耳—迷走神经联系与胆碱能抗炎通路
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摘要
胆碱能抗炎通路(the cholinergic anti-inflammatory pathway, CAP)是近些年来发现的以传出性迷走神经为基础的抑制炎症反应的神经免疫通路。直接刺激迷走神经可以激活此通路,使传出性迷走神经冲动增加,释放乙酰胆碱,进而抑制巨噬细胞等免疫细胞释放炎症相关因子,最终达到控制炎症的目的。内毒素血症(endotoxemia, ETM)由内毒素激活免疫细胞释放炎症因子而引起,常用于制作系统性炎症模型和研究胆碱能抗炎通路的机理。
迷走神经耳支是迷走神经在体表的唯一分支,主要分布在耳廓的耳甲区(耳甲艇和耳甲腔),与孤束核、迷走神经背核、迷走神经之间有密切联系。耳迷走神经投射到孤束核、迷走神经背核及疑核等神经中枢,是耳-迷走神经反射弧的重要组成部分。耳甲区迷走神经末梢与孤束核、迷走神经背核有直接投射关系。针刺耳甲区能激活孤束核和迷走神经背核神经元放电。而胆碱能抗炎通路的初级传入中枢是孤束核,并且通过迷走神经背核发出迷走神经传出冲动。因此,我们设想耳针刺激能够通过激活耳甲区传入性迷走神经进而促使传出性迷走神经传出冲动增加,从而激活胆碱能抗炎通路,最终抑制炎症反应。本研究从系统性炎症因子、NF-κB通路、组织器官炎症因子表达及器官病理状况等层面研究耳针刺激对炎症发生发展的调节作用,探讨耳针激活胆碱能抗炎通路的作用机制。
1.耳针对内毒素血症大鼠血清细胞因子的影响
雄性清洁级Sprague-Dawley大鼠,体重280-300g,中国人民解放军军事医学科学院实验动物中心提供,许可证号：SCXK-(军) 2007-004。动物自由摄食和饮水。
所有大鼠腹腔注射10%乌拉坦(1mL/100g)麻醉。
脂多糖(lipopolysaccharide, LPS. Escherichia coli 0111:B4;Sigma)用生理盐水配制10mg/mL溶液,参照有关文献造模方法,相关各组动物经尾静脉注射脂多糖(10mg/mL,0.5mL/kg)。通过检测血清炎症因子水平、观察肺脏组织病理图片判断大鼠内毒素血症模型制作成功。其余组别经尾静脉注射等量生理盐水。
1.1实验一(静脉给药2h)
实验分组
A组正常对照组(n=12)：经尾静脉注射等量生理盐水。
B组模型组(n=12)：经尾静脉注射脂多糖,造模2h后取材。
C组单纯耳甲迷走神经电针组(简称单纯耳甲电针组,n=12)：经尾静脉注射等量生理盐水,1.5h后进行双侧耳甲刺激,造模2h后取材。
D组耳甲迷走神经电针组(简称耳甲电针组,n=12)：经尾静脉注射脂多糖,1.5h后进行双侧耳甲刺激,造模2h后取材。
E组迷走神经刺激组(n=12)：经尾静脉注射脂多糖,1.5h后进行左侧颈部迷走神经干刺激,造模2h后取材。
F组“后三里”电针组(简称“后三里”组,n=12)：经尾静脉注射脂多糖,1.5h后进行双侧“后三里”刺激,造模2h后取材。
ELISA法检测血清TNF-α、IL-6、IL-1β及IL-10。
实验结果
与正常对照组相比,模型组TNF-α、IL-6、IL-1β及IL-10水平明显升高(P<0.01)；与模型组相比,耳甲电针组和迷走神经刺激组TNF-α、IL-6、IL-1β及IL-10水平明显下降(P<0.01)；“后三里”组‘TNF-α、IL-10水平明显下降(P<0.05,P<0.01)。与迷走神经刺激组相比,耳甲电针组IL-6、IL-1β及IL-10水平有显著差异(P<0.05,P<0.01),“后三里”组TNF-α水平、IL-6水平、IL-1β及IL-10水平有显著差异(P<0.05,P<0.01)。
1.2实验二(静脉给药4h)
1.2.1实验分组
G组正常对照组(n=12)：经尾静脉注射等量生理盐水,给药4h后取材。
H组模型组(n=13)：经尾静脉注射脂多糖,造模4h后取材。
I组耳甲迷走神经电针组(简称耳甲电针组,n=14)：经尾静脉注射脂多糖,1.5h后进行双侧耳甲刺激,造模4h后取材。
J组“后三里”电针组(简称“后三里”组,n=13)：经尾静脉注射脂多糖,1.5h后进行双侧“后三里”刺激,造模4h后取材。
1.2.2指标检测
血清TNF-α、IL-6、IL-1β及IL-10浓度检测采用酶联免疫法,ELISA试剂盒由美国R&D公司提供,检测方法严格按照试剂盒说明书进行。
1.2.3实验结果
与正常对照组相比,模型组TNF-α、IL-6、IL-1β及IL-10水平明显升高(P<0.01)：与模型组相比,耳甲电针组TNF-α、IL-6、IL-1β水平明显下降(P<0.05)；“后三里”组IL-1β水平明显下降(P<0.05)。
13实验三(静脉给药6h)
1.3.1实验分组
K组正常对照组(n=11)：经尾静脉注射等量生理盐水,给药6h后取材。
L组模型组(n=10)：经尾静脉注射脂多糖,造模6h后取材。
M组耳甲迷走神经电针组(简称耳甲电针组,n=11)：经尾静脉注射脂多糖,1.5h后进行双侧耳甲刺激,造模6h后取材。
N组“后三里”电针组(简称“后三里”组,n=10)：经尾静脉注射脂多糖,1.5h后进行双侧“后三里”刺激,造模6h后取材。
1.3.2指标检测
血清TNF-α、IL-6、IL-1β及IL-10浓度检测采用酶联免疫法,ELISA试剂盒由美国R&D公司提供,检测方法严格按照试剂盒说明书进行。
1.3.3实验结果
与正常对照组相比,模型组TNF-α、IL-6、IL-1β水平明显升高(P<0.05,P<0.01)；与模型组相比,耳甲电针组IL-6、IL-1β水平明显下降(P<0.05),IL-10水平明显上升(P<0.05)；“后三里”组TNF-α、IL-1β水平明显下降(P<0.05)。
1.4实验四(迷走神经阻断)
1.4.1实验分组
A组正常对照组(n=11)：经尾静脉注射等量生理盐水。
B组模型组(n=10)：经尾静脉注射脂多糖。
O组迷走神经切断+耳甲迷走神经电针组(简称迷切耳针组,n=11)：经尾静脉注射脂多糖后立即切断双侧颈部迷走神经干,1.5h后进行双侧耳甲刺激。
P组迷走神经切断+“后三里”电针组(简称迷切“后三里”组,n=10)：经尾静脉注射脂多糖后立即切断双侧颈部迷走神经干,1.5h后进行双侧“后三里”刺激。
Q组N受体拮抗+耳甲迷走神经电针组(简称N受体拮抗耳针组,n=10)：造模前经右侧颈静脉注射六烃季铵(10mg/kg),造模1.5h后进行双侧耳甲刺激。
R组N受体拮抗+“后三里”电针组(简称N受体拮抗“后三里”组,n=10)：造模前经右侧颈静脉注射六烃季铵(10mg/kg),造模1.5h后进行双侧“后三里”刺激。
S组N受体α-7亚单位拮抗+耳甲迷走神经电针组(简称α-BGT耳针组,n=10)：造模前经右侧颈静脉注射α-银环蛇毒素(1μg/kg),造模1.5h后进行双侧耳甲刺激。
T组N受体α-7亚单位拮抗+“后三里”电针组(简称α-BGT'后三里”组,n=10)：造模前经右侧颈静脉注射α-银环蛇毒素(1μg/kg),造模1.5h后进行双侧“后三里”刺激。
1.4.2指标检测
血清TNF-α采用酶联免疫法,ELISA试剂盒由美国R&D公司提供,检测方法严格按照试剂盒说明书进行。
1.4.3实验结果
与正常对照组相比,模型组TNF-α水平明显升高(P<0.01)；与模型组相比,O、P、Q、R、S及T组TNF-a水平无限制差异。
2.耳针对NF-κB通路的影响
2.1肺组织NF-κB p65蛋白表达变化
2.1.1实验一(静脉给药2h)
采用第一部分实验一中A、B、C、D、E及F组的肺组织样本(在-80℃超低温冰箱保存的肺右叶组织)。
采用蛋白免疫印记法(Western Blot)测定各组肺脏组织NF-κB p65蛋白表达。
实验结果：与正常对照组(A组)相比,模型组(B组)NF-κB表达明显上调(P<0.01)；与模型组相比,耳甲电针组(D组)及迷走神经刺激组(E组)NF-κB表达明显下调(P<0.01)；与耳甲电针组相比,“后三里”组(F组)NF-κB表达明显增加(P<0.05)：与迷走神经刺激组相比,“后三里”组NF-κB表达明显上调(P<0.01)。
2.1.2实验二(静脉给药4h)
采用第一部分实验二中G、H、I及J组的肺组织样本(在-80。C超低温冰箱保存的肺右叶组织)。
采用蛋白免疫印记法(Western Blot)测定各组肺脏组织NF-κB p65蛋白表达。
实验结果：与正常对照组(G组)相比,模型组(H组)NF-κB表达明显上调(P<0.01)；与模型组相比,耳甲电针组(I组)NF-κB表达明显下调(P<0.05)。
2.1.3实验三(静脉给药6h)
采用第一部分实验三中K、L、M及N组的肺组织样本(在-80℃超低温冰箱保存的肺右叶组织)。
采用蛋白免疫印记法(Western Blot)测定各组肺脏组织NF-κB p65蛋白表达。
实验结果：与正常对照组(K组)相比,模型组(L组)NF-κB表达明显上调(P<0.01)：与模型组相比,耳甲电针组(M组)NF-κB表达明显下调(P<0.01)。
2.2肺脏NF-κB p65活性变化
2.2.1实验一(静脉给药2h)
采用第一部分动物实验一中的A、B、D、E及F组中用甲醛固定的肺组织。
采用免疫组化法测定各组肺脏组织NF-κB p65蛋白表达。
实验结果：与正常对照组(A组)相比,模型组(B组)NF-κB p65表达明显上调(P<0.01)；与模型组相比,耳甲电针组(D组)、迷走神经刺激组(E组)NF-κB p65表达明显下调(P<0.01)；与迷走神经刺激组相比,“后三里”组(F组)NF-κB p65表达明显升高(P<0.05)。
2.2.2实验二(静脉给药4h)
采用第一部分实验一中的G、H、I及J组中用甲醛固定的肺组织。
采用免疫组化法测定各组肺脏组织NF-κB p65蛋白表达。
实验结果：与正常对照组(G组)相比,模型组(H组)NF-κB p65表达明显上调(P<0.01)；与模型组相比,耳甲电针组(I组)、“后三里”组(J组)NF-κB p65表达明显下调(P<0.01,P<0.05)
2.2.3实验三(静脉给药6h)
采用第一部分实验一中的K、L、M及N组中用甲醛固定的肺组织。
采用免疫组化法测定各组肺脏组织NF-κB p65蛋白表达。
实验结果：与正常对照组(K组)相比,模型组(L组)NF-κB p65表达明显上调(P<0.01)；与模型组相比,耳甲电针组(M组)、“后三里”组(N组)NF-κB p65表达明显下调(P<0.05)。
2.2.4实验四(迷走神经阻断)
采用第一部分实验一中的A、B组及实验四中的O、P组用甲醛固定的肺组织。
采用免疫组化法测定各组肺脏组织NF-κB p65蛋白表达。
实验结果：与正常对照组(A组)相比,模型组(B组)NF-κB p65表达明显上调(P<0.01)；与模型组相比,迷切耳针组(O组)、迷切“后三里”组(P组)NF-κB p65表达无明显变化。
3.耳针对内毒素血症大鼠器官组织影响
3.1肝脏组织TNF-αmRNA、IL-10 mRNA表达
3.1.1实验一(静脉给药2h)
采用第一部分动物实验一中A、B、D、E及F组的肝脏组织样本(在-80℃超低温冰箱保存的肝左外叶组织)。
采用定量PCR法测定各组肝脏组织TNF-αmRNA表达变化。
实验结果：与正常对照组(A组)相比,模型组(B组)TNF-αmRNA表达明显增加(P<0.01)：与模型组相比,耳甲电针组(D组)、迷走神经刺激组(E组)TNF-αmRNA表达明显减少(P<0.05,P<0.01)。
3.1.2实验二(静脉给药4h)
采用第一部分动物实验二中G、H、I及J组的肝脏组织样本(在-80℃超低温冰箱保存的肝左外叶组织)。
采用定量PCR法测定各组肝脏组织。TNF-αmRNA、IL-10 mRNA表达变化。
实验结果：与正常对照组(G组)相比,模型组(H组)TNF-αmRNA表达明显增加(P<0.01)：与模型组相比,耳甲电针组(I组)、“后三里”组(J组)TNF-αmRNA表达明显减少(P<0.01)。
与正常对照组(G组)相比,模型组(H组)IL-10 mRNA表达明显增加(P<0.01)；与模型组相比,耳甲电针组(I组)、“后三里”组(J组)IL-10 mRNA表达明显增加(P<0.01,P<0.05)。
3.1.3实验三(静脉给药6h)
采用第一部分动物实验二中K、L、M及N组的肝脏组织样本(在-80℃超低温冰箱保存的肝左外叶组织)。采用定量PCR法测定各组肝脏组织TNF-αmRNA、IL-10mRNA表达变化。
实验结果：各组动物肝TNF-α、IL-10 mRNA表达变化见表X、图X。
与正常对照组(K组)相比,模型组(L组)TNF-αmRNA表达明显增加(P<0.01)：与模型组相比,耳甲电针组(M组)、“后三里”组(N组)TNF-αmRNA表达明显减少(P<0.01,P<0.05)。
与正常对照组(K组)相比,模型组(L组)IL-10 mRNA表达明显增加(P<0.01)；与模型组相比,耳甲电针组(M组)、“后三里”组(N组)IL-10 mRNA表达明显增加(P<0.05)。
3.2肺组织病理学变化
采用第一部分动物实验一中的A、B、D、E及F组中用甲醛固定的肺组织。
采用常规苏木素-伊红(HE)染色法观察各组肺脏组织病理变化。
实验结果：正常对照组(A组)肺泡结构完整,无萎陷,未见出血及炎细胞渗出。模型组(B组)肺泡结构破坏,可见萎陷、不张,肺泡腔内见大量红细胞及渗出液,支气管结构紊乱。耳甲电针组(D组)和迷走神经刺激组(E组)较之模型组损伤程度减轻,肺泡结构破坏明显减少。“后三里”组(F组)较之模型组损伤程度略减轻。
4.结论
本研究从血液循环细胞因子水平、NF-κB亘路、内脏器官细胞因子mRNA水平及器官组织病理变化等几个层面阐述了耳针抑制内毒素血症大鼠炎症发展的效应机制。通过造模后不同时间点炎症因子浓度变化揭示内毒素血症的发展变化过程。同时通过不同时间点相应指标的变化,观察耳针在炎症过程中的干预作用。
实验结果表明：耳针对各时间点的致炎因子都有明显的抑制作用,其效应与直接刺激迷走神经相似,在进行迷走神经阻断后,耳针的抗炎效应消失。同时,耳针抑制了各时间点NF-κB p65的表达,但是这种抑制作用同样有赖于迷走神经的完整。以上结果说明耳针抑制血液循环炎症因子及NF-κB通路可能是通过激活胆碱能抗炎通路来实现的。耳针刺激还有助于肝脏致炎因子TNF-amRNA水平的降低及抗炎因子IL-10mRNA水平的升高,这可能是抑制器官炎症发展的关键环节。此外,耳针刺激改善了肺脏组织的病理状态,说明其对内毒素血症中的器官具有保护作用。以上这些结果为耳针激活胆碱能抗炎通路治疗炎症提供了理论依据。
本研究还进行了直接刺激迷走神经、耳针刺激和“后三里”刺激三种刺激方法抗炎效应的比较。结果发现：直接刺激迷走神经效果优于耳针刺激和“后三里”刺激,耳针刺激效果优于“后三里”刺激。这可能是由于同“后三里”刺激相比,迷走神经刺激和耳针刺激更为直接地激活了胆碱能抗炎通路。
Cholinergic anti-inflammatory pathway (CAP) has been discovered in recent years. Based on efferent vagus nerve, this pathway aims at suppressing inflammatory response. The pathway can be activated by stimulating vagus nerve directly. Then it releases acetylcholine, and suppresses inflammatory cytokines, and control inflammation.
Auricular branch of vagus nerve innervates auricular concha(AC), and its afferent branch projects to nucleus of solitary tract (NTS) and dorsal motor nucleus of vagus (DMV). Neurons in NTS and DMV can be activated by acupuncturing auricular concha. We assume that auricular acupuncture (AA) may activate the cholinergic anti-inflammation pathway via stimulating auricular branch of vagus nerve. This study will focus on systemic inflammatory factors, NF-κB pathway, cytokine expression in organ, and organic pathology in endotoxemia. It aims at reveal mechanism on auricular acupuncture induced CAP.
1. Auricular acupuncture regulates serum cytokines in endotoxeamia rats.
Male SD rats.280-300g, were housed in standard conditions with access to regular chow and water. The permission number is SCXK-(army) 2007-004.
Endotoxemia in animals was induced by administering lipopolysaccharide (LPS, Escherichia coli 0111:B4:Sigma.5mg/kg. i.v.).
1.1 Experiment 1 (2h)
Group A:Control:animals were treated with sterile saline, i.v.
Group B:Model:animals were treated with LPS, i.v.
Group C:Simple AA:animals were treated with sterile saline, i.v.1.5h later, proceed AA.
Group D:AA+LPS:animals were treated with LPS. i.v.1.5h later, proceed AA.
Group E:VNS+LPS:animals were treated with LPS, i.v.1.5h later, treated with vagus nerve stimulation.
Group F:ST36+LPS:animals were treated with LPS, i.v.1.5h later, treated with electroacupuncture at "ST36". 2h after injection, all animals were taken blood. Test serum TNF-α, IL-6, IL-1βand IL-10 with ELISA.
Results:Compared with the control group, serum TNF-α,IL-6, IL-1βand IL-10 contents in the model group were increased significantly (P<0.01). In comparison with the model group, serum TNF-αand IL-10 contents in the AA+LPS, VNS+LPS and ZST36+LPS groups, and IL-6,IL-1βin the AA+LPS and VNS+LPS groups were down-regulated considerably (P<0.01). Compared with the VNS+LPS group, serum IL-6, IL-1βand IL-10 contents in the AA+LPS, and TNF-α, IL-6, IL-1βand IL-10 levels in the ZST36+LPS group were significantly different (P<0.05, P<0.01).
1.2 Experiment 2 (4h)
Group G:Control:animals were treated with sterile saline, i.v.
Group H:Model:animals were treated with LPS, i.v.
Group I:AA+LPS:animals were treated with LPS, i.v.1.5h later, proceed AA.
Group J:ST36+LPS:animals were treated with LPS. i.v.1.5h later, treated with electroacupuncture at "ST36" 4h after injection, all animals were taken blood. Test serum TNF-α, IL-6, IL-1βand IL-10 with ELISA kit.
Results:Compared with the control group, serum TNF-α, IL-6, IL-1βand IL-10 contents in the model group were increased significantly (P<0.01). In comparison with the model group, serum TNF-α, IL-6, IL-1βcontents in the AA+LPS group, and IL-1βin the ST36+LPS were down-regulated considerably (P<0.05).
1.3 Experiment 3 (6h)
Group K:Control:animals were treated with sterile saline, i.v.
Group L:Model:animals were treated with LPS. i.v.
Group M:AA+LPS:animals were treated with LPS. i.v.1.5h later, proceed AA.
Group N:ST36+LPS:animals were treated with LPS. i.v.1.5h later, treated with electroacupuncture at "ST36". 6h after injection, all animals were taken blood. Test serum TNF-α, IL-6, IL-1βand IL-10 with ELISA kit. Results:Compared with the control group, serum TNF-α, IL-6. IL-1βcontents in the model group were increased significantly (P<0.05, P<0.01). In comparison with the model group, serum IL-6,IL-1βcontents in the AA+LPS group,and TNF-α,IL-1βin the ST36+LPS group were down-regulated considerably (P<0.05):serum IL-10 content in AA+LPS group was increased significantly (P<0.05).
1.4 Experiment 4 (vagus nerve block)
Group A:Control:animals were treated with sterile saline, i.v.
Group B:Model:animals were treated with LPS, i.v.
Group O:vagotomy+AA+LPS:animals were treated with LPS, i.v. at the same time apply vagotomy 1.5h later, proceed AA.
Group P:vagotomy +ST36+LPS:animals were treated with LPS. i.v. at the same time apply vagotomy.1.5h later, treated with electroacupuncture at "ST36"
GroupQ:Hexamethonium+AA+LPS:animals were injected with Hexamethonium, i.v. then treated with LPS.1.5h later, proceed AA.
Group R:Hexamethonium-ST36+LPS:animals were injected with Hexamethonium. i.v.. then treated with LPS.1.5h later, stimulate ST36.
Group S:a-BGT+AA+LPS:animals were injected with a-BGT. i.v.. then treated with LPS,1.5h later, proceed AA.
Group T:a-BGT-ST36+LPS:animals were injected with a-BGT. i.v.. then treated with LPS,1.5h later, stimulate ST36. 2h after LPS injection, all animals were taken blood. Test serum TNF-a with ELISA kit.
Results:Compared with the control group, serum TNF-a content in the model group were increased significantly (P<0.01). In comparison with the model group, there was non-significant difference between other groups (P<0.05).
2. Auricular acupuncture influences NF-κB pathway.
2.1 NF-κB p65 protein expression in lung tissue
2.1.1 Experiment 1 (2h)
Take lung tissue from group A, B, C, D, E and F. Detect NF-κB p65 protein expression from each group. Western Blot procedure. Results:Compared with the normal control group, pulmonary NF-κB p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-κB p65 expression levels in the AA+LPS and VNS+LPS groups were down-regulated considerably (P<0.01). Compared with the VNS+LPS group pulmonary NF-κB p65 expression levels in the ZST36+LPS group were significantly different (P<0.01). In comparison with the AA+LPS group, pulmonary NF-κB p65 expression level in the ZST36+LPS group was significantly different (P<0.05).
2.1.2 Experiment 2 (4h)
Take lung tissue from group G, H, I, and J. Detect NF-κB p65 protein expression from each group. Use Western Blot procedure.
Results:Compared with the normal control group, pulmonary NF-κB p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-κB p65 expression levels in the AA+LPS groups were down-regulated considerably (P<0.05).
2.1.3 Experiment 3 (6h)
Take lung tissue from group K, L. M and N. Detect NF-κB p65 protein expression from each group. Use Western Blot procedure. Results:Compared with the normal control group, pulmonary NF-κB p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-κB p65 expression levels in the AA+LPS groups were down-regulated considerably (P<0.01).
2.2 Pulmonary NF-κB p65 activity
2.2.1 Experiment 1 (2h)
Take lung tissue from group A. B, D, E and F. Detect pulmonary NF-κB p65 expression from each group. Use Immunohistochemistry technology.
Results:Compared with the normal control group, pulmonary NF-KBp65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-κB p65 expression levels in the AA+LPS and VNS+LPS groups were down-regulated considerably (P<0.01). Compared with the VNS+LPS group pulmonary NF-κB p65 expression levels in the ZST36+LPS group were significantly increased (P<0.05).
2.2.2 Experiment 2 (4h)
Take lung tissue from group G. H, I and J. Detect pulmonary NF-κB p65 expression from each group. Use Immunohistochemistry technology. Results:Compared with the normal control group, pulmonary NF-κB p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-κB p65 expression levels in the AA+LPS and ST36+LPS groups were down-regulated considerably (P<0.01, P<0.05).
2.2.3 Experiment 3 (6h)
Take lung tissue from group K. L. M and N. Detect pulmonary NF-κB p65 expression from each group. Use Immunohistochemistry technology. Results:Compared with the normal control group, pulmonary NF-κB p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-κB p65 expression levels in the AA+LPS and ST36+LPS groups were down-regulated considerably (P<0.05).
2.2.4 Experiment 4 (vagus nerve block)
Take lung tissue from group A, B, O and P. Detect pulmonary NF-κB p65 expression from each group. Use Immunohistochemistry technology. Results:Compared with the normal control groupm pulmonary NF-κB p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonaiy NF-κB p65 expression levels in the other two groups was non-significant different (P<0.05).
3. Auricular acupuncture protects organs in endotoxemia rats.
3.1 TNF-a mRNA, IL-10 mRNA expression in liver
3.1.1 Experiment 1 (2h)
Take liver sample from group A. B. D. E and F. Detect TNF-a mRNA expression from each group with real time PCR. Results:Compared with the normal control group, TNF-a mRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, TNF-αmRNA expression levels in the AA+LPS and VNS+LPS groups were down-regulated considerably (P<0.05, P<0.01).
3.1.2 Experiment 2 (4h)
Take liver sample from group G, H, I and J. Detect TNF-αmRNA and IL-10 expression from each group with real time PCR. Results:Compared with the normal control group, TNF-αmRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, TNF-αmRNA expression levels in the AA+LPS and ST36+LPS groups were down-regulated considerably (P<0.01).
Compared with the normal control group. IL-10 mRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, IL-10 mRNA expression levels in the AA+LPS and ST36+LPS groups were up-regulated considerably (P<0.01, P<0.05).
3.1.3 Experiment 3 (6h)
Take liver sample from group K, L, M and N. Detect TNF-αmRNA and IL-10 mRNA expression from each group with real time PCR. Results:Compared with the normal control group. TNF-a mRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, TNF-αmRNA expression levels in the AA+LPS and ST36+LPS groups were down-regulated considerably (P<0.01, P<0.05).
Compared with the normal control group, IL-10 mRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, IL-10 mRNA expression levels in the AA+LPS and ST36+LPS groups were up-regulated considerably (P<0.05).
3.2 lung histopathology
Take lung samples from group A,B,D,E and F.
Use HE staining technology.
Result demonstrated that samples from AA+LPS and ST36+LPS groups were much improved in pathological states of lung.
4. Conclusion
This study research the mechanism on inhibitory effect of auricular acupuncture in cytokine regulation. We focus on systemic inflammatory factors, NF-κB pathway, cytokine expression in organ, and organic pathology in endotoxemia rats. The results demonstrate that auricular acupuncture plays an important role in inflammatory response, and inhibits pro-inflammatory cytokines. Meanwhile, auricular acupuncture suppresses NF-κB p65 expression as well. In absence of vagus nerve, the anti-inflammatory effect mentioned above was destroyed. It suggests that auricular acupuncture may activate cholinergic anti-inflammatory pathway to control inflammatory development. In addition, auricular acupuncture regulates TNF-a mRNA and IL-10 mRNA expression in liver, and this might be the key point to suppress inflammatory development.
迷走神经耳支是迷走神经在体表的唯一分支,主要分布在耳廓的耳甲区(耳甲艇和耳甲腔),与孤束核、迷走神经背核、迷走神经之间有密切联系。耳迷走神经投射到孤束核、迷走神经背核及疑核等神经中枢,是耳-迷走神经反射弧的重要组成部分。耳甲区迷走神经末梢与孤束核、迷走神经背核有直接投射关系。针刺耳甲区能激活孤束核和迷走神经背核神经元放电。而胆碱能抗炎通路的初级传入中枢是孤束核,并且通过迷走神经背核发出迷走神经传出冲动。因此,我们设想耳针刺激能够通过激活耳甲区传入性迷走神经进而促使传出性迷走神经传出冲动增加,从而激活胆碱能抗炎通路,最终抑制炎症反应。本研究从系统性炎症因子、NF-κB通路、组织器官炎症因子表达及器官病理状况等层面研究耳针刺激对炎症发生发展的调节作用,探讨耳针激活胆碱能抗炎通路的作用机制。
1.耳针对内毒素血症大鼠血清细胞因子的影响
雄性清洁级Sprague-Dawley大鼠,体重280-300g,中国人民解放军军事医学科学院实验动物中心提供,许可证号：SCXK-(军) 2007-004。动物自由摄食和饮水。
所有大鼠腹腔注射10%乌拉坦(1mL/100g)麻醉。
脂多糖(lipopolysaccharide, LPS. Escherichia coli 0111:B4;Sigma)用生理盐水配制10mg/mL溶液,参照有关文献造模方法,相关各组动物经尾静脉注射脂多糖(10mg/mL,0.5mL/kg)。通过检测血清炎症因子水平、观察肺脏组织病理图片判断大鼠内毒素血症模型制作成功。其余组别经尾静脉注射等量生理盐水。
1.1实验一(静脉给药2h)
实验分组
A组正常对照组(n=12)：经尾静脉注射等量生理盐水。
B组模型组(n=12)：经尾静脉注射脂多糖,造模2h后取材。
C组单纯耳甲迷走神经电针组(简称单纯耳甲电针组,n=12)：经尾静脉注射等量生理盐水,1.5h后进行双侧耳甲刺激,造模2h后取材。
D组耳甲迷走神经电针组(简称耳甲电针组,n=12)：经尾静脉注射脂多糖,1.5h后进行双侧耳甲刺激,造模2h后取材。
E组迷走神经刺激组(n=12)：经尾静脉注射脂多糖,1.5h后进行左侧颈部迷走神经干刺激,造模2h后取材。
F组“后三里”电针组(简称“后三里”组,n=12)：经尾静脉注射脂多糖,1.5h后进行双侧“后三里”刺激,造模2h后取材。
ELISA法检测血清TNF-α、IL-6、IL-1β及IL-10。
实验结果
与正常对照组相比,模型组TNF-α、IL-6、IL-1β及IL-10水平明显升高(P<0.01)；与模型组相比,耳甲电针组和迷走神经刺激组TNF-α、IL-6、IL-1β及IL-10水平明显下降(P<0.01)；“后三里”组‘TNF-α、IL-10水平明显下降(P<0.05,P<0.01)。与迷走神经刺激组相比,耳甲电针组IL-6、IL-1β及IL-10水平有显著差异(P<0.05,P<0.01),“后三里”组TNF-α水平、IL-6水平、IL-1β及IL-10水平有显著差异(P<0.05,P<0.01)。
1.2实验二(静脉给药4h)
1.2.1实验分组
G组正常对照组(n=12)：经尾静脉注射等量生理盐水,给药4h后取材。
H组模型组(n=13)：经尾静脉注射脂多糖,造模4h后取材。
I组耳甲迷走神经电针组(简称耳甲电针组,n=14)：经尾静脉注射脂多糖,1.5h后进行双侧耳甲刺激,造模4h后取材。
J组“后三里”电针组(简称“后三里”组,n=13)：经尾静脉注射脂多糖,1.5h后进行双侧“后三里”刺激,造模4h后取材。
1.2.2指标检测
血清TNF-α、IL-6、IL-1β及IL-10浓度检测采用酶联免疫法,ELISA试剂盒由美国R&D公司提供,检测方法严格按照试剂盒说明书进行。
1.2.3实验结果
与正常对照组相比,模型组TNF-α、IL-6、IL-1β及IL-10水平明显升高(P<0.01)：与模型组相比,耳甲电针组TNF-α、IL-6、IL-1β水平明显下降(P<0.05)；“后三里”组IL-1β水平明显下降(P<0.05)。
13实验三(静脉给药6h)
1.3.1实验分组
K组正常对照组(n=11)：经尾静脉注射等量生理盐水,给药6h后取材。
L组模型组(n=10)：经尾静脉注射脂多糖,造模6h后取材。
M组耳甲迷走神经电针组(简称耳甲电针组,n=11)：经尾静脉注射脂多糖,1.5h后进行双侧耳甲刺激,造模6h后取材。
N组“后三里”电针组(简称“后三里”组,n=10)：经尾静脉注射脂多糖,1.5h后进行双侧“后三里”刺激,造模6h后取材。
1.3.2指标检测
血清TNF-α、IL-6、IL-1β及IL-10浓度检测采用酶联免疫法,ELISA试剂盒由美国R&D公司提供,检测方法严格按照试剂盒说明书进行。
1.3.3实验结果
与正常对照组相比,模型组TNF-α、IL-6、IL-1β水平明显升高(P<0.05,P<0.01)；与模型组相比,耳甲电针组IL-6、IL-1β水平明显下降(P<0.05),IL-10水平明显上升(P<0.05)；“后三里”组TNF-α、IL-1β水平明显下降(P<0.05)。
1.4实验四(迷走神经阻断)
1.4.1实验分组
A组正常对照组(n=11)：经尾静脉注射等量生理盐水。
B组模型组(n=10)：经尾静脉注射脂多糖。
O组迷走神经切断+耳甲迷走神经电针组(简称迷切耳针组,n=11)：经尾静脉注射脂多糖后立即切断双侧颈部迷走神经干,1.5h后进行双侧耳甲刺激。
P组迷走神经切断+“后三里”电针组(简称迷切“后三里”组,n=10)：经尾静脉注射脂多糖后立即切断双侧颈部迷走神经干,1.5h后进行双侧“后三里”刺激。
Q组N受体拮抗+耳甲迷走神经电针组(简称N受体拮抗耳针组,n=10)：造模前经右侧颈静脉注射六烃季铵(10mg/kg),造模1.5h后进行双侧耳甲刺激。
R组N受体拮抗+“后三里”电针组(简称N受体拮抗“后三里”组,n=10)：造模前经右侧颈静脉注射六烃季铵(10mg/kg),造模1.5h后进行双侧“后三里”刺激。
S组N受体α-7亚单位拮抗+耳甲迷走神经电针组(简称α-BGT耳针组,n=10)：造模前经右侧颈静脉注射α-银环蛇毒素(1μg/kg),造模1.5h后进行双侧耳甲刺激。
T组N受体α-7亚单位拮抗+“后三里”电针组(简称α-BGT'后三里”组,n=10)：造模前经右侧颈静脉注射α-银环蛇毒素(1μg/kg),造模1.5h后进行双侧“后三里”刺激。
1.4.2指标检测
血清TNF-α采用酶联免疫法,ELISA试剂盒由美国R&D公司提供,检测方法严格按照试剂盒说明书进行。
1.4.3实验结果
与正常对照组相比,模型组TNF-α水平明显升高(P<0.01)；与模型组相比,O、P、Q、R、S及T组TNF-a水平无限制差异。
2.耳针对NF-κB通路的影响
2.1肺组织NF-κB p65蛋白表达变化
2.1.1实验一(静脉给药2h)
采用第一部分实验一中A、B、C、D、E及F组的肺组织样本(在-80℃超低温冰箱保存的肺右叶组织)。
采用蛋白免疫印记法(Western Blot)测定各组肺脏组织NF-κB p65蛋白表达。
实验结果：与正常对照组(A组)相比,模型组(B组)NF-κB表达明显上调(P<0.01)；与模型组相比,耳甲电针组(D组)及迷走神经刺激组(E组)NF-κB表达明显下调(P<0.01)；与耳甲电针组相比,“后三里”组(F组)NF-κB表达明显增加(P<0.05)：与迷走神经刺激组相比,“后三里”组NF-κB表达明显上调(P<0.01)。
2.1.2实验二(静脉给药4h)
采用第一部分实验二中G、H、I及J组的肺组织样本(在-80。C超低温冰箱保存的肺右叶组织)。
采用蛋白免疫印记法(Western Blot)测定各组肺脏组织NF-κB p65蛋白表达。
实验结果：与正常对照组(G组)相比,模型组(H组)NF-κB表达明显上调(P<0.01)；与模型组相比,耳甲电针组(I组)NF-κB表达明显下调(P<0.05)。
2.1.3实验三(静脉给药6h)
采用第一部分实验三中K、L、M及N组的肺组织样本(在-80℃超低温冰箱保存的肺右叶组织)。
采用蛋白免疫印记法(Western Blot)测定各组肺脏组织NF-κB p65蛋白表达。
实验结果：与正常对照组(K组)相比,模型组(L组)NF-κB表达明显上调(P<0.01)：与模型组相比,耳甲电针组(M组)NF-κB表达明显下调(P<0.01)。
2.2肺脏NF-κB p65活性变化
2.2.1实验一(静脉给药2h)
采用第一部分动物实验一中的A、B、D、E及F组中用甲醛固定的肺组织。
采用免疫组化法测定各组肺脏组织NF-κB p65蛋白表达。
实验结果：与正常对照组(A组)相比,模型组(B组)NF-κB p65表达明显上调(P<0.01)；与模型组相比,耳甲电针组(D组)、迷走神经刺激组(E组)NF-κB p65表达明显下调(P<0.01)；与迷走神经刺激组相比,“后三里”组(F组)NF-κB p65表达明显升高(P<0.05)。
2.2.2实验二(静脉给药4h)
采用第一部分实验一中的G、H、I及J组中用甲醛固定的肺组织。
采用免疫组化法测定各组肺脏组织NF-κB p65蛋白表达。
实验结果：与正常对照组(G组)相比,模型组(H组)NF-κB p65表达明显上调(P<0.01)；与模型组相比,耳甲电针组(I组)、“后三里”组(J组)NF-κB p65表达明显下调(P<0.01,P<0.05)
2.2.3实验三(静脉给药6h)
采用第一部分实验一中的K、L、M及N组中用甲醛固定的肺组织。
采用免疫组化法测定各组肺脏组织NF-κB p65蛋白表达。
实验结果：与正常对照组(K组)相比,模型组(L组)NF-κB p65表达明显上调(P<0.01)；与模型组相比,耳甲电针组(M组)、“后三里”组(N组)NF-κB p65表达明显下调(P<0.05)。
2.2.4实验四(迷走神经阻断)
采用第一部分实验一中的A、B组及实验四中的O、P组用甲醛固定的肺组织。
采用免疫组化法测定各组肺脏组织NF-κB p65蛋白表达。
实验结果：与正常对照组(A组)相比,模型组(B组)NF-κB p65表达明显上调(P<0.01)；与模型组相比,迷切耳针组(O组)、迷切“后三里”组(P组)NF-κB p65表达无明显变化。
3.耳针对内毒素血症大鼠器官组织影响
3.1肝脏组织TNF-αmRNA、IL-10 mRNA表达
3.1.1实验一(静脉给药2h)
采用第一部分动物实验一中A、B、D、E及F组的肝脏组织样本(在-80℃超低温冰箱保存的肝左外叶组织)。
采用定量PCR法测定各组肝脏组织TNF-αmRNA表达变化。
实验结果：与正常对照组(A组)相比,模型组(B组)TNF-αmRNA表达明显增加(P<0.01)：与模型组相比,耳甲电针组(D组)、迷走神经刺激组(E组)TNF-αmRNA表达明显减少(P<0.05,P<0.01)。
3.1.2实验二(静脉给药4h)
采用第一部分动物实验二中G、H、I及J组的肝脏组织样本(在-80℃超低温冰箱保存的肝左外叶组织)。
采用定量PCR法测定各组肝脏组织。TNF-αmRNA、IL-10 mRNA表达变化。
实验结果：与正常对照组(G组)相比,模型组(H组)TNF-αmRNA表达明显增加(P<0.01)：与模型组相比,耳甲电针组(I组)、“后三里”组(J组)TNF-αmRNA表达明显减少(P<0.01)。
与正常对照组(G组)相比,模型组(H组)IL-10 mRNA表达明显增加(P<0.01)；与模型组相比,耳甲电针组(I组)、“后三里”组(J组)IL-10 mRNA表达明显增加(P<0.01,P<0.05)。
3.1.3实验三(静脉给药6h)
采用第一部分动物实验二中K、L、M及N组的肝脏组织样本(在-80℃超低温冰箱保存的肝左外叶组织)。采用定量PCR法测定各组肝脏组织TNF-αmRNA、IL-10mRNA表达变化。
实验结果：各组动物肝TNF-α、IL-10 mRNA表达变化见表X、图X。
与正常对照组(K组)相比,模型组(L组)TNF-αmRNA表达明显增加(P<0.01)：与模型组相比,耳甲电针组(M组)、“后三里”组(N组)TNF-αmRNA表达明显减少(P<0.01,P<0.05)。
与正常对照组(K组)相比,模型组(L组)IL-10 mRNA表达明显增加(P<0.01)；与模型组相比,耳甲电针组(M组)、“后三里”组(N组)IL-10 mRNA表达明显增加(P<0.05)。
3.2肺组织病理学变化
采用第一部分动物实验一中的A、B、D、E及F组中用甲醛固定的肺组织。
采用常规苏木素-伊红(HE)染色法观察各组肺脏组织病理变化。
实验结果：正常对照组(A组)肺泡结构完整,无萎陷,未见出血及炎细胞渗出。模型组(B组)肺泡结构破坏,可见萎陷、不张,肺泡腔内见大量红细胞及渗出液,支气管结构紊乱。耳甲电针组(D组)和迷走神经刺激组(E组)较之模型组损伤程度减轻,肺泡结构破坏明显减少。“后三里”组(F组)较之模型组损伤程度略减轻。
4.结论
本研究从血液循环细胞因子水平、NF-κB亘路、内脏器官细胞因子mRNA水平及器官组织病理变化等几个层面阐述了耳针抑制内毒素血症大鼠炎症发展的效应机制。通过造模后不同时间点炎症因子浓度变化揭示内毒素血症的发展变化过程。同时通过不同时间点相应指标的变化,观察耳针在炎症过程中的干预作用。
实验结果表明：耳针对各时间点的致炎因子都有明显的抑制作用,其效应与直接刺激迷走神经相似,在进行迷走神经阻断后,耳针的抗炎效应消失。同时,耳针抑制了各时间点NF-κB p65的表达,但是这种抑制作用同样有赖于迷走神经的完整。以上结果说明耳针抑制血液循环炎症因子及NF-κB通路可能是通过激活胆碱能抗炎通路来实现的。耳针刺激还有助于肝脏致炎因子TNF-amRNA水平的降低及抗炎因子IL-10mRNA水平的升高,这可能是抑制器官炎症发展的关键环节。此外,耳针刺激改善了肺脏组织的病理状态,说明其对内毒素血症中的器官具有保护作用。以上这些结果为耳针激活胆碱能抗炎通路治疗炎症提供了理论依据。
本研究还进行了直接刺激迷走神经、耳针刺激和“后三里”刺激三种刺激方法抗炎效应的比较。结果发现：直接刺激迷走神经效果优于耳针刺激和“后三里”刺激,耳针刺激效果优于“后三里”刺激。这可能是由于同“后三里”刺激相比,迷走神经刺激和耳针刺激更为直接地激活了胆碱能抗炎通路。
Cholinergic anti-inflammatory pathway (CAP) has been discovered in recent years. Based on efferent vagus nerve, this pathway aims at suppressing inflammatory response. The pathway can be activated by stimulating vagus nerve directly. Then it releases acetylcholine, and suppresses inflammatory cytokines, and control inflammation.
Auricular branch of vagus nerve innervates auricular concha(AC), and its afferent branch projects to nucleus of solitary tract (NTS) and dorsal motor nucleus of vagus (DMV). Neurons in NTS and DMV can be activated by acupuncturing auricular concha. We assume that auricular acupuncture (AA) may activate the cholinergic anti-inflammation pathway via stimulating auricular branch of vagus nerve. This study will focus on systemic inflammatory factors, NF-κB pathway, cytokine expression in organ, and organic pathology in endotoxemia. It aims at reveal mechanism on auricular acupuncture induced CAP.
1. Auricular acupuncture regulates serum cytokines in endotoxeamia rats.
Male SD rats.280-300g, were housed in standard conditions with access to regular chow and water. The permission number is SCXK-(army) 2007-004.
Endotoxemia in animals was induced by administering lipopolysaccharide (LPS, Escherichia coli 0111:B4:Sigma.5mg/kg. i.v.).
1.1 Experiment 1 (2h)
Group A:Control:animals were treated with sterile saline, i.v.
Group B:Model:animals were treated with LPS, i.v.
Group C:Simple AA:animals were treated with sterile saline, i.v.1.5h later, proceed AA.
Group D:AA+LPS:animals were treated with LPS. i.v.1.5h later, proceed AA.
Group E:VNS+LPS:animals were treated with LPS, i.v.1.5h later, treated with vagus nerve stimulation.
Group F:ST36+LPS:animals were treated with LPS, i.v.1.5h later, treated with electroacupuncture at "ST36". 2h after injection, all animals were taken blood. Test serum TNF-α, IL-6, IL-1βand IL-10 with ELISA.
Results:Compared with the control group, serum TNF-α,IL-6, IL-1βand IL-10 contents in the model group were increased significantly (P<0.01). In comparison with the model group, serum TNF-αand IL-10 contents in the AA+LPS, VNS+LPS and ZST36+LPS groups, and IL-6,IL-1βin the AA+LPS and VNS+LPS groups were down-regulated considerably (P<0.01). Compared with the VNS+LPS group, serum IL-6, IL-1βand IL-10 contents in the AA+LPS, and TNF-α, IL-6, IL-1βand IL-10 levels in the ZST36+LPS group were significantly different (P<0.05, P<0.01).
1.2 Experiment 2 (4h)
Group G:Control:animals were treated with sterile saline, i.v.
Group H:Model:animals were treated with LPS, i.v.
Group I:AA+LPS:animals were treated with LPS, i.v.1.5h later, proceed AA.
Group J:ST36+LPS:animals were treated with LPS. i.v.1.5h later, treated with electroacupuncture at "ST36" 4h after injection, all animals were taken blood. Test serum TNF-α, IL-6, IL-1βand IL-10 with ELISA kit.
Results:Compared with the control group, serum TNF-α, IL-6, IL-1βand IL-10 contents in the model group were increased significantly (P<0.01). In comparison with the model group, serum TNF-α, IL-6, IL-1βcontents in the AA+LPS group, and IL-1βin the ST36+LPS were down-regulated considerably (P<0.05).
1.3 Experiment 3 (6h)
Group K:Control:animals were treated with sterile saline, i.v.
Group L:Model:animals were treated with LPS. i.v.
Group M:AA+LPS:animals were treated with LPS. i.v.1.5h later, proceed AA.
Group N:ST36+LPS:animals were treated with LPS. i.v.1.5h later, treated with electroacupuncture at "ST36". 6h after injection, all animals were taken blood. Test serum TNF-α, IL-6, IL-1βand IL-10 with ELISA kit. Results:Compared with the control group, serum TNF-α, IL-6. IL-1βcontents in the model group were increased significantly (P<0.05, P<0.01). In comparison with the model group, serum IL-6,IL-1βcontents in the AA+LPS group,and TNF-α,IL-1βin the ST36+LPS group were down-regulated considerably (P<0.05):serum IL-10 content in AA+LPS group was increased significantly (P<0.05).
1.4 Experiment 4 (vagus nerve block)
Group A:Control:animals were treated with sterile saline, i.v.
Group B:Model:animals were treated with LPS, i.v.
Group O:vagotomy+AA+LPS:animals were treated with LPS, i.v. at the same time apply vagotomy 1.5h later, proceed AA.
Group P:vagotomy +ST36+LPS:animals were treated with LPS. i.v. at the same time apply vagotomy.1.5h later, treated with electroacupuncture at "ST36"
GroupQ:Hexamethonium+AA+LPS:animals were injected with Hexamethonium, i.v. then treated with LPS.1.5h later, proceed AA.
Group R:Hexamethonium-ST36+LPS:animals were injected with Hexamethonium. i.v.. then treated with LPS.1.5h later, stimulate ST36.
Group S:a-BGT+AA+LPS:animals were injected with a-BGT. i.v.. then treated with LPS,1.5h later, proceed AA.
Group T:a-BGT-ST36+LPS:animals were injected with a-BGT. i.v.. then treated with LPS,1.5h later, stimulate ST36. 2h after LPS injection, all animals were taken blood. Test serum TNF-a with ELISA kit.
Results:Compared with the control group, serum TNF-a content in the model group were increased significantly (P<0.01). In comparison with the model group, there was non-significant difference between other groups (P<0.05).
2. Auricular acupuncture influences NF-κB pathway.
2.1 NF-κB p65 protein expression in lung tissue
2.1.1 Experiment 1 (2h)
Take lung tissue from group A, B, C, D, E and F. Detect NF-κB p65 protein expression from each group. Western Blot procedure. Results:Compared with the normal control group, pulmonary NF-κB p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-κB p65 expression levels in the AA+LPS and VNS+LPS groups were down-regulated considerably (P<0.01). Compared with the VNS+LPS group pulmonary NF-κB p65 expression levels in the ZST36+LPS group were significantly different (P<0.01). In comparison with the AA+LPS group, pulmonary NF-κB p65 expression level in the ZST36+LPS group was significantly different (P<0.05).
2.1.2 Experiment 2 (4h)
Take lung tissue from group G, H, I, and J. Detect NF-κB p65 protein expression from each group. Use Western Blot procedure.
Results:Compared with the normal control group, pulmonary NF-κB p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-κB p65 expression levels in the AA+LPS groups were down-regulated considerably (P<0.05).
2.1.3 Experiment 3 (6h)
Take lung tissue from group K, L. M and N. Detect NF-κB p65 protein expression from each group. Use Western Blot procedure. Results:Compared with the normal control group, pulmonary NF-κB p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-κB p65 expression levels in the AA+LPS groups were down-regulated considerably (P<0.01).
2.2 Pulmonary NF-κB p65 activity
2.2.1 Experiment 1 (2h)
Take lung tissue from group A. B, D, E and F. Detect pulmonary NF-κB p65 expression from each group. Use Immunohistochemistry technology.
Results:Compared with the normal control group, pulmonary NF-KBp65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-κB p65 expression levels in the AA+LPS and VNS+LPS groups were down-regulated considerably (P<0.01). Compared with the VNS+LPS group pulmonary NF-κB p65 expression levels in the ZST36+LPS group were significantly increased (P<0.05).
2.2.2 Experiment 2 (4h)
Take lung tissue from group G. H, I and J. Detect pulmonary NF-κB p65 expression from each group. Use Immunohistochemistry technology. Results:Compared with the normal control group, pulmonary NF-κB p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-κB p65 expression levels in the AA+LPS and ST36+LPS groups were down-regulated considerably (P<0.01, P<0.05).
2.2.3 Experiment 3 (6h)
Take lung tissue from group K. L. M and N. Detect pulmonary NF-κB p65 expression from each group. Use Immunohistochemistry technology. Results:Compared with the normal control group, pulmonary NF-κB p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonary NF-κB p65 expression levels in the AA+LPS and ST36+LPS groups were down-regulated considerably (P<0.05).
2.2.4 Experiment 4 (vagus nerve block)
Take lung tissue from group A, B, O and P. Detect pulmonary NF-κB p65 expression from each group. Use Immunohistochemistry technology. Results:Compared with the normal control groupm pulmonary NF-κB p65 expression level in the model group were increased significantly (P<0.01). In comparison with the model group, pulmonaiy NF-κB p65 expression levels in the other two groups was non-significant different (P<0.05).
3. Auricular acupuncture protects organs in endotoxemia rats.
3.1 TNF-a mRNA, IL-10 mRNA expression in liver
3.1.1 Experiment 1 (2h)
Take liver sample from group A. B. D. E and F. Detect TNF-a mRNA expression from each group with real time PCR. Results:Compared with the normal control group, TNF-a mRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, TNF-αmRNA expression levels in the AA+LPS and VNS+LPS groups were down-regulated considerably (P<0.05, P<0.01).
3.1.2 Experiment 2 (4h)
Take liver sample from group G, H, I and J. Detect TNF-αmRNA and IL-10 expression from each group with real time PCR. Results:Compared with the normal control group, TNF-αmRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, TNF-αmRNA expression levels in the AA+LPS and ST36+LPS groups were down-regulated considerably (P<0.01).
Compared with the normal control group. IL-10 mRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, IL-10 mRNA expression levels in the AA+LPS and ST36+LPS groups were up-regulated considerably (P<0.01, P<0.05).
3.1.3 Experiment 3 (6h)
Take liver sample from group K, L, M and N. Detect TNF-αmRNA and IL-10 mRNA expression from each group with real time PCR. Results:Compared with the normal control group. TNF-a mRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, TNF-αmRNA expression levels in the AA+LPS and ST36+LPS groups were down-regulated considerably (P<0.01, P<0.05).
Compared with the normal control group, IL-10 mRNA expression level in the model group were increased significantly (P<0.01). In comparison with the model group, IL-10 mRNA expression levels in the AA+LPS and ST36+LPS groups were up-regulated considerably (P<0.05).
3.2 lung histopathology
Take lung samples from group A,B,D,E and F.
Use HE staining technology.
Result demonstrated that samples from AA+LPS and ST36+LPS groups were much improved in pathological states of lung.
4. Conclusion
This study research the mechanism on inhibitory effect of auricular acupuncture in cytokine regulation. We focus on systemic inflammatory factors, NF-κB pathway, cytokine expression in organ, and organic pathology in endotoxemia rats. The results demonstrate that auricular acupuncture plays an important role in inflammatory response, and inhibits pro-inflammatory cytokines. Meanwhile, auricular acupuncture suppresses NF-κB p65 expression as well. In absence of vagus nerve, the anti-inflammatory effect mentioned above was destroyed. It suggests that auricular acupuncture may activate cholinergic anti-inflammatory pathway to control inflammatory development. In addition, auricular acupuncture regulates TNF-a mRNA and IL-10 mRNA expression in liver, and this might be the key point to suppress inflammatory development.
引文
1. Tracey K J. The inflammatory reflex. Nature,2002,420(6917):853-859.
2. Pavlov V A, Tracey K J. Neural regulators of innate immune responses and inflammation. Cell Mol Life Sci,2004,61(18):2322-2331.
3. Tracey K J. Autonomic neural regulation of immunity. Journal of Internal Medicine, 2005,257(2):156-166.
4. Blalock J E. The immune system as a sensory organ. J Immunol,1984,132(3): 1067-1070.
5. Blalock J E. Shared ligands and receptors as a molecular mechanism for communication between the immune and neuroendocrine systems. Ann N Y Acad Sci.1994,741:292-298.
6. Borovikova LV, I vanova S, Zhang MH, et al. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature,2000,405 (6785):458-462.
7. Tracey K J. Physiology and immunology of the cholinergic anti-inflammatory pathway. J Clin Inves,2007,117(2):289-296.
8. Watkins L R. Blockade of interleukin-1 induced hyperthermia by sub-diaphragmatic vagotomy:evidence for vagal mediation of immune-brain communication. Neurosci Lett,1995,183(1-2):27-31.
9. Hansen M K, O'Connor K A, Goehler L E. et al. The contribution of the vagus nerve in interleukin-1 beta-induced fever is dependent on dose. Am J Physiol Regul Integr Comp Physiol,2001.280(4):929-934.
10. Wang H, Yu M, Ochani M. et al. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature.2003.421(6921):384-388.
11. Wang H, Liao H, Ochani M, et al. Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med,2004.10(11):1216-1221.
12. Huston J M. Ochani M. Rosas-Ballina M, et al. Splenectomy inactivates the cholinergic anti-inflammatory pathway during lethal endotoxemia and polymicrobial sepsis. J Exp Med,2006,203(7):1623-1628.
13. Pavlov V A, Ochani M. Gallowitsch-Puerta M. et al. Central muscarinic cholinergic regulation of the systemic inflammatory response during endotoxemia. Proc Natl Acad Sci.2006,103(13):5219-5223.
14. Pavlov V A, Parrish W R, Rosas-Ballina M, et al. Brain acetylcholinesterase activity controls systemic cytokine levels through the cholinergic anti-inflammatory pathway. Brain Behav and Immun,2009,23(1):41-45.
15. Guarini S, Altavilla D, Cainazzo M M. et al. Efferent vagal fibre stimulation blunts nuclear factor-kappaB activation and protects against hypovolemic hemorrhagic shock. Circulation,2003,107(8):1189-1194.
16. de Jonge W J, van der Zanden E P, The F O, et al. Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway. Nat Immunol.2005,6(8):844-851.
17. Van Westerloo D J, Giebelen I A, Meijers J C. et al. Vagus nerve stimulation inhibits activation of coagulation and fibrinolysis during endotoxemia in rats. J Thromb Haemost,2006,4(9):1997-2002.
18. Eiu C Y, Mueller M H. Grundy D. et al. Vagal modulation of intestinal afferent sensitivity to systemic LPS in the rat. Am J Physiol Gastrointest Liver Physiol. 2007.292(5):1213-1220.
19.黄健.完整迷走神经刺激对大鼠全身炎性反应的影响.中华烧伤杂志,2008,24(2)：99—101.
20. Bernik T R, Friedman S G, Ochani M. et al. Pharmacological stimulation of the cholinergic anti-inflammatory pathway. J Exp Med,2002,195(6):781-788.
21. Mioni C, Bazzani C, Giuliani D. et al. Activation of an efferent cholinergic pathway produces strong protection against my ocardial ischemia/reperfusion injury in rats. Crit Care Med,2005,33(11):2621-2628.
22. Altavilla D. Guarini S,Bitto A, et al. Activation of the cholinergic anti-inflammatory pathway reduces NF-kappa activation, blunts TNF-alpha production, and protects against splanchic artery occlusion shock. Shock,2006, 25(5):500-506.
23. Borovikova L V, Ivanova S, Nardi D. et al. Role of vagus nerve signaling in CNI-1493-mediated suppression of acute inflammation. Auton Neurosci.2000, 85(1-3):141-147.
24. van Westerloo D J, Giebelen I A, Florquin S, et al. The vagus nerve and nicotinic receptors modulate experimental pancreatitis severity in mice. Gastroenterology, 2006,130(6):1822-1830.
25. Saeed R W, Varma S. Peng-Nemeroff T. et al. Cholinergic stimulation blocks endothelial cell activation and leukocyte recruitment during inflammation. J Exp Med,2005,201(7):1113-1123.
26. Matthay MA, Ware LB. Can nicotine treat sepsis? Nat Med,2004, 10(11):1161-1162.
27. Zijlstra FJ. van den Berg-de Lange I, Huygen FJ, et al. Anti-inflammatory actions of acupuncture. Mediators Inflamm.2003,12(2):59-69.
28.袁翔,李建国,黄越,等.电针足三里激活胆碱能抗炎通道抗大鼠感染性休克.武汉大学学报(医学版),2007,28(2)：203—206.
29.李建国,彭周全,杜朝晖,等.电针足三里激活胆碱能抗炎通路抗大鼠失血性休克的研究.中国中西医结合急救杂志,2006,13(1)：27—31.
30.王景杰,黄裕新.C-fos在电针调控大鼠胃运动中的表达及其意义.针刺研究,2001,26(4)：274—278.
31. National Center for Complementary and Alternative Medicine. National Institutes of Health.2004. NCCAM publication D003:Acupuncture. http://nccam.nih.gov/healtli/acupuncture/.
32. Luyer M D, Greve J W, Hadfoune M. et al. Nutritional stimulation of cholecystokinin receptors inhibits inflammation via the vagus nerve. J Exp Med, 2005.202(8):1023-1029.
33. Facchini M. Malfatto G. Sala L. et al. Changes of autonomic cardiac profile after a 3-week integrated body weight reduction program in severely obese patients. J Endocrinol Invest.2003.26(2):138-142.
34. Hoguin F. Tellez-Rojo MM. Lazo M, et al. Cardiac autonomic changes associated with fish oil vs soy oil supplementation in the elderly. Chest,2005, 127(4):1102-1107.
35. Berbert A A, Kondo CR, Almendra CL, et al. Supplementation of fish oil and olive oil in patients with rheumatoid arthritis. Nutrition,2005,21(2):131-136.
36. Christensen J H, Christensen M S, Dyerberg J, et al. Heart rate variability and fatty acid content of blood cell membranes:a dose-response study with n-3 fatty acids. Am J Clin Nutr,1999,70(3):331-337.
37. Christensen J H, Skou H A, Madsen T, et al. Heart rate variability and n-3 polvunsaturated fatty acids in patients with diabetes mellitus. J Intern Med,2001, 249(6):545-552.
38.张诗兴,姜文方.耳穴定位与神经、血管分布的研究.南京中医药大学学报,1998,14(4)：228-229.
39.严振国,糜兢华.耳穴的神经分布及耳廓的形成.上海针灸杂志,1989,1：145-146.
40.戴衡茹,赵学敏.耳廓的神经供应.安徽医科大学学报,.1960,3(2-3)：65-69.
41.雷琦.外耳的神经和动脉的解剖.解剖学报,1963,2(1)：29-37.
42.中国科学院动物研究所.人耳廓及其穴位里的神经分布.针刺麻醉原理的探讨.北京：人民卫生出版社,1974,41-46.
43.中国科学院动物研究所.猫耳廓的神经分布.针刺麻醉原理的探讨.北京：人民卫生出版社,1974,37-40.
44. Hunt Jr. The sensory field of the facial nerve:A further contribution to the symptomatology of the eniculate ganglion. Brain.1915.38:418.
45. Hollinshead WH. Anatomy for Surgeons:The Head-Neck. Harper and Row, 1968,190-191.
46. Hollinshead WH. Anatomy for Surgeons:The Head-Neck,3rd editon. Harper and Row.1982,162.
47. Guild SR. The glomus jugulare, a non chromaffin paraganglion in man. Ann Otol Rhinol Laryngol,1953,62(4):1045-1071.
48. Tekdemir I, Aslan A, Elhan A. A clinico-anatomic study of the auricular branch of the vagus nerve and Arnold's ear-cough reflex. Surg Radiol Anat.1998,20(4): 253-257.
49. Prasad KS. Cardiac depression on syringing the ear. A case report.J Laryngol Otol. 1984.98(10):1013.
50. Moorthy SS, Krishna G, Elliott CL. Is there an auriculovagal reflex producing cardiac dysrhythmias? Arch Otolaryngol.1985,111(9):631.
51. EngelD. The gastroauricular phenomenon and related vagus reflexes. Arch Psychiatr Nervenkr,1979,227(3):271-277.
52. Engel D. Uber das reflektorische Ohrenjucken bei Sodbrennen. Med Klin,1922, 47:1495-1497.
53. Malherbe W.D.F. Otalgia with oesophageal hiatus hernia. Lancet,1958, 1368-1369.
54. Deutsch M. Ein Beitrag zur Symptomatomatologie der beginnenden Lungentuberkulose. Med Klin,1919,43:1090-1091.
55. Bradford F K. The auriculo-genital reflex in cats. J Exp Physiol,1937, 27:272-279.
56. Vasiliu D I. Reflex auriculo-uterin. Spitalul,1932.52:396.
57. Aschner. In:The neurological examination. De Jong. R.N. (ed.), Hober Med Division. New York;London,1967:315.
58. Kalchschmidt, In:Lehrbuch der klinischen Diagnostik, J.mared. J. Mosci (eds); Jena:G. Fischer.1956,293-294.
59. Berlin R. Theagstro-auricular phenomenon. Lancet.1959,734-735.
60. Gordon F J. Effect of nucleus tractus solitarius lesions on fever produced by interleukin-1beta. Auton Neurosci,2000,85(1-3):102-110.
61. Kalia M, Fuxe K. Hokfelt T T, et al. Distribution of neuropeptide immunoreactive nerve terminals within the subnuclei of the nucleus of the tractus solitarius of the rat. J Comp Neurol.1984.222(3):409-444.
62. Kaliam, Mesulam M M. Brainstem projections of sensory and motor components of the vagus complex in the cat:The cervical vagus and nodose ganglion. J Comp Neurol,1980,193(2):453-465.
63. Okuma Y. Osumi Y. Central cholinergic descending pathway to the dorsal motor nucleus of the vagus in regulation of gastric functions. Jpn J Pharmacol,1986, 41(3):373-379.
64. Chien CH, Shieh JY, Ling EA, et al. The composition and central projections of the internal auricular nerves of the dog. J Anat,1996,189(Pt2):349-362.
65. Campbell R, Rodrigo D, Cheung L. Asystole and bradycardia during maxillofacial surgery. Anesth Prog,1994,41(1):13-16.
66. Mantle-St John LA, Tracey DJ. Somatosensory nuclei in the brainstem of the rat: independent projections to the thalamus and cerebellum. J Comp Neurol,1987, 255(2):259271.
67. Menetrey D, Basbaum AI. Spinal and trigeminal projections to the nucleus of the solitary tract:a possible substrate for somatovisceral and viscerovisceral reflex activation. J Comp Neurol,1987.255(3):439-450.
68.高欣妍.耳针疗法与耳-迷走-内脏反射.博士毕业论文,2005.
69.梅志刚.耳-迷走反射与耳针降糖效应机制研究.博士毕业论文,2007.
70.何伟.耳-迷走反射及耳针抗癫痫的效应机制研究.博士毕业论文,2008.
71.赵洁,杨选明,都培双,等.T淋巴细胞抑制急性感染的炎症反应.生物化学与生物物理进展,2008,35(2)：129-132.
72.曹雪涛.免疫学前沿进展.人民卫生出版社,2009,350.
73. Theilgaard-Monch K, Porse BT, Borregaard N. Systems biology of neutrophil differentiation and immune response. Curr Opin Immunol,2006,18(1):54-60.
74. Fierro IM, Serhan CN. Mechanisms in anti- inflammation and resolution:the role of lipoxins and aspirin- triggered lipoxins. Braz J Med Biol Res.2001,34(5):555-566.
75.朱梅,高洪泉,刘瑞丰,等.针刺“足三里”、“关元”穴区对老年大鼠肝脏内巨噬细胞功能影响的实验研究.针灸临床杂志,2003,19(6)：52.
76.朱文莲,刘仁权.艾灸大椎穴对免疫低下小鼠巨噬细胞吞噬功能的影响.北京中医药大学学报,2005,28(1)：89.
77.杨志新,乔跃兵,赵粹英.艾灸对荷瘤小鼠巨噬细胞免疫功能的增强作用.承德医学院学报,2002,19(2)：97.
78.陈国民,黄欣,蒋康平,等.艾灸大椎穴对小鼠巨噬细胞吞噬功能及脾脏重 量的影响.四川中医,1987,(9)：41.
79.李亚东,高洪泉,朱梅,等.针刺老年大鼠足三里、关元穴对NO、SOD、 MDA以及免疫影响的实验研究.中国针灸,2002,22(11)：772.
80.骆文郁.无烟灸疗法对实验性小鼠免疫功能影响的实验研究.针刺研究,2001,18(1)：661.
81.梁繁荣,曾芳,唐勇.针灸在地震后疫病防治中的应用.中国针灸,2008,28(7)：507.
82.逢紫千,王富春,严兴科.针灸天枢穴对脾虚泄泻大鼠免疫功能影响的实验研究.江苏中医药,2005,26(4)：27.
83.严桂珍,郑家铿,许少峰,等.针灸”三阴交”穴择时治疗对脾阳虚家兔免疫功能的影响.中国针灸,2001,21(12)：735.
84.陈雄华,刘又香,王华,等.针灸“足三里”、“关元”穴对阳虚大鼠免疫功能影响比较研究.中国针灸,2000,20(9)：555-557.
85.吕琳.针灸调节肿瘤免疫的临床及实验研究进展.辽宁中医杂志,2001,28(10)：638-640.
86.赵宁侠,高巍,史恒军,等.电针足三里及非经非穴点对大鼠细胞免疫的影响.云南中医学院学报,2001,4(1)：37-39.
87. Yu Y,Kasahara T, Sato T. et al. Enhancement of splenic interferon-gamma, interleukin-2, and NK cyotoxicity by S36 acupoint acu puncture in F344 rats. Jpn J Physiol.1997,42(2):173-178.
88.黄诚,陈汉平,秦秀娣,等.针刺抑制老年大鼠脑与垂体细胞因子基因表达.针刺研究,1998,23(1)：24.
89.谌剑飞,梁浩荣,马雅玲,等.针刺对糖尿病并发急性脑梗死血浆白介素-6及肿瘤坏死因子-α水平的影响.中国中西医结合急救杂志,2001,8(2)：92.
90.吴焕淦,陈汉平,周丽斌.针灸治疗大鼠溃疡性结肠炎的分子机制.上海针灸杂志,1998,17(6)：30.
91. Baldwin. Series introduction:the transcription factor NF-κB and human disease. J Clin Invest,2001;107(1):3-6.
92. Huston JM,Gallowitsch-Puerta M, Ochani M, et al. Transcutaneous vagus nerve stimulation reduces serum high mobility group box 1 levels and improves survival in murine sepsis. Crit Care Med,2007.35(12):2762-2768.
93.傅守延,吉田丰.烟碱对大鼠纹状体儿茶酚氨及吲哚胺含量的影响.沈阳药学院学报,1994,11(3)：186-189.
94. Litscher G. Computer-based quantification of traditional Chinese-ear-and Korean hand acupuncture:needle induced changes of regional cerebral blood flow velocity. Neurol Res,2002,24(4):377-380.
95. Haker E, Eqekvist H, Bjerring P. Effect of sensory stimulation on sympathetic and parasympathetic activities in healthy subjects. J Auton Nerv Syst.2000,79(1): 52-59.
96. Song XY. Torphy TJ,Griswold DE, et al. Coming of Age:anti-Cytokine Therapies. Mol Interv,2002,2(1):36-46.
97. Gosain A, Gamelli RL. A primer in cytokines. J Burn Care Rehabil,2005,26 (2): 7-12.
98. Carswell E A, Old L J, Kassel R L, et al. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci,1975,72(9):3666-3670.
99. Caput D, Beutler B, Hartog K. et al. Identification of a common nucleotide sequence in the 3"-untranslated region of mRNA molecules specifying inflammatory mediators. Proc Natl Acad Sci.1986.83(6):1670-1674.
100. Pennica D, Hayflick JS, Bringman TS, et al. Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor. Proc Natl Acad Sci. 1985.82(18):6060-6064.
101. Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell Death Differ,2003.10(1):45-65.
102. Kwak EL, Larochelle DA. Beaumont C, et al. Role for NF-kappa B in the regulation of ferritin H by tumor necrosis factor-alpha. J Biol Chem,1995. 270(25):15285-15293.
103. Dinarello CA, Cannon JG, Wolff SM, et al. Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1. J Exp Med,1986, 163(6):1433-1450.
104. Manicourt DH, Triki R, Fukuda K, et al. Levels of circulating tumor necrosis factor alpha and interleukin-6 in patients with rheumatoid arthritis. Relationship to serum levels of hyaluronan and antigenic keratin sulfate. Arthritis Rheum,1993, 36(4):490-499.
105. Kupferminc MJ, Peaceman AM, Wigton TR, et al. Tumor necrosis factor-alpha is elevated in plasma and amniotic fluid of patients with severe preeclampsia. Am J Obstet Gynecol,1994,170(6):1752-1757.
106. Harel Y, Silva M, Giroir B, et al. A reporter transgene indicates renal-specific induction of tumor necrosis factor (TNF) by shiga-like toxin. Possible involvement of TNF in hemolytic uremic syndrome. J Clin Invest,1992. 92(5):2110-2116.
107. Wu CJ, Lovett M, Wong-Lee J, et al. Cytokine gene expression in rejecting cardiac allografts. Transplantation,1992,54(2):326-332.
108. Sandborn WJ. A controlled trial of anti-tumor necrosis factor alpha antibody for Crohn's disease. Gastroenterology.1997,113(3):1042-1043.
109. Mizel SB. The interleukins. FASEB J.1989.3(12):2379-2388.
110. Ghosh S. May M J. Kopp E B. NF-kappa B and Rel proteins:evolutionarily conserved mediators of immune responses. Annu Rev Immunol,1998, 16:225-260.
111. Liu SF, Malik AB. NF-κB activation as a pathological mechanism of septic shock and inflammation. Am J Physiol Lung Cell Mol Physiol,2006.290(4):622-645.
112. Han S.T, Ko HM, Choi JH, et al. Molecular mechanisms for lipopolysaccharide-induced biphasic activation of nuclear factor-kappa B (NF-kappa B). J Biol Chem,2002,277(47):44715-44721.
113. Kelly A. Vereker E. Nolan Y, et al. Activation of p38 plays a pivotal role in the inhibitory effect of lipopolysaccharide and interleukin-1 beta on long term potentiation in rat dentate gyrus. J Biol Chem.2003,278(21):19453-19462.
114. Liu SF. Ye X. Malik AB. Pyrrolidine dithiocarbamate prevents I-kappaB degradation and reduces microvascular injury induced by lipopolysaccharide in multiple organs. Mol Pharmacol,1999.55(4):658-667.
115. Pocock J. Gomez-Guerrero C, Harendza S, et al. Differential activation of NF-kappa B, AP-1, and C/EBP in endotoxin-tolerant rats:mechanisms for in vivo regulation of glomerular RANTES/CCL5 expression. J Immunol,2003,170(12): 6280-6291.
116. Pritts TA, Moon MR, Wang Q, et al. Activation of NF-kappaB varies in different regions of the gastrointestinal tract during endotoxemia. Shock,2000. 14(2):118-122.
117. Ge Y, Ezzell RM, Clark BD, et al. Relationship of tissue and cellular interleukin-1 and lipopolysaccharide after endotoxemia and bacteremia. J Infect Dis,1997,176(5):1313-1321.
118. Liu JH, Yan J, Hu JM, et al. Effects of electroacupuncture on gastric myoelectric activity and substance P in the dorsal vagal complex of rats. Neurosci Lett. 2004,356(2):99-102.
119. Li L, Chen Y X, Hong X, et al. Nitric oxide in vPAG mediates the depressor response to acupuncture in stress-induced hypertensive rats. Acupunct Electrother Res.2001; 26(3):165-170.
120.孟卓,吕国蔚.足三里—脊髓背角—孤束核的机能联系.中国科学B辑,1992,4：393-399.
121.胡森,宋琪,盛志勇,等.电针兴奋胆碱能抗炎通路对内毒素引起的细胞因子释放和脏器功能损害作用研究.中国中西医结合急救杂志,2008,15(4)：205-208.
122.石现,宋琪,关玲,等.电针对内毒素引起大鼠肝损伤保护作用的研究.中国针灸,2008;7(4)：290-292.
123. Domenica Altavilla, Salvatore Guarini, Alessandra Bitto. et al. Activation of the cholinergic anti-inflammatory pathway reduces NF-κB activation, blunts TNF-a production, and protects against splanchnic artery occlusion shock. Shock,2006, 25(5):500-506.
124. Valentin A Pavlov. Mahendar Ochan, Li-Hong Yang, et al. Selectivea7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine endotoxemia and severe sepsis. Crit Care Med.2007,35(4):1139-1144.
125.王述菊,孙国杰,杜艳军.孤束核在针刺“足三里”调节胃运动中的作用机制.中国中医药信息杂志,2007,14(9)：28-30.
126.王景杰,黄裕新,郭庆东.电针调节心理性应激状态下胃动力障碍的实验研究.针刺研究,2000,25(4)：267-270.
127.孟卓,吕国蔚.大鼠脊髓背角神经元与孤束核的联系的电生理学研究.科学通报,1990,35(4)：292-295.
128.吴利平,李辉,李云庆.大鼠延髓和脊髓背角的PKCr阳性神经元向孤束核投射.第四军医大学学报,2001,22(23)：2171-2174.
129.李江山,严洁,何军锋,等.针刺对胃扩张模型大鼠孤束核神经元放电的影响.中国医药导报,2009,6(8)：19-21.
130. Goehler L E, Gaykema RP, Hansen MK, et al. Vagal immune-to-brain communication:a visceral chemosensory pathway. Auton Neurosci,2000, 85(1-3):49-59.
131. Hermann G E, Emch G S, Tovar C A. et al. c-Fos generation in the dorsal vagal complex after systemic endotoxin is not dependent on the vagus nerve. Am J Physiol Requl Integr Comp Physiol.2001,280(1):289-299.
132. Emch G S. Hermann G E. Rogers R C. TNF-a activates solitary nucleus neurons responsive to gastric distension. Am J Physiol Gastrointest Liver Physiol,2000, 279(3):582-586.
133. Romanovsky AA. Thermoregulatory manifestations of systemic inflammation: lessons from vagotomy. Auton Neurosci,2000,85(1-3):39-48.
134. Goehler LE, Relton JK, Dripps D. Vagal paraganglia bind biotinylated interleukin-1 receptor antagonist:a possible mechanism for immune-to-brain communication. Brain Res Bull,1997,43(3):357-364.
135.余传霖.细菌内毒素研究现状.国外医学抗生素分册,1997,20(4)：30.
136. MacMicking JD. Nathan C. Hom, et al. Altered responses to bacterial infection and endotoxin shock in mice inducible nitric oxide syntbase. Cell,1995, 81(4):641-501.
137.黄凤,荣培晶,朱兵,等.耳甲迷走神经刺激干预35例糖耐量受损患者临 床观察.中华中医药杂志,2010,25(12)：2185-2186.
138. Bianchi M. Suppression of proinflammatory cytokines in monocytes by a tetravalent guanylhydrazone. J Exp Med.1996; 183:927-936.
139. Bianchi M. Ulrich P. Bloom O, et al. An inhibitor of macrophage arginine transport and nitric oxide production (CNI-1493) prevents acute inflammation and endotoxin lethality. Mol Med,1995,1(3):254-266.
140. Tracey K J. Suppression of TNF and other proinflammatory cytokines by the tetravalent guanylhydrazone CNI-1493. Prog Clin Biol Res,1998,397:335-343.
141. Homines D, van den Blink B, Plasse T, et al. Inhibition of stress-activated MAP kinases induces clinical improvement in moderate to severe Crohn's disease. Gastroenterology,2002,122(1):7-14.
142. Tracey KJ, Czura CJ. Ivanova S. Mind over immunity. FASEB J,2001, 15(9):1575-1576.
143. Pullan RD, Rhodes J, Ganesh S, et al. Transdermal nicotine for active ulcerative colitis. N Engl J Med,1994,330(12):811-815.
144. Ben-Menachem E. Vagus nerve stimulation, side effects,and long-term safety. J Clin Neurophysiol,2001,18(5):415-418.
145. Schachter SC. Vagus nerve stimulation:where are we? Curr Opin Neurol,2002. 15(2):201-206.
2. Pavlov V A, Tracey K J. Neural regulators of innate immune responses and inflammation. Cell Mol Life Sci,2004,61(18):2322-2331.
3. Tracey K J. Autonomic neural regulation of immunity. Journal of Internal Medicine, 2005,257(2):156-166.
4. Blalock J E. The immune system as a sensory organ. J Immunol,1984,132(3): 1067-1070.
5. Blalock J E. Shared ligands and receptors as a molecular mechanism for communication between the immune and neuroendocrine systems. Ann N Y Acad Sci.1994,741:292-298.
6. Borovikova LV, I vanova S, Zhang MH, et al. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature,2000,405 (6785):458-462.
7. Tracey K J. Physiology and immunology of the cholinergic anti-inflammatory pathway. J Clin Inves,2007,117(2):289-296.
8. Watkins L R. Blockade of interleukin-1 induced hyperthermia by sub-diaphragmatic vagotomy:evidence for vagal mediation of immune-brain communication. Neurosci Lett,1995,183(1-2):27-31.
9. Hansen M K, O'Connor K A, Goehler L E. et al. The contribution of the vagus nerve in interleukin-1 beta-induced fever is dependent on dose. Am J Physiol Regul Integr Comp Physiol,2001.280(4):929-934.
10. Wang H, Yu M, Ochani M. et al. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature.2003.421(6921):384-388.
11. Wang H, Liao H, Ochani M, et al. Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med,2004.10(11):1216-1221.
12. Huston J M. Ochani M. Rosas-Ballina M, et al. Splenectomy inactivates the cholinergic anti-inflammatory pathway during lethal endotoxemia and polymicrobial sepsis. J Exp Med,2006,203(7):1623-1628.
13. Pavlov V A, Ochani M. Gallowitsch-Puerta M. et al. Central muscarinic cholinergic regulation of the systemic inflammatory response during endotoxemia. Proc Natl Acad Sci.2006,103(13):5219-5223.
14. Pavlov V A, Parrish W R, Rosas-Ballina M, et al. Brain acetylcholinesterase activity controls systemic cytokine levels through the cholinergic anti-inflammatory pathway. Brain Behav and Immun,2009,23(1):41-45.
15. Guarini S, Altavilla D, Cainazzo M M. et al. Efferent vagal fibre stimulation blunts nuclear factor-kappaB activation and protects against hypovolemic hemorrhagic shock. Circulation,2003,107(8):1189-1194.
16. de Jonge W J, van der Zanden E P, The F O, et al. Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway. Nat Immunol.2005,6(8):844-851.
17. Van Westerloo D J, Giebelen I A, Meijers J C. et al. Vagus nerve stimulation inhibits activation of coagulation and fibrinolysis during endotoxemia in rats. J Thromb Haemost,2006,4(9):1997-2002.
18. Eiu C Y, Mueller M H. Grundy D. et al. Vagal modulation of intestinal afferent sensitivity to systemic LPS in the rat. Am J Physiol Gastrointest Liver Physiol. 2007.292(5):1213-1220.
19.黄健.完整迷走神经刺激对大鼠全身炎性反应的影响.中华烧伤杂志,2008,24(2)：99—101.
20. Bernik T R, Friedman S G, Ochani M. et al. Pharmacological stimulation of the cholinergic anti-inflammatory pathway. J Exp Med,2002,195(6):781-788.
21. Mioni C, Bazzani C, Giuliani D. et al. Activation of an efferent cholinergic pathway produces strong protection against my ocardial ischemia/reperfusion injury in rats. Crit Care Med,2005,33(11):2621-2628.
22. Altavilla D. Guarini S,Bitto A, et al. Activation of the cholinergic anti-inflammatory pathway reduces NF-kappa activation, blunts TNF-alpha production, and protects against splanchic artery occlusion shock. Shock,2006, 25(5):500-506.
23. Borovikova L V, Ivanova S, Nardi D. et al. Role of vagus nerve signaling in CNI-1493-mediated suppression of acute inflammation. Auton Neurosci.2000, 85(1-3):141-147.
24. van Westerloo D J, Giebelen I A, Florquin S, et al. The vagus nerve and nicotinic receptors modulate experimental pancreatitis severity in mice. Gastroenterology, 2006,130(6):1822-1830.
25. Saeed R W, Varma S. Peng-Nemeroff T. et al. Cholinergic stimulation blocks endothelial cell activation and leukocyte recruitment during inflammation. J Exp Med,2005,201(7):1113-1123.
26. Matthay MA, Ware LB. Can nicotine treat sepsis? Nat Med,2004, 10(11):1161-1162.
27. Zijlstra FJ. van den Berg-de Lange I, Huygen FJ, et al. Anti-inflammatory actions of acupuncture. Mediators Inflamm.2003,12(2):59-69.
28.袁翔,李建国,黄越,等.电针足三里激活胆碱能抗炎通道抗大鼠感染性休克.武汉大学学报(医学版),2007,28(2)：203—206.
29.李建国,彭周全,杜朝晖,等.电针足三里激活胆碱能抗炎通路抗大鼠失血性休克的研究.中国中西医结合急救杂志,2006,13(1)：27—31.
30.王景杰,黄裕新.C-fos在电针调控大鼠胃运动中的表达及其意义.针刺研究,2001,26(4)：274—278.
31. National Center for Complementary and Alternative Medicine. National Institutes of Health.2004. NCCAM publication D003:Acupuncture. http://nccam.nih.gov/healtli/acupuncture/.
32. Luyer M D, Greve J W, Hadfoune M. et al. Nutritional stimulation of cholecystokinin receptors inhibits inflammation via the vagus nerve. J Exp Med, 2005.202(8):1023-1029.
33. Facchini M. Malfatto G. Sala L. et al. Changes of autonomic cardiac profile after a 3-week integrated body weight reduction program in severely obese patients. J Endocrinol Invest.2003.26(2):138-142.
34. Hoguin F. Tellez-Rojo MM. Lazo M, et al. Cardiac autonomic changes associated with fish oil vs soy oil supplementation in the elderly. Chest,2005, 127(4):1102-1107.
35. Berbert A A, Kondo CR, Almendra CL, et al. Supplementation of fish oil and olive oil in patients with rheumatoid arthritis. Nutrition,2005,21(2):131-136.
36. Christensen J H, Christensen M S, Dyerberg J, et al. Heart rate variability and fatty acid content of blood cell membranes:a dose-response study with n-3 fatty acids. Am J Clin Nutr,1999,70(3):331-337.
37. Christensen J H, Skou H A, Madsen T, et al. Heart rate variability and n-3 polvunsaturated fatty acids in patients with diabetes mellitus. J Intern Med,2001, 249(6):545-552.
38.张诗兴,姜文方.耳穴定位与神经、血管分布的研究.南京中医药大学学报,1998,14(4)：228-229.
39.严振国,糜兢华.耳穴的神经分布及耳廓的形成.上海针灸杂志,1989,1：145-146.
40.戴衡茹,赵学敏.耳廓的神经供应.安徽医科大学学报,.1960,3(2-3)：65-69.
41.雷琦.外耳的神经和动脉的解剖.解剖学报,1963,2(1)：29-37.
42.中国科学院动物研究所.人耳廓及其穴位里的神经分布.针刺麻醉原理的探讨.北京：人民卫生出版社,1974,41-46.
43.中国科学院动物研究所.猫耳廓的神经分布.针刺麻醉原理的探讨.北京：人民卫生出版社,1974,37-40.
44. Hunt Jr. The sensory field of the facial nerve:A further contribution to the symptomatology of the eniculate ganglion. Brain.1915.38:418.
45. Hollinshead WH. Anatomy for Surgeons:The Head-Neck. Harper and Row, 1968,190-191.
46. Hollinshead WH. Anatomy for Surgeons:The Head-Neck,3rd editon. Harper and Row.1982,162.
47. Guild SR. The glomus jugulare, a non chromaffin paraganglion in man. Ann Otol Rhinol Laryngol,1953,62(4):1045-1071.
48. Tekdemir I, Aslan A, Elhan A. A clinico-anatomic study of the auricular branch of the vagus nerve and Arnold's ear-cough reflex. Surg Radiol Anat.1998,20(4): 253-257.
49. Prasad KS. Cardiac depression on syringing the ear. A case report.J Laryngol Otol. 1984.98(10):1013.
50. Moorthy SS, Krishna G, Elliott CL. Is there an auriculovagal reflex producing cardiac dysrhythmias? Arch Otolaryngol.1985,111(9):631.
51. EngelD. The gastroauricular phenomenon and related vagus reflexes. Arch Psychiatr Nervenkr,1979,227(3):271-277.
52. Engel D. Uber das reflektorische Ohrenjucken bei Sodbrennen. Med Klin,1922, 47:1495-1497.
53. Malherbe W.D.F. Otalgia with oesophageal hiatus hernia. Lancet,1958, 1368-1369.
54. Deutsch M. Ein Beitrag zur Symptomatomatologie der beginnenden Lungentuberkulose. Med Klin,1919,43:1090-1091.
55. Bradford F K. The auriculo-genital reflex in cats. J Exp Physiol,1937, 27:272-279.
56. Vasiliu D I. Reflex auriculo-uterin. Spitalul,1932.52:396.
57. Aschner. In:The neurological examination. De Jong. R.N. (ed.), Hober Med Division. New York;London,1967:315.
58. Kalchschmidt, In:Lehrbuch der klinischen Diagnostik, J.mared. J. Mosci (eds); Jena:G. Fischer.1956,293-294.
59. Berlin R. Theagstro-auricular phenomenon. Lancet.1959,734-735.
60. Gordon F J. Effect of nucleus tractus solitarius lesions on fever produced by interleukin-1beta. Auton Neurosci,2000,85(1-3):102-110.
61. Kalia M, Fuxe K. Hokfelt T T, et al. Distribution of neuropeptide immunoreactive nerve terminals within the subnuclei of the nucleus of the tractus solitarius of the rat. J Comp Neurol.1984.222(3):409-444.
62. Kaliam, Mesulam M M. Brainstem projections of sensory and motor components of the vagus complex in the cat:The cervical vagus and nodose ganglion. J Comp Neurol,1980,193(2):453-465.
63. Okuma Y. Osumi Y. Central cholinergic descending pathway to the dorsal motor nucleus of the vagus in regulation of gastric functions. Jpn J Pharmacol,1986, 41(3):373-379.
64. Chien CH, Shieh JY, Ling EA, et al. The composition and central projections of the internal auricular nerves of the dog. J Anat,1996,189(Pt2):349-362.
65. Campbell R, Rodrigo D, Cheung L. Asystole and bradycardia during maxillofacial surgery. Anesth Prog,1994,41(1):13-16.
66. Mantle-St John LA, Tracey DJ. Somatosensory nuclei in the brainstem of the rat: independent projections to the thalamus and cerebellum. J Comp Neurol,1987, 255(2):259271.
67. Menetrey D, Basbaum AI. Spinal and trigeminal projections to the nucleus of the solitary tract:a possible substrate for somatovisceral and viscerovisceral reflex activation. J Comp Neurol,1987.255(3):439-450.
68.高欣妍.耳针疗法与耳-迷走-内脏反射.博士毕业论文,2005.
69.梅志刚.耳-迷走反射与耳针降糖效应机制研究.博士毕业论文,2007.
70.何伟.耳-迷走反射及耳针抗癫痫的效应机制研究.博士毕业论文,2008.
71.赵洁,杨选明,都培双,等.T淋巴细胞抑制急性感染的炎症反应.生物化学与生物物理进展,2008,35(2)：129-132.
72.曹雪涛.免疫学前沿进展.人民卫生出版社,2009,350.
73. Theilgaard-Monch K, Porse BT, Borregaard N. Systems biology of neutrophil differentiation and immune response. Curr Opin Immunol,2006,18(1):54-60.
74. Fierro IM, Serhan CN. Mechanisms in anti- inflammation and resolution:the role of lipoxins and aspirin- triggered lipoxins. Braz J Med Biol Res.2001,34(5):555-566.
75.朱梅,高洪泉,刘瑞丰,等.针刺“足三里”、“关元”穴区对老年大鼠肝脏内巨噬细胞功能影响的实验研究.针灸临床杂志,2003,19(6)：52.
76.朱文莲,刘仁权.艾灸大椎穴对免疫低下小鼠巨噬细胞吞噬功能的影响.北京中医药大学学报,2005,28(1)：89.
77.杨志新,乔跃兵,赵粹英.艾灸对荷瘤小鼠巨噬细胞免疫功能的增强作用.承德医学院学报,2002,19(2)：97.
78.陈国民,黄欣,蒋康平,等.艾灸大椎穴对小鼠巨噬细胞吞噬功能及脾脏重 量的影响.四川中医,1987,(9)：41.
79.李亚东,高洪泉,朱梅,等.针刺老年大鼠足三里、关元穴对NO、SOD、 MDA以及免疫影响的实验研究.中国针灸,2002,22(11)：772.
80.骆文郁.无烟灸疗法对实验性小鼠免疫功能影响的实验研究.针刺研究,2001,18(1)：661.
81.梁繁荣,曾芳,唐勇.针灸在地震后疫病防治中的应用.中国针灸,2008,28(7)：507.
82.逢紫千,王富春,严兴科.针灸天枢穴对脾虚泄泻大鼠免疫功能影响的实验研究.江苏中医药,2005,26(4)：27.
83.严桂珍,郑家铿,许少峰,等.针灸”三阴交”穴择时治疗对脾阳虚家兔免疫功能的影响.中国针灸,2001,21(12)：735.
84.陈雄华,刘又香,王华,等.针灸“足三里”、“关元”穴对阳虚大鼠免疫功能影响比较研究.中国针灸,2000,20(9)：555-557.
85.吕琳.针灸调节肿瘤免疫的临床及实验研究进展.辽宁中医杂志,2001,28(10)：638-640.
86.赵宁侠,高巍,史恒军,等.电针足三里及非经非穴点对大鼠细胞免疫的影响.云南中医学院学报,2001,4(1)：37-39.
87. Yu Y,Kasahara T, Sato T. et al. Enhancement of splenic interferon-gamma, interleukin-2, and NK cyotoxicity by S36 acupoint acu puncture in F344 rats. Jpn J Physiol.1997,42(2):173-178.
88.黄诚,陈汉平,秦秀娣,等.针刺抑制老年大鼠脑与垂体细胞因子基因表达.针刺研究,1998,23(1)：24.
89.谌剑飞,梁浩荣,马雅玲,等.针刺对糖尿病并发急性脑梗死血浆白介素-6及肿瘤坏死因子-α水平的影响.中国中西医结合急救杂志,2001,8(2)：92.
90.吴焕淦,陈汉平,周丽斌.针灸治疗大鼠溃疡性结肠炎的分子机制.上海针灸杂志,1998,17(6)：30.
91. Baldwin. Series introduction:the transcription factor NF-κB and human disease. J Clin Invest,2001;107(1):3-6.
92. Huston JM,Gallowitsch-Puerta M, Ochani M, et al. Transcutaneous vagus nerve stimulation reduces serum high mobility group box 1 levels and improves survival in murine sepsis. Crit Care Med,2007.35(12):2762-2768.
93.傅守延,吉田丰.烟碱对大鼠纹状体儿茶酚氨及吲哚胺含量的影响.沈阳药学院学报,1994,11(3)：186-189.
94. Litscher G. Computer-based quantification of traditional Chinese-ear-and Korean hand acupuncture:needle induced changes of regional cerebral blood flow velocity. Neurol Res,2002,24(4):377-380.
95. Haker E, Eqekvist H, Bjerring P. Effect of sensory stimulation on sympathetic and parasympathetic activities in healthy subjects. J Auton Nerv Syst.2000,79(1): 52-59.
96. Song XY. Torphy TJ,Griswold DE, et al. Coming of Age:anti-Cytokine Therapies. Mol Interv,2002,2(1):36-46.
97. Gosain A, Gamelli RL. A primer in cytokines. J Burn Care Rehabil,2005,26 (2): 7-12.
98. Carswell E A, Old L J, Kassel R L, et al. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci,1975,72(9):3666-3670.
99. Caput D, Beutler B, Hartog K. et al. Identification of a common nucleotide sequence in the 3"-untranslated region of mRNA molecules specifying inflammatory mediators. Proc Natl Acad Sci.1986.83(6):1670-1674.
100. Pennica D, Hayflick JS, Bringman TS, et al. Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor. Proc Natl Acad Sci. 1985.82(18):6060-6064.
101. Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell Death Differ,2003.10(1):45-65.
102. Kwak EL, Larochelle DA. Beaumont C, et al. Role for NF-kappa B in the regulation of ferritin H by tumor necrosis factor-alpha. J Biol Chem,1995. 270(25):15285-15293.
103. Dinarello CA, Cannon JG, Wolff SM, et al. Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1. J Exp Med,1986, 163(6):1433-1450.
104. Manicourt DH, Triki R, Fukuda K, et al. Levels of circulating tumor necrosis factor alpha and interleukin-6 in patients with rheumatoid arthritis. Relationship to serum levels of hyaluronan and antigenic keratin sulfate. Arthritis Rheum,1993, 36(4):490-499.
105. Kupferminc MJ, Peaceman AM, Wigton TR, et al. Tumor necrosis factor-alpha is elevated in plasma and amniotic fluid of patients with severe preeclampsia. Am J Obstet Gynecol,1994,170(6):1752-1757.
106. Harel Y, Silva M, Giroir B, et al. A reporter transgene indicates renal-specific induction of tumor necrosis factor (TNF) by shiga-like toxin. Possible involvement of TNF in hemolytic uremic syndrome. J Clin Invest,1992. 92(5):2110-2116.
107. Wu CJ, Lovett M, Wong-Lee J, et al. Cytokine gene expression in rejecting cardiac allografts. Transplantation,1992,54(2):326-332.
108. Sandborn WJ. A controlled trial of anti-tumor necrosis factor alpha antibody for Crohn's disease. Gastroenterology.1997,113(3):1042-1043.
109. Mizel SB. The interleukins. FASEB J.1989.3(12):2379-2388.
110. Ghosh S. May M J. Kopp E B. NF-kappa B and Rel proteins:evolutionarily conserved mediators of immune responses. Annu Rev Immunol,1998, 16:225-260.
111. Liu SF, Malik AB. NF-κB activation as a pathological mechanism of septic shock and inflammation. Am J Physiol Lung Cell Mol Physiol,2006.290(4):622-645.
112. Han S.T, Ko HM, Choi JH, et al. Molecular mechanisms for lipopolysaccharide-induced biphasic activation of nuclear factor-kappa B (NF-kappa B). J Biol Chem,2002,277(47):44715-44721.
113. Kelly A. Vereker E. Nolan Y, et al. Activation of p38 plays a pivotal role in the inhibitory effect of lipopolysaccharide and interleukin-1 beta on long term potentiation in rat dentate gyrus. J Biol Chem.2003,278(21):19453-19462.
114. Liu SF. Ye X. Malik AB. Pyrrolidine dithiocarbamate prevents I-kappaB degradation and reduces microvascular injury induced by lipopolysaccharide in multiple organs. Mol Pharmacol,1999.55(4):658-667.
115. Pocock J. Gomez-Guerrero C, Harendza S, et al. Differential activation of NF-kappa B, AP-1, and C/EBP in endotoxin-tolerant rats:mechanisms for in vivo regulation of glomerular RANTES/CCL5 expression. J Immunol,2003,170(12): 6280-6291.
116. Pritts TA, Moon MR, Wang Q, et al. Activation of NF-kappaB varies in different regions of the gastrointestinal tract during endotoxemia. Shock,2000. 14(2):118-122.
117. Ge Y, Ezzell RM, Clark BD, et al. Relationship of tissue and cellular interleukin-1 and lipopolysaccharide after endotoxemia and bacteremia. J Infect Dis,1997,176(5):1313-1321.
118. Liu JH, Yan J, Hu JM, et al. Effects of electroacupuncture on gastric myoelectric activity and substance P in the dorsal vagal complex of rats. Neurosci Lett. 2004,356(2):99-102.
119. Li L, Chen Y X, Hong X, et al. Nitric oxide in vPAG mediates the depressor response to acupuncture in stress-induced hypertensive rats. Acupunct Electrother Res.2001; 26(3):165-170.
120.孟卓,吕国蔚.足三里—脊髓背角—孤束核的机能联系.中国科学B辑,1992,4：393-399.
121.胡森,宋琪,盛志勇,等.电针兴奋胆碱能抗炎通路对内毒素引起的细胞因子释放和脏器功能损害作用研究.中国中西医结合急救杂志,2008,15(4)：205-208.
122.石现,宋琪,关玲,等.电针对内毒素引起大鼠肝损伤保护作用的研究.中国针灸,2008;7(4)：290-292.
123. Domenica Altavilla, Salvatore Guarini, Alessandra Bitto. et al. Activation of the cholinergic anti-inflammatory pathway reduces NF-κB activation, blunts TNF-a production, and protects against splanchnic artery occlusion shock. Shock,2006, 25(5):500-506.
124. Valentin A Pavlov. Mahendar Ochan, Li-Hong Yang, et al. Selectivea7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine endotoxemia and severe sepsis. Crit Care Med.2007,35(4):1139-1144.
125.王述菊,孙国杰,杜艳军.孤束核在针刺“足三里”调节胃运动中的作用机制.中国中医药信息杂志,2007,14(9)：28-30.
126.王景杰,黄裕新,郭庆东.电针调节心理性应激状态下胃动力障碍的实验研究.针刺研究,2000,25(4)：267-270.
127.孟卓,吕国蔚.大鼠脊髓背角神经元与孤束核的联系的电生理学研究.科学通报,1990,35(4)：292-295.
128.吴利平,李辉,李云庆.大鼠延髓和脊髓背角的PKCr阳性神经元向孤束核投射.第四军医大学学报,2001,22(23)：2171-2174.
129.李江山,严洁,何军锋,等.针刺对胃扩张模型大鼠孤束核神经元放电的影响.中国医药导报,2009,6(8)：19-21.
130. Goehler L E, Gaykema RP, Hansen MK, et al. Vagal immune-to-brain communication:a visceral chemosensory pathway. Auton Neurosci,2000, 85(1-3):49-59.
131. Hermann G E, Emch G S, Tovar C A. et al. c-Fos generation in the dorsal vagal complex after systemic endotoxin is not dependent on the vagus nerve. Am J Physiol Requl Integr Comp Physiol.2001,280(1):289-299.
132. Emch G S. Hermann G E. Rogers R C. TNF-a activates solitary nucleus neurons responsive to gastric distension. Am J Physiol Gastrointest Liver Physiol,2000, 279(3):582-586.
133. Romanovsky AA. Thermoregulatory manifestations of systemic inflammation: lessons from vagotomy. Auton Neurosci,2000,85(1-3):39-48.
134. Goehler LE, Relton JK, Dripps D. Vagal paraganglia bind biotinylated interleukin-1 receptor antagonist:a possible mechanism for immune-to-brain communication. Brain Res Bull,1997,43(3):357-364.
135.余传霖.细菌内毒素研究现状.国外医学抗生素分册,1997,20(4)：30.
136. MacMicking JD. Nathan C. Hom, et al. Altered responses to bacterial infection and endotoxin shock in mice inducible nitric oxide syntbase. Cell,1995, 81(4):641-501.
137.黄凤,荣培晶,朱兵,等.耳甲迷走神经刺激干预35例糖耐量受损患者临 床观察.中华中医药杂志,2010,25(12)：2185-2186.
138. Bianchi M. Suppression of proinflammatory cytokines in monocytes by a tetravalent guanylhydrazone. J Exp Med.1996; 183:927-936.
139. Bianchi M. Ulrich P. Bloom O, et al. An inhibitor of macrophage arginine transport and nitric oxide production (CNI-1493) prevents acute inflammation and endotoxin lethality. Mol Med,1995,1(3):254-266.
140. Tracey K J. Suppression of TNF and other proinflammatory cytokines by the tetravalent guanylhydrazone CNI-1493. Prog Clin Biol Res,1998,397:335-343.
141. Homines D, van den Blink B, Plasse T, et al. Inhibition of stress-activated MAP kinases induces clinical improvement in moderate to severe Crohn's disease. Gastroenterology,2002,122(1):7-14.
142. Tracey KJ, Czura CJ. Ivanova S. Mind over immunity. FASEB J,2001, 15(9):1575-1576.
143. Pullan RD, Rhodes J, Ganesh S, et al. Transdermal nicotine for active ulcerative colitis. N Engl J Med,1994,330(12):811-815.
144. Ben-Menachem E. Vagus nerve stimulation, side effects,and long-term safety. J Clin Neurophysiol,2001,18(5):415-418.
145. Schachter SC. Vagus nerve stimulation:where are we? Curr Opin Neurol,2002. 15(2):201-206.