| Objectives To investigate the relationship between microRNA-124 expression and autophagy during cerebral ischemia-reperfusion injury in rats and the possible mechanism.Methods In this study,8-weeks male Sprague-Dawley rats weighing between 250 and280 g ware established the middle cerebral artery occlusion(MCAO)model by the ZeaLonga method.The experimental rats were randomly divided into control group,sham group,CI/R group(3h,6h,12 h,24h),CI/R+miR124 agomir group,CI/R+miR124 antagomir group,CI/R+miR-control group,STAT3 inhibitor group and STAT3 control group.TTC staining and neurological function score were used to evaluate model-making effect.Detection of pathological changes in rat brain by HE staining.The expression of microRNA-124 was detected by qRT-PCR at different time points after reperfusion.Immunohistochemistry and western blot were used to detect autophagy.Luciferase reporter assay was performed to identify the site in the 3’ un-translated region of the target mRNA that was targeted by microRNA-124.Results 1 There was a certain expression of microRNA-124 in brain tissues of early cerebral ischemia/reperfusion(1.00±0.10),and the expression of microRNA-124 in neurons showed dynamic changes with the time of cerebral ischemia-reperfusion.The expression level of microRNA-124 was significantly increased 3h of cerebral ischemiareperfusion in rats(2.86±0.40),and reached the highest peak 12 hours after reperfusion(6.68±0.78).Western blot showed that Beclin1(0.946±0.010)and LC3II/LC3I(0.535±0.006)increased significantly after reperfusion for 3 h,and the expression was further extended with the prolongation of reperfusion time and reached the highest level at 24 h after reperfusion.The expression of Bcl-2 increased significantly at 3h after reperfusion(0.704±0.011),and its expression increased gradually with the reperfusion time,reached a peak at 12 hours after reperfusion(1.286±0.037),and then began to decrease(0.815±0.002).Immunohistochemistry showed that Beclin1,LC3 and Bcl-2 are mainly distributed in cytoplasmCompared with the control group(0.40±0.55),the number of positive cells increased significantly at 3h after reperfusion(4.00±0.70).The difference was statistically significant(P<0.05).Then the number of positive cells increased with the prolongation of reperfusion injury time.2 microRNA-124 agonist can significantly reduce cerebral ischemia-reperfusion injury in rats.In the CI/R+miR124 agomir group,the Zea Longa neurological scores(1.00±0.00 vs 3.00±0.00)and the number of positive cells(4.00± 1.00 vs 14.33±1.15)under an optical microscope in brain tissue of rats in the CI/R+miR124 agomir group were significantly reduced.The results of autophagyassociated protein analysis showed that the expression of Bcl-2(0.578 ± 0.022)in the CI/R+miR124 agomir group was lower than that in control group(0.805±0.080),and Beclin1(0.981±0.028 vs 0.545±0.009)and the ratio of LC3II/LC3I(1.087 ±0.031 vs0.875±0.038)were decreased(P<0.05).Compared with the control group,the microRNA-124 inhibitor group showed an opposite trend(P<0.05).3 Compared with STAT3 negative control,the fluorescence intensity of STAT3-WT group was significantly lower than that of STAT3-MUT group(P < 0.05),but there was no significant change in fluorescence intensity of STAT3-MUT group.At the same time,the STAT3(0.426±0.012 vs0.800±0.029)and p-STAT3(0.140±0.019 vs 0.736±0.036)levels were significantly reduced in the microRNA-124 agonist group compared to the miR-control group.The effect of upregulation of autophagy by microRNA-124 was attenuated after the addition of STAT3 inhibitor.The expression level of Bcl2 was significantly lower in the STAT3 inhibitor group(0.769 ± 0.076 vs 0.436 ± 0.021)(P<0.05),while the expression of Beclin1(0.793 ± 046 vs 1.113 ± 0.0.090)and LC3II/LC3I(0.158 ± 0.006 vs 0.750 ±0.026)was significantly higher(P<0.05).Conclusions In CIRI,microRNA-124 can activate autophagy by STAT3 pathway to protect neurons.It provides a new way for clinical treatment of cerebral ischemiareperfusion injury.Figure27;Table17;Reference 148... |
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