Researches On The Relativity Of Cathepsin L And Cell Autophagy After The Cerebral Ischemia-reperfusion Injury In Rats

Purpose: To establish models of cerebral ischemia-reperfusion injury in rats; to observe the morphological changes of the neurocyte’s autophagosomes and lysosomes in the damaged area after the cerebral ischemia-reperfusion injury in rats and examine the changes of the expression of cathepsin L and the protein LC3 I related to autophagy; and to observe the effect of Z-FY-DMK, the specific inhibitors of cathepsin L, on the neurocyte autophagy after cerebral ischemia-reperfusion injury.Methods: The rats were randomly divided into the normal group, the pseudo-operational group, the model group, and the interference group, and the three latter ones were further divided to the four time points of 3h, 6h, 12 h, and 24 h. The improved Long thread approach was applied to make the cerebral ischemia-reperfusion injury model in rats(modified middle cerebral artery occlusion, MCAO), but no special treatment had been done to the normal group. The depth of the plugged thread in the pseudo-operational group was 9±0.5mm. Z-FY-DMK(1nmol, 5ul DMSO) was injected to the tricorns of patients through penetration 30 minutes before the operation, while DMSO solutions in the same volume had been injected to the tricorns of the patients in the pseudo-operational group and the model group 30 minutes before the operation. Longa’s 5-grade standard scoring method was adopted to evaluate the neurologic impairments of the rats in all groups at various time points after the cerebral ischemia-reperfusion. The TTC dying was adopted to observe the infarct lesions of the brain tissues 24 hours after the cerebral ischemia-reperfusion of the rats in all the groups. Western bolt was taken to examine the changes of the expression of cathepsin L and protein LC3 II related to autophagy at various time points in all the groups. The electron microscope was applied to observe the morphological changes of the autophagosome and lysosome.Results : The rate of successful construction of the cerebral ischemia-reperfusion model in rats is 88.89 %. No infarct lesions of the brain tissues had been seen in the group having normal TTC dying and the pseudo-operational group; pale white infarct lesion was seen in the cortex on the side with ischemia in the model group and the interference group, the size of the infarct in the interference group was smaller than that in the model group, and the scores of the neurological function of the rats in the interference group had been improved compared to those in the model group. The nuclear membranes of the nerve cells were complete and the chromatin structures were normal in the normal group and the pseudo-operational group under the electron microscope, and lysosome could be seen in small amount but there was no autophagosome. In the model group it could be seen that the amount and volume of the lysosomes were increased at the point of the third hour and it could be observed that autophagosome had appeared; the amount of autophagosomes and lysosomes were significantly increased and organelles residues enveloped by autophagosomes as well as autolysosomes in the shape of bubble could be observed during the sixth to the twelfth hour; and at the 24 th hour autophagosomes and lysosomes could still be seen, but the amounts had been reduced compared to the previous ones. In the interference group what had been seen under the electron microscope at various time points was similar to those in the model group, but the structural amount of the autophagosomes and lysosomes had been reduced compare to it. Western bolt was taken to examine cathepsin L: In the normal group and the pseudo-operational groups the expression of cathepsin L at various time points are the same; in the model group the expression of cathepsin L had a rising trend at the time points of the 3rd, 6th, and 12 th hour, after the cerebral ischemia-reperfusion of the rats and reached the top at the 12 th hour but descended at the 24 th hour in addition that the expression amounts at the various time points were all larger than those in the pseudo-operational group. The expression trends of the cathepsin Ls in the interference group at all the time points were identical to those in the model group, and the expression amounts at various time points were all larger than those in the pseudo-operational group but lower than those in the model group. Western bolt was taken to examine protein LC3II: In the normal group and the pseudo-operational groups the expressions of protein LC3 II at various time points were the same; in the model group the expressions of protein LC3 II at the time points of 3 hours, 6 hours, and 12 hours after the cerebral ischemia-reperfusion of the rats and reached the top at the 12 th hour but descended at the 24 th hour in addition that the expression amounts at all various points were all higher than those in the pseudo-operational group. In the interference group the expression trends of protein LC3 II at all the time points were identical to those in the model group, and the expression amounts at all various time points were all higher than those in the pseudo-operational group and lower than those in the model group.Conclusions :(1)The activity of autophagy of the cells had been enhanced after the cerebral ischemia-reperfusion injury of the rats.(2)Cathepsin L took part in the mediation of autophagy in the cerebral ischemia-reperfusion injury of the rats.