Effects Of Interleukin On Oxidative Stress And Autophagy-related Factors During Cerebral Ischemia-reperfusion In Rats

Objectives The brain ischemia and reperfusion model were established in rats.Brain tissue and blood were collected by Elisa method,HE staining,TTC staining,TUNEL staining,immunohistochemistry,Western blot,transmission microscopy,etc,to detect the activity of relevant factors,protein expression and cell autophagy.The results were statistically processed to explore the role of medullone in protecting the brain from the effects of autophagy and oxidative stress during cerebral ischemia-reperfusion injury on the level of protein and ultrastructure.Methods There were 160 healthy male Sprague Dawley rats of SPF grade,6-7 weeks,weight: 260±10g,and the temperature of animal breeding unit was 20-25°C.Relative humidity: 50%-60%;No strong light noise stimulation,appropriate amount of dry feed and clean drinking water,and replacement of litter every 2 days;The state of survival during the feeding process was good and no injuries or epidemics occurred.All rats were randomly divided into 4 groups according to experimental requirements: Sham group(n = 40);I/R group(n = 40),IMD group(100 mg/kg)(n = 40);3-MA(200 nmol)(n = 40);In the sham group(Sham group),only the right general,extracranial,and internal carotid arteries were dissected and no line was inserted.The rest of the groups were pulled out of the line for 1.5 h after ischemia for 24 h.I/R group,IMD group and 3-MA group were injected intracerebroventricularly immediately 1.5 hours after ischemia.Rats in each group were subjected to abdominal aorta surgery.After formalin fixation,30% sucrose was fully dehydrated and cut,brains were frozen for sectioning and electron microscopy sections were used,or fixed with 2.5% glutaraldehyde or perfused with formaldehyde.Neurological deficits were evaluated by ethological changes in rats.Elisa method,HE staining,TTC staining,TUNEL staining,immunohistochemistry,Western blot and transmission microscopy were used to detect relevant factor activities,Protein expression and autophagic expression factors,observe the effects of medullone on oxidative stress and autophagy after cerebral ischemia-reperfusion in rats,and further explore its protective effect on brain.Results 1 Behaviour indicators Through the score of behavioral indicators,it was determined that the cerebral ischemia-reperfusion model was successfully prepared,and the intermediates were initially determined to have a protective effect on cerebral ischemia-reperfusion.2 Elisa assay: Detecting the biochemical metabolic indexes of SOD,MOD and other factors in plasma of rats in each group,reflecting the inhibition of oxidative stress induced by cerebral ischemia-reperfusion in rats;3 HE staining: The results showed that compared with I/R group,the number of injured neurons in IMD group and 3-MA group decreased more normal neurons and lighter edema,indicating that IMD and 3-MA have a protective effect on CIRI.4 TTC staining: The results showed that IMD group and 3-MA group had significant statistical significance compared with I/R group(P<0.05),and there was also significant statistical significance compared with Sham(P<0.05).5 TUNEL staining results showed that the infarct volume of IMD group and 3-AM group was greater than Sham group(P<0.05)but less than I/R group(P<0.05),indicating that autophagy promoted cerebral ischemia-reperfusion injury.IMD has a protective effect against cerebral ischemia-reperfusion.6 Qualitative and quantitative analysis of immunohistochemistry and Western Blot showed that the expression of Beclin1 and LC-3II/LC-3I in IMD group was significantly lower than that in model group,while P62 was increased,indicating that IMD can make cerebral ischemia and reperfusion.24 h reduced the expression of LC-3 and Beclin-1 in hippocampal CA-1 region of rats after cerebral ischemia reperfusion,and reduced the brain damage caused by cerebral ischemiareperfusion.7 Electron microscopic observation: The cells and membranes in the Sham group were intact,the chromatin distribution was uniform,the morphology of various organelles was normal,and no autophagosome appeared;In the I/R group,the number of autophagosomes and autolysosomes at different stages,mitochondria were swollen,membranes were broken,vacuoles were visible,and lysosomes at all levels were significantly increased.The deformed secondary lysosomes and Golgi were seen.Splitting(Figure 7),the results suggest that severe autophagy occurs during cerebral ischemia and reperfusion,Significant increase in lysosomes at all levels and visible deformation of secondary lysosomes;IMD group and 3-AM group also showed different numbers of autophagosomes and autophagy lysosomes in different periods,swelling of mitochondria,membrane rupture,visible vacuoles,significant increase in lysosomes at all levels,visible secondary deformation Enzyme bodies,Golgi division;Compared to IMD group and 3-AM group,I/R group has more autophagosomes and lysosomes at all levels.Conclusions The IMD group has a protective effect on cerebral ischemia-reperfusion,which can reduce the degree of cerebral ischemia-reperfusion injury.Under TEM,we observed that there were significantly more autophagosomes than IMD groups in the I/R group.At the same time,qualitative and quantitative analysis of immunohistochemistry and Western Blot showed that the expression levels of Beclin1 and LC-3II/LC-3I in IMD group were significantly lower than those in model group,while P62 was increased,indicating that IMD can make cerebral ischemia-reperfusion.After 24 hours,it can reduce the autophagy of cerebral ischemia-reperfusion and down-regulate the expression of LC-3 and Beclin-1 in the cortex of reperfusion rats,and reduce the brain damage caused by cerebral ischemia-reperfusion.