The Protective Effect And Mechanism Of HUCMSCs On The Structure And Function Of Genioglossus In OSAHS Rabbits

Objective:In this study,we intend to establish and identify an obstructive sleep apnea hypopnea syndrome(OSAHS)rabbit model with upper airway narrowing by injecting liquid silicon into the soft palate,and explore the protective effect of human umbilical cord mesenchymal stem cells(hUCMSCs)on the structure and function of genioglossus in OSAHS rabbits.The comprehensive mechanism of hUCMSCs in the treatment of genioglossus injury is further clarified from the aspects of oxidative stress,inflammation level,autophagy and endogenous mitochondrial apoptosis pathway,which provides a new perspective and a new important target for the treatment of genioglossus injury,and is expected to become a new therapeutic strategy.Chapter I The establishment and identification of OSAHS rabbit model.Methods:Ten male Japanese large-eared white rabbits were randomly divided into control group and OSAHS group,with five rabbits in each group.Rabbits in the OSAHS group were injected with liquid silicon in submucosa of the soft palate under general anesthesia with 1%pentobarbital sodium 20-25 mg/kg via auricular vein injection,and rabbits in the control group were injected with the same volume of normal saline at the same site.Polysomnography(PSG)and upper airway spiral CT scanning were used to judge the successful establishment of the model.After modeling,the body weight of the rabbits was recorded every day,and the rabbits were given 10%chloral hydrate at a dose of 5-6 ml/kg by oral perfusion to induce sleep.The rabbits were kept in supine position for about 4 hours for 8 weeks.Lowest Oxygen Saturation(LSAT)at 8 weeks was monitored and recorded using a small animal pulse oximeter.An endoscope was applied to observe the oropharyngeal morphology.Results:1.The general state observation of experimental animals.The experimental animals regained consciousness in about 2 hours after modeling under general anesthesia,and were sent back to the cage to eat and drink.Normal urination and defecation.Obstructive sleep apnea was induced in 7 of 10 animals injected with liquid silicon.Of the 7 rabbits in which OSAHS was induced,2 rabbits died of apnea during drug-induced sleep.During the induced sleep,the animals in OSAHS group showed snoring,breath-holding,shallow and rapid breathing,contradictory movement of chest and abdomen,limb movement,cyanosis of lips and auricles to different degrees.There was no significant difference in body weight between OSAHS group and control group before and 2,6 and 8 weeks after modeling(P>0.05).2.Morphological observation of oropharynx assisted by endoscope.After 1-2 days of saline injection in control group,the saline was gradually absorbed and the injection site became flat.After injection of liquid silicon in OSAHS group,the soft palate was obviously elevated and blocked part of the pharyngeal cavity,the liquid silicon was stably confined to the soft palate mucosa without absorption or spreading to the surrounding area,and the local texture of the mucosa at the bulge was hard to the touch,without redness,swelling or breakdown.3.Spiral CT imaging changes of upper airway before and after modeling.Compared with the control group,the upper airway behind the soft palate was narrowed in the OSAHS group.4.The changes of sleep-related indicators before and after modeling.(1)PSG:During the monitoring period,the EEG of experimental animals in the control group was dominated by stage 3 sleep,with smooth respiratory airflow and no significant apnea or hypopnea observed;the EEG of experimental animals in the OSAHS group was dominated by stage 2 or 3 sleep,with apnea and hypopnea,enhanced thoracoabdominal paradoxical movements and decreased oxygen saturation(SAT).One frame(30 s)of PSG sleep monitoring map obtained by EMBLA Systems showed that the experimental animals in the OSAHS group slept in stage N3 with obstructive sleep apnea and apnea hypopnea index(AHI)of 17.14±0.74(times/hour),which was significantly different compared with the control group(P<0.05).The LSAT saturation of rabbits in the control group was above 90%without hypoxemia,while the LSAT of rabbits in the OSAHS group decreased to 83.80±3.11%,which was diagnosed as moderate OSAHS with moderate hypoxemia.(2)LSAT:The results showed that the LSAT was 73.60±2.07%at 8 weeks in OSAHS group,which was statistically different compared with the control group(P<0.05),suggesting that the OSAHS animal model can maintain hypoxia more stably.Conclusions:A rabbit model of OSAHS can be successfully established by injecting liquid silicon into the soft palate,which better simulates the pathophysiology of OSAHS caused by upper airway narrowing.The modeling conditions are simple,and the method is feasible and practical,and lays a good foundation for subsequent experiments.Chapter II The protective effect of hUCMSCs on the structure and function of genioglossus in OSAHS rabbits.Methods:The specific modeling method was the same as that in Chapter I.48 Male Japanese large-eared white rabbits were randomly divided into two major groups,24 rabbits in each group,namely,the early intervention effect group for validation of hUCMSCs and the treatment effect group for validation of hUCMSCs.Among them,the validation of hUCMSCs early intervention effect group consisted of:(i)Control group(Control(early intervention))(n=8);(ii)OSAHS group(OSAHS(early intervention))(n=8);and(iii)hUCMSCs(early intervention)group(OSAHS(early intervention)group applying hUCMSCs for early intervention)(n=8).Validation of hUCMSCs treatment effect group consisted of:(i)Control group(Control(treatment))(n=8);(ii)OSAHS group(OSAHS(treatment))(n=8);(iii)hUCMSCs(treatment)group(OSAHS(treatment)group applying hUCMSCs)(n=8).In the hUCMSCs(early intervention)group,hUCMSCs were injected via auricular vein of rabbits at the early stage of modeling,2.4×10~7 cells/rabbit each time,with an interval of 5 days,for a total of 5times,to observe the early intervention effect.The hUCMSCs(treatment)group was injected with the same dose,cycle and frequency of hUCMSCs after 8 weeks of modeling,and the therapeutic effect of hUCMSCs was observed.The histological changes(Hematoxylin-eosin(HE)staining and MASSON staining)of genioglossus and ultrastructure of mitochondria were observed by optical microscope and transmission electron microscope(TEM).Western Blot,real-time quantitative polymerase chain reaction(RT-q PCR),and genioglossus electrophysiology were used to assess genioglossus myofiber typing,myosin heavy chain(MHC)subtypes,activities of mitochondrial respiratory chain complexes I and IV,and electrophysiology of genioglossus.Results:1.The effect of hUCMSCs on the histomorphology of genioglossus in OSAHS rabbits.Compared with the control(early intervention)group,the normal structure of genioglossus fibers in the OSAHS(early intervention)group disappeared,and the pathological changes such as muscle fiber disorder,the muscle injury score increased significantly(P<0.05),the content of collagen fibers around genioglossus increased(P<0.05),the myofibril and mitochondria of genioglossus were severely damaged;Compared with the control(treatment)group.the histomorphology of the rabbit genioglossus in the OSAHS(treatment)group also showed the above pathological changes,and the above pathological changes were significantly improved after early intervention and treatment of hUCMSCs(P<0.05).2.The effect of hUCMSCs on genioglossus fiber typing and MHC subtype switching in OSAHS rabbits.(1)Western Blot showed that the expression of MHC I protein in genioglossus of rabbits in OSAHS(early intervention)group and OSAHS(treatment)group was lower than those in the corresponding control group(P<0.05),while the expression of MHC II protein was higher than that in the corresponding control group(P<0.05);After early intervention and treatment of hUCMSCs,the expression level of MHC I protein increased(P<0.05),and the expression level of MHC II protein decreased(P<0.05).(2)The m RNA expression of MHC I and MHC IIa in OSAHS(early intervention)group and OSAHS(treatment)group was significantly lower than those in the corresponding control group(P<0.05).The relative expression of MHC IIb and MHC IIx m RNA increased(P<0.05);The relative expression of MHC I and MHC IIa m RNA increased(P<0.05),while the relative expression of MHC IIb and MHC IIx m RNA decreased(P<0.05)after early intervention and treatment of hUCMSCs.3.The effect of hUCMSCs on the activity of mitochondrial respiratory chain complex of genioglossus in OSAHS rabbits.Mitochondrial respiratory chain complex I and IV activity were significantly decreased in the OSAHS(early intervention)and OSAHS(treatment)groups compared with the corresponding control groups(P<0.05);the activities of the above indicators were increased after early intervention and treatment of hUCMSCs(P<0.05).4.The effect of hUCMSCs on the electrophysiology of genioglossus in OSAHS rabbits.The amplitude of action potential and evoked potential of genioglossus in OSAHS(early intervention)group and OSAHS(treatment)group were significantly lower than those in the corresponding control group(P<0.05).The amplitude of action potential and evoked potential of genioglossus in early intervention and treatment of hUCMSCs were significantly higher than those in the corresponding OSAHS group(P<0.05).Conclusion:The early intervention and treatment of hUCMSCs can improve the histomorphology and mitochondrial ultrastructure of genioglossus in OSAHS rabbits,promote the transformation of genioglossus fibers into slow contraction type,improve the anti-fatigue ability of genioglossus,alleviate the structural damage of genioglossus in OSAHS rabbits,and further reverse the fatigue and mitochondrial dysfunction of genioglossus.Chapter III The study on the protective mechanism of hUCMSCs on genioglossus in OSAHS rabbits.Methods:1.The expressions of oxidative damage and inflammation related factors in serum and genioglossus tissue were detected by ELISA and Western Blot.2.The inflammatory reaction of soft palate injected with liquid silicon was observed by HE staining under optical microscope.3.Western Blot,RT-q PCR,mitochondrial membrane potential(MMP)JC-1 probe and TUNEL fluorescence staining were used to evaluate the autophagy and apoptosis of rabbit genioglossus.Results:1.The effect of hUCMSCs on oxidative damage in serum and genioglossus of OSAHS rabbits.Compared with the corresponding control group,the levels of 8-ISO-PG,MDA and HIF-1αin serum and genioglossus of rabbits in OSAHS(early intervention)group and OSAHS(treatment)group were increased(P<0.05),while the levels of SOD and SDH in serum and genioglossus of rabbits in OSAHS group were decreased(P<0.05);After the early intervention and treatment of hUCMSCs,the expression of the above oxidative stress indicators decreased,and the levels of antioxidant enzymes increased(P<0.05).There was a positive correlation between serum and genioglossus,the correlation is strong,and the correlation coefficient was statistically significant(P<0.05).2.The effect of hUCMSCs on the levels of inflammation in serum and genioglossus of OSAHS rabbits.Under the optical microscope,HE staining of the soft palate tissue injected with liquid silicon showed that the squamous epithelium of the soft palate mucosa had obvious hyperplasia,mucous acinus hyperplasia,and no inflammatory cell infiltration was found;Compared with the corresponding control group,the expressions of IL-1βand TNFαin serum and genioglossus of rabbits in OSAHS(early intervention)group and OSAHS(treatment)group were significantly increased(P<0.05),while the expression of IL-10 in serum and genioglossus was significantly decreased(P<0.05);After early intervention and treatment of hUCMSCs,the expression of IL-1βand TNFαdecreased(P<0.05),while the expression of IL-10 increased(P<0.05).There was a positive correlation between serum and genioglossus,the correlation is strong,and the correlation coefficient was statistically significant(P<0.05).3.The effect of hUCMSCs on the autophagy of genioglossus in OSAHS rabbits.Compared with the corresponding control group,the ratio of LC3 II/LC3 I and the expression of Beclin 1 protein were significantly increased in the OSAHS(early intervention)and OSAHS(treatment)groups,respectively(P<0.05);After the early intervention and treatment of hUCMSCs,the protein expression level and m RNA relative expression level of the above autophagy-related indicators were further increased(P<0.05).4.The effect of hUCMSCs on apoptosis of genioglossus in OSAHS rabbits.(1)The MMP of JC-1 probe showed that compared with the corresponding control group,the MMP of OSAHS(early intervention)group and OSAHS(treatment)group was decreased,the fluorescence intensity in Q2 quadrant decreased,and the percentages were 63.66±4.87%and 53.07±6.48%(P<0.05),respectively.The fluorescence intensity of Q4 quadrant increased,and the percentages were 31.30±5.16%and 36.57±3.09%(P<0.05),respectively.After early intervention and treatment of hUCMSCs,the fluorescence intensity in Q2 quadrant increased,and the percentages were 85.17±3.39%and 80.53±6.94%,respectively,while the fluorescence intensity in Q4 quadrant decreased,and the percentages were 12.7±4.03%and 17.07±7.38%,respectively,which significantly increased the MMP of the genioglossus(P<0.05).(2)TUNEL fluorescence staining showed that compared with the corresponding control group,the positive TUNEL apoptosis rate was significantly higher in the OSAHS(early intervention)and OSAHS(treatment)groups,37.73±4.63%and40.80±2.24%,respectively(P<0.05);the positive TUNEL apoptosis rate was significantly lower in hUCMSCs after early intervention and treatment,7.12±1.86%and 8.26±0.85%(P<0.05),respectively.(3)The results of Western Blot and RT-q PCR showed that compared with the corresponding control groups,the expression levels of pro-apoptosis-related proteins Cleaved Caspase 9,Cleaved Caspase 3 and Cytochrome C were increased(P<0.05)and the BCl-2/Bax ratio was decreased(P<0.05)in the OSAHS(early intervention)and OSAHS(treatment)groups;After the early intervention and treatment of hUCMSCs,the expression levels of the above pro-apoptotic proteins were decreased(P<0.05)and the BCL-2/Bax ratio was upregulated(P<0.05).The changes in relative m RNA expression of Cytochrome C in each group were consistent with the changes in protein levels.Conclusion:The early intervention and treatment of hUCMSCs can improve the antioxidant and anti-inflammatory capacity of genioglossus in OSAHS rabbits,further activate autophagy,reverse the endogenous mitochondrial-Cytochrome C-Caspase 3 apoptotic pathway,reduce the apoptosis of genioglossus,and play a protective role in genioglossus of OSAHS rabbits.In summary,we can draw the following conclusions:1.hUCMSCs can protect the structure and function of genioglossus and the ultrastructure of mitochondria in OSAHS rabbits.2.hUCMSCs can ameliorate oxidative damage and inflammatory reaction of genioglossus in OSAHS rabbits.3.The regulation of autophagy and apoptosis may play an important role in the protection of hUCMSCs against genioglossus injury in OSAHS rabbits.