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Öğe An evaluation of ketoprofen as an intranasal anti-inflammatory agent(Verduci Publisher, 2022) Altıntaş, M.; Muluk, Nuray Bayar; Sezer, C. Vejselova; Kutlu, H. M.; Cingi, C.OBJECTIVE: The objective of the study was to evaluate the effect of ketoprofen when locally applied to tissue-cultured nasal epithelium. MATERIALS AND METHODS: Healthy primary nasal epithelial cells were grown in a tissue culture medium. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay was used to evaluate cytotoxicity. Markers of cellular injury revealed by the MTT assay include fragmentation of DNA, condensed nuclei, and changes affecting the cellular outer membrane and cytoskeleton. Epithelial cells at body temperature in cell culture were exposed over a 24- hour period to ketoprofen. Following the MTT assay, the confocal microscopic examination was performed. The extent to which epithelial cells remained capable of proliferating was evaluated by inducing a scratch injury, waiting for the repair to occur, and then examining the result with the ordinary light microscope. RESULTS: Topically applied ketoprofen does not affect the viability of tissue-cultured nasal epithelial cells within a 24-hour period. Furthermore, there were no cellular morphological alterations observed which would indicate toxicity from ketoprofen. In the scratch assay, the cells regained a normal confluent appearance within 24 hours. Thus, ketoprofen neither increases nor alters the rate at which nasal epithelial cells proliferate. CONCLUSIONS: Ketoprofen, when applied topically for 24 hours to nasal epithelial cells in cell culture, does not cause any alterations in cellular appearance which would suggest impairment of the ability to proliferate or indicate a cytotoxic effect. Extrapolating from these results, it appears acceptable to use ketoprofen topically within the nose in cases of rhinosinusitis (acute or chronic) or nasal pain since there is minimal risk of local toxic injury.Öğe Bromelain: a candidate to enhance wound healing after endonasal surgeries(Verduci Publisher, 2023) Esen, E.; Muluk, Nuray Bayar; Sezer, C. Vejselova; Kutlu, H. M.; Cingi, C.OBJECTIVE: In the present study, we investigated the topical bromelain's cytotoxic effects on mouse fibroblast NIH/3T3 cells via cell culture study. MATERIALS AND METHODS: In this cell culture study, Dulbecco's Modified Eagle Medium ( DMEM) with fetal bovine serum (FBS, 10%) and penicillin/streptomycin (1%) was used as a cell growth medium for NIH/3T3 mouse fibroblast cells. MTT test was performed in 96-well plates seeded with NIH/3T3 cells 5x103/ well and under standard cell culture conditions. Bromelain doses of 3.13 to 100 mu M were administered to the wells and incubated for 24, 48, and 72 hours in the same cell culture conditions. For Confocal microscopic evaluation, NIH/3T3 cells were plated on cover slips in 6-well plates (105 cells/ well) and treated with 100 mu M concentration of bromelain for 24 h. Untreated cells were used as controls. RESULTS: M TT r esults s howed t hat b romelain is not cytotoxic on mouse fibroblast NIH/3T3 cells. All three incubation times of 24, 48, and 72 hours bromelain initiated cell growth. A statistically significant rise in cell growth was detected in the only applied highest dose of 100 mu M bromelain for all incubation times except for 24 hours. The nontoxic effect was further investigated by using confocal microscopy by applying the highest bromelain dose of 100 mu M to NIH/3T3 mouse fibroblast cells. Confocal micrographs showed that bromelain did not change the morphology of mouse fibroblast cells at the incubation time of 24h. In untreated cells and bromelain-treated cells, the nucleus of NIH/ 3T3 cells was undamaged and compact, and the cytoskeleton was fusiform and non-fragmented. CONCLUSIONS: Bromelain is not cytotoxic on mouse fibroblast NIH/ 3T3 cells and enhances cell growth. If clinical trials will confirm this, it is possible that bromelain will be used topically in humans to enhance wound healing, in rhinosinusitis and chronic rhinosinusitis with nasal polyps and endonasal surgeries due to its anti-inflammatory effects.Öğe Effects of ceramide C2 application on human laryngeal carcinoma cells: a cell culture study(Verduci Publisher, 2023) Oguz, O.; Manole, F.; Muluk, N. Bayar; Sezer, C. Vejselova; Kutlu, H. M.; Cingi, C.OBJECTIVE: In the present study, we investigated the effects of Ceramide C2 application on human laryngeal carcinoma cells. MATERIALS AND METHODS: Human larynx epidermoid carcinoma HEp-2 (ATCC (R) CCL-23 (TM)) cells were purchased from the American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. Ceramide C2 was prepared, and further dilutions ranging from 3.13 to 100 mu M were prepared in a fresh culture medium. Cells on 96 well plates were exposed to the prepared concentrations of ceramide C2 for 24 and 48 hours. Cytotoxicity evaluation was performed by MTT. Apoptosis profiles of HEp-2 cells were detected by annexin-V analysis. The activated caspases 3/7 on HEp-2 cells after ceramide C2 exposure were evaluated with flow cytometric analysis. The morphological changes on HEp2 cells caused by ceramide C2 were evaluated by staining with phalloidine and acridine orange via confocal microscopy. For the Wound Healing Assay, HEp-2 cells were cultured in 6 well-plates until they became confluent. RESULTS: MTT cytotoxicity test findings revealed that the viability of human laryngeal carcinoma cells decreased with the increased application of ceramide C2 for 24 hours compared to untreated (control) cells. The highest growth inhibition by ceramide C2 for short-term application for 24 hours was detected at the highest concentration of ceramide C2 (100 mu M). Annexin-V findings showed that 98.97 of HEp-2 cells were alive, and 1.63% were detected as early apoptosis for the control group. The results showed that ceramide C2 triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells of 61,40 compared to untreated HEp-2 cells. Cysteine proteases (caspases) 3/7 activation percentages of HEp-2 cells exposed to ceramide C2 for 24 hours were compared to control cells, and the morphology of HEp-2 cells was changed with clear apoptotic signs that underlined the cytotoxicity and pro-apoptotic activity of ceramide C2. Scratch Assay assessed the migration capability of HEp-2 cells before and after the exposure to ceramide C2. It showed that ceramide C2 reduced human laryngeal carcinoma cells' migration capability and proliferation for 24 hours. CONCLUSIONS: Based on all study findings, it can be considered that short-chain ceramide C2 exerted cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner and reduced the viability via inducing caspase-dependent apoptosis. The overall effect might be derived from the elevated intracellular ceramide levels by the exogenous application of ceramide C2. Consequently, it was concluded that ceramide C2 has good potential to cause cytotoxicity and apoptosis in human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anti-cancer drugs with high efficiency.Öğe Efficacy and toxicity of Anatolian propolis on healthy nasal epithelial cells(Verduci Publisher, 2022) Samanci, A. E. Tanugur; Muluk, N. Bayar; Sezer, C. Vejselova; Kutlu, H. M.; Topsakal, V.; Cingi, C.OBJECTIVE: In our study we aimed to evaluate the effects of applying propolis topically to epithelial cells of the nasal cells, to discover whether this causes any toxic effect upon the cells. the cells. MATERIALS AND METHODS: Samples of healthy human primary nasal epithelium harvested during septoplasty from volunteers were incubated in cell culture. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays may be utilized when assessing cellular damage (toxicity), as evidenced by DNA fragmentation, nuclear condensation, alteration in the outer plasma membrane and cytoskeletal alteration. This was the method used in the study. Cultured epithelial cells were incubated with propolis (Bee&You) for 24 hours at 37 degrees C. The MTT assay was then performed, and the cell morphology was examined by confocal microscopy. In addition, via wound healing assay, cellular proliferation was assessed by the artificial scratch method followed by light microscopy. RESULTS: MTT assay results showed that the primary nasal cells were not affected from the topical application of propolis for 24 hours. All of the applied doses not changed significantly the viability of the cells. The agent was not found to cytotoxic to the primary nasal cells in the application time of 24 hours. Our confocal microscopy findings supported the MTT findings. According to the confocal images control cells that were not treated with test agent were with compact morphology and undamaged fusiform cell shape and nucleus. In test group of nasal cells, Propolis found not to be cytotoxic on the cellular morphology and not changed the cells. When evaluating the results from the wound healing assay, the clear area of scratch obtained at the start of incubation (0th) was closed totally with the proliferated primary nasal cells after incubation of 24 hours with propolis. These findings are supported by our MTT findings that imply to the slight induce of proliferation of the primary cells by Propolis. CONCLUSIONS: Topically applied propolis did not have a cytotoxic effect on nasal epithelium cells. Considering its antibacterial and antioxidant effects, it has been concluded that topical application in sinonasal inflammatory diseases (e.g., acute and chronic rhinosinusitis) may have an auxiliary effect in treatment. Moreover, there is a slight induce of proliferation of the primary cells by propolis which may help wound healing in septal surgeries and epistaxis.Öğe Efficacy and toxicity of anise oil as a potential topical wound healer: a cell culture study(Verduci Publisher, 2023) Sungur, I; Muluk, N. Bayar; Sezer, C. Vejselova; Kutlu, H. M.; Cingi, C.OBJECTIVE: We studied the cytotoxic effects of topical anise oil on NIH/3T3 fibroblast cells using a cell culture assay. MATERIALS AND METHODS: NIH/3T3 fibroblast cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10%) and penicillin/streptomycin under standard cell culture conditions in a humidified incubator containing 5% carbon dioxide. For the MTT cytotoxicity experiment, NIH/3T3 cells were plated in triplicate at a concentration of 3x10(3) per well in 96-well plates and incubated for 24 hours. The cells were treated with anise oil concentrations ranging from 3.13 to 100 mu M, and the plates were cultured for 24, 48, and 72 hours under standard cell culture conditions. For assessment by confocal microscopy, NIH/3T3 cells were seeded on sterilized coverslips in 6-well plates at a concentration of 105 cells per well in triplicate. For 24 hours, cells were treated with 100 mu M of anise oil. Three wells that were not treated with anise oil served as the control group. RESULTS: The MTT findings demonstrated that anise oil is not cytotoxic to NIH/3T3 fibroblast cells. Anise oil stimulated cell growth and triggered cell division at all three incubation intervals of 24, 48, and 72 hours. The maximum growth was obtained in the applied highest concentration of 100 mu M anise oil. At doses of 25, 50, and 100 mu M, there was also a statistically significant improvement in cell viability. At 72 hours of incubation, dosages of 6.25 and 12.5 micro of anise oil were shown to be viability-inducing for NIH/3T3 cells. In the confocal microscopy pictures, it was found that anise oil was not cytotoxic on NIH/ 3T3 cells at the applied maximal dose. The experimental group of NIH/3T3 cells exhibited the same cell morphology as the untreated control group. In both sets of NIH/3T3 cells, the nucleus was round and undamaged, and the cytoskeleton was determined to be compact. CONCLUSIONS: Anise oil is not cytotoxic on NIH/ 3T3 fibroblast cells and initiates cell growth. Anise oil could be used topically to enhance wound healing after surgical procedures if clinical trials will confirm experimental data.Öğe Evaluation of ciprofloxacin used as an intranasal antibiotic(Verduci Publisher, 2022) Azizli, E.; Muluk, N. Bayar; Sezer, C. Vejselova; Kutlu, H. M.; Cingi, C.OBJECTIVE: The objective of this research was to examine the effects of topical ciprofloxacin on cultured nasal epithelial cells of human origin. MATERIALS AND METHODS: Human nasal epithelial cells were collected from patients who voluntarily donated tissue left over following septorhinoplasty. The samples were from individuals without any indication of rhinosinusitis. An assay that may be employed to investigate toxic effects at the cellular level is MTT ( 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide). This test reveals where DNA becomes fragmented, the nuclei condense, the outer cell membrane is altered, or the cytoskeleton appears disrupted. The present study employed this technique. Nasal epithelium in cell culture was exposed to ciprofloxacin for 24 hours at a temperature of 37 degrees C, following which the MTT assay was undertaken before examining the cells by confocal microscopy to look for alterations indicating cytotoxicity. Another test of toxicity, the artificial scratch technique, was also used. Cells treated in this way were assessed using a light microscope. RESULTS: The nasal epithelial cells in culture that were exposed to topical ciprofloxacin for 24 hours were less viable than controls and this result was statistically significant. For this length of exposure, the IC50 was calculated as 1.565 mg/mL. The peak reductions in cellular viability occurred with exposure at a concentration of 1.25 mg/mL and 0.625 mg/mL. These was only a mild decrease in viability at other concentrations, but these results were of statistical significance. The MTT assay and confocal microscopy confirmed this result. Cultured nasal epithelium not exposed to ciprofloxacin (i.e., controls) exhibited a compact morphological appearance when examined with confocal microscopy. The epithelial cells had a regular fusiform boundary, and the nuclei were intact. By contrast, the cultures with exposure to the antibiotic were of decreased size and their outline changed from fusiform to round. The epithelial cells cultures where scratch injury was induced were examined by light microscopy, the extent of closure of the denuded area being assessed after 24 hours. It was noted that the area opened up by the experimental scratch was not closed completely by 24 hours later. This result shows that ciprofloxacin decreases the viability of nasal epithelial cells. CONCLUSIONS: Topical application of ciprofloxacin to the nasal lining is not recommended, since this resulted in decreased cellular viability, cellular shrinking and alteration in outline from fusiform to round in cultured nasal epithelial cells. These changes indicate that topically applied ciprofloxacin is toxic to nasal epithelial cells. The outcomes of this study should be studied and correlated in vivo models.Öğe Evaluation of spiramycin for topical applications: a cell culture study(Verduci Publisher, 2023) Aghayarov, O. Yagiz; Muluk, N. Bayar; Sezer, C. Vejselova; Kutlu, H. M.; Cingi, C.OBJECTIVE: Through a cell culture test, we analyzed the cytotoxic effects of topical spiramycin on NIH/3T3 fibroblast cells. MATERIALS AND METHODS: Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin was used for the growth of NIH/3T3 fibroblast cells in a 5% CO 2 incubator. Spiramycin's cytotoxicity was measured using the MTT assay. 5,000 NIH/ 3T3 cells per well of a 96-well plate were seeded in each well, and the cells were treated with spiramycin (3.13-100 mu M) for 24, 48 and 72 hours while the plates were incubated at 37 degrees C in a humidified 5% CO 2 atmosphere. First, 105 NIH/3T3 cells were seeded onto coverslips in 6-well plates for morphological analysis of both untreated and spiramycin-treated cells. For 24 hours, NIH/3T3 cells were exposed to a 100 mu M dosage of spiramycin. The cells in the control group were grown in complete growth media alone. RESULTS: Spiramycin was non- toxic to NIH/3T3 fibroblast cells in a MTT test. The concentration of spiramycin used to stimulate cell growth increased as the concentration was increased. After 24 and 48 hours of treatment with 100 mu M NIH/3T3, the cells showed the most significant increase in size. Cell viability was shown to be significantly reduced at spiramycin doses of 50 and 100 mu M. All MTT findings revealed that spiramycin enhanced cell viability and was not harmful to the fibroblast cells for short-term application of 24 and 48 hours but lowered the viability of fibroblast cells at the doses of 50 and 100 mu M for long-term application duration of 72 hours. Confocal micrographs showed that spiramycin treatment did not affect the cytoskeleton or nucleus of fibroblast cells, in contrast to the control NIH/ 3T3 cells. Both untreated and treated with spiramycin, fibroblast cells were found to be fusiform and compact, with their nuclei remaining unaltered and unreduced in size. CONCLUSIONS: It was concluded that spiramycin has a beneficial effect on fibroblast cells and is safe for use over short periods. Spiramycin reduced fibroblast cell viability when applied for 72 hours. Confocal micrographs showed that fibroblast cell skeletons and nuclei were unharmed and undamaged, that cell shapes were fusiform and compact, and that nuclei were neither broken nor shrunken. Topical spiramycin could be recommended for septorhinoplasty procedures due to anti-inflammatory effects for short-term usage if clinical trials will confirm experimental data.Öğe Investigation of proapoptotic and cytotoxic effects of 2-aminobenzothiazole on human laryngeal carcinoma cells(Verduci Publisher, 2024) Haznedar, B.; Muluk, N. bayar; Sezer, Vejselova; Kutlu, H. M.; Cingi, C.OBJECTIVE: In the present study, we investigated the effects of 2-aminobenzothiazole application on human laryngeal carcinoma cells. MATERIALS AND METHODS: Human larynx epidermoid carcinoma (HEp-2) (ATCC (R) CCL-23 (TM)) cells were purchased from American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. 2-aminobenzothiazole was prepared, and further dilutions ranging from 3.13 to 100 mu M were prepared in fresh culture DMEM. HEp-2 cells on 96 well plates were incubated with the prepared dilutions of 2-aminobenzothiazole for 24, 48, and 72 hours. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test performed cytotoxicity evaluation and viability percentages. The annexin-V staining technique detected 2-aminobenzothiazole-triggered apoptosis of HEp-2 cells. The activated caspases 3/7 on HEp-2 cells after 2-aminobenzothiazole exposure were evaluated with flow cytometric analysis. The membrane potential changing of HEp-2 cells was measured following the Muse (TM) MitoPotential kit manufacturer instructions. RESULTS: MTT cytotoxicity test results showed that the viability of human laryngeal carcinoma cells decreased with an increase in the application of 2-aminobenzothiazole for 24 hours. The highest growth inhibition by 2-aminobenzothiazole for short-term application of 24 hours was detected at the highest concentration of 2-aminobenzothiazole (100 mu M). The results underline that the cytotoxic effect of 2-aminobenzothiazole is dose-dependent. Cytotoxicity test results for an application time of 48 hours showed that the cytotoxicity of 2-aminobenzothiazole is dose-dependent on HEp-2 cells. The required dose of 2-aminobenzothiazole to decrease the cell viability to 50 percent has been 9-fold augmented. Annexin-V findings showed that after exposure to IC50 concentration of 2-aminobenzothiazole for 24 hours, HEp-2 cells underwent the early apoptotic stage (25.99%) and late apoptotic (16.69%), whereas 56.93% of the treated cells were alive. Only 0.39% of 2-aminobenzothiazole treated cells were necrotic. All study results showed that 2-aminobenzothiazole triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells 42.62 compared to untreated HEp-2 cells. Caspase 3/7 activation results showed that only 0.65% of control HEp-2 cells were with activated caspase 3/7, and 99.35% live cells. The analysis data from the Muse cell analyzer revealed that the percentage of cells with intact mitochondrial membranes was 21.30 after 2-aminobenzothiazole application, and 79.9% were cells with depolarized mitochondrial membranes. It has been understood that the depolarization of the inner mitochondrial membrane has been considered a dysfunction in mitochondria as a sign of apoptosis and drug toxicity. CONCLUSIONS: Based on all study findings, 2-aminobenzothiazole has cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner. That means that it decreased viability via inducing caspase-dependent apoptosis. Consequently, it was concluded that 2-aminobenzothiazole has good potential to lead to cytotoxicity and apoptosis on human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anticancer drugs with high efficiency.Öğe Potential protective effect of resveratrol on acoustic trauma: electron microscopy study(Verduci Publisher, 2016) Hanci, D.; Ulusoy, S.; Acar, M.; Burukoglu, D.; Kutlu, H. M.; Muluk, N. Bayar; Cingi, C.OBJECTIVE: To investigate the potential preventive effect of resveratrol in rats exposed to acoustic trauma (AT). MATERIALS AND METHODS: In this experimental study, Wistar albino rats were divided into three groups: Group 1 (Control, n = 6), Group 2 (AT, n = 6), and Group 3 (resveratrol + AT). The rats in Group 2 were exposed to AT. The rats in Group 3 received resveratrol (300 mg/kg/day) via gavage for 7 days. On day 7, the rats were exposed to AT 10 min following resveratrol treatment. Histological sections of the cochleae were examined using light microscopy, transmission (TEM), and scanning electron microscopy (SEM). RESULTS: The cochlear hair cells, stereocilia, and Deiters' cells of the control group appeared normal in all microscopic evaluations. In Group 2, light microscopy revealed predominantly inner hair cell loss, although the outer hair cells were affected. TEM and SEM examination showed severe loss of stereocilia and SEM revealed stereocilia arranged in an asymmetric array. The cochlear structure in Group 3 appeared well preserved under the light microscope, and although TEM and SEM revealed stereocilia loss, the hair cells and stereocilia appeared near normal compared with those of Group 2. CONCLUSIONS: Resveratrol may have a protective effect against AT damage in the cochlea, most likely through its antioxidant activity. Our results may be useful for studies in humans exposed to AT and noise-induced hearing loss related to chronic exposure to occupational noise.
