A Preliminary Research Into The Effects Of The Brain Extract Of Hypoxic-preconditioned Mice On The Tolerance Of Cells To Hypoxia And Into The Possible Effective Constituentes

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. Methods1.1 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 hypoxia1.2.1 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 constituents1.3.1 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 discussionThe 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 preconditioning2.1.1 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 cells2.2.1 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. Conclusions3.1 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.