摘要
目的探讨针刺足三里通过Rho相关卷曲螺旋形成蛋白激酶1(ROCK1)/蛋白激酶B(AKT)信号通路防治脓毒症后肌萎缩的作用及机制。方法8周龄SPF级雄性小鼠45只,随机分为3组,分别为对照(Sham)组、脂多糖(Lipopolysaccharide,LPS)组、针刺(LPS+acupuncture)组,其中LPS组和针刺组分别给予腹腔注射LPS诱导脓毒症模型,对照组腹腔注射等体积生理盐水。苏木素-依红染色法检测小鼠肌肉组织结构变化;酶联免疫吸附剂测定法(Enzyme linked immunosorbent assay,ELISA)测定促炎因子白细胞介素1β(Interleukin1β,IL-1β)、白细胞介素(Interleukin 6,IL-6)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α),抗炎因子白细胞介素10(Interleukin 10,IL-10)、转化生长因子-β(Transforming growth factor-β,TGF-β),以及骨骼肌损伤相关指标肌红蛋白(Myoglobin,MYO)、肌酸激酶MB同工酶(Creatine kinase MB isoenzyme,CK-MB)的变化;实时荧光定量反转录聚合酶链反应(Real-time PCR,RT-PCR)检测小鼠肌肉分解相关指标半胱氨酸天冬氨酸蛋白水解酶-3(Caspase-3)、半胱氨酸天冬氨酸蛋白水解酶-8(Caspase-8)相关基因mRNA的表达水平;蛋白免疫印迹(Western blot,WB)法检测小鼠肌肉组织中ROCK1、磷酸化蛋白激酶B(p-AKT)的表达。结果与对照组相比,LPS组小鼠肌肉组织纤维可见出现断裂、坏死,促炎因子IL-1β、IL-6、TNF-α水平均呈显著上升(P<0.01),抗炎因子IL-10、TGF-β水平均呈显著降低(P<0.01),骨骼肌损伤相关指标MYO、CK-MB的水平均显著升高(P<0.01),Caspase-3、Caspase-8 mRNA水平均显著升高(P<0.01),p-AKT和ROCK1蛋白表达水平均显著升高(P<0.01);与LPS组小鼠相比,针刺组小鼠肌肉结构相对完整,促炎因子IL-1β、IL-6、TNF-α水平均呈显著降低(P<0.01),抗炎因子IL-10、TGF-β水平均呈显著升高(P<0.01),MYO、CK-MB的水平显著降低(P<0.01),Caspase-3、Caspase-8的mRNA水平显著降低(P<0.01),p-AKT和ROCK1的蛋白表达水平显著降低(P<0.01)。结论针刺足三里可以有效地防治脓毒症肌萎缩,其作用机制可能是通过ROCK1/AKT信号通路来实现。
Objective To explore the role and mechanism of acupuncture at“Zusanli”(ST36)through ROCK1/AKT signaling pathway to prevent pyosis.Method Forty-five SPF male mice(8 weeks old)were randomly divided into three groups,which were control group(sham group),lipopolysaccharide(LPS)group and acupuncture group.The LPS group and acupuncture group were given LPS to induce sepsis model by intraperitoneal injection,and the control group was injected with the same volume of normal saline.HE staining method was used to detect the changes in the structure of the muscle tissue of the mice.ELISA method determined the inflammatory factors[interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)],anti-inflammatory factors[interleukin-10(IL-10),transforming growth factor-β(TGF-β)],the changes of myoglobin(MYO)and creatine kinase MB isoenzyme(CK-MB)and indicators related to skeletal muscle injury.RT-qPCR detected the expressions of mice muscle decomposition related indicators Caspase-3 and Caspase-8 related gene mRNA expressions.Western blot method detected ROCK1 and P-AKT in mice muscle tissue expression.Results Compared with those of the control group,the fibers of the muscle tissue in the LPS group showed rupture and necrosis,the levels of IL-1β,IL-6 and TNF-αincreased significantly(P<0.01),the levels of IL-10 and TGF-βwere significantly reduced(P<0.01)and the levels of MYO and CK-MB were significantly increased(P<0.01).The mRNA levels of Caspase-3 and Caspase-8 decreased significantly(P<0.01).P-Akt and ROCK1 protein expressions were significantly increased(P<0.01).Compared with those of the LPS group mice,the muscle structure of the acupuncture group mice was relatively completed,the levels of IL-1β,IL-6 and TNF-αwere significantly reduced(P<0.01),the levels of IL-10 and TGF-βwere increased significantly(P<0.01),the levels of MYO and CK-MB were significantly reduced significantly(P<0.01),the mRNA levels of Caspase-3 and Caspase-8 were significantly reduced(P<0.01),and the protein expression levels of P-Akt and ROCK1 were significantly reduced(P<0.01).Conclusion Acupuncture at“Zusanli”(ST36)can effectively prevent and treat sepsis muscular atrophy,and its mechanism may be achieved through ROCK1/AKT signaling pathway.
作者
陈静
李淑贤
宁怡乐
刘燕燕
赵锋利
林新锋
冼绍祥
江其龙
陈伟焘
CHEN Jing;LI Shuxian;NING Yile;LIU Yanyan;ZHAO Fengli;LIN Xinfeng;XIAN Shaoxiang;JIANG Qilong;CHEN Weitao(The First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong,China)
出处
《中华中医药学刊》
CAS
北大核心
2023年第11期123-126,I0030,I0031,共6页
Chinese Archives of Traditional Chinese Medicine
基金
国家中医临床研究基地建设项目(A6-2-2110100602)
广东省自然科学基金自由申请项目(2019A1515011010)
广东省中医药局面上项目(20192018,20211126)
广东省农村科技特派员重点派驻项目-援疆类(KTPYJ2021027)
广州市科技局-市校院联合资助(登峰医院)基础研究项目(202102010381)
广州市科技局重点项目:广州市第七批健康医疗重大科技专项(202206080015)
广州中医药大学第一附属医院“创新强院”工程科研系列青年科研人才培优项目(2017QN05)。
作者简介
陈静(1988-),女,湖北随州人,主治医师,博士在读,研究方向:中西医结合诊治危重病;通讯作者:江其龙(1986-),男,广东河源人,主治医师,博士,研究方向:神经肌肉病危重症。E-mail:jiangqilong5119@gzucm.edu.cn;通讯作者:陈伟焘(1983-),男,广东广州人,副主任医师,硕士研究生导师,博士,研究方向:中西医结合诊治危重病。E-mail:chenweitao1900@gzucm,edu.cn。
