Abstract
Objective
To evaluate the efficacy of xylitol nasal irrigation (XNI) treatment on chronic rhinosinusitis (CRS) and to investigate the effect of XNI on nasal nitric oxide (NO) and inducible nitric oxide synthase (iNOS) mRNA in maxillary sinus.
Materials and methods
Patients with CRS were enrolled and symptoms were assessed by Visual Analog Scale (VAS) and Sino-Nasal Outcome Test 22 (SNOT-22). Nasal NO and iNOS mRNA in the right maxillary sinus were also examined. Then, they were treated with XNI (XNI group) or saline nasal irrigation (SNI, SNI group) for 30 days, after which their symptoms were reassessed using VAS and SNOT-22, and nasal NO and iNOS mRNA in the right maxillary sinus were also reexamined.
Results
Twenty-five out of 30 patients completed this study. The scores of VAS and SNOT-22 were all reduced significantly after XNI treatment, but not after SNI. The concentrations of nasal NO and iNOS mRNA in the right maxillary sinus were increased significantly in XNI group. However, significant changes were not found after SNI treatment. Furthermore, there were statistical differences in the assessments of VAS and SNOT-22 and the contents of nasal NO and iNOS mRNA in the right maxillary sinus between two groups.
Conclusions
XNI results in greater improvement of symptoms of CRS and greater enhancement of nasal NO and iNOS mRNA in maxillary sinus as compared to SNI.
1
Introduction
Chronic rhinosinusitis (CRS) is an inflammatory disease involving the nasal and paranasal sinus mucosa. It is defined as chronic inflammation when it lasts longer than 3 months without complete symptom resolution. CRS is a common health problem which significantly affects quality of life. The disease has been estimated to affect 12.5% to 15.5% of the total population in the United States and 10.9% in Europe . Saline irrigation has been shown to be beneficial for patients with CRS .
Xylitol is a five-carbon sugar alcohol that has gained extensive attentions in the past decades as a natural antibacterial agent. It decreases the salt concentration of human airway surface liquid that contains many antimicrobial substances, which can contribute to the improvement of the innate immune system, and thereby prevent airway infections . In addition, this agent can exert antibacterial actions through disrupting glucose cell-wall transport and intracellular glycolysis, thus inhibiting bacterial growth . An elegant study reported that xylitol nasal irrigation (XNI) could improve symptoms of CRS clinically . However, the study did not evaluate the inflammatory conditions of the paranasal sinuses of those patients.
It is well known that nitric oxide (NO) provide a first-line defense via its antiviral and antibacterial action and via its upregulation of ciliary motility . High concentrations of NO are found in normal paranasal sinuses, and the lack of NO may lead to the pathogenesis of sinus inflammation . The epithelial cells in the paranasal sinuses were identified as the major source of NO in some studies, and inducible nitric oxide synthase (iNOS) would account for most of this NO production . Measurement of nasal NO may be a useful tool in the diagnosis, management and assessment of patients with CRS .
A previous study demonstrated that xylitol at 5% stimulated NO production from macrophage, which inhibited macrophage infection by Leishmania amazonensis . Macrophage is involved in chronic inflammation of paranasal sinuses though generating NO and cytokines . Therefore, it is reasonable to hypothesize that xylitol treatment may control the development of CRS through regulating the NO concentration produced by macrophage or other cells located in sinus mucosa. Based on the above findings, we sought to explore the therapeutic potential of XNI in treating CRS and the influence of XNI on the inflammatory conditions of paranasal sinuses.
2
Materials and methods
2.1
Study design
This study was designed as a prospective, randomized, double-blinded, controlled pilot study. Recruitment was done in the department of Otorhinolaryngology-Head and Neck Surgery, Huashan Hospital of Fudan University, with all patients enrolled between April and July 2016.
2.2
Study population
Thirty patients with CRS, aged between 35 and 67 years, had undergone bilateral functional endoscopic sinus surgery including at least maxillary antrostomy and anterior ethmoidectomy. Maxillary and ethmoid sinus patency was confirmed endoscopically to ensure adequate exposure to the irrigation solutions. Subjects were excluded if they had a history of allergic rhinitis, asthma, immunocompromise, cystic fibrosis, primary ciliary dyskinesia, active bacterial or fungal infection requiring antibiotics or antifungal medications, history of head and neck irradiation, current smoking, pregnancy, or granulomatous disease.
2.3
Study protocol
There were two study visits. At the first visit, patients underwent the evaluations that included clinical history, nasal endoscopy, Visual Analog Scale (VAS) and Sino-Nasal Outcome Test 22 (SNOT-22). Then, a 30-day treatment regimen was assigned in accordance with an independently generated random code to one of the following groups: xylitol (Acros Organics, Fair Lawn, NJ, USA) was premeasured and packaged in our hospital pharmacy into unlabeled, sealed packets each containing 12 g of the sugar. Participants were given 30 of these packets, and instructed to dissolve the contents of one packet in 240 mL of water (5% wt/vol) in a nasal irrigation bottle (Qiangjian medical instrument Co., Ltd., Yangzhou, China). Then, they were instructed to perform nasal irrigation (37 °C) bilaterally once daily and to use one packet daily for 30 days (xylitol nasal irrigation group, XNI group). Fresh solution was prepared for every use. Standard buffered isotonic salt packets (Nasal Rinse Mix, Qiangjian medical instrument Co., Ltd., Yangzhou, China) were packaged into unlabeled, sealed packets to maintain blinding. Each patient was given 30 of these packets and instructed to dissolve the contents in 240 mL of water in the nasal irrigation bottle. Then, they were instructed to perform nasal irrigation (37 °C) bilaterally once daily and to use one packet daily for 30 days (saline nasal irrigation group, SNI group). Also, fresh solution was prepared for every application. The technique of nasal irrigation was shown in Fig. 1 A and B . At the second visit (after 30-day treatment), the scores of VAS and SNOT-22 were reassessed. During the whole study period, patients were instructed not to use any other drugs for the treatment of CRS.
2.4
Measurement of nasal NO
Nasal NO measurement was performed according to published procedures . Patients had to refrain from eating and drinking at least for 1 h before the measurement. The procedures were performed using the NIOX MINO® Airway Inflammation Monitor (Aerocrine AB, Solna, Sweden). The nasal air was obtained directly from one nostril using the intrinsic flow of the electroluminescence analyzer with a target airflow rate of 0.25 L/min (aspiration/insufflation flows of ~ 5 mL/s). The probe was connected to a polystyrene nasal olive and gently inserted into the vestibulum of one nostril. The contralateral nostril was left open. To avoid contamination by NO originated from the lower airway, the patient was required to exhale orally against a resistance in order to close the soft palate. A nasal NO plateau showed steady state after ~ 20 s. The level of nasal NO was recorded.
2.5
Assessment of iNOS mRNA in the right maxillary sinus
Samples of mucosa in the right maxillary sinus of patients were taken endoscopically using a cup forceps device with local anesthesia and were frozen at − 70 °C for mRNA extraction. Total RNA of samples was extracted with Trizol (Invitrogen, Carlsbad, CA) and treated with RNase-free DNase. For reverse transcription, 2 μg of the previously mentioned RNA was reversely transcribed with random hexamers (Invitrogen, Carlsbad, CA), and complementary DNA (cDNA) was amplified according to the manufacturer’s instructions. Primers were designed using Primer Express Software (Applied Biosystems, Foster City, CA) from sequence available in GenBank and were synthesized (Geneland Biotech, Shanghai, China). Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the mRNA of iNOS. iNOS primers were forward primer 5′-TGGATGCAACCCCATTGTC-3′ and reverse primer 5′-CCCGCTGCCCCAGTTT-3′. Glyceraldehyde-3-phosphate dehydrogenase mRNA was also examined to control the sample-to-sample variation in RNA isolation and integrity by using a pair of primers: forward primer 5′-ACCACAGTCCATGCCATCAC-3′ and reverse primer 5′-TCCACCACCCTGTTGCTGTA-3′. After initial denaturation at 95 °C for 10 min, the amplification profile was 15 s of denaturation at 95 °C and 1 min of annealing and extension at 60 °C for 45 cycles. Negative control RT reaction mixtures contained no reverse transcriptase and no cDNA in the PCR amplification mixtures. For measurement, 2 μL of diluted cDNA was amplified in a total reaction volume of 20 μL by using a 7500 real-time PCR System (Applied Biosystems, Foster City, CA) with 20 × SYBR Green mixture (Invitrogen). Specificity of PCR products was evaluated by melting curve analysis and by size in agarose gels. Using 3 dilutions of cDNA, linearity of PCR amplification was controlled. Evaluation of data was performed using the cycle threshold (ΔCT) method with glyceraldehyde-3-phosphate dehydrogenase as internal standard.
2.6
Adverse effects
There were no adverse effects during the study period whether in XNI group or in NSI group.
2.7
Ethical considerations
The study was approved by the Ethics Committee of Huashan Hospital of Fudan University (no. 2014-249), and written informed consent was obtained from all participants.
2.8
Statistical analysis
The sample size was determined based on the reduction of the score of SNOT-22 in a previous pilot study, which suggested that 13 subjects per group would be required to detect a 2.4 difference in the SNOT-22 score reduction with an error of 0.05 (two tailed) and overall power (1- β ) of 90%. Considering a loss of 10% of patients at follow-up, we recruited 15 participants in each study group. Statistical analysis was performed using a commercially available statistical software prism 6.0 (GraphPad Software Inc., San Diego, Calif., USA). The significance of changes within groups was assessed using the paired Student’s t -test, and changes between groups were assessed using the Mann-Whitney U test. Data were expressed as means ± SEMs. p < 0.05 were considered to be statistically significant.
2
Materials and methods
2.1
Study design
This study was designed as a prospective, randomized, double-blinded, controlled pilot study. Recruitment was done in the department of Otorhinolaryngology-Head and Neck Surgery, Huashan Hospital of Fudan University, with all patients enrolled between April and July 2016.
2.2
Study population
Thirty patients with CRS, aged between 35 and 67 years, had undergone bilateral functional endoscopic sinus surgery including at least maxillary antrostomy and anterior ethmoidectomy. Maxillary and ethmoid sinus patency was confirmed endoscopically to ensure adequate exposure to the irrigation solutions. Subjects were excluded if they had a history of allergic rhinitis, asthma, immunocompromise, cystic fibrosis, primary ciliary dyskinesia, active bacterial or fungal infection requiring antibiotics or antifungal medications, history of head and neck irradiation, current smoking, pregnancy, or granulomatous disease.
2.3
Study protocol
There were two study visits. At the first visit, patients underwent the evaluations that included clinical history, nasal endoscopy, Visual Analog Scale (VAS) and Sino-Nasal Outcome Test 22 (SNOT-22). Then, a 30-day treatment regimen was assigned in accordance with an independently generated random code to one of the following groups: xylitol (Acros Organics, Fair Lawn, NJ, USA) was premeasured and packaged in our hospital pharmacy into unlabeled, sealed packets each containing 12 g of the sugar. Participants were given 30 of these packets, and instructed to dissolve the contents of one packet in 240 mL of water (5% wt/vol) in a nasal irrigation bottle (Qiangjian medical instrument Co., Ltd., Yangzhou, China). Then, they were instructed to perform nasal irrigation (37 °C) bilaterally once daily and to use one packet daily for 30 days (xylitol nasal irrigation group, XNI group). Fresh solution was prepared for every use. Standard buffered isotonic salt packets (Nasal Rinse Mix, Qiangjian medical instrument Co., Ltd., Yangzhou, China) were packaged into unlabeled, sealed packets to maintain blinding. Each patient was given 30 of these packets and instructed to dissolve the contents in 240 mL of water in the nasal irrigation bottle. Then, they were instructed to perform nasal irrigation (37 °C) bilaterally once daily and to use one packet daily for 30 days (saline nasal irrigation group, SNI group). Also, fresh solution was prepared for every application. The technique of nasal irrigation was shown in Fig. 1 A and B . At the second visit (after 30-day treatment), the scores of VAS and SNOT-22 were reassessed. During the whole study period, patients were instructed not to use any other drugs for the treatment of CRS.
