The Impact Study Of Zhilong Huoxue Tongyu Capsule Pretreatment On Transient Focal Cerebral Ischemia And Reperfusion Injury In Rats Brain Cells Autophagy

Abstract:Objective:1.To observe the impact of autophagy in neurological deficit, in model rats of transient focal cerebral ischemia and reperfusion injury.2.To study the impact of experimental rat brain cells autophagy and neurological function by Pretreatment of macrophages induced by agents and inhibitors at different time points.3. To investigate the experimental rat brain cells autophagy and nerve function by Zhilong Huoxue Tongyu Capsule (ZHTC) pretreatmented.Methods:1. Grouping method:120young male SD rats weight between250grams to350grams of4to8weeks, were randomly divided into a, b, c, d, e-five groups, a:sham-operated group (Sham), b: transient focal cerebral ischemia reperfusion injury model group (MCAO group), c:before modeling vermiculite ZHTC in the intervention group (ZHTC+MCAOgroup), d:the intervention group modeling by rapamycin (rapamycin+MCAO group), e:the intervention group modeling by3-MA (3-MA+MCAO group).2. Dosing method:ZHTC+MCAO group to give the ZHTC gavage a month, the MCAO model group were given distilled water as ZHTC group the same volume a month, the rapamycin+MCAO group given intracerebroventricular injection of rapamycin1hour before the modeling,3-MA+MCAO group given intracerebroventricular injection of3-MA1hour before the modeling.3.Detection indicators:①Each group were Neurological scored and TTC staining of infarct area to the projected volume ratio, in cerebral infarction in transient focal cerebral ischemia2hours、after reperfusion3hours and12hours, three time points.②Each group were observed autophagy and lysosomal autophagy using electron microscopy, were detected autophagy-related protein LC3-Ⅱ expression by western blot assay, were observed the autophagy protein Beclinl positive expression by immunohistochemistry, at reperfusion3hours and12hours two time points.Results:1. Use Garcia, JH, s rat nerve function scoring Standard, to compare each group, s neurological score. Compared with the sham operation group, the neurological score of MCAO group, ZHTC+MCAO group, rapamycin+MCAO group and3-MA+MCAO group rats at all experimental time points were significantly reduced, the difference was statistically significant (P<0.01); compared with the MCAO group, the neurological score of all experimental rats of the ZHTC+MCAO group and the rapamycin+MCAO group at three time points were higher, there were significant differences (P<0.01or P<0.05), while3-MA+MCAO group rats the neurological score of three points in time have declined, the difference was statistically significant (P<0.01or P<0.05); compared with ZHTC+MCAO group, rapamycin+MCAO group rats at each time point,the neurological function scores were slightly higher, but the difference was not significant (P>0.05),3-MA+MCAO group rats in the neurological score at each time point, significantly reducing,the differences were statistically significant (P<0.01)2. Use TTC staining to calculate the infarct volume rato.Compared with the sham operation group, MCAO group, ZHTC+MCAO group, rapamycin+MCAO group, and3-MA+MCAO group in transient focal cerebral ischemia2hours、 reperfusion3hours and12hours of the three time points in rats with cerebral infarction volume ratio weresignificantly increased, the difference was statistically significant (p<0.01); Compared with MCAO group, the infarct volume rate of ZHTC+MCAO group and the rapamycin+MCAO group rats were significantly lower, the difference was statistically significant (p<0.01). and the infarct volume rate of the3-MA+MCAO group rats was higher, the difference was statistically significant (P<0.05); compared with ZHTC+MCAO group, the brain infarct volume rate of rapamycin+MCAO group are partial lower at three time points, but the difference was not significant (P>0.05), while the infarct volume ratio of3-MA+MCAO group rats was significantly increased, the difference was statistically significant (P<0.01).3Use electron microscopy to Observed the ultrastructure of autophagic bodies and autophagic lysosomal.In the sham operation group,the morphology of neurons are normal,in these neurons the healthy nucleus were seen, mitochondria and abundant within the endoplasmic reticulum, a small number of lysosomal structures were seen; In the MCAO group, neurons with vacuoles were visible, mitochondria significantly swelling and vacuolization; in the ZHTC+MCAO3h group and rapamycin+MCAO3h group showed double membrane structure containing organelles, autophagy, and rapamycin group slightly more common. In addition, the ZHTC+MCAO group and the rapamycin+MCAO group were seen slightly swollen mitochondria and cysts slight expansion of the endoplasmic reticulum, while in the3-MA+MCAO group the neurons of the nucleus with irregular shape, mitochondrial swelling, endoplasmic reticulum slightly edema, individual cells, organelles, condensation can be seen.4. Use western blot and immunohistochemistry assay autophagy-related proteins.Compared with the sham group, the MCAO group, ZHTC+MCAO group and rapamycin+MCAO group rats at the ischemia-reperfusion3hours and12hours two points time,the autophagy-related protein LC3-II and Beclinl expression was significantly up-regulated (p<0.01), while the difference of3-MA+MCAO group was not significant (P>0.05); Compared with model group, the LC3-II and Beclinl protein expression in ZHTC+MCAO group and the rapamycin+MCAO group were increased, the difference was statistically significant (P<0.0l or P <0.05),while the LC3-II and Beclinl protein expression in3-MA+MCAO group is weak, the difference was statistically significant (P<0.01orP<0.05); compareed with ZHTC+MCAO group, the different expression of these two proteins in rapamycin+MCAO group are not obvious (P>0.05).Conclusion:1. Modified suture method to create transient focal cerebral ischemia-reperfusion rat model, can significantly shorten the operative time and improve the success rate and stability of the modeling.2.This study shows that in transient focal cerebral ischemia reperfusion injury, autophagy was slightly activated;3. ZHTC pretreatment also reducing the cerebral ischemia and reperfusion injury, reduces infarct volume rate in rats, the relative increase in experimental neurological score, a significant increase in the transientfocal cerebral ischemia-reperfusion injury in rat brain ischemic penumbra cortex the expression of macrophage-associated protein LC3-II and beclinel ischemia and at the reperfusion3hour, electron microscopy detected autophagosome, which is similar with rapamycin pretreatment.4. In experimental rat transient focal cerebral ischemia2hours and12hours after reperfusion, the activation extent of autophagy was negatively correlated with infarct volume, was positively correlated with the neurological score. These results show that autophagy activation is the basis of brain protective effect in cerebral ischemia and reperfusion rat, and the ZHTC pretreatment to a certain extent is through this mechanism of brain protection to improve the tolerance of transient focal cerebral ischemia and reperfusion injury experimental rats.