Effect Of PINK1/Parkin Mitochondrial Autophagy Pathway In Genioglossus During The Treatment Of OSA With Mandibular Advancement Device

Objective: To study the improvement of the genioglossus contractile function before and after OSA treatment with mandibular advancement device and the role of mitochondrial autophagy mediated by PINK1(PTEN-induced putative kinase 1)/Parkin(E3 ubinquitin ligases)signaling pathway in the genioglossus injury in OSA rabbits.To investigate the changes of genioglossus function before and after MAD treatment in OSA rabbits and the mechanisms of mitochondrial autophagy.Methods:1.Grouping: 18 male New Zealand white rabbits were randomly divided into 3 groups: the control group,Group OSA and Group MAD,with 6 rabbits in each group.2.Establishment and evaluation of animal models: OSA models in Group OSA and Group MAD were established under general anesthesia.The specific method was as follows: the animals in the two groups were supine,and about2 ml of Sodium hyaluronate for medical use was injected into the soft palate1.5cm away from the junction of soft and hard palate.Subsequently,the models were evaluated by Cone beam Computer Tomography(CBCT)of the upper airway and sleep monitoring with Polysomnography(PSG).After the successful modeling,the MAD rabbit models were established by inserting the mandibular advancement device made of self-coagulable resin for rabbits in Group MAD,and CBCT photography and PSG monitoring methods were also used for model evaluation.3.Sleeping in supine position: After successful modeling,the animals in three groups were given 5-6 m L /kg of chloral hydrate orally by professionals every morning at a fixed time.After sleeping,the animals were placed in self-made wooden boxes and kept in supine sleep for 2-4 h for 8 consecutive weeks.During this period,the vital signs of the animals were closely observed to prevent death due to asphyxia.4.The results of the genioglossus contractile force test: After 8 weeks of supine sleep,the animals were subjected to general anesthesia,the genioglossus was bluntly dissected and exposed.In this study,the BL-420E+biological function experimental system was used to detect the genioglossus in three groups,and the contractile force(unit: g)of single muscle contraction at the frequency of 100 Hz at different time periods(every1 minute)of various single stimulus voltages(5V,10 V,30V)was recorded.5.Specimen collection and processing: the genioglossus specimen was taken,placed in a PVC tube and frozen in a liquid nitrogen tank,and then transferred to a-80° refrigerator for storage.The expression levels of microtubule associated protein light chain 3(LC3)LC3-I,LC3-II,Beclin-1,PINK1 and Parkin were detected by western blot and then compared.Results:1.Sleeping status: in Group OSA,typical symptoms of airway obstruction were obvious,such as lip cyanosis,apnea,snoring and arousing during supine sleep;The symptoms of airway obstruction were significantly relieved in Group MAD;There were no symptoms of airway obstruction and breathing steady in the control group.2.CBCT results of upper airway: sagittal diameter,cross-sectional area and total volume of upper airway in Group OSA were significantly lower than those in Group MAD and the control group,differences between the two groups had statistical significance(P<0.05);There were no significant differences between Group MAD and the control group in the above 3 indexes(P>0.05).3.PSG showed that Oxygen saturation(Sa O2%)and the Lowest Oxygen saturation(LSa O2)were decreased in group OSA,and Apnoea-hypopnea index(AHI)was increased compared with the control group and Group MAD and there were statistically significant differences(P<0.05).There were no significant differences in the above 3 indexes between Group MAD and the control group(P>0.05).4.Genioglossus contractile tension: When the genioglossus was stimulated with 5V,10 V and 30 V,the contractile tension of the genioglossus in Group OSA was significantly lower than that in the control group and Group MAD,there was significant difference(P<0.05);After MAD treatment,the genioglossus contractile tension increased at the level of 5V and 10 V,but there was no significant difference(P>0.05).After MAD treatment,the contractile tension of the genioglossus was significantly increased at the level of 30V(P<0.05),there was significant difference(P<0.05).5.Western blot: The ratio of autophagy related proteins LC3II/LC3 I and the protein expression levels of Beclin-1,Pink1 and Parkin in Group OSA were significantly higher than those in the control group and Group MAD,with significant differences(P<0.05).There was no significant difference between Group MAD and the control group(P >0.05).Conclusions:1.The genioglossus contractility decreased in OSA rabbits,and increased after MAD treatment.2.Hypoxic environment in OSA rabbits activated mitochondrial autophagy mediated by PINK1/Parkin signaling pathway in genoglossus tissue and the expressions of related autophagy proteins LC3II/LC3 I and Beclin-1 were regulated at high levels,leading to functional impairments of the genioglossus.3.MAD can effectively alleviate the hypoxia environment caused by OSA,thereby inhibiting mitochondrial autophagy mediated by PINK1/Parkin signaling pathway in the genioglossus,and thus play a protective role in the genioglossus.