通络干预提高机体缺氧自适应调节能力作用研究
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摘要
目的:自适应是生物体作为复杂系统在长期进化过程中适应环境赖以生存延续的基本能力。低氧或缺氧不仅是缺血性心、脑血管病等常见病的基本病理过程,同时随着西部大开发战略的实施更多的内陆平原地区人群进入高原地区,高原低氧导致的急性高原反应(AMS)、高原肺水肿等疾病严重危害机体健康,机体缺氧自适应调节能力是提高缺氧耐受性的关键机制,因此提高机体缺氧自适应调节能力研究成为寻找抗缺氧有效干预策略和药物的重大课题。本研究在中医脉络学说“承制调平”理论指导下,在既往络病理论代表性药物通心络(以下简称TXL)治疗缺血性心脑血管病取得确切疗效基础上,进一步探讨对提高机体缺氧耐受性的影响及相关机制:观察TXL对平原地区赴藏旅游人群缺氧耐受性的影响;建立急性缺氧兔模型,研究TXL对模型兔缺氧耐受性的影响及其作用机制;建立急性缺氧预适应小鼠模型,观察TXL对进一步提高缺氧自适应调节能力的作用并探讨其机制;研究PI3K/Akt/HIF-1α信号通路在TXL提高人脐静脉内皮细胞(HUVEC)缺氧耐受性中的关键作用。
方法:本研究分为以下四部分内容
1通络干预提高平原地区赴藏旅游人群缺氧耐受性随机、双盲、安慰剂对照研究
以赴藏旅游人群为由平原进入高原地区缺氧人群的代表,采用随机、双盲、安慰剂对照研究,选择纳入复合标准的志愿者120人按1:1随机被分到安慰剂组或TXL组,按随机号进行分组及药物发放,组织进行为期10天的赴藏旅游,旅游3天前开始服用药物,按不同的观测地点填写AMS调查问卷,监测脉搏血氧饱和度,进行动脉血气分析。试验完成后,将数据录入到临床试验远程数据管理系统并校对,确定数据库后,进行首次揭盲,统计分析得出结果,再进行第二次揭盲。
2通络干预对急性缺氧兔缺氧耐受性的影响
将24只新西兰大耳白家兔随机分为对照组、缺氧组和TXL组,先在11.4%氧环境下低氧实验,分别在缺氧前,缺氧5min,30min,60min时进行动脉血气分析,60min后进行密闭缺氧,观察兔密闭缺氧耐受时间,按时间点检测兔动脉血氧分压和血氧饱和度,ELISA法检测血清缺氧诱导因子1-α(HIF-1α)的含量、Western blot法检测主动脉组织HIF-1α和血管内皮生长因子(VEGF)的蛋白表达。
3通络干预对缺氧预适应小鼠缺氧耐受性的作用及机制研究
3.1雄性昆明小鼠随机分为缺氧预适应组(对照组)与TXL组,TXL组按1.52g生药/Kg/d,灌胃给药,两组动物分别进行0次(H0)、1次(H1)、3次(H3)、5次(H5)重复缺氧暴露,小鼠置于低氧密闭瓶中,通过小鼠呼吸消耗瓶内氧造成瓶内低氧,以小鼠出现喘呼吸为低氧耐受极限,然后将小鼠转到另一低氧密闭瓶中,依此类推,连续进行5次低氧。记录它们的缺氧耐受时间,利用Western blot方法检测脑组织HIF-1α、VEGF、Bcl-2、Bax蛋白的表达。
3.2 40只雄性昆明小鼠随机分为正常对照组、低氧组、低氧预适应组和TXL组(低氧预适应+TXL),每组10只,利用重复低氧5次复制低氧预适应小鼠模型后,进行缺氧实验,记录小鼠每次低氧耐受的时间,观察小鼠大脑皮层神经元和血管内皮细胞超微结构的改变。
4 PI3K/Akt/HIF-1α信号通路在TXL提高HUVEC缺氧耐受性中的关键作用
4.1将体外培养的HUVEC分为常氧对照组、TXL组、缺氧组、缺氧+TXL(100μg/ml)组共4组。对照组和TXL组细胞置于常氧条件下:5%CO2+95%空气;缺氧组、缺氧+通心络组细胞置于低氧条件下: 1%O2+5%CO2+94%N2。分别在缺氧12h、24h、48h,利用细胞计数试剂盒-8(CCK-8)检测细胞的增殖率,流式细胞仪检测细胞凋亡率。4.2将体外培养的HUVEC分为对照质粒(GFP)组、DN-HIF组、△p85组、DN-Akt组,每组又分为对照组与TXL组,利用HIF-1α显性负性突变体DN-HIF、PI3K显性负性突变体p85、Akt显性负性突变体DN-Akt瞬时转染相应的HUVEC细胞后,将细胞置入1%O2+5% CO2+94%N2的低氧条件下,分别在缺氧12h、24h、48h,利用CCK-8试剂盒检测细胞的增殖率和存活率,流式细胞仪检测细胞凋亡率。在缺氧4h、12h、24h、36h,利用Western blot方法检测HIF-1α蛋白表达变化及Akt磷酸化水平。
结果:
1通络干预提高平原地区赴藏旅游人群缺氧耐受性随机、双盲、安慰剂对照研究
1.1 TXL对赴藏旅游人群AMS发生率的影响随海拔高度的升高,两组AMS发生率均逐渐增加,后随缺氧时间的延长,两组AMS逐渐降低,表明机体具有缺氧自适应调节能力。与对照组比较,TXL组旅游人群在昆仑山口(观察4)、初到拉萨(观察6)、日喀则(观察10)三地AMS发生率均明显降低(P<0.05),其他观察点TXL有降低趋势,自西宁至拉萨赴藏旅途中总AMS发生率明显低于对照组(P<0.05),往返旅游全程中AMS总发生率亦明显降低(P<0.05),表明TXL提高了平原地区进入高原缺氧环境人群的缺氧耐受性。
1.2 TXL对赴藏旅游人群动脉血氧分压及血氧饱和度的影响与始发地石家庄(观察1)比较,两组人群在拉萨(观察6)动脉血氧分压及血氧饱和度均明显降低(P<0.01),此后随缺氧时间的延长,与观察6比较,离开拉萨前一天(观察14)两组人群动脉血氧分压及血氧饱和度均明显升高(P<0.01),显示了机体具有缺氧自适应调节能力。在拉萨观察6和观察14,TXL组较对照组动脉血氧分压及血氧饱和度均明显升高(P<0.01),表明TXL明显提高了缺氧环境中的机体缺氧耐受能力。
1.3 TXL对赴藏旅游人群脉搏血氧饱和度的影响与低海拔地区比较,随海拔高度的升高,两组人群脉搏血氧饱和度明显降低(P<0.01);随缺氧时间的延长,对照组与TXL组两组人群脉搏血氧饱和度明显升高(P<0.01)。与对照组比较,TXL组脉搏血氧饱和度明显升高(P<0.05或P<0.01)。
2通络干预对急性缺氧兔缺氧耐受性的影响
2.1缺氧耐受时间的比较
从密闭缺氧到死亡,缺氧组家兔密闭缺氧耐受时间为(14.33±5.92)min,TXL组缺氧耐受时间为(22.63±4.75)min,在同一缺氧环境中延长生命达8min,两组具有显著统计学差异(P<0.05)。
2.2血氧分压和血氧饱和度的变化比较
缺氧前缺氧组与TXL组血氧分压和血氧饱和度无明显差异(P>0.05)。与缺氧前比较,缺氧组与TXL组兔在浓度为11.4%的低氧环境5min、30min、60min时,动脉血氧分压及血氧饱和度均明显降低(P<0.01)。与缺氧组比较,TXL组动脉血氧分压和血氧饱和度降低幅度明显减小(P<0.05或P<0.01)。而密闭缺氧后,缺氧组与TXL组动脉血氧分压和血氧饱和度均随缺氧时间延长而下降,缺氧末(呼吸停止即刻)TXL组兔血氧分压、血氧饱和度均明显低于缺氧组(P<0.01),显示TXL不仅明显延长了兔缺氧存活时间,同时在更低血氧浓度时才导致死亡,提高了耐低氧存活能力。
2.3血清HIF-1α的变化比较
HIF-1α为耐缺氧关键蛋白,与缺氧前比较,缺氧组与TXL组血清HIF-1α含量均明显增加(P<0.05或P<0.01),显示了实验兔具有缺氧自适应调节能力;与缺氧组比较,TXL组血清HIF-1α含量增加尤为显著(P<0.05或P<0.01),表明TXL通过增强HIF-1α蛋白表达而明显提高了耐缺氧能力。
2.4主动脉组织HIF-1α、VEGF的变化比较
与对照组比较,缺氧组和TXL组主动脉组织HIF-1α和VEGF表达明显增强(P<0.05或P<0.01)。而与缺氧组比较,TXL组主动脉组织HIF-1α表达增强,VEGF表达明显增强(P<0.05)。
3通络干预对缺氧预适应小鼠缺氧耐受性作用机制研究
3.1两组小鼠缺氧耐受时间的变化
两组小鼠分别与第1次缺氧比较,随着缺氧次数的增加,缺氧耐受时间均明显增加(P<0.05或P<0.01)。而与对照组比较,TXL组各次的缺氧耐受时间明显增加(P<0.05或P<0.01)。
3.2两组小鼠脑组织HIF-1α、VEGF、Bax、Bcl-2蛋白表达的变化
缺氧前,对照组与TXL组小鼠脑组织有微量HIF-1α、VEGF、Bax表达,两组之间无统计学意义(P>0.05),Bcl-2有少量表达,TXL组表达高于对照组(P<0.05)。
与缺氧前比较,对照组与通心络组均随缺氧次数增加,HIF-1α、VEGF、Bcl-2表达逐渐增强(P<0.05或P<0.01);Bax在缺氧1次表达明显增强(P<0.01),但在缺氧3次和缺氧5次逐渐降低,较缺氧1次组明显减少(P<0.05或P<0.01);而与对照组比较,TXL组各次缺氧HIF-1α、VEGF、Bcl-2表达更加明显(P<0.05或P<0.01),各次缺氧小鼠Bax表达明显减弱(P<0.05或P<0.01)。
3.3两组小鼠脑组织HIF-1α表达(免疫组化)
缺氧前,两组小鼠大脑皮层神经元及神经胶质细胞无明显HIF-1α的阳性表达;随缺氧次数的增加,两组小鼠大脑皮层神经元及神经胶质细胞HIF-1α的阳性表达逐渐增强;而与对照组比较,TXL组阳性表达明显增强。
3.4 TXL对缺氧预适应小鼠脑皮层神经元和内皮细胞超微结构的作用
与正常对照组比较,缺氧组小鼠脑皮层内皮细胞和神经元超微结构发生明显改变;与缺氧组比较,缺氧预适应组小鼠脑组织神经元及血管内皮细胞超微结构改善;与缺氧预适应组比较,TXL组脑组织神经元及血管内皮细胞超微结构明显改善。
4 PI3K/Akt/HIF-1α信号通路在TXL提高HUVEC缺氧耐受性中的作用
4.1与正常对照组比较,缺氧组HUVEC增殖率显著降低;与缺氧组比较,经TXL处理后的缺氧+TXL组细胞增殖率明显增高。HUVEC缺氧后细胞凋亡率明显增高,随缺氧时间的延长,细胞凋亡率逐渐增高;与缺氧组比较,经TXL干预的缺氧+TXL组细胞凋亡率明显降低。
4.2与缺氧对照组比较,利用DN-HIF抑制HIF-1α的表达后,缺氧HUVEC的存活率显著下降,细胞凋亡率明显升高,同时TXL提高细胞存活率和降低细胞凋亡率的水平明显受到抑制,表明TXL提高HUVEC耐缺氧存活率和降低凋亡率的作用主要是由HIF-1α介导的。
4.3缺氧条件下,HUVEC细胞HIF-1α蛋白的表达明显上调,且随缺氧时间的延长而增加。与对照组比较,应用TXL干预后,HIF-1α蛋白表达进一步增强。与对照质粒组比较,HUVEC瞬转PI3K的显性负性突变体△p85后,HIF-1α蛋白表达显著下调,同时TXL诱导HIF-1α蛋白表达水平明显被抑制。利用Akt的显性负性突变体DN-Akt瞬转HUVEC,也得出相同的结果。表明PI3K/Akt信号途径介导了HIF-1α蛋白表达。
4.4缺氧条件下,HUVEC随着缺氧时间的延长,P-Akt表达出现明显变化,在缺氧4h增加,8h增加最明显,24h有所减少。与对照组比较,经TXL干预后P-Akt表达明显增加。与对照质粒比较,利用PI3K的显性负性突变体△p85瞬时转染后,P-Akt表达明显减少;同时TXL诱导P-Akt表达水平明显受到抑制。
结论:
1通络干预可有效提高平原赴藏旅游人群和实验动物的缺氧耐受性本研究以平原赴藏旅游人群、急性缺氧兔、缺氧预适应小鼠为研究对象,从临床与基础研究初步证实TXL可明显降低赴藏旅游人群AMS发生率,提高急性缺氧兔密闭缺氧耐受时间,提高缺氧预适应小鼠缺氧耐受时间,表明TXL具有提高缺氧耐受性的作用。
2通络干预通过增强缺氧自适应调节能力提高缺氧耐受性自适应是生物体作为复杂系统在长期进化过程中形成的适应环境延续生存的基本能力,实验动物与平原赴藏旅游人群研究均显示,随缺氧时间延长,生命体可产生耐缺氧自适应调节能力,使缺氧损害明显降低,赴藏旅游人群随海拔高度增加AMS发生率逐渐增高,而随缺氧时间的延长AMS发生率逐渐降低,动脉血氧分压、动脉血氧饱和度、脉搏血氧饱和度明显升高;缺氧预适应小鼠随缺氧次数的增加,缺氧耐受时间逐渐延长。通络干预可明显调动缺氧自适应调节能力,提高赴藏旅游人群动脉血氧分压、动脉血氧饱和度、脉搏血氧饱和度,增加缺氧预适应动物缺氧耐受时间,延长实验动物密闭缺氧到死亡的存活时间,提高了耐低氧存活能力。
3促进HIF-1α蛋白表达是TXL提高缺氧自适应调节能力的核心作用机制,PI3K/Akt/HIF-1α信号通路为其主要途径
对TXL提高缺氧耐受力及动物耐低氧存活能力的作用机制的深入研究证实:TXL可明显促进实验兔血清、主动脉和缺氧预适应小鼠脑组织HIF-1α表达,离体HUVEC实验中利用DN-HIF抑制HIF-1α的表达后,缺氧HUVEC的存活率显著下降,细胞凋亡率明显升高,同时TXL提高细胞存活率和降低细胞凋亡率的作用明显受到抑制,表明TXL提高缺氧耐受力的作用是由HIF-1α介导的。进一步实验证实,PI3K/Akt/HIF-1α信号通路为其主要作用途径。
4“承制调平”对揭示缺氧条件下机体代偿性调节及治疗转归的内在规律指导临床防治具有重要理论指导意义
“承制调平”基于中医阴阳五行学说概括出的脉络学说核心理论,在不同层次上反映了中医学的生理观、病理观、治疗观及预后观,“承”揭示了人体不断与自然界进行着物质交换与信息交流,以维持机体内外环境的和谐平衡,氧供需平衡的自稳调控机制即属“承”的范畴;“制”系指机体对病理损害的代偿性自我调节机制,临床与实验研究显示人体与动物均可在缺氧环境下提高自适应调节能力;由“调”致“平”的通络干预,强调提高机体自适应调节重新恢复自稳平衡态,实验证实通络干预可明显促进耐缺氧关键蛋白HIF-1α与VEGF以及抗凋亡因子Bcl-2的表达,抑制促凋亡因子Bax的表达,重建低氧环境下机体耐缺氧损伤的相对平衡态,其关键机制为提高机体缺氧自适应调节能力,这对缺氧疾病治疗具有重要的临床应用价值。
Objective: Self-adaption is a basic skill of survival, which continued the organism as a complex system acclimatizes in the long process of evolution. Hypoxia is the basic pathological process of ischemic heart, cerebrovascular disease. And with the implementation of the western development strategy, more people enter into the highland from inland areas. Plateau hypoxia dues to acute mountain sickness (AMS), high altitude pulmonary edema. These conditions seriously harm to health of the body. Regulation of hypoxia self-adaptive capacity is the key mechanisms of improving hypoxic tolerance; thereby, the search of increasing hypoxia self-adaptive regulative capacity is a question for discussion of finding effective intervention strategies and drugs. Under the guidance of Chinese medicinal theory of“Cheng-zhi-tiao-ping”, which the core content of Vessels-Collateral Theory, on the basis of Tongxinluo (referred to as TXL, that is the representational drugs of the traditional Chinese medical Theory of Collateral Disease) has achieved exact effect on treatment of ischemic cardio-cerebrovascular disease in the past studies, further to study the effect and mechanism of TXL on improving hypoxic tolerance of a body: to observe the effect of TXL on hypoxic tolerance of the crowd who are from plateau to travel to Tibet plateau with hypobaric hypoxia. To establish acute hypoxia rabbit model and explore the effect and mechanism of TXL on hypoxic tolerance. To establish acute hypoxia preconditioning mouse model, to explore the mechanism of TXL further improving the capacity of hypoxia self-adaptive. To study the key role of PI3K/Akt/HIF-1αsignal pathway in TXL enhances HUVEC hypoxic tolerance.
Methods:
1 A clinical trial study on hypoxic tolerance of crowd in plain area travel to Tibet with dredge collateral intervention randomized, double-blind, placebo -controlled
A crowd who lived plain and had never to plateau travelled to Tibet plateau was the representative of the hypoxia crowd. One hundred and twenty healthy volunteers who were accord with standard were randomly divided into two groups, control and TXL group. Double blind trial was applied in this study. The subjects in the two groups took placebo or TXL capsules respectively for 3 days before going to Tibet, and continued to take for 10 days until the end of the study. On the every observation place, the score of AMS symptoms of volunteers was followed up and recorded by the Lake Louise self-report questionnaire. The heart rate (beats/min) and pulse oxygen saturation (%) were recorded. Arterial blood gas analysis was detected. After the clinical trial, the data was entered in the clinical trial remote data management system and proofed. As soon as the data bank was locked, the first blind was exposed and to analysis,and then second unblinding. 2 Effects of dredge collateral intervention on hypoxia tolerance of acute hypoxic rabbit
Twenty-four healthy flap-eared white rabbits were randomly divided into 3 groups:Control group, hypoxia group and TXL group. The rabbits were in low oxygen condition of 11.4% O2 at first. After 60 minutes, air supply was closed. The level of PO2 and SaO2 of arterial blood were detected. The hypoxia tolerance time of the rabbit was measured. The content of serum hypoxia inducible factor 1alpha (HIF-1α) was detected with ELISA. The expression of HIF-1αand vascular endothelial growth factor (VEGF) in the aorta of rabbit were detected with Western blot.
3 Effects and mechanism of dredge collateral intervention on hypoxic tolerance of hypoxic preconditioning mice
3.1 Mice were randomly divided into groups of hypoxic preconditioning (Control) and TXL group. The mice in TXL group were administered at 1.52g crude drug/kg/d for 5 days. The mice were exposure to acute repetitive hypoxia for 0 run (H0), 1 run (H1), 3 runs (H3) and 5 runs (H5). Mouse was placed in an air-sealed jar and hypoxic environment in the jar which was established through consumption of the oxygen by respiration of the mouse. A gasp breath was regarded as the hypoxic tolerant limit of the mice. And then the mice were transferred to a new jar. The mice were exposed to hypoxia in this way for 5 times. In each run of hypoxic exposure, the hypoxic tolerance time was recorded. The Western blot method was used to measure the expression of hypoxia inducible factor-1α(HIF-1α), vascular endothelial growth factor (VEGF), Bcl-associated X (Bax) and B-cell leukemia/ lymophoma 2 (Bcl-2) in the tissue of cortex.
3.2 Mice were randomly divided into 4 groups: control group, hypoxia group, hypoxia preconditioning (HP) group and TXL group. The hypoxia preconditioning mice were exposure by repetitive hypoxia 5 runs. The animal’s tolerance time of each hypoxia run was recorded. The ultrastructure changes of cerebral neuron and endothelial cell were studied by electron microscope.
4 The role of PI3K/Akt/HIF-1 signal pathway in which TXL enhances the HUVEC hypoxia tolerance.
4.1 Cultured HUVECs were divided into four groups as follows: normoxic control group, TXL group (100μg/ml), hypoxia group, hypoxia+ TXL group (100μg/ml). The HUVEC of control group and TXL group were placed in normal condition: 5% CO2+95% air. The cells of hypoxia group and hypoxia +TXL group were placed in hypoxia condition: 1%O2+5%CO2 + 94% N2. At hypoxia 12h, 24h, 48h, the cell proliferation rate were detected by using Cell Counting Kit -8 (CCK-8) test kit, apoptosis rate were detected with flow cytometry respectively.
4.2 Cultured HUVECs were divided into control GFP, the dominant negative mutant of HIF-1(DN-HIF) group, the dominant negative mutant of PI3K/Akt(△p85) group and the negative mutant of Akt(DN-Akt) group. Every group was divided into control and TXL groups. After the HUVECs were transiently transfected with 5μg of GFP control vector, DN-HIF,△p85 and DN-Akt respectively, they were placed in hypoxia condition: 1%O2+5% CO2 +94% N2. At hypoxia 12h, 24h, 48h, the cell proliferation rate and reveal survival rate were detected by using Cell Counting Kit -8 (CCK-8) test kit, apoptosis rate were detected by using flow cytometry respectively. At hypoxia 4h, 24h, 36h, 48h, the expression of HIF-1αand p-Akt were detected by western blot respectively.
Results:
1 A clinical trial study on hypoxic tolerance of crowd in plain area travelled to Tibet with dredge collateral intervention randomized, double-blind, placebo-controlled
1.1 Effect of the TXL on AMS incidence of the cowed travelled to Tibet
With the increasing altitude, AMS incidence in two groups increased gradually. With the hypoxic time prolonged, the AMS incidence decreased gradually. This indicated that organism has hypoxia self-adaptive regulative capacity. Compared with the control group, at Kunlun Mountain (observation 4), Lhasa (observation 6) and Rikaze (observation 10), the AMS incidence in the TXL group reduced (P <0.05), TXL had a downtrend at the other observation. The total AMS incidence on the way to Tibet by train or during the whole journey decreased greatly (P <0.05). These indicated that the TXL increased the hypoxic tolerance of the crowd from plains to Tibet hypoxic environment.
1.2 Effects of the TXL on PO2 and SaO2 in arterial blood gas analysis of the crowed travelled to Tibet
Compared with Shijiazhuang (observation 1), the PO2 and SaO2 of the two groups decreased significantly in Lhasa (observation 6) (P <0.01). With the prolonged of hypoxia, the PO2 and SaO2 of Lhasa (observation 14, the day before leaving Lhasa) were higher than those of the observation 6 (P <0.01). This indicated that organism has hypoxia self-adaptive regulative capacity. In Lhasa (observation 6 and observation 14), compared with the control group, the PO2 and SaO2 of TXL group was significant higher (P <0.01). These indicated that the TXL increased the hypoxic tolerance capacity of organism in hypoxic environment.
1.3 Effect of the TXL on pulse SaO2 of the crowed travelled to Tibet
With the increasing of altitude, the pulse SaO2 of the two groups decreased gradually (P <0.01). With the prolonged of hypoxic time, the pulse SaO2 of the two groups increased gradually (P <0.01). Compared with the control group, the pulse SaO2 of the TXL groups increased significantly (P <0.01).
2 Effects of dredge collateral intervention on hypoxic tolerance of acute hypoxic rabbit
2.1 Comparison of hypoxia tolerance time
From the closed air supply to death, the hypoxic tolerance time of hypoxia group was (14.33±5.92) min, while time of the TXL group was (22.63±4.75) min. The TXL prolonged the survival time for average 8 minutes in the same hypoxic environment. There was a marked statistical difference between two groups (P <0.05).
2.2 Comparison of the PO2 and SaO2 of arterial blood
The PO2 and SaO2 in arterial blood of both hypoxia and TXL groups were not significant difference before hypoxia (P >0.05). Compared with before hypoxia, the PO2 and SaO2 in arterial blood in 11.4% oxygen environment of hypoxia and TXL groups decreased significantly at the time of hypoxia 5min, 30min, 60min (P <0.05 or P <0.01). Compared with the hypoxia group, the range that the levels of PO2 and SaO2 decreased, which reduced greatly (P <0.05 or P <0.01). After the air supply closed, the PO2 and SaO2 of arterial blood reduced further. Compared with the time of hypoxia 60min, at the end of hypoxia (breathing stops instantly), the PO2 and SaO2 in arterial blood decreased significantly (P <0.05 or P <0.01). The PO2 and SaO2 of rabbits in the TXL group was lower than those of in the hypoxia group (P <0.01). These indicated that the TXL prolonged the survival time of rabbit hypoxic, while tolerance with lower PO2 and SaO2, and improved the survival capacity of organism to tolerance hypoxia.
2.3 Comparison of the expression of serum HIF-1α
HIF-1αis a key protein of tolerance with hypoxia. Compared with before hypoxia, the serum HIF-1αof the hypoxia and TXL groups increased significantly (P<0.05 or P<0.01). This indicated that experiment rabbit has hypoxic self-adaptive regulative capacity. Compared with the hypoxia group, the serum HIF-1αincreased significantly in the TXL group (P <0.05 or P <0.01). These indicated that the TXL obviously improved hypoxic tolerance capacity through enhancing the expression of HIF-1αprotein. 2.4Comparison of the expression of HIF-1αand VEGF in the aorta
Compared with the control group, the expression of HIF-1αand VEGF in the aorta of the hypoxia and TXL groups increased obviously (P <0.05 or P <0.01). Compared with the hypoxia group, the expression of HIF-1αand VEGF in the TXL group increased significantly (P <0.05).
3 Effects and mechanism of dredge collateral intervention on hypoxia tolerance of hypoxic preconditioning mice 3.1Comparsion of hypoxic tolerance time
Compared with the H1 (hypoxia exposed one time) in the same group, the hypoxic tolerance time in control and the TXL groups was gradually increased run by run (P <0.01 or P <0.05). Compared with control group, the tolerance time of the TXL group was increased significantly in 1, 3, 5 runs (P <0.05 or P <0.01). 3.2Comparsion of the HIF-1α, VEGF, Bax, Bcl-2 expression in cerebral tissue There were microscale expression of the HIF-1α, VEGF and Bax protein in cerebral tissue in two groups before hypoxia, while no obvious difference between two groups (P >0.05). There were few Bcl-2 expressions in two groups, the Bcl-2 expression in the TXL group was higher than that of in the control group.
Compared with H0 (no hypoxia exposure) in the same group, the HIF-1α, VEGF and Bcl-2 protein expression were increased gradually (P <0.01 or P <0.05). The Bax in control and the TXL groups was significantly increased at first run hypoxia (H1) (P <0.01). After hypoxia 1 run, the Bax expression decreased step by (P <0.01 or P <0.05). Compared with control group, the expression of HIF-1α, VEGF and Bcl-2 in the TXL group increased in 1, 3, 5 runs (P <0.05 or P <0.01), the expression of the Bax was lower than that of in control group at every run (P <0.01 or P <0.05).
3.3 Comparison of the HIF-1αexpression in cerebral tissue in mice with immunohistochemistry
There were no obvious positive expression of HIF-1αin cortical neurons and glial cells in two group’s mice before hypoxia. With the times of hypoxia exposure runs increased, the expression of the HIF-1αincreased gradually. Compared with the control group, the positive expression in the TXL group increased significantly.
3.4 Comparison of the ultrastructure of cerebral neuron and endothelial cell Compared with control group, the ultrastructure of cerebral neuron and endothelial cell in the hypoxia group changed obviously, with mitochondrion and endoplasmic reticulum destroyed. Compared with the hypoxia group, the destroyed degree of cerebral neuron and endothelial cell in the HP group were slighter than that in control group. The change in the TXL group did show obvious improved, comparison with HP group.
4 The role of PI3K/Akt/HIF-1 signal pathway in which the TXL enhances the HUVEC hypoxia tolerance.
4.1 Compared with the control group, HUVEC proliferation rate in the hypoxic group was significantly reduced. Compared with the hypoxia group, the HUVEC proliferation rate of hypoxia plus TXL group significantly increased. After hypoxia, the HUVEC apoptosis rate was significantly increased. With prolonged the hypoxia time, apoptosis rate increased gradually. The HUVECs apoptosis ratio in the hypoxia plus TXL group was great lower than the hypoxia group.
4.2 Compared with the hypoxia group, after inhibited the expression of HIF-1αwith DN-HIF, the survival rate of the HUVEC decreased significantly, and the apoptosis rate increased obviously, while the degree of which the TXL increased the HUVEC survival rate was significantly inhibited. These indicated that the TXL increased survival rate and decreased apoptosis rate of the HUVEC which mediated by HIF-1α. 4.3 In hypoxic condition, the HIF-1αexpression of HUVECs was significantly increased. And with the prolonged of hypoxia time, the expression of HIF-1αincreased obviously. Compared with the control group, the HIF-1αexpression in TXL group further enhanced.
Compared with the control plasmid group, after the HUVECs were transiently transfected with△p85, the HIF-1αprotein expression was significantly reduced, and the degree of HIF-1αexpression that the TXL increased was inhibited. With DN-Akt, the dominant negative mutant of Akt, the consequence was as well as△p85. These indicated that the HIF-1αexpression was mediated by PI3K/Akt singal pathway.
4.4 With the prolonged of hypoxia time, the expression of the p-Akt changed greatly. It was increased at 4h, and was obvious higher at 8h, but it was reduced at 24h. Compared with the control plasmid group, the p-Akt expression in the TXL group was significantly increased.
Compared with the control plasmid group, after the HUVECs were transiently transfected with the△p85, the P-Akt expression was significantly reduced, and the degree of P-Akt expression that TXL increased was inhibited.
Conclusion:
1 Dredge collateral intervention may improve the hypoxia tolerance of crowd which travelled to Tibet and experimental animals With the crowed travelled to Tibet, acute hypoxia rabbits, and the hypoxic preconditioned mice as the research objects, from the initial clinical and basic research, the study initially confirms that the TXL could significantly reduce the AMS incidence in crowed which travelled to Tibet, improve the hypoxic tolerance time in acute hypoxia rabbits and hypoxia preconditioning mice. It indicated that the TXL could improve the hypoxia tolerance.
2 Dredge collateral intervention improved the hypoxic tolerance with enhancing hypoxic self-adaptive regulative capacity
Self-adaption is a basic skill of survival and continued that the organism as a complex system acclimatizes in the long process of evolution. The study of crowed which travelled to Tibet and experimental animals showed that organism could produce self-adaptive regulative capacity to tolerance hypoxia with prolonged the hypoxia time. The capacity could reduce the damage of hypoxia significantly. With the rising of altitude, the AMS incidence of the crowd which travelled to Tibet increased. With the prolonged of the hypoxic time, the AMS incidence decreased, the PO2, SaO2 in arterial blood and pulse SaO2 increased significantly. The hypoxic tolerance time of hypoxic preconditioning mice increased run by run. The TXL intervention could mobilize the hypoxic self-adaptive regulative capacity, increase the PO2, SaO2 in arterial blood, pulse SaO2 and hypoxic tolerance time in hypoxic preconditioning mice, prolong the survival time of experimental animals in closeness hypoxia device, improve the survival ability of hypoxia. 3 Promoting the HIF-1αexpression is the key mechanism of the TXL intervention improving the hypoxic self-adaptive regulative capacity, and the PI3K/Akt/HIF-1αsignal pathway is the major pathway.
The depth study of mechanism that intervention of the TXL in hypoxic tolerance ability and animal’s viability to resist lower hypoxia confirmed: The TXL could promote the serum HIF-1αexpression, aorta of experimental rabbits and cerebral tissue of hypoxic preconditioning mice. After expression of the HIF-1αwas inhibited by DN-HIF, the survival rate of HUVEC was decreased significantly in hypoxic condition and the apoptosis rate of HVEC was increased significantly. But the effect of the TXL on increasing survival rate and decreasing apoptosis rate was significantly inhibited. These indicated that the TXL increases survival rate and decreases apoptosis rate of the HUVEC, which is mediated by HIF-1α. Further experiments confirmed that the PI3K/Akt/HIF-1αis the major signaling pathway.
4 It is important that the theory of“Cheng-zhi-tiao-ping”on revealing the inherent laws of body’s compensatory adjustment in hypoxic conditions, therapy outcome and on directing clinical preventive treatment.
“Cheng-zhi-tiao-ping”is the core of Vessels-Collateral Theory, based on the traditional Chinese medicine theory of yin-yang and five-element. It reflects the physiological, pathology, treatment and prognosis concepts of the traditional Chinese medicine.“Cheng”reveals that human body constantly exchanges substance and information with the nature, in order to maintain a harmonious balance between internal and external environment. The self-stability regulation mechanism of the oxygen supply-demand balance is longs to "Cheng" category. "Zhi" means the body’s self-regulating mechanisms for compensatory pathological damages. Clinical and experimental studies had shown that humans and animals could improve self-adaptive regulative capacity in hypoxic conditions.
Dredge collateral intervention, from“Tiao”to“Ping”, emphasizes that enhancing self-adaptive regulative capacity of organism and to regain homeostasis. Experiments confirmed that dredge collateral intervention can promote the expression of HIF-1α(the key protein of hypoxia), VEGF and Bcl-2 (anti-apoptotic factor), inhabits the expression of Bax (pro-apoptotic factor), rebuild the relative equilibrium state to against hypoxic injury in hypoxic condition. The key mechanism was that dredge collateral intervention can improve hypoxic self-adaptive regulative capacity of organism. This had an important value of clinical application for treatment of hypoxic disease.
方法:本研究分为以下四部分内容
1通络干预提高平原地区赴藏旅游人群缺氧耐受性随机、双盲、安慰剂对照研究
以赴藏旅游人群为由平原进入高原地区缺氧人群的代表,采用随机、双盲、安慰剂对照研究,选择纳入复合标准的志愿者120人按1:1随机被分到安慰剂组或TXL组,按随机号进行分组及药物发放,组织进行为期10天的赴藏旅游,旅游3天前开始服用药物,按不同的观测地点填写AMS调查问卷,监测脉搏血氧饱和度,进行动脉血气分析。试验完成后,将数据录入到临床试验远程数据管理系统并校对,确定数据库后,进行首次揭盲,统计分析得出结果,再进行第二次揭盲。
2通络干预对急性缺氧兔缺氧耐受性的影响
将24只新西兰大耳白家兔随机分为对照组、缺氧组和TXL组,先在11.4%氧环境下低氧实验,分别在缺氧前,缺氧5min,30min,60min时进行动脉血气分析,60min后进行密闭缺氧,观察兔密闭缺氧耐受时间,按时间点检测兔动脉血氧分压和血氧饱和度,ELISA法检测血清缺氧诱导因子1-α(HIF-1α)的含量、Western blot法检测主动脉组织HIF-1α和血管内皮生长因子(VEGF)的蛋白表达。
3通络干预对缺氧预适应小鼠缺氧耐受性的作用及机制研究
3.1雄性昆明小鼠随机分为缺氧预适应组(对照组)与TXL组,TXL组按1.52g生药/Kg/d,灌胃给药,两组动物分别进行0次(H0)、1次(H1)、3次(H3)、5次(H5)重复缺氧暴露,小鼠置于低氧密闭瓶中,通过小鼠呼吸消耗瓶内氧造成瓶内低氧,以小鼠出现喘呼吸为低氧耐受极限,然后将小鼠转到另一低氧密闭瓶中,依此类推,连续进行5次低氧。记录它们的缺氧耐受时间,利用Western blot方法检测脑组织HIF-1α、VEGF、Bcl-2、Bax蛋白的表达。
3.2 40只雄性昆明小鼠随机分为正常对照组、低氧组、低氧预适应组和TXL组(低氧预适应+TXL),每组10只,利用重复低氧5次复制低氧预适应小鼠模型后,进行缺氧实验,记录小鼠每次低氧耐受的时间,观察小鼠大脑皮层神经元和血管内皮细胞超微结构的改变。
4 PI3K/Akt/HIF-1α信号通路在TXL提高HUVEC缺氧耐受性中的关键作用
4.1将体外培养的HUVEC分为常氧对照组、TXL组、缺氧组、缺氧+TXL(100μg/ml)组共4组。对照组和TXL组细胞置于常氧条件下:5%CO2+95%空气;缺氧组、缺氧+通心络组细胞置于低氧条件下: 1%O2+5%CO2+94%N2。分别在缺氧12h、24h、48h,利用细胞计数试剂盒-8(CCK-8)检测细胞的增殖率,流式细胞仪检测细胞凋亡率。4.2将体外培养的HUVEC分为对照质粒(GFP)组、DN-HIF组、△p85组、DN-Akt组,每组又分为对照组与TXL组,利用HIF-1α显性负性突变体DN-HIF、PI3K显性负性突变体p85、Akt显性负性突变体DN-Akt瞬时转染相应的HUVEC细胞后,将细胞置入1%O2+5% CO2+94%N2的低氧条件下,分别在缺氧12h、24h、48h,利用CCK-8试剂盒检测细胞的增殖率和存活率,流式细胞仪检测细胞凋亡率。在缺氧4h、12h、24h、36h,利用Western blot方法检测HIF-1α蛋白表达变化及Akt磷酸化水平。
结果:
1通络干预提高平原地区赴藏旅游人群缺氧耐受性随机、双盲、安慰剂对照研究
1.1 TXL对赴藏旅游人群AMS发生率的影响随海拔高度的升高,两组AMS发生率均逐渐增加,后随缺氧时间的延长,两组AMS逐渐降低,表明机体具有缺氧自适应调节能力。与对照组比较,TXL组旅游人群在昆仑山口(观察4)、初到拉萨(观察6)、日喀则(观察10)三地AMS发生率均明显降低(P<0.05),其他观察点TXL有降低趋势,自西宁至拉萨赴藏旅途中总AMS发生率明显低于对照组(P<0.05),往返旅游全程中AMS总发生率亦明显降低(P<0.05),表明TXL提高了平原地区进入高原缺氧环境人群的缺氧耐受性。
1.2 TXL对赴藏旅游人群动脉血氧分压及血氧饱和度的影响与始发地石家庄(观察1)比较,两组人群在拉萨(观察6)动脉血氧分压及血氧饱和度均明显降低(P<0.01),此后随缺氧时间的延长,与观察6比较,离开拉萨前一天(观察14)两组人群动脉血氧分压及血氧饱和度均明显升高(P<0.01),显示了机体具有缺氧自适应调节能力。在拉萨观察6和观察14,TXL组较对照组动脉血氧分压及血氧饱和度均明显升高(P<0.01),表明TXL明显提高了缺氧环境中的机体缺氧耐受能力。
1.3 TXL对赴藏旅游人群脉搏血氧饱和度的影响与低海拔地区比较,随海拔高度的升高,两组人群脉搏血氧饱和度明显降低(P<0.01);随缺氧时间的延长,对照组与TXL组两组人群脉搏血氧饱和度明显升高(P<0.01)。与对照组比较,TXL组脉搏血氧饱和度明显升高(P<0.05或P<0.01)。
2通络干预对急性缺氧兔缺氧耐受性的影响
2.1缺氧耐受时间的比较
从密闭缺氧到死亡,缺氧组家兔密闭缺氧耐受时间为(14.33±5.92)min,TXL组缺氧耐受时间为(22.63±4.75)min,在同一缺氧环境中延长生命达8min,两组具有显著统计学差异(P<0.05)。
2.2血氧分压和血氧饱和度的变化比较
缺氧前缺氧组与TXL组血氧分压和血氧饱和度无明显差异(P>0.05)。与缺氧前比较,缺氧组与TXL组兔在浓度为11.4%的低氧环境5min、30min、60min时,动脉血氧分压及血氧饱和度均明显降低(P<0.01)。与缺氧组比较,TXL组动脉血氧分压和血氧饱和度降低幅度明显减小(P<0.05或P<0.01)。而密闭缺氧后,缺氧组与TXL组动脉血氧分压和血氧饱和度均随缺氧时间延长而下降,缺氧末(呼吸停止即刻)TXL组兔血氧分压、血氧饱和度均明显低于缺氧组(P<0.01),显示TXL不仅明显延长了兔缺氧存活时间,同时在更低血氧浓度时才导致死亡,提高了耐低氧存活能力。
2.3血清HIF-1α的变化比较
HIF-1α为耐缺氧关键蛋白,与缺氧前比较,缺氧组与TXL组血清HIF-1α含量均明显增加(P<0.05或P<0.01),显示了实验兔具有缺氧自适应调节能力;与缺氧组比较,TXL组血清HIF-1α含量增加尤为显著(P<0.05或P<0.01),表明TXL通过增强HIF-1α蛋白表达而明显提高了耐缺氧能力。
2.4主动脉组织HIF-1α、VEGF的变化比较
与对照组比较,缺氧组和TXL组主动脉组织HIF-1α和VEGF表达明显增强(P<0.05或P<0.01)。而与缺氧组比较,TXL组主动脉组织HIF-1α表达增强,VEGF表达明显增强(P<0.05)。
3通络干预对缺氧预适应小鼠缺氧耐受性作用机制研究
3.1两组小鼠缺氧耐受时间的变化
两组小鼠分别与第1次缺氧比较,随着缺氧次数的增加,缺氧耐受时间均明显增加(P<0.05或P<0.01)。而与对照组比较,TXL组各次的缺氧耐受时间明显增加(P<0.05或P<0.01)。
3.2两组小鼠脑组织HIF-1α、VEGF、Bax、Bcl-2蛋白表达的变化
缺氧前,对照组与TXL组小鼠脑组织有微量HIF-1α、VEGF、Bax表达,两组之间无统计学意义(P>0.05),Bcl-2有少量表达,TXL组表达高于对照组(P<0.05)。
与缺氧前比较,对照组与通心络组均随缺氧次数增加,HIF-1α、VEGF、Bcl-2表达逐渐增强(P<0.05或P<0.01);Bax在缺氧1次表达明显增强(P<0.01),但在缺氧3次和缺氧5次逐渐降低,较缺氧1次组明显减少(P<0.05或P<0.01);而与对照组比较,TXL组各次缺氧HIF-1α、VEGF、Bcl-2表达更加明显(P<0.05或P<0.01),各次缺氧小鼠Bax表达明显减弱(P<0.05或P<0.01)。
3.3两组小鼠脑组织HIF-1α表达(免疫组化)
缺氧前,两组小鼠大脑皮层神经元及神经胶质细胞无明显HIF-1α的阳性表达;随缺氧次数的增加,两组小鼠大脑皮层神经元及神经胶质细胞HIF-1α的阳性表达逐渐增强;而与对照组比较,TXL组阳性表达明显增强。
3.4 TXL对缺氧预适应小鼠脑皮层神经元和内皮细胞超微结构的作用
与正常对照组比较,缺氧组小鼠脑皮层内皮细胞和神经元超微结构发生明显改变;与缺氧组比较,缺氧预适应组小鼠脑组织神经元及血管内皮细胞超微结构改善;与缺氧预适应组比较,TXL组脑组织神经元及血管内皮细胞超微结构明显改善。
4 PI3K/Akt/HIF-1α信号通路在TXL提高HUVEC缺氧耐受性中的作用
4.1与正常对照组比较,缺氧组HUVEC增殖率显著降低;与缺氧组比较,经TXL处理后的缺氧+TXL组细胞增殖率明显增高。HUVEC缺氧后细胞凋亡率明显增高,随缺氧时间的延长,细胞凋亡率逐渐增高;与缺氧组比较,经TXL干预的缺氧+TXL组细胞凋亡率明显降低。
4.2与缺氧对照组比较,利用DN-HIF抑制HIF-1α的表达后,缺氧HUVEC的存活率显著下降,细胞凋亡率明显升高,同时TXL提高细胞存活率和降低细胞凋亡率的水平明显受到抑制,表明TXL提高HUVEC耐缺氧存活率和降低凋亡率的作用主要是由HIF-1α介导的。
4.3缺氧条件下,HUVEC细胞HIF-1α蛋白的表达明显上调,且随缺氧时间的延长而增加。与对照组比较,应用TXL干预后,HIF-1α蛋白表达进一步增强。与对照质粒组比较,HUVEC瞬转PI3K的显性负性突变体△p85后,HIF-1α蛋白表达显著下调,同时TXL诱导HIF-1α蛋白表达水平明显被抑制。利用Akt的显性负性突变体DN-Akt瞬转HUVEC,也得出相同的结果。表明PI3K/Akt信号途径介导了HIF-1α蛋白表达。
4.4缺氧条件下,HUVEC随着缺氧时间的延长,P-Akt表达出现明显变化,在缺氧4h增加,8h增加最明显,24h有所减少。与对照组比较,经TXL干预后P-Akt表达明显增加。与对照质粒比较,利用PI3K的显性负性突变体△p85瞬时转染后,P-Akt表达明显减少;同时TXL诱导P-Akt表达水平明显受到抑制。
结论:
1通络干预可有效提高平原赴藏旅游人群和实验动物的缺氧耐受性本研究以平原赴藏旅游人群、急性缺氧兔、缺氧预适应小鼠为研究对象,从临床与基础研究初步证实TXL可明显降低赴藏旅游人群AMS发生率,提高急性缺氧兔密闭缺氧耐受时间,提高缺氧预适应小鼠缺氧耐受时间,表明TXL具有提高缺氧耐受性的作用。
2通络干预通过增强缺氧自适应调节能力提高缺氧耐受性自适应是生物体作为复杂系统在长期进化过程中形成的适应环境延续生存的基本能力,实验动物与平原赴藏旅游人群研究均显示,随缺氧时间延长,生命体可产生耐缺氧自适应调节能力,使缺氧损害明显降低,赴藏旅游人群随海拔高度增加AMS发生率逐渐增高,而随缺氧时间的延长AMS发生率逐渐降低,动脉血氧分压、动脉血氧饱和度、脉搏血氧饱和度明显升高;缺氧预适应小鼠随缺氧次数的增加,缺氧耐受时间逐渐延长。通络干预可明显调动缺氧自适应调节能力,提高赴藏旅游人群动脉血氧分压、动脉血氧饱和度、脉搏血氧饱和度,增加缺氧预适应动物缺氧耐受时间,延长实验动物密闭缺氧到死亡的存活时间,提高了耐低氧存活能力。
3促进HIF-1α蛋白表达是TXL提高缺氧自适应调节能力的核心作用机制,PI3K/Akt/HIF-1α信号通路为其主要途径
对TXL提高缺氧耐受力及动物耐低氧存活能力的作用机制的深入研究证实:TXL可明显促进实验兔血清、主动脉和缺氧预适应小鼠脑组织HIF-1α表达,离体HUVEC实验中利用DN-HIF抑制HIF-1α的表达后,缺氧HUVEC的存活率显著下降,细胞凋亡率明显升高,同时TXL提高细胞存活率和降低细胞凋亡率的作用明显受到抑制,表明TXL提高缺氧耐受力的作用是由HIF-1α介导的。进一步实验证实,PI3K/Akt/HIF-1α信号通路为其主要作用途径。
4“承制调平”对揭示缺氧条件下机体代偿性调节及治疗转归的内在规律指导临床防治具有重要理论指导意义
“承制调平”基于中医阴阳五行学说概括出的脉络学说核心理论,在不同层次上反映了中医学的生理观、病理观、治疗观及预后观,“承”揭示了人体不断与自然界进行着物质交换与信息交流,以维持机体内外环境的和谐平衡,氧供需平衡的自稳调控机制即属“承”的范畴;“制”系指机体对病理损害的代偿性自我调节机制,临床与实验研究显示人体与动物均可在缺氧环境下提高自适应调节能力;由“调”致“平”的通络干预,强调提高机体自适应调节重新恢复自稳平衡态,实验证实通络干预可明显促进耐缺氧关键蛋白HIF-1α与VEGF以及抗凋亡因子Bcl-2的表达,抑制促凋亡因子Bax的表达,重建低氧环境下机体耐缺氧损伤的相对平衡态,其关键机制为提高机体缺氧自适应调节能力,这对缺氧疾病治疗具有重要的临床应用价值。
Objective: Self-adaption is a basic skill of survival, which continued the organism as a complex system acclimatizes in the long process of evolution. Hypoxia is the basic pathological process of ischemic heart, cerebrovascular disease. And with the implementation of the western development strategy, more people enter into the highland from inland areas. Plateau hypoxia dues to acute mountain sickness (AMS), high altitude pulmonary edema. These conditions seriously harm to health of the body. Regulation of hypoxia self-adaptive capacity is the key mechanisms of improving hypoxic tolerance; thereby, the search of increasing hypoxia self-adaptive regulative capacity is a question for discussion of finding effective intervention strategies and drugs. Under the guidance of Chinese medicinal theory of“Cheng-zhi-tiao-ping”, which the core content of Vessels-Collateral Theory, on the basis of Tongxinluo (referred to as TXL, that is the representational drugs of the traditional Chinese medical Theory of Collateral Disease) has achieved exact effect on treatment of ischemic cardio-cerebrovascular disease in the past studies, further to study the effect and mechanism of TXL on improving hypoxic tolerance of a body: to observe the effect of TXL on hypoxic tolerance of the crowd who are from plateau to travel to Tibet plateau with hypobaric hypoxia. To establish acute hypoxia rabbit model and explore the effect and mechanism of TXL on hypoxic tolerance. To establish acute hypoxia preconditioning mouse model, to explore the mechanism of TXL further improving the capacity of hypoxia self-adaptive. To study the key role of PI3K/Akt/HIF-1αsignal pathway in TXL enhances HUVEC hypoxic tolerance.
Methods:
1 A clinical trial study on hypoxic tolerance of crowd in plain area travel to Tibet with dredge collateral intervention randomized, double-blind, placebo -controlled
A crowd who lived plain and had never to plateau travelled to Tibet plateau was the representative of the hypoxia crowd. One hundred and twenty healthy volunteers who were accord with standard were randomly divided into two groups, control and TXL group. Double blind trial was applied in this study. The subjects in the two groups took placebo or TXL capsules respectively for 3 days before going to Tibet, and continued to take for 10 days until the end of the study. On the every observation place, the score of AMS symptoms of volunteers was followed up and recorded by the Lake Louise self-report questionnaire. The heart rate (beats/min) and pulse oxygen saturation (%) were recorded. Arterial blood gas analysis was detected. After the clinical trial, the data was entered in the clinical trial remote data management system and proofed. As soon as the data bank was locked, the first blind was exposed and to analysis,and then second unblinding. 2 Effects of dredge collateral intervention on hypoxia tolerance of acute hypoxic rabbit
Twenty-four healthy flap-eared white rabbits were randomly divided into 3 groups:Control group, hypoxia group and TXL group. The rabbits were in low oxygen condition of 11.4% O2 at first. After 60 minutes, air supply was closed. The level of PO2 and SaO2 of arterial blood were detected. The hypoxia tolerance time of the rabbit was measured. The content of serum hypoxia inducible factor 1alpha (HIF-1α) was detected with ELISA. The expression of HIF-1αand vascular endothelial growth factor (VEGF) in the aorta of rabbit were detected with Western blot.
3 Effects and mechanism of dredge collateral intervention on hypoxic tolerance of hypoxic preconditioning mice
3.1 Mice were randomly divided into groups of hypoxic preconditioning (Control) and TXL group. The mice in TXL group were administered at 1.52g crude drug/kg/d for 5 days. The mice were exposure to acute repetitive hypoxia for 0 run (H0), 1 run (H1), 3 runs (H3) and 5 runs (H5). Mouse was placed in an air-sealed jar and hypoxic environment in the jar which was established through consumption of the oxygen by respiration of the mouse. A gasp breath was regarded as the hypoxic tolerant limit of the mice. And then the mice were transferred to a new jar. The mice were exposed to hypoxia in this way for 5 times. In each run of hypoxic exposure, the hypoxic tolerance time was recorded. The Western blot method was used to measure the expression of hypoxia inducible factor-1α(HIF-1α), vascular endothelial growth factor (VEGF), Bcl-associated X (Bax) and B-cell leukemia/ lymophoma 2 (Bcl-2) in the tissue of cortex.
3.2 Mice were randomly divided into 4 groups: control group, hypoxia group, hypoxia preconditioning (HP) group and TXL group. The hypoxia preconditioning mice were exposure by repetitive hypoxia 5 runs. The animal’s tolerance time of each hypoxia run was recorded. The ultrastructure changes of cerebral neuron and endothelial cell were studied by electron microscope.
4 The role of PI3K/Akt/HIF-1 signal pathway in which TXL enhances the HUVEC hypoxia tolerance.
4.1 Cultured HUVECs were divided into four groups as follows: normoxic control group, TXL group (100μg/ml), hypoxia group, hypoxia+ TXL group (100μg/ml). The HUVEC of control group and TXL group were placed in normal condition: 5% CO2+95% air. The cells of hypoxia group and hypoxia +TXL group were placed in hypoxia condition: 1%O2+5%CO2 + 94% N2. At hypoxia 12h, 24h, 48h, the cell proliferation rate were detected by using Cell Counting Kit -8 (CCK-8) test kit, apoptosis rate were detected with flow cytometry respectively.
4.2 Cultured HUVECs were divided into control GFP, the dominant negative mutant of HIF-1(DN-HIF) group, the dominant negative mutant of PI3K/Akt(△p85) group and the negative mutant of Akt(DN-Akt) group. Every group was divided into control and TXL groups. After the HUVECs were transiently transfected with 5μg of GFP control vector, DN-HIF,△p85 and DN-Akt respectively, they were placed in hypoxia condition: 1%O2+5% CO2 +94% N2. At hypoxia 12h, 24h, 48h, the cell proliferation rate and reveal survival rate were detected by using Cell Counting Kit -8 (CCK-8) test kit, apoptosis rate were detected by using flow cytometry respectively. At hypoxia 4h, 24h, 36h, 48h, the expression of HIF-1αand p-Akt were detected by western blot respectively.
Results:
1 A clinical trial study on hypoxic tolerance of crowd in plain area travelled to Tibet with dredge collateral intervention randomized, double-blind, placebo-controlled
1.1 Effect of the TXL on AMS incidence of the cowed travelled to Tibet
With the increasing altitude, AMS incidence in two groups increased gradually. With the hypoxic time prolonged, the AMS incidence decreased gradually. This indicated that organism has hypoxia self-adaptive regulative capacity. Compared with the control group, at Kunlun Mountain (observation 4), Lhasa (observation 6) and Rikaze (observation 10), the AMS incidence in the TXL group reduced (P <0.05), TXL had a downtrend at the other observation. The total AMS incidence on the way to Tibet by train or during the whole journey decreased greatly (P <0.05). These indicated that the TXL increased the hypoxic tolerance of the crowd from plains to Tibet hypoxic environment.
1.2 Effects of the TXL on PO2 and SaO2 in arterial blood gas analysis of the crowed travelled to Tibet
Compared with Shijiazhuang (observation 1), the PO2 and SaO2 of the two groups decreased significantly in Lhasa (observation 6) (P <0.01). With the prolonged of hypoxia, the PO2 and SaO2 of Lhasa (observation 14, the day before leaving Lhasa) were higher than those of the observation 6 (P <0.01). This indicated that organism has hypoxia self-adaptive regulative capacity. In Lhasa (observation 6 and observation 14), compared with the control group, the PO2 and SaO2 of TXL group was significant higher (P <0.01). These indicated that the TXL increased the hypoxic tolerance capacity of organism in hypoxic environment.
1.3 Effect of the TXL on pulse SaO2 of the crowed travelled to Tibet
With the increasing of altitude, the pulse SaO2 of the two groups decreased gradually (P <0.01). With the prolonged of hypoxic time, the pulse SaO2 of the two groups increased gradually (P <0.01). Compared with the control group, the pulse SaO2 of the TXL groups increased significantly (P <0.01).
2 Effects of dredge collateral intervention on hypoxic tolerance of acute hypoxic rabbit
2.1 Comparison of hypoxia tolerance time
From the closed air supply to death, the hypoxic tolerance time of hypoxia group was (14.33±5.92) min, while time of the TXL group was (22.63±4.75) min. The TXL prolonged the survival time for average 8 minutes in the same hypoxic environment. There was a marked statistical difference between two groups (P <0.05).
2.2 Comparison of the PO2 and SaO2 of arterial blood
The PO2 and SaO2 in arterial blood of both hypoxia and TXL groups were not significant difference before hypoxia (P >0.05). Compared with before hypoxia, the PO2 and SaO2 in arterial blood in 11.4% oxygen environment of hypoxia and TXL groups decreased significantly at the time of hypoxia 5min, 30min, 60min (P <0.05 or P <0.01). Compared with the hypoxia group, the range that the levels of PO2 and SaO2 decreased, which reduced greatly (P <0.05 or P <0.01). After the air supply closed, the PO2 and SaO2 of arterial blood reduced further. Compared with the time of hypoxia 60min, at the end of hypoxia (breathing stops instantly), the PO2 and SaO2 in arterial blood decreased significantly (P <0.05 or P <0.01). The PO2 and SaO2 of rabbits in the TXL group was lower than those of in the hypoxia group (P <0.01). These indicated that the TXL prolonged the survival time of rabbit hypoxic, while tolerance with lower PO2 and SaO2, and improved the survival capacity of organism to tolerance hypoxia.
2.3 Comparison of the expression of serum HIF-1α
HIF-1αis a key protein of tolerance with hypoxia. Compared with before hypoxia, the serum HIF-1αof the hypoxia and TXL groups increased significantly (P<0.05 or P<0.01). This indicated that experiment rabbit has hypoxic self-adaptive regulative capacity. Compared with the hypoxia group, the serum HIF-1αincreased significantly in the TXL group (P <0.05 or P <0.01). These indicated that the TXL obviously improved hypoxic tolerance capacity through enhancing the expression of HIF-1αprotein. 2.4Comparison of the expression of HIF-1αand VEGF in the aorta
Compared with the control group, the expression of HIF-1αand VEGF in the aorta of the hypoxia and TXL groups increased obviously (P <0.05 or P <0.01). Compared with the hypoxia group, the expression of HIF-1αand VEGF in the TXL group increased significantly (P <0.05).
3 Effects and mechanism of dredge collateral intervention on hypoxia tolerance of hypoxic preconditioning mice 3.1Comparsion of hypoxic tolerance time
Compared with the H1 (hypoxia exposed one time) in the same group, the hypoxic tolerance time in control and the TXL groups was gradually increased run by run (P <0.01 or P <0.05). Compared with control group, the tolerance time of the TXL group was increased significantly in 1, 3, 5 runs (P <0.05 or P <0.01). 3.2Comparsion of the HIF-1α, VEGF, Bax, Bcl-2 expression in cerebral tissue There were microscale expression of the HIF-1α, VEGF and Bax protein in cerebral tissue in two groups before hypoxia, while no obvious difference between two groups (P >0.05). There were few Bcl-2 expressions in two groups, the Bcl-2 expression in the TXL group was higher than that of in the control group.
Compared with H0 (no hypoxia exposure) in the same group, the HIF-1α, VEGF and Bcl-2 protein expression were increased gradually (P <0.01 or P <0.05). The Bax in control and the TXL groups was significantly increased at first run hypoxia (H1) (P <0.01). After hypoxia 1 run, the Bax expression decreased step by (P <0.01 or P <0.05). Compared with control group, the expression of HIF-1α, VEGF and Bcl-2 in the TXL group increased in 1, 3, 5 runs (P <0.05 or P <0.01), the expression of the Bax was lower than that of in control group at every run (P <0.01 or P <0.05).
3.3 Comparison of the HIF-1αexpression in cerebral tissue in mice with immunohistochemistry
There were no obvious positive expression of HIF-1αin cortical neurons and glial cells in two group’s mice before hypoxia. With the times of hypoxia exposure runs increased, the expression of the HIF-1αincreased gradually. Compared with the control group, the positive expression in the TXL group increased significantly.
3.4 Comparison of the ultrastructure of cerebral neuron and endothelial cell Compared with control group, the ultrastructure of cerebral neuron and endothelial cell in the hypoxia group changed obviously, with mitochondrion and endoplasmic reticulum destroyed. Compared with the hypoxia group, the destroyed degree of cerebral neuron and endothelial cell in the HP group were slighter than that in control group. The change in the TXL group did show obvious improved, comparison with HP group.
4 The role of PI3K/Akt/HIF-1 signal pathway in which the TXL enhances the HUVEC hypoxia tolerance.
4.1 Compared with the control group, HUVEC proliferation rate in the hypoxic group was significantly reduced. Compared with the hypoxia group, the HUVEC proliferation rate of hypoxia plus TXL group significantly increased. After hypoxia, the HUVEC apoptosis rate was significantly increased. With prolonged the hypoxia time, apoptosis rate increased gradually. The HUVECs apoptosis ratio in the hypoxia plus TXL group was great lower than the hypoxia group.
4.2 Compared with the hypoxia group, after inhibited the expression of HIF-1αwith DN-HIF, the survival rate of the HUVEC decreased significantly, and the apoptosis rate increased obviously, while the degree of which the TXL increased the HUVEC survival rate was significantly inhibited. These indicated that the TXL increased survival rate and decreased apoptosis rate of the HUVEC which mediated by HIF-1α. 4.3 In hypoxic condition, the HIF-1αexpression of HUVECs was significantly increased. And with the prolonged of hypoxia time, the expression of HIF-1αincreased obviously. Compared with the control group, the HIF-1αexpression in TXL group further enhanced.
Compared with the control plasmid group, after the HUVECs were transiently transfected with△p85, the HIF-1αprotein expression was significantly reduced, and the degree of HIF-1αexpression that the TXL increased was inhibited. With DN-Akt, the dominant negative mutant of Akt, the consequence was as well as△p85. These indicated that the HIF-1αexpression was mediated by PI3K/Akt singal pathway.
4.4 With the prolonged of hypoxia time, the expression of the p-Akt changed greatly. It was increased at 4h, and was obvious higher at 8h, but it was reduced at 24h. Compared with the control plasmid group, the p-Akt expression in the TXL group was significantly increased.
Compared with the control plasmid group, after the HUVECs were transiently transfected with the△p85, the P-Akt expression was significantly reduced, and the degree of P-Akt expression that TXL increased was inhibited.
Conclusion:
1 Dredge collateral intervention may improve the hypoxia tolerance of crowd which travelled to Tibet and experimental animals With the crowed travelled to Tibet, acute hypoxia rabbits, and the hypoxic preconditioned mice as the research objects, from the initial clinical and basic research, the study initially confirms that the TXL could significantly reduce the AMS incidence in crowed which travelled to Tibet, improve the hypoxic tolerance time in acute hypoxia rabbits and hypoxia preconditioning mice. It indicated that the TXL could improve the hypoxia tolerance.
2 Dredge collateral intervention improved the hypoxic tolerance with enhancing hypoxic self-adaptive regulative capacity
Self-adaption is a basic skill of survival and continued that the organism as a complex system acclimatizes in the long process of evolution. The study of crowed which travelled to Tibet and experimental animals showed that organism could produce self-adaptive regulative capacity to tolerance hypoxia with prolonged the hypoxia time. The capacity could reduce the damage of hypoxia significantly. With the rising of altitude, the AMS incidence of the crowd which travelled to Tibet increased. With the prolonged of the hypoxic time, the AMS incidence decreased, the PO2, SaO2 in arterial blood and pulse SaO2 increased significantly. The hypoxic tolerance time of hypoxic preconditioning mice increased run by run. The TXL intervention could mobilize the hypoxic self-adaptive regulative capacity, increase the PO2, SaO2 in arterial blood, pulse SaO2 and hypoxic tolerance time in hypoxic preconditioning mice, prolong the survival time of experimental animals in closeness hypoxia device, improve the survival ability of hypoxia. 3 Promoting the HIF-1αexpression is the key mechanism of the TXL intervention improving the hypoxic self-adaptive regulative capacity, and the PI3K/Akt/HIF-1αsignal pathway is the major pathway.
The depth study of mechanism that intervention of the TXL in hypoxic tolerance ability and animal’s viability to resist lower hypoxia confirmed: The TXL could promote the serum HIF-1αexpression, aorta of experimental rabbits and cerebral tissue of hypoxic preconditioning mice. After expression of the HIF-1αwas inhibited by DN-HIF, the survival rate of HUVEC was decreased significantly in hypoxic condition and the apoptosis rate of HVEC was increased significantly. But the effect of the TXL on increasing survival rate and decreasing apoptosis rate was significantly inhibited. These indicated that the TXL increases survival rate and decreases apoptosis rate of the HUVEC, which is mediated by HIF-1α. Further experiments confirmed that the PI3K/Akt/HIF-1αis the major signaling pathway.
4 It is important that the theory of“Cheng-zhi-tiao-ping”on revealing the inherent laws of body’s compensatory adjustment in hypoxic conditions, therapy outcome and on directing clinical preventive treatment.
“Cheng-zhi-tiao-ping”is the core of Vessels-Collateral Theory, based on the traditional Chinese medicine theory of yin-yang and five-element. It reflects the physiological, pathology, treatment and prognosis concepts of the traditional Chinese medicine.“Cheng”reveals that human body constantly exchanges substance and information with the nature, in order to maintain a harmonious balance between internal and external environment. The self-stability regulation mechanism of the oxygen supply-demand balance is longs to "Cheng" category. "Zhi" means the body’s self-regulating mechanisms for compensatory pathological damages. Clinical and experimental studies had shown that humans and animals could improve self-adaptive regulative capacity in hypoxic conditions.
Dredge collateral intervention, from“Tiao”to“Ping”, emphasizes that enhancing self-adaptive regulative capacity of organism and to regain homeostasis. Experiments confirmed that dredge collateral intervention can promote the expression of HIF-1α(the key protein of hypoxia), VEGF and Bcl-2 (anti-apoptotic factor), inhabits the expression of Bax (pro-apoptotic factor), rebuild the relative equilibrium state to against hypoxic injury in hypoxic condition. The key mechanism was that dredge collateral intervention can improve hypoxic self-adaptive regulative capacity of organism. This had an important value of clinical application for treatment of hypoxic disease.
引文
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