| Beta-asarone, a major component of Acorus tatarinowii Schott, has significant pharmacological effects on the central nervous system. Our previous studies suggested that it can attenuate ischemia-reperfusion brain injury via attenuating the neuronal apoptosis and infarction. Autophagy plays important roles in cell survival in ischemia-reperfusion brain injury. But Beta-asarone's effects on autophagy have not been reported yet.Our study was a four-stage procedure. The first stage was a study on the preparation of β-asarone. The second stage is a study on the time-course analysis of the Beclin1in the rat brain's core, penumbra, and unaffected areas and anlysis the differences among the core, penumbra, and unaffected areas after middle cerebral artery occlusion. The third stage is an evaluation of the β-asarone effects on ischemia-reperfusion induced brain injury and on the Beclin1levels in the rat brain's core, penumbra, and unaffected areas. The last stage is an analysis of the possible mechanism of3-asarone' effects on autophagy in the rat brain's core, penumbra, and unaffected areas. The details of each stage are as follows:1. The details of study in the first stage1.1Experimental purposes of this study:Experimental purposes of first stage are in order to extract the β-asarone from the Acorus tatarinowii Schott and then use the gas chromatography-mass spectrometry method to evaluate the purity of β-asarone.1.2Methods related this study:The β-asarone was obtained by four steps. The first step:the Acorus tatarinowii Schott identified as from the Araceae was purchased from the first affiliated hospital of Guangzhou University of Chinese Medicine. The second step, the volatile oil was extracted from the Acorus tatarinowii Schott. The third step:the volatile oil was purified by the freezing crystallization method and the β-asarone was obtained. The last step:the purity of β-asarone was confirmed with the gas chromatography-mass spectrometry, infrared spectrum and nuclear magnetic resonance detection.1.3Results:The β-asarone with a purity of99.55%is extracted from the Acorus tatarinowii Schott. The stability, precision and reproducibility of GC-MS analysis conditions were obtained by the precision test and the reproducibility test. This method prompts that the β-asarone can show peak completely within30minutes. In the precision test, the precision of GC-MS system was fine according that the coefficient variation of relative amount and retention time were less than1%and0.2%, respectively. In the stability test, the stability of β-asarone solution was good in120h according that the coefficient variation of relative amount and retention time were less than1%and0.3%, respectively. In the reproducibility test, the reproducibility of β-asarone was fine according that the coefficient variation of relative amount and retention time were less than1%and0.3%, respectively1.4Conclusions:We can extract β-asarone with high purity from the Acorus tatarinowii Schott and the gas chromatography-mass spectrometry method used to confirm the purity is simple and stable.2The details of study of the second stage2.1Experimental purposes of this study:Some data suggested that the ischemia-reperfusion brain injury can up-regulate autophagy level in vivo. However, little was known about the time-courses expression of the Beclin1levels in the rat Brain's the core (ipsilateral parietal cortex and ipsilateral lateral part of striatum), penumbra (ipsilateral frontal and ipsilateral motor cortex), and unaffected (contralateral parietal cortex) areas. The purposes of study in the second stage are to evaluate the time-courses expression of the Beclin1levels in the rat Brain's core, penumbra, and unaffected areas and analyze the differences among the three areas. 2.2Methods related this study:The Middle cerebral artery occlusion model was adopted to make the ischemia-reperfusion brain injury. Rats were randomly divided into seven groups:the sham group and six treated groups according to the reperfusion time:group A (reperfusion time=0hr), group B (reperfusion time=0.5hr), group C (reperfusion time=4hr), group D (reperfusion time=12hr), group E (reperfusion time=24hr), and group F (reperfusion time=72hr). The rats in the treated groups underwent the MCAO while the rats in the sham group underwent the same surgical procedures except for the MCAO. The neurologic findings were scored on a five-point scale:a score of0indicated no neurologic deficit, a score of1(failure to extend left forepaw fully) a mild focal neurologic deficit, a score of2(circling to the left) a moderate focal neurologic deficit, and a score of3(falling to the left) a severe focal deficit; rats with a score of4did not walk spontaneously and had a depressed level of consciousness. Scores were recorded when the sutures were removed. The rats died during the experiment and rats with a score of0or4were excluded without further analysis. Then, we divided the brains into three areas:core (ipsilateral parietal cortex and ipsilateral lateral part of striatum), penumbra (ipsilateral frontal and ipsilateral motor cortex), and unaffected (contralateral parietal cortex) areas and analyzed the time-course levels of the Becl in1in the three areas, respectively. Beclin1levels were evaluated. Enzyme linked immunosorbent assay was used to evaluate levels of the neuron-specific enolase, which is often used to evaluate the brain injury levels. Additionally, transmission electron was employed to observe the autophagy directly. Immunohistochemistry was used to evaluate the Beclin1qualitatively.2.3Results:2.3.1. Time-courses of Beclin1levels in the rat Brain's core, penumbra, and unaffected areas in MCAO ratsIn the core area, Beclin1was up-regulated beginning at0hr reperfusion injury peaking at24hr and lasting for72hr. Beclin1levels increased significantly in the MACO-treated groups. Compared to group A (reperfusion time=0hr), the groups with reperfusion (reperfusion time=0.5,4,12,24, and72hr) resulted in a significant increase of Beclin1levels (P<0.05). Compared to the group E (reperfusion time=24hr), the other groups (reperfusion time=0,0.5,4,12, and72hr) resulted in a significant decrea of Beclin1levels (P<0.05). But there was no significant increase among the three groups (reperfusion time=0.5, and4,12hr). In the penumhra and unaffected areas, the time course levels of Beclin1were almost similar with the core area. Beclin1was also up-regulated beginning at0hr reperfusion injury peaking at24hr and lasting for72hr.2.3.2. D i f f erences of the Beclin1levels between the i schem i a and reperfus i on injuryCompared to the sham group, the group A (reperfuion time=0hr) resulted in a significant increase of Beclin1levels(P<0.05). And compared to the group A (reperfusion time=0hr), the groups with reperfusion (reperfusion time>0hr) resulted in a significant increase of Beclin1levels (P<0.05).2.3.3. Autophagosome recorded by the transmission electronWe can find that there were many autophagosome in the core, penumbra, and unaffected areas in treated groups. But there are few autophagosome in the sham group.2.3.4. Beclin1recorded by the ImmunohistochemistryWe can find that there were many positive expressions of Beclin1in treated groups, which suggested that the Ischemia and reperfusion injury can up-regulate the Beclin1levels2.4Conclusions:Beclin1levels in the core, penumbra, and unaffected areas are up-regulated beginning at0hr reperfusion injury peaking at24hr and lasting for72hr.The up-regulation of Beclin1levels are related to the ischemia and reperfusion injury. At the early stage (without reperfusion), the up-regulation of Beclin1levels is mainly related to ischemia injury. At the later stage (reperfusion>0.5hr), the up-regulation of Beclin1levels is mainly related to reperfusion injury.Ischemia and reperfusion injury can induce autophagosome in the core, penumbra, and unaffected areas.3The details of study of the third stage3.1Experimental purposes of this study:The β-asarone, a major component of Acorus tatarinowii Schott, has significant pharmacological effects on the central nervous system. In our previous study, we found that β-asarone can decrease the ischemia-reperfusion brain injury by decreasing the apoptosis and necrosis in brain. But β-asarone's effects on the brain ischemia-reperfusion-induced autophagy have not been reported yet. Could the β-asarone be able to decrease the ischemia-reperfusion induced brain injury by decreasing autophagy? To answer this question, we did this study.Meanwhile, our previous data also suggest that β-asarone can pass through the blood-brain barrier and be widely distributed without target areas in the brain. Could the β-asarone be able to attenuate autophagy in a widespread manner or in a targeted area? To answer this question, we divided the brains of MCAO rats into three areas:the core (ipsilateral parietal cortex and ipsilateral lateral part of striatum), penumbra (ipsilateral frontal and ipsilateral motor cortex), and unaffected (contralateral parietal cortex) areas, and analyzed their Beclin1levels, respectively3.2Methods related this study:The middle cerebral artery occlusion model was adopted to make the ischemia-reperfusion injury. Rates were randomized into groups of10animals. The treatment was as follows:group I (sham),2mL/kg water intraperitoneally per day for4days; group II (model control),2mL/kg water intraperitoneally per day for4days; group III (low dose),2mL/kg of β-asarone (7.5mg/mL) intraperitoneally per day for4days; group IV (medium dose),2mL/kg of β-asarone (15mg/mL) intraperitoneally per day for4days; and group Ⅴ (high dose),2mL/kg of β-asarone (30mg/mL) intraperitoneally per day for4days. At1hr after the last administration, rats were anesthetized with intraperitoneal injection of3%chloral hydrate (350mg/kg) and then treated with MCAO model. The neurologic findings were scored on a five-point scale: a score of0indicated no neurologic deficit, a score of1(failure to extend left forepaw fully) a mild focal neurologic deficit, a score of2(circling to the left) a moderate focal neurologic deficit, and a score of3(falling to the left) a severe focal deficit; rats with a score of4did not walk spontaneously and had a depressed level of consciousness. Scores were recorded when the sutures were removed. The rats died during the experiment and rats with a score of0or4were excluded without further analysis. The brains were harvested and then divided into three areas:core (ipsilateral parietal cortex and ipsilateral lateral part of striatum), penumbra (ipsilateral frontal and ipsilateral motor cortex), and unaffected (contralateral parietal cortex) areasThe Beclin1levels were evaluated and the enzyme linked immunosorbent assay was used to evaluate levels of the neuron-specific enolase, which is often used to evaluate the brain injury levels.3.3Results:3.3.1. Evaluation of the β-asarone effects on autophagyCompared to the sham group, the Beclin1levels in the model control, low dose, medium dose, and high dose were significantly increased (P<0.05). Compared to both the model control and low dose groups, the Beclin1levels in the medium and high dose groups were significantly decreased. Meanwhile, there was no significant expression difference between the model control and low dose groups.3.3.2. The β-asarone can decrease the Beclin1levels in the core, penumbra, and unaffected areas at the same time.There were no significant differences among the core, penumbra and unaffected areas.3.3.3. Evaluation of β-asarone effects on NSE and neurologic scoresCompared to the sham group, the NSE levels and neurologic scores in the model control, low dose, medium dose, and high dose groups were significantly increased (P<0.05). Compared to both the model and low dose groups, the NSE levels and neurologic scores in the medium and high dose groups were significantly decreased. There was no significant difference between the model control and low dose groups. Meanwhile, there was no significant difference between the medium and high dose group.3.4Conclusions:The β-asarone can attenuate the ischemia-reperfusion-induced autophagy in a dose-dependent manner in MCAO rats. The rats with the medium dose (15mg/mL) and high dose (30mg/mL) are with significant decrease of Beclin1.The β-asarone can attenuate the ischemia-reperfusion-induced brain injury in a dose-dependent manner in MCAO rats. The rats with the medium dose (15mg/mL) and high dose (30mg/mL) are with significant decrease of NSE levels.The β-asarone can attenuate autophagy in a widespread manner in MCAO rats. There were no significant differences among its effects on the core, penumbra, and unaffected areas.4The details of study of the forth stage4.1Experimental purposes of this study:The data in the third stage suggest that the β-asarone can decrease ischemia-reperfusion-induced autophagy in a dose-dependent manner (medium dose,15mg/mL, and high dose,30mg/mL). The β-asarone also can decrease the ischemia-reperfusion-induced brain injury by decreasing the autophagy. Meanwhile, there were no significant differences among its effects on the core, penumbra, and unaffected areas in MCAO rats. But the possible mechanism of the β-asarone on autophagy has not been reported yet. The purpose of this study is to analyze its possible mechanism related to autophagy and brain injury.The β-asarone, a major component of Acorus tatarinowii Schott, has significant pharmacological effects on the central nervous system. We have reported that the β-asarone can increase the c-fos levels in rat brain. And we also found the Acorus tatarinowii Schott can increase the expression of the c-Jun, which is related to autophagy. We also found that the β-asarone can decrease the loss of nerve cells and can increase the levels of Bcl-2in brain. So we hypothesized that the mechanism might be related to modulating the JNK, p-JNK, and Bcl-2and Beclin1levels. Meanwhile, we divided the brain into three areas:core (ipsilateral parietal cortex and ipsilateral lateral part of striatum), penumbra (ipsilateral frontal and ipsilateral motor cortex), and unaffected (contralateral parietal cortex) areas, and analyzed their mechanisms respectively to see if there are any differences among the three areas.4.2Methods related this study:In order to analyze the possible mechanism of β-asarone effects on the autophagy, rates were randomized into groups of10animals. The treatment was as follows:model control group,2mL/kg water intraperitoneally per day for4days;β-asarone group,2mL/kg of β-asarone (the dose was decided by the first stage study,15mg/mL) intraperitoneally per day for4days; and JNK inhibitor group,2mL/kg of SP1600125(sc-200635, Santa Cruz Biotechnology, California, American)(7.5mg/mL) intraperitoneally per day for4days. At 1hr after the last administration, rats were anesthetized with intraperitoneal injection of3%chloral hydrate (350mg/kg) and then treated with MCAO model. The neurologic findings were scored on a five-point scale: a score of0indicated no neurologic deficit, a score of1(failure to extend left forepaw fully) a mild focal neurologic deficit, a score of2(circling to the left) a moderate focal neurologic deficit, and a score of3(falling to the left) a severe focal deficit; rats with a score of4did not walk spontaneously and had a depressed level of consciousness. Scores were recorded when the sutures were removed. The rats died during the experiment and rats with a score of0or4were excluded without further analysis. The brains were divided into three areas:core, penumbra and unaffected areas. The JNK, p-JNK, Bcl-2and Beclin1levels were evaluated, respectively. Additionally, the transmission electron microscope was employed to observe the autophagy.4.3Results:4.3.1.β-asarone's effects on the JNK, p-JNK, Bcl-2and Beclin1levelsCompared to the model control group, the Beclin1, JNK and p-JNK levels in the β-asarone and JNK inhibitor groups were significantly decreased (P <0.05), but the Bcl-2levels were significantly increased (P<0.05). Meanwhile, there was no significant expression difference between β-asarone group and JNK inhibitor group. The correlations among Beclin1, Bcl-2, and p-JNK/JNK were calculated. The correlations of Beclin1with Bcl-2and p-JNK/JNK were-0.494and0.519. Meanwhile, the correlation of Bcl-2and p-JNK/JNK was-0.328. All correlations had significance (P<0.05).4.3.2. Beclin1, JNK, p-JNK and Bcl-2levels in the three areasIn the β-asarone and JNK inhibitor groups, the Beclin1, JNK, p-JNK and Bcl-2levels in core, penumbra, and unaffected areas had no significant differences, respectively.4.3.3. The β-asarone and JNK inhibitor can significantly decrease the scores of the neurologic findings.Compared to the control group, the scores of the neurologic findings in the β-asarone and JNK inhibitor groups were significantly increased (P <0.05). There was no significant expression difference between the β-asarone and JNK inhibitor groups.4.4Conclusions:The β-asarone attenuates ischemia-reperfusion-induced autophagy in rat brains via modulating JNK, p-JNK, Bcl-2and Beclin1levels in the core, penumbra and unaffected areas. Details as follow:the β-asarone can decrease the JNK and p-JNK levels at first, and then increase Bcl-2level, finally interfere with the functions of Beclin1during the execution of autophagy, which leads to decrease the ischemia-reperfusion-induced brain injury.The possible mechanisms of β-asarone on autophagy in the core, penumbra and unaffected areas are very similar. The β-asarone can decrease the JNK, p-JNK, Bcl-2and Beclin1levels at the same time, which suggest that the3-asarone can attenuate autophagy in a widespread manner.Final Conclusions:We can extract β-asarone with high purity from the Acorus tatarinowii Schott and the gas chromatography-mass spectrometry method used to confirm the purity is simple and stable.Beclin1levels in core, penumbra, and unaffected areas are up-regulated beginning at0hr reperfusion injury peaking at24hr and lasting for72hr.The β-asarone can attenuate brain ischemia-reperfusion-induced autophagy in a dose-dependent manner. The mechanism by which β-asarone attenuates the autophagy is likely that β-asarone can modulate JNK, p-JNK, Bcl-2and Beclin1. Details as follow:the β-asarone can decrease the JNK and p-JNK levels at first, and then increase Bcl-2level, finally interfere with the functions of Beclin1during the execution of autophagy. The correlations of Beclin1with Bcl-2and p-JNK/JNK are-0.494and0.519. Furthermore, the β-asarone effects on core (ipsilateral parietal cortex and ipsilateral lateral part of striatum), penumbra (ipsilateral frontal and ipsilateral motor cortex), and unaffected (contralateral parietal cortex) areas have no significant differences supporting that β-asarone can attenuate autophagy in a widespread manner.
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