| Objective:To explore the effect and mechanism of metformin on cerebral ischemia-reperfusion injury in rats through autophagy.Methods: Male SD rats were selected,whose weight was maintained at 240g～250g,and the PSI blood perfusion instrument was used to establish cerebral ischemia reperfusion model(ischemia for 2h,reperfusion for 6h).They were stochastically separate into sham operation group,ischemia reperfusion model group(I/R group),ischemia reperfusion model+metformin(I/R+Metformin group)(100mg/kg);The cerebral infarction of rats in each group was checked by way of TTC staining;The pathological damage of nerve cells was observed by means of hematoxylin-eosin staining(HE);The situation of autophagy-related proteins such as p-AMPK,AMPK,Beclin-1,LC3-Ⅱ/LC3-Ⅰ,p62 and the content of TFEB-related pathway proteins in brain tissue of each group were checked by way of Western blot(WB).Transmission electron microscopy(TEM)was used to observe the autophagy of neurons in brain tissue and the damage of mitochondria.The rats were divided into I/R group,I/R+Metformin group,I/R+Metformin+sh TFEB group.The expression of autophagy-related proteins such as p-AMPK(Thr172),AMPK,Beclin-1,LC3-Ⅱ/LC3-Ⅰ,p62,and the expression of TFEB,p-ULK1(Ser757),and ULK1 in brain tissue of each group were detected by Western blot(WB).Result:Part 11.Change trend of AMPK expression level during cerebral ischemia-reperfusion injuryWB results showed that compared with Sham group,the level of p-AMPK protein decreased after 2h ischemia and 6h reperfusion(P<0.05).2.Metformin concentration screeningWB results showed that compared with the 0mg/kg group,the activity of p-AMPK protein in rat brain tissue was significantly activated when the dose of 100mg/kg was pretreated with metformin(P<0.01).Part 21.The model of cerebral ischemia-reperfusion injury in rats was established by PSI blood perfusion instrumenThe cerebral blood perfusion image of the sham operation group showed that the blood perfusion was full,the blood vessel distribution was normal and clear,and all data indicators were normal.After 2 hours of ischemia in MCAO group,the right cerebral blood flow was interrupted due to middle artery occlusion,and the volume of blood flow perfusion was significantly decreased,which was statistically significant compared with that in sham operation group(P<0.05).The right cerebral blood flow perfusion was restored after 6 hours of blood flow restoration,but some regions still could not return to normal level(P<0.05).2.cerebral infarction of rats in each groupCompared with Sham group,the infarct focus of ischemia reperfusion group was significantly(P<0.01).Compared with ischemia reperfusion group,the infarct focus of Met Pretreatment group was reduced(P<0.001).3.Brain histopathological changes in each groupIn the sham group,the morphology of brain nerve cells was intact,and the cytoplasm and nucleolus were clearly visible;Compared with the sham group,the cells in the ischemia-reperfusion group were light stained,showing a sieve network,accompanied by liquefaction necrosis of cells,showing vacuolar shape;Compared with ischemia-reperfusion group,Met pretreatment group had less cell damage,more orderly arrangement and fewer necrotic cells.4.Changes of autophagy-related protein level in brain tissue of each groupWB results showed that compared with Sham group,the situation of Beclin-1,LC3-Ⅱ/LC3-Ⅰ protein in I/R group decreased(P<0.05),and the situation of p62 protein increased(P<0.001).Compared with I/R group,the expression situation of Beclin-1 protein and LC3-Ⅱ/LC3-Ⅰ protein in I/R+Metformin group increased(P<0.05),while the expression of p62 protein decreased(P<0.01).5.Expression of AMPK and TFEB proteins in rats of each groupWB results showed that compared with Sham group,the situation of p-AMPK(Thr172)protein and TFEB protein in I/R group both decreased(P<0.05);Compared with the I/R group,the situation of p-AMPK(Thr172)protein and TFEB protein in the I/R+Metformin group both increased(P<0.05);There was no obvious discrepancy in the expression of AMPK protein in each group(P>0.05).6.Observe the autophagy of nerve cells of rats in each group by transmission electron microscopeThe observation of autophagy or autophagy lysosome by electron microscope is considered as the gold standard for recognizing autophagy.Therefore,we have detected the formation of autophages in neurons of ischemic cortex by electron microscopy.The results showed that compared with Sham group,autophagy and autophagy lysosome in I/R group decreased,mitochondria swelled abnormally,and cell morphology was seriously affected.Compared with I/R group,the number of autophagosomes and autophagic lysosomes in I/R+Metformin group increased,the morphology of mitochondria was relatively normal,the nucleus and membrane of neurons were clearly visible,and the damage of neuron ultrastructure was reduced.Part 31.The level Situation of AMPK and autophagy protein in brain tissue of rats after TFEB knockdownSet up subgroups: I/R,I/R+Met,I/R+Met+sh TFEB.According to the WB experiment,testing the expression changes of autophagy proteins such as Beclin-1,LC3-Ⅱ/LC3-Ⅰ,p62,as well as related pathway proteins TFEB,p-AMPK(Thr172),AMPK,p-ULK1(Ser757),ULK1.The results showed that compared with I/R group,the expression level of Beclin-1 protein and LC3-Ⅱ/LC3-Ⅰ protein in brain tissue of I/R+Met group increased(P<0.05),while the expression level of p62 protein decreased(P<0.05);In addition,the expression level of TFEB protein,p-AMPK(Thr172)protein and p-ULK1(Ser757)protein also increased(P<0.05);Compared with I/R+Met group,the expression level of Beclin-1 protein and LC3-Ⅱ/LC3-Ⅰprotein in brain tissue of I/R+Met+sh TFEB group both decreased(P<0.05),while the expression of p62 protein increased(P<0.05);The protein expression of TFEB was decreased(P<0.05),while the protein expression of p-ULK1(Ser757)was decreased(P<0.05),and the protein expression of p-AMPK(Thr172)was not different(P>0.05).There was no obvious discrepancy in the expression of AMPK and ULK1 protein in each group(P>0.05).Conclusion: Metformin activates AMPK-TFEB pathway to improve autophagy,thus alleviating cerebral ischemia-reperfusion injury in rats. |
