| BackgroundVisible light is an indispensable information carrier for the formation of vision,but long-term excessive light exposure is considered an important pathogenic factor for various retinal degenerative diseases.For example,age-related macular degeneration(AMD)has become one of the important eye diseases that cause blindness in the elderly population in developed countries,and long-term chronic photochemical damage to the retina is closely related to the occurrence and development of AMD.With the rapid development of technology,many electronic screens and artificial lighting are widely used in daily life.However,excessive light exposure and pollution can cause retinal photochemical damage.Retinal photochemical damage is mainly caused by photooxidation.The retina,as a visual organ,contains a large number of photosensitive groups that can perceive visible light.When high-energy photons are absorbed by photosensitive groups,they can undergo chemical structural changes,such as electron orbital transitions or chemical bond breaks,resulting in chemiluminescence damage.In addition,when photon energy is transferred to photosensitive molecules,it also transfers to oxygen molecules,leading to electron orbital transitions and the production of reactive oxygen species(ROS),such as singlet oxygen,oxygen radicals,hydroxyl radicals,nitric oxide(NO),etc.Subsequently,the reaction between ROS and surrounding molecules will disrupt molecular bonds,a process known as photooxidation.Visual cells are rich in mitochondria to produce a large amount of adenosine triphosphate(ATP)for visual physiological activities.However,severe redox imbalance can cause mitochondrial damage and further trigger programmed cell death.In addition,NO can penetrate the nuclear membrane,causing oxidative deoxyribonucleic acid(DNA)damage in the nucleus.The bases,nucleotides,and single or double stranded DNA molecules in the nuclear DNA structure can all become ROS damage targets.Autophagy is one of the main pathways for clearing damaged proteins and organelles in cells.Autophagy transports protein aggregates and damaged organelles into lysosomes to complete the degradation and recycling of cellular components.Autophagy has long been considered a non-selective degradation pathway,but recent studies have shown that it can selectively remove unnecessary cytoplasmic substances such as organelles and proteins.Mitophagy is an important form of autophagy that can selectively degrade aging and damaged mitochondria,reducing the abnormal accumulation of dysfunctional mitochondria.When mitophagy is disrupted and aging and damaged mitochondria cannot be cleared in a timely manner,dysfunctional mitochondria will produce excessive ROS and release apoptosis related factors such as cytochrome C,thereby triggering programmed cell death.In mammalian cells,mitophagy is mainly mediated through two signaling pathways:the ubiquitin dependent pathway and the receptor dependent pathway.Mitophagy is strictly regulated to cope with various stresses and maintain mitochondrial and cellular homeostasis.Mitophagy dysfunction has been proven to be related to various diseases,such as neurodegenerative diseases、metabolic diseases and ischemia-reperfusion injury.Poly(ADP ribose)polymerase(PARP-1)is a ribozyme responsible for repairing DNA damage.When DNA damage occurs,PARP-1 is quickly activated,and the activated PARP-1 can synthesize poly(ADP ribose)(PAR)to recruit various related factors for DNA repair.However,in the case of extensive DNA damage,excessive activation of PARP-1 can synthesize a large amount of PAR,which can enhance the permeability of the mitochondrial outer membrane and promote the hydrolysis and activation of Apoptosis inducing factor(AIF)anchored in the mitochondrial inner membrane,and translocation to the nucleus leading to PAR dependent cell death.However,the regulatory relationship between PARP-1 and mitophagy signals still needs further clarification.This study investigated the role of mitophagy induced by excessive light exposure in retinal light injury through in vitro and in vivo experiments,and verified that inhibiting mitophagy can play a protective role in retinal light injury.In addition,we investigated for the first time the regulatory effect of PARP-1 activation on mitophagy in retinal light injury,and confirmed that inhibiting PARP-1 signaling and inhibiting mitophagy can synergistically play a protective role against retinal light injury.This study lays an experimental foundation for the further development of targeted drugs for the treatment of retinal degenerative diseases related to light damage.Objective1.To establish an in vitro photoreceptor(661W)photoinjury model and detect the activation of mitophagy pathway.Using the mitophagy inhibitor Cyclosporin A(CsA)to treat cells,we validated the photoinduced damage of 661W cells and the role of mitophagy signal activation in light damage through in vitro experiments.2.To establish a PARP-1 knockdown cell line of 661W cells,and investigate the regulatory effect of PARP-1 signaling pathway on mitophagy and its impact on cell photodamage in 661W cells.3.To establish a retinal light injury model in C57BL/6J mice,and investigate the role of mitophagy in the mechanism of retinal light injury in vivo by intraperitoneal injection of mitophagy inhibitor CsA.4.To explore the effect of PARP-1 signaling pathway on photoinduced mitophagy activation in the retina through in vivo experiments,as well as the synergistic protective effect of inhibiting PARP-1 signaling pathway and mitophagy in retinal light damage.Methods1.To explore the role of mitophagy inin the light-induced damage of 661W cells.(1)To irradiate 661W cells for 1-3 days in visible light(1500Lux).CCK8 was used to detect the activity of cells,Hochester/PI staining was used to detect the death of cells,and Western Blot was used to detect the expression levels of mitophagy signaling related proteins PINK1,p-Parkin,BECN1,and LC3B II.(2)Cells were treated with mitophagy inhibitor CsA,and the effects of CsA on the death percentage and mitophagy related protein expression of 661W cells under light injury conditions were detected by Hochester/PI staining and Western Blot.Co staining with Mitotracker Green and Lysotacker Red was used to detect the co localization of mitochondria and lysosomes.Additionally,transmission electron microscopy was used to detect cellular mitophagy levels.2.To detect the regulatory effect of PARP-1 on mitophagy and its impact on 661W cells in the light-induced damage.(1)661W cells was exposed to visible light at 1500Lux intensity for 1-3 days.Western Blot was used to detect the expression levels of PARP-1 protein and its downstream substance PAR protein in the cells.(2)The PARP-1 knockdown cell line was prepared by infecting 661W cells with shRNA mediated by lentivirus,and its expression levels of PARP-1 and downstream protein PAR were detected by Western blot to validate the PARP-1 knockdown cell line.(3)Using PARP-1 knockdown cells or PARP-1 inhibitor NU1025 to treat 661W cells and conducting light damage experiments,the mitophagy level of cells was observed through Western Blot,cell fluorescence co localization,and transmission electron microscopy.The effect of PARP-1 inhibition on the mitophagy level of 661W cells under light damage conditions was detected.(4)Hochester/PI staining was used to exploring the effect of PARP-1 knockdown combined with CsA treatment on 661W cells in the light-induced damage.3.To explore the role of mitophagy on mouse retina under light injury.Establish a C57BL/6J mouse retinal light injury model and divide it into a normal control group,a light-vehicle group,and a light-CsA treatment group.Retinal mitophagy levels were detected using Western Blot and transmission electron microscopy.HE staining and transmission electron microscopy were used to detect changes in the thickness of ONL and number of nuclei,and ultrastructure of the ONL of the retina.Western blot and tissue immunofluorescence staining were used to detect changes in the content ofrhodopsin in the retina.Electroretinogram(ERG)was used to detect the retinal function of mice.4.To explore the protective effects of inhibiting PARP-1 and mitophagy on mouse retina under light injury.(1)Establish a C57BL/6J mouse retinal light injury model and divide it into a normal control group,a light-vehicle group,and a light-NU1025 treatment group.The regulatory effect of inhibiting PARP-1 on mitophagy in retinal light injury was detected by Western blot and transmission electron microscopy.(2)Establish a C57BL/6J mouse retinal light injury model and divide it into a normal control group,a light-vehicle group,a light-CsA group,a light-NU1025 group,and a light-CsA-NU1025 treatment group.The effects of inhibiting PARP-1 and mitophagy on retinal light damage were detected by ERG,HE staining,and transmission electron microscopy.Results1.The results of CCK8 showed that compared with the normal control group,the cell activity of 661W cells gradually decreased after 1-3 days of light exposure,and significantly decreased on the 3rd day.The results of Hochester/PI staining showed that compared with the normal control group,661W cells did not show significant cell death percentage after 2 days of continuous light exposure,and the cell cell death percentage significantly increased on the 3rd day.The results of Western blot showed that light damage caused a gradual increase in the expression levels of mitophagy related proteins PINK1,p-Parkin,BECN1,and LC3B II,and reached their peak on the third day of light exposure.The treatment of cells with mitophagy inhibitor CsA significantly reduced the expression of mitophagy related proteinss after light exposure.The results of confocal microscope showed that compared with the normal control group,light exposure induced an increase in co localization of mitochondria and lysosomes,and CsA treatment inhibited co localization of mitochondria and lysosomes in cells after light exposure.The results of transmission electron microscopy showed that compared with the normal control group,autophagic vesicles containing mitochondria were found in the cells after light damage,while CsA treatment inhibited the formation of mitophagy.In addition,CsA treatment significantly reduced the cell death percentage of light injury in 661W cells.2.Light exposur of 661W cells for 1-3 days resulted in an increase in the expression levels of PARP-1 protein and its downstream substance PAR within the cells over time.Successfully constructed a PARP-1 knockdown cell line,which can inhibit the expression of PARP-1 protein and mitophagy related proteins light exposure.And PARP-1 knockdown blocked the co localization of mitochondria and lysosomes,inhibiting the formation of autophagic vesicles containing mitochondria.In addition,compared with the light-vehicle group,CsA treatment or PARP-1 knockdown alone can reduce the cell death percentage of 661W cells after light exposure,while the combination of PARP-1 knockdown and CsA treatment can significantly reduce the cell death percentage compared to single treatment.3.In vivo experiments showed that compared with the normal control group,excessive light induced an increase in the expression of mitophagy related proteins PINK1,p-Parkin,BECN1,and LC3B II in the mouse retina.Western blot and immunofluorescence staining results showed that compared with the normal control group,the expression of Rhodopsin in photoreceptor cells in the light damaged group decreased,while CsA treatment was effective in maintaining the content of Rhodopsin in the light damaged group.Retinal transmission electron microscopy shows that after light injury,autophagic vesicles containing mitochondria can be seen in the inner segment of the retina.The photoreceptor cell body becomes smaller,the nucleus shrinks,the capsule ruptures,and the outer segment disc membrane swells and is arranged in disorder.The thickness of retinal ONL becomes thinner and the number of nuclei significantly decreases.ERG showed a significant decrease in the amplitude of a-wave and b-wave in the mouse retina.The CsA treatment group inhibited the increase in mitophagy related protein expression caused by light exposure.The transmission electron microscopy of retina showed that CsA treatment inhibited the formation of autophagic vesicles containing mitochondria,with intact photoreceptor cell bodies and neatly arranged outer segments.The HE staining results of retinal paraffin sections showed that CsA treatment improved the thinning of retinal ONL thickness and the reduction of nuclear number caused by light exposure.ERG showed that CsA treatment effectively alleviated the decrease in amplitude of a-wave and b-wave.4.The results of Western blot showed that the PARP-1 inhibitor NU1025 can effectively inhibit the expression of PAR downstream of the PARP-1 signaling pathway,and can also inhibit the increase in expression levels of mitophagy related proteins PINK1,p-Parkin,BECN1,and LC3B II induced by light exposure.Transmission electron microscopy showed that NU1025 treatment inhibited the formation of autophagic vesicles containing mitochondria after light exposure.ERG and retinal HE staining were performed on the normal control group,light-vehicle group,light-CsA-group,light-NU 1025-group,and light-CsA-NU 1025 group,respectively.The results showed that NU1025 or CsA treatment could effectively reduce the amplitude reduction of a wave and b wave,and CsA combined with NU1025 treatment had better protective effects.The HE staining results of retinal paraffin sections showed that the thickness of retinal ONL significantly decreased and the number of cells significantly decreased in the light-vehicle group of mice.The thickness of retinal ONL significantly improved in the NU1025 or CsA treatment group,but the number of nuclei still significantly decreased compared to the normal control group.However,there was no significant difference in the thickness of retinal ONL and the number of cells between the combined treatment group and the normal group.Conclusion1.In the model of retinal light injury in photoreceptor cell(661W),mitophagy signals are activated,and CsA inhibition of mitophagy has a protective effect on photoreceptor cell in light damage.2.In the model of retinal light injury in photoreceptor cell(661W),inhibiting the PARP-1 signaling pathway can inhibit mitophagy activation induced by light and have a synergistic protective effect.3.In an in vivo model of light injury in mouse retina,inhibiting PARP-1 can inhibit mitophagy and have a synergistic protective effect on retina. |
PDF Full Text Request