Protective Effects And Mechanisms By Which Fullerenol Protects Cornea Damaged By UVB Radiation And Degenerative Retina

The human eye consists of the anterior segment mainly including the sclera,iris,conjunctiva,and cornea,and the posterior segment made up of the vitreous humor,retina,and choroid.As the light traverses the eye cavity,it is refracted,absorbed,and converted into an electrical signal through optical-electrical conversion.Subsequently,the propagation of light out of the optic to the cortex occurs,allowing for visual and circadian perception.The quality of vision is determined not only by the photorefractive media comprised of the cornea and lens which focus the light on the correct places but also by photoelectric transformation as well as integrating and processing visual signals within the retina.In this process light rays are refracted,and ultraviolet B（UVB）rays are absorbed by the cornea,which is sufficient to block them from reaching the ocular structures,consequently exerting tissue-protective effects.UVB exposure triggers the generation of reactive oxygen species（ROS）and reactive nitrogen species（RNS）in the cornea.An imbalance between ROS/RNS production and oxidant defense leads to oxidative stress（OS）,which plays an important role in the damage and degeneration of ocular tissues,resulting in impaired vision and blindness.As a result of excessive UVB absorption,oxidative stress appears in keratocytes,followed by photokeratitis,carcinogenesis,and other complications.This can cause refractive visual impairment,and ocular symptoms like blurred vision and even blindness are present.The generation and transmission of electrical activities require an increased metabolic rate in the retina during the formation of vision.Nevertheless,exuberant energy metabolism in the retina is often accompanied by endogenously produced ROS.If left uncleared and accumulating in the retina,ROS can cause oxidative damage to both cellular macromolecules including lipids,proteins,DNA,and cell-component structure such as photoreceptor cell membranous disks.Genetic abnormalities,light exposure,and aging overwhelm the cellular antioxidant capacity,affect the intracellular repair of damage,and lead to oxidative stress-induced apoptosis,initiating retinal degeneration（RD）.RD is one of the leading causes of nonrefractive visual impairment and irreversible blindness worldwide,during which degeneration and loss of photoreceptors as well as other retinal cells are the main pathological changes.Oxidative stress induced by excessive ROS has been acknowledged as a hallmark of RD across a background of different genetic alterations.Moreover,ROS-related pathological activation of microglia can exacerbate inflammation,thereby further accelerating the progression of RD.To reduce oxidative stress in corneal and retinal tissues,antioxidant therapies are crucial.Small molecule antioxidants represented by glutathione（GSH）,dietary antioxidants,and antioxidant enzymes are used to treat ocular diseases.However,the majority of the traditional antioxidants act as scavengers for specific radical species,the clinical application is hampered by their inadequate oxidation resistance under pathophysiological conditions.Searching for an antioxidant with bio-safety,stability,and high potency to be applied in ophthalmopathy against oxidative stress remains an urgent problem to be solved.Fullerenol（fullerol）is known to be one type of water-soluble polyhydroxylated derivative of fullerene.The presence of numerous largeπbonds and hydroxyl groups is capable of quenching multiple radical species including ROS like superoxide anion-radicals（O₂·^-）,hydroxyl radicals（·OH）,lipid radical,and even RNS,making fullerenol widely used in antioxidant therapy.Fullerenol can activate the expression of Nrf2/ARE signaling pathway-induced antioxidant enzymes under various oxidative stresses,thereby enhancing the antioxidative capacity of the cells.Compared to other ROS quenchers,fullerenol is known to possess a higher free radical quenching ability and preserve high stability in different pathophysiological settings or under the stimulation of radiation.It has been applied for the prevention and treatment of various oxidative stress-related diseases,including Alzheimer’s Disease and myocardial infarction,but at present,there is no evaluation of the therapeutic effect for ocular diseases of fullerenol in vivo.Whether fullerenol can exert antioxidative effects in the retina and cornea to counteract UVB-induced corneal photodamage as well as RD is therefore unclear and needs further proof by experimental research.Construction of the SD rat corneal UVB-induced injury model and B6.C3-Pde6brd1Hps4le（rd1）autosomal recessive retinitis pigmentosa model was performed for the present study.The protective effects of fullerenol on cornea damaged by UVB radiation and degenerative retina were assessed after electrophysiological experiments,OCT,and immunohistochemical analysis.The antioxidant in ophthalmologic clinical applications glutathione was added as a negative control,and ex vivo experiments were performed for validation of the effects and mechanisms of fullerenol on damaged cornea and retina.The results of our study have practical significance for the treatment of UVB-induced corneal injury and retinal degeneration.Methods and resultsPart One:Protective effects and mechanisms of fullerenol on cornea damaged by UVB radiation1.The restoration effect of fullerenol on cornea damaged by UVB radiation in SD ratsThe corneal injury induced by UVB was observed by fluorescein sodium staining,and the corneal thickness was continuously monitored by Anterior Segment Optical Coherence Tomography（AS-OCT）to compare the corneal edema at different time points between all these groups.The modeling was successful,as the green area represented corneal epithelial defect by positive fluorescein staining was shown after UVB radiation.A smaller area of fluorescein staining represented faster repair of corneal injury after treatment of fullerenol.The damaged area shrunk and gradually disappeared within 16 days.The thickness of the cornea was significantly increased due to UVB irradiation,as shown by AS-OCT.Although the thickness of the cornea decreased after GSH addition at each time point,the cornea maintained swelling.Quicker alleviation and elimination of corneal edema as well as corneas with normal thickness were observed in the fullerenol-treated group.2.The mechanism of fullerneol to exert protective effects on cornea damaged by UVB radiationCell apoptosis in all layers of the cornea was observed by immunofluorescence staining,as well as the expressions of proliferation marker PH3 and limbal stem cell marker CK15.High numbers of TUNEL-positive nuclei and Caspase3-positive cells were detected in the cornea of the UVB rats.GSH significantly decreased the number of apoptotic cells.Fullerenol treatments resulted in almost complete evasion of apoptosis.Quantification of PH3-positive cells showed that fullerenol increased the number of proliferative cells in the central cornea and limbal region.The corneal limbal stem cells（LSCs）are located in the basal layer of limbus epithelium and a decline in the number of LSCs was found in the UVB group as the decrease in fluorescence of CK15.Fullerenol led to a proliferation in the stem cell population.3.Protective effects and mechanisms of fullerenol on h CECs exposed to UVBIn the cellular experiment,the influence of fullerenol and GSH on morphology and viability of the h CECs irradiated by UVB radiation was evaluated by light microscopy,CCK-8 test,and Ki67 immunofluorescence staining.Intracellular ROS/RNS,DNA oxidation,and double-strand DNA breaks were detected using immunofluorescence cytochemistry.The expression of Nrf2 and HO-1 in the h CECs and effect of fullerenol were measured by staining and western blot.Mitochondrial membrane potential was assessed by JC-1 staining on the cellular organelle level.The key molecular targets by which fullerenol exerted protective effects were identified by RNA-seq analysis.UVB radiation-induced severe cytotoxicity.The cell viability were elevated significantly under the fullerenol treatment.Intracellular ROS was monitored by DCFH-DA as a fluorescent probe and the numbers of DCFH-DA positive cells were significantly lowered in the fullerenol-treated cells compared to UVB alone.The quantitative evaluation of flow cytometric results showed fullerenol diminished the intracellular accumulation of ROS.ONOO^--staining results indicated RNS generation was suppressed in cells by fullerenol.Nuclear staining for 8-OH-d G andγH2AX was rare in fullerenol-treated group,which represented the complete suppression of fullerenol on DNA oxidation,and double-strand DNA breaks.Western blot revealed increased levels of Nrf2 and HO-1 proteins in the fullerenol group compared with other groups.Results of JC-1 staining expressed that the abnormal mitochondria with low MMP emerged in the h CECs exposed to UVB.There was a slight further reduction in the proportion of abnormal mitochondria of the GSH-treated h CECs.Treatment with fullerenol reversed the mitochondrial membrane potential changes.RNA-seq analysis showed that fullerenol influenced different cellular processes in UVB-damaged h CECs,including cell apoptosis,oxidative stress,DNA repair,and cell proliferation.RT-PCR and WB are applied to further evaluate related gene expression.Fullerenol treatment led to the up-regulation of FOXO1 and RAD51 transcriptional activity,contributing to the antioxidant capacity and recombinational DNA repair.Part Two:Protective effects and mechanisms of fullerenol on the degenerative retina1.The rescue of fullerenol for visual function and photoreceptors in rd1 mice with retinal degenerationThe rd1 mice were divided into three groups,and their vitreous chambers were injected with fullerenol,GSH,and PBS respectively on day 7 and 11 after birth.The visual function was assessed by flash electroretinography analysis tests and black/white transition box behavioral experiments.The morphological alterations in the retina were monitored by immunofluorescent staining.The rd1mice exhibited a significant decrease in electroretinography wave amplitudes from P14 to P28 compared to wild-type mice.Waveform of electrophysiological signals tended to improve in the GSH group,but not significantly.Quantification of the ERG amplitudes showed a strong increase after fullerenol treatment,black/white transition box behavioral experiments indicated that the fullerenol-treated rd1 mice responded to the light stimulus and spent significantly more time in the dark chamber.Histomorphological staining revealed that TUNEL-positive cells mainly expressed in the ONL,which thinned with photoreceptor cell death on day 14.The retinas treated with fullerenol had reduced apoptotic cell numbers,and ONL appeared to be thicker.For photoreceptors,fullerneol facilitated their functional proteins expression of Rhodopsin,Gnat-1,and Arrestin.2.The mechanisms underlying the protective effects of fullerenol on the retina in rd1miceThe transcriptomes of wild-type and degenerating rd1 mouse retinas after different treatments were analyzed by RNA sequencing,screening key genes and signaling pathways through which fullerenol exerted its actions.The results were also verified by RT-PCR and WB technology.The heatmap showed total gene expression,which indicated that there was a high degree of similarity in gene expression profiles of the normal retina and fullerenol-treated degenerative retina.GO enrichment analysis showed that the differential genes between the injured retina and the normal retina mainly contributed to mitochondrial structure and function.Relative quantification of gene expression was performed and indicated that fullerenol reversed the expression of mt DNA and n DNA genes concentrated in mitochondria,especially functional pathways including ETC.The transcription of the mt DNA genes（e.g.mt-Nd4l and mt-CYTB）was extremely up-regulated,while the transcription of the n DNA genes（e.g.Ndufa1 and Atp5g3）was down-regulated compared to the rd1 group but was consistent with the normal transcription of the genes.3.Protective effects and mechanisms of fullerenol on oxidative stress-induced degeneration of photoreceptorsThe photoreceptor cell line 661W was treated with H₂O₂to mimic the oxidative retinal photoreceptor injury.The free radical levels increased in the oxidative process were evaluated by fluorescence staining and flow cytometric analysis.The total antioxidant capacity assay was performed and SOD activity was determined for comparing the effects of different treatments on the antioxidant capacities of the photoreceptor.The influence of fullerenol and GSH on apoptosis and cell viability of the 661W were also evaluated by light microscopy,CCK-8 test,and immunofluorescence staining.Changes in mitochondrial membrane potential and cytochrome c release were determined with JC-1 loaded and cytochrome c-stained cells,for validating that fullerenol protected against oxidative damage to mitochondria at organelle level.The oxidative modification of bases in DNA was detected by 8-OHd G level.Fullerenol inhibited the production of intracellular ROS after H₂O₂stimulation,lifted the total antioxidant capacity,especially the enzyme activities of SOD,reduced photoreceptor apoptosis,and maintained cell viability.At the level of organelle,the integrity of the mitochondrial membrane of 661W was maintained after fullerenol treatment,and the loss of mitochondrial functionality was suppressed.The 8-OH-d G level in the DNA of fullerenol-treated 661W reduced to normal levels.Conclusion1.This study demonstrated the effectiveness of fullerenol in repairing UVB-induced corneal injury and treating retinal degeneration.Results revealed that fullerenol had more substantial protection efficacy in alleviating corneal edema caused by UVB radiation and functional rescue of the degenerative retina,reflecting its advantage over GSH,the antioxidant in ophthalmologic clinical applications.2.This study clarified that fullerenol promoted repair in the area of corneal epithelial defects in morphology,possibly by inhibiting cell apoptosis,maintaining cell stemness,and promoting cell proliferation.This study took the lead to report its strength to facilitate homologous recombination,which promoted the repair of UVB-induced DNA fragmentation and cell death suppression.3.This study confirmed that fullerenol was more powerful than GSH in the regulation of redox balance,alleviated,and even eliminated the mitochondrial and DNA damage caused by oxidative stress.4.This study clarified that fullerenol played an essential role in maintaining the integrity of the outer nuclear layer in the retina of rd1 mice by protecting photoreceptors against apoptosis and enhancing the expression of functional proteins to ameliorate the retinal function.In the present study,for the first time,fullerenol was found to contribute to the reversion of mitochondrial gene expression patterns,conducive to the maintenance of normal mitochondrial morphology and function,and lead to retinal protection.5.This study provided a new idea for the treatment of corneal damage caused by UVB irradiation as well as RD and opened discourse about its prescription in clinical practice.