“肺应秋”生理机制的实验研究

【作者】 顾晓静；

【导师】 郭霞珍；

【作者基本信息】 北京中医药大学 ， 中医基础理论， 2009， 博士

【副题名】四时季节变化对肺脏细胞信号转导系统的影响

【摘要】 “四时五脏阴阳”理论体现出人是一个有机的统一整体,“肺应秋”是中医学“四时五脏阴阳”理论的重要组成部分,秋季肺如何应时地适应外界环境的变化,以维持自身稳定的生理状态,是“肺应秋”理论研究的重要内容。近年来,季节节律对人体生理病理变化影响的研究已引起学者的重视,研究季节气候变化对人体生理病理的影响对于临床治疗肺系疾病和指导人体养生具有重要的意义。1目的以“肺应秋”为切入点,通过研究肺脏细胞信号转导的季节性变化特点,从而揭示中医“肺应秋“的本质内涵,为“四时五脏阴阳”理论的研究提供科学依据和途径,也可指导养生并为治疗目前临床常见的季节性肺部疾患的研究提供理论和实验依据。2方法2.1理论研究本研究采用文献资料法和逻辑分析法,探讨“肺应秋”的中医理论内涵以及“肺应秋”调控机制与细胞信号转导理论的相关性,说明秋季脏腑的适应性调控具有一定的理论依据,进一步充实了“四时五脏阴阳”的理论内涵。2.2实验研究2.2.1实验动物:健康Wistar大鼠,雄性24只,体重150-170g,分别于2006年12月22日（冬至）、2007年3月21日（春分）、6月22日（夏至）和9月23日（秋分）前四十天由北京维通利华实验动物技术有限公司购入。随机分为生理组、模型组（松果腺摘除模型组）和伪手术组,每组各8只。饲养条件:自然光照,室温（冬春季室温为17℃±2℃,夏秋季室温为25℃±2℃）。自由摄取水及饲料,饲料为普通鼠全价颗粒饲料。模型组、伪手术组分别于春分、秋分、夏至、冬至（简称二分、二至）前一个月施行手术造模,与生理组动物在相同条件下饲养到二分、二至当日晚8:00以后处死取材。2.2.2检测方法:肺组织中G蛋白采用Western-blot法;肺组织中cAMP、cGMP采用ELISA法;肺组织IP₃采用放射免疫法;肺组织中c-fosmRNA、c-junmRNA采用原位杂交法。2.2.3实验结果统计学分析方法:所有数据均用均数±标准差表示,运用SPSS11.5统计软件分析。各季节组内及四季节组间比较均采用单因素方差分析（One-Way Anova）检验,以P＜0.05为差异有显著性。3结果3.1理论研究结果3.1.1在秋季,肺通过其肃降功能的增强,不但可以影响肺脏自身的生理功能,而且可以影响其他四脏的功能活动以适应外界环境的变化,这是“肺应秋”理论的生理内涵。3.1.2本研究通过对“肺应秋”调控机制和细胞信号转导机制进行分析发现,二者都强调外界环境对机体的影响,机体内存在整体调控机制,调控目的是为了实现“整体稳态”,认为理论上“肺应秋”调控机制与细胞信号转导存在相关性,并提出了从细胞信号转导角度来研究“肺应秋”调控机制的研究思路。3.2实验研究结果3.2.1季节变化对肺脏细胞信号转导物质的影响在正常生理状态下,季节变化对肺脏细胞信号转导物质可产生显著影响,表现在:（1）全年大鼠肺组织中Gq含量春季显著地低于夏季、秋季（P＜0.05）,非常显著地低于冬季（P＜0.01）;大鼠肺组织中Gs含量春季非常明显地低于夏季和冬季（P＜0.01）,夏季非常明显地高于春季和秋季（P＜0.01）,秋季非常明显地低于夏季（P＜0.01）、明显地低于冬季（P＜0.05）。（2）肺组织中第二信使cAMP含量夏季明显高于秋季（P＜0.05）;肺组织中cGMP的含量春季非常明显地低于秋季（P＜0.01）,明显低于冬季（P＜0.05）;肺组织中IP₃的含量四季生理组之间无明显差异。（3）肺组织中第三信使c-fos mRNA、c-jun mRNA表达的平均光密度四季生理组之间无明显差异;c-fos mRNA表达的面积百分数春季非常明显地高于再秋冬季（P＜0.01）;c-jun mRNA表达的面积百分数春季非常明显地高于夏秋冬季（P＜0.01）,夏季明显高于冬季（P＜0.05）。3.2.2松果腺对肺脏细胞信号转导物质的影响在摘除松果腺的情况下,肺脏细胞信号转导物质的节律会发生不同程度的变化,表现在:（1）春季大鼠肺组织中Gq含量生理组明显低于模型组（P＜0.05）,生理组与伪手术组之间没有差异,摘除松果腺后四季肺组织中Gq的含量无明显差异,伪手术组四季肺组织中Gq的含量冬季非常明显地高于春季和秋季（P＜0.01）;夏季肺组织中Gs的含量生理组明显高于模型组（P＜0.05）,生理组与伪手术组之间没有差异,摘除松果腺后四季肺组织中Gs的含量无明显差异,伪手术组四季肺组织中Gs的含量冬季非常明显地高于春季、夏季、秋季（P＜0.01）。（2）肺组织中IP₃的含量秋季模型组明显高于生理组和伪手术组（P＜0.05）。（3）肺组织中c-fos mRNA的表达平均光密度冬季生理组明显低于模型组（P＜0.05）,生理组与伪手术组之间没有差别;肺组织中c-fos mRNA的表达面积百分数夏、秋季生理组明显低于模型组（P＜0.05）,生理组与伪手术组之间没有差别。肺组织中c-jun mRNA的表达平均光密度冬季生理组明显高于模型组（P＜0.05）,且生理组与伪手术组之间没有差别;秋季c-jun mRNA的表达面积百分数模型组非常明显地高于生理组、伪手术组（P＜0.01）。这些都表明了松果腺在上述物质的季节变化中起了调节作用。4结论4.1在正常生理状态下,肺脏细胞信号转导呈现季节性变化节律。4.2四季肺脏细胞信号转导的节律性变化与松果腺的高位调节作用有关,这种调节具有复杂性和季节选择性。4.3“肺应秋”秋季肺脏中褪黑素影响肺脏局部免疫功能可能通过肺脏胞内Gs→（+）Ac→cAMP（?）→（+）PKA→（+）CREB→（+）c-fos细胞信号通路的抑制来实现。更多还原

【Abstract】 Theory of "Yinyang of five Zang-organs and four seasons" express that man is an integrated whole,and man and nature is an integrated whole.Theory of "Lung correspondence to autumn "is the important content of theory of "Yinyang of five Zang-organs and four seasons".The researches on theory of "Lung correspondence to autumn " mainly focus on how lung adapt the changes of nature to maintain the balance of physiological function.Recently,seasonal rhythm influencing on man’s physiology and pathology becomes an interesting direction for many researchers. Studying on the seasonal climate influencing on physiological function is of great importance to clinical diagnosis,theory and preventing diseases.1ObjectiveTaking the idea of"lung correspondence to autumn" in TCM as a breaking point, we research on the seasonal changes of lung cell signal transduction to discuss the truth of theory of "Yinyang of five Zang-organs and four seasons",which can enrich the scientific methods of theory of "Yinyang of five Zang-organs and four seasons". Through this research,we can give some theoretic and experimental proofs on clinical diagnosis,theory and preventing diseases.2Methods2.1Theoretical methodsThe methods of the documentary materials and logical analysis were used to explain and analyze the physiological mechanism of "lung correspondence to autumn" and the relationship of "lung correspondence to autumn" and cell signal transduction.It showed that "Zang Fu proper to nature" had theoretical basis,and enriched the theoretical connotation of "Yinyang of five Zang-organs and four seasons".2.2Experimental methods2.2.1 Experimental animalsTwenty-four Wistar male rats,weighted 150-170g,brought from Beijing Weitong Lihua animal study center forty days before vernal equinox,summer solstice,autumnal equinox and winter solstice,were randomly divided into three groups:normal group,fake group and operation group（pinealectomy group）.There were eight rats in every group.All rats received natural light,ate and drank freely,and were controlled under certain room temperature was 17℃±2℃in winter and spring,and it was 25℃±2℃in summer and autumn.Fake group and operation group are operated a month before vernal equinox,summer solstice,autumnal equinox and winter solstice.All the rats would be raised till vernal equinox,summer solstice,autumnal equinox and winter solstice,on these days at 8 o’clock at night,they would be beheaded.2.2.2 Test methodsTo assay the contents of G protein in lung,westen-blot was engaged.To assay the contents of cAMP cGMP in lung,ELISA was engaged.To assay the contents of IP₃ in lung,Radio-immunity was engaged.To assay the express of c -fos mRNA and c-jun mRNA in lung,Tissue in situ hybridization（TISH） was engaged.2.2.3 Analysis method:All the data were showed by mean±standard error.The differences of groups were tested by SPSS 11.0.The values were calculated with Duncan’s multiple range test or Student’s test.P＜0.05 meant obvious difference.3Results3.1 Results of theoretical study3.1.1 In autumn,lung can adjust itself to correspond autumn,it also can influence the other four zang-organs to adapt to the changes of the nature,and this is the physiology of theory of "Lung correspondence to autumn "3.1.2 After analysis of the pertinence between the modulating mechanism of ’Lung correspondence to autumn’ and cell signal transduction theoretically in this study,we think they all emphasize on nature’s influence on human being,and there are whole control mechanism which aimed at modulating.So we can from the point view of cell signal transduction accords with the essence of’Lung correspondence to autumn’ in TCM.3.2 Results of experiments3.2.1 Seasonal change can influence cell signal transduction of lungIn normal stasis,the cell signal transduction have the character of seasonal changes.（1） Gq in lung of spring was lower than it of summer and autumn （P＜0.05）,significantly lower than it of winter（P＜0.01）;Gs in lung of spring was significantly lower than it of summer and winter（P＜0.01）,Gs in lung of summer was significantly higher than it of spring and autumn,Gs in lung of autumn was significantly lower than it of summer（P＜0.01） and lower than it of winter（P＜0.05）.（2）cAMP in lung of summer was higher than it of autumn（P＜0.05）;cGMP in lung of spring was significantly lower than it of autumn（P＜0.01 ） and lower than it in winter（P＜0.05）（3）Seasonal change can not influence on Density mean of c-fos mRNA and c-jun mRNA in lung.But Per Area of c-fos mRNA in lung of spring was significantly lower than it of summer,autumn and winter（P＜0.01）.Per Area of c-jun mRNA in lung of spring was significantly lower than it of summer,autumn and winter（P＜0.01 ）, and if of summer was higher than it of winter（P＜0.05）. 3.2.2Pineal gland can influence on the substances of cell signal transduction in lungIf we remove the Pineal gland,the substances of cell signal transduction in lung can changes:（1）Normal group of Gq in lung of spring was lower than operation group of it （P＜0.05）.Fake group of Gq in lung of winter was significantly lower than it of spring and autumn（P＜0.01 ）.Normal group of Gs in lung of summer was higher than operation group of it（P＜0.05）.Fake group of Gs in lung of winter was significantly higher than it of spring,summer and autumn（P＜0.01）.（2）Operation group of IP₃ in lung of autumn was higher than normal group and fake group of it（P＜0.05）.（3）Normal group of Density mean of c-fos mRNA in lung of winter was lower than operation group of it（P＜0.05）.Normal group of Per Area of c-fos mRNA in lung of summer and autumn was lower than operation group of it（P＜0.05）.Normal group of Density mean of c-jun mRNA in lung of winter was lower than operation group of it（P＜0.05）.Normal group of Per Area of c-jun mRNA in lung of autumn was higher than operation group and fake group of it（P＜0.01）.Above all,Pineal gland can effect on the substances of cell signal transduction in lung.4 Conclusions4.1 Cell signal transduction in lung have seasonal changes..4.2 Pineal gland can effect on the substances of cell signal transduction in lung.4.3 The modulating mechanism of "Lung correspondence to autumn" maybe through the routine of Gs- cAMP-c-fos.cell in lung effect on the immune system.更多还原