摘要
目的:
肠易激综合征(irritable bowel syndrome,IBS)已经成为最常见的胃肠功能性疾病之一。IBS的发病涉及多方面因素,其病因和发病机制迄今尚未完全阐明,一般认为该病是一种具个体差异性的多病因、异质性疾病。近年多数研究发现,内脏敏感性增高是IBS最重要的病理生理特征之一,是IBS病人症状产生的基础和症状多样化的原因。
目前尚没有一种西药或单一疗法对IBS完全有效。而中药因具有多靶点、整体调节及副作用小的特点,在治疗IBS当中具独特优势,临床报道疗效显著。痛泻要方是中医经典方剂,其主治脾虚肝旺之“痛泻”与现代医学之IBS的症状特点很相似,因此该方被广泛应用于IBS的治疗,并取得良好疗效。
本课题参考目前较为成熟的IBS大鼠模型制作经验,建立了内脏高敏感性IBS大鼠模型。在此模型的基础上进一步探讨痛泻要方对实验性肠易激综合征的治疗作用及作用机制。不仅为IBS临床用药提供实验依据,而且对古方的开发及研制具有重要意义。
方法:本课题分为三部分
第一部分:内脏高敏感性IBS大鼠模型的建立及评价。
采用结肠醋酸慢性刺激法制作内脏高敏感性的IBS大鼠模型。将新生大鼠分为正常组和模型组。模型组:从出生第8d至21d每天予以直肠内醋酸刺激。正常组:每天直肠内予生理盐水作为对照。测量两组大鼠每周的体重,并分别于刺激停止2周及4周后进行肠道敏感性指标检测,旨在评价大鼠模型的复制是否成功。
第二部分:痛泻要方对内脏高敏性IBS大鼠的治疗作用。
造模方法同第一部分。造模成功后,将大鼠分为正常组(A)、模型组(B)、痛泻要方低剂量组(C)、痛泻要方高剂量组(D)及得舒特组(E),第36d龄始予以相应药物灌胃2周。分别于灌胃前后检测各组大鼠肠道敏感性指标,以及观察灌胃前后各组大鼠排便粒数及粪便含水量,旨在评价痛泻要方的治疗效果。
第三部分:痛泻要方治疗IBS的作用机制。
造模及动物分组给药同第二部分。运用免疫组织化学方法对模型大鼠回盲部、结肠及脊髓SP、VIP含量进行检测。旨在初步阐明痛泻要方治疗IBS的部分作用机制。
结果:
第一部分:两组大鼠实验后均未出现异常活动,未见明显腹泻、粪便不成形。第35d和第50d龄模型组大鼠抬腹和拱背的压力容量阈值明显低于正常组(P<0.01),在注水量为1.0ml时腹壁反射次数高于正常组(P<0.01),说明模型组的肠道敏感性较正常组明显增高,且此肠道高敏感性长时间存在。模型复制成功。
第二部分:模型组大鼠粪便表面较为粘滞、边缘松散,排便粒数增多,粪便含水量升高,与正常组相比有显著性差异(P<0.05或P<0.01)。而各治疗组则有不同程度的改善,尤以痛泻要方高剂量组更明显。第35d各实验组大鼠肠道敏感性指标异常提示造模成功。药物干预后(第50d),痛泻要方低、高剂量组及得舒特组大鼠肠道敏感性均有不同程度的降低,与模型组相比均有显著性差异(P<0.05或P<0.01)。
第三部分:模型组大鼠肠道SP、VIP及脊髓背角SP表达均较正常组显著增高(P<0.01),而各组脊髓背角VIP表达无明显差异。痛泻要方低剂量组脊髓背角SP表达较模型组降低(P<0.05),痛泻要方高剂量组、得舒特组肠道SP、VIP及脊髓SP表达均较模型组降低(P<0.05或P<0.01)。
结论:
1.采用结肠慢性醋酸刺激法制作的内脏高敏性IBS大鼠模型稳定性好,重复性高,较好地模拟出临床IBS患者慢性内脏痛觉过敏状态。
2.痛泻要方能改变IBS大鼠内脏高敏感状态,改善应激引起的排便量增多及粪便含水量增高的症状,且作用时间较长,综合疗效优于得舒特,并以大剂量更甚。
3.内脏高敏性大鼠模型中肠道SP、VIP及脊髓背角SP表达异常,提示SP、VIP与IBS胃肠动力异常及肠道痛觉过敏有关。SP可能在脊髓水平参与IBS内脏痛觉的调节。
4.本研究证实大剂量痛泻要方能抑制肠道SP、VIP及脊髓背角SP的异常表达。调节肠道内SP和VIP的分泌,减少伤害性信息传入,抑制脊髓内SP的释放,减弱背角神经兴奋性可能是痛泻要方治疗IBS的作用机理。
Objective
Irritable bowel syndrome(IBS) has become one of the most frequent functional gastrointestinal disorders. The incidence of IBS involves many aspects. The cause and pathogenesis of IBS is not yet expounded until now. Some researches indicate that IBS is a multi-cause and heterogenic disease which is different individual. At present we thought that visceral hypersensitivity is one of IBS most important pathophysiological characteristics, which is the foundation and reason for symptom diversification of the IBS patients.
Now there is not any kind of medicine or monotherapy fully good for IBS. Multi-target, integrity effect and few side effects are superiority of traditional Chinese medicine(TCM). The clinical reports showed that curative effect of TCM was superior. TXYF is a typical decoction of TCM, and it is mainly used to cure“diarrhea involves abdominal pain”which is similar to the symptom of IBS. So Tongxieyaofang is widely used in curing IBS, and received well clinical effects.
Referring to the experiences on making a rat model of IBS ripely, we has established a rat model of visceral hypersensitivity associated with IBS, based on which we investigated the therapic effects and mechanism of TXYF. The investigation could not only provide a experimental proof of guiding clinical medication of IBS, but also conduce to exploit and manufacture the typical decoctions of TCM.
Methods: This article is composed of three parts.
PART ONE: Establishment and evaluation of a visceral hypersensitivity rat model of IBS.
A visceral hypersensitivity rat model of IBS was manufactured by acetic acid chronic stimulation in colon. Childhood rats were randomly divided into two groups. The rats of model group were stimulated by intrarectal administration of acetic acid daily between postnatal 8 and 21 days, while the control one were stimulated by saline instead. The body weight was measured every week. The threshold of abdominal withdrawal reflex(AWR) was evaluated during rectal distension at the second week and forth week after stimulation, in order to confirm the success of rat model.
PART TWO: The therapic effects of TXYF on visceral hypersensitivity model rat of IBS.
The rat model was established the same as PART ONE. After modeling, rats were randomly divided into five groups: normal control group(A), model group (B), the low-dose TXYF group (C), the high-dose TXYF group (D), the dicetel group (E). Rats of each groups received their two weeks treatments at postnatal 36 days. The threshold of abdominal withdrawal reflex (AWR) was evaluated before and after their administration. Also the stool and its water content was measured to evaluate the therapic effects of TXYF.
PRAT THREE: The mechanism of TXYF on IBS.
The rat model and their administration was established the same as PART TWO. This part we observed the content of SP and VIP in ileocecal junction, colon and spinal cord by immunohistochemistry, in order to expound the mechanism of TXYF on IBS.
Results
PART ONE: Rats of both groups did not active abnormally and have diarrhea. The threshold of abdomen-uplifting and back-arching and the time of contract of abdomen muscle of model group showed an obvious decrease compared with the normal group at postnatal 35 days and 50 days (P<0.01). It implied that the visceral sensitivity of model group was much higher than normal group, and the hypersensitivity lasted for a long time. The model group was reproduced successfully.
PART TWO: The stool of model group was more and softer than normal group in everytime. And the stool water content was higher than normal group. The threshold of abdomen-uplifting and back-arching and the time of contract of abdomen muscle of C group, D group and E group showed an obvious increase compared with the model group at postnatal 50 days (P<0.05 or P<0.01).
PART THREE: Model group’s mean density of SP and VIP in ileocecal junction and colon and mean density of SP in posterior horn of spinal cord was increased compared with normal group’s (P<0.01). Mean density of VIP in posterior horn of spinal cord showed no difference between each group (P>0.05). C group’s mean density of SP in posterior horn of spinal cord was decreased (P<0.05), D group’s, E group’s mean density of SP and VIP in intestine and mean density of SP in posterior horn of spinal cord was decreased compared with model group’s (P<0.05或P<0.01).
Conclusions
1. Visceral hypersensitivity Rat Model of IBS which was manufactured by the acetic acid chronic stimulation in colon is steady, practicable and repeatable, which is a good imitation of IBS patients who have chronic visceral hypersensitivity.
2. With the effects of TXYF, the threshold of AWR could be increase, the stool and stool water content could be decreased. The effects of high-dose TXYF are more obvious.
3. The express of SP and VIP in intestines and express of SP in spinal cord is abnormal, which implies that SP and VIP both relate to the happening of abnormal gastrointestinal dynamia and visceral hypersensitivity. And the SP can moderate the visceral hypersensitivity on the level of spinal cord.
4. This study indicated that high-dose TXYF could decrease the content of SP and VIP in intestine and content of SP in spinal cord. To regulate the secretion of SP and VIP in intestine which could inhibit importation of harm message, decrease the content of SP in spinal cord which could depress the excitability of nerve cells, should be one of the mechanisms of TXYF on visceral hypersensitivity of IBS.
引文
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