| Traumatic brain injury(TBI)is a major cause of death and disability worldwide and its incidence is rising rapidly.However,there is no effective treatment for TBI.Acute cell death can result from primary brain injury and is difficult to intervene.Secondary damage induced by primary injury involves numerous cellular and molecular mechanisms,such as inflammation,activation of glial cell,oxidative stress,cell metabolism,apoptosis,autophagy and mitophagy,which could be the targets for medical intervention.Sirt1 is the most studied member of the deacetylase family.The deacetylase family is a group of homologous proteins of the yeast silencing information regulator 2(SIRT2).These enzymes perform lysine deacetylation and are associated with intermediate products of the cellular energy metabolism process,namely nicotinamide adenine dinucleotide(NAD+).Therefore,the activity of sirtuins can be dependent on and regulated by NAD+.Many studies have shown that some substances,such as resveratrol(Res),play an important role by regulating the expression or activity of Sirt1,which is involed in cell survival,metabolism and inflammation.Furthermore,Sirt1 is neuroprotective in neurodegenerative diseases,and its activity is related to autophagy.Autophagy is a highly conserved intracellular mechanism in the process of evolution,and it plays an important role in maintaining cell stability and removing abnormal proteins or organelles in cells through the process of self-catabolism and recycling in the state of stress or nutritional deficiency.Once autophagy is activated by autophagy related proteins,abnormal cytoplasmic proteins or organelles are encapsulated by a membrane to form autophagosomes,which fuse with lysosomes to form autolysosomes,and then the abnormal cytoplasmic proteins or organelles are degraded by lysosomal enzymes.Microtubule-associated protein light chain 3(LC3)has been widely used as a key marker for autophagosome.LC3 is mainly synthesized in raw form,namely pro LC3.When autophagy was activated,pro LC3 was cracked by ATG4 to form LC3-?,then LC3-? and phosphatidyl ethanolamine(PE)combine to form fat changes LC3(LC3-?).p62 promotes ubiquitination to autophagosomes and is degraded by autolysosomes.Therefore,down-regulation of p62 indicates the occurrence of autophagy flux.ROS produced by damaged or dysfunctional mitochondria in most cells will be cleared by mitophagy.The loss of mitochondrial membrane potential induces the accumulation of putative kinase 1(Pink1)by PTEN in the mitochondrial outer membrane,where Pink1 recruites ubiquitin ligase parkin and phosphorylated and activates it.Activated parkin recruites p62 into the damaged mitochondria,which are then encapsulated by LC3 on the autophagosome,and the damaged or dysfunctional mitochondria are then degraded in the lysosome.In recent years,studies have found that Sirt1 is associated with autophagy and mitophagy.However it is unclear if Sirt1 recovers the neuological function in the severe brain trauma by autophagy and mitophagy.In the current study,we aim to explore the mechanism of the interaction between Sirt1 and autophagy or mitophagy and look for new therapeutic targets.Methods: Animal weight-drop models of severe traumatic brain injury were established,and tissues were collected at different time points after TBI.Lateral ventricular administration of Resveratrol,intraperitoneal administration of mitochondrial division inhibitor mdivi-1 and rapamycin were used for intervention,and the sham group and the lateral ventricular administration of DMSO solvent group were used as controls.Western blot was used to detect the time-dependent expression of Sirt1,LC3 and p62,as well as the expression of LC3,p62 and PINK in each group,and the expression of LC3 in neurons was detected by immunofluorescence double staining.Rotarod experiment was used to detect the motor coordination ability of animals in different groups.The experimental data were expressed as meanąSD(xąs).The results were analyzed and processed by the Graph Pad prism6 software,and one-way anova was used among multiple groups.P < 0.05 was considered statistically significant.Results: Western blot results showed that the relative expression of Sirt1 in the damaged cortex and surrounding tissues was decreased with time after severe brain injury,and it began to decrease significantly after 1 hour,and then remained in a low expression state,although it was increased slightly at 24 hours.Sirt1 expression in subcortical tissue was decreased significantly from 0.5 hour after injury,and to the lowest level 3 hours after injury,recovered 3 days after injury,and continued to be at low expression level until 14 days later.In the damaged cortex and its surrounding area,the expression of LC3-?/LC3-? was increased significantly in 0.5 hour,then dropped significantly,and began to rise again after 12 hours until 3 days later,and then gradually went back to control level until 14 days;in the damaged subcortical tissue,the expression of LC3-?/LC3-? was increased significantly in 0.5 hour after injury,and then went back to control level,then began to increase in 12 hours,to the peak at 24 hours,and was gradually reduced to the control level 3 days after injury until 14 days.The expression of autophagy connexin p62 in the injured cortex and surrounding area was decreased gradually with time,and decreased significantly at 1 hour,reached a minimum at 3 hours,increased at 12 hours,and recovered to the level of sham group at 24 hours,decreased again at 3 to 7 days,and recovered to the level of control at 14 days.The expression of p62 of subcortical tissue was significantly reduced in 0.5 hour,and then it was similar to the control group,decreased slightly in 7 days,and recovered to the level of the sham group in 14 days.Animal behavior experiments showed that the motor coordination ability of animals was significantly reduced 24 hours after brain injury,and there was no difference in the motor duration between the pretreatment group and the sham group.Resveratrol plays an important role in improving neurological function,especially motor coordination.Western blot results showed that pretreatment with resveratrol prevented the rise of LC3-?,and restored the expression of p62.Resveratrol can prevent the occurrence of excessive autophagy.Fluorescence double-labeled LC3 B and Neu N showed that autophagy in neurons existed in the sham operation group to some extent,but the expression of LC3 around the injury was significantly increased after the injury,especially neurons.However,the expression of LC3 in neurons of the resveratrol group was significantly lower than that of the injury group.Autophagy,which is localized to neurons,also decreased.Western blot results showed that PINK,a key protein of mitophagy,was decreased significantly after severe brain injury,and mitophagy was significantly impaired.The administration of resveratrol partially restored the expression of PINK.Through drug intervention on mitophagy,it was found that inhibition of mitochondrial division could partially restore PINK's expression,but it could block the recovery effect of resveratrol on PINK.Rapamycin activates mitophagy,but in this study,rapamycin did not significantly improve the reduction of PINK caused by injury,and rapamycin did not affect the recovery of the expression of PINK by resveratrol.Therefore,inhibition of mitochondrial autophagy by mdivi-1 could weaken the recovery effect of resveratrol on PINK,while stimulation of mitophagy by rapamycin did not affect the reversal effect of resveratrol on PINK during traumatic stress.In the same time,we found that inhibition of the mitophagy by mdivi-1 can block the inhibitory effect of resveratrol on the expression of LC3-?/LC3-?.This suggests that resveratrol alleviates autophagy without synergistic effects with mitochondrial dynein inhibitors.Rapamycin does not affect the inhibitory effect of resveratrol on LC3-?/LC3-? expression.Western blot results showed that the administration of resveratrol increased the expression of p62,and the mitophagy inhibitor mdivi-1 could assist the recovery of p62 expression of resveratrol to the level of sham operation.Rapamycin did not affect the effect of resveratrol on the expression of p62.The results of motor behavior showed that the duration of exercise of the animals in each group on the first day after the injury was basically the same as that of the sham operation group,indicating that the application of these drugs alone or in combination can have a recovery effect on the reduction of motor ability caused by brain trauma.Conclusions:1.Autophagy was activated after traumatic brain injury in our animal model.2.Resveratrol can improve the decline of motor coordination caused by TBI in our animal model.3.Resveratrol can improve autophagy disorders induced by TBI in our animal model.4.Up-regulation of Sirt1 can improve the impaired mitophagy function caused by severe brain injury.5.Inhibition of mitochondrial division affects the recovery of autophagy and mitophagy by resveratrol after TBI.6.mTOR pathway has no effect on the regulation of autophagy and mitophagy by resveratrol. |
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