缺氧预适应小鼠脑匀浆液对离体细胞缺氧耐受性的影响及其可能有效组分的初步研究
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摘要
缺氧时,机体会出现各种代偿反应以对抗缺氧的影响。机体这种与缺氧做斗争的能力,即缺氧的耐受力显然是有一定限度的。近年来的研究表明,机体的这种对缺氧的耐受性可以通过缺氧预适应等措施得到加强。缺氧预适应是指机体经短暂时间或亚致死量的缺氧后,对后续的更长时间或更严重缺氧性损伤产生明显耐受的能力。其机制除了与缺氧引发的由缺氧诱导因子-1等转录因子介导的各种适应代偿性反应蛋白表达改变有关外,组织、细胞的能量代谢产物,特别是ATP的分解代谢产物等在缺氧预适应中也起重要作用。
脑是对缺氧最敏感的器官,是影响机体缺氧耐受能力的主要限制因素。因此中枢神经系统的机能或代谢变化可能是机体缺氧耐受形成的主要机制。研究表明,缺氧时脑组织的物质变化不仅对于机体缺氧耐受能力的建立具有重要作用,而且缺氧预适应小鼠的脑匀浆液也能提高动物甚至离体细胞对缺氧的耐受能力。积极寻找并研究急性重复缺氧预适应小鼠脑匀浆液中的可提高机体缺氧耐受能力的生物活性物质具有重要的理论和实际意义。本研究通过观察不同实验条件对急性重复缺氧预适应小鼠模型的影响,建立稳定可靠、效果显著的小鼠缺氧预适应模型。由于动物对缺氧的耐受性个体差异较大,影响因素较多,因此为便于控制实验条件和探索增强缺氧耐受性的物质特性,本研究采用离体细胞观察急性重复缺氧预适应小鼠脑匀浆液对离体细胞缺氧耐受性的影响。在确定急性重复缺氧预适应小鼠脑匀浆液对不同离体培养细胞(未用NGF诱导的PC12细胞、HepG2细胞)缺氧耐受性具有双相效应的基础上,以NGF诱导分化的PC12细胞作为观察对象,探索急性重复缺氧预适应小鼠脑匀浆液对分化PC12细胞只起保护作用、无损伤作用的最适浓度,并进一步将该提取液分成蛋白部分和非蛋白部分,探讨其中存在的增强细胞缺氧耐受性的可能组分。实验分三部分进行。
一、方法
第一部分急性重复缺氧预适应小鼠模型的复制
(1).使小鼠一次性缺氧致死,以观察小鼠缺氧致死前的变化规律。
(2).将小鼠连续4次重复缺氧,以观察小鼠每次缺氧耐受时间的变化。
(3).将小鼠分成2组,均经过连续4次缺氧,2组小鼠每次结束缺氧的标准分别是呼吸频率约40次/min或出现第1次喘呼吸,以观察每次不同时间缺氧对小鼠缺氧耐受性的影响。
(4).将小鼠分成2组,均经过连续4次缺氧,2组小鼠每次结束缺氧的标准分别是第一次喘呼吸或末次喘呼吸,以观察每次不同时间缺氧对小鼠缺氧耐受性的影响。
(5).将小鼠分成4组,均经过连续4次缺氧,但4组小鼠连续4次缺氧的环境温度分别是:9-11℃、13-15℃、17-19℃、21-23℃,以观察不同环境温度对小鼠缺氧耐受性的影响。
(6).将小鼠分成2组,均经连续4次缺氧,2次缺氧间的换瓶操作时间分别是5-15s或20-30s,以观察不同复氧时间对小鼠缺氧耐受性的影响。
(7).将经过连续4次缺氧的小鼠恢复常氧, 30分钟后再放回缺氧瓶缺氧1次,以观察小鼠复氧30分钟后缺氧耐受性的变化。
第二部分缺氧预适应小鼠脑匀浆液对未用NGF诱导分化的PC12细胞、HepG2细胞以及NGF诱导分化的PC12细胞缺氧耐受性的影响
1.以不同浓度的小鼠脑匀浆液分别作用于未用NGF诱导的PC12细胞,观察缺氧24h、48h、72h时PC12细胞的活力、LDH透出率、早期凋亡率、晚期凋亡率的变化规律,以确定小鼠脑匀浆液对细胞缺氧耐受性的影响。
2.以不同浓度的小鼠脑匀浆液分别作用于HepG2细胞,观察缺氧24h、48h、72h缺氧时HepG2细胞的活力、早期凋亡率、晚期凋亡率的变化规律,以了解小鼠脑匀浆液对非神经元模型细胞的影响。
3.根据上述第二部分实验的结果,仅以较低浓度范围小鼠脑匀浆液分别作用于NGF诱导分化的PC12细胞,观察缺氧24h、48h、72h时分化PC12细胞的活力、LDH透出率,晚期凋亡率的变化规律,以探索缺氧预适应小鼠脑匀浆液对缺氧、NGF诱导分化的PC12细胞只起保护作用、没有损伤作用的最适浓度。
第三部分:缺氧预适应小鼠脑匀浆液不同组分对NGF诱导分化的PC12细胞缺氧耐受性的影响及其可能有效成分的初步探讨
(1).以高氯酸除蛋白法制备缺氧预适应小鼠脑匀浆去蛋白液,HPLC法测定其腺苷含量。用小鼠脑匀浆去蛋白液作用于NGF诱导分化PC12细胞,以确定去蛋白液对缺氧的分化PC12细胞有无保护作用;以小鼠脑匀浆去蛋白液合并腺苷A1或A2A受体阻断剂作用于分化PC12细胞,以确定小鼠脑匀浆去蛋白液的作用是否与腺苷有关。
(2).透析法除去缺氧预适应小鼠脑匀浆液中小分子物质,并观察透析保留液对缺氧的分化PC12细胞有无保护作用;分别用RT-PCR、Western blot方法检测缺氧预适应小鼠脑组织中VEGF mRNA、VEGF的蛋白水平是否增加;用重组VEGF纯品作用于分化PC12细胞,以确定透析保留液对分化PC12细胞的保护作用是否与VEGF有关。
二、结果与讨论
通过上述3部分的工作获得以下结果
(一)影响急性重复缺氧模型的因素
1.小鼠经4次急性重复缺氧后,每次缺氧耐受时间显著增加。这一结果提示,小鼠在每次缺氧后,均在体内产生了增强缺氧耐受性的物质,并且各次缺氧后缺氧耐受的物质有积累效应。
2.在小鼠出现第一次喘呼吸时将小鼠换入下一个缺氧瓶与小鼠呼吸频率在40次/min时换瓶比较,可显著提高小鼠每次耐受时间。这是因为小鼠在呼吸频率为40次/min时的缺氧时间比出现喘呼吸时的缺氧时间短,缺氧程度尚未达到缺氧耐受形成的程度,因此对缺氧的耐受性也不强,因为缺氧预适应的形成需要足够程度的缺氧。
3.在小鼠出现第一次喘呼吸时将小鼠换入下一个缺氧瓶与小鼠出现末次喘呼吸时换瓶比较,缺氧耐受时间无显著差异。其原因是,不同小鼠喘呼吸次数不同,有的小鼠喘呼吸3-5次即死亡,有的小鼠喘呼吸却可达30次以上,因此小鼠的个体差异较大,使组间无显著差异;并且由于末次喘呼吸后小鼠随即死亡,因此末次喘呼吸难以判断,以其作为结束缺氧标准导致小鼠死亡率增加。
4.随着环境温度的增加,小鼠的缺氧耐受时间显著降低。这可能是因为低温可减少小鼠体内有害物质的产生、降低代谢,有利于缺氧预适应的形成。
5.每次对小鼠换瓶操作时间长于15秒会显著降低小鼠的缺氧耐受时间。其原因可能是复氧时间太长,因为复氧时间短暂有利于预适应的形成。由于经过重复4次缺氧的小鼠恢复常氧30分钟后,其缺氧耐受性还可以部分保留,说明小鼠4次缺氧耐受性的完全消失需要至少30分钟以上的时间,因此赋予预适应小鼠缺氧耐受性的物质,可能部分对机体恢复常氧供应较为敏感,但部分能维持较长时间。
(二)缺氧预适应小鼠脑匀浆液对未用NGF诱导的PC12细胞、HepG2细胞、NGF诱导分化的PC12细胞缺氧耐受性的影响
1.缺氧预适应小鼠脑匀浆液对缺氧、未用NGF诱导的PC12细胞、HepG2细胞缺氧耐受性的影响是:缺氧预适应小鼠脑匀浆液可显著提高细胞对缺氧的耐受性。在缺氧初期(24h),较高浓度的保护作用显著强于低浓度的保护作用,这提示缺氧预适应小鼠脑组织中产生了某种或某些可以增强细胞缺氧耐受性的物质或使脑组织中抗缺氧成分表达增加。随着缺氧时间延长,保护作用下降,高浓度的下降更快。至缺氧72h,高浓度提取液的缺氧损伤作用显著高于低浓度组,该结果提示,缺氧预适应小鼠脑组织中可能产生了半衰期较短的抗缺氧物质及在高浓度时才显示作用的、作用时间持久的促缺氧细胞损伤的物质,即提示缺氧预适应有着双相效应,缺氧预适应小鼠脑匀浆的作用存在量效关系和时间效应。但是由于本研究的目的是为了寻找缺氧预适应小鼠脑匀浆液中增强缺氧耐受性的物质,因此实验中应避免损伤性物质的影响。为此,在后续缺氧预适应小鼠脑匀浆液对分化PC12细胞的作用研究中,以蛋白浓度对提取液进行定量,并降低实验中所用提取液的浓度范围,以期找到提取液对NGF诱导分化的PC12细胞只起保护作用,无损伤作用(即只增强NGF诱导分化的PC12细胞缺氧耐受性,对分化PC12细胞无损伤作用)的最适浓度,为进一步研究缺氧预适应小鼠脑匀浆液的保护效应及保护性物质奠定基础。
2.经较低浓度的缺氧预适应小鼠脑匀浆液对NGF诱导分化的PC12细胞的作用实验发现,蛋白终浓度为100.0μg/mL的缺氧预适应小鼠脑匀浆液与同浓度正常小鼠脑匀浆液比较,可显著增强NGF诱导分化的PC12细胞的缺氧耐受性,并且此浓度的缺氧预适应小鼠脑匀浆液对分化PC12细胞未显示出损伤作用,为增强分化PC12细胞缺氧耐受性的适合浓度。
(三)缺氧预适应小鼠脑匀浆液不同组分对NGF诱导分化的PC12细胞缺氧耐受性的影响及其可能有效成分的初步探讨
1.向离体培养的NGF诱导分化的PC12细胞中加入不同浓度的腺苷,随着腺苷浓度增加,细胞的缺氧耐受性增高,10.0μmol/L的腺苷对细胞的保护作用可维持24h。急性4次重复缺氧可使小鼠脑组织腺苷含量上升。缺氧预适应小鼠脑匀浆去蛋白液只在缺氧的24h内显著增加分化PC12细胞的缺氧耐受性,腺苷A2A受体阻断剂可阻断其保护作用。正常小鼠脑匀浆去蛋白液对分化PC12细胞无保护作用。因此,缺氧预适应小鼠脑匀浆液的非蛋白组分的抗缺氧作用时间较短,腺苷可能是其中一个重要的活性物质。
2.在缺氧24h和48h,缺氧预适应小鼠脑匀浆透析保留液与未经透析的缺氧预适应小鼠脑匀浆液对分化PC12细胞均有保护作用,且经两种孔径(3.5kD、7.0kD)透析袋透析的缺氧预适应小鼠脑匀浆透析保留液和未经透析的缺氧预适应小鼠脑匀浆液对缺氧细胞的保护作用无显著差异,说明缺氧预适应小鼠脑组织中起保护作用的主要是分子量大于7.0kD的蛋白成分。急性重复缺氧预适应小鼠脑组织中VEGF表达虽增加,但应用重组VEGF直接作用于分化的PC12细胞发现,不同浓度的VEGF对NGF诱导分化的、不同缺氧时间的PC12细胞均无保护作用。这一结果提示:缺氧预适应小鼠脑匀浆液的蛋白组分抗缺氧作用较为持久,其中VEGF表达量虽增高,但并不参与NGF诱导分化的PC12细胞的抗缺氧作用。由于缺氧预适应小鼠脑匀浆液的蛋白组分抗缺氧作用时间持久,提示蛋白质组学方法可能是研究其中抗缺氧成分的有效方法,因此本研究结果为今后进一步研究缺氧预适应小鼠脑匀浆液中抗缺氧物质提示了方向。
三、结论
1.赋予重复缺氧小鼠缺氧耐受性的物质,部分对机体恢复常氧供应较为敏感,部分则能持续较长时间;
2.缺氧预适应小鼠脑匀浆液在浓度较高时对离体缺氧培养细胞的作用表现为双相性--缺氧早期,脑匀浆液对缺氧细胞具有保护效应,延长缺氧时间,其损伤作用逐渐显现,这种双相作用存在量效关系和时间效应;其只起保护作用的浓度是在较低浓度;
3.缺氧预适应小鼠脑匀浆液可显著提高不同来源离体培养细胞的缺氧耐受性,提示对缺氧细胞具有普遍的保护作用;
4.缺氧预适应小鼠脑匀浆液的蛋白组分抗缺氧作用较为持久,其中VEGF表达量虽增高,但并不参与NGF诱导分化的PC12细胞的抗缺氧作用;
5.缺氧预适应小鼠脑匀浆液的非蛋白组分的抗缺氧作用时间较短,腺苷可能是其中一个重要的活性物质。
Hypoxia can lead to various kinds of reactions in animal or human bodies to compensate for hypoxic influence. But the extent to which animal or human bodies fight against hypoxia, namely tolerate hypoxia is limited. However, recent investigations have showed that the tolerance of the bodies to hypoxia can be enhanced through such measures as hypoxic preconditioning (HP). HP referrs to the phenomenon that a short duration of moderate hypoxia or sub-lethal hypoxia enables animals, tissues or cells to tolerate subsequent longer time of hypoxia or more severe hypoxic insult, whose mechanism is related not only to the changes in expressions of various accommodative proteins mediated via HIF-1, but also to energy metabolites. In particular, ATP catabolite is involved in HP mechanism.
Brain is the most vulnerable organ in animal and human bodies and the main limiting factor influencing tolerance of the bodies to hypoxia. Therefore, the changes in functions and substances in central nervous system may be implicated in the main mechanism of tolerance formation. Researches have indicated that not only the alteration in substances in brain plays important roles in hypoxia tolerance formation, but also the brain homogenate of hypoxic-preconditioned mice (HP mice) could improve the hypoxic tolerance of animals and in vitro cells that do not undergo hypoxia. It is of both great theoretical and practical significance to search for the bioactive substances in the brain homogenate of HP mice, which can enhance the capacity of tolerating hypoxia.
For this purpose, the stable, reliable and significantly effective model of HP mice was reproduced based on observing the influence of different experimental conditions on the model of acute repetitive hypoxic preconditioning of mice Because individual animals differ in tolerance to hypoxia and their individual tolerance is easily affected by many other influential factors, cells in vitro were used to investigate the influence of brain extract of acute repetitive hypoxic-preconditioned mice (BEAHPM) on the tolerance of the cells in order to easily control experimental conditions and to explore the properties of the tolerance-enhancing substances. On the basis of certifying the fact that BEAHPM had biphasic effects on non-NGF-differentiated PC12 cells and HepG2 cells, NGF-differentiated PC12 cells alone was used in the subsequent experiments to determine the optimal concentration only showing protective effects on differentiated PC12 cells. Moreover, the extract of HP mice was further prepared in the form of protein portion and in the form of non-protein portion respectively to study the possible constituents enhancing cell tolerance to hypoxia. The experiments consist of three parts.
1. Methods
1.1 Part I. The mice model of acute repetitive hypoxic preconditioning was reproduced.
1.1.1 Mice were exposed to hypoxia till death to show their activity changes before their death.
1.1.2 Mice were subjected to four times of consecutive hypoxia to show the changes in hypoxic tolerance time.
1.1.3 Mice were divided into two groups to undergo four times of consecutive hypoxia, and hypoxia was stopped for the first group when the respiratory frequency of mice was about 40 times per minute but hypoxia was stopped for the second group when the first asthmoid respiration of mice appeared respectively, aiming at observing the influence of different hypoxia duration on mice tolerance.
1.1.4 Mice were divided into two groups to undergo four times of consecutive hypoxia. The standard of ending hypoxia was when the first asthmoid respiration appeared or when the last asthmoid respiration appeared respectively.
1.1.5 Mice were divided into four groups to experience four times of consecutive hypoxia, while the environmental temperature was 9-11℃, 13-15℃, 17-19℃and 21-23℃respectively to determine the influence of different environmental temperatures on the tolerance to hypoxia.
1.1.6 Mice were divided into two groups for four times of consecutive hypoxia, the operating time between two hypoxias was 5-15s or 20-30s respectively to determine the influence of different re-oxygenation time on mice tolerance.
1.1.7 The mice exposed to four times of consecutive hypoxia were transferred to normoxic environment. 30 minutes later, they were subjected to hypoxia once again to observe the change in tolerance time.
1.2 Part II. The effects of BEAHPM on tolerance of PC12, HepG2 and NGF-differentiated PC12 cells to hypoxia
1.2.1 BEAHPM at different concentrations were used to treat non-NGF-differentiated PC12 cells and the changes in cell vitality, release rate of lactate dehydrogenase (LDH), apoptotic rates at early and late stages of hypoxia were determined to find out the effects of BEAHPM on the cells.
1.2.2 BEAHPM at different concentrations were used to treat HepG2 cells and the changes in cell vitality, apoptotic rates of HepG2 cells at early and late stages of hypoxia were observed to investigate the effects of BEAHPM on non-neuronal cells.
1.2.3 According to the results of avove experiments in Part II, BEAHPM at relatively low concentrations were used to treat NGF-differentiated PC12 cells and the changes in cell vitality, LDH release rate, apoptotic rates at late stages of hypoxia were examined to determine the optimal concentration of BEAHPM to protect NGF-differentiated PC12 cells against hypoxia without showing injurious effects simultaneously.
1.3. Part III. The effects of different components of BEAHPM on tolerance of NGF-differentiated PC12 cells to hypoxia and preliminary researches into possible effective constituents
1.3.1 Deproteinized BEAHPM was prepared by deproteinization with perchloric acid and adenosine in deproteinized BEAHPM was quantified by HPLC (high performance liquid chromatography). The effects of deproteinized BEAHPM on NGF-differentiated PC12 cells were determined to clarify whether it had protective effects on the cells. Then the effects of deproteinized BEAHPM plus antagonists of adenosine A1 or of A2A receptor were tested to show whether the effects were relevant to adenosine.
1.3.2 Dialysis method was used to remove small molecules from BEAHPM to observe whether protein-retained solution has protective effects on NGF-differentiated PC12 cells. RT-PCR and Western blot were adopted to check levels of VEGF (vascular endothelial growth factor) mRNA and VEGF protein in the brain of HP mice. Recombined VEGF was used to investigate whether VEGF alone had protective effects on differentiated PC12 cells in hypoxia.
2 Results and discussion
The following results are obtained from above three parts of work.
2.1 The factors influencing the mice tolerance in the model of acute repetitive hypoxic preconditioning
2.1.1 After four times of acute repetitive hypoxia, the hypoxic tolerance time of mice was obviously increased, revealing that after each time of hypoxia, the substance (or substances) for tolerance was produced and they were accumulative in HP mice.
2.1.2 The tolerance time of the group which was transferred into the next hypoxic container when the first asthmoid respiration appeared was significantly longer than that of the group which was put into the next hypoxic container when the respiration frequency was 40 times per minute. The cause of this result was that the hypoxic duration when the respiration frequency was 40 times per minute was shorter than that when the first asthmoid respiration appeared, and that the formation of tolerance to hypoxia needs sufficient degrees of exposure to hypoxia. Therefore, hypoxic degree was not enough for mice to form tolerance if mice were transferred into the next hypoxic container when the respiration frequency was 40 times per minute.
2.1.3 The tolerance time of the group which was transferred into the next hypoxic container when the first asthmoid respiration appeared was not significantly different from that of the group which was put into the next hypoxic container when the last asthmoid respiration appeared. One cause of the results was that asthmoid respiration times were different in different mice. Some mice died after 3 or 5 times of asthmoid respirations, but some mice died after 30 times of asthmoid respirations, resulting in great individual differences in tolerance time within the latter group. The second cause was that mice died almost immediately after the last asthmoid respiration so it is difficult to judge the last asthmoid respiration, which increased mice mortality in the latter group.
2.1.4 With the increase of environmental temperature, the tolerance time was decreased significantly. The cause might be that low temperature could reduce the production of detrimental substances in mice and could also decrease the metabolism and be helpful to tolerance formation.
2.1.5 If the time for transferring mice into the next container was longer than 15 seconds, the tolerance time was significantly decreased. The reason for this result may be that when re-oxygenation time is too long, the tolerance degree was decreased because short time for re-oxygenation is favorable for tolerance formation. Because the effects of tolerance could be partly retained 30 minutes after the mice exposed to four times of acute repetitive hypoxia returned to nomorxia, the complete disappearance of tolerance needed more than 30 minutes and it could be deduced that some tolerance-enhancing substances remained stable but some were susceptible to re-oxygenation.
2.2 Part II. The effects of BEAHPM on tolerance of non-NGF-differentiated PC12 cells, HepG2 cells and NGF-differentiated PC12 cells
2.2.1 The effects of BEAHPM on non-NGF-differentiated PC12 cells and HepG2 cells was that it could significantly enhance the tolerance of these two cell strains to hypoxia. At the initiate time of hypoxia (24 h of hypoxia), the protective effect of BEAHPM at relatively high concentrations was stronger than that at the relatively low concentrations, indicating that some tolerance-enhancing substances were produced or the expressions of anti-hypoxia substances were up-regulated in the brain of HP mice. With the prolongation of hypoxic time, protective effects of BEAHPM decreased. And the effects of BEAHPM at high concentrations decreased faster than those at low concentrations. After 72-hour hypoxia, BEAHPM at high concentrations showed injurious effects on these two cells. This result indicated that anti-hypoxia substances with short half-time and detrimental substances showing long-lasting injurious effects at high concentrations were produced. Namely, acute hypoxic preconditioning had biphasic effects and the biphasic effects of BEAHPM were concentration-dependent and time-dependent. Because the objiective of this research is to try to roughly locate the tolerance-enhancing substances in BEAHPM, the effects of damage-enhancing substances should be avoided.
For this purpose and based on above results, protein concentration was used to quantify BEAHPM and BEAHPM in a lower concentration range was used in the subsequent experiments on the effects of BEAHPM on NGF-differentiated PC12 cells in order to find out the optimal concertration of BEAHPM only showing protective effects on NGF-differentiated PC12 cells and not showing damage effects on NGF-differentiated PC12 cells. Namely, the optimal concentration only enhances tolerance of NGF-differentiated PC12 cells and does not show injurious effects on NGF-differentiated PC12 cells, the experimental design aiming at laying a foundation for further researches into the protective effects and protective substances in BEAHPM.
2.2.2 The results of the effects of BEAHPM in a relatively lower concentration range on NGF-differentiated PC12 cells showed that BEAHPM at final protein concentration of 100.0ug/mL could significantly enhance the tolerance of differentiated PC12 cells to hypoxia without showing harmful effects on the cells compared with the brain extract of normal mice at the same protein concentration.
2.3 Part III. The effects of deproteinized BEAHPM, dialyzed protein-retained solutions of BEAHPM, adenosine and VEGF on hypoxic tolerance of NGF-differentiated PC12 cells.
2.3.1 The tolerance of NGF-differentiated PC12 cells could be enhanced by addition of adenosine at different concentrations. Besides, the tolerance of differentiated PC12 cells could be increased as the concentrations of adenosine increased. Adenosine at 10.0μmol/L could protect cells only within 24-hour hypoxia. The content of adenosine was up-regulated in mice brain through four times of acute repetitive hypoxia. The deproteinized BEAHPM showed its protective effects on differentiated PC12 cells only within 24 hours of hypoxia and deproteinized brain extract of normal mice showed no protective effects on differentiated PC12 cells. Antagonist of adenosine A2A receptor could inhibit the protective action of deproteinized BEAHPM and the protective action of adenosine, so adenosine might be an active protective constituent.
2.3.2 After 24-hour and 48-hour hypoxia, the dialyzed protein-retained solutions of BEAHPM, and non-dialyzed BEAHPM protected NGF-differentiated PC12 cells against hypoxia. Moreover, the protective effects of the dialyzed protein-retained solution through 3.5kD bag filter or through 7.0kD bag filter did not significantly differ from the effects of non-dialyzed BEAHPM, suggesting that the protective substances in the brain of HP mice were proteins with molecular weight larger than 7.0kD. Although the expression of VEGF in the brain of HP mice was significantly increased, recombined VEGF at different concentrations had no protective effects on differentiated PC12 cells after different durations of hypoxia. These results revealed that the protein constituents in BEAHPM had long-lasting anti-hypoxia action, but the up-regulation of VEGF expression was not involved in the anti-hypoxia effects on differentiated PC12 cells. The long-lasting effects of the protein constituents in BEAHPM suggest that proteomics methods might be the effective ways of investigating the anti-hypoxia substances in BEAHPM, giving insight into the focus of future research efforts.
3. Conclusions
3.1 Some substances endowing mice exposed to acute repetitive hypoxia with tolerance were relatively susceptibly to re-oxygenation, but some had long-lasting protective effects.
3.2 BEAHPM had biphasic effects on in vitro cells subjected to hypoxia. Namely, at the early stage of hypoxia, BEAHPM protected cells against hypoxia. As time for hypoxia prolongs, it gradually showed damage action. This biphasic effects were concentration-dependent and time-dependent.
3.3 BEAHPM could significantly improve the tolerance of in vitro cells of diverse origins, indicating that BEAHPM had universal protective effect on hypoxic cells.
3.4 The protein constituents in BEAHPM had long-lasting protective effects. VEGF expression in BEAHPM increased significantly, but VEGF was not involved in anti-hypoxia effects on differentiated PC12 cells.
3.5 The non-protein constituents in BEHPM only had short-lived protective action and adenosine might be an important active substance.
脑是对缺氧最敏感的器官,是影响机体缺氧耐受能力的主要限制因素。因此中枢神经系统的机能或代谢变化可能是机体缺氧耐受形成的主要机制。研究表明,缺氧时脑组织的物质变化不仅对于机体缺氧耐受能力的建立具有重要作用,而且缺氧预适应小鼠的脑匀浆液也能提高动物甚至离体细胞对缺氧的耐受能力。积极寻找并研究急性重复缺氧预适应小鼠脑匀浆液中的可提高机体缺氧耐受能力的生物活性物质具有重要的理论和实际意义。本研究通过观察不同实验条件对急性重复缺氧预适应小鼠模型的影响,建立稳定可靠、效果显著的小鼠缺氧预适应模型。由于动物对缺氧的耐受性个体差异较大,影响因素较多,因此为便于控制实验条件和探索增强缺氧耐受性的物质特性,本研究采用离体细胞观察急性重复缺氧预适应小鼠脑匀浆液对离体细胞缺氧耐受性的影响。在确定急性重复缺氧预适应小鼠脑匀浆液对不同离体培养细胞(未用NGF诱导的PC12细胞、HepG2细胞)缺氧耐受性具有双相效应的基础上,以NGF诱导分化的PC12细胞作为观察对象,探索急性重复缺氧预适应小鼠脑匀浆液对分化PC12细胞只起保护作用、无损伤作用的最适浓度,并进一步将该提取液分成蛋白部分和非蛋白部分,探讨其中存在的增强细胞缺氧耐受性的可能组分。实验分三部分进行。
一、方法
第一部分急性重复缺氧预适应小鼠模型的复制
(1).使小鼠一次性缺氧致死,以观察小鼠缺氧致死前的变化规律。
(2).将小鼠连续4次重复缺氧,以观察小鼠每次缺氧耐受时间的变化。
(3).将小鼠分成2组,均经过连续4次缺氧,2组小鼠每次结束缺氧的标准分别是呼吸频率约40次/min或出现第1次喘呼吸,以观察每次不同时间缺氧对小鼠缺氧耐受性的影响。
(4).将小鼠分成2组,均经过连续4次缺氧,2组小鼠每次结束缺氧的标准分别是第一次喘呼吸或末次喘呼吸,以观察每次不同时间缺氧对小鼠缺氧耐受性的影响。
(5).将小鼠分成4组,均经过连续4次缺氧,但4组小鼠连续4次缺氧的环境温度分别是:9-11℃、13-15℃、17-19℃、21-23℃,以观察不同环境温度对小鼠缺氧耐受性的影响。
(6).将小鼠分成2组,均经连续4次缺氧,2次缺氧间的换瓶操作时间分别是5-15s或20-30s,以观察不同复氧时间对小鼠缺氧耐受性的影响。
(7).将经过连续4次缺氧的小鼠恢复常氧, 30分钟后再放回缺氧瓶缺氧1次,以观察小鼠复氧30分钟后缺氧耐受性的变化。
第二部分缺氧预适应小鼠脑匀浆液对未用NGF诱导分化的PC12细胞、HepG2细胞以及NGF诱导分化的PC12细胞缺氧耐受性的影响
1.以不同浓度的小鼠脑匀浆液分别作用于未用NGF诱导的PC12细胞,观察缺氧24h、48h、72h时PC12细胞的活力、LDH透出率、早期凋亡率、晚期凋亡率的变化规律,以确定小鼠脑匀浆液对细胞缺氧耐受性的影响。
2.以不同浓度的小鼠脑匀浆液分别作用于HepG2细胞,观察缺氧24h、48h、72h缺氧时HepG2细胞的活力、早期凋亡率、晚期凋亡率的变化规律,以了解小鼠脑匀浆液对非神经元模型细胞的影响。
3.根据上述第二部分实验的结果,仅以较低浓度范围小鼠脑匀浆液分别作用于NGF诱导分化的PC12细胞,观察缺氧24h、48h、72h时分化PC12细胞的活力、LDH透出率,晚期凋亡率的变化规律,以探索缺氧预适应小鼠脑匀浆液对缺氧、NGF诱导分化的PC12细胞只起保护作用、没有损伤作用的最适浓度。
第三部分:缺氧预适应小鼠脑匀浆液不同组分对NGF诱导分化的PC12细胞缺氧耐受性的影响及其可能有效成分的初步探讨
(1).以高氯酸除蛋白法制备缺氧预适应小鼠脑匀浆去蛋白液,HPLC法测定其腺苷含量。用小鼠脑匀浆去蛋白液作用于NGF诱导分化PC12细胞,以确定去蛋白液对缺氧的分化PC12细胞有无保护作用;以小鼠脑匀浆去蛋白液合并腺苷A1或A2A受体阻断剂作用于分化PC12细胞,以确定小鼠脑匀浆去蛋白液的作用是否与腺苷有关。
(2).透析法除去缺氧预适应小鼠脑匀浆液中小分子物质,并观察透析保留液对缺氧的分化PC12细胞有无保护作用;分别用RT-PCR、Western blot方法检测缺氧预适应小鼠脑组织中VEGF mRNA、VEGF的蛋白水平是否增加;用重组VEGF纯品作用于分化PC12细胞,以确定透析保留液对分化PC12细胞的保护作用是否与VEGF有关。
二、结果与讨论
通过上述3部分的工作获得以下结果
(一)影响急性重复缺氧模型的因素
1.小鼠经4次急性重复缺氧后,每次缺氧耐受时间显著增加。这一结果提示,小鼠在每次缺氧后,均在体内产生了增强缺氧耐受性的物质,并且各次缺氧后缺氧耐受的物质有积累效应。
2.在小鼠出现第一次喘呼吸时将小鼠换入下一个缺氧瓶与小鼠呼吸频率在40次/min时换瓶比较,可显著提高小鼠每次耐受时间。这是因为小鼠在呼吸频率为40次/min时的缺氧时间比出现喘呼吸时的缺氧时间短,缺氧程度尚未达到缺氧耐受形成的程度,因此对缺氧的耐受性也不强,因为缺氧预适应的形成需要足够程度的缺氧。
3.在小鼠出现第一次喘呼吸时将小鼠换入下一个缺氧瓶与小鼠出现末次喘呼吸时换瓶比较,缺氧耐受时间无显著差异。其原因是,不同小鼠喘呼吸次数不同,有的小鼠喘呼吸3-5次即死亡,有的小鼠喘呼吸却可达30次以上,因此小鼠的个体差异较大,使组间无显著差异;并且由于末次喘呼吸后小鼠随即死亡,因此末次喘呼吸难以判断,以其作为结束缺氧标准导致小鼠死亡率增加。
4.随着环境温度的增加,小鼠的缺氧耐受时间显著降低。这可能是因为低温可减少小鼠体内有害物质的产生、降低代谢,有利于缺氧预适应的形成。
5.每次对小鼠换瓶操作时间长于15秒会显著降低小鼠的缺氧耐受时间。其原因可能是复氧时间太长,因为复氧时间短暂有利于预适应的形成。由于经过重复4次缺氧的小鼠恢复常氧30分钟后,其缺氧耐受性还可以部分保留,说明小鼠4次缺氧耐受性的完全消失需要至少30分钟以上的时间,因此赋予预适应小鼠缺氧耐受性的物质,可能部分对机体恢复常氧供应较为敏感,但部分能维持较长时间。
(二)缺氧预适应小鼠脑匀浆液对未用NGF诱导的PC12细胞、HepG2细胞、NGF诱导分化的PC12细胞缺氧耐受性的影响
1.缺氧预适应小鼠脑匀浆液对缺氧、未用NGF诱导的PC12细胞、HepG2细胞缺氧耐受性的影响是:缺氧预适应小鼠脑匀浆液可显著提高细胞对缺氧的耐受性。在缺氧初期(24h),较高浓度的保护作用显著强于低浓度的保护作用,这提示缺氧预适应小鼠脑组织中产生了某种或某些可以增强细胞缺氧耐受性的物质或使脑组织中抗缺氧成分表达增加。随着缺氧时间延长,保护作用下降,高浓度的下降更快。至缺氧72h,高浓度提取液的缺氧损伤作用显著高于低浓度组,该结果提示,缺氧预适应小鼠脑组织中可能产生了半衰期较短的抗缺氧物质及在高浓度时才显示作用的、作用时间持久的促缺氧细胞损伤的物质,即提示缺氧预适应有着双相效应,缺氧预适应小鼠脑匀浆的作用存在量效关系和时间效应。但是由于本研究的目的是为了寻找缺氧预适应小鼠脑匀浆液中增强缺氧耐受性的物质,因此实验中应避免损伤性物质的影响。为此,在后续缺氧预适应小鼠脑匀浆液对分化PC12细胞的作用研究中,以蛋白浓度对提取液进行定量,并降低实验中所用提取液的浓度范围,以期找到提取液对NGF诱导分化的PC12细胞只起保护作用,无损伤作用(即只增强NGF诱导分化的PC12细胞缺氧耐受性,对分化PC12细胞无损伤作用)的最适浓度,为进一步研究缺氧预适应小鼠脑匀浆液的保护效应及保护性物质奠定基础。
2.经较低浓度的缺氧预适应小鼠脑匀浆液对NGF诱导分化的PC12细胞的作用实验发现,蛋白终浓度为100.0μg/mL的缺氧预适应小鼠脑匀浆液与同浓度正常小鼠脑匀浆液比较,可显著增强NGF诱导分化的PC12细胞的缺氧耐受性,并且此浓度的缺氧预适应小鼠脑匀浆液对分化PC12细胞未显示出损伤作用,为增强分化PC12细胞缺氧耐受性的适合浓度。
(三)缺氧预适应小鼠脑匀浆液不同组分对NGF诱导分化的PC12细胞缺氧耐受性的影响及其可能有效成分的初步探讨
1.向离体培养的NGF诱导分化的PC12细胞中加入不同浓度的腺苷,随着腺苷浓度增加,细胞的缺氧耐受性增高,10.0μmol/L的腺苷对细胞的保护作用可维持24h。急性4次重复缺氧可使小鼠脑组织腺苷含量上升。缺氧预适应小鼠脑匀浆去蛋白液只在缺氧的24h内显著增加分化PC12细胞的缺氧耐受性,腺苷A2A受体阻断剂可阻断其保护作用。正常小鼠脑匀浆去蛋白液对分化PC12细胞无保护作用。因此,缺氧预适应小鼠脑匀浆液的非蛋白组分的抗缺氧作用时间较短,腺苷可能是其中一个重要的活性物质。
2.在缺氧24h和48h,缺氧预适应小鼠脑匀浆透析保留液与未经透析的缺氧预适应小鼠脑匀浆液对分化PC12细胞均有保护作用,且经两种孔径(3.5kD、7.0kD)透析袋透析的缺氧预适应小鼠脑匀浆透析保留液和未经透析的缺氧预适应小鼠脑匀浆液对缺氧细胞的保护作用无显著差异,说明缺氧预适应小鼠脑组织中起保护作用的主要是分子量大于7.0kD的蛋白成分。急性重复缺氧预适应小鼠脑组织中VEGF表达虽增加,但应用重组VEGF直接作用于分化的PC12细胞发现,不同浓度的VEGF对NGF诱导分化的、不同缺氧时间的PC12细胞均无保护作用。这一结果提示:缺氧预适应小鼠脑匀浆液的蛋白组分抗缺氧作用较为持久,其中VEGF表达量虽增高,但并不参与NGF诱导分化的PC12细胞的抗缺氧作用。由于缺氧预适应小鼠脑匀浆液的蛋白组分抗缺氧作用时间持久,提示蛋白质组学方法可能是研究其中抗缺氧成分的有效方法,因此本研究结果为今后进一步研究缺氧预适应小鼠脑匀浆液中抗缺氧物质提示了方向。
三、结论
1.赋予重复缺氧小鼠缺氧耐受性的物质,部分对机体恢复常氧供应较为敏感,部分则能持续较长时间;
2.缺氧预适应小鼠脑匀浆液在浓度较高时对离体缺氧培养细胞的作用表现为双相性--缺氧早期,脑匀浆液对缺氧细胞具有保护效应,延长缺氧时间,其损伤作用逐渐显现,这种双相作用存在量效关系和时间效应;其只起保护作用的浓度是在较低浓度;
3.缺氧预适应小鼠脑匀浆液可显著提高不同来源离体培养细胞的缺氧耐受性,提示对缺氧细胞具有普遍的保护作用;
4.缺氧预适应小鼠脑匀浆液的蛋白组分抗缺氧作用较为持久,其中VEGF表达量虽增高,但并不参与NGF诱导分化的PC12细胞的抗缺氧作用;
5.缺氧预适应小鼠脑匀浆液的非蛋白组分的抗缺氧作用时间较短,腺苷可能是其中一个重要的活性物质。
Hypoxia can lead to various kinds of reactions in animal or human bodies to compensate for hypoxic influence. But the extent to which animal or human bodies fight against hypoxia, namely tolerate hypoxia is limited. However, recent investigations have showed that the tolerance of the bodies to hypoxia can be enhanced through such measures as hypoxic preconditioning (HP). HP referrs to the phenomenon that a short duration of moderate hypoxia or sub-lethal hypoxia enables animals, tissues or cells to tolerate subsequent longer time of hypoxia or more severe hypoxic insult, whose mechanism is related not only to the changes in expressions of various accommodative proteins mediated via HIF-1, but also to energy metabolites. In particular, ATP catabolite is involved in HP mechanism.
Brain is the most vulnerable organ in animal and human bodies and the main limiting factor influencing tolerance of the bodies to hypoxia. Therefore, the changes in functions and substances in central nervous system may be implicated in the main mechanism of tolerance formation. Researches have indicated that not only the alteration in substances in brain plays important roles in hypoxia tolerance formation, but also the brain homogenate of hypoxic-preconditioned mice (HP mice) could improve the hypoxic tolerance of animals and in vitro cells that do not undergo hypoxia. It is of both great theoretical and practical significance to search for the bioactive substances in the brain homogenate of HP mice, which can enhance the capacity of tolerating hypoxia.
For this purpose, the stable, reliable and significantly effective model of HP mice was reproduced based on observing the influence of different experimental conditions on the model of acute repetitive hypoxic preconditioning of mice Because individual animals differ in tolerance to hypoxia and their individual tolerance is easily affected by many other influential factors, cells in vitro were used to investigate the influence of brain extract of acute repetitive hypoxic-preconditioned mice (BEAHPM) on the tolerance of the cells in order to easily control experimental conditions and to explore the properties of the tolerance-enhancing substances. On the basis of certifying the fact that BEAHPM had biphasic effects on non-NGF-differentiated PC12 cells and HepG2 cells, NGF-differentiated PC12 cells alone was used in the subsequent experiments to determine the optimal concentration only showing protective effects on differentiated PC12 cells. Moreover, the extract of HP mice was further prepared in the form of protein portion and in the form of non-protein portion respectively to study the possible constituents enhancing cell tolerance to hypoxia. The experiments consist of three parts.
1. Methods
1.1 Part I. The mice model of acute repetitive hypoxic preconditioning was reproduced.
1.1.1 Mice were exposed to hypoxia till death to show their activity changes before their death.
1.1.2 Mice were subjected to four times of consecutive hypoxia to show the changes in hypoxic tolerance time.
1.1.3 Mice were divided into two groups to undergo four times of consecutive hypoxia, and hypoxia was stopped for the first group when the respiratory frequency of mice was about 40 times per minute but hypoxia was stopped for the second group when the first asthmoid respiration of mice appeared respectively, aiming at observing the influence of different hypoxia duration on mice tolerance.
1.1.4 Mice were divided into two groups to undergo four times of consecutive hypoxia. The standard of ending hypoxia was when the first asthmoid respiration appeared or when the last asthmoid respiration appeared respectively.
1.1.5 Mice were divided into four groups to experience four times of consecutive hypoxia, while the environmental temperature was 9-11℃, 13-15℃, 17-19℃and 21-23℃respectively to determine the influence of different environmental temperatures on the tolerance to hypoxia.
1.1.6 Mice were divided into two groups for four times of consecutive hypoxia, the operating time between two hypoxias was 5-15s or 20-30s respectively to determine the influence of different re-oxygenation time on mice tolerance.
1.1.7 The mice exposed to four times of consecutive hypoxia were transferred to normoxic environment. 30 minutes later, they were subjected to hypoxia once again to observe the change in tolerance time.
1.2 Part II. The effects of BEAHPM on tolerance of PC12, HepG2 and NGF-differentiated PC12 cells to hypoxia
1.2.1 BEAHPM at different concentrations were used to treat non-NGF-differentiated PC12 cells and the changes in cell vitality, release rate of lactate dehydrogenase (LDH), apoptotic rates at early and late stages of hypoxia were determined to find out the effects of BEAHPM on the cells.
1.2.2 BEAHPM at different concentrations were used to treat HepG2 cells and the changes in cell vitality, apoptotic rates of HepG2 cells at early and late stages of hypoxia were observed to investigate the effects of BEAHPM on non-neuronal cells.
1.2.3 According to the results of avove experiments in Part II, BEAHPM at relatively low concentrations were used to treat NGF-differentiated PC12 cells and the changes in cell vitality, LDH release rate, apoptotic rates at late stages of hypoxia were examined to determine the optimal concentration of BEAHPM to protect NGF-differentiated PC12 cells against hypoxia without showing injurious effects simultaneously.
1.3. Part III. The effects of different components of BEAHPM on tolerance of NGF-differentiated PC12 cells to hypoxia and preliminary researches into possible effective constituents
1.3.1 Deproteinized BEAHPM was prepared by deproteinization with perchloric acid and adenosine in deproteinized BEAHPM was quantified by HPLC (high performance liquid chromatography). The effects of deproteinized BEAHPM on NGF-differentiated PC12 cells were determined to clarify whether it had protective effects on the cells. Then the effects of deproteinized BEAHPM plus antagonists of adenosine A1 or of A2A receptor were tested to show whether the effects were relevant to adenosine.
1.3.2 Dialysis method was used to remove small molecules from BEAHPM to observe whether protein-retained solution has protective effects on NGF-differentiated PC12 cells. RT-PCR and Western blot were adopted to check levels of VEGF (vascular endothelial growth factor) mRNA and VEGF protein in the brain of HP mice. Recombined VEGF was used to investigate whether VEGF alone had protective effects on differentiated PC12 cells in hypoxia.
2 Results and discussion
The following results are obtained from above three parts of work.
2.1 The factors influencing the mice tolerance in the model of acute repetitive hypoxic preconditioning
2.1.1 After four times of acute repetitive hypoxia, the hypoxic tolerance time of mice was obviously increased, revealing that after each time of hypoxia, the substance (or substances) for tolerance was produced and they were accumulative in HP mice.
2.1.2 The tolerance time of the group which was transferred into the next hypoxic container when the first asthmoid respiration appeared was significantly longer than that of the group which was put into the next hypoxic container when the respiration frequency was 40 times per minute. The cause of this result was that the hypoxic duration when the respiration frequency was 40 times per minute was shorter than that when the first asthmoid respiration appeared, and that the formation of tolerance to hypoxia needs sufficient degrees of exposure to hypoxia. Therefore, hypoxic degree was not enough for mice to form tolerance if mice were transferred into the next hypoxic container when the respiration frequency was 40 times per minute.
2.1.3 The tolerance time of the group which was transferred into the next hypoxic container when the first asthmoid respiration appeared was not significantly different from that of the group which was put into the next hypoxic container when the last asthmoid respiration appeared. One cause of the results was that asthmoid respiration times were different in different mice. Some mice died after 3 or 5 times of asthmoid respirations, but some mice died after 30 times of asthmoid respirations, resulting in great individual differences in tolerance time within the latter group. The second cause was that mice died almost immediately after the last asthmoid respiration so it is difficult to judge the last asthmoid respiration, which increased mice mortality in the latter group.
2.1.4 With the increase of environmental temperature, the tolerance time was decreased significantly. The cause might be that low temperature could reduce the production of detrimental substances in mice and could also decrease the metabolism and be helpful to tolerance formation.
2.1.5 If the time for transferring mice into the next container was longer than 15 seconds, the tolerance time was significantly decreased. The reason for this result may be that when re-oxygenation time is too long, the tolerance degree was decreased because short time for re-oxygenation is favorable for tolerance formation. Because the effects of tolerance could be partly retained 30 minutes after the mice exposed to four times of acute repetitive hypoxia returned to nomorxia, the complete disappearance of tolerance needed more than 30 minutes and it could be deduced that some tolerance-enhancing substances remained stable but some were susceptible to re-oxygenation.
2.2 Part II. The effects of BEAHPM on tolerance of non-NGF-differentiated PC12 cells, HepG2 cells and NGF-differentiated PC12 cells
2.2.1 The effects of BEAHPM on non-NGF-differentiated PC12 cells and HepG2 cells was that it could significantly enhance the tolerance of these two cell strains to hypoxia. At the initiate time of hypoxia (24 h of hypoxia), the protective effect of BEAHPM at relatively high concentrations was stronger than that at the relatively low concentrations, indicating that some tolerance-enhancing substances were produced or the expressions of anti-hypoxia substances were up-regulated in the brain of HP mice. With the prolongation of hypoxic time, protective effects of BEAHPM decreased. And the effects of BEAHPM at high concentrations decreased faster than those at low concentrations. After 72-hour hypoxia, BEAHPM at high concentrations showed injurious effects on these two cells. This result indicated that anti-hypoxia substances with short half-time and detrimental substances showing long-lasting injurious effects at high concentrations were produced. Namely, acute hypoxic preconditioning had biphasic effects and the biphasic effects of BEAHPM were concentration-dependent and time-dependent. Because the objiective of this research is to try to roughly locate the tolerance-enhancing substances in BEAHPM, the effects of damage-enhancing substances should be avoided.
For this purpose and based on above results, protein concentration was used to quantify BEAHPM and BEAHPM in a lower concentration range was used in the subsequent experiments on the effects of BEAHPM on NGF-differentiated PC12 cells in order to find out the optimal concertration of BEAHPM only showing protective effects on NGF-differentiated PC12 cells and not showing damage effects on NGF-differentiated PC12 cells. Namely, the optimal concentration only enhances tolerance of NGF-differentiated PC12 cells and does not show injurious effects on NGF-differentiated PC12 cells, the experimental design aiming at laying a foundation for further researches into the protective effects and protective substances in BEAHPM.
2.2.2 The results of the effects of BEAHPM in a relatively lower concentration range on NGF-differentiated PC12 cells showed that BEAHPM at final protein concentration of 100.0ug/mL could significantly enhance the tolerance of differentiated PC12 cells to hypoxia without showing harmful effects on the cells compared with the brain extract of normal mice at the same protein concentration.
2.3 Part III. The effects of deproteinized BEAHPM, dialyzed protein-retained solutions of BEAHPM, adenosine and VEGF on hypoxic tolerance of NGF-differentiated PC12 cells.
2.3.1 The tolerance of NGF-differentiated PC12 cells could be enhanced by addition of adenosine at different concentrations. Besides, the tolerance of differentiated PC12 cells could be increased as the concentrations of adenosine increased. Adenosine at 10.0μmol/L could protect cells only within 24-hour hypoxia. The content of adenosine was up-regulated in mice brain through four times of acute repetitive hypoxia. The deproteinized BEAHPM showed its protective effects on differentiated PC12 cells only within 24 hours of hypoxia and deproteinized brain extract of normal mice showed no protective effects on differentiated PC12 cells. Antagonist of adenosine A2A receptor could inhibit the protective action of deproteinized BEAHPM and the protective action of adenosine, so adenosine might be an active protective constituent.
2.3.2 After 24-hour and 48-hour hypoxia, the dialyzed protein-retained solutions of BEAHPM, and non-dialyzed BEAHPM protected NGF-differentiated PC12 cells against hypoxia. Moreover, the protective effects of the dialyzed protein-retained solution through 3.5kD bag filter or through 7.0kD bag filter did not significantly differ from the effects of non-dialyzed BEAHPM, suggesting that the protective substances in the brain of HP mice were proteins with molecular weight larger than 7.0kD. Although the expression of VEGF in the brain of HP mice was significantly increased, recombined VEGF at different concentrations had no protective effects on differentiated PC12 cells after different durations of hypoxia. These results revealed that the protein constituents in BEAHPM had long-lasting anti-hypoxia action, but the up-regulation of VEGF expression was not involved in the anti-hypoxia effects on differentiated PC12 cells. The long-lasting effects of the protein constituents in BEAHPM suggest that proteomics methods might be the effective ways of investigating the anti-hypoxia substances in BEAHPM, giving insight into the focus of future research efforts.
3. Conclusions
3.1 Some substances endowing mice exposed to acute repetitive hypoxia with tolerance were relatively susceptibly to re-oxygenation, but some had long-lasting protective effects.
3.2 BEAHPM had biphasic effects on in vitro cells subjected to hypoxia. Namely, at the early stage of hypoxia, BEAHPM protected cells against hypoxia. As time for hypoxia prolongs, it gradually showed damage action. This biphasic effects were concentration-dependent and time-dependent.
3.3 BEAHPM could significantly improve the tolerance of in vitro cells of diverse origins, indicating that BEAHPM had universal protective effect on hypoxic cells.
3.4 The protein constituents in BEAHPM had long-lasting protective effects. VEGF expression in BEAHPM increased significantly, but VEGF was not involved in anti-hypoxia effects on differentiated PC12 cells.
3.5 The non-protein constituents in BEHPM only had short-lived protective action and adenosine might be an important active substance.
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