基于衰老雌鼠模型探讨铁皮石斛“补肾”功效的科学内涵

发布时间：2018-05-07 07:18

本文选题：铁皮石斛 + 补肾　；参考：《广州中医药大学》2016年博士论文

【摘要】：衰老是生命无可避免且复杂的一种自然规律,随着年龄的增加,体内新陈代谢下降和各器官的功能逐渐衰退,形体外观和内脏衰老征象变得更加明显[1]。中医药学对衰老理论和延缓衰老具有深刻的阐述和丰富的实践,《素问·上古天真论》中明确指出机体的“生、长、壮、老、已”,受肾中精气的调节,提出肾虚是衰老的主要原因。老年期中耳鸣耳聋、腰酸腿软、夜尿频多等均为肾气衰退的表现,因此,在调摄养生的前提下,应注重补肾培元。虽然我们不可能违背生命衰老这个自然规律,但是希望通过探索人类衰老的机理,合理采用保健措施,重视防护,可有效降低衰老相关疾病的发病率,提高生活质量,延缓衰老。传统中医对石斛功效评价很高,《本草通玄》记载石斛“咸能益肾,多功于水土(肾、脾)二脏”；《本草思辨录》记载石斛为肾药、为肺药、为肠胃药；《神农本草经》认为石斛新鲜或干燥茎入药,有滋阴清热、补肾益精之功效：《药性论》中石斛“主治腰脚软弱,骨中虚损,补肾积精,养肾气,益力”；《本草纲目》评价铁皮石斛“强阴益精,补肾益力,壮筋骨,益智除惊,补五脏虚劳赢瘦,轻身延年”；《道藏》更将铁皮石斛列为“中华九大仙草”之首,因其具有独特的药用价值和保健功效,成为历代养生补品,说明石斛的延缓衰老作用早已被认识。近来研究证实mTOR (mammalian target of rapamycin,哺乳动物雷帕霉素靶蛋白)通路的调节作用与机体衰老密切相关,可通过活化NF-κB、促炎症因子释放等而参与炎症衰老的过程。线粒体功能紊乱致氧自由基生成增加是衰老调控机制之一,线粒体功能受损及数量减少与诸多年龄相关疾病密切联系。mTORCl受抑制后可致发挥保护功能的基因群活化,通过限制线粒体呼吸减少氧自由基造成的损伤,从而致机体寿命延长。我们前期研究发现铁皮石斛抗雌鼠卵巢衰老的机制可通过增强血液中的抗氧化酶活性、抑制促炎症因子的释放、抑制NF-κB炎症通路的活化来调控,因此,本研究进一步提出“铁皮石斛可通过抑制mTOR信号通路过度活化,参与调控线粒体介导的细胞凋亡通路,改善生殖衰老,从而影响多种老年疾病的相关进程,发挥“补肾、延年益寿”的作用。目的：基于铁皮石斛“补肾、延年益寿”的功效及结合“肾主骨生髓、主藏精、主生殖”的中医理论,本研究通过构建去势致骨质疏松小鼠模型、D-半乳糖致亚急性衰老小鼠及自然衰老小鼠模型,分别探讨铁皮石斛“壮筋骨、益智、强阴益精、补五脏轻身延年”等功效。方法：一、从“肾主骨”的角度探讨铁皮石斛“补肾益力、壮筋骨”的功效昆明小鼠,雌性,1月龄。进行去势手术,术后1个月将造模组小鼠随机分为：去势模型组、雌二醇组(0.17mg/kg)、铁皮石斛榨汁组(2 g生药(鲜品)/kg,含多糖约130mg/kg)、铁皮石斛多糖组(140mg/kg,含多糖约130mg/kg),另取同批空白小鼠作为假手术组,连续给药1个月。检测指标包括：(1)血清生化指标：钙(Ca)、磷(P)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)和丙二醛(MDA)含量；(2)组织病理检查：子宫组织；(3)骨组织形态学观察：骨组织计量学及骨密度考察。二、从“肾生髓”的角度探讨铁皮石斛“益智”的功效昆明小鼠,雌性,1月龄。通过皮下注射1g/kg D-半乳糖连续8周建立D-半乳糖致亚急性衰老小鼠模型,造模2周后随机分为D-半乳糖模型组、雌二醇组(0.17mg/kg)、铁皮石斛榨汁组(2 g生药(鲜品)/kg,含多糖约130mg/kg)、铁皮石斛多糖组(140mg/kg,含多糖约130mg/kg)。另取同批空白小鼠作为正常组,连续给药3个月。去势模型采用上一章节中同样造模方法进行模型构建,术后6个月进行分组给药,给药剂量不变,给药周期为13个月。检测指标包括：(1)行为学考察：Morris水迷宫实验；(2)脏器病理检查：脑组织。三、从“肾藏精”探讨铁皮石斛“补五脏、延年益寿”的功效采用第三章第二节D-半乳糖致亚急性雌性衰老模型小鼠；将9月龄衰老动物随机分为：自然衰老模型组、铁皮石斛榨汁组(2 g生药(鲜品)/kg,含多糖约130mg/kg)、铁皮石斛多糖组(140mg/kg,含多糖约130mg/kg),连续给药至小鼠全部死亡；野生型黑腹果蝇,由广州中医药大学中药学院李耿老师馈赠,收集8h内羽化的果蝇随机分为空白对照组、3%红景天组、雌二醇组、1%铁皮石斛榨汁组、3%铁皮石斛榨汁组、9%铁皮石斛榨汁组、0.1%铁皮石斛多糖组、0.3%铁皮石斛多糖组、0.9%铁皮石斛多糖组。将药液配于基础培养基中,每5天更换培养基,连续培养至果蝇全部死亡。检测指标包括：(1)D-半乳糖致亚急性衰老小鼠：体温变化,脏器病理检查：心、肝、脾、胸腺、肺、肾、肾上腺,重要脏器线粒体膜电位检测：脑、脾、胸腺、肾上腺；(2)自然衰老小鼠：体重、体温变化,寿命观察；(3)黑腹果蝇：寿命观察。四、从“肾主生殖”的角度初步探讨铁皮石斛“补肾、强阴益精”功效的作用机制昆明小鼠,雌性,1月龄。D-半乳糖致亚急性衰老模型采用第三章第二节中同样造模方法进行模型构建,造模两周后随机分为：D-半乳糖模型组、雌二醇组(0.17 mg/kg)、铁皮石斛榨汁组(2g生药(鲜品)/kg,含多糖约130mg/kg)、铁皮石斛多糖组(140mg/kg,含多糖约130mg/kg),另取同批空白小鼠作为正常组,连续给药4个月。检测指标包括：(1)脏器病理检查：子宫及卵巢组织；(2)子宫及卵巢线粒体功能检测：ATP含量、线粒体膜电位分析及线粒体介导凋亡相关基因及线粒体融合蛋白-2基因表达检测；(3)子宫及卵巢组织mTOR通路相关基因表达检测。结果：一、从“肾主骨”的角度探讨铁皮石斛“补肾益力、壮筋骨”的功效铁皮石斛能提高血清磷、钙、碱性磷酸酶含量,对钙磷平衡具有一定的调节作用,并能降低MDA含量,提高SOD活性,对子宫组织、骨小梁骨皮质变薄、形态结构性差等病变程度也具有改善作用,榨汁液更能显著升高子宫系数,增加骨小梁面积百分比、骨小梁数目以及降低骨小梁分离度,多糖亦能不同程度地改善上述骨小梁形态变化,提示铁皮石斛能促进骨形成,可能通过增强机体的抗氧化能力,有效改善氧自由基代谢异常,从而起到抗骨质疏松作用。二、从“肾生髓”的角度探讨铁皮石斛“益智”的功效D-半乳糖造模8周后,小鼠血清SOD活性明显下降,MDA含量明显升高,表明机体造成氧化损伤,模型建立成功。去势老年小鼠与青年组比较,学习能力明显低下,与去势青年小鼠比较差异不明显,提示经过去势手术后会对定位搜索及空间搜索学习能力均有一定的影响,进一步说明雌激素缺乏可以导致学习记忆功能减退、加重脑衰老。在D-半乳糖模型中,与模型组相比,榨汁组潜伏期和搜索距离均明显缩短,且空间探索实验中的目标象限停留时间比及游泳距离比均显著增加,提示铁皮石斛对D-半乳糖模型定位航行的学习能力有一定增强作用,对空间探索中的记忆巩固能力作用更为明显；在去势模型中,铁皮石斛榨汁液能缩短去势初老小鼠搜索距离,对去势初老模型定位航行中的学习能力有一定作用,对去势老年模型的学习记忆能力改善不明显。提示铁皮石斛对氧化损伤及激素缺失造成的脑记忆功能障碍有一定改善作用,可能通过清除自由基,改善氧化损伤造成的脑组织神经元细胞数量减少及神经元纤维缠结现象,促进受损神经元的恢复,提高脑神经元的细胞活力,改善脑组织和延缓脑的老化,从而激发和提高脑的智能,使动物的学习能力提高。但对激素缺失的老年机体作用不明显,提示在激素缺乏及年龄增长双重刺激下所导致的脑记忆功能障碍程度较严重,铁皮石斛对此改善作用不明显。三、从“肾藏精”探讨铁皮石斛“补五脏、延年益寿”的功效铁皮石斛对D-半乳糖致亚急性衰老小鼠局部温度影响并不明显,而多糖则对自然衰老小鼠的肩胛部、背部及腹部温度升高较明显,表明铁皮石斛多糖对老年基础代谢生命活动有一定的增强作用。铁皮石斛对D-半乳糖致亚急性衰老雌鼠中各重要脏器病变均有不同程度的改善作用,对脑、脾脏及肾上腺组织ATP含量有升高的作用,并能明显升高脑及脾脏组织线粒体膜电位,改善去极化现象,对脏器的凋亡坏死有一定的保护作用。在自然衰老小鼠模型及黑腹果蝇模型中,铁皮石斛平均寿命及半数死亡时间均有一定的延长；且以榨汁组在自然衰老小鼠模型中延长最为明显。表明铁皮石斛具有一定补五脏功效及延长寿命的作用。四、从“肾主生殖”的角度初步探讨铁皮石斛“补肾、强阴益精”功效的作用机制铁皮石斛可升高子宫系数,改善子宫萎缩现象；脏器病理检查分析表明能改善子宫、卵巢等病理变化。铁皮石斛可升高雌性衰老小鼠子宫及卵巢组织ATP含量,通过下调卵巢组织Mfn2、上调子宫及卵巢组织Bcl-2、下调子宫组织Bax基因表达水平,增加Bcl-2/Bax的表达比率,保护线粒体膜并升高线粒体膜电位,缓解D-半乳糖对子宫及卵巢组织线粒体的损伤,抑制子宫及卵巢组织的凋亡从而延缓衰老。铁皮石斛对卵巢组织mTOR基因表达水平也具有下调作用,但目前对mTOR信号通路的了解不十分完全且细胞凋亡机制尚未研究明确,因此铁皮石斛是否通过抑制mTOR信号通路发挥抗凋亡保护作用仍需进一步的实验研究。结论：本研究以铁皮石斛为研究对象,采用去势致骨质疏松小鼠模型、D-半乳糖致亚急性衰老小鼠模型、自然衰老小鼠模型及黑腹果蝇模型,从“肾主藏精、主骨、生髓、主生殖”多角度探讨铁皮石斛“强阴益精、壮筋骨、益智、补五脏、轻身延年”等功效,发现铁皮石斛可能通过补益肾脏系统,改善生殖功能的衰退,而使精力增强,强健筋骨,增强人的智力清醒头脑,延缓骨质疏松症、老年性痴呆和更年期综合征等多种老年疾病的相关进程,从而延长一定的生存时间来达到抗衰老的目的,其通过调控mTOR信号通路及线粒体介导的细胞凋亡延缓生殖器官衰老的作用可能是其对机体整体发挥抗衰老的作用基础之一。本研究为铁皮石斛“补肾、延年益寿”的功效提供科学性依据并为其在老年保健品中的开发应用阐明价值。
[Abstract]:Aging is a natural law that is inevitable and complex in life . As the age increases , the body ' s metabolism decreases and the functions of organs gradually fade , and the appearance of the body and the signs of visceral aging become more obvious . Traditional Chinese medicine has a profound effect on aging theory and delaying aging , and it is pointed out that the body ' s " life , long , strong , old and old " is the main cause of aging .
According to the invention , the dendrobium stem is used as a kidney medicine , and is a lung medicine , and is used as a stomach - stomach medicine ;
The fresh or dried stem of Dendrobium nobile is used as medicine for nourishing yin , clearing away heat , invigorating kidney and replenishing essence , and is mainly used for treating weakness of waist and foot , deficiency of bone , tonifying kidney and essence , nourishing kidney and qi , and tonifying kidney .
Objective : To evaluate the effect of Dendrobium officinale " on strengthening yin and replenishing essence , invigorating kidney and tonifying body , strengthening muscles and bones , benefiting intelligence , relieving convulsion , tonifying five viscera , and losing weight , and prolonging life ;
Objective : To study the effect of Dendrobium officinale on aging in mice by inhibiting the activation of NF - 魏B and pro - inflammatory factors . The serum biochemical indexes were as follows : ( 1 ) serum biochemical indexes : calcium ( Ca ) , phosphorus ( P ) , alkaline phosphatase ( ALP ) , superoxide dismutase ( SOD ) and malondialdehyde ( MDA ) ;
( 2 ) histopathological examination : uterine tissue ;
( 3 ) Bone histomorphology observation : Bone histometrology and bone mineral density were investigated . The effect of Dendrobium officinale on " intelligence " of Dendrobium officinale was investigated from the angle of " kidney marrow " . The model of D - galactose - induced subacute aging mice was established by subcutaneous injection of 1 g / kg D - galactose for 8 weeks . The same batch of blank mice were taken as the normal group and the drug was administered for 3 months . The castration model was constructed by the same method as in the previous chapter . After 6 months of operation , the dose of the drug was unchanged . The administration period was 13 months . The detection index included : ( 1 ) Behavior study : Morris water maze test ;
( 2 ) pathological examination of organs : brain tissue . 3 . The effect of " tonifying the five viscera and prolonging the life " of Dendrobium officinale from " kidney essence " was used to study the efficacy of the second section of D - galactose - induced subacute female aging model mice ;
Nine - month - old aging animals were randomly divided into : natural aging model group , Dendrobium officinale juice group ( 2 g crude drug ( fresh ) / kg , polysaccharide ( about 130mg / kg ) , Dendrobium officinale polysaccharide group ( 140mg / kg , polysaccharide containing about 130mg / kg ) , continuous administration to all mice dead ;
The wild type black flies were donated by the Chinese Academy of Traditional Chinese Medicine ( TCM ) of Guangzhou University of Traditional Chinese Medicine ( Chinese Academy of Traditional Chinese Medicine ) . The flies were randomly divided into blank control group , 3 % salidrotic group , estradiol group , 1 % Dendrobium nobile juice group , 3 % dendrobium stem juice group , 9 % dendrobium stem juice group , 0.1 % Dendrobium officinale polysaccharide group , 0.3 % Dendrobium officinale polysaccharide group and 0.9 % Dendrobium officinale polysaccharide group .
( 2 ) natural aging mice : body weight , body temperature change and life observation ;
( 3 ) The effect mechanism of Dendrobium officinale " kidney tonifying , strengthening yin and replenishing essence " was preliminarily discussed from the angle of " kidney main reproduction " . The model was constructed by the same molding method as in section II of chapter III . The model was divided into two groups : D - galactose model group , estradiol group ( 0.17 mg / kg ) , Dendrobium officinale polysaccharide group ( 140mg / kg , polysaccharide about 130mg / kg ) , Dendrobium officinale polysaccharide group ( 140mg / kg , polysaccharide about 130mg / kg ) , and the same batch of blank mice as normal group .
( 2 ) Detection of mitochondrial function in uterus and ovary : ATP content , mitochondrial membrane potential analysis and mitochondria - mediated apoptosis - related genes and mitochondrial fusion protein - 2 gene expression detection ;
The results showed that Dendrobium nobile could improve the content of serum phosphorus , calcium and alkaline phosphatase and improve the content of serum phosphorus , calcium and alkaline phosphatase .
The effect of Dendrobium officinale on brain memory dysfunction caused by oxidative damage and hormone deficiency is not obvious . It is suggested that Dendrobium officinale has some effects on brain memory dysfunction caused by oxidative damage and hormone deficiency .
The results show that Dendrobium officinale has the functions of tonifying kidney and strengthening yin and replenishing essence , and can raise the uterine factor and improve the uterine contraction .
Objective To study the effect of D - galactose on the expression of Mfn2 in uterus and ovary , and to decrease the expression of Bcl - 2 / Bax .

【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R285.5;R-332

【相似文献】