| Stress is the body’s normal response to anything that disturbs its natural physical, emotional, or mental balance. Stress disturb the immune status by the hypothalamic-pituitary-adrenal axis (HPA axis) and the sympathetic nervous system activity. HPA axis and sympathetic nervous system regulate the immune system by neurotransmitters, hormones, and cytokines, such as glucocorticoids, epinephrine, norepinephrine, IL-1, endorphins, etc. By acting on the different receptors, these factors play differents role in regulating the immune system. Stress brings a series of changes to the function of the body system through the nerve-endocrine-immune network. In the central nervous system, the prefrontal cortex, hippocampus, hypothalamus has been proved to be closely related to stress. Surgery is a common clinical treatment, also as a stress source, which can affect the immune function. Clinical study confirms that stress caused by surgery can result in immunosuppression. Stress successfully induced rat’s immune suppression in animal traumatic stress model. In addition, surgery induced immune suppression is more obvious in older group with prolonged recovery time, which result in the increased probability or even life-threatening. Fing a good way to improve immune function after surgery stress in older group is very important in clinical treatment.Acupuncture is an important part of Chinese traditional medicine, which has advantages of safety, effective, simple, and almost no side effects. Acupuncture’s immunoregulation effects have been driving more and more attention. It’s regulating role also has been validated in clinic. The regulation of acupuncture on the immune system include improving the number and activity of lymphocytes, leukocytes, and NK cells, etc. The previous work in our laboratory also confirmed that lymphocyte proliferation, NK cell activity decreased after traumatic stress, and electroacupuncture significantly reverse the immunosuppression after single treatment in adult rats. In clinical, the healthcare function of acupuncture has been proved, regularly given long-term acupuncture treatments could modulate immunity of older group, such as increasing appetite, improving sleep quality, and prevention of disease. Here, we compared the differences of single EA treatment and long-term regularly multiple EA treatments to further explore the mechanism of electroacupuncture’s immune regulation.Src protein kinase belongs to non-receptor protein tyrosine kinase family, including eight kinds of highly homologous protein c-Src, Fyn, Yes, Lck, Lyn, Hck, Fgr and Blk. It is an important intracellular signaling molecule, involved in the regulation of cell proliferation, differentiation, migration, and cell morphology maintained, also involved in T cell receptor activation by regulating protein tyrosine phosphorylation. Therefore, Src protein play an important role in the peripheral immune system and central nervous system. Src, Fyn, Yes, abundantly express in the central nervous system. Activation of Fyn protein in the hypothalamus could affect the rat’s energy metabolism. Fyn also involved in synaptic plasticity in the hippocampus,and closely related to the integrity of the hippocampal formation, playing an important role in the process of learning and memory. In the cerebral cortex, fyn is closely related to aging-related neuron circuit remodeling. However, fyn play an important role in stress-related brain areas associated with aging. ERK1/2is a major component of the Src protein kinase signaling pathway, Fyn can activate ERK1/2through G protein-coupled receptors and growth factor receptor, therefore, fyn-ERK1/2acts as a coincidence point of the interactive activation of signal transduction and participates in multiple signal transduction pathways. Src-ERK1/2signal transduction pathway has also been shown to be involved in neuroimmune regulation, improving the immune suppression caused by stress or morphine dependence and withdrawal. So, fyn-ERK1/2signaling pathway is the focus of this research, we try to find the role of this pathway in neuro-immune regulation and EA immunomodulation effect.Src family kinase could be activated by many upstream molecules, including NMDA receptors, growth factor receptors, G protein-coupled receptors and calcium. TrkB is a neurotrophic factor receptor, known to its specific ligands, including brain-derived neurotrophic factor BDNF and an NT4/5. Phosphorylation of various growth factor receptors by fyn has been shown, fyn is also involved in the activation of TrkB in different conditions, for example, fyn can facilitate BDNF-induced TrkB’s activation in the presence of BDNF; with the mediation of G protein-receptor coupled, fyn can activate TrkB in the absence of BDNF;, Src and Fyn’s participation is required in Zn2+activated TrkB. BDNF or Zn2+can phosphorylate Tyr-705/706sites of TrkB, while fyn is more likely to involve in the phosphorylation of the Tyr-515sites of TrkB. Different forms of TrkB activation can regulate the expression of hypothalamic CRH, which take fyn participate in HPA axis through TrkB-mediated activation. In this study,we assume fyn as a signaling point, play a role in the regulation of traumatic stress mediated immune changes. And we also try to further investigate the immunomodulation mechanism of EA in traumatic stress.In summary, in this study, we investigate the changes of stress-related brain areas in traumatic stress rats of different ages,inclued the prefrontal cortex, hippocampus and hypothalamus.(1) By detecting lymphocyte proliferation and natural killer (NK) cell activity, we observed traumatic stress induced immunosuppression in rats of different ages. And evaluate single or multiple electro-acupuncture treatment’s therapeutic effection on traumatic stress rats of different age;(2) After traumatic stress,observe Fyn/NeuN and pERK1/2expression changes by immunohistochemistry, Western blot and other molecular biology methods, and observe the related changes in the adjustment of EA;(3) By the regulation of fyn or downstream pERK1/2in the CNS, observe the immunomodulation in traumatic stress rats;(4)By immunohistochemistry, Western blot and other molecular biological methods, observe changes of co-expressive Fyn/TrkB cells, BDNF expression, and expression of TrkB phosphorylation sites after traumatic stress and EA treatment;(5) In primary cultured neurons, we investigate Src inhibitor PP2’s effect in BDNF induced TrkB phosphorylation.The results are as follows:1. Traumatic stress induced immunosuppression in rats of different ages and the immunomodulation effect of EA treatment.1-month-old and12-month-old rats were randomly divided into:control group (non-stress group TO), Traumatic stress1day group (T1), Traumatic Stress3day group (T3), Traumatic Stress7day group (T7). Both1-month-old and12-month-old rats showed dramatic decrease in lymphocyte proliferation and NK cell activity after1day of trauma. After3day of trauma, immuno-suppression began to improve, with lower level in12-month-old compared with1-month-old ones. By7day of trauma, fully recovery from the immuno-depression could be detected on1-month-old rats. But12-month subjects exhibited longer duration of imuno-suppression.1-month-old and12-month-old rats were randomly divided into:control group (non-stress group TO), traumatic stress1day group (T1), traumatic stress1day+single EA group (T1+SEA), traumatic stress1day+multiple EA group (T1+MEA), traumatic stress1day+single sham EA group (T1+sham SEA), traumatic stress1day+multiple sham EA group (T1+sham MEA). SEA could reverse the inhibition of lymphocyte proliferation and NK cell activity by the traumatic stress in1-month-old rats. In12-month-old cohorts, there was slightly increase, but there was not detectable difference compared with control group. MEA treatment could noticeably improve the traumatic stress mediated immuno-suppression in1-month-old rats, the level was similar to that of SEA. In contrast, MEA also had obvious improvement in12-month-old rats, lymphocyte proliferation and NK cell activity was increased. Sham EA didn’t show obvious change with trauma group.2. Alteration of Fyn-NeuN double staining cells and the downstream ERK1/2expression by traumatic stress and EA treatmentIn the prefrontal cortex, hippocampus CA3/DG, hypothalamic paraventricular nucleus (PVN), by double immunofluorescent labeling, we observed an increasing trend of the brain of Fyn/NeuN co-expression cells after stress.1day after traumatic stress Fyn/NeuN co-expression cells began to increase, the number of cells reached maximum by3days after trauma.The trend of Fyn/NeuN co-expression was attenuated in12-month-old rats. p-ERK1/2expression in prefrontal cortex of different ages rat was increased after traumatic stress. p-ERK1/2expression began to increase1day after traumatic stress, by3days after trauma the p-ERK1/2expression reached its peak. Compared with1-month-old rats, the p-ERK1/2expression of12-month-old rats in an overall reduction in traumatic stress recovery process. After Traumatic stress, the trend of p-ERK1/2expression consistent with the expression of Fyn.7days later, the expression of p-ERK1/2in all ages rats were not restored to the level before trauma.After EA treatment, in the same CNS region, we observed that EA increased the number of Fyn/NeuN co-expression cells in different age rats. Compared with12-month-old rats,1-month-old rats’Fyn/NeuN co-expression cells increased more significantly by SEA treatment. However, MEA could significantly improve the number of Fyn/NeuN cells in12-month-old rats. EA also increased the expression of Fyn downstream signaling pERKl/2, the expression level of pERKl/2consistent with Fyn.3. Fyn-ERK1/2participate in the trauma stress-induced immunosuppression recovery processTwo age groups of rats were randomly divided into:control group (non-stress group TO), traumatic stress3day group (T3), traumatic stress3day+Fyn antibody (T3+Fyn Ab), traumatic stress3day+active Fyn (T3+active Fyn), traumatic stress3day+PD98059,(T3+PD98059), traumatic stress3day+U0126,(the T3+U0126), traumatic stress3day+PD98059&active Fyn (T3+PD98059&active Fyn)After icv injection of Fyn antibody, lymphocyte proliferation and NK cell activity were attenuated in1-month-old and12-month-old rats. In contrast, recombinant adenovirus expressing active Fyn resulted in the improvement in immuno-depression. In1-month-old rats, lymphocyte proliferation and NK cell activity were increased.12-month-old rats exhibited similar change but with lower level than the1-month-old subjects. Fyn up-regulation led to increased in lymphocyte proliferation and NK cell activity At3days of the trauma, we found that icv injection of Fyn antibody caused marked decrease in pERK1/2expression level. When treatment with recombinant adenovirus expressing active Fyn, pERK1/2expression level was transformed into increase. Additionally, lymphocyte proliferation and NK cell activity were reduced in rats of different age respectively when treatment with PD98059, a ERK inhibitor. This down-regulation was also found when ERK phosphorylation was blocked by U0126suggesting that ERK activation was implicated in traumatic stress induced neuro-immune modulation. Interestingly, improvement in lymphocyte proliferation and NK cell activity was then attenuated by concurrently application of Fyn and PD98059both in1-month-old and12-month-old rats.4. The changes of co-expression Fyn/TrkB cell number,BDNF expression and phosphorylated TrkB expression after traumatic stress and EA treatmentIn prefrontal cortex, hippocampus CA3/DG region, hypothalamic PVN region, we observed an increasing trend of the brain of Fyn/TrkB co-expression cells after stress.1day after traumatic stress Fyn/TrkB co-expression cells began to increase, the number of cells reached maximum by3days after trauma.The trend of Fyn/TrkB co-expression was attenuated in12-month-old rats. The trend of Fyn/TrkB expression consistent with the expression of Fyn/NeuN cells. The shape of TrkB positive cells changed when co-expressed with Fyn, the fluorescence signal is concentrated and enhanced. Immunohistochemical DAB staining found that,after traumatic stress, cortex, hippocampus and hypothalamus of BDNF positive cells appeared vacuoles phenomenon, but Western blot analysis suggested BDNF protein did not show significant changes, reminded that Fyn/TrkB activation after traumatic stress may exist other mechanisms. In the hypothalamic, p-TrkB (Tyr-705/706) and p-TrkB (Tyr-515) increased after traumatic stress, by3day after traumatic stress,they reach the peak. p-TrkB (Tyr-515) of1-month-old rat returned to normal levels by7days after the traumatic stress, but the expression of p-TrkB (Tyr-705/706) still increased slightly than control group. The expression levels of p-TrkB (Tyr-705/706) and p-TrkB (Tyr-515) were not return to the control level by7days after the traumatic stress in12-month-old rats.In the same CNS region, EA increased the number of Fyn/TrkB co-expression cells in different age rats. Compared with12-month-old rats,1-month-old rats’ Fyn/TrkB co-expression cells increased more significantly by SEA treatment. However, MEA could significantly improve the number of Fyn/TrkB cells in12-month-old rats. Sham EA were not significant increase the number of Fyn/TrkB positive cells. In the hypothalamic, p-TrkB (Tyr-705/706) and p-TrkB (Tyr-515) increased after EA treatment. Compared with SEA, MEA significantly improved p-TrkB (Tyr-515) expression on12-month-old rats.5. The effect of Src kinase inhibitor PP2in phosphorylation of TrkB in primary cultured neuronsFetal rat cortical primary cultured neurons were divided into three groups: control group, BDNF treatment group, PP2+BDNF treatment group. By Western blot, we found that BDNF significantly induced phosphorylation of Tyr-705/706and Tyr-515point. Pretreatment with PP2one hour early, phosphorylation of the TrkB Tyr-705/706sites which induced by BDNF could be partially reversed, and PP2could completely inhibited the phosphorylation of TrkB Tyr-515sites. The present study suggested:1. Traumatic stress induced immuno-suppression and EA immunomodulation displayed with age dependent manner.2. Fyn and downstream pERK1/2participated in neuroimmune modulation process in traumatic stress rats. Fyn-pERK1/2expression changed by EA treatment. The regulating effect of EA associated with expression of Fyn and pERK1/2. Neuron maybe one of the effector cells in EA treatment.3.Traumatic stress could induce phosphorylation on different sites of TrkB. TrkB might be acted as one of fyn upstream signal during the immune recovery process.4. EA tended to regulate the Tyr-515site of TrkB, which suggested fyn might be one of the targeting point in EA’s immunoregulation effect. |
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