Cytotoxic effect and possible mechanisms of Tetracaine on human corneal epithelial cells in vitro

AIM: To demonstrate the cytotoxic effect and possible mechanisms of Tetracaine on human corneal epithelial (HCEP) cells in vitro. METHODS: In vitro cultured HCEP cell were treated with Tetracaine hydrochloride at different doses for different times, and their morphology, viability, and plasma membrane permeability were detected by light microscopy, methyl thiazolyl tetrazolium (MTT) assay, and acridine orange (AO)/ethidium bromide (EB) staining, respectively. Their cell cycle progression, phosphatidylserine orientation in plasma membrane, and mitochondrial membrane potential (MTP) were assessed by flow cytometry. DNA fragmentation, ultrastructure, caspase activation, and the cytoplasmic apoptosis inducing factor (AIF) and cytochrome c (Cyt. c) along with the expression of B-cell lymphoma-2 (Bcl-2) family proteins were examined by gel electrophoresis, transmission electron microscope, enzyme linked immunosorbent assay (ELISA), and Western blot, respectively. RESULTS: After exposed to Tetracaine at doses from 10.0 to 0.3125 g/L, the HCEP cells showed dose- and time-dependent morphological abnormality and typical cytopathic effect, viability decline, and plasma membrane permeability elevation. Tetracaine induced phosphatidylserine externalization, DNA fragmentation, G1 phase arrest, and ultrastructural abnormality and apoptotic body formation. Furthermore, Tetracaine at a dose of 0.3125 g/L also induced caspase-3, -9 and -8 activation, MTP disruption, up-regulation of the cytoplasmic amount of Cyt. c and AIF, the expressions of Bax and Bad, and down-regulation of the expressions of Bcl-2 and Bcl-xL. CONCLUSION: Tetracaine above 0.3125 g/L (1/32 of its clinical applied dosage) has a dose- and time-dependent cytotoxicity to HCEP cells in vitro, with inducing cell apoptosis via a death receptor-mediated mitochondrion-dependent pathway.     

Cytotoxicity of pilocarpine to human corneal stromal cells and its underlying cytotoxic mechanisms

AIM: To examine the cytotoxic effect of pilocarpine, an anti-glaucoma drug, on human corneal stromal (HCS) cells and its underlying cytotoxic mechanisms using an in vitro model of non-transfected HCS cells. METHODS: After HCS cells were treated with pilocarpine at a concentration from 0.15625 g/L to 20.0 g/L, their morphology and viability were detected by light microscopy and MTT assay. The membrane permeability, DNA fragmentation and ultrastructure were examined by acridine orange (AO)/ethidium bromide (EB) double-staining. DNA electrophoresis and transmission electron microscopy (TEM), cell cycle, phosphatidylserine (PS) orientation and mitochondrial transmembrane potential (MTP) were assayed by flow cytometry (FCM). And the activation of caspases was checked by ELISA. RESULTS: Morphology observations and viability assay showed that pilocarpine at concentrations above 0.625 g/L induced dose- and time-dependent morphological abnormality and viability decline of HCS cells. AO/EB double-staining, DNA electrophoresis and TEM noted that pilocarpine at concentrations above 0.625 g/L induced dose- and/or time-dependent membrane permeability elevation, DNA fragmentation, and apoptotic body formation of the cells. Moreover, FCM and ELISA assays revealed that 2.5 g/L pilocarpine also induced S phase arrest, PS externalization, MTP disruption, and caspase-8, -9 and -3 activation of the cells. CONCLUSION: Pilocarpine at concentrations above 0.625 g/L (1/32 of its clinical therapeutic dosage) has a dose- and time-dependent cytotoxicity to HCS cells by inducing apoptosis in these cells, which is most probably regulated by a death receptor-mediated mitochondrion-dependent signaling pathway.     

Parthenolide inhibits the proliferation and induces the apoptosis of human uveal melanoma cells

AIM: To explore the effect of parthenolide (PTL) on human uveal melanoma (UM) cells (C918 and SP6.5 cells) and its molecular mechanism. METHODS: Carboxyfluorescein succinimidyl amino ester (CFSE) assays and cell counting kit-8 (CCK-8) were performed to detect the cell viability. Flow cytometry was used to analyze cell cycle and apoptosis. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays were performed to measure proliferation-related and apoptosis-related factors. RESULTS: Firstly, PTL decreased the viability of C918 and SP6.5 cells in a dose-dependent manner, and the effect of PTL on C918 cells was stronger than on SP6.5; however, it did not affect normal cells. Secondly, PTL increased the proportion of cell number at cell cycle G1 phase in C918 cells, and decreased the proportion of cell number at S phase, but the proportion did not change at G2 phase. In addition, PTL induced the apoptosis of C918 cells, and decreased the expressions of Cyclin D1, B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-XL). Also, PTL increased Cyclin inhibition protein 1 (P21), Bcl-2-associated X protein (Bax), Cysteinyl aspartate specific proteinas-3 (Caspase-3) and Caspase-9 expression. However, the expression of Caspase-8 was not changed. CONCLUSION: PTL inhibites proliferation and induces apoptosis in UM cells by arresting G1 phase and regulating mitochondrial pathway, however, it does not affect normal cells.     

Crucial role of apoptosis in the resolution of experimental autoimmune anterior uveitis

PURPOSE: Experimental autoimmune anterior uveitis (EAAU) serves as an animal model of human idiopathic anterior uveitis. This study was undertaken to investigate the role of apoptosis in the resolution of EAAU. METHODS: EAAU was induced in Lewis rats by bovine melanin-associated antigen (MAA). Animals were killed at different time points during EAAU, and apoptosis of the inflammatory cells within the eye was monitored. RESULTS: Flow cytometry, TUNEL staining, and light microscopy demonstrated that CD11b/c(+) and CD4(+) T cells undergo apoptosis during EAAU. Electron microscopic analysis demonstrated that the macrophages remove these apoptotic infiltrating cells from the eye by phagocytosis. Caspase-3 levels peaked during the resolution of EAAU, and the upregulation of caspase-8 and -9 preceded that of caspase-3, suggesting that both extrinsic and intrinsic pathways of apoptosis are involved. There was an inverse relationship between the expression of proapoptotic protein Bax and antiapoptotic protein Bcl-2 during EAAU. Cytochrome c was present in the cytoplasm of the infiltrating cells undergoing apoptosis. CONCLUSIONS: These results demonstrate that extrinsic and intrinsic pathways of apoptosis are involved in the resolution of EAAU. They further suggest that apoptosis followed by phagocytosis plays a critical role in the clearance of infiltrating cells from eyes with uveitis and leads to the resolution of EAAU.     

Acute energy reduction induces caspase-dependent apoptosis and activates p53 in retinal ganglion cells (RGC-5)

The energy reduction-induced death of retinal ganglion cells is associated with many ophthalmic diseases. The present study was designed to investigate the apoptosis pathway of retinal ganglion cells (RGC-5) following acute ATP reduction by using glucose deprivation (GD). RGC-5 cells were cultured in glucose-free or normal DMEM for 3 days. The changes in intracellular ATP and cell viability were monitored by ATP assay and MTT assay. APOPercentage™ and in situ TUNEL assays were used to determine the cell death pattern. The involvement of oxidative stress was assessed by measuring intracellular ROS generation, the HO-1 expression, the effect of antioxidants, and the ratio of GSSG to total GSH. The activation of p53 and apoptosis markers was evaluated by Western blotting. We found that glucose deprivation caused an acute decline of intracellular ATP level, concomitantly decreasing cell viability. The cell death exhibited typical features indicative of apoptosis, including cell shrinkage, phosphatidylserine externalization and DNA fragmentation. Oxidative stress was involved in the cell death process; an antioxidant significantly protected the cells against glucose deprivation. p53 and apoptosis markers, caspase-3 and PARP-1 were activated after RGC-5 cells were cultured in glucose-free media for 32 h. Z-VAD-fmk, a pan-caspase inhibitor, was sufficient to prevent apoptosis. These results suggest that acute energy reduction induced by glucose deprivation triggers caspase-dependent apoptosis and activates p53. Blocking the critical steps in this cell death pathway may have therapeutic effects, rescuing the retinal ganglion cells from damages associated with acute energy reduction.     

Protective effects of upregulated HO-1 gene against the apoptosis of human retinal pigment epithelial cells in vitro

AIM: To assess the correlation between disorganization of the retinal inner layers (DRIL) and best-corrected visual acuity (BCVA) in patients with uveitis and macular edema (UME) who underwent systemic treatment using optical coherence tomography (OCT). METHODS: A retrospective clinical study of 23 patients (30 eyes) with DRIL and 23 patients (31 eyes) without DRIL secondary to UME were included. All patients underwent comprehensive ophthalmic examinations at baseline, 3, 6, and 12mo after local and systemic treatment. The OCT-based parameters included: foveal center point thickness (FCPT), mean thickness (MT), and diameters of DRIL in horizontal and vertical directions. BCVA and OCT-based parameters were compared between the two groups. The relationship between each OCT parameter and BCVA was evaluated using linear correlation and regression analysis. RESULTS: At the initial visit, the mean baseline FCPT was 441.03±128.68 μm in the eyes with DRIL and 337.26±99.31 μm in the eyes without DRIL (P=0.001). No significant differences were observed in MT (P=0.357). The mean size of transverse and vertical diameters of DRIL was 684.07±267.51 μm, and 267.07±104.61 μm at baseline, respectively. There was significant improvement in BCVA and OCT-based parameters at baseline, 3, 6, and 12mo in all cases (P<0.001 for each timepoint). In addition, significant differences were detected in BCVA and OCT parameters between eyes with and without DRIL at each time point (P<0.01 for each timepoint). A greater DRIL range at baseline was associated with a worse baseline BCVA (transverse diameter of DRIL: r=0.875, P<0.001; vertical diameter of DRIL: r=0.622, P<0.001). The transverse diameter of baseline DRIL was found to be significantly correlated with the final BCVA (P=0.003). CONCLUSION: The improvement in BCVA is associated with DRIL in patients with UME. DRIL is an easy-to-determine and robust imaging biomarker that could help predict BCVA prognosis in eyes with UME.     

Caspase and proteasome activity during staurosporin-induced apoptosis in lens epithelial cells

PURPOSE: To determine what caspases are activated during staurosporin-induced apoptosis in cultured bovine lens epithelial cells (BLECs), to study the time course of caspase activation in relation to morphologic changes, and to investigate the effect of caspase and/or proteasome inhibition on apoptosis. METHODS: BLECs were incubated with staurosporin at different concentrations or for different times. Phosphatidylserine (PS) externalization was detected by annexin-V labeling, nuclear morphology was studied by staining with Hoechst 33342 stain (Hoechst, Frankfurt, Germany), and the percentage of apoptotic cells was determined by the TdT-dUTP terminal nick-end labeling (TUNEL) assay. The activity of caspase-1, -2, -3, -4, -8, and -9 as well as the chymotrypsin-like activity of the proteasome was measured by the use of fluorogenic peptide substrates. Inhibition of the proteasome was performed by incubation with 10 microM lactacystin, and caspases were inhibited by 1 microM Z-DEVD-FMK or 20 microM Z-VAD-FMK. RESULTS: Staurosporin treatment caused a dose- and time-dependent increase in the number of apoptotic cells and in caspase-3 activity. Activation of caspase-2, -4, -8, and -9 was also seen. Caspase activity was increased after 3 hours' incubation with 1 microM staurosporin, which is also the time when most cells became annexin-V-positive. Nuclear changes indicative of apoptosis, viewed with both Hoechst and TUNEL staining, appeared after 4 to 6 hours of staurosporin incubation. Incubation of BLECs with lactacystin caused reduction of proteasome activity and increased apoptosis, evidenced in both the TUNEL assay and caspase-3 activation. Preincubation of lens epithelial cells with caspase inhibitors caused complete inhibition of lactacystin- or staurosporin-induced caspase-3 activation (Z-DEVD-FMK/Z-VAD-FMK) and also of caspase-2, -4, -8, and -9 (Z-VAD-FMK), but the reduction in TUNEL-positive cells was only partial. PS translocation and DNA fragmentation after staurosporin treatment occurred despite complete caspase blockade. CONCLUSIONS: Staurosporin-induced apoptosis in BLECs involves activation of several caspases. Inhibition of the proteasome causes caspase-3 activation and apoptosis. Both staurosporin- and lactacystin-induced apoptosis can be executed in a caspase-independent manner. The present data are useful for understanding of proteolytic mechanisms during apoptosis in lens epithelial cells, which may be an important event in normal lens development as well as in some types of cataract.     

Mitomycin-C induces the apoptosis of human Tenon's capsule fibroblast by activation of c-Jun N-terminal kinase 1 and caspase-3 protease

PURPOSE: To investigate whether mitochondrial dysfunction and mitogen-activated protein kinase family proteins are implicated in apoptotic signaling of human Tenon's capsule fibroblasts (HTCFs) by mitomycin-C. METHODS: Apoptosis was determined by Hoechst nuclei staining, agarose gel electrophoresis, and flow cytometry in HTCFs treated with 0.4 mg/mL mitomycin-C for 5 minutes. Enzymatic digestion of florigenic biosubstrate assessed the catalytic activity of caspase proteases, including caspase-3, caspase-8, and caspase-9. Phosphotransferase activity of c-Jun N-terminal kinase (JNK) 1 was measured by in vitro immune complex kinase assay using c-Jun(1-79) protein as a substrate. Mitochondrial membrane potential transition (MPT) was measured by flow cytometric analysis of JC-1 staining. RESULTS: Mitomycin-C (0.4 mg/mL) induced the apoptosis of HTCFs, which was characterized as nucleic acid and genomic DNA fragmentation, chromatin condensation, and sub-G(0)/G(1) fraction of cell cycle increase. The catalytic activity of caspase-3 and caspase-9 was significantly increased and was accompanied by cytosolic release of cytochrome c and MPT in response to mitomycin-C. Treatment with mitomycin-C resulted in the increased expression of Fas, FasL, Bad, and phosphorylated p53 and a decreased level of phosphorylated AKT. Treatment with mitomycin-C also increased the phosphotransferase activity and tyrosine phosphorylation of JNK1, whose inhibitor significantly suppressed the cytotoxicity of mitomycin-C. CONCLUSIONS: Mitomycin-C induced the apoptosis of HTCFs through the activation of intrinsic and extrinsic caspase cascades with mitochondrial dysfunction. It also activated Fas-mediated apoptotic signaling of fibroblasts. Furthermore, the activation of JNK1 played a major role in the cytotoxicity of mitomycin-C.     

Differential mechanisms of conjunctival cell death induction by ultraviolet irradiation and benzalkonium chloride

PURPOSE: To determine the molecular mechanisms of conjunctival cell death on exposure to the quaternary ammonium preservative benzalkonium chloride (BAC) and ultraviolet (UV) irradiation. METHODS: Chang conjunctival cells, either wild-type or stably transfected with various constructs encoding antiapoptotic molecules or transiently transfected with siRNA targeting the beclin-1 gene, were exposed to BAC or UV radiation Cell death was analyzed morphologically with fluorescence and electron microscopy, and molecular mechanisms of death were studied by using immunofluorescence, cell fractionation, caspase substrates, and immunoblot analysis, with or without immunoprecipitation. The main results were controlled in IOBA-NHC cells. RESULTS: Both agents induced cytochrome c release from the mitochondria, caspase activation, and nuclear chromatin condensation, suggesting caspase-dependent apoptosis. These events are prevented by stable expression of Bcl-2 protein. Both agents also induced a redistribution of Fas in plasma membrane rafts and the Fas-ligand-independent formation of a death-inducing complex leading to caspase-8 activation. Stable expression of either a dominant negative construct of Fas-associated death domain (FADD) or the long or short isoform of FADD-like interleukin-1-beta-converting enzyme inhibitory protein (FLIP) inhibited caspase-8 activation in response to both UV radiation and BAC. However, these proteins, as well as permeant peptides and baculovirus p35 caspase-inhibitors, delayed more efficiently the UV irradiation-induced than the BAC-induced nuclear chromatin condensation. BAC specifically activated a caspase-independent pathway by inducing the mitochondrial release of apoptosis-inducing factor. BAC-treated cells contain autophagosomes/autolysosomes, a characteristic feature of autophagy, and siRNA-mediated downregulation of the beclin-1 gene, whose product is crucial for autophagy, increases BAC toxicity. CONCLUSIONS: UV irradiation induces typical, caspase-dependent cell death, whereas death induced by BAC associates features of caspase-dependent and -independent apoptosis counteracted by an autophagic process.     

阿司匹林对体外培养的人晶状体上皮细胞凋亡调节基因表达的影响

目的:研究阿司匹林(Asp)对培养的人晶状体上皮细胞(humanlens epithelial cells,hLEC)Bax,Bcl-2,caspase-3表达的影响。方法:人晶状体上皮细胞系SRA01/04传代培养,MTT比色法检测Asp对bFGF诱导的hLEC增殖的影响;Western-blot方法检测Asp作用1,3,6,12,24h后凋亡相关因子Bcl-2,Bax,caspses-3蛋白的表达。结果:Asp5mmol/L可明显抑制bFGF诱导的hLEC增殖(P<0.01),且抑制作用呈浓度依赖性。Western-blot结果显示,10mmol/LAsp作用1h后Bax蛋白表达增强,随着时间的延长,表达逐渐增加,6h达到高峰,24h后恢复至基础水平;而Bcl-2的表达与其相反,Asp作用1h后Bcl-2蛋白表达明显下降,6h降到最低点,然后有上升趋势。caspase-3的表达与Bax相似,Asp作用1h后caspase-3表达增加,6h达到高峰,持续24h仍有较高的表达。结论:凋亡因子Bcl-2,Bax,caspase-3参与了Asp诱导的人晶状体上皮细胞凋亡途径。Asp通过调节凋亡基因蛋白产物的表达,诱导hLEC的凋亡。     

Aldose reductase prevents aldehyde toxicity in cultured human lens epithelial cells

Aldehydes are widespread environmental and industrial compounds, which cause cytotoxicity, tissue damage, mutagenicity, and carcinogenicity leading to various disease conditions such as cardiovascular, bronchial, and visual complications. We have shown earlier that aldose reductase (AR) besides reducing glucose to sorbitol, efficiently reduces various toxic lipid-derived aldehydes, generated under oxidative stress, with K(m) in the physiological range. We have identified the role of AR in the prevention of various lipid aldehyde-induced cytotoxic signals leading to apoptosis in human lens epithelial cells (HLEC). HLEC were cultured without or with AR inhibitors followed by addition of various saturated and unsaturated lipid aldehydes with a carbon chain length varying from C3 to C10. The cell viability was assessed by cell counts and MTT assay, and apoptosis was measured by evaluating nucleosomal degradation and caspase-3 activation using specific ELISA kits. Although all the aldehydes caused apoptosis of HLEC, the unsaturated aldehydes were more toxic than saturated aldehydes. Inhibition of AR by sorbinil potentiated while the over-expression of AR prevented the apoptosis induced by various lipid aldehydes. AR over-expression also prevented the lipid aldehyde-induced activation of caspase-3, MAPK, JNK and the expression of Bcl-2 family of proteins in HLEC. The results indicate that the lipid aldehydes generated under oxidative stress are cytotoxic to HLEC leading to apoptosis and that the reduction of lipid aldehydes by AR would prevent it.     

银杏内酯B联合视网膜干细胞移植对青光眼大鼠的影响及机制研究

目的：研究银杏内酯B联合干细胞移植对青光眼大鼠的影响及相关机制。

方法：实验共分为五组：正常对照组(Control)、青光眼模型组(GLA)、干细胞组(RSCs)、银杏内酯B组(GKB)与混合组(RSCs+ GKB)。眼球组织进行HE染色,TUNEL染色检测视网膜神经节细胞的凋亡,免疫印迹法(Western blot)检测Bcl-2、Bax以及Cleaved caspase-3和Cleaved caspase-9的蛋白表达水平,实时荧光定量PCR(RT-qPCR)分别检测各组组织中Bcl-2、Bax mRNA表达水平。

结果：HE染色结果显示,给予干细胞或银杏内酯B处理,减少纤维间质水肿与空泡的出现,混合组纤维间质少见水肿与空泡; 银杏内酯B联合干细胞处理后显著减少视网膜神经节细胞的凋亡,上调Bcl-2的蛋白与mRNA表达水平,并下调Bax的蛋白与mRNA表达水平、Cleaved caspase-3和Cleaved caspase-9的蛋白表达水平。

结论：银杏内酯B联合干细胞能够抑制青光眼大鼠视网膜神经节细胞的凋亡,改善青光眼,其作用机制可能与调控Bcl-2、Bax、Cleaved caspase-3和Cleaved caspase-9因子的表达有关。     

Activation of caspase-8 and caspase-12 pathways by 7-ketocholesterol in human retinal pigment epithelial cells

PURPOSE: To determine whether caspase or cathepsin pathways are activated in human retinal pigment epithelial cells (ARPE-19) after exposure to 7-ketocholesterol (7kCh). METHODS: ARPE-19 cells were exposed to 7kCh with or without z-VAD-fmk, a pan-caspase inhibitor. Caspase-3, -8, and -9 activities were measured by a fluorochrome inhibitor of caspase (FLICA) assay. Caspase-12 activity was detected by Western blotting. RT-PCR was performed for 18s, mortalin-2, cathepsins B, D, and L/V2. RESULTS: At 24 hours, 7kCh-treated cultures had increased caspase-8 (P < 0.001) and caspase-3 (P < 0.001) activities compared with vehicle-treated cultures. 7kCh-induced caspase-3 activation was blocked by z-VAD-fmk (P < 0.001). Caspase-9 was not activated by 7kCh treatment (P > 0.05). Procaspase-12 was cleaved into its active form after treatment with 7kCh for 24 hours. At 6 hours, the RNA level for mortalin-2, a pro-survival gene, was upregulated. ARPE-19 cells did not express RNA for cathepsins B, D, or L/V2 under any conditions. CONCLUSIONS: In ARPE-19 cells, 7kCh-induced apoptosis uses the receptor-mediated caspase-8 pathway and the endoplasmic reticulum stress-induced caspase-12 pathway but not the mitochondrial caspase-9 pathway. The cathepsin pathways are not involved in 7kCh-induced cell death. These data demonstrate that 7kCh causes a loss of cell viability through caspase-dependent apoptosis and can act as an oxidative stressor leading to retinal pigment epithelial cell atrophy. Elucidating the specific apoptotic pathways involved may have therapeutic potential for AMD and other retinal diseases.     

丹酚酸B对高糖诱导的视网膜Müller细胞凋亡、自噬及AMPK信号通路的影响

目的 探讨丹酚酸B对高糖诱导的视网膜Müller细胞凋亡、自噬及AMPK信号通路的影响.方法 提取大鼠视网膜原代Müller细胞,随机分为对照组、高糖组、高糖+丹酚酸B组、高糖+AMPK抑制剂Dorsomorphin(DS)组、高糖+DS+丹酚酸B组、高糖+AICAR组和高糖+AICAR+丹酚酸B组.其中,对照组细胞给...     

薯蓣皂苷对高糖诱导的视网膜色素上皮细胞损伤的保护作用

目的 探讨薯蓣皂苷(Dio)对高糖(HG)诱导的视网膜色素上皮(RPE)细胞损伤的保护作用,并分析其机制.方法 用细胞计数试剂8(CCK-8)法筛选葡萄糖浓度和无细胞毒性的Dio剂量范围.将ARPE-19细胞分为对照组(5 mmol·L-1葡萄糖处理48 h)、模型组(50 mmol·L-1葡萄糖处理48 h)和低、中...     

血管内皮生长因子-B对高糖环境下人视网膜色素上皮细胞凋亡的保护作用

目的探讨血管内皮生长因子-B(vascular endothelial growth factor-B,VEGF-B)对高糖环境下诱导的人视网膜色素上皮细胞(cultured human retinal pigmentepithelial cells-19,ARPE-19)凋亡的保护作用及其机制。方法将ARPE-19细胞分为正常组(体积分数10%胎牛血清+5.6 mmol·L^-1葡萄糖)、高糖组(体积分数10%胎牛血清+30 mmol·L^-1葡萄糖)、药物组(100μg·L^-1 VEGF-B+体积分数10%胎牛血清+30 mmol·L^-1葡萄糖)、抑制组[20μmol·L^-1 PD98059(ERK1/2阻滞剂)+100μg·L^-1 VEGF-B+体积分数10%胎牛血清+30 mmol·L^-1葡萄糖]。倒置显微镜观察细胞的形态变化;CCK-8法检测细胞存活率;流式细胞仪检测细胞凋亡率;Western blot检测蛋白Bcl-2、Bax...