RESUMO
Corneal stromal wound healing is a well-balanced process promoted by overlapping phases including keratocyte proliferation, inflammatory-related events, and tissue remodeling. L-carnitine as a natural antioxidant has shown potential to reduce stromal fibrosis, yet the underlying pathway is still unknown. Since transient receptor potential vanilloid 1 (TRPV1) is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing, we investigated if L-carnitine can mediate inhibition of the fibrotic response through suppression of TRPV1 activation in human corneal keratocytes (HCK). We determined TRPV1-induced intracellular calcium transients using fluorescence calcium imaging, channel currents by planar patch-clamping, and cell migration by scratch assay for wound healing. The potential L-carnitine effect on TRPV1-induced myofibroblast transdifferentiation was evaluated by immunocytochemical detection of alpha smooth muscle actin. RT-PCR analysis confirmed TRPV1 mRNA expression in HCK. L-carnitine (1 mmol/l) inhibited either capsaicin (CAP) (10 µmol/l), hypertonic stress (450 mOsmol/l), or thermal increase (>43 °C) induced Ca2+ transients and corresponding increases in TRPV1-induced inward and outward whole-cell currents. This was accompanied by suppression of injury-induced increases in myofibroblast transdifferentiation and cell migration. In conclusion, L-carnitine contributes to inhibit stromal scarring through suppressing an injury-induced intrinsic TRPV1 activity that is linked with induction of myofibroblast transdifferentiation in HCK cells.
Assuntos
Carnitina/uso terapêutico , Transdiferenciação Celular/efeitos dos fármacos , Ceratócitos da Córnea/efeitos dos fármacos , Substância Própria/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Carnitina/farmacologia , Células Cultivadas , Substância Própria/citologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Miofibroblastos , Canais de Cátion TRPV/efeitos dos fármacosRESUMO
Plants and their extracts have been used especially in China for more than ten centuries for preventing and treating disease. However, there are only few reports describing their use in animal cell culture and tissue transplantation. In this study, onion epithelial membranes (OEM) is used as scaffolds to support cultures of a variety of cells such as fibroblasts and epithelial cells notably; they maintain the phenotypic characteristics of corneal epithelial cells. This improvement includes preservation of the proliferative potential and stemness of rabbit corneal epithelial cells (RCECs). Such an outcome suggests that this cost-effective technology warrants further evaluation to determine if OEM is a viable candidate for use as scaffolds in corneal epithelial transplantation surgery. To test this possibility, rabbit corneal epithelial cells expanded on OEM are transplanted to treat corneal epithelial defects in limbal stem cell deficient rabbits. This procedure is successful because it shortens the time required for wound healing to restore losses in corneal epithelial integrity, and forms a more compact and stratified epithelium framework than the untreated group. Ultimately, should they be proven to be effective in other relevant animal model systems, their usefulness for treating wounds in a clinical setting warrants consideration.
Assuntos
Limbo da Córnea , Animais , Células Cultivadas , China , Córnea , Células Epiteliais , Cebolas , Coelhos , Células-TroncoRESUMO
PURPOSE: To determine whether hypertonic stress promotes increases in inflammatory cytokine release through transient receptor potential vanilloid channel type 1 (TRPV1) signaling pathway activation in human corneal epithelial cells (HCECs). METHODS: Hyperosmotic medium was prepared by supplementing isotonic Ringers solution with sucrose. Ca2+ signaling was measured in fura2-AM-loaded HCECs using a single-cell fluorescence imaging system. Western blot analysis evaluated the phosphorylation status of EGFR, ERK, p38 MAPK, and nuclear factor (NF)-κB. ELISA assessed the effect of TRPV1 activation on the release of IL-6 and IL-8. RESULTS: A 450 mOsm hypertonic stress elicited 2-fold Ca2+ transients that were suppressed by the TRPV1-selective antagonists capsazepine and JYL 1421. Such transients were enhanced by PGE2. Hypertonicity-induced EGF receptor (EGFR) transactivation was suppressed by preincubating HCECs with capsazepine, matrix metalloproteinase 1 (MMP1) inhibitor TIMP-1, broad-spectrum MMP inhibitor GM 6001, heparin-bound (HB)-EGF inhibitor CRM 197, or EGFR inhibitor AG 1478. ERK and p38 MAPK and NF-κB activation after EGFR transactivation occurred in tonicity and in a time-dependent manner. Hypertonicity-induced increases in IL-6 and IL-8 releases were suppressed by exposure to capsazepine, AG 1478, ERK inhibitor PD 98059, p38 inhibitor SB 203580, or NF-κB inhibitor PDTC. CONCLUSIONS: Hypertonic stress-elicited TRPV1 channel stimulation mediates increases in a proinflammatory cytokine IL-6 and a chemoattractant IL-8 by eliciting EGFR transactivation, MAPK, and NF-κB activation. Selective drug modulation of either TRPV1 activity or its signaling mediators may yield a novel approach to suppressing inflammatory responses occurring in dry eye syndrome.
Assuntos
Epitélio Corneano/efeitos dos fármacos , Soluções Hipertônicas/farmacologia , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Canais de Cátion TRPV/metabolismo , Western Blotting , Cálcio/metabolismo , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/metabolismo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fura-2/análogos & derivados , Humanos , Microscopia de Fluorescência , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Fosforilação , Transdução de Sinais , Estresse Fisiológico , Sulfonamidas/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Tioureia/análogos & derivados , Tioureia/farmacologia , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE: To determine whether a Chinese herbal medicine component, emodin, suppresses inflammatory/fibrogenic reaction in cultured subconjunctival fibroblasts and reduces injury-induced increases in ocular surface inflammation in mice. METHODS: Effects of emodin were measured in human subconjunctival fibroblasts on proliferation and migration with colorimetry and scratch wound assay, respectively. Neovascularization was evaluated using an endothelial cell-fibroblast coculture model. Proinflammatory mediator and extracellular matrix component gene and protein expression was characterized with real-time reverse transcription-polymerase chain reaction, enzyme immunoassay, and immunocytochemistry, respectively. Western blotting and immunohistochemistry evaluated the activation of nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK). In a mouse corneal alkali-burn model, the effects of emodin on ocular surface inflammation and fibrosis were evaluated. RESULTS: Emodin suppressed tumor necrosis factor alpha (TNF-alpha)-induced fibroblast migration and fibronectin deposition in vitro. VEGF induced neovascularization but did not affect cell proliferation and collagen type 1 production. Monocyte/macrophage-chemoattractant protein-1 gene and protein expression declined. Emodin inhibited TNF-alpha-induced NF-kappaB p65 and JNK activation but did not affect transforming growth factor beta1-induced Smad2/3 signaling. In vivo, emodin inhibited proinflammatory and fibrogenic reactions. CONCLUSIONS: Emodin suppressed in vitro TNF-alpha-induced stimulation of proinflammatory reaction. In a mouse ocular alkali burn model, this herbal component lessened inflammation and scarring. Additional studies are warranted to evaluate the therapeutic potential of emodin in lessening ocular tissue inflammation and resultant fibrosis after injury.
Assuntos
Túnica Conjuntiva/efeitos dos fármacos , Conjuntivite Alérgica/prevenção & controle , Medicamentos de Ervas Chinesas/farmacologia , Emodina/farmacologia , Animais , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Técnicas de Cocultura , Colágeno Tipo I/metabolismo , Túnica Conjuntiva/citologia , Túnica Conjuntiva/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/metabolismoRESUMO
We characterized the dependence of hypotonicity-induced regulatory volume decrease (RVD) responses on mitogen-activated protein kinase (MAPK) pathway signaling in SV40-immortalized rabbit corneal epithelial cells (RCEC). Following calcein-AM loading, RVD was monitored using a microplate fluorescence reader. Western blot analysis determined MAPK activation. After 30 min, the RVD response restored the relative cell volume to nearly isotonic values, whereas it was inhibited when cells were bathed either in a Cl- -free solution or with the Cl- -channel inhibitors: 5-nitro-2-(3-phenylpropylamino)benzoic acid or niflumic acid. Similar declines occurred with either a high-K+ (20 mM) supplemented solution or the K+ channel inhibitor 4-aminopyridine. Activation of extracellular signal-regulated kinase (ERK), p38, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) was time and tonicity-dependent. Stimulation of ERK and SAPK/JNK was maximized earlier than that of p38. Activation of ERK and SAPK/JNK was insensitive to Cl- and K+ channel inhibitors, whereas inhibition with either PD98059 or SP600125, respectively, blocked RVD. However, inhibition of p38 with SB203580had no effect on RVD. Suppression of RVD instead blocked p38 activation. Differences in the dependence of RVD activation on Erk1/2 and p38 signaling were validated in dominant negative (d/n)-Erk1 and d/n-p38 cells. Volume-sensitive Cl- and K+ channel activation contributes, in concert, to RVD in RCEC. Therefore, swelling-induced ERK and SAPK/JNK stimulation precedes Cl- and K+ channel activation, whereas p38 activation occurs as a consequence of RVD.
Assuntos
Tamanho Celular , Epitélio Corneano/citologia , Proteínas do Olho/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Animais , Western Blotting/métodos , Linhagem Celular Transformada , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Cloro/fisiologia , Ativação Enzimática/fisiologia , Epitélio Corneano/enzimologia , Soluções Isotônicas , MAP Quinase Quinase 4/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Pressão Osmótica , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/fisiologia , Coelhos , Solução de Ringer , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE: Inchin-ko-to is a herbal medicine which has therapeutic effects in ameliorating liver fibrosis or cholestatic liver diseases. Its main bioactive component is genipin, which is an intestinal bacterial metabolite of this medication. Accordingly, we determined whether or not Inchin-ko-to suppresses in a wound healing model subconjunctival fibroblast (SCF) migration proliferation and myofibroblast transdifferentiation since an inhibitory effect could be of value in improving trabeculotomy outcome. METHODS: Effects of genipin on SCF cell migration were examined subsequent to wounding confluent monolayer cultures. Alamar blue staining evaluated the effects of genipin (0-50 microg/ml) on fibroblast cell proliferation. Immunostaining determined alpha-smooth muscle actin (alphaSMA) expression. Western blotting evaluated (alphaSMA) expression and phospho-Smad2 formation. Real-time RT-PCR evaluated TGFbeta1 and collagen Ialpha2 mRNA expression. Enzyme-immunoassay determined culture medium collagen I content. RESULTS: Genipin suppressed wound-induced cell migration and proliferation. It also decreased collagen type I TGFbeta1 and alphaSMA mRNA and protein expression. Smad2 signaling was inhibited by genipin in a dose-dependent manner. CONCLUSION: Genipin suppresses injury-induced fibrogenic responses in SCFs. This result suggests that the herbal medicine Inchin-ko-to might have therapeutic value following trabeculotomy.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Túnica Conjuntiva/citologia , Fibroblastos/citologia , Iridoides/farmacologia , Actinas/metabolismo , Western Blotting , Células Cultivadas , Colágeno Tipo I/genética , Medicamentos de Ervas Chinesas/farmacologia , Humanos , Glicosídeos Iridoides , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta1/genética , Cicatrização/efeitos dos fármacosRESUMO
PURPOSE: To determine in a lens epithelial cell line, alpha-TN4, whether genipin, an intestinal metabolite component of the herbal medicine inchin-ko-to, suppresses profibrogenic myofibroblast generation and upregulation of fibrogenic cytokines and to evaluate the potential benefit of the medicine in preventing posterior capsule opacification (PCO). SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: In this study, alpha-TN4 cell proliferation, migration, and expression of alpha-smooth muscle actin (alpha-SMA), the hallmark of myofibroblast generation, were assayed with a colorimetric assay, scratch wound assay, immunohistochemistry, and Western blot analysis. Gene expression of transforming growth factor-beta1 (TGF-beta1) and connective tissue growth factor (CTGF) was characterized with real-time reverse transcription-polymerase chain reaction. In addition, p38 mitogen-activated protein kinase (p 38 MAPK), extracellular signal-regulated kinase (ERK) limb, and Smad signalings were evaluated by Western blotting and immunohistochemistry. Cytotoxicity of genipin was evaluated using a commercial colorimetric assay kit for nuclear matrix protein 41/7 (NMP41/7) in culture medium. RESULTS: Genipin suppressed cell proliferation and migration in association with inhibition of Smad and p38 MAPK phosphorylation, although ERK signaling was enhanced. Genipin suppressed mRNA expression of TGF-beta1 and CTGF. Cytoplasmic fiber formation declined based on less intense alpha-SMA immunocytochemical staining. However, alpha-SMA protein expression was actually not altered. This negative result suggests that genipin attenuated formation of alpha-SMA-containing cytoskeleton. Treatment of the cells with genipin for 48 hours did not increase the release of NMP41/7 to the medium, indicating this compound is not cytotoxic. CONCLUSION: Because genipin suppressed alpha-TN4 lens cell fibrogenic behaviors, it may be of therapeutic value in preventing PCO.
Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Células Epiteliais/efeitos dos fármacos , Cristalino/citologia , Piranos/farmacologia , Actinas/metabolismo , Antígenos Nucleares/metabolismo , Western Blotting , Proteínas de Ciclo Celular , Linhagem Celular , Fator de Crescimento do Tecido Conjuntivo , Medicamentos de Ervas Chinesas/toxicidade , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Glicosídeos Iridoides , Iridoides , Proteínas Associadas à Matriz Nuclear/metabolismo , Piranos/toxicidade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1 , Regulação para Cima , Cicatrização/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE: To characterize in SV40-immortalized human corneal epithelial cells (tHCEC) osmosensitive taurine transporter gene and protein expression as well as its functional activity. To evaluate whether medium supplementation with taurine improves cell viability during a hypertonic challenge. METHODS: tHCEC were preincubated for up to 48 hours in hypertonic DMEM medium (i.e., up to 500 mosmol/kg). Taurine uptake was monitored through measurements of intracellular [3H]taurine accumulation. Gene and protein expression was detected by Northern and Western blot analyses, respectively. An amino acid analyzer measured intracellular cold taurine content. The live/dead assay evaluated with confocal microscopy determined cell viability. RESULTS: Na+-dependent taurine uptake occurred in an isotonic (310 mosmol/kg) medium. The apparent Michaelis-Menten constant, K(t), for taurine was 4.6 micro M, and uptake increased as a function of exposure time and rises in osmolality. Exposure for 12 hours to a 450 mosmol/kg medium increased uptake by 4.1-fold. However, after 48 hours of exposure to this medium, taurine uptake returned to its isotonic level. With time, biphasic changes occurred in taurine transporter gene and protein expression and taurine transport activity as well as elevating intracellular taurine content by 4.5-fold. Taurine medium supplementation for 48 hours improved cell viability. CONCLUSIONS: tHCEC express Na+-dependent osmosensitive taurine transport activity. The hypertonic-induced biphasic effects on gene and protein expression as well as transport activity suggest feedback regulation of these responses. Rises in intracellular taurine do not appear to be essential for osmoregulation, but instead enhance cell survival perhaps through a membrane stabilizer or an antioxidant effect.