Effects Of Tongue Muscle Group Training On Genioglossus Muscle And Its Central Regulation In Patients With Obstructive Sleep Apnea And Chronic Intermittent Hypoxia Rats

Background:Patients with obstructive sleep apnea（OSA）have recurrent upper airway collapse at night,which often causes hypertension,coronary heart disease and other diseases,and is a chronic disease that seriously endangers social health.The main muscle that keeps the upper airway open is the upper airway dilator,which is represented by the genioglossus muscle.The function of genioglossus muscle is often decreased in OSA patients.The main pathophysiological feature of OSA is chronic intermittent hypoxia,which can further damage the genioglossal muscle and cortex motor center.Previous studies have found that upper airway muscle group training（oropharyngeal muscle training,playing training）can reduce the occurrence of OSA to a certain extent,but most of them are small sample studies,with different methods and individual differences.Therefore,in the current context of precision medicine,how to establish a suitable training mode and select patients suitable for upper airway muscle group training to benefit from training is very important.On the basis of previous animal experiments and previous studies,our research group has established a new functional training model of animal tongue muscle group,and found that this model can improve the upper airway function of rats and reduce the collapsibility of the upper airway of rats.Through further combination of medicine and engineering,our research group has built a new training instrument and functional training mode of human tongue muscle group-a precise treatment mode of upper airway with quantifiable intensity,and carried out a small sample study,which has a certain effect.However,it is still unclear which patients are suitable for this training and how this training mode affects the function of genioglossus muscle and the mechanism of its central regulation.Sirtuins is a NAD+dependent histone deacetylase family,and an important regulator of neuroprotection,energy metabolism,etc.Among them,SIRT1 is the key metabolic sensor in this family,which is widely expressed in human brain,muscle,liver and other tissues.Previous studies have confirmed that single exercise can increase the expression of SIRT1 protein,and the relationship between muscle exercise,lactic acid and the expression of SIRT1 in the brain has been established.The expression of SIRT1 induced by exercise has a potential impact on a variety of metabolic activities of the body,especially the central nervous system.Because of its positive regulatory role in the brain,the therapeutic potential of SIRT1 in the nervous system is currently being widely studied.In the early stage,the research team found that tongue muscle group training improved the upper airway dynamics of rats by improving central regulation.Objective:To investigate the effect of tongue task training group on the genioglossus muscle and its central regulation in OSA patients;Taking chronic intermittent hypoxia rats as models,the role and possible mechanism of SIRT1 in the influence of this training mode on the genioglossal muscle group and its central regulation were discussed.Methods:（1）Effect of tongue task training on the excitability of genioglossus muscle and its motor cortex in OSA patients:subjects（n=58）were selected from the sleep center of the hospital.They were randomly divided into functional training group of lingual muscle group（n=30）and sham training group（n=28）for one week.Before and after training,the PSG and related questionnaires of the two groups of patients were tested respectively to evaluate the improvement of their disease degree subjectively and objectively;At the same time,TMS was improved to evaluate the dynamic changes of central excitability in the genioglossal cortex;Analyze the factors affecting the curative effect.（2）The effect and mechanism of tongue task training on genioglossal muscle and central regulation of chronic intermittent hypoxia rats:1)24 8-week-old male SD rats（SPF level）were selected and divided into normoxia control group（NC,n=6）,normoxia training group（NT,n=6）,intermittent hypoxia control group（CC,n=6）,and intermittent hypoxia training group CIH group（CT,n=6）according to random number table method.After 4 weeks of air/hypoxia treatment,rats in the training group were treated with"hunger and thirst→accurate tongue extension→reward"mode to establish a rat model of functional training of tongue muscle group.The dynamic changes of genioglossus muscle and its central reactivity（amplitude and latency）were measured by electromyography（EMG）and transcranial magnetic stimulation（TMS）after training for 2,4 and 8 weeks,respectively,The dynamic characteristics of the upper airway were measured by the negative pressure suction method（Pcrit,etc.）.After 8weeks of training,the rats were killed immediately after the functional training of the tongue muscle group.The muscle tissue of the rat genioglossus muscle was made into electron microscope samples,and the mitochondrial membrane potential of the genioglossus muscle was measured,and the myosin heavy chain typing of the genioglossus muscle was analyzed.At the same time,AMPK-PGC1α-SIRT1-NF-κB in the rat genioglossus muscle was measured The expression level of each protein;The rats were perfused with formaldehyde for internal fixation,the brain tissue was taken and sectioned,and the expression of noradrenergic neurons,SIRT1 in the locus coeruleus and the hypoglossal nucleus were observed by immunohistochemistry or immunofluorescenceα_1Areceptor expression.2)Twenty-four 8-week-old male SD rats（SPF level）were randomly divided into normoxic training+DMSO group（NT+DMSO,n=6）,normoxic training+Ex527 group（NT+Ex527,n=6）,intermittent hypoxic training+DMSO group（CT+DMSO,n=6）,intermittent hypoxic training+Ex527 group（CT+Ex527,n=6）.After 8 weeks of functional training of tongue muscle group,all rats were killed and sampled,and the structure,function,central reactivity（EMG and TMS）and upper airway dynamics（pcrit）of rat genioglossal muscle were detected.The model of functional training of tongue muscle,preparation of electron microscope specimen,determination of mitochondrial membrane potential,WB detection of peripheral protein expression,immunohistochemistry or immunofluorescence technology detection of central protein expression were the same as before.Result:1.The effect of tongue task training on the genioglossal muscle and its central regulation in OSA patients（1）Compared with the control group,the PSG-related indexes in the tongue muscle group training function group were significantly improved after training,which showed that AHI,ODI,supine AHI,REM-AHI decreased,snoring index decreased,and the lowest blood oxygen saturation at night increased（p<0.05）.（2）The TMS response amplitude of the genioglossus muscle in the functional training group increased after training（p<0.05）,indicating that the genioglossus muscle center reactivity increased.（3）The results of univariate analysis and logistic regression showed that the effect of tongue task training group was closely related to the TMS response amplitude at 100%stimulation intensity before training（p<0.05）.The higher the response amplitude,the better the training effect.2.The effect and mechanism of tongue task training on the genioglossus muscle and its central regulation in rats with chronic intermittent hypoxia（1）Part I:1)After the tongue task training,the maximum tongue extension strength of rats increased,the proportion of rats falling into the pressure window increased significantly（p<0.05）,and the tongue extension frequency also increased significantly compared with the baseline value（p<0.05）,suggesting that the tongue muscle strength increased after training,and the accuracy and flexibility of tongue muscle for strength control increased.2)In terms of structure,compared with NC group,the normal structure of genioglossus muscle in CC group disappeared under electron microscope,and a large number of vacuoles degenerated in mitochondria;The mitochondrial membrane potential activity of genioglossal muscle was decreased by flow cytometry;Through WB,the content of MHC protein in genioglossus muscle decreased,and the proportion of type I muscle fiber decreased significantly.Compared with CC group,the structure of genioglossal muscle in CT group was basically normal,the pathological damage was mild,and the mitochondria were partially denatured;Mitochondrial membrane potential activity increased;The content of MHC protein in genioglossus muscle increased,and the proportion of type I muscle fiber increased.3)In function,compared with NC group,the myoelectric activity of genioglossus muscle in CC group decreased;The reactivity of genioglossal motor cortex decreased;The negative value of the closing pressure of the upper airway decreases,and the collapsibility increases.Compared with CC group,the myoelectric activity of genioglossus muscle in CT group was increased;The central reactivity of the genioglossal muscle was enhanced,which was characterized by shortened latency and increased amplitude;The negative value of pcrit increased,and the collapsibility of the upper airway decreased.4)The mechanism was verified by WB,immunohistochemistry,immunofluorescenceand immunoprecipitation.The results showed that compared with NC group,the expression of AMPK and PGC1αand SIRT1 were reduced in genioglossus muscle of rats in CC group,expression of NF-κB were increased,NF-κB deacetylation level decreased;The expression of SIRT1 and NE in locus coeruleus decreased,and the expression of SIRT1 and NE in hypoglossal nucleusα1 receptor activity decreased.Compared with CC group,the expression of SIRT1 in the genioglossal muscle of rats in CT group was significantly higher,and the expression of NF-κB is decreased,and the functional training of tongue muscle group can make the deacetylation level of NF-κB in the genioglossal muscle of rats increased;The expression level of SIRT1 and NE in the locus coeruleus nucleus increased,the average optical density ofα1 receptor increased.（2）Part II:After the administration of exogenous Ex527,the tongue task training group did not improve the genioglossal muscle structure and central excitability of chronic intermittent hypoxia rats,and the expression level of SIRT1 in genioglossal muscle did not increase,the expression level of NF-κB did not decrease,while the expression level of SIRT1 and NE in the central locus coeruleus did not increase,the activity of receptorα1 was not enhanced.Conclusion:1.Tongue task training can reduce the severity of disease in OSA patients and improve the excitability of genioglossal motor center.Those with high excitability of the motor center of the genioglossal cortex before training are more suitable for functional training of the tongue muscle group.2.The tongue task training can improve the damage of genioglossus muscle and central regulation caused by chronic intermittent hypoxia in rats,and reduce the collapse of upper airway.3.The improvement of genioglossus muscle and its central regulation of chronic intermittent hypoxia rats by tongue task training may by up-regulating AMPK-PGC1α-SIRT1 in genioglossus muscle and enables NFκB deacetylation,while up-regulating SIRT1-NE in the locus coeruleus nucleus and increasing the activity ofα_1Areceptor in the hypoglossal nucleus.