| Objective To explore the mechanism of SIRT3 mediated mitochondrial autophagy in the aggravation of cerebral ischemia-reperfusion injury by high glucoseMethods Sprague Dawley male rats aged 8-10 weeks(230-280g)were randomly divided into three groups: normal group(39 NG),diabetes group(39 DG)and rapamycin pretreatment group(39 DRG),Each group was divided into three groups: sham operation group(sham group),ischemia / reperfusion 1 day group(1d group),3 days group(3d group).In the DG group,2% STZ(streptozotocin)(50mg / kg)was injected intraperitoneally once,and blood glucose was measured in the tail vein three days later.If the blood glucose value was more than 12 mmol /L,the diabetes model was successful.In DRG group,rapamycin was injected intraperitoneally 3 days before and after operation,once a day.The middle cerebral artery occlusion(MCAO)model was made by the modified thread occlusion method in the ischemia/reperfusion injury model,which was reperfusion after 2 hours of ischemia.According to the longa scoring method,the neural function of rats was scored,and 2-4 points were selected as the successful model of MCAO.In the sham group,only the carotid artery was exposed,and no embolization or ligation was performed.Four rats were used for TTC(2,3,5-triphenyltetrazolium chloride)staining to observe the volume of cerebralinfarction,five rats were used for pathological detection and immunohistochemistry after perfusion and fixation,four rats were used to extract the whole cell protein of hippocampus for Western blotting,To observe the expression of rapamycin target protein(mTOR)and autophagy related proteins.Results 1.One week after STZ intraperitoneal injection,the blood glucose value was more than 12 mmol / L,and the model was successful.Compared with DG group,the blood glucose value in DRG group did not increase(P > 0.05),suggesting that rapamycin pretreatment had no hypoglycemic effect.Results 2.The neurological score of the DG group(2.378 ± 07941)was significantly higher than that of the NG group(1.351 ± 0.5877)(P< 0.01),while that of the DRG group(1.757 ± 0.5480)was significantly lower than that of the DG group(P< 0.01).It is suggested that hyperglycemia aggravates brain injury,while rapamycin pretreatment reduces brain injury.Results 3.TTC staining display: Compared with NG group(0.1320 ±0.02295),the volume of cerebral infarction in DG group(0.4026 ± 0.04459)increased(P< 0.01),while that in DRG group(0.2442 ± 0.02882)decreased(P<0.01).HE staining showed that the hippocampal neurons in the sham group were relatively neat and uniform in size,with no obvious eosinophilic changes in the cytoplasm,relatively complete cell membrane,uniform cytoplasm,clear nuclear membrane,uniform chromatin particles,and no significant changes among the three groups.Compared with sham group,the cells in N1 d and N3 d group were necrosis,irregular shape,eosinophilic change,nuclear pyknosis,unclear nuclear membrane and nucleolus.The number of necrotic cells increased with time(P <0.01).Compared with NG group,the necrosis rate of D1 d [(47.60 ± 5.217)%] and D3 d [(57.95 ± 6.008)%] was significantly higher than that of N1 d [(17.97 ±3.215)%] and N3 d [(32.15 ± 4.693)%],with statistical significance(P < 0.01).Compared with DRG group,the necrosis rate of DR1 d [(33.36 ± 4.886)%] and DR3 d [(40.70 ± 3.760)%] cells in DG group was significantly lower(P<0.01)Results 4.Immunohistochemistry results: the expression of P-S6 protein in DG group was higher than that in ng group(P < 0.01),while that in DRG group was lower than that in DG group(P<0.01).Results 5.Western blot showed that the expression of p-mTOR increased after ischemia-reperfusion,especially in the DG group(P< 0.01),the expression of DGR was inhibited significantly(P< 0.01).The expression of Beclin1,LC3 II and OPA1 protein increased after ischemia-reperfusion(P< 0.05).The expression of Beclin1,LC3 II and OPA1 protein in DG group was significantly higher than that in NG group(P<0.05),especially in DRG group(P<0.05).The expression of p-DRP1 and TOM20 increased after ischemia-reperfusion,especially in D1 d group(P<0.05),while DGR group significantly inhibited the expression(P<0.05).The expression of Nix/BNIP3 and SIRT3 increased after ischemia-reperfusion,but the protein expression of DG group decreased compared with ng and DRG(P< 0.05).There was no significant change in the expression of MFF(P>0.05).Conclusion1.High glucose can activate mTOR pathway and aggravate cerebral ischemia-reperfusion injury;rapamycin can reduce brain injury by inhibiting mTOR pathway.2.High glucose may aggravate cerebral ischemia-reperfusion injury due to mitochondrial dynamic imbalance.3.SIRT3 may be the key molecule to regulate the dynamic balance ofmitochondria,and its upstream regulatory pathway may be mTOR pathway.4.Nix / BNIP3 may be the main pathway of mitochondrial autophagy. |
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