“逆针灸”对更年期大鼠热休克蛋白及应激相关机制影响的研究

【作者】 郑玲；

【导师】 李晓泓；

【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2007， 硕士

【摘要】 “治未病”是中医基础理论的重要组成部分,是《内经》以预防为主的学术观点的具体体现,对后世影响深远。“逆针灸”作为中医“治未病”的重要方法之一,即在机体无病、疾病发生之前或疾病发展轻浅之时,预先应用针灸的方法,以激发经络之气,扶助正气,调动机体内在调衡阴阳的能力,从而达到防止疾病的发生、减轻随后疾病的损害或保健延年的目的。逆针灸防病保健的作用已被大量研究证实,但其确切的作用机制尚在探询中。随着现代应激理论研究的深入,机体具有内源性保护机制已被广泛证实,利用人为手段使机体适度应激,预防或减轻随后疾病正引起医学界的高度重视。其特点在于:并非向机体补充大量的外源性物质,而是采取一些理化的预处理手段激发机体应激,启动机体内源性保护机制,加强机体对随后疾病损伤的抵抗与耐受力,是一种充分重视机体自身潜力激发与调动的整体调节方法。这正是“逆针灸”防病保健的特色所在,因而极具研究与开发潜力。更年期是妇女衰老进程中的关键启动期,此期由于肾元渐衰,天癸将竭,导致机体内环境急剧变化,多系统出现动荡与紊乱,机体处于一种生理性应激状态,在调整过程中不能适应而造成的由生理性应激转变成的病理性应激,致使70%～90%的更年期妇女出现繁杂多变的更年期综合征。更年期综合征的发生机理尚未完全明了,而应激反应中诱导生成的热休克蛋白与更年期时机体诸多环节如神经内分泌、免疫、抗氧化、雌激素受体、胰岛素受体功能等密切相关,因此探讨热休克蛋白在更年期机体中的作用,对于防治更年期综合征具有重要意义。本课题结合中医“治未病”的学术思想和现代应激医学理论,以自然更年期大鼠为模型,采用免疫组化、原位杂交、放射免疫、免疫比浊等方法,观察逆针灸关元穴对随后不同月龄自然更年期大鼠热休克蛋白、应激激素、神经递质、抗氧化相关因子及免疫因子等的影响,试图从HSP70及HSP70mRNA的角度探询逆针灸关元穴防治更年期综合征的相关应激机制。主要实验结果及结论如下:1.大鼠进入更年期后下丘脑不同核团HSP70及mRNA的表达随着月龄的增长表现不同的分布趋势,即视上核呈波动性升高,而室旁核和弓状核升高的幅度随着月龄的增长有下降的趋势。提示下丘脑不同核团在介导更年期中枢应激反应中发挥着不同的作用。下丘脑CRH含量的波动性降低和血浆ACTH、皮层5-HT含量的波动性升高,提示更年期大鼠HPA轴处于异常活跃状态,应激激素可能通过受体结合途径激活中枢吲哚类神经递质的大量合成。同时下丘脑抑制性应激激素β-EP含量却呈增龄性波动下降,皮层NE含量呈波动性升高,提示机体抑制性激素降低增强应激兴奋性的同时,也削弱了对儿茶酚胺神经元的紧张性抑制作用。预先针灸关元穴可不同程度的下调视上核HSP70及mRNA的表达,上调弓状核HSP70及mRNA的表达。提示逆针灸能够降低应激兴奋性因素,提高抑制性因素,有利于减缓应激中枢异常活跃状态。而对于应激反应中心核团的室旁核作用复杂,表现为各月龄HSP70mRNA的表达增强,说明逆针灸能够提高下丘脑室旁核应激调节中枢的警觉状态,转录大量HSP70mRNA以增强细胞应激保护能力。而12、14月龄HSP70表达的下调,可能与逆针灸提前诱导的HSP70在组织细胞中发挥保护效应减轻组织损伤、减缓应激反应程度有关;16月龄HSP70表达的升高,可能由于细胞内有害物质蓄积增多,逆针灸作用后使得机体翻译合成更多的HSP70以加强机体防御适应反应的能力,从而表现出双向性的调节作用。逆针灸下调CRH的作用主要表现在12、14月龄,对于血浆ACTH的调节表现为12月龄升高,14月龄降低,以缓和14月龄骤然升高的ACTH水平。而对于β-EP的上调和NE、5-HT的下调作用则主要体现在12月龄。提示逆针灸对中枢应激激素及神经递质的调节以早期效应为主。而逆针灸是否通过调节下丘脑重要核团HSP70及mRNA的表达及功能活动影响相应核团应激激素的合成和释放从而调节中枢神经递质的水平及其他神经内分泌功能,改善更年期精神及植物神经紊乱状态,有待于进一步研究。2.更年期机体应激反应使得CRH大量释放,能够造成对GnRH、垂体促性腺激素及卵巢雌激素合成的抑制作用,同时也可诱导细胞内热休克蛋白的生成。本实验观察到随着大鼠月龄的增长子宫HSP70及mRNA的表达呈先增后降的变化,早期热休克蛋白的增加可能与CRH的增加及局部雌激素水平下降对HSP抑制作用的降低有关,但随着组织器官的衰退,其蛋白合成能力下降,因而表现出晚期热休克蛋白及基因表达下调。同时随着机体进入更年期子宫SOD、NOS活性明显降低,而经逆针灸作用能增强各月龄组HSP70及mRNA的表达;且提高SOD、NOS的活性,主要表现在12、16月龄。提示逆针灸关元穴对随后更年期大鼠子宫产生保护作用机制可能通过减缓应激中枢的异常活跃状态,提高不同月龄外周子宫组织细胞HSP70及mRNA的表达,以促进组织SOD、NOS活性的增强,对抗应激激素及卵巢功能衰退引起的子宫靶器官的结构及功能的改变,提高机体抗氧化能力,增强NO/NOS的局部调节作用,减轻随后更年期衰老过程中自由基对组织器官的损害及组织器官功能紊乱,对于维持子宫正常的结构和功能,延缓子宫退行性变具有重要作用。3.更年期时机体的应激状态不仅引起机体神经内分泌系统的应答,同时也对免疫系统功能活动产生重要影响。本实验观察到更年期早期血清免疫因子IL-2、TNF-α的含量的下降,可能与机体应激导致血液和组织中神经递质及肽类激素含量升高,对免疫细胞产生的抑制性调节作用有关。而同时这些神经递质和肽类激素也能够诱导免疫细胞HSP的表达,因而表现出更年期大鼠早期脾脏HSP70mRNA表达的增高。随着月龄的增长,脾脏HSP70及mRNA的表达、血清IL-2的含量呈增龄性下降,而血清TNF-α的含量显著升高,提示机体在更年期衰老进程中由于自身保护能力和免疫功能的降低,可能导致有害物质在体内的蓄积,刺激TNF-α大量合成。而预先运用逆针灸的方法后,不同月龄更年期大鼠脾脏HSP70及mRNA的表达明显增强,血清IL-2含量显著升高,以14、16月龄尤为明显,且能提高更年期早期血清TNF-α的含量,降低更年期晚期TNF-α的过度表达。提示逆针灸关元穴对随后更年期大鼠免疫系统功能调整作用的机制可能与其调节应激中枢的功能状态,增强脾脏组织细胞HSP70及其基因表达,进而调节IL-2、TNF-α的合成、分泌及其生物学效应等相关,从而提高机体对外源性抗原的免疫应答能力,降低自身免疫炎性反应和组织器官损伤,减轻更年期应激状态下机体免疫系统功能的抑制或紊乱状态,对于延缓免疫系统退变和功能紊乱造成的机体衰老加速具有重要意义,并表现出良性、双向性的调整作用。综上所述:逆针灸关元穴可能通过调节中枢(下丘脑)及外周(子宫、脾脏)组织细胞热休克蛋白及基因的表达,调动了机体内在的抗病与应变能力;从而减缓中枢应激过度活跃状态,一定程度上降低脑内神经递质的异常分泌,提高子宫SOD、NOS的活性,调节血清免疫因子IL-2、TNF-α的含量,对于维持机体内环境稳定,调整更年期植物神经及免疫功能紊乱,改善子宫退行性变化具有一定作用。更多还原

【Abstract】 The opinion of“treat disease before it arises”is an import part of basic theory of chinese medicine. It embodies the preventive opinion of Neijing and has a profound influence to the afterworld.“Preventive acupuncture and preventive moxibustion”is one of the methods of“treat disease before it arises”. It refers to stimulation of qi and blood within meridians when a person is healthy or before the disease arises, and the internal potentiality of banlancing yin and yang within the body will be moderated so that health protection or alleviation of diseases and disorders could be achieved.The preventive function of preventive acupuncture and preventive moxibustion has been approved, but the exact mechanism is still in research.As the developing of modern stress theory, the internal protective mechanism has been widely validated. Proper stress induced by some manmade means can prevent or relieve diseases, and the opinion attracts much attention. It has its own character: It does not depend on adding many external materials into the body, but stimulates stress to activate the internal protective mechanism with some physical chemical pretreatment methods, so as to resist the damage of diseases. It is a method which pays much more attention to stimulating self potentiality and adusting from global level. Preventive acupuncture and preventive moxibustion has the same character so it is worth researching and developing.Menopause is an important transitional period to women from young to old. Insufficiency of kidney essence and exhaustion of“tiangui”during this period cause an sudden change of internal environment and disorder of many systems. If the body of menopause can not adapt the change, the physiological stress in this time will turn into pathological stress and as a result, about 70%～90% women will catch complexed menopause syndrome. The mechanism of menopause syndrome is still unclear. Heat shock protein(HSP) is induced by stress response and it has an intinate relationship with many links during menopause such as neutroendocrine, immunity, antioxidation, estrogen receptor, and insulin receotor, so discussing the function of HSP will benefit the prevention of menopause syndrome.The combination of the opinion of“treat disease before it arises”in Chinese medicine and stress theory in morden westen medicine was the start point of our research. Natual aging rats were chosen to be the model of menopause. A lot of advanced techniques such as immuno-histochemistry, in situ hybridization, radio-immunity, immunoturbidimetric assay and the like methods were used to observe the effect on HSP, stress hormones, neurotransmitters and factors of antioxidation and immunity of different month-aged menopausal rats with preventive acupuncture and preventive moxibustion, so as to discuss the stress mechanism of prevtenting menopause syndrome with preventive acupuncture and preventive moxibustion from the expression of HSP70and HSP70mRNA.Main results and conclusions are as following:1. As the growing of month, the expression of HSP70 and HSP70mRNA in different hypothalamus nuclei of menopausal rats presented different distribution: It displayed a wave like increasing in supraoptic nucleus, however, the growth range of the expression of HSP70 and HSP70mRNA in paraventricular nucleus and arcuate nucleus descended as the growing of month. It indicated different nuclei had different functions in central stress response during menopause. The descending of CRH in hypothalamus and ascending of ACTH in blood plasma and 5-HT in cerebral cortex showed that the HPA axis was abnormally active during menopause and stress hormones might stimulate the secretion of indoles neurotransmitter through receptor binding.β-EP was one of the inhibiting stress hormones in hypothalamus. It decreased during menopause. Meanwhile, NE in cerebral cortex increased. It suggested that the reducion of inhibiting stress hormones could strengthen the exiting of stress and weaken the inhibitory effects on catecholaminergic neurons. The expression of HSP70 and HSP70mRNA in supraoptic nucleus came down and that in arcuate nucleus went up with preventive acupuncture and preventive moxibustion. It demonstrated that the therapy could decrease the exciting factors, increase the inhibiting factors and alleviate the abnormal activity of centre of stress. Paraventricular nucleus was the central nucleus in stress response, as a result, the effect on it with preventive acupuncture and preventive moxibustion was complicated. The expression of HSP70mRNA was elevated in every month-aged group. It indicated that preventive acupuncture and preventive moxibustion could strengthen the vigilance of paraventricular nucleus so that much more HSP70mRNA was transcribed to protect the body. However, the expression of HSP70 in 12 and 14 month-aged groups decreased. It might be associated with that HSP70 induced by preventive acupuncture and preventive moxibustion in advance had alleviated tissue damage and stress response. The expression of HSP70 in 16 month-aged group rised. It might be associated with that intracellular harmful substances accumulated at this time and as a result preventive acupuncture and preventive moxibustion promoted the the expression of HSP70 to protect the body. The down-regulation of CRH with preventive acupuncture and preventive moxibustion was mainly reflected in 12th and 14th month. ACTH in blood plasa increased in 12th month and decreased in 14 th month. The up-regulation ofβ-EP and down-regulation of NE and 5-HT were maily reflected in 12th month. It demonstrated that the effect on central stress hormones and neurotransmitters with preventive acupuncture and preventive moxibustion mainly appeared at early time. However,the effect on the expression of HSP70 and HSP70mRNA in hypothalamus nuclei with prevetive acupuncture and preventive moxibustion and the relationship with the secretion of stress hormones and neurotransmitters still need to research further.2. The release of CRH in stress response during menopause inhibited the secretion of GnRH, gonadotropic hormone and estrogen and induced the expression of intracellular HSP. As the growing of month, the expression of HSP70 and HSP70mRNA in uterus went up at the beginning and came down in the end. The result might be caused by the following:The increase of CRH and the decrease of estrogen during early menopause weakened the inhibitory effects on HSP. However, tissues and organs declined in the end of menopause, as a result, the determination of protein synthesis decreased. The activity of SOD and NOS in uterus during menopause was weakened significantly. The expression of HSP70 and HSP70mRNA and the activity of SOD and NOS in uterus were elevated with preventive acupuncture and preventive moxibustion and the influence on the activity of SOD and NOS was mainly refected in 12th and 16th month. It indicated that preventive acupuncture and preventive moxibustion on Guanyuan point could relieve the abnormal activity of center of stress, promote the expression of HSP70 and HSP70mRNA and the activity of SOD and NOS in uterus so as to maintain the normal structure and function and prolong the retrograde degeneration of uterus. It might be one of mechanisms of protecting uterus with preventive acupuncture and preventive moxibustion.3. The stress response during menopause not only influenced neuroendocrine system, but also effected immunity system. The level of IL-2 and TNF-αin blood serum of menopausal rats decreased. It was possibly caused by inhibitory effects on immunity cell of neurotransmitters and peptide hormones induced by stress response. Meanwhile, the neurotransmitters and peptide hormones could stimulate the expression of HSP in immunity cell, as a result, the expression of HSP70mRNA in spleen during early menopause rised. However, as the growing of month, the expression of HSP70 and HSP70mRNA in spleen and the level of IL-2 in blood serum decreased and at the same time, the level of TNF-αin blood serum increased obviously. It showed that self-protected ability and immunity function declined during menopause. And harmful substances accumulated at this time so as to stimulate the synthesis of TNF-α. With preventive acupuncture and preventive moxibustion, the expression of HSP70 and HSP70mRNA in spleen and the level of IL-2 in blood serum went up apparently in 14th and 16th month and the level of TNF-αin blood serum presented an adverse change compared to the model group. It indicated that preventive acupuncture and preventive moxibustion could regulate the center of stress, strengthen the expression of HSP70 and HSP70mRNA and adjust the synthesis and secretion of IL-2 and TNF-αso as to promote immune response to exogenous antigen, alleviate the autoimmune reaction and damages of tissues and organs and relieve the disorder of immunity function. The effect on immunity during menopause with preventive acupuncture and preventive moxibustion was benign and bidirectional. And it could prolong the aging through improving the decline of the structure and funcition of immunity system. In summary, preventive acupuncture and preventive moxibustion on Guanyuan point could stimulate the ability of resisting diseases and meeting emergencies through adjusting the expression of HSP70 and HSP70mRNA in center and periphery of body. It could also alleviate the abnormal activity of center of stress, decrease the secretion of neurotransmitters in brain, promote the activity of SOD and NOS in uterus and regulate the level of IL-2 and TNF-αin blood serum. As a result, preventive acupuncture and preventive moxibustion had benign effects on maintaining homeostasis, moderating the funcition of neuroendocrine and immunity and improving the decline of uterus.更多还原