【作者】 杜广中；

【导师】 王华；

【作者基本信息】 湖北中医药大学 ， 针灸推拿学， 2011， 博士

【摘要】 目的：深入观察针灸足三里对感染后肠易激综合征(PI-IBS)模型大鼠的调节效应,探索针灸足三里不同结构及不同针灸方法对结肠高敏感和肠动力紊乱调节的作用机制及其科学方法。发掘文献记载的有创的瘢痕灸法,引入临床上具有成熟经验的射频热凝技术。方法：选用Wistar雌性大鼠,建立TNBS感染后腹泻型IBS大鼠模型,随机分为4组：模型组、模型+电针组、模型+瘢痕灸组和模型+射频针灸组以及空白对照组,其中电针与射频针灸的针尖位于足三里胫骨前肌、瘢痕灸位于局部皮肤。大鼠模型采用直肠TNBS灌肠,而对照组应用生理盐水灌肠。所有实验均在灌肠后第28天进行。电针,瘢痕灸,射频针灸均取后三里；其参数为电针采用HansLH202型电针治疗仪,左右双穴,刺入3mm,疏密波,2/15HZ,强度1mA,以大鼠双下肢轻微抖动为宜,15min/次,1次/d,连续6d；瘢痕灸右侧穴位,采用正四面体形、重40mg、边长6.26mm、比重1.38mg/m3艾炷,灸3壮,只灸1次；射频针灸右侧穴位,刺入3mm,时间60s+60s+40s+40s,参数75℃,阻抗640Ω,只射频1次。第33天检测内脏敏感性AWR积分,全胃肠传输功能,结肠纵行肌条平均张力、最大振幅、平均频率,酶联免疫吸附法测定血浆5-HT、SP、CGRP等,来观察针灸逆转肠道内脏高敏感和肠动力紊乱的调节效应,探索其作用机制,并对足三里穴不同结构及不同针灸方法加以比较。结果：1、AWR评分。模型组大于对照组(P<0.005)；针灸足三里的不同结构后,均不同程度地降低评分(P<0.05-0.005)；三组间没有显著差异(P>0.05)；但射频针灸组在20mmHg直结肠球囊扩张时,没有显著改善(P>0.05)。2、针灸后足三里局部组织病理。对照组基本正常；造模后出现部分肌纤维变性、肌纤维断裂；电针后发现少量炎细胞,间皮轻度增生；瘢痕灸后出现皮肤组织坏死并慢性炎症；射频针灸后致其局部组织坏死。3、全胃肠传输功能。大鼠全胃肠活性炭黑便排出时间,模型组小于对照组(P<0.05)；针灸足三里的不同结构后,均不同程度地延长了全胃肠活性炭黑便排出时间(P<0.05-0.01)；三组间射频针灸(P<0.05)>瘢痕灸≥电针。4、大鼠结肠纵行肌条平均张力。模型组大于对照组(P<0.05)；针灸足三里的不同结构后,均不同程度地降低了大鼠结肠纵行肌条的平均张力(P<0.05-0.01),三组间没有显著差异(P>0.05)。Ach作用后,随着浓度累积,其平均张力逐渐增高；其中对照组与模型组在10-6以后,增高极显著(P<0.005)；而针灸干预的三组,在10-5以后,始显著增高(P<0.05-0.005)；三组间比较,没有显著差异。5、大鼠结肠纵行肌条最大振幅。模型组大于对照组(P<0.005)；针灸足三里的不同结构后,均不同程度地降低了大鼠结肠纵行肌条的最大振幅(P<0.05-0.01),三组间比较射频针灸组较电针、瘢痕灸组更显著降低(P<0.05)。Ach作用后,随着浓度累积,其最大幅度逐渐增大,于10-8到最大值；随后,从10-7始肌条逐渐发生强制性收缩,其最大幅度逐渐减小。其中对照组与模型组在10-8,增高显著(P<0.025-0.005),在10-6以后直至10-4,显著减小(P<0.025-0.005)；而针灸干预的三组,电针与瘢痕灸组10-5以后,显著减小(P<0.01-0.005),射频针灸组则在10-6以后,显著减小(P<0.05-0.005)。三组间比较,10-9、10-8射频针灸组显著低于电针与瘢痕灸组(P<0.025-0.01),10-7射频针灸组显著低于电针组(P<0.01),10-6、10-5三组间没有显著差异(P>0.05),10-4瘢痕灸和射频针灸法两组显著低于电针组(P<0.01)。6、大鼠结肠纵行肌条平均频率。实验的5组间没有显著差异(P>0.05)。Ach作用后,随着浓度累积,其平均频率逐渐增大,基本在其10-6达到最大值(P>0.05)；随后,从10-5始肌条逐渐发生强制性收缩,其频率逐渐减小；其中对照组在10-6以后极显著变化(P<0.005),模型组与电针、瘢痕灸三组只有10-4极显著变化(P<0.005),而射频针灸组只有变化趋势(P>0.05)。三组间比较,10-9至10-5三组间没有显著差异(P>0.05),10-4瘢痕灸和射频针灸两组显著低于电针组(P<0.025-0.005),瘢痕灸组与电针、射频针灸组间也有变化(P<0.025)。7, PI-IBS模型大鼠血浆5-HT、SP水平显著增高,CGRP显著降低(P<0.01-0.005)；针灸后三组均可显著降低5-HT、SP水平,显著增高CGRP水平(P<0.05-0.005)；三组间5-HT、SP、CGRP比较没有明显差异。射频针灸组SP、CGRP水平与对照组比较有改善,但没有恢复到正常水平(P<0.05-0.025)。8、不同质量艾炷灸的温度时间(mass-temperature-time,MTT)变化规律：艾炷燃烧的温度最大值及其时刻、艾灸持续时间,与艾炷质量成正相关。即随着艾炷质量的增加,其燃烧的温度最大值和艾灸持续时间也增大,出现温度最大值时刻的时间延长。9、艾炷灸温度-时间(Temperature-time, TT)变化规律：呈单波峰,均有缓慢上升期、急速上升期、急速下降期和缓慢下降期。随着艾炷质量的增大,缓慢上升期和缓慢下降期均延长,其中缓慢下降期更明显；同时,波峰稳定时间随艾炷质量的增大而延长,呈现平台期。结论：1、针灸足三里可以有效逆转IBS结肠内脏高敏感和运动功能紊乱异常状态。2、针灸足三里逆转IBS结肠运动功能紊乱主要通过调节其纵行肌条张力和振幅来实现,而频率没有显著变化。3、针灸足三里解剖结构的皮肤,尤其是瘢痕灸可有效逆转IBS结肠内脏高敏感和运动功能紊乱,以前者更明显。4、针灸足三里解剖结构的胫骨前肌,尤其是射频针灸可有效逆转IBS结肠运动功能紊乱和内脏高敏感,以前者更明显。5、5-HT、SP和CGRP是针灸足三里调控IBS已经失常的神经-免疫-内分泌网络的中间介质。6、艾炷灸燃烧的温度最大值及其时刻、艾灸持续时间,与其质量正相关；艾炷灸温度-时间变化规律：单波峰,有缓慢上升期、急速上升期、急速下降期和缓慢下降期。更多还原

【Abstract】 Objective:To observe the effective regulation of acupuncture-moxibustion on ST36 for post infectious irritable bowel syndrome in rats, and investigate the method and working mechanism of different organizational structure for colonic visceral hypersensitivity and gut disfunction. To search the scar-producing moxibustion in classical documents, and draw the technique of radio frequency into acupuncture.Methods:Wistar famale rats have been used in this study, and then the animals had been randomly divided into 5 groups:control, post infectious irritable bowel syndrome model, electro-acupuncture, scar-producing moxibustion and radio frequency group. The needlepoint of electroacupuncture and radio frequency was penetrated in tibialis anterior, and the moxa cone was puted on skin. Post infectious irritable bowel syndrome was induced by intracolonic instillation of trinitro-benzene-sulfonic acid in diarrhea model group and the control group was using same method by intracolonic instillation of normal salt solution. All experiments were conducted at the 28th day of intracolonic instillation. The point ST36 was used in this study for 15min in electro-acupuncture or 3 moxa cone with 40mg in scar-producing moxibustion or 75℃and 200sec in radio frequency. The scores of abdorminal withdrawal reflex induced by colorectal distention for visceral hypersensitivity was used in this study at the 33th day of intracolonic instillation, and total gastrointestinal transit times was used for gut disfunction at same day, also mean tone and frequency or maximum amplitude of longitudinal colonic smooth muscle strips was used like just before to observe the effective regulation of acupuncture-moxibustion on ST36. The rats concentrations of substance P and 5-HT and calcitonin gene-related peptide in plasma were detected by a double-antibody sandwich enzyme-linked immunosorbent assay to investigate working mechanism of acupunture-moxibustion.Result:1. The scores of abdorminal withdrawal reflex (AWR). The scores of model group was more than control group signif icantly(P<0.005), scores of EA or SP-Moxi or RF group have decerased significantly (P<0.05-0.005), and then did not change significantly among last 3 groups (P>0.05), but it was on significant change between the group RF and cotrol in 20mmHg by colorectal distention (P>0.05)2. The histopathology of acupoint ST36 after acupuncture and moxibustion. There are some inflammatory cells and a few mesothelium hyperplasia after eletroacupuncture, tissue necrosis of epithelium and chronic inflammation after scar-producing moxibustion, and tissue necrosis of tibialis anterior after radio frequency. 3. Total gastrointestinal transit function. The times of activated carbon discharged of model group was less than control group significantly (P<0.05), times of EA or SP-moxi or RF group were longer significantly (P<0.05-0.01) than model group, and then it was better significantly between RF and the other two group (P<0.05)4. Mean tone of longitudinal colonic smooth muscle strips. The tone of model increased significantly than control (P<0.05), by means of acupuncture-moxi bust ion therapy on ST36 acupoint, The tone decreased significantly (P<0.05-0.01),and then did not change significantly among last 3 groups (P>0.05). In Acetylcholine solution, the mean tone increased gradually due to more and more concentration, after 10-6, it increased significantly (P<0.005) than basic in group of control and model, but after 10-5, it began to increase significantly(P<0.05-0.005) than basic in group of EA and SP-Moxi and RF, and then did not change significantly among last 3 groups (P>0.05)5. Maximum amplitude of longitudinal colonic smooth muscle strips. Group of model increased significantly than control (P<0.005),the maximum amplitude in group of EA and SP-Moxi and RF decreased significantly (P<0.05-0.01), and then it decreased significantly between RF and the other two group (P<0.05)In Acetylcholine solution, the maximum amplitude increased gradually due to more and more concentration until 10-8, its maximum was in 10-8 Acetylcholine solution. After 10-7, the maximum amplitude decreased gradually due to tetanic contraction of strips. In 10-8, it increased significantly (P<0.05-0.01) in group of control and model, After 10-6, it decreased significantly than basic (P<0.025-0.005).But After 10-5, it decreased significantly than its basic in group of EA and SP-moxi (P<0.05-0.005), After 10-6, it decreased significantly than its basic in group of RF (P<0.05-0.005).Compared among 3 groups, the group of RF decreased significantly than the group of EA and SP-moxi in 10-9 and 10-8 (P<0.025-0.01), the group of RF decreased significantly than the group of EA in 10-7 (P<0.01), no change significantly among 3 groups in 10-6 and 10-5 (P>0.05), the group of SP-moxi and RF decreased significantly than the group of EA in 10-4 (P<0.01)6. Mean frequency of longitudinal colonic smooth muscle strips. It did not effect the mean frequency among 5 groups (P>0.05). In Acetylcholine solution, the mean frequency increased gradually due to more and more concentration until 10-6. After 10-5, the mean frequency decreased gradually due to tetanic contraction of strips. After 10-5, it changed significantly (P<0.005) in group of control, but in 10-6, it changed significantly than basic (P<0.005), the group of RF did not effect. Contrasted among 3 groups, there were not much difference in Acetylcholine solution concentration from 10-9 to 10-5 (P>0.05), but in 10-4, the group of SP-moxi and RF decreased significantly than the group of EA (P<0.025-0.005), the group of SP-moxi changed significantly than the group of EA and RF in 10-4 (P<0.025).7. The plasma 5-hydroxytryptamine and substance P increased significantly than befor in PI-IBS model rats (P<0.01-0.005), calcitonin gene-related peptide decreased significantly than beforc. The plasma 5-HT and SP decreased significantly than before in the group of EA and SP-moxi and RF rats, CGRP increased significantly than before (P<0.05-0.005). there were not much difference contrasted among last 3 groups, the RF improved than control group in SP and CGRP,but did not still recover (P<0.05-0.025)8. The rule of variation tendency of mass-temperature-time in moxa cone. The maximun temperature or moment or duration of moxa-cone were positive in pertinency beween mass and others.9. The rule of variation tendency of temperature-time. The curve of temperature-time of one moxa cone was a single-crest wave, it consisted of slowly rising stage and quickly rising stage and slowly descent stage and quickly descent stage.Conclusion:1. It can treat colonic visceral hypersensitivity and gut disfunction of IBS with acupuncture and moxibustion.2. By means of regulation of tone and amplitude of longitudinal colonic smooth muscle strips, it can treat gut disfunction of IBS with acupuncture and moxibustion.3. The skin of ST36 with scar-producing moxibustion can treat colonic visceral hypersensitivity and gut disfunction of IBS, but anterior one is better than post.4. The tibialis anterior of ST36 with radio frequency can treat gut disfunction and colonic visceral hypersensitivity of IBS, but anterior one is better than post.5. By means of ST36 of IBS with acupuncture and moxibuston,5-HT, SP and CGRP were intermedia of disturbed nerve-internalsecretion-immunity network.6. The maximun temperature or moment or duration of moxa-cone were positive in pertinency beween mass and others. Curve of variation tendency of temperature-time was a single-crest wave, it consisted of slowly rising stage and quickly rising stage and slowly descent stage and quickly descent stage.更多还原