ABSTRACT
Bucillamine is used for the treatment of rheumatoid arthritis. This study investigated the protective effects of bucillamine against cisplatin-induced damage in auditory cells, the organ of Corti from postnatal rats (P2) and adult Balb/C mice. Cisplatin increases the catalytic activity of caspase-3 and caspase-8 proteases and the production of free radicals, which were significantly suppressed by pretreatment with bucillamine. Bucillamine induces the intranuclear translocation of Nrf2 and thereby increases the expression of gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GSS), which further induces intracellular antioxidant glutathione (GSH), heme oxygenase 1 (HO-1) and superoxide dismutase 2 (SOD2). However, knockdown studies of HO-1 and SOD2 suggest that the protective effect of bucillamine against cisplatin is independent of the enzymatic activity of HO-1 and SOD. Furthermore, pretreatment with bucillamine protects sensory hair cells on organ of Corti explants from cisplatin-induced cytotoxicity concomitantly with inhibition of caspase-3 activation. The auditory-brainstem-evoked response of cisplatin-injected mice shows marked increases in hearing threshold shifts, which was markedly suppressed by pretreatment with bucillamine in vivo. Taken together, bucillamine protects sensory hair cells from cisplatin through a scavenging effect on itself, as well as the induction of intracellular GSH.
Subject(s)
Animals , Male , Mice , Rats , Antioxidants/metabolism , Apoptosis/drug effects , Caspase 3/metabolism , Caspase 8/metabolism , Cell Line , Cisplatin/toxicity , Cysteine/analogs & derivatives , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , Glutathione/metabolism , Heme Oxygenase-1/genetics , Intracellular Space/metabolism , Metabolic Detoxication, Phase II/genetics , NF-E2-Related Factor 2/genetics , Nitric Oxide/biosynthesis , Organ of Corti/drug effects , RNA Interference , Reactive Oxygen Species/metabolism , Superoxide Dismutase/geneticsABSTRACT
No abstract available.
ABSTRACT
BACKGROUND AND OBJECTIVES: The stable cell line system of middle ear epithelial cells is essential for studying molecular pathogenesis of otitis media. Recently, we succeeded in establishing the human middle ear epithelial cell line (HMEEC) using a retrovirus. The cell line retains many of the phenotypic and morphological properties of the non-transformed, parental cultures such as the expression of cytokeratin and tight junctions. We aimed to show the conservation of mucosal characteristics and subcellular mechanisms of transcriptional regulation in this cell line. MATERIALS AND METHOD: RT-PCR was performed using mucin gene specific primers and total RNA extracted from HMEEC. The luciferase-expressing vector containing 5' flanking region of human beta defensin 2 (hBD-2), an inducible antimicrobial peptide, was transfected to HMEEC. After starvation of serum, HMEEC was treated with interleukin 1 alpha (IL-1alpha) and subsequently harvested 10 hrs later. Luciferase activity was measured using luminometer after the corresponding substrate was supplemented to the cell lysate. RESULTS: Expression of mucin genes (MUC1, 2 and 5B) in HMEEC was demonstrated by RT-PCR. Luciferase assay showed that IL-1alpha up-regulates the promoter activity of hBD-2 more than 10 fold. This transcriptional regulatory mechanism was also demonstrated in the well established reference cell lines, HeLa cells and A549 cells. CONCLUSION: We demonstrated the conservation of mucin gene expression and transcriptional regulatory mechanism of hBD-2 in HMEEC. The proposed cell line can serve as a useful experimental model for elucidating the pathogenesis of middle ear mucosa-related diseases.
Subject(s)
Humans , 5' Flanking Region , Cell Line , Defensins , Ear, Middle , Epithelial Cells , Gene Expression , HeLa Cells , Interleukin-1alpha , Keratins , Luciferases , Models, Theoretical , Mucins , Otitis Media , Parents , Retroviridae , RNA , Starvation , Tight JunctionsABSTRACT
BACKGROUND AND OBJECTIVES: It is believed that the innate immunity plays a critical role in protecting the tubotympanum from being infected because the middle ear cavity is normally sterile despite of a paucity of immune cells. Among known antibacterial molecules, defensins have been shown to contribute significantly to innate immunity. However, it is still unclear whether or not beta defensins are expressed in human middle ear mucosa. MATERIALS AND METHOD: Immunolabeling and RT-PCR were performed with the mucosal specimen from normal subjects and otitis media patients, respectively. Expression of beta defensin 2 mRNA was compared between the control group and experimental group that was treated by inflammatory stimuli in the animal models using RT-PCR. RESULTS: beta defensin 1 was expressed in both normal and inflamed middle ear mucosa of human, but beta defensin 2 and 3 were found only in the inflamed mucosa. The expression of beta defensin 2 mRNA was up-regulated when the interleukin-1alpha (IL-1alpha) or lipopolysaccharide (LPS) was treated in the middle ear mucosa of the experimental animals. CONCLUSION: We could show that beta defensins are expressed in the human middle ear mucosa and that beta defensin 2 is up-regulated by the inflammatory stimuli, IL-1alpha or LPS.
Subject(s)
Animals , Humans , beta-Defensins , Defensins , Ear, Middle , Immunity, Innate , Interleukin-1alpha , Models, Animal , Mucous Membrane , Otitis Media , RNA, MessengerABSTRACT
No abstract available.
ABSTRACT
The purpose of this study was to describe the luminal development of the murine eustachian tube and middle ear. Thirty specimens, aging from gestational day 11 to postnatal day 21, were investigated through the light microscopic observations. The present study also used digitizer, computer, and serially sectioned temporal bone specimens for three-dimensional reconstruction to measure the volume of the eustachian tube and middle ear cavity at different gestational and postnatal ages. The first pharyngeal pouch elongated during gestational day 12 to form the tubotympanic recess. Between gestational day 13 and 14 this tubotympanic recess extended to the middle ear area. A rapid increment in the volume of the tubotympanic recess was noted between gestational day 15 and 16. At this age, a definite division of the tubotympanic recess into the eustachian tube and middle ear cavity was observed. During the postnatal period, the maximum change of the middle ear volume was noted on postnatal day 11 when the mesenchymal tissue in the middle ear cavity disappeared completely.
Subject(s)
Female , Male , Mice , Pregnancy , Animals , Ear, Middle/anatomy & histology , Eustachian Tube/anatomy & histology , Mice, Inbred BALB CABSTRACT
In order to describe the developmental anatomy of the murine eustachian tube and its related structures, seventy six mice of ages ranging from gestational day 11 to postnatal day 21 were investigated through the light and electron microscopic observations. Development of the ciliated cells was seen concurrently in both the eustachian tube and middle ear on the 16 th gestation day, one day earlier than the epithelial secretory cells appeared in both the eustachian tube and middle ear. The number of ciliated cells and secretory cells increased rapidly after birth. Tubal glands were well identified with evidence of secretory activity around the time of birth. Thus, the findings of this study indicate that the mucociliary defense system starts to develop during the fetal stage and is well established immediately after birth.