| 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 -controlledA 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 rabbitTwenty-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 mice3.1 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-controlled1.1 Effect of the TXL on AMS incidence of the cowed travelled to TibetWith 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 TibetCompared 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 TibetWith 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 rabbit2.1 Comparison of hypoxia tolerance timeFrom 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 bloodThe 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-1αHIF-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 aortaCompared 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 timeCompared 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 immunohistochemistryThere 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 capacitySelf-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.
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