Effect Of Autophagy Mediated By HIF-1?/BNIP3 Signal Pathway On Genioglossus Muscle-derived Stem Cells Under Hypoxia

ObjectiveObstructive sleep apnea-hypopnea syndrome(OSAHS)is an increasing disorder characterized by repeated partial of complete collapse of the upper airway during the sleep,leading to interrupting and reducing flow of the air.Common symptoms include loud snoring,excessive daytime sleepiness,observed apneas and waking up with a choking sensation.Several factors may contribute to the cause and development of OASHS,while the main cause is reduction of the opening forces of the pharyngeal dilator muscles,(especially,the genioglossus muscle dysfunction),anatomical malformations,hormonal causes and discoordination between the inspiratory activity of the muscle and respiratory effect.The response and adaption of cells to hypoxia are controlled by transcription factors by a low partial pressure of O2.The most important transcription factor in this homeostasis is the hypoxia-inducible factor-1(HIF-1).HIF-1?,as the activity subunit of HIF-1,is regulated by the oxygen signal,which controls the activity of HIF-1.HIF-1? transfers to the nucleus to activate more than 100 genes(such as glucose transorter-1,3,VEGF,insulin-like growth factor,erythropoietin,matrix metalloproteinases)that affect cell metabolism,growth,proliferation,angiogenesis,biosynthesis,and apoptosis.HIF-1 is regulated by the change of oxygen concentration and is involved in hypoxic response.As being one of the most important transcription factors,HIF-1,is a heterodimer composed of two subunits of HIF-1? and HIF-1?.And HIF-1? is the functional subunit and HIF-1? is the structural subunit.Autophagy is an evolutionally conserved catabolic process,induced by hunger,hypoxia,infection,to degrade the damaged organelle,redundant or misfolded proteins.According to the different pathways of phagocytosis into lysosomes,autophagy is divided three categories:macrophage,microautophagy and chaperone-mediated autophagy.Autophagy plays an important role in the metabolism of a variety of cells,mainly in the following aspects:First of all,when cells are stimulated by hypoxia,nutritional deficiency,oxidative stress and infection,all kinds of degradation metabolites,such as free amino acids,fatty acids and nucleotides,can be recycled to provide raw materials for material and energy metabolism of cells.Second,autophagy can remove some damaged organelles,haploproteins and aging cells,and maintain homeostasis.Moreover,autophagy can participate in the process of specific fusion of some tissues.In normal physiological conditions,autophagy can help cells survive in poor nutrition.Moderate autophagy level can effectively clear the damaged organelles and proteins in skeletal muscle,which is beneficial to maintain the stability of skeletal muscle environment.Too high or too low autophagy is harmful to the physiological function of skeletal muscle.The molecular regulatory mechanism of autophagy is very complicated and is regulated by the ATG(autophagy related genes)and Atgs(autophagy related proteins).In mammals,more than 30 ATG have been found to be involved in regulation,and regulatory proteins include three core complexes:ATG/ULK1 complex and its regulatory protein Beclin-1/? PI3Kand two ubiquitin like systems(Atg5-Atgl2-Atgl6L and Atg8-PE).In 1998,Beth Levine cloned Beclin-1,the homologue of autophagy related gene Atg6 in yeast,which is a very important positive regulator in the process of autophagy.Under normal conditions,the activation of BH3 domain proteins or BH3 structure-like proteins(JNK1-mediated Bcl-2 phosphorylation)promotes the binding of Beclin-1 to Bcl-2 and negatively regulates autophagy.When starvation,hypoxia and so on,the interaction between Bcl-2 and Beclin-1 decreased,and the autophagy level is upregualated.One of the most important roles of HIF-1? is to activate the transcription of BNIP3(Bcl-2 and adenovirus E1B 19-kDa interacting protein 3)gene,encoding the BNIP3 protein,which can induce autophagy.Several studies have proved that is a link between hypoxia and autophagy,and our previous studies also demonstrate that the HIF-la/BNIP3 signal pathway plays an important role in the activation of autophagy in the cells of adenoid cystic carcinoma.HIF-1 a interacts with the BH3 domains of BNIP3,then BNIP3 disrupts the Beclin-1/Bcl-2 or Beclin-1/Bcl-xl complex,displaces the Beclin-1 and subsequently releases Beclin-1 to trigger autophagy,however the role of HIF-la in OSAHS is still unclear.Skeletal muscle is the largest organ in the body,and there are several adult stem cells within the skeletal muscles,such as Muscle Satellite Cell,Side Population cells,Muscle Derived Stem Cells.The classical stem cells in the skeletal muscles are satellite cells,referred as myogenic precursors that are capable of regenerating muscles and demonstrating self-renewal characteristics.To our knowledge,MDSCs are considered as a predecessor of the satellite cells,possessing a higher capacity of regeneration and resistance to stress,exhibiting better cell survival abilities and a broader range of multilineage capabilities.These distinguish features make MDSCs ideal stem cells for tissue regeneration and engineering.Normally,MDSCs can differentiate into myogenic cells spontaneously when cultivated in basic medium;While,when stimulated by inductive factors,MDSCs are able to differentiate into multilineage and regenerate muscle,bone,cartilage,nerve and cardiac tissue types.Clinical applications of MDSCs translation are firstly started in the patients with Duchenne muscular dystrophy(DMD)and stress urinary incontinence(SUI).With the development of tissue engineering and genomics,great progress has been made in the range of regeneration of bone,cartilage and cardiac muscle after infarction in the animal models.At present,a large amount of research shows that there is a correlation between autophagy induced by hypoxia and progression of tumors.However,it is unclear how the hypoxia influences the genioglossus MDSCs.In this study,we investigated whether and how the autophagy occurred in the genioglossus MDSCs under hypoxic conditions.Methods1 The isolation,purification and identification of genioglossus MDSCsSprague-Dawley rats were dissected under sterile conditions,and the genioglossus muscle was obtained,then successive enzymatic digestion was performed with collagenase type ? and trypsin.The genioglossus MDSCs were isolated by a modified preplate technique,which based on the different adhesive capacity.In order to identify the genioglossus MDSCs,we induced the cells differentiate into myotubes and performed the immunofluorescence staining with anti-Sca-1?anti-CD34?anti-CD45?anti-Desmin.2 Detection the level of autophagy in the Genioglossus MDSCs under hypoxic conditionsIn this section,we seeded and incubated the genioglossus MDSCs separately in different flasks under normoxia and under hypoxia for 3h,6h,12h,24h.After incubation,the cells were fixed,rinsed.postfixed,dehydrated and embedded,thus the specimens were obtained.Then we used the transmission electron microscopy to observe the autophagosome formation.As the meantime,another group cells were lysed,and the total proteins were separated,transferred onto polyvinylidene difluoride(PVDF)membranes,then,the Western blot was used to detect the expression of HIF-1??Beclin-1?microtubule-associated protein 1 light chain 3(LC3),which are the important proteins in analyzing the level of autophagy.3 Detection the expression of HIF-1? and BNIP3 in the Genioglossus MDSCs under hypoxic conditions.In this part of the experiment,we seeded and incubated the genioglossus MDSCs separately in different flasks under normoxia and under hypoxia for 3h,6h,12h,24h.The Western blot analysis was conducted as the previous described procedures to detect the expression of HIF-la and BNIP3.Another group cells were used to isolate the total RNA and synthesize cDNA.Afterwards,the real-time quantitative PCR was carried out to detect the expression of HIF-1? and BNIP3 in mRNA.Results1 The genioglossus MDSCs of SD rats could be obtained with high purityWe usd successive enzymatic digestion with collagenase type ? and trypsin and preplate technique to get the genioglossus MDSCs.The characteristics of genioglossus MDSCs are as following:low adhesion,small circle,short fusiform and strong refraction.The cells adhered to the wall and extended into polygonal shape after incubated 48h.The cells proliferate logarithmically,and 90%of cells are positive of Sca-1,CD34 and Desmin,and negative of CD45.The genioglossus MDSCs can differentiate into myotubes when incubated in differentiation medium for 7 days.2 Hypoxic stress induced autophagy in Genioglossus MDSCsIn this part,we seeded and incubated the genioglossus MDSCs separately in different flasks under normoxia and under hypoxia for 3h,6h,12h,24h,respectively.The expression of HIF-1? protein was detected by Western Blot assay.The data showed that the expression of HIF-1? protein was low under normoxic conditions,while the expression of HIF-1? protein was up-regulated after incubated under hypoxic conditions 6h.It showed that we had successfully simulated the hypoxic microenvironment.After 12 hours of hypoxic incubation,the Golgi apparatus and endoplasmic reticulum were observed to dilate,and the vacuolar structure increased obviously,and a large number of double-membrane structure containing organelles were observed.We measured the autophagy molecular marker LC3-? and Beclin-1 used Western blot assay.Compares to the normoxic group,the expression of LC3-?and Beclin-1 in hypoxic group increased significantly in a time-dependent manner.These results indicated that hypoxia enhanced autophagy in the genioglossus MDSCs3 HIF-1?/BNIP3 signal pathway regulated autophagy activation in the genioglossus MDSCs under hypoxiaOn the one hand,we detected the mRNA expression of HIF-1? and BNIP3 under different hypoxia time.The mRNA expression of HIF-1? and BNIP3 increased after incubation 6h under hypoxic conditions.On the other hand,we also detected the protein expression of HIF-1? and BNIP3.The protein expression of HIF-1? and BNIP3 were also up-regulated after incubation 6h under hypoxic conditions,especially the multiple bands of BNIP3 appeared after incubation 12h under hypoxia,and the expression increased significantly.Moreover,the mRNA and protein expression of HIF-1? and BNIP3 increased in a time-dependent manner.ConclusionsThe genioglossus MDSCs of SD rats could be obtained by using successive enzymatic digestion with collagenase type ? and trypsin and preplate technique with high purity.Under the suitable culture conditions,the proliferation activity of the genioglossus MDSCs was good and the passage was stable.The results of this paper revealed that hypoxia promoted autophagy in genioglossus MDSCs,and HIF-1?/BNIP3 signal pathway might be involved in upregulating in this process.These findings give insight into the critical role of autophagy in the pathogenesis of OSAHS.