耳电针缓解功能性消化不良大...规律及对神经生长因子的影响_周静珠.pdf
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1、耳电针缓解功能性消化不良大鼠胃部高敏的效应规律及对神经生长因子的影响周静珠1,程宏亮2,陈欢1,徐万里1,朱伟坚1,周帅1,张朝晖1(1 南京医科大学第一附属医院,江苏 南京 210029;2 南京中医药大学附属医院,江苏 南京 210029)摘要 目的观察耳电针在降低功能性消化不良(FD)大鼠胃部高敏感性中的作用规律,并探讨其对神经生长因子(NGF)的影响。方法实验一:将 16 只 10 d 龄 SD 雄性幼鼠随机分为对照组和耳电针组,每组 8 只。耳电针组幼鼠通过三硝基苯磺酸灌肠诱导 FD 胃部高敏模型,第 8周龄时选取双侧耳穴“胃”穴进行连续 5 d 电针刺激;对照组幼鼠仅给予生理盐水灌
2、肠。分别记录2 组大鼠第 8 周龄干预前颈部斜方肌肌电积分、腹部回撤反射评分和耳电针组大鼠单次耳电针后、5 次耳电针后及耳电针结束后第 3,5,10 天颈部斜方肌肌电积分,分析耳电针的即时效应、累加效应和维持效应。实验二:将 40 只 10 d 龄 SD 雄性幼鼠随机分为对照组、模型组、耳电针组、电针对照组,每组 10 只。除对照组外,其余组幼鼠通过三硝基苯磺酸灌肠诱导 FD 胃部高敏模型。第 8周龄时,耳电针组选取双侧耳穴“胃”穴、电针对照组选取双侧耳垂进行连续 5 d 电针刺激。干预结束后处死各组大鼠,HE 染色观察各组胃底及远端结肠的病理改变,ELISA 法检测血浆去甲肾上腺素(NE)水
3、平,T PC 和 Western blot 法检测胃底 NGFmNA 和蛋白表达情况。结果耳电针组大鼠第 8 周龄电针前胃部球囊扩张压力为 60 和 80 mmHg 时的肌电积分和腹部回撤反射评分均显著高于对照组(P 均 0 05)。胃部球囊扩张压力为 60 和 80 mmHg 时,单次耳电针后的肌电积分均明显低于第 8 周龄电针前(P 均 0 05),5 次耳电针后的肌电积分均明显低于单次耳电针后(P 均 0 05),耳电针结束后第 3 天的肌电积分与 5 次耳电针后比较差异均无统计学意义(P 均 0 05),耳电针结束后第 5 天的肌电积分仍明显低于第 8 周龄电针前(P 均 0 05),
4、耳电针结束后第 10 天的肌电积分与第 8 周龄电针前比较差异均无统计学意义(P 均 0 05)。各组大鼠胃底、结肠 HE 染色均未见明显炎症反应。模型组和电针对照组大鼠血浆 NE 水平、胃底组织中 NGFmNA 和蛋白相对表达量均明显高于对照组(P 均 0 05),耳电针组大鼠血浆 NE 水平、胃底组织中 NGF mNA 和蛋白相对表达量均明显低于模型组和电针对照组(P 均 0 05)。结论耳电针可有效缓解功能性消化不良大鼠胃部高敏,多次干预效果更显著,且有一定的维持效应,其镇痛机制可能与调控血浆 NE 和胃底 NGF 表达有关。关键词 耳穴疗法;功能性消化不良;内脏高敏;神经生长因子doi
5、:10 3969/j issn 1008 8849 2022 22 004 中图分类号 332 文献标识码 A 文章编号 1008 8849(2022)22 3083 07 作者简介 周静珠,女,硕士,副主任中医师,研究方向为功能性胃肠病的诊治。基金项目 江苏省自然科学基金项目(BK20191065)Effect laws of ear electroacupuncture in relieving gastric hypersensitivity in rats with functionaldyspepsia and its effect on nerve growth factorZHO
6、U Jingzhu1,CHENG Hongliang2,CHEN Huan1,XU Wanli1,ZHU Weijian1,ZHOU Shuai1,ZHANG Zhaohui1(1 The First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,Jiangsu,China;2 Affiliated Hospital ofNanjing University of Traditional Chinese Medicine,Nanjing 210029,Jiangsu,China)Abstract:Objecti
7、ve It is to observe the laws of ear electroacupuncture in reducing gastric hypersensitivity in rats withfunctional dyspepsia(FD),and to explore its effect on nerve growth factor(NGF)Methods Experiment I:Sixteen10-day-old male SD rats were randomly divided into control group and ear electroacupunctur
8、e group,with 8 rats in each3803现代中西医结合杂志 Modern Journal of Integrated Traditional Chinese and Western Medicine 2022 Nov,31(22)group The young rats in the ear electroacupuncture group were given trinitrobenzene sulfonic acid by enema to induce FDgastric hypersensitivity models The EMG scores of cervi
9、cal rhomboid muscle and abdominal retraction reflex scores of therats at the 8 th week of age in the two groups before the intervention,and the EMG scores of cervical rhomboid muscle ofrats in the ear acupuncture group after once ear acupuncture,5 times ear acupuncture and on the 3 rd,5 th and 10 th
10、 dayafter the end of ear acupuncture were recorded respectively,to analyze the immediate,cumulative and maintenance effectsof ear acupuncture Experiment II:Forty 10-day-old male SD rats were randomly divided into control group,modelgroup,ear electroacupuncture group and electroacupuncture control gr
11、oup,with 10 rats in each group The FD gastric hy-persensitivity model was induced by enema with trinitrobenzene sulfonic acid in the pups of all groups except the controlgroup At the age of 8 weeks,the ear acupuncture group selected bilateral ear acupuncture points“stomach”and the con-trol group sel
12、ected bilateral ear lobes for 5 d of continuous electroacupuncture stimulation After the intervention,the rats ineach group were executed,and the pathological changes of gastric fundus and distal colon were observed by HE staining,plasma norepinephrine(NE)level was measured by ELISA,and mNA and prot
13、ein expression of gastric fundic nervegrowth factor(NGF)were detected by T-PC and Western blot esults The EMG scores and abdominal retraction re-flex scores of the rats in the ear electroacupuncture group were significantly higher than those in the control group at 60 and80 mmHg of gastric balloon d
14、ilatation pressure before ear electroacupuncture at the 8 th week of age(P 0 05)The EMGscores after once ear electroacupuncture were significantly lower than those before electroacupuncture at the 8 th week of ageat gastric balloon dilatation pressures of 60 and 80 mmHg(all P 0 05),the EMG scores af
15、ter 5 times ear electroacu-puncture were significantly lower than those after once(all P 0 05),the differences between the EMG scores on the 3 rdday after the end of the ear electroacupuncture and after 5 times ear electroacupuncture were not statistically significant(allP 0 05),and the EMG scores o
16、n the 5 th day after the end of the ear electroacupuncture were still significantly lowerthan those before ear electroacupuncture at the 8 th week of age(all P 0 05),and the differences between the EMGscores on the 10th day after the end of the ear electroacupuncture and those before the electroacup
17、uncture at 8 th week ofage were not statistically significant(all P 0 05)No obvious inflammatory reaction was found in HE staining of stom-ach fundus and colon of rats in each group The level of plasma NE and the relative expression of NGF mNA and proteinin the gastric fundus tissue of rats in the m
18、odel group and the electroacupuncture control group were significantly higher thanthose in the control group(all P 0 05),the level of plasma NE and the relative expression of NGF mNA and protein inthe gastric fundus tissue of rats in the ear electroacupuncture group were significantly lower than tho
19、se in the model groupand the electroacupuncture control group(all P 0 05)Conclusion Auricular electroacupuncture can effectively relievethe gastric hypersensitivity in rats with functional dyspepsia,and the effect of multiple interventions is more significant,andhas a certain maintenance effect Its
20、analgesic mechanism may be related to the regulation of the expression of NE in plasmaand NGF in gastric fundusKeywords:ear point therapy;functional dyspepsia;visceral hypersensitivity;nerve growth factor慢性内脏痛是功能性消化不良(functional dys-pepsia,FD)患者最常见的症状之一,其中内脏高敏是 FD 慢性内脏痛关键机制之一1 3。神经生长因子(NGF)在启动以及长期加剧
21、炎症反应的疼痛敏感性中起着关键作用,其能引起传入神经敏化,从而引起内脏高敏4。相关实验研究发现,幼年大鼠的肠道轻度炎症刺激可引起成年后出现自主神经功能紊乱,交感神经活性增加而迷走神经低张,血浆去甲肾上腺素(NE)水平升高,胃底部肌层 NGF 表达上调,继而出现内脏高敏5。在 FD 患者中也检测到胃底部 NGF mNA 表达升高6。目前内脏痛的治疗手段非常有限且效果不佳,长期服用镇痛药物易出现成瘾性,并可能出现继发性消化道损伤。耳穴疗法是祖国传统外治疗法,广泛运用临床各科疾病。基于耳郭上独特的体表迷走神经耳支分布,耳穴疗法可以通过刺激耳迷走神经来调控内脏感觉、运动7 8。耳迷走神经主要分布在耳郭
22、的耳甲区,本课题组前期研究显示单次耳电针耳甲区“胃”穴可以有效缓解 FD 大鼠内脏高敏,且此效应与调节迷走神经功能密切相关9。本研究通过建立幼鼠三硝基苯磺酸灌肠诱导 FD 胃部高敏模型,观察肌电图、动物行为学、血浆 NE、胃底 NGF mNA 和蛋白表达情况,进一步研究了耳电针的效应规律及机制。1实验材料与方法1.1实验动物购入 SPF 级别孕鼠 辽宁长生生物技术股份有限公司,许可证号:SCKX(辽)2020 4803现代中西医结合杂志 Modern Journal of Integrated Traditional Chinese and Western Medicine 2022 Nov,
23、31(22)0001,选取新生雄性大鼠进入实验。实验动物饲养条件为室温22 24,湿度50%,光照周期12 h/12 h,母鼠自由进食,幼鼠与母鼠同笼饲养。本研究获得 南京医科大学实验动物福利伦理委员会 审查同意(IACUC 2004001)。1.2主要试剂及仪器华佗牌一次性无菌针灸针(0 25 mm 25 mm,苏州医疗用品厂有限公司,中国),电针仪(CMNS6 2 型,无锡佳键,中国);肌电图电极线(AE Medical,Farmingdale,NJ,美国);肌电图放大器(EMG 100C;Biopac systems,Inc,SantaBarbara,CA,美国);大鼠 NE 酶联免疫分
24、析试剂盒(MM 203021,酶免,中国);酶标仪(MultiskanFC,Thermo,美国);低温离心机(D3024,SCILO-GEX,美国);eal time PC 仪(TL998 IV,天隆科技,中国);梯度 PC 仪(TC1000 G,SCILOGEX,美国);DYCZ 24DN 型垂直电泳装置(北京市六一仪器厂,中国);IPA 裂解液,BCA 试剂盒、GAPDHpolyclonal antibody、NGF abbit pAb(南京恩晶生物,中国)。1.3实验方法按照实验目的,为了减少实验操作对观察指标的影响,分为 2 部分实验。1.3.1实验一将 16 只 10 d 龄雄性幼鼠
25、随机分为对照组和耳电针组,每组 8 只。耳电针组幼鼠参照文献 4 制备 FD 胃部高敏模型:用 1 mL 注射器抽取配置好的三硝基苯磺酸(TNBS)/乙醇灌肠溶液(用量为 130 mg/kg,溶解入含 10%乙醇的生理盐水0 2 mL 中),然后将1 mL 注射器连接2 cm 的细软管插入幼鼠远端结肠灌肠,灌肠后保持头朝下位置 2min,以防止液体流出。对照组幼鼠灌入等量生理盐水。造模后,所有幼鼠与母鼠继续共同饲养。2 组大鼠第 7 周龄时,埋置胃部气囊(腹腔注射 2%戊巴比妥钠 30 mg/kg 麻醉后进行腹部剃毛,用外科刀片做前正中切口,打开腹腔,找到胃底,做荷包缝合,用眼科剪剪开小口,插
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