosis and caused the premature aging of the whole corneal endothelium and DM.In vitro blue light irradiation resulted in decreased proliferation of CECs,increased cell cycle arrest in G0/G1 phase,increased senescence related βgalactosidase activity,increased expression of p16,p21 and SASP,that is,the proportion of senescent cells increased significantly,and apoptosis also increased.The mechanism was related to oxidative stress.Blue light irradiation induced ROS production in CECs and activated antioxidant system through Nrf2 signaling pathway.With the extension of blue light exposure time,the balance between oxidation system and antioxidant system in CECs was disrupted,the accumulation of ROS and various oxidation products led to mitochondrial dysfunction,decreased membrane potential,and even mitochondrial structure destruction,thereby accelerating cell senescence and increasing cell apoptosis of CECs.The use of exogenous ROS scavenger improved the dysfunction of CECs and inhibited cell senescence.Activation of Nrf2 signaling pathway was an antioxidant defense mechanism of self-protection for CECs,which reduced ROS to inhibit cell senescence.In the in-vitro model,after the cessation of blue light irradiation,Nrf2 signaling pathway was continuously activated,and the activity and function of CECs could basically recover,but part of the abnormality of endothelial morphology could not be reversed,while the decrease of CECs density in the in-vivo model could partially recover after the cessation of blue light irradiation.Conclusion:Blue light can induce the increase of ROS in corneal endothelial cells and oxidative stress in mitochondria,leading to cell senescence and cell dysfunction.even cell apoptosis,and finally cause the aging of corneal endothelium and accelerated cell loss.The increase of ROS can induce the activation of Nrf2 signaling pathway in the early stage,which can protect CECs and inhibit cell senescence.After stopping blue light irradiation,the destruction of CECs can be improved through continuous activation of Nrf2 signaling pathway.