| RESEARCH BACKGROUNDKeloid represents an abnormal healing response to injuries, characterized by excess accumulation of disproportionate extracelluar matrix (ECM) and fibroblast proliferation. But the pathogenesis of keloid formation has been unknown. There is no effective method to treat it on clinic. Keloid is the attractive research problem in the department of plastic surgery.Recent studies indicate that the Rel homology domain/ nuclear factor-kappaB (NF-κB) signal transduction pathway play a crucial role in skin biology. It exists in the whole biological process of wound healing; transduces signal from cell surface to intracellular; participate in cell growth, development, apoptosis and other biological functions. More and more studies demonstrate that NF-κB activation has been involved in the pathogenesis of many types of human cancers and fibrosis. So targeting the NF-κB signaling pathway has been the focus to treat or release disease.NF-κB has been an attractive target for the therapeutic strategies against diseases including cancer. Acetylsalicylic acid (ASA) is one important drug of nonsteroidal anti-inflammatory drugs (NSAIDs). It inhibits prostaglandin E2 synthesis to treat chronic inflammatory diseases by blocking cyclooxygenase (COX). Recent reports suggest that aspirin show anti-tumor activity and prevention of carcinogenesis through the inhibition of NF-κB activation in a dose- and time-dependent manner.Recent studies demonstrated that there are similarity between keloid and cancer such as clinical feature, gene mutation, cytokines and its acceptors, and treatment methods. There is a significant difference in apoptotic profiles between normal skin and keloid fibroblasts, whereas keloid fibroblasts are more resistant to cell death than their normal skin counterparts.At present, there have been no related reports about the effect of NF-κB signaling pathway in keloid pathogensis and aspirin inhibits NF-κB activation on keloid formation.AIMThe aim of the paper is to study the effect of NF-κB signaling pathway in the pathogenesis of keloid and to search effective inhibitory agent which will be adopted to prevent and treat keloid.Keloid tissue and normal skin tissue will be collected from clinic. To observe the expression level of the related gene to the NF-κB signaling pathway such as NF-κB p65, IκB-αand cyclinD1 which is the target gene of activated NF-κB p65 in these tissue and to compare the difference expression of NF-κB signaling pathway between the keloid and normal shin.We choose fibroblast which plays a crucial role in the pathogenesis of keloid as the object of our research, and primary cultured KFs. Tumor necrosis factor-α(TNF-α) as the stimulation agent of NF-κB signaling pathway will be used to stimulate KFs and NFs. To observe the effect on the proliferation of KFs, to observe the difference of dynamic diversity of p65, IκB-αand cyclinD1 on the NF-κB signaling pathway; to compare the variability change between the KFs and NFs.We choose aspirin as the inhibitory agent of NF-κB signaling pathway. We pretreat the KFs with a serious of concentration of aspirin and then with TNF-αstimulation. To examine the effects of aspirin on related gene such as p65, IκB-αand cyclinD1 on the NF-κB signaling pathway, cellular growth rates, cell cycle analysis and apoptosis in keloid fibroblasts (KFs).METHODSKeloid and normal skin samples were collected from patients in the Department of Plastic Surgery of the Second Affiliation Hospital of Shandong University, 15 cases respectively, nuclear protein and total mRNA were prepared, the protein expression of NF-κB p65 in tissues was detected by immunohistochemistry, the transcriptional level of IκB-αmRNA and NF-κB p65 DNA-binding activity were examined with real time quantitative RT-PCR and TransAM? NF-κB Kit respectively. Nuclear proteins were prepared and cyclinD1 protein level was examined with Western blot technique.Primary KFs and NFs were cultured and the low passages (4-8th) were employed in the study. The KFs were treated with various concentrations TNF-α(10ng/mL; 50ng/mL; 100ng/mL) for 24h, 48h, 72h, 96h, or 120h. The proliferative effects were measured by the MTT assay; the cells were stimulated with 50ng/mL TNF-αfor 15 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours, to observe the location of NF-κB P65 and IκB-αin KFs and NFs at quiescent condition and the nuclear translocation of NF-κB P65 after TNF-αstimulation by immunofluorescence technique; the treated KFs were collected and prepared for protein, to detect the expression of NF-κB p65 DNA-binding activity with TransAM?NF-κB p65 Kit and to investigate the IκB-αprotein level by means of Western blot technique.Primary KFs were cultured; KFs were divided into six groups: control group (group A); treated with 50ng/ml TNF-αgroup (group B); pretreated with 1.0mM (group C), 2.5 mM (group D), 5.0mM (group E), 10.0mM aspirin (group F) for 2 hours and then with TNF-αstimulation for 15minutes, Nuclear and cytoplasmic proteins were extracted from each treated cells group. To observe the subcellular location of NF-κB P65 and IκB-αby immunofluorescence technique; to detect the expression of NF-κB p65 DNA binding activity with TransAM? NF-κB p65 Kit; to investigate the change of IκB-αand p-IκBαprotein level by means of Western Blot technique. Cells were plated at a density of 3×103 cells/well of 96-well microtiter plates. Twenty-four hours after seeding, the cells were treated with various concentrations aspirin (1.0mM, 2.5mM, 5.0mM, 10.0mM) and 50ng/ml TNF-α-containing culture medium for 24h, 48h, 72h, 96h, or 120h. The anti-proliferative effects were measured by the MTT assay. KFs were pre-treated with 1.0mM; 5.0mM; 10.0mM aspirin for 2h and then with TNF-αfor 24h to observe the apoptosis rate and the change of cell cycle with flow cytometer (FCM). RESULTSThe clinical samples show that the protein expression level of NF-κB p65 and its DNA Binding Activity in Keloids tissues at quiescent condition were higher than those in normal skin tissues. The mRNA transcription level and protein expression level of IκB-αas the upstream target gene in the NF-κB p65 signaling pathway in Keloid tissues were lower than their counterparts from normal skin. The protein expression level of cyclinD1 as the downstream target in the NF-κB p65 signaling pathway in keloid tissue was higher than in normal skin.Low concentration of TNF-αcan promote the KFs proliferation and the max promote concentration is 50ng/ml TNF-α, but 100ng/ml TNF-αinhibits the KFs proliferation; after TNF-αstimulation, TNF-αinduced IκB-αphosphorylation inceases at 15 minutes and becomes undetectable in cytoplasm after 4 hours; NF-κB p65 translocation into the nucleus, NF-κB p65 DNA binding activity arrived its maximum at 1 hour and was close to normal at 4 hours; TNF-αinduces most degradation of IκB-αat 15 minutes and becomes detectable in cytoplasm after 4 hours. At each time point, the change degree of IκB-αand NF-κB p65 DNA binding activity in KFs are higher than in NFs. KFs showed more sensitive ability to TNF-αstimulations than NFs.TNF-αinduces most phosphorylation and degradation of IκB-αand NF-κB p65 translocation into the nucleus and NF-κB p65 DNA binding activity increase. Aspirin prevents TNF-α-induced phosphorylation and degradation of IκB-αprotein and NF-κB p65 translocation into the nucleus and decreases NF-κB p65 DNA binding activity. In addition, aspirin preloading sensitizes keloid fibroblasts to TNF-α-induced apoptosis and inhibits KFs proliferation in a dose- and time-dependent manner. Aspirin inhibits KFs proliferation by retaining the cells at the GO phase in a dose-dependent manner.CONCLUSION1. The expression level of the related genes to NF-κB signal pathways in keloid such as NF-κB p65, NF-κB DNA binding activity, and cyclinD1 are much higher than in normal skin. But the up-stream inhibitory factor IκB-αexpress lower level compared with normal skin. The abnormal expression suggests that NF-κB signal pathway may play a role in keloid pathogenesis.2. The raction to the TNF-αstimulation of KFs is different. Low concentration TNF-αcan promote the KFs proliferation and high concentration TNF-αinhibits the KFs proliferation.3. KFs show a more intensive reaction to TNF-αstimulation, which displays a higher change degree of the related genes to the NF-κB signal pathway than the change in NFs. The difference may be the reason of KFs which posses the abnormal proliferation and anti-apoptosis ability.4. Aspirin can effectively inhibit the phosphorylation and degration of IκB-α, the nuclear translocation of NF-κB, and decrease NF-κB DNA binding activity induced by TNF-α.5. Aspirin sensitizes KFs to TNF-α-induced apoptosis, inhibits KFs proliferation, and arrests the cell cycle in GO phase in a dose- and time-dependent manner.
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