Effect Of Electroacupuncture At "Point" On Proliferation Of PI3K / Akt / MTOR Signaling Pathway

The multifidus muscles, located nearest to the spine, is one of the largest vertebral side muscle. It is composed of multiple muscle packages, and arise from the lumbar vertebral plate and spines, end with the back of the sacrum. It is very important to maintain the stability of lumbar spine. Injury, atrophy and dysfunction of the multifidus muscles is closely related to the occurrence of various acute or chronic low back pain. Thus, there is a significant importance to promote regeneration and functional reconstruction after injury. Muscle satellite cells belong to skeletal muscle stem cell, and during the process of muscle repair, it is essential of the muscle satellite cells on activation, proliferation, differentiation and myogenic fusion. In physiological conditions, MSCs is usually in a stationary state, once the cells of the basement membrane is destroyed, MSCs began to active, proliferate, differentiate, and fuse with each other to form new muscle fibers. Therefore, study on MSCs has an important value to further understand the pathogenesis and repair mechanism of muscle diseases.In recent years, majority of researches have verified that acupuncture can bring forward the peak period of muscle satellite cell proliferation to promote regeneration of the muscle and accelerate the damage healing process. The "Weizhong" acupoints belong to the bladder meridian and have a special effect for the disease of back and low back. "Treating lumbar-back problems by Weizhong" is a summary of experience of the "Weizhong" acupoints for the disease of back and low back. Our previous studies have proved that electroacupuncture on "Weizhong" acupoints could accelerate proliferation, differentiation, and fusion of the muscle satellite cells, however, the mechanism still need further exploration.In our last study, we found that it was better on 4th day of electroacupuncture on "Weizhong" acupoint to improve the expression of Myod, which indicated that it was more active of the "Weizhong" acupoint at the beginning of the muscle recovery. Therefore, this research will focus on the effect of "Weizhong" acupoints for the regeneration of multifidus muscle injury on 4d from in vivo and in vitro. Through the detect the expression of protein Pax7, Myod and PI3K/Akt/mTOR signaling molecule, we will further explore the mechanism of "Weizhong" acupoint for the regeneration of multifidus muscle injury.Part 1:The Effect of Electroacupuncture on "Weizhong" Acupoints for theExpression of Pax7 and Myod during Regeneration of Multifidus MuscleObjective:To explore the effect of acupuncture on "Weizhong" acupoint for the regeneration of multifidus muscle injury in vitro.Methods:Male SD rats were randomly divided into normal group (NG), model group (MG), model control group (CG), "Weizhong" acupoint (BL40) electroacupuncture group (WG), "Shenshu" acupoint (BL23) electroacupuncture group (SG). Multifidus muscle (L4 and L5 sections) toxicity was established by injecting 0.5% bupivacaine hydrochloride, and normal saline injection as control. The WG and LG group accepted electro-acupuncture for 4d after 24h of modeling,1 time per day. The multifidus muscle of each rodent was stained with HE to observe pathological changes, and western-blot was used to observe Pax7 and Myod expression.Results:(1) HE staining analysis indicated that the thin and even muscle fibers were in alignment, even there were slight edema in some of the CG group cells. Widely muscle fibers degeneration and necrosis with macrophage infiltration could be seen in MG group. Large muscle fibers regeneration with the nucleus migrated to the round of the cell could be seen in WG and LG group.(2) Western-blot analysis showed that no significant difference was found between MG group and ZG、CG group in terms with Myod expression (P>0.05). The Myod expression in WG and SG group were superior to that of other three groups (P<0.05) while WG group was superior to SG group (P<0.05). No difference was found among all the groups when it came to Pax7 expression.Conclusions:(1) Both electroacupuncture at "Weizhong" and "Shenshu" acupoint can obviously increase the expression of Myod to promote myogenic differentiation of muscle satellite cells. However, no obvious effect of Pax7 expression was found, probably because the Pax7 protein expression appeared down-regulated during the beginning of differentiation while the Myod protein was still high expressed.(2) "Weizhong" acupoints is better than "Shenshu" acupoints at the beginning of the muscle recovery.Part 2:Isolation, Culture and Identification of Muscle Satellite Cells from Single Muscle FibersObjective:To isolate and identify muscle satellite cells from single muscle fibersMethods:Isolate single muscle satellite cells from the multifidus muscle of rat using the method of single muscle fibers, and induce differentiation by low serum. Identify the isolated muscle satellite cells through different muscles related specific protein markers during proliferation and differentiation stage.Results:(1) Morphological observation:Primary cell are round and high refraction under microscope after digestion. After 12h of culture, most of the cells have adhered, and stretched as spindle. After 3 days, cells begin proliferate and appear directional arrangement on 5 days. After 8 days, cells could fuse with each other to form along a definite direction and differentiate to myotubes.(2) Immunofluorescent identification:Pax7 and Myod were positively expressed in nuclear in the process of proliferation while Desmin was positively expressed in cytoplasm at the beginning of differentiation. Myotubes could be seen after 7 days of settle, the same as after cultured with 2% horse serum.Conclusions:Mutifidus muscle satellite cells could be successfully isolated by single muscle fibers. Muscles related specific protein markers are positively expressed, and the cells could differentiate to muscle cells after myogenic inducement.Part 3:Effect of Electroacupuncture Serum on Proliferation of Multifidus Muscle Satellite Cells and PI3K/Akt/mTOR pathwayObjective:To observe the effect of electroacupuncture serum on proliferation of multifidus muscle satellite cells and PI3K/Akt/mTOR pathway.Methods:Serum were selected from Part 1. Multifidus muscle satellite cells were randomly assigned into control group (CG), model group (MG), weizhong (BL40) serum group (WG), weizhong (BL40) serum plus LY294002 group(WLG), shenshu(BL23) serum group (SG) and shenshu(BL23) serum plus LY294002 group (SLG). LY294002 was added 2h in advance. CCK-8 and Edu were applied to explore the effect of electroacupuncture serum on proliferation of multifidus muscle satellite cells. Western-blot and RT-PCR were used to detect the expression of Pax7、Myod and PI3K/Akt/mTOR pathway signal molecule.Results:(1) CCK-8 analysis indicated that OD value in CG、MG、WG and SG group was better than that of blank group (P<0.01), MG group was better than CG group (P<0.01), WG and SG group were better than MG group (P<0.01). The OD value was significant reduced (P<0.01) in WG and SG group when LY294002 was added. No significant difference was found between WG and SG group (P>0.05).(2) Edu analysis showed that the percent of edu positive cells in MG、WG and SG group was superior to CG group (P<0.05, P<0.01, P<0.01), WG and SG group was superior to MG group (P<0.01, P<0.05).The percent of edu positive cells was significant reduced (P<0.01, P<0.05) in WG and SG group when LY294002 was added. No significant difference was found between WG and SG group (P>0.05).(3) Western-blot analysis showed that no difference was found of Pax7 among all groups. The expression of Myod in MG group was better than CG group (P<0.05), WG and SG group was superior to MG group (P<0.05, P<0.01). The expression of Myod was significant reduced (P<0.05, P<0.01) in WG and SG group when LY294002 was added. No significant difference was found between WG and SG group (P>0.05).The expression of p-Akt in MG group was better than CG group (P<0.05), WG and SG group was superior to MG group (P<0.01, P<0.05). The expression of p-Akt was significant reduced (P<0.01, P<0.01) in WG and SG group when LY294002 was added. No significant difference was found between WG and SG group (P>0.05).The expression of p-mTOR in MG group was better than CG group, No significant difference was found among MG、WG and SG group (P>0.05).(4) RT-PCR analysis indicated that no significant difference was found among all the groups of Pax7 and Myod. On the contrary to western-blot, the expression of Pax7 mRNA and Myod mRNA in MG、WG and SG group existed a tendency to below the ZG group; the WG and SG group also showed the same tendency when LY294002 was added.Conclusions:Both the "Weizhong" (BL40) serum and "Shenshu" (BL23) serum could promote proliferation and differentiation of multifidus muscle satellite cells, which may be related with PI3K/Akt pathway. However, the effect of electroacupuncture serum for Pax7 mRNA and Myod mRNA need further study.(2) There maybe other pathways of the two acupoints except for serum.