RESUMO
Diclofenac sodium (DFS), a nonsteroidal anti-inflammatory drug, is frequently used in ophthalmology, but it causes negative effects on corneas. The mechanisms underlying the toxicities to corneas remains unclear. The present study was designed to assess the cytotoxicity of DFS to human corneal epithelial (HCEP) cells in vitro and further investigate its related mechanisms. The HCEP cells were treated with DFS at different concentrations ranging from 0.003â¯125% to 0.1%. DFS showed a dose- and time-dependent cytotoxicity to HCEP cells including abnormal morphology and declined viability. The 0.05% DFS-treated HCEP cells presented cell cycle arrest at S phase, reactive oxygen species (ROS) overproduction, and positive staining of phosphorylated H2AX, suggesting that DFS caused ROS-mediated DNA damage. The upregulation of p53 expression, formation of apoptotic body, phosphatidylserine externalization, and DNA ladder demonstrated that the p53-dependent apoptosis pathway was involved in the cytotoxicity of DFS. Furthermore, DFS activated caspase-8, caspase-9, and caspase-3 altered the expression levels of Bcl-2 family proteins including tBid, Bax, and Bcl-2, as well as increased poly(ADP-ribose) polymerase (PARP) cleavage. DFS also induced ΔΨm disruption, resulting in the release of cytochrome c and apoptosis-inducing factor into the cytoplasm. Additionally, the DFS-induced apoptosis was alleviated by p53 inhibitor. Taken together, DFS triggered p53-dependent apoptosis in HCEP cells via ROS-mediated crosstalk between the extrinsic and intrinsic pathways.
Assuntos
Apoptose/efeitos dos fármacos , Diclofenaco/farmacologia , Epitélio Corneano/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dano ao DNA , Diclofenaco/química , Epitélio Corneano/metabolismo , Humanos , Espécies Reativas de Oxigênio/análiseRESUMO
PURPOSE: Tetracaine is a local anesthetic widely used in ocular diagnosis and ophthalmic surgery and may lead to some adverse effects and complications at a clinical dose. To assess the cytotoxicity and molecular toxicity mechanisms of tetracaine, we used human corneal stromal (HCS) cells as an in vitro model to study the effects of tetracaine on HCS cells. MATERIALS AND METHODS: The cytotoxicity of tetracaine on HCS cells was investigated by examining the changes of cell growth, morphology, viability and cell cycle progressing when HCS cells were treated with tetracaine at concentrations from 10 g/L to 0.078125 g/L. To prove the hypothesis that the cytotoxicity of tetracaine on HCS cells was related with apoptosis induction, we further detected multiple changes in HCS cells, including plasma membrane (PM) permeability, phosphatidylserine (PS) orientation, genomic DNA integrality, and cell ultrastrcuture after treated with tetracaine. Furthermore, the pro-apoptotic signalling pathway induced by tetracaine was explored through detecting the activation of various caspases, the changes of mitochondrial transmembrane potential (MTP), the expression level of Bcl-2 family proteins and the amount of mitochondria-released apoptosis regulating proteins in cytoplasm. RESULTS: Tetracaine at concentrations above 0.15625 g/L had a dose- and time-dependent cytotoxicity to HCS cells, which resulted cell growth inhibition, proliferation retardation, morphological abnormalities and decreased viability. Meanwhile, we found that the HCS cells treated with tetracaine had typical features associated with apoptosis, including an increase in PM permeability, PS externalization, DNA fragmentation and apoptotic body formation. Tetracaine not only resulted in caspase-3, caspase-8 and caspase-9 activation and disruption of MTP but also downregulated Bcl-2 and Bcl-xL and upregulated Bad and Bax, along with the upregulation of cytoplasmic cytochrome c (Cyt. c) and apoptosis-inducing factor (AIF). CONCLUSIONS: These results suggested that tetracaine-induced apoptosis might be triggered through Fas death receptors and mediated by Bcl-2 family proteins in the mitochondria-dependent pathway. Our findings identified the cytotoxicity and molecular mechanisms of tetracaine, which could provide a reference value for the safety of this medication and prospective therapeutic interventions in eye clinic.
Assuntos
Anestésicos Locais/toxicidade , Apoptose/efeitos dos fármacos , Córnea/patologia , Mitocôndrias/efeitos dos fármacos , Células Estromais/patologia , Tetracaína/toxicidade , Caspases/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Córnea/efeitos dos fármacos , Córnea/ultraestrutura , Fragmentação do DNA , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Estudos Prospectivos , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/ultraestruturaRESUMO
A tissue-engineered human corneal stroma (TE-HCS) has been developed as a promising equivalent to the native corneal stroma for replacement therapy. However, there is still a crucial need to improve the current approaches to render the TE-HCS equivalent more favorable for clinical applications. At the present study, we constructed a TE-HCS by incubating non-transfected human corneal stromal (HCS) cells in an acellular porcine corneal stromata (aPCS) scaffold in 20% fetal bovine serum supplemented DMEM/F12 (1:1) medium at 37 °C with 5% CO2in vitro. After 3 days of incubation, the constructed TE-HCS had a suitable tensile strength for transplantation, and a transparency that is comparable to native cornea. The TE-HCS had a normal histological structure which contained regularly aligned collagen fibers and differentiated HCS cells with positive expression of marker and functional proteins, mimicking a native HCS. After transplantation into rabbit models, the TE-HCS reconstructed normal corneal stroma in vivo and function well in maintaining corneal clarity and thickness, indicating that the completely biological TE-HCS could be used as a HCS equivalent. The constructed TE-HCS has promising potentials in regenerative medicine and treatment of diseases caused by corneal stromal disorders.
Assuntos
Doenças da Córnea/cirurgia , Substância Própria/citologia , Transplante de Córnea/métodos , Engenharia Tecidual/métodos , Análise de Variância , Animais , Células Cultivadas , Edema da Córnea/patologia , Modelos Animais de Doenças , Humanos , Coelhos , Suínos , Alicerces TeciduaisRESUMO
Pranoprofen (PPF), a non-steroidal anti-inflammatory drugs (NSAIDs), is often used in keratitis treatment in clinic. Several studies have assessed in vitro the cytotoxicity of topical NSAIDs to corneal epithelial cells due to its importance for predicting human corneal toxicity. Damage by cytotoxic drugs can result in excessive loss of human corneal endothelial (HCE) cells which lead to decompensation of the endothelium and eventual loss of visual acuity. However, the endothelial cytotoxicity of PPF has not yet been reported using an in vitro model of HCE cells. This study assessed the cytotoxicity of PPF to HCE cells and its underlying mechanism. Cellular viability was determined using inverted phase contrast light microscopy, and plasma membrane permeability, genomic DNA fragmentation, and ultrastructure were detected by acridine orange/ethidium bromide staining, DNA agarose gel electrophoresis, and transmission electron microscopy (TEM), respectively. The results on cellular viability showed that PPF at concentrations ranging from 0.0625 to 1.0 g/l had poignant cytotoxicity to HCE cells, and the extent of its cytotoxicity was dose- and time-dependent. Further characterization indicated that PPF induced plasma membrane permeability elevation, DNA fragmentation, and apoptotic body formation, proving its apoptosis inducing effect on HCE cells. In conclusion, PPF above 0.0625 g/l has poignant cytotoxicity on HCE cells in vitro by inducing cell apoptosis, and should be carefully employed in eye clinic.
Assuntos
Anti-Inflamatórios não Esteroides , Apoptose/efeitos dos fármacos , Benzopiranos , Células Endoteliais/efeitos dos fármacos , Endotélio Corneano/efeitos dos fármacos , Propionatos , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/toxicidade , Benzopiranos/administração & dosagem , Benzopiranos/toxicidade , Técnicas de Cultura de Células , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais/ultraestrutura , Endotélio Corneano/ultraestrutura , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Contraste de Fase , Propionatos/administração & dosagem , Propionatos/toxicidade , Fatores de TempoRESUMO
Betaxolol is commonly used to manage glaucoma in clinical practice. However, its long-term use may damage the cornea. Thus, the cytotoxicity and mechanisms of betaxolol in human corneal stromal cells (HCSCs) warrant further study. In this study, we used in vitro HCSCs and in vivo rabbit corneal models to investigate betaxolol cytotoxic effects and mechanism of action. At near-clinical concentrations (0.28% and 0.14%), betaxolol inhibited caspase-8 activity, activated receptor-interacting protein kinase (RIPK)1, RIPK3, and mixed-spectrum kinase-like domain (MLKL), and phosphorylated MLKL to induce necroptosis in HCSCs. Similarly, moderate concentrations of betaxolol (0.07%-0.0175%) activated caspase-8 to trigger the exogenous apoptotic pathway. Through the intrinsic apoptotic pathway, betaxolol upregulated the expression of Bcl-2 family apoptotic proteins Bax and Bad and downregulated that of anti-apoptotic proteins Bcl-2 and Bcl-xL. This subsequently disrupted the mitochondrial membrane potential and cytoplasmic transfer of cytochrome c and apoptosis-inducing factor, activated caspase-9, and induced apoptosis in HCSCs. Furthermore, continuous treatment with low betaxolol concentrations (0.00875%) for three generations of HCSCs prevented apoptosis by promoting the expression of Bcl-xL and suppressing that of Bax. However, its toxic effects initiated cellular senescence by increasing reactive oxygen species, leading to the disruption of energy metabolism and DNA damage. Finally, clinical concentrations of betaxolol had a pro-apoptotic effect on rabbit corneal stromal cells in vivo. These results suggest that betaxolol induces cytotoxicity in a concentration-dependent manner in HCSCs, and that caspase-8 and Bcl-2 family proteins may be critical switches in the conversion of different HCSC death mechanisms.
Assuntos
Betaxolol , Necroptose , Animais , Humanos , Coelhos , Betaxolol/metabolismo , Betaxolol/farmacologia , Caspase 8/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células Estromais/metabolismoRESUMO
Carteolol is a commonly-used topical medication for primary open-angle glaucoma. However, long-term and frequent ocular application of carteolol entails its residuals at low concentration in the aqueous humor for a long duration and may exert latent toxicity in the human corneal endothelial cells (HCEnCs). Here, we treated the HCEnCs in vitro with 0.0117% carteolol for 10 days. Thereafter, we removed the cartelolol and normally cultured the cells for 25 days to investigate the chronical toxicity of carteolol and the underlying mechanism. The results exhibited that 0.0117% carteolol induces senescent features in the HCEnCs, such as increased senescence-associated ß-galactosidase positive rates, enlarged relative cell area and upregulated p16INK4A and senescence-associated secretory phenotypes, including IL-1α, TGF-ß1, IL-10, TNF-α, CCL-27, IL-6 and IL-8, as well as decreased Lamin B1 expression and cell viability and proliferation. Thereby, further exploration demonstrated that the carteolol activates ß-arrestin-ERK-NOX4 pathway to increase reactive oxygen species (ROS) production that imposes oxidative stress on energetic metabolism causing a vicious cycle between declining ATP and increasing ROS production and downregulation of NAD+ resulting in metabolic disturbance-mediated senescence of the HCEnCs. The excess ROS also impair DNA to activate the DNA damage response (DDR) pathway of ATM-p53-p21WAF1/CIP1 with diminished poly(ADP-Ribose) polymerase (PARP) 1, a NAD+-dependent enzyme for DNA damage repair, resulting in cell cycle arrest and subsequent DDR-mediated senescence. Taken together, carteolol induces excess ROS to trigger HCEnC senescence via metabolic disturbance and DDR pathway.
Assuntos
Carteolol , Glaucoma de Ângulo Aberto , Humanos , Espécies Reativas de Oxigênio/metabolismo , Senescência Celular , Transdução de Sinais/fisiologia , Células Endoteliais/metabolismo , beta-Arrestinas/metabolismo , NAD/metabolismo , NADPH Oxidase 4/metabolismoRESUMO
The human corneal endothelial cells (HCEnCs) play a vital role in the maintenance of corneal transparency and visual acuity. In our daily life, HCEnCs are inevitably exposed to ultraviolet B (UVB) radiation leading to decreases of visual acuity and corneal transparency resulting in visual loss eventually. Therefore, understanding the UVB-induced cytotoxicity in HCEnCs is of importance for making efficient strategies to protect our vision from UVB-damage. However, in-depth knowledge about UVB-induced cytotoxicity in HCEnCs is missing. Herein, we pulse-irradiated the HCEnCs in vitro with 150 mJ/cm2 UVB (the environmental dose) at each subculture for 4 passages to explore the insights into UVB-induced phototoxicity. The results showed that the UVB-treated HCEnCs exhibit typical senescent characteristics, including significantly enlarged relative cell area, increased senescence-associated ß-galactosidase positive staining, and upregulated p16INK4A and senescence associated secretory phenotypes (SASPs) such as CCL-27, IL-1α/6/8/10, TGF-ß1 and TNF-α, as well as decreased cell proliferation and Lamin B1 expression, and translocation of Lamin B1. Furthermore, we explored the causative mechanisms of senescence and found that 150 mJ/cm2 UVB pulse-irradiation impairs DNA to activate DNA damage response (DDR) pathway of ATM-p53-p21WAF1/CIP1 with downregulated DNA repair enzyme PARP1, leading to cell cycle arrest resulting in DDR-mediated senescence. Meanwhile, UVB pulse-irradiation also elicits a consistent increase of ROS production to aggravate DNA damage and impose oxidative stress on energy metabolism leading to metabolic disturbance resulting in metabolic disturbance-mediated senescence. Altogether, the repeated pulse-irradiation of 150 mJ/cm2 UVB induces HCEnC senescence via both DDR pathway and energy metabolism disturbance.
Assuntos
Senescência Celular , Dano ao DNA , Células Endoteliais , Estresse Oxidativo , Raios Ultravioleta , Células Cultivadas , Senescência Celular/efeitos da radiação , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos da radiação , Humanos , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta/efeitos adversos , beta-Galactosidase/metabolismoRESUMO
Limbal stem cells (LSCs) are crucial for corneal transparency and vision. Any damages to LSCs might lead to limbal stem cell deficiency resulting in corneal opacification and even blindness. Here, we investigated the cytotoxicity of timolol and its underlying mechanisms in rabbit LSCs (rLSCs) in vitro. High concentrations of 0.5% and 0.25% timolol induced necroptosis in rLSCs to upregulate receptor interacting protein kinase (RIPK)1, RIPK3, mixed lineage kinase domain-like (MLKL) and phosphorylated MLKL along with downregulation of caspase-8 and caspase-2 within 4 h. While, median concentrations of 0.125% to 0.0625% timolol induced apoptosis in the rLSCs within 28 h. The apoptotic mechanism in the median-concentration timolol-treated rLSCs is probably via extrinsic apoptosis pathway by activating caspase-2, caspase-8 and caspase-3 and intrinsic apoptosis pathway triggered by excessive generation of ROS and subsequent DNA damage to upregulate Bax and Bad, downregulate Bcl-2 and Bcl-xL, subsequently disrupt mitochondrial membrane potential, cytosolically translocate cytochrome c and apoptosis-inducing factor, and activate caspase-9. In addition, low concentration of 0.03125% timolol induced senescence in the rLSCs by elevating ROS level and increasing number of senescence associated ß-galactosidase positive cells at 28 h. Our findings reveal that timolol induces necroptosis, apoptosis and senescence concentration-dependently in rLSCs in vitro.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Apoptose , Senescência Celular , Limbo da Córnea/patologia , Necroptose , Células-Tronco/patologia , Timolol/farmacologia , Animais , Técnicas In Vitro , Limbo da Córnea/efeitos dos fármacos , Limbo da Córnea/metabolismo , Masculino , Fosforilação , Coelhos , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Norfloxacin (NOR) is applied clinically to treat keratitis. However, NOR has brought severe side-effects for human corneal epithelium (HCEP) due to overdose and potential toxicity. In this study, two in vitro experimental models including monolayer HCEP cells and tissue-engineered human corneal epithelium (TE-HCEP) were used to explore the cytotoxicity and its related mechanisms. The HCEP cells treated with NOR at concentrations from 0.1875 to 3.0 mg/mL displayed abnormal morphology, declined viability, and increased plasma membrane permeability. Moreover, 0.75 mg/mL NOR induced chromatin condensation, S phase arrest, phosphatidylserine externalization, and formation of apoptotic body through activation of caspase-2/-8/-9/-3, downregulation of Bcl-2 and Bcl-xL, upregulation of Bad and Bax, mitochondrial transmembrane potential disruption and release of cytochrome c and apoptosis inducing factor into cytosol, whereas 1.5 mg/mL and 3.0 mg/mL NOR upregulated the expressions of receptor-interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL) together with inactivation of caspase-2/-8. Furthermore, 0.1875-3.0 mg/mL NOR destroyed the multilayer structure of TE-HCEP model due to a dose-dependent cytotoxicity, which validated the above results. Overall, low-dose (0.1875-0.75 mg/mL) NOR induced apoptosis through mitochondrion-dependent and death receptor-mediated pathways, and high-dose (1.5-3.0 mg/mL) NOR triggered necroptosis via RIPK1-RIPK3-MLKL cascade in HCEP cells.
Assuntos
Antibacterianos/toxicidade , Apoptose/efeitos dos fármacos , Córnea/citologia , Células Epiteliais/efeitos dos fármacos , Necroptose/efeitos dos fármacos , Norfloxacino/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Humanos , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Engenharia TecidualRESUMO
Carteolol is a non-selective ß-adrenoceptor antagonist used for the treatment of glaucoma, and its abuse might be cytotoxic to the cornea. However, its cytotoxicity and underlying mechanisms need to be elucidated. Herein, we used an in vivo model of feline corneas and an in vitro model of human corneal endothelial cells (HCECs), respectively. In vivo results displayed that 2% carteolol (clinical dosage) could induce monolayer density decline and breaking away of feline corneal endothelial (FCE) cells. An in vitro model of HCECs that were treated dose-dependently (0.015625-2%) with carteolol for 2-28 h, resulted in morphological abnormalities, declining in cell viability and elevating plasma membrane (PM) permeability in a dose- and time- dependent manner. High-dose (0.5-2%) carteolol treatment induced necrotic characteristics with uneven distribution of chromatin, marginalization and dispersed DNA degradation, inactivated caspase-2/-8, and increased RIPK1, RIPK3, MLKL, and pMLKL expression. The results suggested that high-dose carteolol could induce necroptosis via the RIPK/MLKL pathway. While low-dose (0.015625-0.25%) carteolol induced apoptotic characteristics with chromatin condensation, typical intranucleosomal DNA laddering patterns, G1 cell-cycle arrest, phosphatidylserine (PS) externalization, and apoptotic body formation in HCECs. Meanwhile, 0.25% carteolol treatment resulted in activated caspase-2, -3, -8, and -9, downregulation of Bcl-2 and Bcl-xL, upregulation of Bax and Bad, ΔΨm disruption, and release of cytoplasmic cytochrome c (Cyt.c) and AIF into the cytoplasm. These observations suggested that low-dose carteolol could induce apoptosis via a caspase activated and mitochondrial-dependent pathway. These results suggested that carteolol should be used carefully, as low as 0.015625% cartelol caused apoptotic cell death in HCECs in vitro.
RESUMO
Norfloxacin, a frequently used ocular antibiotic, might have cytotoxic effect on human corneal endothelial cells (HCECs), subsequently damage the cornea and finally impair human vision. However, the possible mechanisms of cytotoxicity of norfloxacin to HCEC line are unclear. Herein, we investigated the cytotoxicity of norfloxacin and its underlying cellular and molecular mechanisms using in vitro cultured non-transfected HCECs and verified the cytotoxicity with cat corneal endothelium in vivo. In the present study, the cytotoxicity of norfloxacin in the in vitro cultured HCECs was recognized by causing abnormal morphology such as cell shrinkage and detachment from plate bottom, and decline of viability of in vitro cultured HCECs. Then, its cytotoxicity was verified by inducing reduction of cell density and morphological abnormality of in vivo cat corneal endothelial cells. Furthermore, the cytotoxicity of norfloxacin in HCECs was corroborated as apoptosis by elevation of plasma membrane permeability, S phase arrest, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation in in vitro cultured HCECs and apoptosis-like swollen cells in the in vivo model. Moreover, norfloxacin induced extrinsic death receptor-mediated apoptosis pathway by activating caspase-2/-8/-3 and intrinsic mitochondrion-dependent apoptosis pathway by downregulating anti-apoptotic Bcl-2 and upregulating of pro-apoptotic Bad, which disrupted mitochondrial transmembrane potential, subsequently upregulated cytoplasmic cytochrome c and apoptosis-inducing factor and finally activated caspase-9/-3. Generally, norfloxacin induces HCE cell apoptosis via a death receptor-mediated and mitochondrion-dependent signaling pathway.
Assuntos
Antibacterianos/farmacologia , Córnea/citologia , Células Endoteliais/efeitos dos fármacos , Norfloxacino/farmacologia , Animais , Apoptose/efeitos dos fármacos , Gatos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Células Endoteliais/ultraestrutura , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Eletrônica de TransmissãoRESUMO
PURPOSE: To provide scientific data for clinical practice in making strategies for accelerating corneal endothelial wound healing, we investigated the impact of UVA on the corneal endothelial wound healing process and the underlying mechanism using an in vitro cell model. MATERIALS AND METHODS: An in vitro cell model for corneal endothelial wound healing was established by scratching the in vitro cultured human corneal endothelial cell (HCEnC) confluent layer. Then, we investigated the impacts of UVA irradiation and Ascorbic acid-2-phosphate (Asc-2p) on the wound healing process of the in vitro HCEnC model by examining wound-healing index, F-actin+ rate, Ki-67+ rate, and ROS production. RESULTS: After scratching, the Ki-67+ and F-actin+ HCEnCs occupied the scratching gap. Furthermore, the F-actin+ rates were significantly higher than Ki-67+ rates in the wound closure area. After irradiated with UVA, the wound-healing indexes, Ki-67+ rates and F-actin+ rates of the wound-healing model significantly reduced, whereas the ROS production significantly increased in a dose-dependent manner. Pretreatment with Asc-2p significantly reduced the ROS production as well as increased the wound-healing indexes, Ki-67+rates and F-actin+ rates of the UVA irradiated wound-healing model. CONCLUSION: The migration of HCEnC plays a major role in the wound healing process of the established cell model, which is like the wound healing process in vivo. UVA decreases the wound closure of the in vitro HCEnC model dose-dependently, while antioxidant Asc-2p can attenuate the damage to UVA to HCEnCs probably via reducing ROS to improve their migration.
Assuntos
Endotélio Corneano/efeitos da radiação , Raios Ultravioleta , Cicatrização/efeitos da radiação , Actinas/metabolismo , Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/farmacologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Endotélio Corneano/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismoRESUMO
To obtain water-soluble holothurian glycosides with high tumor suppressing activities from Apostichopus japonicus, macroporous resin, silica gel and gel-filtration column chromatograghy were used to purify the water-soluble holothurian glycosides, and their tumor suppressing activity and inducing apoptosis of tumor cells were examined in this study. The 70% ethanol fraction of macroporous resin column, the pSC-2 and pSC-3 fractions from silica gel column showed very strong tumor suppressing activity towards HeLa cells, A-549 lung cancer cells, SGC-7901 stomach cancer cells and Bel-7402 liver cancer cells. SC-2 and SC-3 fraction purified from Sephadex LH-20 gel-filtration column chromatography, with a purity above 99.6%, all had the properties of triterpenoid glycosides. Purified SC-2 fraction had remarkable tumor suppressing activity on HeLa cells in a dose- and time-dependent manner, and had prominent tumor suppressing activity to mouse S180 solid tumors in a dose-dependent manner. Besides, the SC-2 fraction also had remarkable ability in elevating mouse thymus index and spleen index. The purified SC-2 fraction could induce apoptosis of HeLa cells in a dose-dependent manner, and DNA fragmentation of HeLa cells occurred after treated 12 h with 10 mg x L(-1) and 50 mg x L(-1) of SC-2 fractions. From the results, it can be concluded that the purified SC-2 fraction of water-soluble holothurian glycosides has extremely strong tumor suppressing activity, and the suppression is realized by inducing tumor cells to undergo apoptosis. This study lays solid foundation for development of highly effective new natural anticancer agents from sea cucumbers.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Glicosídeos/farmacologia , Sarcoma 180/patologia , Stichopus/química , Animais , Antineoplásicos/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glicosídeos/isolamento & purificação , Células HeLa , Humanos , Concentração Inibidora 50 , Camundongos , Transplante de Neoplasias , Distribuição Aleatória , Carga Tumoral/efeitos dos fármacosRESUMO
In the present study, the toxicity of phenylephrine, a selective α1-adrenergic receptor agonist, in corneal epithelial cells and its underlying mechanisms were investigated using an in vitro model of human corneal epithelial cells (HCEPCs) and an in vivo model of New Zealand white rabbit corneas. The HCEPCs treated with phenylephrine at concentrations from 10% to 0.078125% displayed abnormal morphology, decline of cell viability and elevation of plasma membrane permeability time- and dose-dependently. Moreover, 10%-1.25% phenylephrine induce necrosis characteristics of marginalization and uneven distribution of chromatin through up-regulation of RIPK1, RIPK3 and MLKL along with inactivation of caspase-8 and caspase-2, whereas 0.625% phenylephrine induced condensed chromatin, S phase arrest, phosphatidylserine externalization, DNA fragmentation and apoptotic body formation in the HCECs through activation of caspase-2, -8, -9 and -3 as well as down-regulation of Bcl-2, up-regulation of Bad, ΔΨm disruption and release of cytochrome c and AIF into cytosol. At last, 10% phenylephrine induced destruction of the corneal epithelia and apoptosis of corneal epithelial cells in rabbit corneas. In conclusion, 10% to 1.25% phenylephrine cause necroptosis via RIPK1-RIPK3-MLKL axis and 0.625% phenylephrine induce apoptosis via a mitochondrion-dependent and death receptor-mediated signal pathway in HCEPCs.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 1/toxicidade , Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Necroptose/efeitos dos fármacos , Fenilefrina/toxicidade , Animais , Ciclo Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Córnea/citologia , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Humanos , Masculino , Coelhos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fatores de TempoRESUMO
AIM: To reveal the cytotoxicity and related mechanisms of gatifloxacin (GFX) to stromal fibroblasts (SFs) in vitro. METHODS: SFs were treated with GFX at different concentrations (0.009375%-0.3%), and their viability was detected by MTT method. The cell morphology was observed using light/transmission electron microscope. The plasma membrane permeability was measured by AO/EB double-staining. Then cell cycle, phosphatidylserine (PS) externalization, and mitochondrial transmembrane potential (MTP) were analyzed by flow cytometry. DNA damage was analyzed by electrophoresis and immunostaining. ELISA was used to evaluate the caspase-3/-8/-9 activation. Finally, Western blotting was applied for detecting the expressions of apoptosis-related proteins. RESULTS: Morphological changes and reduced viability of GFX-treated SFs demonstrated that GFX above 0.009375% had cytotoxicity to SFs with dependence of concentration and time. GFX-treating cells also showed G1 phase arrest, increased membrane permeability, PS externalization and DNA damage, which indicated that GFX induced apoptosis of SFs. Additionally, GFX could activate the caspase-8, caspase-9, and caspase-3, induce MTP disruption, downregulate B-cell leukemia-2 (Bcl-2) and B-cell leukemia-XL (Bcl-XL), and upregulate Bcl-2 assaciated X protein (Bax), Bcl-2-associated death promoter (Bad), Bcl-2 interacting domain (Bid) and cytoplasmic cytochrome C in SFs, suggesting that caspase-dependent extrinsic and intrinsic pathways were related to GFX-contributed apoptosis of SFs. CONCLUSION: The cytotoxicity of GFX induces apoptosis of SFs through triggering the caspase-dependent extrinsic and intrinsic pathways.
RESUMO
Clonidine, an α2-adrenoreceptor agonist, is an anti-glaucoma drug clinically used in many developing countries, and its abuse might damage the cornea and impair human vision. However, its cytotoxicity and precise mechanisms need to be elucidated. Herein, we investigated the cytotoxicity of clonidine and its underlying mechanisms, using an in vitro model of human corneal epithelial (HCEP) cells and an in vivo model of cat corneas, respectively. HCEP cells were treated with various doses of clonidine for 1-28 h, resulting in abnormal morphology, decline of cell viability and G1 phase arrest in a time- and/or dose-dependent manner. Moreover, clonidine treatment induced elevation of plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation in HCEP cells. Furthermore, we found that clonidine treatment resulted in activated caspase-2, -3, -8, and -9, disruption of the mitochondrial transmembrane potential, downregulation of Bcl-2, and upregulation of Bad, cytoplasmic cytochrome c and apoptosis inducing factor, suggesting that clonidine-induced apoptosis is triggered through Fas/TNFR1 death receptors and Bcl-2 family proteins-mediated mitochondria-dependent pathways. Finally, our in vivo results displayed that 0.25% clonidine could induce DNA fragmentation of cat corneal epithelial cells. In summary, our findings suggest that clonidine above 1/32 of its clinical therapeutic dosage is cytotoxic to corneal epithelial cells by inducing cell apoptosis both in vitro and in vivo, and its pro-apoptotic effect on HCEP cells is triggered by a Fas/TNFR1 death receptors-mediated, mitochondria-dependent signaling pathway.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/efeitos adversos , Apoptose/efeitos dos fármacos , Clonidina/efeitos adversos , Epitélio Corneano/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/agonistas , Transdução de Sinais/efeitos dos fármacos , Receptor fas/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/administração & dosagem , Animais , Caspases/química , Caspases/metabolismo , Gatos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/patologia , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clonidina/administração & dosagem , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Fase G1/efeitos dos fármacos , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismoRESUMO
PURPOSE: To investigate the cytotoxic effect of latanoprost on corneal stroma and its underlying cellular and molecular mechanisms using non-transfected human corneal stromal (HCS) cells as an in vitro model. METHODS: After HCS cells were treated with latanoprost at concentrations varying from 50 mg/l (clinical therapeutic dosage) to 0.78125 mg/l, and cell morphology, cell viability, and cell cycle were detected by light microscopy, methyl thiazolyl tetrazolium assay, and flow cytometry (FCM) with propidium iodide (PI) staining, respectively. Meanwhile, alterations in plasma membrane permeability, phosphatidylserine (PS) orientation, DNA integrality, and cell ultrastructure were examined by acridine orange (AO)/ethidium bromide (EB) double staining, FCM with Annexin-V/propidium iodide (PI) staining, DNA electrophoresis, and transmission electron microscopy. Furthermore, caspase activation, mitochondrial transmembrane potential (MTP), and expression of pro-apoptotic regulators were determined by ELISA, FCM with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethybenzimida (JC-1) staining, and Western blot, respectively. RESULTS: Latanoprost above concentrations of 3.125 mg/l can induce dose- and time-dependent morphological abnormality, growth retardation, viability decline, and plasma membrane permeability elevation of HCS cells. Moreover, latanoprost can arrest the cell cycle of these cells at S phase and induce PS externalization, DNA fragmentation, and apoptotic body formation of the cells. Furthermore, latanoprost can induce activation of caspase-3, -8 and -9; disruption of MTP; downregulation of anti-apoptotic Bcl-2; upregulation of pro-apoptotic Bax; and cytoplasmic cytochrome c release. CONCLUSIONS: Latanoprost above and at 3.125 mg/l (1/16 of its clinical therapeutic dosage) has a dose- and time-dependent cytotoxicity to HCS cells by inducing death receptor-mediated mitochondria-dependent apoptosis, which should be used with great caution in clinical situations to avoid undesired damages to HCS cells.
Assuntos
Anti-Hipertensivos/toxicidade , Ceratócitos da Córnea/efeitos dos fármacos , Substância Própria/efeitos dos fármacos , Prostaglandinas F Sintéticas/toxicidade , Apoptose/efeitos dos fármacos , Western Blotting , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ceratócitos da Córnea/metabolismo , Ceratócitos da Córnea/patologia , Substância Própria/metabolismo , Substância Própria/patologia , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Latanoprosta , Fatores de TempoRESUMO
Using chorion of Paralichthys as a specific substrate, hatching enzyme (HE) from Paralichthys olivaceus (PHE) was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular weight and enzymatic properties in this study. It was found that the molecular size of PHE is about 34.8 kDa in SDS-PAGE. The PHE had obvious choriolytic activity, which was optimal at pH 7.0 and temperature of 35 degrees C, respectively. The Km value of the PHE for casein was 4.28 mg ml(-). The PHE was very sensitive to trypsin-specific inhibitors, especially serine protease-specific inhibitors, such as LBTI, SBTI, bestatin and p-APMSF, leupeptin, ovomucoid, PMSF, pepstatin A and TLCK, indicates that it is a trypsin-type serine protease. The PHE was also extremely sensitive to Cu(2+) and Ca(2+), combined with the results that it was inhibited by EDTA in a dose-dependent manner, indicates this PHE is also a kind of metalloprotease.
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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.
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Carteolol is a frequently used nonselective ß-adrenoceptor antagonist for glaucoma and ocular hypertension treatment, and its repeated/prolonged usage might be cytotoxic to the cornea, especially the outmost human corneal epithelium (HCEP). The aim of the present study was to characterize the cytotoxicity of carteolol to HCEP and its underlying cellular and molecular mechanisms using an in vitro model of HCEP cells. After HCEP cells were treated with carteolol at concentrations varying from 2% to 0.015625%, the cytotoxicity, apoptosis-inducing effect and pro-apoptotic pathway was investigated, respectively. Our results showed that carteolol at concentrations above 0.03125% induced time- and dose-dependent growth retardation, cytopathic morphological changes and viability decline of HCEP cells. Moreover, carteolol induced G1 phase arrest, plasma membrane permeability elevation, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation of HCEP cells. Furthermore, carteolol also induced activation of caspase-9 and -3, disruption of mitochondrial transmembrane potential, up-regulation the cytoplasmic amount of cytochrome c and apoptosis-inducing factor, and up-regulation of pro-apoptotic Bax and Bad, down-regulation of anti-apoptotic Bcl-2 and Bcl-xL. In conclusion, carteolol above 1/64 of its clinical therapeutic dosage has a time- and dose-dependent cytotoxicity to HCEP cells, which is achieved by inducing apoptosis via triggering Bcl-2 family protein-mediated mitochondrial pro-apoptotic pathway.