基于PI3K/Akt通路探讨电针血清对肌卫星细胞自噬的影响  

Effects of electroacupuncture serum on autophagy of muscle satellite cells based on PI3K/Akt signaling pathway

作　　者：刘通[1] 于佳妮[2] 刘悦[1] 邝伟川[1] 陈欢 王小寅[1] 文希[1] 江烨[1] 邱晓佳[1] 曾遥 邹汉宏 LIU Tong;YU Jia-ni;LIU Yue;KUANG Wei-chuan;CHEN Huan;WANG Xiao-yin;WEN Xi;JIANG Ye;QIU Xiao-jia;ZENG YAO;ZOU Han-hong(Department of Acupuncture and Rehabilitation,Guangdong Second Hospital of Traditional Chinese Medicine,Guangzhou 510095,China;Department of Rehabilitation,Guangdong Hospital of Traditional Chinese Medicine,Guangzhou 510120,China;Department of Acupuncture,The First Affiliated Hospital of Nanjing Medical University,Nanjing 210000,China)

机构地区：[1]广东省第二中医院针灸康复科,广州510095 [2]广东省中医院康复科,广州510120 [3]南京医科大学第一附属医院针灸科,南京210000

出　　处：《中华中医药杂志》2020年第7期3642-3646,共5页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金青年科学基金项目(No.81704179,No.81503641);广东省中医药局面上项目(No.20191018,No.20191019)。

摘　　要：目的:观察电针血清调控磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)通路对肌卫星细胞自噬的影响。方法:原代培养多裂肌卫星细胞,选择传至第3代的肌卫星细胞,按本课题组前期的实验方法获取电针血清,先以空白血清(无血清,仅含培养基)干预细胞12h诱导肌卫星细胞自噬,后将各组细胞随机分为空白血清组、10%电针血清组、10%电针血清+LY294002组、3-MA组,各组分别干预12、24h后,采用CCK-8检测各组细胞的增殖能力,Western blot检测各组细胞蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)、Beclin1、微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)、Pax7蛋白的表达变化。结果:CCK-8结果显示,培养12h时,10%电针血清组细胞增殖能力优于其余3组(P<0.05),培养24h时,10%电针血清组细胞增殖能力优于其余3组及12h时电针血清组(P<0.01,P<0.05),10%电针血清+LY294002组和3-MA组优于空白血清组(P<0.01)。Western blot结果显示:12h后,与空白血清组、10%电针血清+LY294002组比较,10%电针血清组和3-MA组p-Akt、Pax7蛋白表达显著升高(P<0.01,P<0.05),Beclin1和L C3-Ⅱ蛋白表达显著降低(P<0.01,P<0.05)。24h后,与空白血清组、10%电针血清+LY294002组比较,10%电针血清组和3-MA组p-Akt蛋白表达显著升高(P<0.01),Beclin1和LC3-Ⅱ蛋白表达显著降低(P<0.01),10%电针血清组Pax7蛋白表达显著升高(P<0.01);3-MA组Pax7蛋白表达显著高于空白血清组(P<0.01)。与12h时本组比较,空白血清组Beclin1、LC3-Ⅱ蛋白表达均显著升高(P<0.01),Pax7蛋白表达显著降低(P<0.01),10%电针血清组Pax7蛋白表达显著升高(P<0.05)。结论:电针血清可改善肌卫星细胞的过度自噬,保护肌卫星细胞的增殖,该作用可能与激活PI3K/Akt通路有关。Objective:To observe the effects of electroacupuncture serum on autophagy of muscle satellite cells based on PI3K/Akt signaling pathway.Methods:Primary multifidus muscle satellite cells were isolated and cultured,and the third generation of muscle satellite cells were selected.The electroacupuncture serum was obtained according to experimental methods as our previous research.The blank serum(culture medium only without serum)was used for 12h to induce autophagy of muscle satellite cells.Then the cells were randomly divided into blank serum group,10% electroacupuncture serum group,10% electroacupuncture serum+LY294002 group and 3-MA group.After 12h and 24h of intervention,the proliferation capacity was detected by CCK-8,and expressions of Akt,p-Akt,Beclin1,LC3-Ⅱ,Pax7 were detected by Western blot.Results:CCK-8 results indicated that proliferation capacity in 10% electroacupuncture serum group at 12h was better than other groups(P<0.05).The proliferation capacity in 10%electroacupuncture serum group at 24h was better than that of the other three groups and the electroacupuncture serum group at 12h(P<0.01,P<0.05),and 10%electroacupuncture serum group+LY294002 group and 3-MA group were better than blank serum group(P<0.01).Western blot results demonstrated that,after 12h intervention,the expression of p-Akt and Pax7 in the 10%electroacupuncture serum group and 3-MA group were significantly higher than blank serum group and 10% electroacupuncture serum group+LY294002 group(P<0.01,P<0.05),while the expressions of Beclin1 and LC3-Ⅱwere significantly decreased(P<0.01,P<0.05).After 24h intervention,the expression of p-Akt in the 10% electroacupuncture serum group and 3-MA group were significantly higher than blank serum group and 10% electroacupuncture serum group+LY294002 group(P<0.01),while the expressions of Beclin1 and LC3-Ⅱsignificantly decreased(P<0.01).The expression of Pax7 in 10% electroacupuncture serum group was higher than blank serum group and 10% electroacupuncture serum group+LY294002 group(P<0.01),wh

关 键 词：电针血清 肌卫星细胞 PI3K/AKT通路 自噬 BECLIN1 LC3-Ⅱ Pax7 

分 类 号：R245[医药卫生—针灸推拿学]