Effect Of Mandible Advanced Device Upon NF-κb And TNF-α, Il-6 In Genioglossus Of Rabbit In OSAHS

Objective: By setting up the animal model of New Zealand white rabbits with obstructive sleep apnoea-hypopnea syndrome（OSAHS）, to research the effects of OSAHS upon the expression of nuclear NF-κB P65 and the concentration of inflammatory cytokines TNF-α and IL-6 in the genioglossus（GG） and the changes of the-above biomarkers of OSAHS rabbits after treated with mandibular advancement device（MAD）, to investigate the possible inflammatory mechanisms of GG damaged, which provided corresponding theoretical base for the pathogenesis of GG injury induced by OSAHS and the influence on GG of OSAHS with the treatment of MAD.Methods:1 Animals groupedEighteen male New Zealand white rabbits in six months old were randomly divided into three groups and there were six rabbits in each group, their body weights ranged from 3.0kg to 3.5kg. Three groups were obstructive sleep apnoea-hypopnea syndrome group（Group OSAHS）, mandibular advancement device group（Group MAD） and control group respectively.2 Animal model establishedAll animals in three groups were given spiral CT（USA Light Speed 16-detector spiral CT scanner） of the upper airway and polysomnography（US Bond Monet polysomnography system） in the supine position before modeling to exclude congenital OSAHS or anatomical upper airway stenosis. After general anaesthesia with 1% sodium pentobarbitone（20mg/kg²5mg/kg, intravenously）, rabbits in Group OSAHS and Group MAD were injected with 2ml polyacrylamide hydrogel mixture, via the submucous muscular layer in the soft palate, about 1.5cm away from the junction of the hard and soft palates. To confirm the success of setting up the OSAHS animal model, spiral CT scanning and polysomnography recordings were conducted according to the same methods as above after the injection. After 3 days of adaption, OSAHS rabbits in Group MAD were worn mandiblular advancement device（MAD）, which was made up of self-curing denture powder and liquid. The front slope of MAD was positoned 60 degrees to the maxillary occlusal plane and was glued to the upper anterior teeth with glass ionomer, with 3mm⁴mm of mandibular advancement and 2mm³mm of jaw opening. Finally, spiral CT and polysomnography were conducted as described above to evaluate the effectiveness of MAD.3 Sleep in supine positionThere were no break and no infection while checking the wound of the experimental animals on the fourth day after the animal model was set up. And no animals had difficulty in taking food or water. All subjects were given 10% chloral hydrate through mouth by 5ml/kg⁶ml/kg and slept 2 hours⁴ hours every morning in supine position during the next eight weeks. Food and water were available ad lib at rest time.4 Collection and disposal of samples8 weeks later, food and water were forbidden for 24 hours before the operation. The animals were given general anaesthesia with 1% sodium pentobarbitone（2ml/kg） via the vein of ear. Then they were placed in a supine positon, fixed the limbs, opened the mouth to lift the tougue and separated hypoglossal fascia and muscle layer to expose genioglossus, which were carefully cut off and washed with pre-cooling saline. Rapidly, the genioglossus was cut into two pieces of similar size and volume and accepted quick-frozen with liquid nitrogen. Lastly, put the samples in -80 to store.℃5 Determine the expression of NF-κB P65 protein in the nuclear of genioglossusNuclear proteins were extracted from the genioglossus of animals according to the instructions of nuclear protein extraction kit and the protein concentration was determined with BCA method to ascertain the quality of the samples. The relative expression of NF-κB P65 protein to nuclear reference histone 3 was measured with Western Blot.6 Determine the concentration of inflammatory cytokines TNF-α and IL-6The absorbance value of TNF-α and IL-6 in genioglossus at the wavelength of 450 nm was determined according to the instructions of enzyme linked immuno sorbent assay（ELISA） kit and standard curve was drawn in order to calculate the concentration of TNF-α and IL-6 in genioglossus.7 Correlation analysisThe correlation analysis was made between the relative expression of NF-κB P65 protein and the concentration of TNF-α and IL-6 in genioglossus. Meanwhile, the correlation analysis was made between NF-κB P65 and AHI, SaO2 respectively in three groups.Results:1 The general situation of animals3 days after modeled, animals in every group had no difficulty in taking food or water, and they had activity freely. At baseline, 2 weeks and 8 weeks post-experiment, there were no significant differences in body weight among the three groups. While sleeping in supine position, animals of Group OSAHS had obvious, uneven snore and waked several times. Animals in Group MAD were stable, except for heavier breath sounds or light snore in a few animals, animals of control group breathed regularly and smoothly.2 CT scans of upper airwayThe CT results showed that the upper airway cross-sectional area and the sagittal space in 1/4 point, 1/2 point and 3/4 point of soft palate were decreased significantly in Group OSAHS compared with Group MAD and control group（P<0.05）, and there was no significant difference between Group MAD and the control group（P>0.05）.3 PSG recordingsThe PSG results indicated that apnoea hypopnea index was increased significantly and both oxygen saturation and sleep efficiency were decreased significantly in Group OSAHS compared with Group MAD and control group（P<0.05）, and there was no significant difference between Group MAD and the control group（P>0.05）.4 Relative expression of NF-κB P65 protein in the nuclear of genioglossusThe relative expression of NF-κB P65 protein in genioglossus in Group OSAHS, Group MAD and control group was 0.44±0.08, 0.30±0.09, 0.24±0.07 respectively. The data in Group OSAHS was significantly higher than Group MAD and control group（P<0.05）, while there was no significant difference between Group MAD and the control group（P>0.05）.5 Concentration of TNF-α and IL-6 in geniglossusThe concentration of TNF-α in genioglossus in Group OSAHS, Group MAD and control group were 96.80±18.00pg/ml, 71.16±20.18 pg/ml, 64.80±19.51pg/ml respectively. The data in Group OSAHS was significantly higher than Group MAD and control group（P<0.05）, while there was no significant difference between Group MAD and the control group（P>0.05）.The concentration of IL-6 in genioglossus in Group OSAHS, Group MAD and control group were 93.30±16.97pg/ml, 69.25±14.28pg/ml, 63.49±13.15pg/ml respectively. The data in Group OSAHS was significantly higher than Group MAD and control group（P<0.05）, while there was no significant difference between Group MAD and the control group（P>0.05）.6 Correlation analysis between NF-κB P65 and TNF-α, IL-6The relative expression of NF-κB P65 protein in the nuclear of genioglossus was significantly positively correlated with the concentration of TNF-α and IL-6 in genioglossus, with the correlation coefficient 0.886 and 0.862 respectively（P=0.000）.7 Correlation analysis between NF-κB P65 and AHI, SaO₂The relative expression of NF-κB P65 protein was significantly positively correlated with AHI, with the correlation coefficient 0.722（P=0.000）. The relative expression of NF-κB P65 protein was significantly negatively correlated with SaO2, with the correlation coefficient -0.685（P=0.000）.Conclusion:1 OSAHS can cause the increased activation of NF-κB in genioglossus and the concentration of TNF-α and IL-6 also rises. The elevation of these inflammatory indicators may be one of the mechanisms that lead to genioglossus fatigue.2 Treating OSAHS with mandibular advancement device（MAD） can reduce the activation of NF-κB and lower the the concentration of TNF-α and IL-6, which plays a significant role in protecting genioglossus.