Mechanism Of RIP1/RIP3 And AIF Mediated Necroptosis Pathway In Retinal Light Injury

Purpose: To establish cell and animal models of retinal photodamage in R28 cells and mice,respectively,and to explore the molecular mechanisms underlying photodamage.Methods: R28 cells were selected as the in vitro model,and were placed under 2000 Lux LED white light for 1,2,and 3 days.Then,the CCK-8 kit was used to detect the viability of the R28 cells at different time points.Western Blot(WB)was used to detect the expression levels of RIP1,RIP3,MLKL,AIF,and cleaved AIF in each group,and HoechstPI staining was used to detect the proportion of necrotic changes.RIP1 and RIP3 inhibitors and AIF/RIP3 siRNA were used to detect the relationship between RIP1/RIP3 and AIF activation following light damage.Additionally,the localization of RIP3 and AIF in the cells was detected by IF.Male Bal B/C mice that were 6-8 weeks of age with body weights of approximately 20-30 g were selected as the light damage animal model,and were placed under an LED white light of 7000 Lux.After exposure to light for 1,3,7,and 14 days,the retinal thickness of each group was determined by paraffin sectioning and HE staining.The expression of RIP3 and AIF genes in each group was detected using RTqPCR.Results:After visible light irradiation,the viability of R28 cells decreased with increasing illumination time.Compared to the control group(100±5.232%),the cell viability of the 3-day light group decreased significantly to only 35.27±4.051% of the control group(P<0.001),and the difference was statistically significant.The expression of RIP1 and RIP3 in R28 cells increased after 1 day of visible light irradiation,AIF was activated on day 2 and 3 of visible light irradiation,and the expression of cleaved AIF significantly increased,with the most obvious elevation in the 3-day light group(9.552±3.564 folds,P<0.001).The PI staining positive rate of R28 cells after illumination increased with increasing illumination time.Compared to the control group(2.305±1.444%),the PI staining positive rate in the 3-day light group increased to 26.68±9.392%(P<0.001).After exposure to visible light,the retinal thickness of the mouse eyeballs decreased,especially in the outer nuclear layer.Compared to the control group(45.06±1.304μm),the thickness of the outer nuclear layer in the 3-day light group significantly decreased(14.40±1.032μm,P<0.001).Visible light irradiation increased AIF and RIP3 transcription in Bal B/C mice.Compared to the control group,the transcriptional levels of AIF and RIP3 in mice were increased after 3days illumination((1.608±0.02910 folds,P<0.001)and(2.811±0.1328 folds,P<0.001)),respectively,and the difference was statistically significant.The RIP1 inhibitor Nec-1 and RIP3 inhibitor GSK’872 attenuated the decrease of R28 cells viability that was induced by light damage.Compared to the control group(100.0±7.169%),the viability of R28 cells after irradiation decreased(29.78±6.046%,P=0.0008),while the viability of R28 cells pretreated with Nec-1 and GSK’872 was higher than that in the light group.The corresponding increases were(68.03±21.88%,P<0.001)and(51.33±15.92%,P=0.0166),respectively,and the elevation were statistically significant.Nec-1 and GSK’872 inhibit light-induced necroptosis.Compared to the light+DMSO group(39.60±17.73% PI-positive cells),the number of PIpositive cells in the light+Nec-1 and light+GSK’872 groups were significantly decreased to 2.861±2.201% and 3.945±2.109%,respectively(P<0.001).The WB results showed that compared with the light+DMSO group,the expression level of Cleaved AIF decreased(2.134±1.582 folds,P=0.0018)after RIP1 inhibitor treatment and RIP3 inhibitor treatment.Furthermore,RIP3 expression decreased(0.8229±0.2605 folds,P=0.0009),as did the expression of cleaved AIF(3.439±3.061 folds,P<0.001),with statistical significance.RIP3 siRNA specifically interfered with RIP3 translation.In compared with the light+NC group(4.032±1.211 folds),the expression level of cleaved AIF in the RIP3 siRNA group decreased(2.425±0.9617 folds,P=0.0178).Furthermore,using AIF siRNA interference,we found that AIF siRNA reduced the expression of AIF and cleaved AIF,but did not reduce the expression levels of RIP1 and RIP3.The immunofluorescence results showed that compared to the dark group,AIF and RIP3 in the light group co-localized and underwent nuclear translocation.Conclusion: In this study,cell and animal models of retinal photodamage were successfully established.Using these models,we found that 2000 Lux light induced the activation of RIP1/RIP3 and AIF signaling pathways in R28 cells.After exposure to 7000 Lux of visible light,the retina of mice became thinner,resulting in increased transcript levels of AIF and RIP3.Furthermore,the RIP1/RIP3 signaling pathway was found to be involved in the regulation of AIF activation during visible light irradiation-induced necroptosis in R28 cells.