The Effect Of Electrical Stimulation On Muscle Satellite Cells Proliferation Signaling

Background:Obstructive sleep apnea-hypopnea syndrome (OSAHS) is a common sleep apnea otolaryngology disease. Because of narrow pharyngeal airway and muscle relaxation, patients repeatedly occurred airway obstruction and collapse in the process of sleep at night. It lead to low ventilation and apnea, finally causing hypoxemia and hypercapnia.Its pathogenesis is complex and not only with many complications, but the potential risk.The treatment of OSAHS used to be surgery and drug therapy. According to study, however, not all of the patients with OSAHS can be treated with operation. As we know, Frideman classification can effectively predict the result of operation. For patients in Frideman stage I, the surgical curative result can reach 80.6%, and those in stage Ⅱ, the surgical curative result was 37.9%, but in stage Ⅲ the surgical curative result was lower to 8%. For patients in Frideman stage Ⅲ or Ⅳ, the surgery is pointless. For these patients, most effective treatment has been continuous positive airway pressure (continuous positive airway pressure, CPAP). However, Patients who want to accept the treatment of CPAP need to wear a mask at night, which is noisy and seriously influences patients’ rest. Despite its significant advantage, it is still hard to be accepted by partial patients, and the percentage of patients who refused CPAP therapy was as high as 40-50%.Hypoglossal nerve stimulation (hypoglossal nerve stimulation, HGNS) had been used in clinical trials in 2001,with the development of pathogenesis, anatomy and clinical equipment of OSAHS. But there are still limitation of clinical applications.Miki and other researchers used a needle electrode to stimulate sober experimental canine genioglossus, and confirmed that the stimulation on airway dilator can steadily enlarge pharyngeal cavity, to achieve the goal of treatment for OSAHS in the early 1980s. Genioglossus is one of the most important muscle on airway dilator. It is from chin spines, fanshaped muscle fibers forward expanded, end in the tip of the tongue, tongue back, with little muscle to hyoid body bone edge. Genioglossus is mainly responsible for the movement of the tongue, and its contraction can move body of the tongue to the contral side and forward, to enlarge pharyngeal cavity and keep the airway open. So it is called "airway guard". When insufficiency genioglossus could not resist on airway inspiratory pressure, collapse oropharyngeal airway cause sleep apnea or low ventilation. Genioglossus is different skeletal muscle from other muscles. Its blood supply is rich, and muscle fiber is relatively small, and contraction is also more quickly. These characteristics easily result in muscle loose and fatigue, not suitable for continuous and strong contraction. Through functional electrical stimulation on genioglossus, patients can controllbility contract and achieve the goal of treatment for OSAHS。Current research of electrical stimulation on genioglossus is only limited at laboratory. Odeh studied pharyngeal expansion muscles on experimental dogs undergoing anesthesia, and found genioglossus mechanical response is more prominent to stimulation than other muscles. The response is not related with head side. Oliven found the response of oropharyngeal muscle to stimulate genioglossus is samliar to hypoglossal nerves.Therefore, muscles pacemaker for OSAHS developed. Known as functional electrical stimulation (functional electrical stimulation FES) has been widely used in other fields and obtain success, such as cardiac pacemaker, diaphragm pacemaker and bladder detrusor pacemakers. In our otolaryngology department, there are throat pacemaker. As new treatment, muscle pacemaker is a kind of functional muscle stimulator, which can can stimulate muscle and restore the activities after pacemaker. Zealear and Dedo from United States firstly applied the concept to larynx in lab. In 1996, the pacemaker is first applied in the treatment of bilateral vocal cord paralysis by Zealear. He successfully applied pace on the laryngeal muscle of innervation in dog which can also be applied to the laryngeal muscle stimulation on people.With the growing application of muscle of pacemaker, studies of electrical stimulation on muscle is also more and more important. Muscle satellite cells is most important source of muscle regeneration and repair, and some parameters of electrical stimulation can promote satellite stem cell proliferation, so studying the effect of electrical stimulation on muscle satellite cell proliferation is more important.Muscle satellite cells, a kind of pluripotent stem cells in skeletal muscle injury, plays an important role in repairing and maintenance. Under normal circumstances, however, muscle satellite cells is in a stationary state, only when the muscle is damaged, such as innervation, hypoxia, pull or electrical stimulation, satellite cells will be activated and starts multiplying differentiation. It is complex after muscle satellite cells activated and proliferated. The signaling pathway used to mediate the proliferation of muscle satellite cells under electrical stimulation is unknown. Currently, NO/HGF/c-met pathway may be involved in the signal path, and promote the proliferation of satellite cells other intrinsic pathways, such as Wnt pathways, Sphingolipid signaling pathways, in vivo. Notch signaling pathways are not seen.This study intends to study aforementioned three signaling pathways in vitro, in hope of finding a certain satellite channel for electrical stimulation on the stem cells proliferation.for clinical research and development of the muscle pacemaker.Objective:Using mice body sources of C2C12 cell lines and primary cultured youth beagle chin tongue muscle satellite cells as the research object. We used the fixed voltage, fixed duration, fixed pulse width, different frequency pulse electrical stimulation on the cultured cells, and to find proliferation/differentiation regulation of signaling pathways undergoing different electrical stimulation frequency on C2C12 cell line and muscle satellite cells.Methods:1. We used electrical stilumation on C2C12 cell lines for 72 hours, which parameter is fixed voltage (5 v), train stimulation(5s,3 s-on,2s-off), the different frequency (0 Hz,10 Hz,80Hz three groups).Total mRNA were collected,. After reverse transcription, singal pathways mRNA expression were measured with qRT PCR. We also observe cell proliferation.2. Using Percoll discontinuous density gradient centrifugation to separate and purificate muscle satellite cells from beagle dog genioglossus muscle. We used immunofluorescence method to identify the original generation of cultured muscle satellite cells. We also used electrical stilumation on C2C12 cell lines for 72 hours, which parameter is fixed voltage (5 v), train stimulation(5s,3 s-on,2s-off), the different frequency (0 Hz,10 Hz,80Hz three groups).72 hours. The mRNA expression and protein concentration were measured with qRT-PCR and Western blot to quantitative/semi-quantitative cell proliferation, differentiation related molecular expression and signal path.3. Use SPSS 19.0 statistical software to analysis.Independent sample T test to analysis the different mRNA expression among different frequency electrical stimulate. With P<0.05 for the difference was statistically significant.Results:1. The purity of primary cultured beagle chin tongue muscle satellite cells is above 80%;2. Undergoing train(5 s,3 s-on,2 s-off) electrical stimuation(5v,1 ms,80Hz), the expression of mRNA of MyoD, Myc, Cyclin D1, Wnt signaling pathways(RFZ, mfzl, mfz2, mfz3, mfz4, mfz6, mfz7, mfz8 and Wnt4, Wnt5a, Wnt7a, Wnt8, Wntl0, Wntl1,lef-1 and DVL-1) relatively decreased in C2C12.The Timpl and Timp3 and β-catenin expression increased. Change to 10 Hz and 80 Hz, Nothc pathways(Notchl, Notch2, Notch3, HERP, DEC) mRNA expression relatively increase. The mRNA expression of Sphingolipid signaling pathways(Sphkl, Sphk2, Sgpl1,Slpr1,Slpr2, Slpr5, cadherin, Pax7) also increases accordingly.3. Undergoing train(5 s,3 s-on,2 s-off) electrical stimulation(5v,1 ms,80Hz), there is no proliferation of muscle satellite cells by qRT-PCR results. The mRNA expression of Sphingolipid signaling pathways(Sphk1, Sphk2, Sgpl1, Slpr1, Slpr2, Slpr5, cadherin, Pax7) also increases undergoing lOHz train electrical stimulation, results show that the experimental group and control group proliferated andThere are no different between experimental group and control group in Pax7 gray through Western Blot. The MyoD gray level is highest in control group, followed by 80Hz group and 10Hz group.Conclusion:PERCOLL discontinuous density gradient centrifugation of primitive culture is best for the purification of beagle genioglossus satellite cells..Undergoing a fixed voltage, pulse width and duration electrical stimulation, genioglossus satellite cells has different response to in high,low frequency electric stimulation reaction from C2C12. The C2C12 proliferation and differentiation were increased undergoing electrical stimulation. Genioglossus satellite cells differentiation decreased undergoing low or high frequency electrical stimulation,The Wnt, Notch and Sphingolipid signaling pathways was activated by electrical stimulation in C2C12 cells proliferation On the contrary, genioglossus satellite cell differentiation decreased undergoing low frequency electrical stimulation., which is result from decreased Notch and increase Sphingolipid signal pathway. Undergoing high frequency stimulation, low genioglossus satellite cell differentiation resulted from Notch and Sphingolipid pathways inhibition.