The Conversion Of Fiber Type In OSAS Patients' Upper Airway Expansive Muscles And The Fuction Of Estrogen-Related Receptor-?

BackgroundObstructive sleep apnea hypopnea syndrome(OSAS)is a hotspot in recent years,as its potential risk,high mobidity and involved multiple departments.But the detailed pathophysiological process of pharyngeal space's collaps still remains unknown.Abnormal anatomic structure and upper airway expansive muscles dysfunction are two most important pathogenic factors.As the main pathogenic factor are individual,the long term effects of surgery in some patients are poor.The research due to anatomic factor in OSAS is profound,while the research focused on specific muscular pathologic change in OSAS patients' upper airway expansive muscles is rare,much less in discussion on mechanism of muscular dysfunction.In addition,epidemiological investigation had shown that estrogen is related with female low morbidity of OSAS,but its regulative pathway still remains unknown.Estrogen related receptor-?(ERR-?)expresses mainly in tissue with high metabolic and plays a certain role in mitochondria metabolism in myotube as transcription factor.So we hypothesised that ER/ERR-? may participate in the conversion of fiber typers in OSAS,leading to the change of muscular strength in upper air way and collaps of pharyngeal space.ER/ERR-a may play important role in pathogenesis of OS AS.So,we will use ER stimulation?ER specific antagonist?chronic intermittent hypoxia and SiRNA interference test in celled level,employ ovariectomized and ER reversion animal model,complete tissue sample target gene detection,so to verify the molecular mechanism of the conversion of fibre type in OSAS patients' upper airway expansive muscles which maybe mediatd by ERR-a.This project will discuss that ER/ERR-? may play certain roles in the pathogenic process of OSAS.It will illustrate the mechanism of low morbidity of femal OSAS in a wholly new view.It can provide theoretical and experimental basis to the precaution and hormone replacement therapy for female with high risk of OSAS.Materials and Methods1.Clinical specimensPrimary biopsy specimens and normal biopsies of palatopharyngeal muscle were obtained from otolaryngological department in Nanfang Hospital(Southern Medical University,Guangzhou,China).There are 28 cases of adult male OSAS patients and 14 case of chronic tonsillitis but not OSAS male as control group during 2011.2 to 2013,7.Both tissues were histologically confirmed by H&E(hematoxylin and eosin)staining.All cases have not the history of local operation or radiotherapy.Informed consent was obtained from each patient,and the research protocols were approved by the Ethics Committee of Nanfang Hospital.The project was approval by Chinese ClinicalTrials.gov,number ChiCTR-CCC-13003415.Diagnose and surgical treatment for OSAS was according to the guideline of in Obstructive sleep apnea hypopnea syndrome which was hosted by Chinese Medical Association.The serious of disease was judged by PSG2.Muscular pathological stainingMuscle samples were stood in cork,fixed by tragacanth gum,frozen serial section.Except conventional hematoxylin(HE)staining,we still use other three enzyme histochemical stained,including NADH-TR(nicotinamide adenine dinucleotide-tetrazolium reductase)?MGT(modified Gomori trichrome)and ATPase(adenosine triphosphatase)stained.3.Common cell cultureThe mus musculus cell C2C12 was cultured in DMEM high glocuse with 10%FBS and 1%Penicillin-Streptomycin antibiotic solution.All cells were cultured in a humidified atmosphere of 95%air and 5%CO2 at 37?.It was used in good growing status.4.Chronic intermittent hypoxia cell cultureThe mus musculus cell C2C12 were cultured in three gas incubator(Thermo 3111,USA),3%intermittent oxygen,35min hypoxia,25min normal oxygen,8h/day.Control cell were cultured in a humidified atmosphere of 95%air and 5%CO2 at 37?.5.Cellular ER stimulateAdded different indicator ER suited for cell culture,set blank control.Added different indicator XCT-790 which is the special antagonist for ERR-a to make sure the best blocking effect.Add different indicator ER also 10M XCT-790 to reverse the effect of ER.So we identified that ER lead to shift of fibre type exact through ERR-a.6.Cellular instant transfectioninstant SiRNA against ERR-a:We infected with instant SiRNA against ERR-a with lipo2000,direction for LipofectamineTM 2000 reagent was as reference.Quantity for transfection of SiRNA is 100 nM in 6 pore plate.7.Real-time PCRTotal RNA and small RNA were extracted from cells and tissues.The RNA was reversely transcribed into cDNA.Quantitative real-time PCR(qPCR)was performed using SYBR Green PCR master mix.All samples were normalized to internal controls and fold changes were calculated through relative quantification(2-??Ct).8.Western blotTotal protein was isolated and quantitated using BCA assay.The protein lysates were separated by 10%SDS-PAGE,and electrophoretically transferred to PVDF membrane.Then,the membrane was incubated with antibodies and detected by chemiluminescence.The intensity of protein fragments was quantified with the Quantity One software.9.Animal experimentEach group contained 5 mice and the experiment was repeated 3 times.Ovariectomy and shamed surgery were done after successful paralyzed.Stemohyoideus muscle biopsy were carried before and 2w after surgery in both group as selfcontrol.Total RNA were extracted from tissues in both groups.Myhc and ERR-a were measured by quantitative real-time PCR(qPCR).Method were same as before.10.ImmunohistochemistryFormalin-fixed,paraffin-embedded tissues of muscular sample were sectioned at 4-mm thickness and analyzed for ERR-a and Myhc slow expression.Visualization was achieved using the EnVisionlp peroxidase system.A sample was considered positive if more than 50%of the cells retained nuclear staining,and 5 fields were randomly selected according to semiquantitative scales.The intensity of staining was scored manually(high,3;medium,2;low,1;no staining,0)by 2 independent experienced pathologists.11.Statistical analysisSPSS 13.0 software was used for statistical analysis.Data were presented as mean±SEM.All experiments were performed in triplicate at least.One-sample Kolmogorov-Smirnov test were analyzed for normal distribution.Levene's Test were tested for homogeneity of variance.Two-tailed Student's t test was used for comparisons of two independent groups.One way ANOVA and Dunnett T3 test were used for comparisons of multiple independent groups.Two-tailed Student's t test was used for comparisons of two independent groups.P values of<0.05 were considered statistically significant.Results1?Skeletal muscle pathologyImmuno-histochemistry showed that palatopharyngeal muscle of OSAS patients had pathological lesion.The pathological changes included muscular lesion and abnormal distribution of fiber types,the rate of type I fiber which to maintain the opening of upper air way decreased.There were distinctive pathologic change for OSAS including lobular or motheaten appearance(?2 19.091,P=0.000<0.05)%ragged red fiber(RRF,?2 3.977,P=0.046<0.05)?Clusters for common fibre type(?217.348,P=0.000<0.05).Centralized located nuclei could be observed in both group,but it was more common in OSAS(?2 3.938,P=0.047<0.05).Fibrous necrosis?endomysium connective tissue proliferation?target-like fibers were only observed in some OSAS patients,but there became no statistically significant.The characteristic round appearance by NADH-tetrazolium reductase?obvious versatile diameter of fiber in hematoxylin-eosin were general change in both groups.2.Expression of ERR-a and distribution of different fibre types in palatopharyngeal muscle of OSAS patientsThe results showed that the expression of ERR-? in palatopharyngeal muscle of OSAS was only 15%compared with control which were measured by qRT-PCR,The value of ERR-? ?CT between control and OSAS had statistical significant(13.62±4.08 vs 16.42±3.06,t=-2.495,P=0.07<0.05).The results of fibrous distribution displayed that the percentage of type I fibre decreased in OSAS(23.89±24.20vs44.45± 16.27,t=3.257,P =0.002<0.05),while the percentage of type ?x increased(41.91±21.98vs24.09±10.85,t=-2.799,P= 0.008<0.05).There were no statistical differences in percentage of ?a and ?b fibre type.3.Expression of ERR-a during differentiation in C2C12 myocytes compared with MBThe results indicated a time-dependent increase of ERR-? mRNA levels in MT-1(2.202-fold T=-4.578,P=0.001<0.05)?MT-3(2.71-fold T=-4.326,P=0.001<0.05)and MT-5(1.56-fold,T=-4.588,P=0.006<0.05)cells respectively.It were examined by quantitative real-time PCR at the indicated time points during differentiation and normalized to 18s mRNA levels.Protein expression levels of ERR-a were examined by western blotting at the indicated time points during differentiation.MB was regarded as control group.4.ERR-a SiRNA instant trial:ERR-a induced change of Myhc slow levels(0.31-fold,T=-11.275,P=0.001<0.05)while not Myhc fast(1.01-fold,T=-0.029,P=0.977>0.05)during differentiation which were determined by infection with instant SiRNA against ERR-a(0.48-fold,T=-4.367,P=0.005<0.05)with lipo2000.NC group was as control while transcripts was normalized to 18s.Protein expression levels were examined by western blotting.5.cellular intermittent hypoxia trial:Differentiations in C2C12 were obviously depressed and less myotube conformed in 3%chronic intermittent hypoxia compared with normal culture,and less myotube conformed.We showed that expression of Myhc slow levels in MT-1 decreased(16.573±1.407vs 26.852±3.881,T=4.313,P=0.013<0.05),also in MT-3(180.14±57.75vs335.72±21.93,T=4.362,P=0.012<0.05)and MT-5(341.18±8.97vs484.33±33.39,T=3.582,P=0.023<0.05)respectively,which were determined in 3%chronic intermittence hypoxia compared with normal group.The expression of ERR-? in MT-1 decreased(1.457±.567vs3.628±.884,T=3.582,P=0.023<0.05),while the myhc fast had no significant change all time in 3%ICH compared with normal culture.6.ER stimulated trial;We proved that ERR-a and Myhc slow both increased obviously under different ER stimulated,especially in 10--10M ER condition(ERR-a 4.378-fold,T=-4.69,P=0.009<0.05;Myhc slow 3.517-fold,T=-4.304,P=0.013<0.05).Myhc fast decreased in ER10-7M(0.73-fold,T=4.588,P=0.038<0.05)and increased in 10-10M,the trend is unclear.Reverse trial of ER+XCT790 identified that level of ERR-??Myhc slow were significantly decreased compared with control in 10-7?10-8?10-9M ER+XCT790,while the level of Myhc fast only decreased in ER10-8M+XCT790.Under 10-10ER condition which promoted differentiation mostly,when added XCT790,all the three genes had no significant change.So ER can promote differentiation in C2C12 due to the increase expression level of Myhc slow,this effect can be reversed by specific antagonist of ERR-a.7.Animal trial:Uterus,s weight of 2 weeks postoperative in ovariectomized group decreased significantly compared with shamed surgery group(T=6.812,P=0.000<0.05).Compared with preoperative,express of ERR-a(paired t test,1.324±0.596vs0.534±0.710,t=5.468,P=0.032<0.05).Myhc slow(1.494±0.716.s0.694 ±0.296,t=3.038,P-=0..038<0.05)levels decreased in SD rat' s stemohyoideus muscle 2 weeks postoperative measured by real-time PCR.There were no change for Myhc fast in paired t test in ovariectomized group.In shamed surgery group,there were no changes for these 3 genes between preoperative and 2 weeks postoperative.Immumohistochemical staining was used to identisfied the change in protein.Conclusions1.There existed remodeling in expansive muscle in upper air way of OSAS,palatopharyngeal muscle of OSAS patients had pathological lesion.The pathological changes included muscular lesion and abnormal distribution of fiber type.2.Compared with control group,OSAS patients' type I fiber which to maintain the opening of upper air way decreased,while percentage of ?x increased.There are no significant difference in ?a and ?b fiber type.3.The expression of ERR-? decreased in OSAS patients compared with control.It maybe associated with abnormal distribution of fiber type in OSAS.4.ERR-a participate in myotube differentiated progress,if it expressed abnormal,the result of differentiation would change.5.Both animal trial and cellular experiment demonstrated that change level of ER could lead to shift of fibre type,however this effect could be conversed by specific antagonist of ERR-a.6.ERR-a may play a certain role in physiopathologic process of OSAS,as it regulate the express of Myhc slow and lead to the functional change of upper air way expansive muscle.