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Objective To investigate the correlation between the proportion of peripheral blood follicular T helper cells(Tfh cells)and intracellular interleukin-21(IL-21)content with blue light retinal injury.Methods Brown Norway(BN)rats were randomly divided into 4 groups and were exposed to blue light for 3 hours a day to establish retinal light damage model.According to the duration of illumination,the rats was divided into 0 days(control group),3 days(3 d group),7 days(7 d group)and 14 days(14 d group).The proportion of Tfh cells and content of IL-21 in Tfh cells in peripheral blood of each group was detected by flow cytometry and ELISA sepa-rately after illumination.Electroretinogram(ERG)was used to evaluate retinal function.The changes of fundus in rats were observed by fundus photography.The thickness of outer nuclear layer of retina was analyzed by HE staining.Results After retinal blue light injury,with the extension of illumination time,the proportion of Tfh cells in peripheral blood and intracellular IL-21 content both increased(P<0.05).ERG showed that retinal function decreased after light damage and aggravated with the extension of illumination time,the latency and ampli-tudes of A-wave and B-wave increased and decreased respectively(P<0.05).The retinal fundus of rats showed depigmentation in 3 d,and the retinal vessels became thinner and exudate with the extension of illumination time.HE staining showed that the outer nuclear layer of retina(ONL)became thinner(P<0.05).Correlation analy-sis indicated that the proportion of Tfh cells in peripheral blood and the intracellular IL-21 content could jointly reflect the degree of injury(P<0.000 1),and the proportion of Tfh cells in peripheral blood was negatively corre-lated with ONL thickness and the amplitude of a and b waves,positively correlated with the peak time of a and b waves,(P<0.0001).Conclusion The proportion of Tfh cells in peripheral blood and the intracellular IL-21 content were increased after blue light damage to retina,and were significantly increased with the extension of light time with a certain correlation.
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Objective To research the role of mitochondrial DNA mediate the cultured human retinal pigment epithelium (hRPE) cell apoptosis induced by blue light and the relationship with time.Methods Established the blue light damage model of cultured hRPE cells in vitro with light emitting diode (LED) blue light density of (4.0-±0.5)mW/cm2 adjusted by FL-1D blue light illumination meter,and the illumination time was set as 0,0.5,1,2,4,6,12 and 24 hours,then the cells were grouped according to the illumination time.Immunofluorescence were used to identify the cells;the expressions of caspase-3,cleaved caspase-3,caspase-9,cleaved caspase-9,bax and bcl-2 were detected with Western blot.Quantitative PCR was used to detect the copy number of mitochondrial DNA and PCR was used to detect mitochondrial DNA 4977bp common deletion.Results Immunofluorescence results showed that the RPE65 protein was expressed in the cytoplasm.The expressions of bax were upregulated after illumination for 1 hour,cleaved caspase-3 were upregulated after illumination for 2 hours,caspase-3,caspase-9,cleaved caspase-9 were upregulated after illumination for 4 hours,while the expression of bcl-2 was downregulated after illuminated for 2 hours,with significant differences compared to the normal control group (all at P＜0.05).The copy number of mitochondrial DNA in 0.5,1,2,4,6,12 and 24 hours groups was downregulated,with significant differences compared to the normal control group (all at P＜0.05).The expressions of 4977bp common deletion in 0.5,1,2,4,6,12 and 24 hours groups were increased,with significant differences compared with the normal control group (all at P＜0.05).Conclusions Blue light can cause cell apoptosis,especially mitochondrial apoptosis,in hRPE probably motivated by mitochondrial DNA damage.
ABSTRACT
Mitochondrial DNA (mtDNA) is a genetic effect DNA molecule of double closed loop, and is crucial for cells and their functions. Mitochondria take an active part in physiological activities of retinal pigment epithelium (RPE) cells. The oxidative stress is usually occurred in RPE for its active metabolism, which can lead to mitochondria and mtDNA dam?age. Once mitochondria and mtDNA lesions have not been repaired timely, the lesions can be accumulated, which can cause dysfunctions and damaged-structures of RPE and mitochondria, and can motivate the progression of cell apoptosis. In the end it can result in some ocular related diseases such as aged-related macular degeneration (AMD). This study reviewed the functional relationship between mtDNA and RPE, and repair and detection methods of mtDNA damage.
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Background Researches showed that mitochondria and oxidative stress play a crucial role in retinal photochemical injury,but the relationship between the damage of human retinal pigment epithelium (RPE) cell-induced by blue light and light-irradiated time is less studied.Objective The aim of this study was to research the possible mechanism of RPE oxidative damage induced by blue light in vitro.Methods Human RPE cells were isolated from healthy donors and cultured.The cells were divided into the normal control group and the light exposure group.The cells of light exposure group were irradiated using the blue light of (4.0±0.5) mW/cm2 for 0.5,1,2,3,4,5,6,12 and 24 hours,respectively,and the cells of the normal control group were cultured in dark environment.Cellular viability was detected by MTT method,and the ultrastructure change of subcellular organelles in RPE cells was examined under the transmission electron microscope (TEM).The content of reactive oxygen species (ROS) was assayed by flow cytometry for the assessment of oxidative stress reaction.The relative expressions of nicotinamide adenine dinucleotide phosphate (NADPH) mRNA and cyclooxygenase 1 (COX1) mRNA in the cells were detected by real-time fluorescence quantitative PCR to evaluate the mitochondria function.Results The percentages of cellular viability were (100.00±20.00) ％,(95.73±0.89) ％,(94.67±2.56) ％,(84.23±0.16) ％,(78.57±3.09)％,(75.43±2.18)％,(66.13±1.42)％,(53.43±1.91)％ and (47.97±1.36)％ in the normal control group and light exposure for 1-hour,2-hour,3-hour,4-hour,5-hour,6-hour,12-hour and 24-hour groups,respectively,showing a significant difference among the groups (F =172.270,P =0.000),and the percentages of light exposure for the more than 3 hours groups were significantly lower than those of the normal control group (all at P＜ 0.05).The vacuoles-like degeneration,mitochondrial swelling,decreased microvilli were seen under the TEM.The contents of ROS in RPE cells were (14.75±2.49)％,(19.04± 1.02) ％,(22.81 ±3.20)％,(28.75±2.15)％,(33.06±0.96) ％,(40.64±2.11) ％,(48.25±2.50) ％ and (60.44±2.68) ％ in the normal control group and light exposure for 0.5-hour,1-hour,2-hour,3-hour,4-hour,5-hour,6-hour groups,and with significant increases in ROS contents in various light exposure groups compared with the normal control group (all at P＜0.05).The relative expression levels of NAPDH mRNA in the cells were gradually elevated 3 hours after light exposure with the increase of time in comparison with the normal control group (all at P＜0.05),and the relative expression levels of COX1 mRNA in the cells were higher in the light exposure for 2-hour,3-hour,4-hour and 5-hour group compared with the normal control group (all at P＜0.05),and after that the COX1 mRNA levels were gradually declined and were close to the normal level.Conclusions Blue light irradiation for more than 3 hours causes oxidative stress damage of mitochondria in RPE in vitro,and the damage was more obvious after irradiation for 5-6 hours.
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Objective To investigate the regulations of Bax , Bcl-2 in the protection of lipoic acid-niacin diad in acrolein-induced apoptosis in ARPE-19 cells. Methods The ARPE-19 cells were cultured in medium containing 10% fetal bovine serum , at 37 ℃ with 5% CO2. The ARPE-19 was transferred to 6-well plate after reaching to 70% confluence. After starvation for 24 h , the cells in 6-well plates were divided into three groups , including the blank control group , the acrolein treatment group with 50 μmol/L acrolein for 24 h , and the protection group with 100 μmol/L lipoic acid-niacin diad for 24 h and with the acrolein for another 24 h. The apoptotic cells were detected by flow cytometry assay , and expressions of Bcl-2 , Bax protein were detected by Western Blot assay. Results The percentages of normal healthy cells were 94.8%, 60.98%, and 91.34% in the blank control group , 50 μmol/L acrolein group and 100 μmol/L diad contained of lipoic acid and niacin group , respectively. The ratios of Bax/Bcl-2 protein expression were 0.293 9, 1.389 2, and 0.555 8 in the blank control group, 50 μmol/L acrolein group and 100 μmol/L diad contained of lipoic acid and niacin group, respectively. Conclusion The protective effect of lipoic acid-niacin diad on acrolein-induced apoptosis in ARPE-19 cell through promoting Bcl-2 expression and inhibiting Bax expression.