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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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ObjectiveThis study aimed to predict the pharmacodynamic material basis and core targets of Bailing capsules in the treatment of chronic obstructive pulmonary disease （COPD） based on network pharmacology and molecular docking， which were further verified by cell experiments to explore the mechanism. MethodThe main active ingredients and related targets of Bailing capsules were screened in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） and SwissTargetPrediction. The main COPD targets were searched from GeneCards， DrugBank， Online Mendelian Inheritance in Man （OMIM） and Therapeutic Target Database （TTD）. The protein-protein interaction （PPI） network was constructed by STRING and Cytoscape 3.6.1. Gene Ontology （GO） function annotation and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis were performed by the Database for Annotation， Visualization and Integrated Discovery （DAVID）. Molecular docking verification was carried out using AutoDock Vina. The cell viability was detected by 3-（4，5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium （MTS） assay， and the mRNA level of the targets was detected by real-time polymerase chain reaction （Real-time PCR）. ResultA total of 11 active ingredients of Bailing capsules such as cerevisterol， 270 related drug targets， and 1 020 COPD target proteins were obtained， with 74 intersection targets. The visualization analysis of the PPI network showed that the core targets of Bailing capsules in the treatment of COPD were tumor protein P53 （TP53）， catenin beta 1 （CTNNB1）， tumor necrosis factor （TNF）， interleukin-6 （IL-6） and insulin （INS）. Further， 20 signaling pathways were screened by KEGG enrichment analysis as the main pathways for Bailing capsules to treat COPD， involving phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt）， cyclic adenosine monophosphate （cAMP）， forkhead box O （FoxO）， TNF， and hypoxia inducible factor-1 （HIF-1） signaling pathways. Molecular docking validation demonstrated that four active ingredients had stable binding to IL-6， with the lowest energy. Bailing capsules could reduce the mRNA level of IL-6 in RAW264.7 cells induced by lipopolysaccharide （LPS） （P<0.01） compared with the control group. ConclusionThe pharmacological mechanism of Bailing capsules in the treatment of COPD might be that its main active ingredients improved the inflammatory response by acting on TP53， CTNNB1， TNF， IL-6 and other targets and regulating PI3K/Akt， cAMP and other signaling pathways， thereby ameliorating COPD symptoms. This study provided experimental basis for subsequent in-depth research， and provided a diagnosis and treatment direction for disease-related clinical treatment.
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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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[Objective] This prospective study was conducted to assess the influence of overnight orthokeratology (OK) on low,middle and high myopia in southern Chinese teenagers.[Methods] Fifty-four subjects (107 eyes,age 11.65 ± 2.44 years,mean±SD)who matched the inclusion criteria for OK were enrolled in the study.The subjects were divided into three groups according to the different refraction baseline (low myopia group:0.75 ～ 3.00 D,51 eyes;middle myopia group:3.25 ～ 5.00 D,36 eyes;high myopia group:5.25 ～ 9.00 D,20 eyes).Refraction,central corneal thickness,corneal keratometry,anterior chamber depth and axial length (AL) were measured at baseline and after 1 years using ocular biometry.The changes were evaluated and compared among the groups.Results were analyzed by SPSS 17.0 software.[Results] Fifty-four subjects (total 107 eyes) completed the 1-year follow-up examinations.At baseline,the spherical equivalent refractive error (SER) was-2.15 D ± 0.63 D,-4.20 D ± 0.54 D,and-6.63 D ±1.18 D in three groups.The axial length was (24.50-± 0.66) mm,(25.16-± 0.65) mm,and (26.35 ± 0.87) mm in the three groups,respectively.There were no significant differences among the groups in initial central corneal thickness,anterior chamber depth,central,flat and steep kerotometry.The increase in axial length during the 1-year study period was (0.22 ± 0.27) mm,(0.09-± 0.22)mm and (0.02-± 0.22) mm in three groups,respectively,and the difference was significant between low myopia group and middle myopia group (P =0.04),low myopia group and high myopia group (P =0.008),whereas no significant difference was found between middle myopia group and high myopia group (P =0.35).Spherical equivalence decreased dramatically in three groups after one year (all P ＜ 0.0001).And the difference was significant between low myopia group and middle myopia group (P ＜ 0.0001),low myopia group and high myopia group (P ＜ 0.0001).Central,flat and steep kerotometry after one year became flatten in all group (all P ＜0.0001).There were no significant differences in the change of central corneal thickness,anterior chamber depth and corneal diameter.[Conclusions] OK lens is more effective in middle and high myopia control than in the low myopia control.The change in corneal keratometry may be one of the main factors influencing the OK treatment effect.
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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.