不同频率电针介导PINK1/Parkin通路治疗STC模型大鼠的机制研究  

Mechanism of electroacupuncture with different frequencies in the regulation of PINK1/Parkin pathway activity in a rat model of slow transit constipation(STC)

作　　者：吴姝雯 刘敏 楼毅杰 崔宇胜 刘承浩[1] WU Shu-wen;LIU Min;LOU Yi-jie;CUI Yu-sheng;LIU Cheng-hao(Department of Acupuncture and Moxibustion,Zhejiang integrated traditional Chinese and Western Medicine Hospital,Zhejiang 310003,China)

机构地区：[1]浙江省中西医结合医院针灸科,杭州310003 [2]浙江中医药大学第一临床医学院,杭州310053

出　　处：《浙江中西医结合杂志》2023年第2期106-111,共6页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

基　　金：杭州市科技发展计划项目(No.20191231Y048V)。

摘　　要：目的 探讨不同频率电针介导线粒体自噬相关蛋白(PINK1)/帕金蛋白(Parkin)通路治疗慢传输型便秘(STC)模型大鼠的作用。方法 取34只8周龄雄性健康SD大鼠,采用随机数字表分为正常对照组7只和建模组27只。建模组采用洛哌丁胺混悬液灌胃法制备STC模型大鼠,建模成功后采用随机数字表分为模型对照组,2 Hz、50 Hz电针组和假电针组。2 Hz、50 Hz电针组予以电子针疗仪频率2 Hz、50 Hz,强度10 mA刺激“后三里”,假电针组针刺“后三里”但不通电,模型对照组和正常对照组仅固定处理,每天30 min,每天1次,持续14 d。碳素墨汁灌胃法检测肠道推进率;苏木素-伊红(HE)染色观察结肠组织病理改变;免疫荧光共聚焦显微镜观察结肠Cajal间质细胞(ICC)形态;免疫组化及蛋白质免疫印迹(WB)法检测结肠组织原癌基因(C-Kit)、PINK1、Parkin、自噬效应蛋白1(Beclin1)、核孔糖蛋白(p62)、自噬相关蛋白3(LC3)蛋白表达。结果 与模型对照组、假电针组比较,50 Hz电针组、2 Hz电针组增加肠道推进率[(60.63±10.21)%、(55.84±9.04)%比(48.43±7.02)%、(49.60±8.19)%,P<0.05]、ICC数量[(29.78±4.19)个、(20.45±4.37)个比(10.50±2.11)个、(11.02±2.28)个,P<0.05]、免疫荧光积分光密度(IOD)值[(2101.35±320.33)、(1936.04±307.26)比(1615.82±276.33)、(1635.97±281.52),P<0.05]、C-kit蛋白表达[(1.26±0.21)、(0.92±0.17)比(0.45±0.07)、(0.48±0.09),P<0.05]、p62蛋白表达[(1.06±0.19)、(0.87±0.13)比(0.54±0.09)、(0.55±0.10),P<0.05]。与模型对照组、假电针组比较,50 Hz电针组、2 Hz电针组降低PINK1蛋白表达[(0.37±0.08)、(0.59±0.11)比(0.85±0.17)、(0.83±0.15),P<0.05]、Parkin蛋白表达[(0.55±0.09)、(0.78±0.12)比(1.02±0.21)、(1.04±0.22),P<0.05]、Beclin1蛋白表达[(0.61±0.11)、(0.90±0.13)比(1.07±0.21)、(1.09±0.23),P<0.05]、LC3蛋白表达[(0.69±0.12)、(0.82±0.15)比(1.14±0.18)、(1.11±0.20),P<0.05]。正常对照组大鼠结肠组织、ICC形态均�Objective To evaluate the effect of electroacupuncture different frequencies on the regulation of the phosphatase and tensin homolog(PTEN)-induced kinase 1(PINK1)/Parkin pathway activity in a rat model of slow transit constipation(STC).Methods Thirty-four male Sprague-Dawley(SD) rats of 8-week-old were purchased and randomly divided into normal control(n=7) and model groups(n=27).The STC model rats were prepared by intragastric administration of loperamide suspension(3.0 mg/kg daily for 6 days)and after successfully establishing the model,these rats were further randomly divided into model control,2 or 50 Hz electroacupuncture,and sham electroacupuncture groups.The electroacupuncture groups of rats were stimulated by the electronic acupuncture instrument with a frequency of 2 Hz or 5 Hz and an intensity of 10 mA after puncturing "hou san li",whereas the sham electroacupuncture group of rats only received the "hou san li" acupuncture without adding electricity and normal and model control rats were only fixed for 30 min daily for 14 days.After that,rat intestinal propulsion rate was measured by using carbon ink gavage(2 ml) and clearance duration of time.Hematoxylin eosin(H&E) staining was used to assess changes in the colon histology.The morphology of the interstitial cells of Cajal(ICC) was also observed by immunofluorescence confocal microscope.Level of c-Kit,PINK1,Parkin,autophagy effector protein 1(Beclin1),p62,and autophagy related protein 3(LC3) proteins in the colon was assayed using immunohistochemistry and Western blotting.Results Compared with the model control and sham electroacupuncture groups of rats,both50 and 2 Hz electroacupuncture treatments induced the intestinal propulsion rate(60.63±10.21 and 55.84±9.04 vs.48.43±7.02 and 49.60±8.19%,respectively;P<0.05),ICC number(29.78±4.19 and 20.45±4.37 vs.10.50±2.11 and11.02±2.28 cells/200 time view,respectively;P<0.05),immunofluorescence integral optical density(IOD;2101.35±320.33 and 1936.04±307.26 vs.1615.82±276.33 and 1635.97±281.52,respect

关 键 词：大鼠 线粒体自噬相关蛋白 帕金蛋白 慢传输型便秘 CAJAL间质细胞 电针 

分 类 号：R245[医药卫生—针灸推拿学]