| AimsObstructive sleep apnea hypopnea syndrome(OSAHS) is one of the common sleep-related breathing disorders. It is mainly manifested as recurrent obstruction of upper airway and ventilation dysfunction during sleep. As the most typical pathological characteristic of OSAHS, intermittent hypoxia(IH) easily causes hypoxemia, hypercapnia, etc. IH is an underlying risk for dysbolism and cardiovascular diseases. Researches have shown that, the abnormal contractile function of upper airway dilator, especially genioglossus(GG), is an important pathogenesis of OSAHS, and IH further aggravates the injury. It’s well known that the muscle satellite cells(Mu Scs) are the chief participants which involved in the regeneration of repaired skeletal muscle. Even so, it’s still unlikely to reverse the changes of muscle fiber types and functional abnormalities in genioglossus among OSAHS. Therefore, we speculate that, IH may has anomalous effects on Mu Scs of genioglossus which reducing their capacity to repair.As a focus in medical research, autophagy is closely involved in numerous diseases related to hypoxia. Hypoxia can induce autophagy. Autophagy is generally considered to be an important regulatory mechanism of cellular environmental homeostasis. Then what is the correlation between autophagy and GG dysfunction in IH conditions? It is still an unanswered question at this timeTherefore, we attempt to study the activities and effects of autophagy on GG Mu SCs under IH first, and then to further explore the pathogenesis of genioglossus dysfunction,.and ultimately to find a new way to treat OSAHS. Methods1.We applied the modified oxygen chamber to simulate the conditions of IH. Then we evaluated whether these rat models with IH had been established successfully with the aid of blood gas analysis or not.2. We observed the histopathologic changes of genioglossus by electron microscopy, HE dying, immunochemistry and immunofluorescence.3.The enzyme digestion and the purification method which depends on different velocity of adherent were applied to culture the primary generation of GG Mu SCs. Immunofluorescent techniques were applied to identify the Mu SCs.4.We tested the Multi-differentiation potential of GG Mu SCs by inducing the cytodifferentiation into myotubes, adipogenic and osteoblast.5.The GG Mu SCs models in IH conditions were simulated by using three gas incubators. Western blot was applied to detect the expressions of autophagy-related proteins and apoptosis–related proteins at different time point of GG Mu SCs. Then we select the time point which showed the maximum expressions of autophagy-related protein as the IH processing time for subsequent experiments.6.The methods including MTT assay, western blot and acridine orange(AO) staining were used to screen the most suitable concentration of chloroquine for subsequent experiments. Then we can simulate states of different activities of autophagy for GG Mu SCs in IH conditions by using this concentrations of chloroquine.7. Western blot and flow cytometry were respectively applied to detect the expression of mitochondria-dependent-apoptosis-related proteins and the apoptosis rate for GG Mu SCs with different autophagic activities.8.The mitochondrial ultrastructures of GG Mu SCs with different autophagic activities were detected by means of transmission electron microscopy, and the mitochondrial membrane potential was measured by flow cytometry.9.The effects of autophagy on intracellular calcium(Ca2+) of GG Mu SCs were observed through laser scanning confocal microscopy. Results1.The result of blood gas analysis showed that, compared with the control group, the blood oxygen saturation and the partial pressure of oxygen were obviously decreased in rats which were exposed to IH.2.HE staining showed that, the fibers of genioglossus were obviously hypertrophy, and the spacing between muscle fibers were narrowed in IH group. The observations through transmission electron microscope showed markedly abnormal morphology in mitochondria with swollen shape, unclear boundary and abnormal internal structure in conditions of IH. The results of immunohistochemistry showed a significant increase in Cyt c protein expression in genioglossus in conditions of IH. Immunofluorescence staining showed that, the expression of LC3 protein significantly increased in IH group. All of the above was compared with the control group.3.The immunofluorescence staining of anti-Pax7 antibody and anti-MHC antibody showed more than 95% positivity for the cells what we cultured. It indicated that these cells are what we required for subsequent researches.4.The identifications showed that the cells of what we cultured own the ability to differentiate into myotubules, adipose and osteogenesis.5.The results of western blot showed that, in conditions of IH, the expressions of autophagy-related proteins generally increased, and autophagy had reached its peak in about six hours. Then it began to decline after twelve hours. At the same time, the expressions of apoptosis–related proteins also generally increased obviously, but there is no significant changes during the first six hours. Then the expression of apoptosis–related proteins significantly increased in about twelve hours and it continued to increase over time. Above all, we take six hours as the default duration for subsequent experiments under IH.6.According to the results of MTT assay, western blot and acridine orange(AO) staining, we concluded that the suitable dose of chloroquine(CQ) is about 25 μmol/L on GG Mu SCs.7.The results of western blot showed that, compared with the control group, the expressions of Cyt c and caspase 3 protein obviously increased in IH group. Compared with the IH group, the expressions of Cyt c and caspase 3 protein significantly increased. With the aid of flow cytometry, the apoptosis rate of GG Mu SCs were recorded as follows: IH+ CQ group >IH group > control group. There was significant difference among three groups.8.With the aid of transmission electron microscope, our observations showed that there were no obvious abnormalities in mitochondria of the control group. Compared with the control group, the mitochondria became swelling and hypertrophy in IH groups. Compared with the IH group, the mitochondria of IH+ CQ group shrank obviously. With the aid of flow cytometry, the mitochondrial membrane potential of each group was recorded as follows: control group > IH group > IH+ CQ group group. There was significant difference among three groups.9. The intracellular fluorescence intensity of calcium(Ca2+) were constantly monitored by laser scanning confocal microscope. In the light of fluorescence intensity, the results showed as follows: IH+ CQ group> IH group > control group. The difference was statistically significant among them. Given the KCL stimulation, the transition of Ca2+ intensity quickly went in the control group. Compared with the control group, the transition rate of intracellular Ca2+ intensity reduced when was in IH group. Compared with the IH group, the transition rate of intracellular Ca2+ intensity was obviously retarded, and there was no striking fluctuations of fluorescence intensity in IH+ CQ group. Soon afterwards, there was an abnormal quenching of fluorescence intensity. The difference was statistically significant among each group. Conclusions1.We successfully established the rat models with IH.2. IH could cause the injury of genioglossus tissue, and it also can trigger autophagy.3.We successfully established the model of GG Mu SCs with high purity in vitro.4.IH can trigger autophagy and apoptosis in GG Mu SCs.5.The appropriate concentration of CQ could effectively inhibit the autophagy which was induced by IH6.Inhibit the autophagy occurred in IH conditions will increase the apoptosis of GG Mu SCs. Autophagy might be a significantly protective mechanism for GG Mu SCs response to IH damage and cell apoptosis.7.IH could induced the damage on mitochondria and cells’ function of GG Mu SCs. It may aggravate the damage of mitochondria in GG muscle when inhibit the autophagy which induced by IH. |
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