【摘要】：目的:以補(bǔ)益腎陰名方左歸飲為研究對(duì)象,從中醫(yī)臨床功效出發(fā),采用現(xiàn)代藥理學(xué)研究方法,探討左歸飲對(duì)衰老模型大鼠肝腎器官功能的影響,并對(duì)其作用機(jī)制進(jìn)行初步研究,為左歸飲延緩機(jī)體衰老的理論提供更多實(shí)驗(yàn)依據(jù),同時(shí)也為左歸飲臨床合理用藥提供科學(xué)指導(dǎo)。方法:將實(shí)驗(yàn)大鼠分正常對(duì)照組、模型組、給藥組(維生素E組、六味地黃丸組、左歸飲高、低劑量組),采用D-半乳糖頸部皮下注射方法復(fù)制亞急性衰老大鼠模型,造模同時(shí),各給藥組灌胃給予相應(yīng)藥物進(jìn)行干預(yù),連續(xù)給藥8周后,進(jìn)行以下實(shí)驗(yàn)研究:(1)采用行為學(xué)實(shí)驗(yàn)方法(水迷宮和跳臺(tái)實(shí)驗(yàn))對(duì)衰老模型大鼠進(jìn)行評(píng)價(jià),考察左歸飲對(duì)衰老模型大鼠學(xué)習(xí)記憶能力的影響;(2)通過對(duì)衰老模型大鼠肝腎功能指標(biāo)及其組織中自由基進(jìn)行檢測(cè),考察左歸飲對(duì)衰老模型大鼠肝腎功能及自由基代謝水平的影響;(3)采用病理學(xué)實(shí)驗(yàn)方法對(duì)衰老模型大鼠肝腎組織病理進(jìn)行研究,考察左歸飲對(duì)衰老模型大鼠肝腎組織細(xì)胞的形態(tài)結(jié)構(gòu)和病理變化的影響;(4)采用免疫組化方法對(duì)肝腎組織中細(xì)胞凋亡相關(guān)蛋白Bax、Bcl-2、Caspase-3進(jìn)行研究,考察左歸飲對(duì)衰老模型大鼠肝腎組織細(xì)胞凋亡相關(guān)蛋白表達(dá)水平的影響;(5)采用代謝組學(xué)研究方法,通過LC-MS技術(shù)對(duì)衰老模型大鼠尿液進(jìn)行分析,比較左歸飲對(duì)衰老模型大鼠尿液中內(nèi)源性生物標(biāo)記物的調(diào)節(jié)作用,考察左歸飲對(duì)衰老模型大鼠尿液代謝組學(xué)的影響。結(jié)果:(1)在大鼠行為學(xué)評(píng)價(jià)中,左歸飲能縮短衰老模型大鼠在水迷宮、跳臺(tái)實(shí)驗(yàn)的潛伏時(shí)間,增加水迷宮實(shí)驗(yàn)穿越隱藏平臺(tái)次數(shù),減少跳臺(tái)實(shí)驗(yàn)錯(cuò)誤次數(shù);(2)在生化指標(biāo)測(cè)試中,除ALT外,左歸飲能夠降低衰老模型大鼠血清中AST、ALP、CREA、BUN指標(biāo)水平,提高衰老模型大鼠肝腎組織中SOD和T-AOC活性,降低MDA含量;(3)肝腎組織形態(tài)的病理學(xué)實(shí)驗(yàn)結(jié)果表明,左歸飲在一定程度上能夠減輕衰老模型大鼠肝腎組織細(xì)胞的病理?yè)p傷,保持組織細(xì)胞形態(tài)結(jié)構(gòu)的完整性;(4)肝腎組織免疫組化實(shí)驗(yàn)結(jié)果表明,左歸飲能夠增強(qiáng)衰老模型大鼠肝腎組織細(xì)胞中Bcl-2的陽(yáng)性表達(dá),降低肝腎組織細(xì)胞中Bax、Caspase-3的陽(yáng)性表達(dá);(5)大鼠尿液代謝組學(xué)分析結(jié)果共鑒定出29個(gè)生物標(biāo)記物,找出9個(gè)相關(guān)代謝通路,結(jié)果表明,左歸飲能夠使衰老模型大鼠尿液代謝輪廓趨于正常,對(duì)23個(gè)生物標(biāo)記物有回調(diào)作用,且對(duì)7個(gè)相關(guān)代謝通路產(chǎn)生影響。結(jié)論:左歸飲能夠通過提高衰老模型大鼠學(xué)習(xí)記憶能力,改善衰老模型大鼠肝腎功能,增強(qiáng)對(duì)衰老模型大鼠體內(nèi)自由基的代謝及清除能力,保護(hù)肝腎組織細(xì)胞免于損傷,發(fā)揮延緩衰老作用。其作用可能通過影響肝腎組織細(xì)胞中相關(guān)凋亡蛋白的表達(dá),調(diào)整衰老模型大鼠機(jī)體的異常代謝等機(jī)制起到延緩機(jī)體衰老作用。
[Abstract]:Objective: to study the effect of Zuo Gui Yin, a famous prescription of tonifying kidney yin, on the function of liver and kidney organs in aging model rats. It provides more experimental basis for the theory of Zuo Gui Yin to delay the aging of organism, and also provides scientific guidance for the rational use of Zuo Gui Yin in clinic. Methods: the experimental rats were divided into normal control group, model group, administration group (vitamin E group, Liuwei Dihuang pill group, Zuo Guiyin high dose group, low dose group). Subacute aging rat model was induced by subcutaneous injection of D-galactose. After 8 weeks of continuous administration, the following experimental studies were carried out: (1) the aging model rats were evaluated by behavioral experiment (water maze and jumper test). To investigate the effect of Zuoguiyin on learning and memory ability of aging model rats. (2) to detect the indexes of liver and kidney function and free radicals in tissues of aging model rats. To investigate the effects of ZuoGuiyin on liver and kidney function and free radical metabolism in aging model rats. (3) to study the pathological changes of liver and kidney tissues in aging model rats by pathological experiment. To investigate the effect of Zuoguiyin on the morphology and pathological changes of liver and kidney cells in aging model rats. (4) to study the apoptosis related protein Baxia Bcl-2Caspase-3 in liver and kidney by immunohistochemical method. To investigate the effect of Zuoguiyin on the expression of apoptosis-related protein in liver and kidney tissues of aging model rats. (5) to analyze the urine of aging model rats by LC-MS with the method of metabolomics. To compare the effects of Zuoguiyin on endogenous biomarkers in urine of aging model rats, and to investigate the effect of Zuogui Yin on urine metabolism of aging model rats. Results: (1) in the behavioral evaluation of rats, Zuo Gui Yin can shorten the latency time in water maze and platform jumping experiment, increase the number of water maze experiment traversing hidden platform, reduce the number of errors in platform jumping experiment; (2) in biochemical index test, In addition to alt, Zuoguiyin could decrease the serum level of ASTPPA-CREAEABUN, increase the activities of SOD and T-AOC and decrease the content of MDA in the liver and kidney of aging model rats. (3) the pathological results of liver and kidney morphology showed that Zuo Gui Yin can alleviate the pathological injury of liver and kidney cells in aging model rats to some extent and maintain the integrity of histocyte morphology. (4) Immunohistochemical experiment of liver and kidney tissue showed that, ZuoGuiyin could enhance the expression of Bcl-2 and decrease the positive expression of Baxfen Caspase-3 in the liver and kidney of aging model rats. (5) 29 biomarkers were identified by the results of urine metabolism in rats. Nine related metabolic pathways were identified. The results showed that Zuoguiyin could make the metabolic profile of urine of aging model rats normal, have a callback effect on 23 biomarkers, and have an effect on 7 related metabolic pathways. Conclusion: ZuoGuiyin can improve the learning and memory ability of aging model rats, improve the liver and kidney function of aging model rats, enhance the metabolism and scavenging ability of free radicals in aging model rats, and protect liver and kidney tissue cells from injury. Play a role in delaying senescence. Its effect may delay senescence by affecting the expression of apoptosis protein in liver and kidney tissues and regulating the abnormal metabolism of aging model rats.
【學(xué)位授予單位】：黑龍江中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285.5;R-332

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