The Research Of Mechanism About T Lymphocyte Immunoregulation Of Aging Model Rats By Means Of Electro-acupuncture Therapy On Guanyuan (CV4) And Zusanli (ST36) Points

With the increasing problem of aging of the population all over the world,it gets more and more urgent and important to gain deep insight into the mechanism of aging and anti-aging in order to improve the life quality of the elders.Acupuncture study is one of the most characteristic and typical subjects in Traditional Chinese Medicine,and it has accumulated valuable experiences in human health care with its long history.The thought of "Preventive Treatment" which was originated from the HuangDi NeiJing(Huangdi's Classic of Internal Medicine) embodies the core values of the adjustment of the current medical purpose and the change of medical pattern.The theoretical system of "Preventive Treatment",which includes the ideas of " preventive treatment before disease onset, preventing the disease to change,and preventing relapse after restoration",has provided an important theoretical foundation and specific means for the diagnoses and treatment of the diseases and health care in modern medicine.The principle of this study is the Treatment Determination Based on Syndrome Differentiation and the thought of "Preventive Treatment" in TCM.The subjects of this study are D-galactose-induced aging rats.The instrument of the study is to use electro-acupuncture on Important Tonifying Points such as Guanyuan(CV4),Zusanli(ST36) as intervention means based on Prof.Wanghua's "Shuang Gu Yi Tong"(Tonifying congenital and acquired essence,eliminating pathogen acupuncture therapy).The study analyzed the effect and mechanism of acupuncture on adjusting immunity and preventing aging in the aspect of T cell-mediated immunity,which can provide a new scientific evidence for acupuncture to prevent aging.PurposeTo investigate the working mechanism of electro-acupuncture on Guanyuan and Zusanli for anti-aging with respect to Neuroendocrine-immuuno-modulation network.Methods:Based on the scientific understanding of aging in Traditional Chinese Medicine and Modern Medicine,in the present study,80 D-galactose-induced aging rats were taken as an aging model which was then divided into 8 groups:Aging Group(Model)×2,Aging Electro-acupuncture Group(EA)×2,Aging Immuno- suppressive Group (ISU),Aging Electro-acupuncture plus Immunosuppressive Group (ISU+EA),Aging mifepristone groups(RU486),RU486 plus Aging Electric-acupuncture Group(RU486 + EA),each having 10 rats.20 three-month old rats were taken as the Control Group(Normal)×2(5 males and 5 females).The study used electro-acupuncture (HANS,LH202-type electric apparatus,continuous wave,frequency of 2Hz,intensity of 1mA,electricity for 15 minutes) on Guanyuan(CV4) and Zusanli(ST36) points,once a day,six days a week, and the treatment period was 27 days.The study also used the methods to observe target-related changes of neuroendocrine and the immune system of rats in each group,such as radioimmunoassay(radioimmunity) for testing the reproductivity of T cells,flow cytometry,enzyme-linked immunosorbent assay (ELISA) for inducible level of Interleukin-2(IL-2) and Interleukin-2 receptor(IL-2R),real time quantitative reverse transcription-polymerase chain reaction(RT-PCR), radioimmunoassayfor the content of IL-6,ACTH,CORT in the serum, flow cytometry for the expression of CD8+CD28+T,ABC method of immunohistochemistry for expression of IL-6R,β-end and CRH, RT-PCR for the expression of GRmRNA,etc.In order to explore the efficiency of "preventive treatment theory", with the aid of intraperitoneal injection with cytoxan and the impact of the electro-acupuncture at important Tonifying Points on the capability of immune and anti-disease of the aging was examined in this study.In addition,the effect of hippocampus' s negative feedback regulation under the adjustment of electric acupuncture in immune response on the hypothalamic-pituitary-adrenal(HPA) axis is investigated by injecting subcutaneously with glucocorticoid receptor,antagon,and Mifepristone(RU486). Results:1.Thymus Cell(T Cell) indices,such as reproductivity,secretion level of Interleukin-2(IL-2) and Interleukin-2 receptor(IL-2R) and CD28 expression of surface antigen in Model Group,were found to be significantly lower than the Normal Group(P<0.01).After electro-acupuncture treatment at Important tonifying points, Thymus Cell(T Cell) ' s reproductivity,inducible level of Interleukin-2(IL-2) and Interleukin-2 receptor(IL-2R) and CD28 expression of surface antigen in EA Group were found to be significantly improved(P<0.01).The adjustment of electric acupuncture to T Cell' s reproductivity of EA Group was positively correlated to IL-2 and its receptor and the density of T cell (CD8+CD28+).2.Compared with Model Group,T Cell' s properties,such as proliferation index,inducible level of Interleukin-2(IL-2) and secretion level of Interleukin-2 receptor(IL-2R),and T Cell' s density(CD8+CD28+) in ISU Group after intraperitoneal injection with immunosuppressant decreased significantly(P<0.01,P_IL-2<0.05); while,the index levels mentioned above in EA+ISU Group after electro-acupuncture treatment had no significant differences compared with Model Group(P>0.05),but had significant differences (P<0.01) compared with EA+ISU Group without electro-acupuncture treatment.3.IL-6 secretion of peripheral blood and IL-6R expression in the hippocampus area in Model Group were found to be significantly higher than that of the Normal Group(P <0.01).Electro-acupuncture at Important tonifying points could improve the T Cell ' s reproductivity in Model Group and at the same time reduced IL-6 secretion of peripheral blood and IL-6R expression levels in hippocampus area(P<0.01)4.The immunohistochemistry results ofβ-end immunohisto-chemistry of arcuate nucleus of the hypothalamus are as follows:β-end Immunoreactive material were mainly distributed in arcuate nucleus of the hypothalamus(nucleus arcuatus),and it could be seen under the High-power microscope that tan Positive granules in the intracytoplasm of the nerve cell and part of the neuroepithelial cells,and immunoreactive objects of Axons of neurons presented the shape of a string of pearls.The number of neurons in Model Group under light microscope was less than that of the Normal Group,but it showed a trends to increase in EA Group after electro-acupuncture at Important tonifying points;OD value analysis showed that this number of Model group was significantly high compared with the Normal group,(P<0.01);OD value ofβ-end positive reaction material in EA Group with electro-acupuncture treatment were significantly increased(P<0.05) compared with Model Group,while, there was no significant differences between that in EA Group and that in the Normal Group(P> 0.05).5.The immunohistochemistry results of Hypothalamus orticotro-phin releasing hormone(CRH) are as follows:CRH immune was mainly distributed in the arcuate nucleus and periventricular nucleus. Especially in the periventricular nucleus CRH immunopositive material appeared to be in tan colour and distributed in the intracytoplasm of megacell nerve cell if a high-power microscope is used.The CRH in the intracytoplasm in D-galactose-induced Group reduced significantly compared with that in Nomal Group,while,CRH in EA Group with electro-acupuncture treatment tended to increase. OD value analysis showed that,the OD value of CRH immunopositive object was significantly reduced compared in the case of normal rats (P<0.05);The value in EA Group with electro-acupuncture treatment was significantly increased(P <0.05).There was no significant difference between the OD value of CRH immunoposi tive object in EA group and the Normal group(P>0.05).6.Compared with the Nomal Group,T cell's reproductivity in Model Group decreased,and the content of serum Adrenocorticotropic Hormone(ACTH) and corticosterone(CORT) were significantly increased(P<0.01).The content of ACTH and CORT in EA Group was significantly decreased and it was significantly different from that in the Model Group(P<0.01).The adjustment of electro-acupuncture to the effect of T Cell proliferation of rats in EA Group was negatively correlated to the secretion level of ACTH, CORT in HPA axis,and there was no any obvious relationship with CRH.7.The GRmRNA expression of Hippocampus were integrity,and had not degraded by RNA enzyme;gene amplification products only had one single peak and it had its own specificity;the length of fragment of the target gene product coincided with the designed length.Compared with the Normal Group,in Model Group T Cell reproductivity decreased,and serum CORT level was significantly increased.In the meanwhile,the GRmRNA expression of hippocampus were significantly decreased(P<0.01);with the electro-acupuncture treatment,EA Group was significantly different from the Model Group(P< 0.01),that is,T Cell reproductivity in EA Group increased,and serum CORT level decreased.In addition,the GRmRNA expression of hippocampus were significantly increased(P<0.01);the GRmRNA expression of hippocampus were in RU486 Group were significantly decreased compared with the Model Group(P<0.01,or P<0.05).the GRmRNA expression of hippocampus in EA+RU486 Group were increased compared with RU486 group,but there were no significant differences between these two groups(P>0.05).GRmRNA expression in EA + RU486 Group were decreased significantly(P<0.01) compared with the EA Group. Conclusion: 1.Electro-acupuncture at Important tonifying points can improve proliferation of T cell,inducible level of IL-2 and IL-2R and T cell surface antigen expression of CD28.This implies that Electro-acupuncture at Important tonifying points can be used for anti-aging purpose through modifying immune function of T cell.2.Electro-acupuncture at Important tonifying points can be used to fight with Immuno-suppressive cytoxan through enhancing T Cell immune function in the aspects of T cell reproductivity,secretion level of IL-2 and IL-2R,and the T cell surface antigen expression of CD28,suggesting that this cure-to-prevent-disease strategy can enhance the immune defense ability of the elders,and electro-acupuncture at Important tonifying points on the old organism has the adjustment effect of "preventive treatment".3.Electro acupuncture at Important tonifying points enhancing T cell immune function is related to the adjustment of Electro acupuncture to the neuroendocrine system:(1) electro-acupuncture can reduce aging model rats' IL-6R expression of hippocampus,and increased GRmRNA expression of hippocampus,and then enhance negative feedback regulation of the hippocampus in the HPA axis and reduce immune suppression of high-level ACTH and GC in serum.(2) electro-acupuncture can improve hypothalamic beta endorphin(β-end) expression and reduce the suppressive effect of T Cell immune function resulting from excessive secretion ofβ-end,which may be related to improving the negative feedback regulation capability ofβ-end on the peripheral high-level ACT and GC.4.RU486,to some extent,can block the electro-acupuncture on regulation of the hippocampus on the HPA axis negative feedback regulation,thus reducing the efficiency of electro-acupuncture on reproductivity of T cell.To sum up,electro-acupuncture on important tonifying points was proved to have the capability to improve the regulation of T cells immunological function and anti-aging,which was completed through Neuroendocrine-immuuno-modulation network,during the process, where serum IL-6,hippocampal IL-6R,hypothalamicβ-end,and the CRH,ACTH and CORT of hypothalamus-pituitary-adrenal(HPA) axis participated in the specific regulation of the network..The present study investigated the mechanism of electro-acupuncture in anti-aging purpose with reference to T cell-mediated immunity,Neuroendocrine-immuuno-modulation network and contributed to this research field with valuable experimental evidence.The general discussion from the Neuroendocrine-immuuno-modulation(NIM) on the network will have a certain degree of inspiration and innovative for finding out mechanism of acupuncture' s immunological regulation.We stimulate the aging model in advance,which demonstrate the feasibility and science of the advance stimulate on important tonifying points can improve immuno-competence preventing disease.This study could not observe the secretion property ofβ-end in external blood.This should be recommended for the future study in this field.