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
|
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。
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 rat
1.1The effects of eye acupuncture on general state of the liver-stagnation and spleen-deficiency IBS rat
The 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 rat
Compared 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 rat
2.1The effects of eye acupuncture on the content and expression of5-HT in colon tissue ofthe liver-stagnation and spleen-deficiency IBS rat
Compared 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 rat
Compared 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 rat
Compared 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 rat
Compared 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 IBSrat
3.1The effects of eye acupuncture on the expression of CA3area of hippocampus and5-HTin hypothalamus tissue of the liver-stagnation and spleen-deficiency IBS rat
Compared 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 rat
Compared 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.
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。
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 rat
1.1The effects of eye acupuncture on general state of the liver-stagnation and spleen-deficiency IBS rat
The 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 rat
Compared 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 rat
2.1The effects of eye acupuncture on the content and expression of5-HT in colon tissue ofthe liver-stagnation and spleen-deficiency IBS rat
Compared 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 rat
Compared 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 rat
Compared 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 rat
Compared 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 IBSrat
3.1The effects of eye acupuncture on the expression of CA3area of hippocampus and5-HTin hypothalamus tissue of the liver-stagnation and spleen-deficiency IBS rat
Compared 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 rat
Compared 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.
引文
[1]湖南医学院附一院中医基础理论研究室.肝郁脾虚的理论与实验研究[J].湖南医学院学报,1979,4(3):131-143.
[2]郭振秋,赵晓威.肝纤宁对肝郁脾虚大鼠的保肝抗纤作用[J].湖南中医学院学报,1998,18(2):10-12.
[3]顾永华,彭绵.中医肝郁证研究概况[J].成都中医药大学学报,1987,10(3):561-562.
[4]须惠仁,傅湘琦,向丽华等.肝郁证的动物实验研究--激怒刺激对大白鼠血液流变学的影响[J].中医杂志,1991,(6):44-47.
[5]陈小野.实用中医证候动物模型学[M].北京:中国协和医科大学、北京医科大学联合出版社,1993:263.
[6]丁杰,陈家旭,饶红梅.逍遥散对肝郁脾虚证模型大鼠中枢GluR1、G luR2表达的影响[J].北京中医药大学学报,2009,32(6):389-393.
[7]岳利峰,丁杰,陈家旭.肝郁脾虚证大鼠模型的建立与评价[J].北京中医药大学学报,2008,31(6):396-400.
[8]唐亚平,龚超奇,黄秋粤.逍遥散对肝郁脾虚证大鼠治疗作用的观察[J].甘肃中医,2008,21(10):53-54.
[9]郭建丽，冯玛莉，宋美卿等.情志刺激致大鼠肝郁脾虚证模型的研究[J].中华中医药杂志,2012,27(11):2976-2978.
[10]尚向永.肝郁证候患者的心理研究[J].中国健康心理学杂志,2007,15(11):983-984.
[11]周力,蒲琴,申理等.利用不良情志刺激建立肝郁证动物模型初探[J].中药药理与临床,2008,4(3):114-115.
[12]张国萍，郭蕾，窦志芳.逍遥散和痛泻药方煎剂对肝郁脾虚证大鼠模型的改善作用[J].山西中医学院学报,2012,13(1):18-20.
[13]乔明琦,陈雨振.肝郁证动物模型研究的理论思考[J].中国医药学报,1997,32(5):43-45.
[14]韩秋艳．肝郁脾虚证动物模型的建立[J]．贵阳中医学院学报，2001，23(3):59-61.
[15]雷云霞,刘新,蔡淦.两种肝郁脾虚动物模型的对比研究[J].新疆中医药,2012,30(1):10-12.
[16]郑小伟,王颖,宋红.肝郁脾虚证胃溃疡大鼠胃窦组织差异蛋白质表达及柴黄胃溃宁的干预研究.中华中医药杂志,2011,26(12):2840-2842.
[17]钱锋，卜平.痛泻要方干预大鼠肠易激综合征肝郁脾虚证的作用及机制[J].苏州大学学报,2010,30(6):1179-1181.
[18]张国梁,丰广宁,陶永.消化复宁汤对肝郁脾虚大鼠胃泌素、胃动素和胃肠运动功能的影响[J].安徽中医学院学报,2009,28(3):35-38.
[19]张燕,王威.针刺预处理及不同配穴对肝郁脾虚证大鼠血管活性肽、胃泌素影响的实验研究[J].光明中医,2012,27(2):309-311.
[20]严灿，徐志伟，吴丽丽等．建立中医情志致病动物模型的思考[J]．中国临床康复，2006，10(3):155-157．
[1]韩秋艳,吴文尧,王敏.肝郁脾虚证动物模型的建立[J].贵阳中医学院学报,2001,23(3):59-61.
[2]戴芳,唐亚平,龚超奇等.肝郁脾虚证大鼠模型的研制[J].甘肃中医,2009,22(10):69-70.
[3]岳利峰,丁杰,陈家旭等.肝郁脾虚证大鼠模型的建立与评价[J].北京中医药大学学报.2008,31(6):396-340.
[4]于丰彦,周福生.肝郁脾虚动物模型的研究概况[J].现代消化及介入治疗,2009,14(1):57-59.
[5]彭延娟,彭成.肝郁脾虚证动物模型的研究进展[J].实验动物科学与管理,2004,21(1):32-34.
[6]金光亮，王胜兰.关于建立肝郁证动物模型的思考[J].山东中医药大学学报.2004，28(6):408-409.
[7]于琦，金光亮.三种复方对慢性应激肝郁模型大鼠海马5-HT，A受体基因表达的影响[J].北京中医药.2008，27(l):57-59.
[8]林文娟，王玮雯，邵枫.慢性情绪应激对大鼠行为、神经内分泌和免疫反应的影响：一个新的情绪应激模型[J].科学通报,2003,48(9):926-928.
[9] Murua VS, Gomez RA, Andrea ME, et al. Shttle-box deficits induced by chronic variablestress: reversal by imipramine administration [J]. Pharmacology Biochemistry&Behavior,1991,38:125-130.
[10] Platt JE, Stone EA. Chronic restraint stress elicits a positive antidepressant response on theforced swim test [J]. Eur J Pharmacol,1982,82:179-181.
[11] Garcia-Marquez C, Armario A. Chronic stress depresses exploratory activity andbehavioral performance in the forced swimming test without alterning ACTH response toa novel acute stressor [J]. Physiol Behav,1987,40:33-38.
[12]乔明琦，张慧云，陈雨振，等.肝郁证动物模型研究的理论思考[J].中国医药学报.1997，12(5):42-44.
[13]吕志平，刘承才.“肝郁”大鼠的脂质过氧化反应及逍遥散的保护作用[J].山东中医学院学报,1995,19(3):199-221.
[14]唐已婷，陈家旭.慢性束缚应激动物模型的研究进展[J].北京中医药大学学报,2002，25(1):25-28.
[15]严灿，徐志伟，吴丽丽等.建立中医情志致病动物模型的思考[J].中国临床康复,2006，10(3):155-157.
[16]焦格，元海军，陈向伟.加味逍遥散对慢性束缚应激BALB/C小鼠行为学变化的调节研究[J].山西中医学院学报,2007,8(5):9-11.
[17]严灿，吴丽丽，徐志伟．应用心理应激理论和方法开展中医药实验研究的思路[J]．广州中医药大学学报,2004,21(4):325-328.
[18]黄柄山．中医证候与应激反应的关系（续二）[J]．黑龙江中医药,2003,(5):13-17.
[19] Mantyh PW. Neurobiology of substance P and the NK1receptor1[J]. Clin Psychiatry,2002,63(1):6-10.
[20] De W ied D, Sigling HO. Neuropeptides involved in the pathophysiology of schizophreniaand major depression[J]. Neurotox Res,2002,4:453-468.
[21] Bondy B, Baghai TC, M inov C, et al. Substance P serum levels are in creased in majordepression: preliminary results[J]. Biol Psychiatry,2003,53:538-542.
[22]杨斌王有德张兰等.抑郁症患者血浆P物质含量变化及其相关性研究[J].中华精神科杂志,2006,39(2):78-80.
[23] Datar P, Srivastava S, Coutinho E, et al. Substance P: structure, function, andtherapeutics[J]. Curr Top Med Chem,2004,4:75-103.
[24] Herpfer I, Lieb K. Substance P and Substance P receptor antagonistsin the pathogenes isand treatment of affective disorders[J]. World J Biol Psychiatry,2003,4:56-63.
[25] Duffy RA. Potential therapeutic targets for neurokinin-1receptor antagonists[J]. ExpertOpin Emerg Drugs,2004,9:9-21.
[26] Holmes A, Heilig M, Rupniak NM, et al. Neuropeptide systems as novel therapeutictargets for depression and anxiety disorders[J]. Trends Pharmacol Sci,2003,24:580-588.
[27] Santarelli L, Saxe MD. Substance P antagonists: meet the new drugs, same as the olddrugs: insights from transgenic an imalmodels[J]. CNS Spectr,2003,8:589-596.
[28] Adell A. Antidepressant properties of substance P antagonists: relationship tomonoaminergic mechanisms?[J]. Curr Drug Targets CNS Neurol Disord,2004,3:113-121.
[29]钱锋，卜平.痛泻要方干预大鼠肠易激综合征肝郁脾虚证的作用及机制[J].苏州大学学报（医学版）,2010,30(6):1179-1181.
[30]须惠仁,傅湘琦,向丽华等.肝郁证的动物实验研究[J].中医杂志,1991.(6):44-46.
[31]谢建群,郑昱,吴大正.疏肝饮对腹泻型肠易激综合征大鼠血管活性肠肽和P物质的影响[J].上海中医药大学学报,2004,18(3):36-39.
[32]汪正芳，郭前坤，张声生.疏肝健脾方对腹泻型肠易激综合征大鼠血浆及结肠组织5-HT、SP、VIP的影响[J].天津中医药,2012,29(5):459-461.
[33]董海燕,董卫国,王高华等.抑郁模型大鼠血浆、脑垂体和结肠组织中P物质和血管活性肠肽的变化[J].中国行为医学科学,2005,14(3):208-209.
[34]詹丽杏,许国铭,李兆申等.肠易激综合征患者活动期和缓解期血浆5-HT、5-HIAA的变化[J].第二军医大学学报,2003,24(2):152-153.
[35]黄适,林寿宁,谈驰等.安肠汤结肠水疗对腹泻型肠易激综合征患者血浆胃动素和P物质的影响[J].辽宁中医杂志,2009,36(7):1150-1151.
[36]潘明.抑郁症患者血浆P物质含量检测及临床意义[J].中国热带医学,2008,8(7):1147-1148.
[37]彭静山．眼针疗法[M]．沈阳：辽宁科学技术出版社，1991:31.
[38]林燕,李兴广.基于肠易激综合征的脑-肠互动与肝郁脾虚实质探讨[J].世界中医药,2009,4(6):304-306.
[39]马瑞军,王俊平,陈星,等.腹泻型肠易激综合征患者结肠黏膜AQP4、AQP8相关性表达[J].国际内科学杂志,2008,3,35(3):129-131.
[40] Wang JP and Hou XH. Expression of aquaporin8in colonic epithelium withdiarrhea-predominant irritable bowel syndrome. Chinese Medical Journa [J].2007,120(4):313-316.
[41]王德山,张宇,王哲等.脾虚模型大鼠结肠黏膜上皮细胞水通道蛋白8表达变化[J].中国中西医结合消化杂志,2008,16(2):71-73.
[42] Yamamoto T, Kuramoto H, Kadowaki M. Downregulation in aquaporin4and aquaporin8expression of the colon associated with the induction of allergic diarrhea in a mousemodel of food allergy [J]. Life Sci,2007,81(2):115–120.
[43] Calamita G, Spalluto C, Mazzone A, et al. Cloning, structural organization andchromosomal localization of the mouse Aquaporin-8water channel gene (Aqp8)[J].Cytogenetic and Genome Research,1999,85(3-4):237-241.
[44]马瑞军,王俊平,陈星,等.腹泻型肠易激综合征患者结肠黏膜AQP4、AQP8相关性表达[J].国际内科学杂志,2008,3,35(3):129-131.
[45]王晓玲,王俊平.腹泻大鼠结肠水通道蛋白8表达的研究[J].中国药物与临床,2008,48(4):295-297.
[46]冯景，王俊平，贾泽明.血管活性肠肽VIP对人结肠黏膜上皮细胞系HT29细胞AQP8表达调节的研究[J].中国现代医学杂志，2007,17(19):2345-2348.
[47] Gradilone SA，Garcia F，Huebert RC. Glucagon induces the plasma Membraneinsertion of functional aquapor-in-8water channels in isolated Rat hepatocytes [J].Hepatology,2003,37(6):1435-1441.
[48] Zahn A, Moehle C, Langmann T. Aquaporin-8expression is reduced in ileum andinduced in colon of patients with ulcerative colitis [J]. World J Gastroenterol,2007,13:1687-1695.
[49] Van DerVeek Patrick, Biemond I, Masclee A, et al. Proximal and distal gut hormonesecretion in irritable bowel syndrome [J]. Scandinavian Journal of Gastroenterology,2006,41(2):170-177.
[50] Morise K, Furusawa A, Yamamoto H, et al. Role of gut hormones in irritable bowelsyndrome [J]. Nippon Rinsho,1992,50(11):2697-2702.
[51]李兆申,董文,邹多武等.肠易激综合征肠黏膜SP、VIP、CGRP变化的研究[J].第二军医大学学报,2003,(24)2:147-151.
[52] Zhang H, Yan Y, Shi R, et al. Correlation of gut hormones with irritable bowelsyndrome [J]. Digestion,2008,78(2-3):72-76.
[53] Gradilone SA, Garcia F, Huebert RC, et al. Glucagon induces the plasma membraneinsertion of functional aquaporin-8water channels in isolated rat hepatocytes [J].Hepatology,2003,37(6):1435-1441.
[54] Palsson OS, Morteau O, Bozymski EM, et al. Elevated vasoactive intestinal peptideconcentrations in patients with irritable bowel syndrome[J]. Dig Dis Sci，2004，49(7/8):1236-1243.
[55]陈芝芸，严茂祥,项柏康.调肝实脾方对肝郁脾虚型肠易激综合征患者胃肠激素的影响[J].临床报道,2003,44(12):928.
[56]李艳彦,谢鸣,陈禹等.一种运用复合病因造模法复制大鼠肝郁脾虚证模型的研究[J].中国中医基础医学杂志,2006,12(6):439-442.
[57]姜敏,凌立平,傅宝玉.5-羟色胺在肠易激综合征中作用的研究[J].中国医科大学学报,2006,35(2):171-172.
[58]李运红,朱晓蕾,徐肇敏.腹泻型肠易激综合征患者结肠黏膜5-羟色胺和5-羟色胺3受体的研究[J].胃肠病学,2006,11(8):477-479.
[59]李兆申,詹丽杏,邹多武等.肠易激综合征患者分泌5-羟色胺的肠嗜铬细胞形态及功能的改变[J].中华消化杂志,2004,24:94-97.
[60]雷云霞,蔡淦.5羟色胺在肠易激综合征中的作用[J].中国中西医结合消化杂志,2005,13:410-413.
[61] Pan H,Gershon M D. Activation of intrinsic afferent pathways in submucosal ganglia ofthe guinea pig small intestine[J]. the Journal of Neuroscience,2000,20,3295-3309.
[62] Wyke SM，Tisdale MJ．NF-kappaB mediates proteolysis-inducing factor induced proteindegradation and expression of the ubiquitin-proteasome system in skeletal muscle[J]．BrJCancer,2005,92(4):711-721.
[63] Aznar S, Qian Z, Shah R, et al. The5-HT1A serotonin receptor is located oncalbindin-and parvalbumin-containing neurons on the rat brain[J]. Brain Res，2003，959(1):58-67.
[64] Costelli P，Llovera M，Carbo N, et al. Interleukin-1receptor antagonist(IL-1ra) is unableto reverse cachexia in rats bearing an ascites hepatoma (Yoshida AH-130)[J]．CancerLett，1995，95(1-2):33-38.
[65] Baker DE. Rationale for using serotonergic agents to treat irritable bowel syndrome [J].Am JH ealth S yst Pharm,2005,62(7):700-711.
[66] Joanna C, Michael C and John M. Vasoactive intestinal peptide is a neuropeptide mediatorof the secretory response to serotonin in rat[J]. Journal of Surgical Research,2000,91:118–122.
[67] Lacolley P, Ow en JR, Sandock K, et al.5-HT activates vagal afferentcell bodies in vivo:role of5-HT (2) and5-HT (3) rece tors [J]. Neuroscience,2006,143(1):273-287.
[68]隋红,陈卫,陆杰.5-羟色胺对大鼠膈下迷走神经传入放电的影响及机制[J].第三军医大学学报,2007,29(17):1706-1708.
[69] B lackshaw L A, Grundy D. Effects of5-Hydroxytryptamine on discharge of vagalmucosal afferen t fibres from th e upper gastro intestin altract of the ferret [J]. JAu tonNerv Syst,1993,45(1):41-50.
[70] H illsley K, Grundy D. Seroton in and cho lecystok in in act ivate d ifferent populations ofratmesenteric vagal afferents [J]. Neurosci Lett,1998,255(2):63-66.
[7]郑红，郑容，何岸平.肠易激综合征与抑郁症的关系[J].现代医药卫生,2011,27(10):1466-1467.
[72]岳广欣,陈家旭.肝主疏泄生理学基础探讨[J].北京中医药大学学报,2005,2(1):22-24
[73]岳利峰,陈家旭,霍素坤等.逍遥散对肝郁脾虚证模型大鼠海马CA1区和杏仁核BLA区GluR2阳性细胞数变化的影响[J].中华中医药杂志,2010,25(8):842-845.
[74]高亮,高德.人体信息控制系统生理学[M].呼和浩特:内蒙古人民出版社,1997.1~23,151,332.
[75] Talley NJ, Howell S, Poulton R. The irritable bowel syndrome and psychiatric disorders inthe communty: is there a link?[J]. Am J Gastroenterol,2001,96(4):1072-1079.
[76]霍清萍.从肠易激综合征脑-肠轴学说谈肝郁脾虚病理机制[J].中国中西医结合消化杂志,2005,13(5):318-320.
[77]刘松林,梅国强,赵映前,等.功能性消化不良的中医临床辨证规律研究[J].中国医药学报,2004,19(8):499-501.
[78]周福生,吴文江,张庆宏.胃肠功能性疾病证型分布的统计分析[J].中华国际医学杂志,2002,2(5):438-442.
[79]危北海.中医脾胃学说研究[M].北京:中国中医药出版社,2000:9.
[80]李瑜元.功能性胃肠疾病的精神心理因素及治疗[J].国外医学内科学分册,1998,25(3):105-107.
[81]张瑜,卜平,孔桂美,等.肠易激综合征中医病证与肠胆囊收缩素、细胞癌基因fos、P物质的相关性[J].中医杂志,2008,49(9):803-805.
[82] Sarkar S, Hobson AR, Furlong PL, et al. Central neural mechanisms mediating humanvisceral hypersensitivity [J]. Am J Physiol Gastrointest Liver Physiol,2001,281(5):G1196-1202.
[83] MertzH, Morgan V, Tanner G, et al. Regional cerebral activation inirritable bowelsyndrome and control subjects with painful and nonpainful rectal distention [J].Gastroenterology,2000,118(5):842-848.
[84] Ringel Y, Drossman DA, Turkington TG, et al. Regional brain activation in response torectal distension in patients with irritable bowel syndrome and the effect of a history ofabuse [J]. Dig Dis Sci2003,48(9):1774-1781.
[85] Saper CD. Direct hypothalamo-autonomic connections[J]. Brain Res,1976,117:305-312.
[86] Akmaer IG. The orgin of the hypothealamic-vagal descending pathway: a experimentrltrastructural study[J]. Brain Res,1981,230:342-349.
[87] Swanson LW and Kuypers HGJM. The paraventricular nucleus of the hypothalamus:cytoarchitectonic subdivisiona and organization of projections to the pitultary, dorsal vagalcomplex, and spinal cord as demonstrated by retrograde fluorescencs double-labellingmethods[J]. J Control,1998,194:555-563.
[88] Swanson LW and Kuypers HGJM. A direct projection from the ventromedical nucleus andChiasmatic area of hypothalamus to the medulla and spinal cord of the cat[J]. NeurosciLet1980,307-315.
[89] Posserud I, Agerforz P, Ekman R, et al. Altered visceral perceptual and neuroendocrineresponse in patients with irritable bowel syndrome during mental stress[J]. Gut,2004,53(8):1102-1109.
[90]岳利峰,陈家旭王大伟等.逍遥散对肝郁脾虚证模型大鼠海马和杏仁核AMPA受体亚基基因表达调节机制。北京中医药大学学报,2009,32(8):513-517.
[91] Brezun JM, Daszuta A. Serotonin may stimulate granule cell proliferation in the adulthippocampus, as observed in rats grafted with foetal raphe neurons. Eur J Neurosci,2000,12(1):391-396.
[92] Gold E, Tanapat P, Rydel T, et al. Regulation of hippocampul neurogenesis inadulthood[J]. Biol Psychiatry.2000,48(8):715-720.
[93] Sarkar S, Hobson AR, Furlong PL, et al. Central neural mechanisms mediating humanvisceral hypersensitivity [J]. Am J Physiol Gastrointest Liver Physiol,2001,281(5):1196-1202.
[94]肖爱娇,闵建新,王莉等.抑郁症模型大鼠血清雌激素水平与海马5-HT含量之间的关系[J].天津医药,2009,37(11):944-947.
[95]唐久余,曾元山,陈巧格等.缬草对慢性应激导致的抑郁大鼠大脑海马5-羟色胺水平、细胞增殖及神经元数量的影响[J].中西医结合学报,2008,6(3):283-288.
[96]赵歆，陈家旭，杨建新等．疏肝中药复方对慢性束缚应激大鼠下丘脑－垂体－肾上腺轴的调节[J]．实验动物科学与管理,2003,20(2):6-12.
[97]郭军,耿强,王福等.翘芍止泄合剂对肝郁模型大鼠性行为及中枢5－羟色胺的影响[J].中国性科学,2012,21(2):28-30.
[98]严灿,徐志伟,李艳等.调肝、补肾、健脾方药对慢性心理应激大鼠单胺类神经递质影响的比较研究[J].中国中西医结合杂志,2002,22(12):925-927.
[99] Whitwo rth EJ, Kosti O, Renshaw D, et al. Adr enal neur opeptides: Regulat ion and interaction with ACTHand o ther adr enal regulators [J]. M icrosc Res Tech,2003,61(4):259-267.
[100]石君杰.调肝健脾法治疗束缚应激IBS大鼠的实验研究[J].现代中西医结合杂志,2005,14(6):722-723.
[101]谢建群,郑昱,吴大正等.疏肝饮对腹泻型肠易激综合征大鼠血管活性肠肽和P物质的影响[J].上海中医药大学学报,2004,18(3):37.
[102] Carter Wr, Herman J, Stokes K. Promotion of diabetes onset by stress in BB rat [J].Diabetologia,1987,30(1):674.
[103]潘锋,陈建永,张涛.痛泻要方对脾虚肝郁型肠易激综合征大鼠内脏敏感性干预作用的研究[J].中国中医药科技,2009,16(5):361-362.
[104]张铮铮,邝卫红,张进领.痛泻要方对慢性应激大鼠肠功能及下丘脑和结肠VIP的影响[J].安徽中医学院学报,2010,29(5):53-55.
[105]祝淑贞.5-HT与肠易激综合征发病的内在相关性[J].临床消化病杂志,2004,16(5):229-230.
[108] Nakai A, Kumaku ra Y, Boivin M, et al. Sex differences of brain serotonin synthesis inpatients with irritable bowel syndrome using alpha-[11C] methyl tryptophan,positronemission tomography and statistical parametric mapping. Can J Gastroenterol,2003,17:191-196.
[109]何侠垠,戴宁.5-羟色胺与肠易激综合征关系的研究进展[J].国际消化病杂志.2010,30(1):1-3.
[110] William H, Christine T, Bruces M. Modulation by vasoactive intestinal peptide (VIP) ofserotonin receptors in membranes from rat hippocampus[J]. The Journal of Neuroscience,1983,3(12):2415-2419.
[111] Micheline H, Marie-Claude B, Maxime F, et al. Effect of vasoactive intestinal peptide onserotonin metabolism in the suprachiasmatic area of the rat: mechanism of action[J].Peptides,1986,7:183-188.
[112] Seiji H, Masashi U, Fumimasa A, et al. Serotonin modulates expression of VIP and GRPmRNA via the5-HT1B receptor in the suprachiasmatic nucleus of the rat[J].Experimental Neurology,2001,171:285-292.
[113] Fumio K, Hitoshi O, Yoshitaka T, et al. Changes in vasoactive intestinal peptide mRNAlevels in the rat suprachiasmatic nucleus following pchlorophenylalanine (PCPA)treatment under light/dark condition s [J]. Neuroscience Letters,1995,200:171-174.
[2]郭振秋,赵晓威.肝纤宁对肝郁脾虚大鼠的保肝抗纤作用[J].湖南中医学院学报,1998,18(2):10-12.
[3]顾永华,彭绵.中医肝郁证研究概况[J].成都中医药大学学报,1987,10(3):561-562.
[4]须惠仁,傅湘琦,向丽华等.肝郁证的动物实验研究--激怒刺激对大白鼠血液流变学的影响[J].中医杂志,1991,(6):44-47.
[5]陈小野.实用中医证候动物模型学[M].北京:中国协和医科大学、北京医科大学联合出版社,1993:263.
[6]丁杰,陈家旭,饶红梅.逍遥散对肝郁脾虚证模型大鼠中枢GluR1、G luR2表达的影响[J].北京中医药大学学报,2009,32(6):389-393.
[7]岳利峰,丁杰,陈家旭.肝郁脾虚证大鼠模型的建立与评价[J].北京中医药大学学报,2008,31(6):396-400.
[8]唐亚平,龚超奇,黄秋粤.逍遥散对肝郁脾虚证大鼠治疗作用的观察[J].甘肃中医,2008,21(10):53-54.
[9]郭建丽，冯玛莉，宋美卿等.情志刺激致大鼠肝郁脾虚证模型的研究[J].中华中医药杂志,2012,27(11):2976-2978.
[10]尚向永.肝郁证候患者的心理研究[J].中国健康心理学杂志,2007,15(11):983-984.
[11]周力,蒲琴,申理等.利用不良情志刺激建立肝郁证动物模型初探[J].中药药理与临床,2008,4(3):114-115.
[12]张国萍，郭蕾，窦志芳.逍遥散和痛泻药方煎剂对肝郁脾虚证大鼠模型的改善作用[J].山西中医学院学报,2012,13(1):18-20.
[13]乔明琦,陈雨振.肝郁证动物模型研究的理论思考[J].中国医药学报,1997,32(5):43-45.
[14]韩秋艳．肝郁脾虚证动物模型的建立[J]．贵阳中医学院学报，2001，23(3):59-61.
[15]雷云霞,刘新,蔡淦.两种肝郁脾虚动物模型的对比研究[J].新疆中医药,2012,30(1):10-12.
[16]郑小伟,王颖,宋红.肝郁脾虚证胃溃疡大鼠胃窦组织差异蛋白质表达及柴黄胃溃宁的干预研究.中华中医药杂志,2011,26(12):2840-2842.
[17]钱锋，卜平.痛泻要方干预大鼠肠易激综合征肝郁脾虚证的作用及机制[J].苏州大学学报,2010,30(6):1179-1181.
[18]张国梁,丰广宁,陶永.消化复宁汤对肝郁脾虚大鼠胃泌素、胃动素和胃肠运动功能的影响[J].安徽中医学院学报,2009,28(3):35-38.
[19]张燕,王威.针刺预处理及不同配穴对肝郁脾虚证大鼠血管活性肽、胃泌素影响的实验研究[J].光明中医,2012,27(2):309-311.
[20]严灿，徐志伟，吴丽丽等．建立中医情志致病动物模型的思考[J]．中国临床康复，2006，10(3):155-157．
[1]韩秋艳,吴文尧,王敏.肝郁脾虚证动物模型的建立[J].贵阳中医学院学报,2001,23(3):59-61.
[2]戴芳,唐亚平,龚超奇等.肝郁脾虚证大鼠模型的研制[J].甘肃中医,2009,22(10):69-70.
[3]岳利峰,丁杰,陈家旭等.肝郁脾虚证大鼠模型的建立与评价[J].北京中医药大学学报.2008,31(6):396-340.
[4]于丰彦,周福生.肝郁脾虚动物模型的研究概况[J].现代消化及介入治疗,2009,14(1):57-59.
[5]彭延娟,彭成.肝郁脾虚证动物模型的研究进展[J].实验动物科学与管理,2004,21(1):32-34.
[6]金光亮，王胜兰.关于建立肝郁证动物模型的思考[J].山东中医药大学学报.2004，28(6):408-409.
[7]于琦，金光亮.三种复方对慢性应激肝郁模型大鼠海马5-HT，A受体基因表达的影响[J].北京中医药.2008，27(l):57-59.
[8]林文娟，王玮雯，邵枫.慢性情绪应激对大鼠行为、神经内分泌和免疫反应的影响：一个新的情绪应激模型[J].科学通报,2003,48(9):926-928.
[9] Murua VS, Gomez RA, Andrea ME, et al. Shttle-box deficits induced by chronic variablestress: reversal by imipramine administration [J]. Pharmacology Biochemistry&Behavior,1991,38:125-130.
[10] Platt JE, Stone EA. Chronic restraint stress elicits a positive antidepressant response on theforced swim test [J]. Eur J Pharmacol,1982,82:179-181.
[11] Garcia-Marquez C, Armario A. Chronic stress depresses exploratory activity andbehavioral performance in the forced swimming test without alterning ACTH response toa novel acute stressor [J]. Physiol Behav,1987,40:33-38.
[12]乔明琦，张慧云，陈雨振，等.肝郁证动物模型研究的理论思考[J].中国医药学报.1997，12(5):42-44.
[13]吕志平，刘承才.“肝郁”大鼠的脂质过氧化反应及逍遥散的保护作用[J].山东中医学院学报,1995,19(3):199-221.
[14]唐已婷，陈家旭.慢性束缚应激动物模型的研究进展[J].北京中医药大学学报,2002，25(1):25-28.
[15]严灿，徐志伟，吴丽丽等.建立中医情志致病动物模型的思考[J].中国临床康复,2006，10(3):155-157.
[16]焦格，元海军，陈向伟.加味逍遥散对慢性束缚应激BALB/C小鼠行为学变化的调节研究[J].山西中医学院学报,2007,8(5):9-11.
[17]严灿，吴丽丽，徐志伟．应用心理应激理论和方法开展中医药实验研究的思路[J]．广州中医药大学学报,2004,21(4):325-328.
[18]黄柄山．中医证候与应激反应的关系（续二）[J]．黑龙江中医药,2003,(5):13-17.
[19] Mantyh PW. Neurobiology of substance P and the NK1receptor1[J]. Clin Psychiatry,2002,63(1):6-10.
[20] De W ied D, Sigling HO. Neuropeptides involved in the pathophysiology of schizophreniaand major depression[J]. Neurotox Res,2002,4:453-468.
[21] Bondy B, Baghai TC, M inov C, et al. Substance P serum levels are in creased in majordepression: preliminary results[J]. Biol Psychiatry,2003,53:538-542.
[22]杨斌王有德张兰等.抑郁症患者血浆P物质含量变化及其相关性研究[J].中华精神科杂志,2006,39(2):78-80.
[23] Datar P, Srivastava S, Coutinho E, et al. Substance P: structure, function, andtherapeutics[J]. Curr Top Med Chem,2004,4:75-103.
[24] Herpfer I, Lieb K. Substance P and Substance P receptor antagonistsin the pathogenes isand treatment of affective disorders[J]. World J Biol Psychiatry,2003,4:56-63.
[25] Duffy RA. Potential therapeutic targets for neurokinin-1receptor antagonists[J]. ExpertOpin Emerg Drugs,2004,9:9-21.
[26] Holmes A, Heilig M, Rupniak NM, et al. Neuropeptide systems as novel therapeutictargets for depression and anxiety disorders[J]. Trends Pharmacol Sci,2003,24:580-588.
[27] Santarelli L, Saxe MD. Substance P antagonists: meet the new drugs, same as the olddrugs: insights from transgenic an imalmodels[J]. CNS Spectr,2003,8:589-596.
[28] Adell A. Antidepressant properties of substance P antagonists: relationship tomonoaminergic mechanisms?[J]. Curr Drug Targets CNS Neurol Disord,2004,3:113-121.
[29]钱锋，卜平.痛泻要方干预大鼠肠易激综合征肝郁脾虚证的作用及机制[J].苏州大学学报（医学版）,2010,30(6):1179-1181.
[30]须惠仁,傅湘琦,向丽华等.肝郁证的动物实验研究[J].中医杂志,1991.(6):44-46.
[31]谢建群,郑昱,吴大正.疏肝饮对腹泻型肠易激综合征大鼠血管活性肠肽和P物质的影响[J].上海中医药大学学报,2004,18(3):36-39.
[32]汪正芳，郭前坤，张声生.疏肝健脾方对腹泻型肠易激综合征大鼠血浆及结肠组织5-HT、SP、VIP的影响[J].天津中医药,2012,29(5):459-461.
[33]董海燕,董卫国,王高华等.抑郁模型大鼠血浆、脑垂体和结肠组织中P物质和血管活性肠肽的变化[J].中国行为医学科学,2005,14(3):208-209.
[34]詹丽杏,许国铭,李兆申等.肠易激综合征患者活动期和缓解期血浆5-HT、5-HIAA的变化[J].第二军医大学学报,2003,24(2):152-153.
[35]黄适,林寿宁,谈驰等.安肠汤结肠水疗对腹泻型肠易激综合征患者血浆胃动素和P物质的影响[J].辽宁中医杂志,2009,36(7):1150-1151.
[36]潘明.抑郁症患者血浆P物质含量检测及临床意义[J].中国热带医学,2008,8(7):1147-1148.
[37]彭静山．眼针疗法[M]．沈阳：辽宁科学技术出版社，1991:31.
[38]林燕,李兴广.基于肠易激综合征的脑-肠互动与肝郁脾虚实质探讨[J].世界中医药,2009,4(6):304-306.
[39]马瑞军,王俊平,陈星,等.腹泻型肠易激综合征患者结肠黏膜AQP4、AQP8相关性表达[J].国际内科学杂志,2008,3,35(3):129-131.
[40] Wang JP and Hou XH. Expression of aquaporin8in colonic epithelium withdiarrhea-predominant irritable bowel syndrome. Chinese Medical Journa [J].2007,120(4):313-316.
[41]王德山,张宇,王哲等.脾虚模型大鼠结肠黏膜上皮细胞水通道蛋白8表达变化[J].中国中西医结合消化杂志,2008,16(2):71-73.
[42] Yamamoto T, Kuramoto H, Kadowaki M. Downregulation in aquaporin4and aquaporin8expression of the colon associated with the induction of allergic diarrhea in a mousemodel of food allergy [J]. Life Sci,2007,81(2):115–120.
[43] Calamita G, Spalluto C, Mazzone A, et al. Cloning, structural organization andchromosomal localization of the mouse Aquaporin-8water channel gene (Aqp8)[J].Cytogenetic and Genome Research,1999,85(3-4):237-241.
[44]马瑞军,王俊平,陈星,等.腹泻型肠易激综合征患者结肠黏膜AQP4、AQP8相关性表达[J].国际内科学杂志,2008,3,35(3):129-131.
[45]王晓玲,王俊平.腹泻大鼠结肠水通道蛋白8表达的研究[J].中国药物与临床,2008,48(4):295-297.
[46]冯景，王俊平，贾泽明.血管活性肠肽VIP对人结肠黏膜上皮细胞系HT29细胞AQP8表达调节的研究[J].中国现代医学杂志，2007,17(19):2345-2348.
[47] Gradilone SA，Garcia F，Huebert RC. Glucagon induces the plasma Membraneinsertion of functional aquapor-in-8water channels in isolated Rat hepatocytes [J].Hepatology,2003,37(6):1435-1441.
[48] Zahn A, Moehle C, Langmann T. Aquaporin-8expression is reduced in ileum andinduced in colon of patients with ulcerative colitis [J]. World J Gastroenterol,2007,13:1687-1695.
[49] Van DerVeek Patrick, Biemond I, Masclee A, et al. Proximal and distal gut hormonesecretion in irritable bowel syndrome [J]. Scandinavian Journal of Gastroenterology,2006,41(2):170-177.
[50] Morise K, Furusawa A, Yamamoto H, et al. Role of gut hormones in irritable bowelsyndrome [J]. Nippon Rinsho,1992,50(11):2697-2702.
[51]李兆申,董文,邹多武等.肠易激综合征肠黏膜SP、VIP、CGRP变化的研究[J].第二军医大学学报,2003,(24)2:147-151.
[52] Zhang H, Yan Y, Shi R, et al. Correlation of gut hormones with irritable bowelsyndrome [J]. Digestion,2008,78(2-3):72-76.
[53] Gradilone SA, Garcia F, Huebert RC, et al. Glucagon induces the plasma membraneinsertion of functional aquaporin-8water channels in isolated rat hepatocytes [J].Hepatology,2003,37(6):1435-1441.
[54] Palsson OS, Morteau O, Bozymski EM, et al. Elevated vasoactive intestinal peptideconcentrations in patients with irritable bowel syndrome[J]. Dig Dis Sci，2004，49(7/8):1236-1243.
[55]陈芝芸，严茂祥,项柏康.调肝实脾方对肝郁脾虚型肠易激综合征患者胃肠激素的影响[J].临床报道,2003,44(12):928.
[56]李艳彦,谢鸣,陈禹等.一种运用复合病因造模法复制大鼠肝郁脾虚证模型的研究[J].中国中医基础医学杂志,2006,12(6):439-442.
[57]姜敏,凌立平,傅宝玉.5-羟色胺在肠易激综合征中作用的研究[J].中国医科大学学报,2006,35(2):171-172.
[58]李运红,朱晓蕾,徐肇敏.腹泻型肠易激综合征患者结肠黏膜5-羟色胺和5-羟色胺3受体的研究[J].胃肠病学,2006,11(8):477-479.
[59]李兆申,詹丽杏,邹多武等.肠易激综合征患者分泌5-羟色胺的肠嗜铬细胞形态及功能的改变[J].中华消化杂志,2004,24:94-97.
[60]雷云霞,蔡淦.5羟色胺在肠易激综合征中的作用[J].中国中西医结合消化杂志,2005,13:410-413.
[61] Pan H,Gershon M D. Activation of intrinsic afferent pathways in submucosal ganglia ofthe guinea pig small intestine[J]. the Journal of Neuroscience,2000,20,3295-3309.
[62] Wyke SM，Tisdale MJ．NF-kappaB mediates proteolysis-inducing factor induced proteindegradation and expression of the ubiquitin-proteasome system in skeletal muscle[J]．BrJCancer,2005,92(4):711-721.
[63] Aznar S, Qian Z, Shah R, et al. The5-HT1A serotonin receptor is located oncalbindin-and parvalbumin-containing neurons on the rat brain[J]. Brain Res，2003，959(1):58-67.
[64] Costelli P，Llovera M，Carbo N, et al. Interleukin-1receptor antagonist(IL-1ra) is unableto reverse cachexia in rats bearing an ascites hepatoma (Yoshida AH-130)[J]．CancerLett，1995，95(1-2):33-38.
[65] Baker DE. Rationale for using serotonergic agents to treat irritable bowel syndrome [J].Am JH ealth S yst Pharm,2005,62(7):700-711.
[66] Joanna C, Michael C and John M. Vasoactive intestinal peptide is a neuropeptide mediatorof the secretory response to serotonin in rat[J]. Journal of Surgical Research,2000,91:118–122.
[67] Lacolley P, Ow en JR, Sandock K, et al.5-HT activates vagal afferentcell bodies in vivo:role of5-HT (2) and5-HT (3) rece tors [J]. Neuroscience,2006,143(1):273-287.
[68]隋红,陈卫,陆杰.5-羟色胺对大鼠膈下迷走神经传入放电的影响及机制[J].第三军医大学学报,2007,29(17):1706-1708.
[69] B lackshaw L A, Grundy D. Effects of5-Hydroxytryptamine on discharge of vagalmucosal afferen t fibres from th e upper gastro intestin altract of the ferret [J]. JAu tonNerv Syst,1993,45(1):41-50.
[70] H illsley K, Grundy D. Seroton in and cho lecystok in in act ivate d ifferent populations ofratmesenteric vagal afferents [J]. Neurosci Lett,1998,255(2):63-66.
[7]郑红，郑容，何岸平.肠易激综合征与抑郁症的关系[J].现代医药卫生,2011,27(10):1466-1467.
[72]岳广欣,陈家旭.肝主疏泄生理学基础探讨[J].北京中医药大学学报,2005,2(1):22-24
[73]岳利峰,陈家旭,霍素坤等.逍遥散对肝郁脾虚证模型大鼠海马CA1区和杏仁核BLA区GluR2阳性细胞数变化的影响[J].中华中医药杂志,2010,25(8):842-845.
[74]高亮,高德.人体信息控制系统生理学[M].呼和浩特:内蒙古人民出版社,1997.1~23,151,332.
[75] Talley NJ, Howell S, Poulton R. The irritable bowel syndrome and psychiatric disorders inthe communty: is there a link?[J]. Am J Gastroenterol,2001,96(4):1072-1079.
[76]霍清萍.从肠易激综合征脑-肠轴学说谈肝郁脾虚病理机制[J].中国中西医结合消化杂志,2005,13(5):318-320.
[77]刘松林,梅国强,赵映前,等.功能性消化不良的中医临床辨证规律研究[J].中国医药学报,2004,19(8):499-501.
[78]周福生,吴文江,张庆宏.胃肠功能性疾病证型分布的统计分析[J].中华国际医学杂志,2002,2(5):438-442.
[79]危北海.中医脾胃学说研究[M].北京:中国中医药出版社,2000:9.
[80]李瑜元.功能性胃肠疾病的精神心理因素及治疗[J].国外医学内科学分册,1998,25(3):105-107.
[81]张瑜,卜平,孔桂美,等.肠易激综合征中医病证与肠胆囊收缩素、细胞癌基因fos、P物质的相关性[J].中医杂志,2008,49(9):803-805.
[82] Sarkar S, Hobson AR, Furlong PL, et al. Central neural mechanisms mediating humanvisceral hypersensitivity [J]. Am J Physiol Gastrointest Liver Physiol,2001,281(5):G1196-1202.
[83] MertzH, Morgan V, Tanner G, et al. Regional cerebral activation inirritable bowelsyndrome and control subjects with painful and nonpainful rectal distention [J].Gastroenterology,2000,118(5):842-848.
[84] Ringel Y, Drossman DA, Turkington TG, et al. Regional brain activation in response torectal distension in patients with irritable bowel syndrome and the effect of a history ofabuse [J]. Dig Dis Sci2003,48(9):1774-1781.
[85] Saper CD. Direct hypothalamo-autonomic connections[J]. Brain Res,1976,117:305-312.
[86] Akmaer IG. The orgin of the hypothealamic-vagal descending pathway: a experimentrltrastructural study[J]. Brain Res,1981,230:342-349.
[87] Swanson LW and Kuypers HGJM. The paraventricular nucleus of the hypothalamus:cytoarchitectonic subdivisiona and organization of projections to the pitultary, dorsal vagalcomplex, and spinal cord as demonstrated by retrograde fluorescencs double-labellingmethods[J]. J Control,1998,194:555-563.
[88] Swanson LW and Kuypers HGJM. A direct projection from the ventromedical nucleus andChiasmatic area of hypothalamus to the medulla and spinal cord of the cat[J]. NeurosciLet1980,307-315.
[89] Posserud I, Agerforz P, Ekman R, et al. Altered visceral perceptual and neuroendocrineresponse in patients with irritable bowel syndrome during mental stress[J]. Gut,2004,53(8):1102-1109.
[90]岳利峰,陈家旭王大伟等.逍遥散对肝郁脾虚证模型大鼠海马和杏仁核AMPA受体亚基基因表达调节机制。北京中医药大学学报,2009,32(8):513-517.
[91] Brezun JM, Daszuta A. Serotonin may stimulate granule cell proliferation in the adulthippocampus, as observed in rats grafted with foetal raphe neurons. Eur J Neurosci,2000,12(1):391-396.
[92] Gold E, Tanapat P, Rydel T, et al. Regulation of hippocampul neurogenesis inadulthood[J]. Biol Psychiatry.2000,48(8):715-720.
[93] Sarkar S, Hobson AR, Furlong PL, et al. Central neural mechanisms mediating humanvisceral hypersensitivity [J]. Am J Physiol Gastrointest Liver Physiol,2001,281(5):1196-1202.
[94]肖爱娇,闵建新,王莉等.抑郁症模型大鼠血清雌激素水平与海马5-HT含量之间的关系[J].天津医药,2009,37(11):944-947.
[95]唐久余,曾元山,陈巧格等.缬草对慢性应激导致的抑郁大鼠大脑海马5-羟色胺水平、细胞增殖及神经元数量的影响[J].中西医结合学报,2008,6(3):283-288.
[96]赵歆，陈家旭，杨建新等．疏肝中药复方对慢性束缚应激大鼠下丘脑－垂体－肾上腺轴的调节[J]．实验动物科学与管理,2003,20(2):6-12.
[97]郭军,耿强,王福等.翘芍止泄合剂对肝郁模型大鼠性行为及中枢5－羟色胺的影响[J].中国性科学,2012,21(2):28-30.
[98]严灿,徐志伟,李艳等.调肝、补肾、健脾方药对慢性心理应激大鼠单胺类神经递质影响的比较研究[J].中国中西医结合杂志,2002,22(12):925-927.
[99] Whitwo rth EJ, Kosti O, Renshaw D, et al. Adr enal neur opeptides: Regulat ion and interaction with ACTHand o ther adr enal regulators [J]. M icrosc Res Tech,2003,61(4):259-267.
[100]石君杰.调肝健脾法治疗束缚应激IBS大鼠的实验研究[J].现代中西医结合杂志,2005,14(6):722-723.
[101]谢建群,郑昱,吴大正等.疏肝饮对腹泻型肠易激综合征大鼠血管活性肠肽和P物质的影响[J].上海中医药大学学报,2004,18(3):37.
[102] Carter Wr, Herman J, Stokes K. Promotion of diabetes onset by stress in BB rat [J].Diabetologia,1987,30(1):674.
[103]潘锋,陈建永,张涛.痛泻要方对脾虚肝郁型肠易激综合征大鼠内脏敏感性干预作用的研究[J].中国中医药科技,2009,16(5):361-362.
[104]张铮铮,邝卫红,张进领.痛泻要方对慢性应激大鼠肠功能及下丘脑和结肠VIP的影响[J].安徽中医学院学报,2010,29(5):53-55.
[105]祝淑贞.5-HT与肠易激综合征发病的内在相关性[J].临床消化病杂志,2004,16(5):229-230.
[108] Nakai A, Kumaku ra Y, Boivin M, et al. Sex differences of brain serotonin synthesis inpatients with irritable bowel syndrome using alpha-[11C] methyl tryptophan,positronemission tomography and statistical parametric mapping. Can J Gastroenterol,2003,17:191-196.
[109]何侠垠,戴宁.5-羟色胺与肠易激综合征关系的研究进展[J].国际消化病杂志.2010,30(1):1-3.
[110] William H, Christine T, Bruces M. Modulation by vasoactive intestinal peptide (VIP) ofserotonin receptors in membranes from rat hippocampus[J]. The Journal of Neuroscience,1983,3(12):2415-2419.
[111] Micheline H, Marie-Claude B, Maxime F, et al. Effect of vasoactive intestinal peptide onserotonin metabolism in the suprachiasmatic area of the rat: mechanism of action[J].Peptides,1986,7:183-188.
[112] Seiji H, Masashi U, Fumimasa A, et al. Serotonin modulates expression of VIP and GRPmRNA via the5-HT1B receptor in the suprachiasmatic nucleus of the rat[J].Experimental Neurology,2001,171:285-292.
[113] Fumio K, Hitoshi O, Yoshitaka T, et al. Changes in vasoactive intestinal peptide mRNAlevels in the rat suprachiasmatic nucleus following pchlorophenylalanine (PCPA)treatment under light/dark condition s [J]. Neuroscience Letters,1995,200:171-174.