电针对肝部分切除术后小鼠肝再生的影响及Notch信号通路在其中的作用

Effect of electroacupuncture on liver regeneration after partial hepatectomy in mice and role of Notch signaling pathway

目的评价电针对肝部分切除术后小鼠肝再生的影响及Notch信号通路在其中的作用。方法SPF级健康雄性C57BL/6小鼠36只,6周龄,体质量20~22 g。采用随机数字表法分为6组(n=6):假手术组(S组)、肝部分切除术组(PH组)、非穴位电针+肝部分切除术组(NPH组)、肝部分切除术+Fli-06组(PH+F组)、穴位电针+肝部分切除术组(EPH组)和穴位电针+肝部分切除术+Fli-06组(EPH+F组)。除S组以外,其余组小鼠均接受肝部分切除术;PH+F组和EPH+F组术前2 d开始腹腔注射Fli-064.8 mg/kg,1次/d,直至小鼠处死,其余组注射等体积0.9%氯化钠溶液;EPH组术后即刻开始电针刺激,选择双侧足三里穴,连续波,频率2 Hz,强度1 mA,以后肢出现轻微颤动为度,每次15 min,1次/d,连续3 d。肝部分切除术后第2天时麻醉小鼠,眼球取血样,采用全自动生化分析仪测定血清ALT和AST浓度,ELISA法测定表皮生长因子(EGF)和肝细胞生长因子(HGF)浓度。随后处死小鼠取肝组织,计算肝质量/体质量比,免疫组织化学染色法检测肝脏Ki-67表达;Western blot法检测增殖细胞核抗原(PCNA)、G1/S-物异性同期蛋白-D1(CCND1)、Notch胞内结构域(NICD)和缺氧诱导因子-1α(HIF-1α)的表达;RT-PCR法检测Notch1、锯齿状典型Notch配体1(Jagged1)和Hes家族bHLH转录因子1(Hes1)mRNA表达。结果与S组比较,PH组血清ALT、AST、EGF和HGF浓度升高,肝脏Notch1、Jagged1、Hes1 mRNA表达和Ki-67、PCNA、CCND1、NICD表达上调(P<0.05或0.01);与PH组比较,EPH组肝质量/体质量比、血清EGF和HGF浓度升高,血清ALT和AST浓度降低,肝脏Notch1、Jagged1、Hes1 mRNA表达和Ki-67、PCNA、CCND1、NICD、HIF-1α表达上调,PH+F组肝质量/体质量比、血清HGF浓度降低,血清ALT和AST浓度升高,肝脏Jagged1、Hes1 mRNA和Ki-67、PCNA、CCND1、NICD、HIF-1α表达下调(P<0.05或0.01);与EPH组比较,EPH+F组肝质量/体质量比、血清EGF和HGF浓度降低,血清ALT和AST浓度升高,肝脏Notch1、Jagged1、Hes1 mRNA和Ki-67、PCNA、CCND1、NICD和HIF-1α表达下调(P<0.05或0.01)。结论电针足三里穴促进肝部分切除术后小鼠肝再生,其机制可能与激活Notch信号通路有关。

Objective To evaluate the effect of electroacupuncture on liver regeneration after partial hepatectomy in mice and the role of the Notch signaling pathway.Methods Thirty-six SPF healthy male C57BL/6 mice,aged 6 weeks,weighing 20-22 g,were divided into 6 groups(n=6 each)using a random number table method:sham operation group(group S),partial hepatectomy group(group PH),non-acupoint electroacupuncture+partial hepatectomy group(group NPH),partial hepatectomy+Fli-06 group(group PH+F),acupoint electroacupuncture+partial hepatectomy group(group EPH),and acupoint electroacupuncture+partial hepatectomy+Fli-06 group(group EPH+F).All the mice except for group S underwent partial hepatectomy.Fli-064.8 mg/kg was intraperitoneally injected starting from 2 days before surgery,once a day,until the mice were sacrificed in group PH+F and group EPH+F,while the equal volume of 0.9%sodium chloride solution was injected in the other groups.In EPH group,electroacupuncture of bilateral"Zusanli"acupoints lasting for 15 min was performed using continuous waves with a frequency of 2 Hz and an intensity of 1 mA once a day starting from the time point immediately after surgery for 3 consecutive days.Mice were anesthetized at day 2 after partial hepatectomy,and blood samples were taken from the eyeball for determination of the serum alanine transaminase(ALT)and aspartate aminotransferase(AST)concentrations(using a fully automated biochemical analyzer)and concentrations of serum epidermal growth factor(EGF)and hepatocyte growth factor(HGF)(by enzyme-linked immunosorbent assay).The mice were subsequently sacrificed and liver tissues were taken for calculation of the liver mass to body mass ratio and for determination of the expression of liver proliferation marker Ki-67(by immunohistochemical staining),proliferating cell nuclear antigen(PCNA),cyclin D1(CCND1),Notch Intracellular Domain(NICD),and hypoxia-inducible factor-1alpha(HIF-1α)(using Western blot)and Notch1,jagged canonical Notch ligand 1(Jagged1)and hairy and enhancer of split 1(Hes1)mRNA(by real-time polymerase chain reaction).Results Compared with group S,the serum ALT,AST,EGF and HGF concentrations were significantly increased,and the expression of hepatic Notch1,Jagged1 and Hes1 mRNA and Ki-67,PCNA,CCND1 and NICD was up-regulated in group PH(P<0.05 or 0.01).Compared with group PH,the liver mass to body mass ratio and serum EGF and HGF concentrations were significantly increased,the serum ALT and AST concentrations were decreased,and the expression of hepatic Notch1,Jagged1,Hes1 mRNA and Ki-67,PCNA,CCND1,NICD and HIF-1αwas up-regulated in group EPH,and the liver mass to body mass ratio and the serum HGF concentrations were significantly decreased,the serum ALT and AST concentrations were increased,and the expression of hepatic Jagged1 and Hes1 mRNA and Ki-67,PCNA,CCND1,NICD,and HIF-1αwas down-regulated in group PH+F(P<0.05 or 0.01).Compared with group EPH,the liver mass to body mass ratio and serum EGF and HGF concentrations were significantly decreased,the serum ALT and AST concentrations were increased,and the expression of hepatic Notch1,Jagged1,Hes1 mRNA and Ki-67,PCNA,CCND1,NICD and HIF-1αwas down-regulated in group EPH+F(P<0.01).Conclusions Electroacupuncture at Zusanli acupoint promotes liver regeneration after partial hepatectomy in mice,and the mechanism may be related to the activation of the Notch signaling pathway.