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Objective:To observe the effect of the antioxidant N-acetylcysteine (NAC) and selective endoplasmic reticulum stress response inhibitor salubrinal on the apoptosis of retinal pigment epithelial cells induced by all-trans-retinoic acid (ATRA).Methods:Human ARPE-19 cell line was used as the experimental cell line, and was divided into normal control group cultured with complete medium, model control group cultured with complete medium containing 10 μmol/L ATRA, NAC treatment group cultured with complete medium containing 10 μmol/L ATRA+ 5 mmol/L NAC, salubrinal group cultured with complete medium containing 10 μmol/L ATRA+ 40 μmol/L salubrinal, NAC+ salubrinal group cultured with complete medium containing 10 μmol/L ATRA+ 5 mmol/L NAC+ 40 μmol/L salubrinal.After 24-hour culture, apoptosis rate, multicaspase level and reactive oxygen species (ROS) level of ARPE-19 cells were detected by flow cytometry.The expressions of vascular endothelial growth factor A (VEGF-A), C/EBP-homologous protein (CHOP), cleaved-caspase 3 in cells were detected by Western blot.Results:There were significant differences in the apoptosis rate, multicaspase and ROS levels among the five groups ( F=113.23, 602.41, 160.39; all at P<0.001). The apoptosis rate, multicaspase and ROS levels of normal control group, NAC treatment group, salubrinal group and NAC+ salubrinal group were significantly lower than those of model control group (all at P<0.05). There were significant differences in the expression levels of VEGF-A, CHOP and cleaved-caspase 3 among the five groups ( F=24.62, 36.35, 60.25; all at P<0.001). The protein expression levels of VEGF-A, CHOP and cleaved-caspase 3 of normal control group, NAC treatment group, salubrinal group and NAC+ salubrinal group were significantly lower than those of model control group (all at P<0.05). Conclusions:ATRA can induce RPE cells to produce oxidative stress and endoplasmic reticulum stress injury, which leads to apoptosis.NAC and salubrinal can effectively reduce the RPE cell apoptosis by inhibiting stress response.
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Background The chronic injury of the hematopoietic system caused by ionizing radiation (IR) is often ignored. The essential cause of this injury is the damage of hematopoietic stem and progenitor cells (HSPCs). Objective To explore the long-term effects of IR at different radiation doses and at different radiation fractions of the same radiation dose on HSPCs in the bone marrow of mice, and to provide a scientific basis for reducing the chronic damage to the hematopoietic system caused by IR. Methods A total of 16 male C57BL/6 mice aged 8-10 weeks were randomly divided into four groups that received different doses or fractions of total body X-ray irradiation, including 1.5 Gy×4 irradiation group (n=5), 3 Gy irradiation group (n=4), 6 Gy irradiation group (n=4), and non-irradiation group (n=3). Two months after irradiation, bone marrow cells from each mouse were collected and counted. The clone forming ability of bone marrow cells was analyzed by cobblestone area-forming cell (CAFC) assay. The proportion of HSPCs was measured by flow cytometry. The cell cycle of HSPCs was assessed by antigen identified by monoclonal antibody Ki 67 (Ki-67) and 7-amino-actinomycin D (7-AAD) double staining. The reactive oxygen species (ROS) levels of HSPCs were estimated with a 2,7-dichlorodihydrofluorescein diacetate (DCFDA) probe. The cellular senescence of HSPCs was evaluated with a 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG) probe. The expression of senescence related genes such as P16, P19, P21, and P27 was measured by real-time fluorescence quantitative PCR. Results There was no significant change in the numbers of bone marrow cells 2 months after different doses and fractions of radiation (P>0.05). The clone forming ability of bone marrow cells was significantly decreased after 3 Gy and 6 Gy irradiation when compared to non-irradiated mice (P<0.01). HSPCs responded inconsistently to different doses and fractions of irradiation. Overall, there was no significant change in long-term hematopoietic stem cells (LT-HSCs) proportion after irradiation (P>0.05), the proportions of hematopoietic progenitor cells (HPCs), hematopoietic stem cells (HSCs), short-term hematopoietic stem cells (ST-HSCs), and multipotent progenitors 2 (MPP2) increased after irradiation (P<0.05), and the proportions of LSK, MPP1, MPP3, and MPP4 cells decreased after irradiation (P<0.05); except for HPCs and MPP2, the proportion of HSPCs in G0 phase was decreased (P<0.05). The ROS production in HSPCs was increased significantly after 6 Gy irradiation (P<0.05), while the ROS levels after 3 Gy and 1.5 Gy×4 irradiation were similar to that of the non-radiation group (P>0.05). The cellular senescent proportion of HPCs, LSK, and HSCs increased after irradiation (P<0.05). The expression levels of senescence related genes P16, P19, and P21 in HSCs were significantly increased (P<0.05). Conclusion The responses of HSPCs in bone marrow to IR vary depending on doses and fractions of irradiation. Increased ROS production and cellular senescence may be involved in the damage process of HSPCs under radiation settings.
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Objective:To investigate all-trans retinoic acid(ATRA)-induced apoptosis signaling pathway in ARPE-19 cells in vitro. Methods:The APRE-19 cell was treated with different concentrations of ATRA for 24 hours and 48 hours.Cell counting kit-8 (CCK-8) was used to detect the cell viability in order to determine the experimental concentration range.Flow cytometry and Western blot method were performed to evaluate the apoptosis and caspase related protein levels in ARPE-19 cells treated with 0, 2.5, 5, 10, 15 and 20 μmol/L of ATRA for 24 hours.Flow cytometry was used to detect the reactive oxygen species (ROS) and multicaspase levels and quantitative real-time PCR was carried out to determine the mRNA relative expression levels of caspase related proteins in ARPE-19 cells treated with 0, 2.5, 5, 10 and 20 μmol/L of ATRA, and 0 μmol/L ATRA group was used as the blank control group.Results:CCK-8 test showed that the half maximal inhibitory concentration of ARPE-19 cells treated with different concentrations of ATRA for 24 hours and 48 hours were 13.88 μmol/L and 11.99 μmol/L, respectively.The cell survival rates of ARPE-19 cells treated with different concentrations of ATRA for 24 hours and 48 hours were significantly different ( F=176.60, 350.30; both at P<0.01). When cultured for 24 hours, the cell survival rates of ARPE-19 cells in the 2 μmol/L and 6 μmol/L of ATRA groups were higher than that of the blank control group (both at P<0.05), and the cell survival rates of ARPE-19 cells in the 12, 14, 16, 18 and 20 μmol/L of ATRA groups were lower than that of the blank control group (all at P<0.05). Flow cytometry showed that there were significant differences in the apoptosis, ROS and multicaspase level among ARPE-19 cell groups treated with different concentrations of ATRA ( F=86.39, 116.84, 101.40; all at P<0.01). The apoptosis rates of APRE-19 cells in the 2.5 μmol/L and 5 μmol/L of ATRA groups were significantly decreased than that of the blank control group, and the apoptosis rate of APRE-19 cells in the 10, 15 and 20 μmol/L of ATRA groups were significantly increaseded than that of the blank control group (all at P<0.01). The relative expression levels of multicaspase and ROS were significantly higher in the 2.5, 5, 10 and 20 μmol/L of ATRA groups than that of the blank control group (all at P<0.01). Western blot assay showed that the relative expression level of caspase 9 was increased in the 2.5, 5, 10, 15 and 20 μmol/L of ATRA groups than that of the blank control group (all at P<0.05). Compared with the blank control group, the relative expression levels of caspase 12 were increased in the 2.5 μmol/L of ATRA group and reduced gradually in the 5, 10, 15 and 20 μmol/L of ATRA groups, among which there were significant differences between the blank control group and 2.5, 15, and 20 μmol/L of ATRA groups (all at P<0.05). The relative expression level of caspase 3 was significantly increased in the 5, 10 and 20 μmol/L of ATRA groups than that of the blank control group (all at P<0.05). The relative expression level of cleaved caspase 3 was significantly increased in the 2.5, 5, 10, 15 and 20 μmol/L of ATRA groups than that of the blank control group (all at P<0.01). Quantitative real-time PCR assay showed that the relative expression levels of caspase 9 and caspase 12 mRNA were significantly higher in the 2.5, 5, 10 and 20 μmol/L of ATRA groups than that of the blank control group (all at P<0.01). The relative expression levels of caspase 3 mRNA were significantly higher in the 5 μmol/L and 10 μmol/L of ATRA groups than that of the blank control group (both at P<0.01). When the concentration of ATRA was lower than 10 μmol/L, the relative expression levels of caspase 9 and caspase 12 mRNA were elevated in a concentration-dependent manner.When the concentration of ATRA reached 20 μmol/L, the relative expression levels of caspase 9 and caspase 12 mRNA were markedly decreased, but it was still higher than that of the blank control group. Conclusions:ATRA induces apoptosis in ARPE-19 cells in vitro through activating the reactive oxygen species and endogenous caspase-dependent apoptotic pathway.
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Background Excessive elongation of axis and expansion of sclera is one of the hot topics in the study of the pathogenesis of high myopia.To establish a human scleral fibroblasts (HSFs)-collagen matrix culture model is helpful for understanding the reciprocal and adaptive interactions between HSFs and the collagen matrix in tissue.Objective The aim of this study was to establish a HSFs-seeded collagen three-dimension culture system that may mimic the sclera remolding in myopia.Methods HSFs were isolated and cultured from donor eyes by explant culture and purified by passages culture in vitro.The expressions of vimentin and keratin in the cells were detected by immunofluorescence technique to identify the cells.Rat tail tendon was obtained from 8-week-old SPF SD rats to prepare the collagen matrix.The mixed solution of 400 μl collagen matrix and 1 100 μl PBS,200 μ1 nutrient medium,50 μ1 NaOH and HSF suspension were mixed to prepare the collagen gel three-demension culture system.The growth and morphology of the cells in the culture system were observed under the inverted phase contrast microscope,and IPP-5 software was used to measure the contraction area of collagen gel,and the mechanical creep properties of the HSFs-seeded collagen matrix were measured by a biomechanics test instrument.Results HSFs emigrated from tissue 7 days after culture and passage could be performed 14 days after culture.The expression of keratin was absent in HSFs,while vimentin was positively expressed.The free-cell collagen gel was clear and unchanged in the experimental duration.However,the cells were obviously increased on the three-demension culture system and showed a tissue-like structure of net-like arrangement on dozens of layers.In 7-14 days after culture,the collagen gel area in a three-demension collagen matrix revealed a decrease of 90％.Duotriode-like and fusiform cells were seen 24 hours after culture.The biomechanical creep curve of HSFs-seeded collagen matrix consisted of the nonlinear section (0-100 seconds) and linear section (100-600 seconds),and the former appeared to be an elastic change of the gel under the temporal stress,and the latter was the creeping of the gel with the time.Conclusions Rat tail collagen appears to have a good biocompatibility to HSFs.HSFs-seeded collagen matrix can retain the mechanical creep properties,and it may be a good tool for the study on the relationship between HSFs and extracellular matrix or intercellular biological behaviour for scleral remodeling.
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AIM: To explore the role of PI3K /PKB pathway in the growth inhibition effect of rabbit lens epithelial cells (LECs) induced by (-)-epigallocatechin-3-gallate (EGCG). METHODS: Rabbit LECs were obtained by tissue culture; MTT colormetric assay was used to study growth of LECs; Western blotting was used to study the kinsae phosphorylation- and nonphosphorylation- level of PKB. RESULTS: (1) When LECs were preincubated with 25, 50 ?mol/L LY294002 for 1 h, 50 ?mol/L EGCG had little influence on the proliferation of LECs. It showed significant inhibition when EGCG increased to 100, 200 ?mol/L ( P