摘要
目的：在中医学理论指导下,应用现代生物医学技术探讨不同频率电针刺激对免疫功能的调节作用及其神经生物学机制,为进一步明确和扩展电针的临床应用提供理论和实践依据。
方法：通过文献研究,从祖国医学和现代医学角度认识针灸与免疫、针灸与穴位、针灸与免疫调节的关系以及针灸的临床应用；应用神经药理学、细胞生物学、MTT、ELISA.放射免疫、免疫荧光和流式细胞术等技术和方法,在人体和小鼠层面上检测不同频率电针刺激对NK细胞活性、吞噬细胞功能、T细胞增殖能力和细胞因子等免疫学指标的影响；在小鼠上测定电针对T细胞亚群、β=内啡肽(β-EP)含量的影响以及α7尼古丁受体(α7nAChR)在免疫细胞上的表达情况；应用α7riAChR抗体和特异性的α7nAChR拮抗剂α-银环蛇毒(α-Bgt)研究α7nAChR在电针对NK细胞活性中的作用。
结果：
1.文献研究：针灸是祖国传统医学的瑰宝,其应用已有几千年的历史；针灸以调和气血、平衡阴阳、疏通经脉而达到增强人体“正气”和“扶正祛邪”之功效；针灸调节机体免疫功能的机理在于扶正固本,取穴重点在于与免疫相关的强壮要穴；针灸对机体免疫功能调节具有整体性和双向性；由针灸发展而来的电针,其调节免疫功能的作用与神经内分泌免疫网络系统密切相关。
2。人体实验
(1)不同频率电针(EA)对NK细胞活性、吞噬功能(吞噬率和吞噬指数)的影响：2Hz和2/100Hz EA在末次EA当天和停EA后第1d的NK细胞活性均明显升高(P<0.05)；而100Hz EA仅在停EA后第1d的NK细胞活性明显升高(P<0.05)。2Hz EA在末次EA当天和停EA后第1d的吞噬百分率率和吞噬指数均明显升高(P<0.01)且保持同一水平；2/100Hz EA在末次EA当天和停EA后第1d的吞噬百分率和吞噬指数均显著升高(P<0.05, P<0.01)且有上升趋势(P<0.01);100Hz EA在末次EA当天和停EA后第1d的吞噬百分率均显著升高(P<0.01,P<0.05),而吞噬指数仅在末次EA当天的吞噬指数显著升高(P<0.01),且与末次EA当天相比均有下降趋势(P<0.01)。
(2)2/100Hz电针对NK细胞活性、细胞因子和吞噬功能作用的后效应：在EA的第1、3d和停EA后的前2d，NK细胞活性明显升高(P<0.01),在停EA后的第3d恢复到EA前水平；在末次EA的当天和停EA后的第1d IL-2和IL=6均有明显升高(P<0.05),且在停EA后的第2d恢复到EA前水平,而IL-4、IL-10、IL-12、TNF-α及IFN-γ均没有明显变化；在EA的第1、3d及停EA后3d,吞噬百分率均明显高于EA前水平(P<0.01),且基本保持在同一个水平；在EA的第1、3d和及停EA后3d,吞噬指数均明显高于EA前水平(P<0.01),且有上升趋势。2/100Hz电针对白细胞计数没有明显影响。
(3)2/100Hz电针刺激不同穴位组合对NK细胞活性和吞噬指数的影响：右曲池-右中渚、左承山-左昆仑穴位组合的NK细胞活性和吞噬指数均无明显变化；而大椎-右曲池、左足三里-左三阴交穴位组合在末次EA的当天和停EA后的第1d的NK细胞活性和吞噬指数均有明显升高(P<0.01)。
3.动物实验
(1)不同频率电针单次刺激对正常小鼠免疫功能的影响：电针1次后,分别在0、24和48h时间点检测小鼠的脾指数、脾脏NK细胞活性及T细胞增殖能力,2HZ、100Hz和2/100Hz EA均无明显变化。
(2)不同频率电针连续刺激对正常小鼠免疫功能的影响：连续电针3d(每天1次)后,2Hz、100Hz和2/100Hz EA对脾指数均无明显影响；100Hz EA的NK细胞活性显著降低(P<0.05),2/100Hz EA的NK细胞活性明显升高(P<0.05),而2Hz EA的NK活性无明显变化;2Hz EA的脾脏T细胞增殖能力显著升高(P<0.01),而100Hz和2/100Hz EA均无明显变化；100Hz和2/100Hz EA的血清IL-6水平显著升高(P<0.05，P<0.001),而2Hz EA无明显变化。
(3)2/100HZ电针连续刺激对小鼠血清细胞因子的影响：2/100Hz EA连续刺激3d后,外周血IL-6水平逐渐升高,于第3d达高峰,第6d恢复至正常水平,第1d及第3d与对照组相比有显著差异(P<0.01)；而IL-2、TNF-a及IFN-y含量无明显变化。
(4)不同频率电针连续刺激对环磷酰胺小鼠T细胞亚群的影响：连续电针3d(每天1次)后,2Hz、100Hz和2/100Hz EA对外周血中CD4+及CD8+均无明显影响；而2/100Hz EA可明显升高CD4+/CD8+(P<0.05),但2Hz和100Hz EA则没有。
(5)不同频率电针连续刺激对环磷酰胺小鼠β-内啡肽的影响：环磷酰胺组与对照组相比,下丘脑的p-EP明显降低(P<0.05),延髓和脾脏的p-EP均无明显变化；与环磷酰胺组相比,2Hz EA和100Hz EA的下丘脑、延髓和脾脏的p-EP均变化不明显；而2/100HzEA在下丘脑及延髓的p-EP均变化不明显,但脾脏的p-EP显著升高(P<0.01),而且与2Hz EA和100Hz EA的p-EP相比均明显升高(P<0.05, P<0.01)。
(6)小鼠巨噬细胞上α7nAChR的定位：免疫荧光方法(α7nAChR抗体)发现在小鼠腹腔液中巨噬细胞表面有α7nAChR的表达。
(7) α7nAChR抗体对100Hz电针作用脾脏NK细胞活性的影响：给予小鼠α7nACh-R抗体后,与100Hz EA组相比,100Hz EA+α7nAChR-Ab组的NK细胞活性明显增强(P<0.05)
(8)特异性的α7nAChR拮抗剂α-Bgt对100Hz电针作用脾脏NK细胞活性的影响：给予小鼠不同剂量的特异性α7nAChR拮抗剂α-Bgt后,与100Hz EA组相比,1.5和3.0μgα-Bgt+100HzEA组均明显增强NK细胞活性(P<0.01)
结论：
1.针灸扶正固本和调整机体功能具有整体性和双向性,其针效强弱与穴位配合密切相关；
2.电针具有调节免疫功能的作用并存在频率响应性和后效应,而且其效应与刺激频度有关；
3.电针刺激不同穴位组合影响其调节免疫功能的效应,即穴位特异性;
4.β-内啡肽与细胞因子之间相互作用可能介导电针调节免疫功能；
5. α7nAChR可能参与100Hz电针调节免疫功能的作用。
Objective:
To explore the modulation for immune function and its mechanism of electroacupuncture (EA) at different frequencies under the guidance of Traditional Chinese Medicine (TCM), and to provide the theoretical and practical basis for further clarifying and expanding the clinical application of EA.
Methods:
Through literature study, to understand the relations of immune and acupuncture, acupuncture and acupoints, acupuncture and immune modulation as well as the clinical application of acupuncture in the view of traditional medicine and modern medical; Neuropharmacology, cytobiology, MTT, ELISA, RIA, immunofluorescence and flow cytometry techniques were used to detect the effect of EA at different frequency on the immunological indexes of NK cell activity, phagocyte function, T cell proliferation and cytokines in humans and mice; To confirm the effect of EA on the level of splenic T cell subsets,(3-endorphin and the expression of a7nAChR in immune cells in mice; Applying a7nAChR antibody and the specific a7nAChR antagonist a-Bgt to study the action of a7nAChR on NK cell activity in EA.
Results:
1. First, literature study
Acupuncture, which has been practiced for thousands of years, is a gem of traditional Chinese medicine. By regulation of qi and blood, balancing Yin and Yang, dredge the meridians, acupuncture plays a role of enhancing human righteousness and strengthen body resistance and eliminating; The mechanisms that acupuncture modulates immune function is to strengthen the body resistance to consolidate the constitution. The acupoints selection focus on the strong immune-related main point; Acupuncture mediated immune modulation has the characters of integrity and bidirection; EA, developed from acupuncture, has the role of immune modulation which is closely related to the neuroendocrine-immune network systems.
2. Second, human experiments
(1) The effect of EA of different frequencies on the NK cell activity and phagocytosis (phagocytic rate and phagocytic index):In and1d after the last day of EA with2Hz and2/100Hz, NK cell activity was significantly increased (P<0.05); Only in the first day after EA with a frequence of100Hz, NK cell activity was significantly increased (P<0.05). In and1d after the last day of EA with a frequence of2Hz, the phagocytic percentage rate and phagocytic index were significantly enhanced (P<0.01) and maintain the same level; In and1d after last day of EA with frequence of2/100Hz, the phagocytic percentage and phagocytic index were significantly higher (P<0.05, P<0.01) and there is an upward trend (P<0.0l); In and1d after the last day of EA with a frequence of100Hz, the phagocytic percentage were significantly higher (P<0.01, P<0.05), and only in the last day of EA with a frequent of100Hz the phagocytic index significantly increased (P<0.01), and compared with the last day of EA showed a downward trend (P<0.01).
(2) The after-effects of2/100Hz EA on NK cell activity, cytokines and phagocytic function:In the1,3d and the last2d of EA treatment, NK cell activity was significantly increased (P<0.01), and In the3rd day after EA restore to the level before the EA treatment; In and1d after last day of EA, IL-2and IL-6were significantly enhanced (P<0.05), and recovered in the2nd day after EA. while IL-4, IL-10, IL-12, IFN-y and TNF-α had no significant changes; in the1,3d and3days after EA, the phagocytic percentage was significantly higher than the EA level (P<0.01) with the same level in each day and phagocytic index were significantly higher, with an upward trend. There was no significant effect on the white blood cell count treated by2/100Hz EA.
(3) The effect of2/100Hz EA at different acupoints on NK cell activity and phagocytic index:NK cell activity and phagocytic index did not change significantly when stimulating the acupoint combination such as right Quchi-right Zhongzhu and left Chengshan-left Kunlun; and when stimulating acupoint combination such as Dazhui-right Quchi and left Zusanli-left Sanyinjiao in the last day of and the3rd day after EA treatment, NK cell activity and phagocytic index changed significantly (P<0.01).
3. Third, animal experiments
(1) The effect of Single EAS with different frequencies on the immune function of normal mice:After one time of EA, The spleen index, spleen NK cell activity and T cell proliferative ability in mice were detected respectively at0,24and48h time points, the result showed that there was no significant change in the2Hz,100Hz and2/100Hz EA.
(2) The effect of successive EAS with different frequencies on immune function of normal mice:After three days of successive EAS with frequence of2Hz,100Hz and2/100Hz, the splenic index did not significantly changed; NK cell activity significantly reduced when treated with100Hz EA (P<0.05), and significantly increased when applied with2/100Hz EA (P<0.05), while there was no significant change with2Hz EA on NK cell activity; Splenic T cell proliferative ability was significantly increased when applied with2Hz EA (P<0.01), while did not change significantly to100Hz and2/100Hz EA; IL-6levels in serum was significantly increased when treated with100Hz and2/100Hz EA (P<0.05, P<0.001), but had no significant change to2Hz EA.
(3) The effect of successive2/100Hz EAS on serum cytokines:After treated with successive2/100Hz EAS for3days, the IL-6levels in peripheral blood gradually increased and reached a peak on the3rd day, returned to normal levels on the6th day, and showed significant differences compared with control group on the1st and3rd day (P<0.01), but had no significant changes to the content of IL-2, TNF-a and IFN-y.
(4) The effect of successive EAS of different frequencies on T cell subsets in CP mice:After three days of successive EAS with frequence of2Hz,100Hz and2/100Hz, blood of CD4+and CD8+did not significantly changed in2Hz,100Hz and2/100Hz; while CD4+/CD8+was significantly increased when treated with2/100Hz EA (P<0.05), but not in2Hz and100Hz.
(5) The effect of successive EAS with different frequencies on β-EP in CP mice:Compared with the control group, in the CP group, β-EP significantly reduced in the hypothalamus (P<0.05), and did not change significantly in medulla oblongata and spleen; compared with the CP group, β-EP did not significantly change in hypothalamus, medulla oblongata and spleen in2Hz EA and100Hz EA, as to2/100Hz EA β-EP did not significantly change in hypothalamus, medulla oblongata, but significantly changed in the spleen (P<0.01), and the level of β-EP were significantly higher than2Hz EA and100Hz EA (P<0.05, P<0.01).
(6) The localization of α7nAChR in the mouse macrophage cells:The expression of a7nAChR was found on the surface of macrophages in the Intraperitoneal Fluid of mice with immunofluorescence method (a7nAChR antibody).
(7) The effect of antibody against a7nAChR on splenic NK cell activity in100Hz EA:After given a7nAChR antibody to mice, compared with100Hz EA group, NK cell activity was significantly enhanced in100Hz EA plus a7nAChR-Ab group (P<0.05).
(8) The effect of specific a7nAChR antagonist a-Bgt on splenic NK cell activity in100Hz EA:After given different doses of specific a7nAChR antagonist a-Bgt to mice, compared to100Hz EA group,1.5and3.0μg specific a7nAChR antagonist a-Bgt plus100Hz EA group were significantly enhanced NK cell activity, respectively (P<0.01).
Conclusion:
1. Acupuncture has integrity and bidirection for strengthen the body resistance to consolidate the constitution and modulation of body function, its efficacy of acupuncture is closely related to the selection of acupoint;
2. EA has immune modulation function and the existence of frequency responsiveness and post-effects, its efficacy is closely related to the number of stimuli;
3. The efficacy of EA immune modulation varied with different acupoints combination, ie. acupoint speciality;
4. Interaction between β-endorphin and cytokines is likely to mediate immune modulation function of EA;
5.a7nAChR may be involved in100Hz EA modulation for immune function.
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