| ObjectiveStroke is currently the world’s leading cause of long-term disability, resulting in the third common causes of death in Chinese urban residents. According to statistics, ischemic stroke makes up 85%～90% of all the stroke, and the ischemia reperfusion injury is a major cause of disability. Recent studies show that in addition to inflammation and oxidative stress reaction, excessive activation of autophagy is also involved in the process of ischemia reperfusion injury. Clinical study results show that inhibiting the excessive activation of autophagy can alleviate cerebral ischemia reperfusion injury and exert a neuroprotective effect. As a clinical recommendation neuroprotective agent, statins has wide application. Researcher found that it not only has the function of the anti-inflammatory and anti oxidative stress, but also could regulate cell autophagy level in the cardiac ischemia reperfusion injury. This study investigated whether rosuvastatin can also affect the regulation of autophagy level to play neuroprotective effect. Rosuvastatin is kinds of the classic statins, belonging to selective HMG CoA reductase inhibitors, and has long half-life, higher blood drug concentration in the body. Studies have shown that rosuvastatin pretreatment could protect the nerve function of cerebral ischemia reperfusion injury rats, and its mechanism may be associated with inhibition of nerve cell apoptosis and local inflammatory response. However, is cell autophagy involved in nervous system damage mechanism of cerebral ischemia reperfusion injury, or is the pathological process regulated by rosuvastatin? There is barely report inland. We established a middle cerebral artery occlusion (MCAO) model in rats with acute focal cerebral ischemia reperfusion in this study, and observed pathological morphology and the change of autophagy level of neurons and glial cells in the ischemic penumbra. We also selected rosuvastatin as neuroprotectant to pretreatment the models, and analyzed whether rosuvastatin can play a role of nerve protection by adjusting the cell autophagy level, so as to provide data support for its further mechanisms study.Methods1 Model preparation of acute focal cerebral ischemia-reperfusion rats75 healthy adult male SD rats were selected and divided into 5 groups (the control group, the sham-operation group, MCAO model group, rosuvastatin pretreatment group and DMSO pretreatment group) randomly with 15 cases in each group. Rats in the control group were raised as normal. Rats in MCAO model group, rosuvastatin pretreatment group and DMSO pretreatment group were MCAO acute focal cerebral ischemia-reperfusion model by the middle cerebral artery occlusion. Rats in the sham-operation group were given the surgical procedure, but the middle cerebral artery was not occluded. In addition, rats in rosuvastatin pretreatment group and DMSO pretreatment group were given rosuvastatin/DMSO intraperitoneal injection 30 min before occlusion.90 min after occlusion, pulled plug and completed the ischemia reperfusion model. At last, we used modified neural injury severity score (mNSS) to evaluate model preparation results.2 Morphological observations of neurons and glial cells in ischemic penumbra90 min after occlusion, the cerebral infarction area was evaluated by TTC staining method, and the neurons and glial cells morphology as well as autophagy bubble number were observed under electron microscope in ischemia penumbra.3 Autophagy activation positioning and level measurementDouble standard image of LC-3B and NeuN, LC-3B and GFAP, LC-3B and Iba-1 were observed after immunofluorescence to determine the position of autophagy activation. At the same time, the protein expression levels of microtubule-associated protein 1 lightchain 3A/3B (LC-3A/LC-3B) and pre-autophagy body membrane protein Beclinl in ischemia penumbra were detected by Western Blot assay.4 Data statistical analysisThe statistical package programme SPSS 19.0 was used to analyze data. The measurement data to mean+ /- standard deviation, said One-way analysis or LSD-t analyse. The count data to the proportion or percentage, said chi square test. And the ranked data said Kruskal-Wallis. P< 0.05 was considered that statistical significance was obvious.Results1 model evaluationThere were no neurologic damage symptoms appeared in the control group and the sham-operation group, and their mNSS score were all zero. The mNSS score in MCAO model group, rosuvastatin pretreatment group and DMSO pretreatment group were respectively 11.2±1.26,10.9±1.12 and 11.4±1.31, and there was statistically significant between groups (P> 0.05).2 Morphological characteristics of neurons and glial cells in ischemic penumbraThe cerebral TTC staining results in cerebral infarction region showed pale, and non infarction area was dyed red, and the pale areas in the brain were consistent to the regions supplied blood by middle cerebral artery. The infarction area of rosuvastatin pretreatment group was smaller than those of MCAO model group and DMSO pretreatment group, and its differences were statistically significant (P< 0.05), but there was no statistical difference between MCAO model group and DMSO pretreatment group (P> 0.05). Further more, the apoptotic cells and autophagy bubble numbers of rosuvastatin pretreatment group were less than those of MCAO model group and DMSO pretreatment group, and its differences were statistically significant (P< 0.05), but there was no statistical difference between MCAO model group and DMSO pretreatment group (P> 0.05).3 Autophagy activation positioning and levelIn the confocal microscope of immunofluorescence, LC-3B showed green fluorescence, NeuN showed red fluorescence, ischemic penumbra showed orange fluorescence, which declared that LC-3B mainly locate in the neurons. Further more, the Western blot assay results showed that LC3-Ⅰ、LC3-Ⅱ and Beclinl protein expression levels of MCAO model group, rosuvastatin pretreatment group and DMSO pretreatment group were obviously higher than those of the control group and the sham-operation group (P< 0.05), but there was no significant difference between the control group and the sham-operation group (P> 0.05). The LC3-Ⅰ、LC3-Ⅱ and Beclinl protein expression levels of rosuvastatin pretreatment group were obviously lower than those of MCAO model group and DMSO pretreatment group (P< 0.05), but there was no significant difference between MCAO model group and DMSO pretreatment group (P> 0.05).Conclusion1. Ischemia injury can promote the formation of autophagic vacuole and autolysosome in penumbra, so as to start the autophagy reaction, and it exists with apoptosis at the same time.2. The autophagy factor LC is mainly expressed in neuron, and there is a small amount of distribution in astrocyte and microglia too, so the reasonable regulations of those cells’ autophagy transduction pathways all can protect neurological function.3. The expression of LC-3A, LC-3B and Beclinl of penumbra are increased, and rosuvastatin has ability to decrease their expression levels, so as to inhabit autophagic reaction and protect neurological function, which may be one of its critical mechanisms to treat ischemia reperfusion injury. |
PDF Full Text Request