眼针介导中枢5-HT、VIP递质途径对肝郁脾虚型IBS大鼠结肠AQP8表达的影响

【作者】 刘慧慧；

【导师】 王德山；

【作者基本信息】 辽宁中医药大学 ， 中西医结合基础， 2013， 博士

【摘要】 目的：观察肝郁脾虚型IBS模型大鼠海马、下丘脑、结肠组织5-HT、VIP、VIP-R与AQP8的表达变化，以及眼针干预对其影响。从中枢与外周两方面探讨肝郁脾虚型IBS病症结合模型的病理生理学基础，以及眼针干预的生物学效应，为阐明肝郁脾虚型IBS的发生及眼针疗法的干预机制提供实验依据。材料与方法：1. SPF级雄性Wistar大鼠40只，随即选取28只采用“精神刺激+饮食失宜+劳倦”方法建立肝郁脾虚型IBS动物模型。利用11±1℃冷水游泳至力竭（沉入水下10s不浮出）、禁食24h、禁水24h、45℃高温烤箱15min、夹尾5min、电击（30V，电击5s，间歇5s，共进行3min）六种刺激，每日随机选取一种刺激结合自制铁笼束缚1h，共进行3轮即18d。观察大鼠一般状态、体重变化及体重增长率等的变化对模型进行评价，通过评价后选取造模成功的大鼠24只，按照随机数字法分为正常组、模型组及眼针组，每组12只。眼针组造模后采用31号（直径13mm）毫针，针刺大鼠眼眶眶缘上1mm处的下焦区、大肠区、肝区、脾区进行干预7d。2.采用ELISA法检测血清和结肠组织中5-HT、SP和VIP含量。3.采用RT-PCR法检测结肠组织中VIP、VIP-R及AQP8的mRNA表达。4.采用SABC免疫组化法检测海马、下丘脑及结肠中5-HT、VIP和VIP-R的表达并进行分析。结果：1.眼针对肝郁脾虚型IBS模型大鼠一般状态及血清5-HT和SP含量的影响1.1眼针对肝郁脾虚型IBS模型大鼠一般状态的影响造模成功的大鼠，表现为精神萎靡、烦躁、皮毛干枯散乱无光泽、扎堆或蛰伏在角落、明显消瘦、进食减少，大便变软或不成形，排便次数逐渐增多、便质变稀，肛门部多有粪便附着等。肝郁日久，木郁乘土，形成“肝郁脾虚证”表现。经7d针刺治疗后，眼针组大鼠上述情志方面表现有所好转，活动次数、进食量有所增加，排便次数明显减少，便质向正常好转；模型组则无明显变化。与正常组相比，造模结束时（第18d）模型组及眼针组大鼠的体重增长率显著降低（P<0.01），但二者之间差异无统计学意义（P>0.05）；在第25d时，与正常组相比，模型组大鼠的体重增长率显著降低（P<0.01）；与模型组相比，眼针组大鼠的体重增长率显著增加（P<0.01）。与第18d正常组相比，模型组和眼针组大鼠的粪便含水率显著增加（P<0.01），但二者之间无显著无统计学意义（P>0.05）；与第25d正常组相比，模型组和眼针组大鼠的粪便含水率显著增加（P<0.01），与第25d模型组相比，经过眼针干预后粪便含水率显著降低（P<0.01）。1.2眼针对肝郁脾虚型IBS模型大鼠血清5-HT和SP含量的影响与正常组血清相比，模型组和眼针组大鼠血清5-HT含量显著增加（P<0.01），与模型组血清相比，眼针组大鼠血清5-HT含量显著降低（P<0.01）；与正常组大鼠血清相比，模型组大鼠血清SP含量显著增加（P<0.01），与模型组大鼠血清相比，眼针组大鼠血清5-HT含量明显降低（P<0.05）。2.眼针对肝郁脾虚型IBS模型大鼠结肠组织5-HT、VIP、VIP-R及AQP8的影响2.1眼针对肝郁脾虚型IBS模型大鼠结肠组织5-HT含量及表达的影响与正常组结肠相比，模型组与眼针组结肠组织5-HT含量显著增加（P<0.01），与模型组大鼠结肠组织相比，经过眼针干预后其含量显著减少了（P<0.01）。与正常组相比，模型组大鼠结肠组织5-HT的表达显著上调（P<0.01），眼针组大鼠结肠组织5-HT的表达明显上调（P<0.05）；与模型组相比，眼针组大鼠结肠组织5-HT的表达显著下调（P<0.01）。2.2眼针对肝郁脾虚型IBS模型大鼠血清、结肠组织VIP含量及表达的影响与正常组大鼠血清相比，模型组大鼠血清VIP含量显著增加（P<0.01）；与模型组大鼠血清相比，眼针组大鼠血清VIP含量显著降低（P<0.01）。与正常组大鼠结肠相比，模型组与眼针组大鼠结肠组织VIP含量显著增加（P<0.01），与模型组大鼠结肠组织相比，经过眼针干预后其表达显著下调了（P<0.01）。与正常组大鼠相比，模型组大鼠结肠组织VIP mRNA的表达显著上调（P<0.01），眼针组大鼠结肠组织VIP mRNA的表达上调不明显（P>0.05）；与模型组大鼠相比，眼针组大鼠结肠组织VIP mRNA的表达明显下调了（P<0.05）。与正常组相比，模型组结肠组织VIP的表达显著上调（P<0.01），与模型组相比，经过眼针干预后其表达显著下调了（P≤0.01）。2.3眼针对肝郁脾虚型IBS模型大鼠VIP-R表达的影响与正常组大鼠相比，模型组大鼠结肠组织VIP-R mRNA的表达显著上调（P<0.01）；与模型组大鼠相比，眼针组大鼠结肠组织VIP-R mRNA的表达显著下调了（P<0.01）。与正常组大鼠相比，模型组大鼠结肠组织VIP-R的表达显著上调（P<0.01），眼针组大鼠结肠组织VIP-R的表达显著下调（P<0.01）；与模型组大鼠相比，眼针组大鼠结肠组织VIP-R的表达显著下调了（P<0.01）。2.4眼针对肝郁脾虚型IBS模型大鼠AQP8表达的影响与正常组大鼠相比，模型组及眼针组大鼠结肠组织AQP8mRNA的表达显著下调（P<0.01）；与模型组大鼠相比，眼针组大鼠结肠组织AQP8mRNA的表达显著上调了（P<0.01）。与正常组大鼠相比，模型组大鼠结肠组织AQP8的表达显著下调（P<0.01），眼针组大鼠结肠组织AQP8的表达变化不明显，差异无统计学意义（P>0.05）；与模型组相比，眼针组大鼠结肠组织AQP8的表达显著上调（P<0.01）。3.眼针对肝郁脾虚型IBS模型大鼠海马CA3区及下丘脑5-HT、VIP及VIP-R表达的影响3.1眼针对肝郁脾虚型IBS模型大鼠海马CA3区及下丘脑5-HT表达的影响与正常组大鼠海马CA3区相比，模型组大鼠海马CA3区5-HT的表达显著下调（P<0.01）；与模型组大鼠海马CA3区相比，眼针组大鼠海马CA3区5-HT的表达显著上调（P<0.01）。与正常组大鼠下丘脑相比，模型组大鼠下丘脑5-HT的表达有所减少但差异无统计学意义（P>0.05），眼针组大鼠下丘脑5-HT的表达明显上调（P<0.05）；与模型组大鼠下丘脑相比，眼针组大鼠下丘脑5-HT的表达显著上调（P<0.01）。3.2眼针对肝郁脾虚型IBS模型大鼠海马CA3区及下丘脑VIP表达的影响与正常组大鼠海马CA3区相比，模型组大鼠海马CA3区VIP的表达显著上调（P<0.01），眼针组大鼠海马CA3区VIP的表达变化不明显，差异无统计学意义（P>0.05）；与模型组大鼠海马CA3区相比，眼针组大鼠海马CA3区VIP的表达明显下调（P<0.05）。与正常组大鼠下丘脑相比，模型组大鼠下丘脑VIP的表达显著上调（P<0.01），眼针组大鼠下丘脑VIP的表达无显著性变化（P>0.05）；与模型组下丘脑相比，眼针组大鼠下丘脑VIP的表达显著下调（P<0.01）。3.3眼针对肝郁脾虚型IBS模型大鼠海马CA3区及下丘脑VIP-R表达的影响与正常组大鼠海马CA3区相比，模型组和眼针组大鼠海马CA3区VIP-R的表达显著上调（P<0.01）；与模型组大鼠海马CA3区相比，眼针组大鼠海马CA3区VIP-R的表达变化不明显，差异无统计学意义（P>0.05）。与正常组大鼠下丘脑相比，模型组大鼠下丘脑VIP-R的表达明显上调（P<0.05），眼针组大鼠VIP-R的表达变化不明显，差异无统计学意义（P>0.05）；与模型组大鼠下丘脑相比，眼针组大鼠VIP-R的表达显著下调了（P<0.01）。结论：1.本研究采用“精神刺激+饮食失宜+劳倦”方法，成功建立了符合肝郁脾虚型IBS病证结合的动物模型。2.肝郁脾虚型IBS模型大鼠血清5-HT及SP含量显著增加；眼针能够改善肝郁脾虚型IBS模型大鼠的证候；降低血清5-HT及SP含量。3.肝郁脾虚型IBS模型大鼠结肠组织5-HT、VIP及VIP-R的表达明显上调，AQP8的表达下调；眼针干预将其逆转。4.眼针“疏肝健脾”的可能机制之一，是下调结肠组织5-HT、VIP及VIP-R的表达，上调AQP8的表达。5.肝郁脾虚型IBS模型大鼠海马及下丘脑5-HT表达下调，VIP及VIP-R表达上调；眼针干预能够使其逆转。6.海马及下丘脑可能是“肝郁脾虚”证候的中枢反应区之一，5-HT及VIP递质系统参与了肝郁脾虚型IBS的形成。7.眼针的“疏肝健脾”作用之一，是上调海马及下丘脑5-HT表达，下调VIP及VIP-R的表达。更多还原

【Abstract】 Purpose：To observe the change of the liver-stagnation and spleen-deficiency irritable bowelsyndrome (IBS) rat model hippocampus, hypothalamus, colon tissue5-hydroxytryptamine(5-HT), vasoactive intestinal peptide (VIP) and vasoactive intestinal peptide receptor (VIP-R),the expression of aquaporin8(AQP8) in colon, and the influence of the eye acupoint (EA). Toexplore the pathophysiology basis of the liver-stagnation and spleen-deficiency IBS diseasecombine model from the central and peripheral two aspects, and the biological effect of the eyeacupoint. To provide the experimental basis for illustrating the happening of theliver-stagnation and spleen-deficiency IBS and the mechanisms of the eye acupuncture.Material and method：1.28Wistar SPF male rats were randomly chose from40to establish the liver-stagnation andspleen-deficiency IBS animal model with the combination methods of “the mentalstimulation and improper diet and overstrain”. To use six kinds of stimulation as follows: coldwater swimming to exhaustive, fasting for24h, water deprivation for24h,45℃hyperthermyoven for15min, clip tail for5min, electric shock (30V, shock for5s, pause for5s, process for3min in all), randomly selected a kind of stimulation everyday, combining with homemadecages bound for1h, process for3round,18d in all. Observed the change of the rats’ generalstate, body weight change and growth rate of body mass, and evaluated the model. Afterpassing the evaluation, selected24successfully molding rats, randomly divided into threegroups as Normal, Model, Eye acupoint (EA),12per group. After the EA group molded, used31size (diameter13mm) acupuncture needle, needled the rats’ lower energizer, large intestine,hepatic region, spleen intervention for7d.2. Enzyme-linked immuno sorbent assay (ELISA) was used to detect the content of5-HT, SPand VIP in serum and colon tissue.3. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNAof VIP, VIP-R and AQP8in colon tissue.4. Strept actividin-biotin complex (SABC) immunohistochemistry was used to detect and analyze the expressions of5-HT, VIP and VIP-R in hippocampus, hypothalamus and colon.Results：1. The effects of eye acupuncture on general state and the content of5-HT and SP in serum ofthe liver-stagnation and spleen-deficiency IBS rat1.1The effects of eye acupuncture on general state of the liver-stagnation and spleen-deficiency IBS ratThe molded rats appeared depression, agitated, lose flesh, stool soft or deformed, anddefecate times increased gradually and stool turned thin. Expressed as liver-stagnation andspleen-deficiency, after acupuncture treatment for7d, the eye acupuncture rats looked likebetter and the stool improved to be normal, while model group had no obvious change.Compared to the Normal, in the18th day, the growth rate decreased significantly (P<0.01)in Model and EA; Compared to the Normal in the25th day, the growth rate of the EA setincreased dramatically (P<0.01). Compared to the Normal in the18th day, the rate of fecesmoisture content increased significantly (P<0.01) in Model and EA; Compared to the Normalin the25th day, the rate of feces moisture content decreased dramatically (P<0.01) in EA.1.2The effects of eye acupuncture on the content of5-HT and SP in serum of theliver-stagnation and spleen-deficiency IBS ratCompared to the Normal, the content of5-HT and SP in serum increased dramatically inModel and EA (P<0.01). Compared to the Model, the content of5-HT and SP in serumdecreased dramatically in eye acupuncture (P<0.01).2. The effects of eye acupuncture on the colon tissue5-HT, VIP, VIP-R and AQP8of theliver-stagnation and spleen-deficiency IBS rat2.1The effects of eye acupuncture on the content and expression of5-HT in colon tissue ofthe liver-stagnation and spleen-deficiency IBS ratCompared to the Normal, the content of5-HT in colon tissue increased significantly inModel and EA (P<0.01). Compared to the Model, the expression of5-HT dramaticallydecreased (P<0.01) in eye acupuncture rat colon tissue.2.2The effects of eye acupuncture on serum, the content and expression of VIP in colontissue of the liver-stagnation and spleen-deficiency IBS ratCompared to Normal set serum, the content of serum VIP increased significantly in Model (P<0.01). Compared to the Model, the content of serum VIP decreased significantly ineye acupuncture (P<0.01), the expression of VIP mRNA in colon tissue increaseddramatically in Model (P<0.05or P≤0.01).2.3The effects of eye acupuncture on the expression of VIP receptor in colon tissue of theliver-stagnation and spleen-deficiency IBS ratCompared to Normal, the expression of VIP receptor mRNA in colon tissue increasedsignificantly in Model (P<0.01). Compared to the Model, the expression of VIP receptormRNA in colon tissue decreased dramatically in eye acupuncture (P<0.01).2.4The effects of eye acupuncture on the expression of AQP8in colon tissue of theliver-stagnation and spleen-deficiency IBS ratCompared to Normal, the expression of AQP8mRNA in colon tissue decreasedsignificantly in Model (P<0.01). Compared to Model, the expression of AQP8mRNA incolon tissue increased dramatically in eye acupuncture (P<0.01).3. The effects of eye acupuncture on the expression of CA3area of hippocampus and5-HT,VIP, VIP receptor in hypothalamus tissue of the liver-stagnation and spleen-deficiency IBSrat3.1The effects of eye acupuncture on the expression of CA3area of hippocampus and5-HTin hypothalamus tissue of the liver-stagnation and spleen-deficiency IBS ratCompared to Normal, the expression of CA3area of hippocampus and5-HT inhippocampus decreased significantly in Model (P<0.01). Compared to Model, the expressionof CA3area of hippocampus and5-HT in hypothalamus tissue increased dramatically in eyeacupuncture (P<0.01).3.2The effects of eye acupuncture on the expression of CA3area of hippocampus and VIP inhypothalamus tissue of the liver-stagnation and spleen-deficiency IBS ratCompared to Normal, the expression of CA3area of hippocampus and VIP inhypothalamus tissue increased significantly in Model (P<0.01). Compared to Model, theexpression of CA3area of hippocampus and VIP in hypothalamus tissue decreaseddramatically in eye acupuncture (P<0.05or P<0.01).3.3The effects of eye acupuncture on the expression of CA3area of hippocampus and VIPreceptor in hypothalamus tissue of the liver-stagnation and spleen-deficiency IBS rat Compared to the Normal, the expression of CA3area of hippocampus and VIP receptorin hypothalamus tissue increased significantly in Model and eye acupuncture (P<0.01orP<0.05). Compared to Model, the expression of VIP receptor in hypothalamus tissuedecreased dramatically in eye acupuncture (P<0.01).Conclusion：1. The liver-stagnation and spleen-deficiency IBS rat model was established successfully withthe combination methods of “mental stimulation and improper diet and overstrain”.2. The content of5-HT and SP in serum increased significantly in Model. Eye acupuncture canimprove the syndrome and decrease the content of5-HT and SP in serum.3. The expression of5-HT, VIP and VIP receptor in colon tissue increased significantly and theexpression of AQP8decreased markedly in Model. Eye acupuncture can changeover them.4. One of the possible mechanisms of soothing liver and strengthening spleen with eye acupu-ncture is down-regulation the expression of5-HT, VIP and VIP receptor and up-regulation theexpression of AQP8in colon tissue.5. The expression of5-HT in hippocampus and hypothalamus tissue decreased significantly inModel, and the expression of VIP and VIP receptor increased significantly. Eye acupuncturecan changeover them.6. There are two possible central zones of hippocampus and hypothalamus with theliver-stagnation and spleen-deficiency IBS rat, and there are two neurotransmitter of5-HT andVIP involved in the formation of the liver-stagnation and spleen-deficiency IBS.7. One of the possible mechanisms of soothing liver and strengthening spleen with eye acupu-ncture is up-regulation the expression of5-HT in hippocampus and hypothalamus tissue anddown-regulation the expression of VIP and VIP receptor.更多还原