| With the development of economy and the reason of military, more and more people go to west plateau in our homeland. To most people high altitude exposure will cause hypoxia. Hypoxia is a state of oxygen deficiency in the body which is sufficient to lead to body function impairment and it may cause a variety of psychological and neurological abnormalities. So for many years researchers are working on finding out anti-hypoxia medicine. Studies indicate that many chemical drugs, such as acetazolamide, dexamethasone and nimodipine, show the effects of preventing or alleviating symptoms of mountain sickness. But most of these drugs also bring about a lot of side effects and so can't be used in some conditions. Many traditional Chinese medicines, such as ginseng, rhodlola eoccinea and gingko, have anti-hypoxia effects too. But most of them are expensive and rare and so limited their uses.Portulaca oleracea L. (PO) has a cosmopolitan distribution and is one of the most common traditional Chinese medicines. It has been reported to be rich in noradrenaline,α-linolenic acid,β-carotene, vitamin E and carotene. Modern researches have showed that this plant exhibited a wide range of pharmacological effects, such as anti-bacteria, anti-virus, anti-inflammatory, wound-healing, regulating serum lipids, anti-aging and so on. In our previous research we found that the Portulaca oleracea L. aqueous extracts significantly lengthened the survival time of hypoxic mouse exposed to the normal pressure closed bottles or potassium cyanide. Our preliminary experiment showed that PO aqueous extracts enhanced the activities of pyruvate kinase (PK), phosphofructokinase (PFK), and the level of ATP and HIF-1αof the hypoxic mouse cortices. But Portulaca oleracea L. aqueous extracts was a very complex mixture. It would cause a lot of difficulties in our later research work. In present study, we examined further the effects of the extraction on hypoxia models mice and neuron induced by the normobaric and chemical hypoxia so as to elucidate the anti-hypoxia activity, possible underlying mechanism and provide scientific basis for the clinical use of Portulaca ObjectiveTo study further the effects of the extraction on hypoxia models mice and neuron induced by the normobaric and chemical hypoxia so as to elucidate the anti-hypoxia activity, possible underlying mechanism and provide scientific basis for the clinical use of Portulaca oleracea.Method1. Portulaca oleracea L. activity screening and dose-effect relationship1.1 The Portulaca oleracea L. sample preparationThe aerial parts of Portulaca oleracea L. were collected in Henan province PR China in October 2006 and authenticated by Prof. Zheng HanChen. A voucher specimen has been deposited in Department of Traditional Chinese Medicine (TCM), Second Military Medical University. The powdered of aerial parts of Portulaca oleracea L.40 kg was refluxed with eight times of 80% ethanol solution for 2 times, one hr each time. The extract was concentrated under reduced pressure to 80 L, then to centrifuge at 5000 rpm for 4 min, and the precipitation part (680.8 g) as tested Material.1.2 normobarie hypoxia testIn the normobarie hypoxia test, groups of overnight fasted mice (n= 10) were treated orally with the PO, vehicle (10ml/kg) or ginseng which was included as a positive control. Drugs were treated once a day for a week.30 minutes after last treatment, each mouse was put into a 200ml airtight container with some medical soda lime inside. The survival time of oxygen deprivation was recorded.1.3 Different doses of normobarie hypoxia testGroups of overnight fasted mice (n= 10) were treated orally with Different doses of the PO, vehicle (10ml/kg) or ginseng which was included as a positive control. Drugs were treated once a day for a week.30 minutes after last treatment, each mouse was put into a 200ml airtight container with some medical soda lime inside. The survival time of oxygen deprivation was recorded.1.4 Sodium nitrite toxicosis testIn the sodium nitrite toxicosis test, groups of overnight fasted mice (n= 10) were treated orally with PO, vehicle (10ml/kg) or ginsen once a day for a week.30 minutes after last treatment, each mouse was given sodium nitrite (800mg/kg) into vena caudalis. Then the survival time was recorded.1.6 GC-MS analysis on Major constituents from the PO extract.GC/MS analyses were performed using the Finnigan Vayager equipped with a VF-5 ms fused-silica capillary column. The column temperature was programmed at 100℃(2 min), increasing to 300℃at rate of 15℃/min, injector temperature was 250℃; Carrier gas (helium) was set at a flow rate of 1.0 ml/min; ionization energy 70 eV, and scan mode EI.1 ml of sample was injected with split ratio of 1:30 (v/v) and the compounds were identified by matching their mass spectra and retention times with standard obtained from NIST whenever possible.2. Protective effect of PO extracts on hypoxic nerve tissue2.1 Preparation of herb-containing serumAccording to the literature, SD rats were used for the preparation of herb-containing serum. Being orally administrated with the PO extracts, twice a day for five days, the rats were anesthetized with diethylether and sacrificed one hour after the last drug administration. The blood was collected and stored at 4℃for 24 hours, then centrifuged for 10 min at 1000 r/m. The serum was collected and inactivated at 56℃for half an hour, and stored for future use.2.2 Cell studyFor in vitro experiments the PC-12 cells or primary cultured newborn rats cortex neuron cells were adapted in the incubator filled with the mixture of nitrogen and oxygen, or cultured in the medium coupled with CoCl2. The cell viability was determined by CCK-8 methods. The LDH concentration in the supernatant in the culture medium was determined by LDH detection kit. Cell apoptosis was analyzed by flow cytometry.2.3 Animal stydyICR mice (20-24 g) were purchased from the Shanghai-BK Ltd. Co and were bred in a temperature-controlled ((24±1)℃), (55±5)% humidity room with a 12-hour light and 12-hour dark cycle. After adaptation for three days, the mice were divided into control group,6 hour hypoxia exposure group,12-hour hypoxia exposure group and 24-hour hypoxia exposure group. Each group was sub-divided into distilled water group, PO group, and the mice were orally administrated with distilled water and PO, one milliliter every day for seven days, respectively. One hour after the last drug administration, mice in the hypoxia exposure groups were adopted normobaric low oxygen environment (8% oxygen and 92% nitrogen) for 6,12 or 24 hours. Mice in the control group were sacrificed one hour after the last drug administration, and mice in the hypoxia exposure groups were sacrificed immediately after hypoxia administration, and the mouse cortices were collected for analysis. The evaluation of cerebral cortex damages was performed by normal histopathological methods and scored in a blind way. The serum was collected and the concentration of neuronspecific enolase in the serum was determined by immunosorbent assay. Part of the cortex tissues were selected for cell apoptosis detection by ways of determination of caspase-3 activity.3. Study on mechanisms of the protective effect of PO extract on hypoxic nerve tissue3.1 Cell studyFor primary cultured newborn rats cortex neuron cells were adapted in the incubator filled with the mixture of nitrogen and oxygen. Cells were divided into four groups. (1) No-hypoxic control group, (2) Hypoxic group, (3) Hypoxic group treat with PO, (3) Hypoxic group treat with PO and LY294002. The cell viability was determined by CCK-8 methods. HIF-1α,P-AKT,AKT level were determined by Western blot. 3.2 Animal studyICR mice (20-24 g) were purchased from the Shanghai-BK Ltd. Co and were bred in a temperature-controlled ((24±1)℃), (55±5)% humidity room with a 12-hour light and 12-hour dark cycle. After adaptation for three days, the mice were divided into no-hypoxic control group, Hypoxic group, Hypoxic group treat with low, middle, high doses of PO groups. One hour after the last drug administration, mice in the hypoxia exposure groups were adopted normobaric low oxygen environment (8% oxygen and 92% nitrogen) for 12 hours. Mice in the control group were sacrificed one hour after the last drug administration, and mice in the hypoxia exposure groups were sacrificed immediately after hypoxia administration, and the mouse cortices were collected for analysis. The activities of pyruvate kinase (PK), phosphofructokinase (PFK), lactic acid (LD), the level of lactate dehydroenase (LDH) and adenosine triphosphate (ATP) were detected by the common methods and ATP kits. HIF-1α,P-AKT level were determined by Western blot.4. Statistical analysisAll results were expressed as mean±SD. Statistical analysis was carried out by using SPSS 11.0. All values below the detection limits were set to zero and absolute values without correction for recovery rate were used in analyses. P value less than 0.05 was considered statistically significant.Results1. Portulaca oleracea L. activity screening and dose-effect relationship1.1 Effect on the normobarie hypoxia testIn the normobarie hypoxia test, the treatment with the extraction prolonged significantly the survival time of oxygen deprivation (p<0.01). The data showed that the extraction had an effect of increasing the survival time in mice exposed to hypoxia.1.2 Effects of different doses of PO on the normobarie hypoxia test, the sodium nitrite toxicosis testIn the normobarie hypoxia test, the treatment with different doses of PO prolonged significantly the survival time of oxygen deprivation. At the same time, in the sodium nitrite toxicosis test, the treatment with different doses of PO could also significantly prolonged the survival time (P<0.05). Data showed that the PO had a dose-dependent effect of increasing the survival time in mice in different tests.1.3 Main Chemical compounds in the of PO extractsThe results of GC-MS analysis on the showed predominance of terpenoids and unfatty acids. Forty-four constituents were identified. We can got their concentration, according to the area they occupied. Terpenoids and unfatty acids might be the main antihypoxic ingredients2. Protective effect of PO extracts on hypoxic nerve tissue2.1 Protective effect of PO extracts on hypoxic PC-12 cell and neuronPO extracts enhanced the viability of the PC-12 cells or primarily cultured newborn rats cortex nerve cells exposed to hypoxia, at the same time decreased the LDH level of the supernatant in the cultured medium. PO extracts can also reduced cell apoptosis in some extent.2.2 Protective effect of PO extracts on hypoxic mice brainIn the hypoxic conditions, the degree of the mouse brain damage and the activity of neuronspecific enolase increased with the prolongation of hypoxic time. PO extracts reduced the damage of cortices of hypoxia mouse. The degree of the mouse brain damage and the activity of neuronspecific enolase were significant lower than that in the corresponding control group. PO extracts lowered the serum concentration of caspase-3 in hypoxia mice brain.3. Study on mechanisms of the protective effect of PO extract on hypoxic nerve tissue3.1 Effect of PO extracts on activities of hypoxic primarily cultured newborn rats cortex nerve cellsCCK-8 test showed that the activities of primarily cultured newborn rat cortex nerve cells got lower under hypoxic condition (0% O2,6h). But the group treated with PO was higher than hypoxia group (p<0.05). At the same time the group treated with PO and LY294002 was lower than the group treated with PO only, but still higher than hypoxia group.3.2 Effect of PO extracts on PI3K/AKT signal way of hypoxic primarily cultured newborn rats cortex nerve cellsThe study showed that the level of P-AKT in hypoxic group was higher than normal culture cells. P-AKT of the group treated with PO was higher than hypoxia group (p<0.05). At the same time P-AKT of the group treated with PO and LY294002 was lower than the group treated with PO only, but still higher than hypoxia group. Interestingly, total AKT was not changed by PO or LY294002 (p>0.05).3.3 Effect of PO extracts on HIF-la of hypoxic primarily cultured newborn rats cortex nerve cellsHIF-1αwas not detected under normal primarily cultured newborn rats cortex nerve cells. Under hypoxic condition, HIF-1αof primarily cultured newborn rats cortex nerve cells accumulated. HIF-1αof the group treated with PO was higher than hypoxia group (p<0.05). At the same time HIF-1αof the group treated with PO and LY294002 was lower than the group treated with PO only, but still higher than hypoxia group. 3.4 Effect of PO extracts on HIF-la and P-AKT of hypoxic mice brainHIF-1αwas not detected in normal mice. Under hypoxic condition, HIF-1αof mice brain accumulated. HIF-1αof the group treated with different doses of PO was higher than hypoxia group (p<0.05). It has dose-dependent effect. The change of P-AKT was just like the same with HIF-1α. 3.5 Effect of PO extracts on activities of PFK, PK, and LDH in hypoxic mice brainActivities of PFK, PK, and LDH in hypoxia group were higher than normal group. Activities of PFK, PK, and LDH in the group treated with different doses of PO were higher than hypoxia group (p<0.05). It has dose-dependent effect.3.6 Effect of PO extracts on level of ATP in hypoxic mice brainLevel of ATP in hypoxia group were higher than normal group. Level of ATP in the group treated with different doses of PO were higher than hypoxia group (p<0.05). The data showed that PO extracts had a dose-dependent effect.Conclusions1. Our research showed that the treatment with different doses of PO prolonged significantly the survival time of oxygen deprivation in the normobarie hypoxia test. At the same time, in the sodium nitrite toxicosis test, the treatment with different doses of PO could also prolonged significantly the survival time(P<0.05). Data showed that the PO had a dose-dependent effect of increasing the survival time in mice in different tests.2. Our study demonstrated that PO extracts had Protective effect on hypoxic nerve tissue in vivo/vitro. In vitro, PO extracts enhanced the viability of the PC-12 cells or primarily cultured newborn rats cortex nerve cells exposed to hypoxia, at the same time decreased the LDH level of the supernatant in the cultured medium. PO extracts can also reduced cell apoptosis in some extent. In vivo, the degree of the mouse brain damage and the activity of neuronspecific enolase increased with the prolongation of hypoxic time. PO extracts reduced the damage of cortices of hypoxia mouse. The degree of the mouse brain damage and the activity of neuronspecific enolase were significant lower than that in the corresponding control group. PO extracts could also decrease the serum concentration of caspase-3 in hypoxia mice brain.3. We believed that PO extract could increase level of P-AKT in hypoxic mice, which leaded to activate the signal way of PI3K/AKT. The level of HIF-1αbecame stable because of the activation of this signal way, which was house-keeping gene of hypoxia. But the LY294002 could block the effect of PO in some extend, which was the specific blocker of PI3K/AKT. Furthermore, PO could not only increase Activities of PFK, PK, and LDH, but also improve the level of ATP in hypoxic mice. These results demonstrated that the PO extract possessed notable anti-hypoxic activity, which might be related to promoting the activity of PI3K/AKT signal way and than stabilize HIF-1α, which lead to active hypoxic adaptive target gene in hypoxic mice.In conclusion, our study found that PO extract could prolonged the survival time of oxygen deprivation and possessed notable protective effect on hypoxic nerve tissue in vivo/vitro, which might be related to promoting the activity of PI3K/AKT signal way and than stabilize HIF-1α, which lead to active hypoxic adaptive target gene in hypoxic mice.
|
PDF Full Text Request