The Neuroprotection Mechanisms Of Metformin Inducing The Expression Of Autophagy By Chronic Preconditioning

BACKGROUND: Stroke is the second leading cause of death and the major causes of disability in world, leading to a series of complications such as hemiplegia, aphasia,which bring a heavy burden on the patient’s family and society, As well it have an lot of effect about their life quality. Although there is advance in understanding for stroke pathogenesis,it still lacks of some effective treatments in clinic. Currently It was found metformin,the first-line drug for the treatment of type II diabetes, can reduce incidence of stroke and infarct size after stroke while its neuroprotective effect has little associated with hypoglycemic. In contrast, Metformin,as AMPK activator, has attracted a lot of attention. According to a number of literature,the AMPK as major energy receptor and regulator,play an important role and can regulate a variety of its downstream pathways after stroke, including inhibition of microglia activity and NF-k B, reducing the release of inflammatory mediators and inflammation damage effect; down-regulating the expression of MMP-9, increasing ZO-1and releasing blood-brain barrier damage; up-regulating PCG-a and its downstream molecules TFAM and NRF-1 function, increasing mitochondrial regeneration to inhibit neuronal apoptosis. But autophagy,as an important regulatory AMPK downstream target, had not been reported by chronic metformin preconditioning after stroke. And when AMPK is active, how it might affect brainmetabolism and the expression of heat shock proteins is unclear.In this study, after middle cerebral artery occlusion(MCAO) model, the C57 BL /6 mice were used to study neuroprotective role of autophagy during chronic metformin preconditioning through the use of 3-MA and compound C inhibitors.Experiment 1: With chronic metformin preconditioning, to investigate AMPK levels and lactic acid levels after I/R 6h.Objective : with chronic metformin preconditioning,AMPK levels and lactic acid were observed after I/R 6h:Methods: 1) to confirm the neuroprotective role for chronic metformin preconditioning after cerebral ischemic. C57 BL / 6 mice were randomly divided into two groups including MCAO group and Met+MCAO group receiving chronic metformin. After MCAO model was established, infarct volume and neurological behavior score were detected 24 h after ischemia-reperfusion 2)To detect AMPK lever and latic acid level after ischemia-reperfusion 6h. C57 BL / 6 mice were used. Mice were randomly divided into four groups: sham group, metformin group、 MCAO group and Met + MCAO group. After MCAO model were established. Western bolt was used to analysis AMPK level and lactic kit were used to detect the lactic lever. 4) C57 BL / 6 mice were used. Mice were randomly divided into three groups: Met group、MCAO group and Met + MCAO group.After MCAO model were established, apoptotic cells were counted after ischemia-reperfusion 24 h with TUNEL methods.Results: Chronic metformin preconditioning can significantly reduce brain infarct and improve neurological behavior score after ischemia-reperfusion 24 h. It was surprised to find that after ischemia-reperfusion 6h AMPK levels in Met+MCAO group lower comparing to Met group and MCAO group. As well in Met+MCAO group lactate levels and apoptosis cells reduced in contrast to MCAO group.Conclusion: Chronic metformin preconditioning can play a protective role in the CNS, which reduce AMPK level and lactate levels after ischemia-reperfusion 6h.Experimental 2 To study whether the autophagy expression were up-regulated and played a neuroprotective role after stroke by chronic metformin preconditioning.Objective:To investigate the expression of autophagy and the neuroprotective role after chronic metformin preconditioning.Method: 1)The autophagy was observed after chronic metformin preconditioning. C57 BL / 6 mice were used. Mice were randomly divided into five groups: Sham group、 Met group 、MCAO group and Met + MCAO group. After MCAO model were established, autophagy were observed after ischemia-reperfusion 6h, 24 h.With immunofluorescence expression of LC3 were detected. western blot were used to analysis LC3, Beclin-1and p62 expression 2) To test the neuroprotective effects about autophagy with chronic metformin preconditioning. C57 BL / 6 mice were used. Mice were randomly divided into 3 groups: Met+MCAO grou、 Met+Veh+MCAO grou and 3-MA+Met+MCAO group. Intracerebroventricular injection was administered using 3-MA before MCAO model were established. To detect infarct volume and neurological behavior score after ischemic-reperfusion 24 h 3) Autophagy was observed after the use of 3-MA inhibitor. To detect the changes about the level of autophagy after ischemic-reperfusion 6h, 24 h. C57 BL / 6 mice were used. Mice were randomly divided into 3 groups: Met+MCAO group、Met+Veh+MCAO group and 3-MA+Met+MCAO group. Intracerebroventricular injection was administered using 3-MA before MCAO model were established. Western blot method was applied to detect the LC3, Beclin-1 and p62 expression. 4) To observe Hsp70 expression levels after use of 3-MA inhibitor. C57 BL / 6 mice were used. Mice were randomly divided into 3 groups: Met+MCAO group、Met+Veh+MCAO group and 3-MA+Met+MCAO group. Intracerebroventricular injection was administered using 3-MA before MCAO model were established. Western blot method were adopted to observe Hsp70 expression after schemic-reperfusion 6h,24 h. 5)To confirm the neuroprotective effects about autophagy with preconditioning with chronic metformin. C57 BL / 6 mice were used. Mice were randomly divided into 3 groups: Met+MCAO group、Met+Veh+MCAO group and CC+Met+MCAO group. Intraperitoneal injection were administered using CC before MCAO model were established. To detect infarct volume and neurological behavior score after ischemic-reperfusion 24 h. 6)To detect the AMPK lever after using Compound C inhibition. C57 BL / 6 mice were used. Mice wererandomly divided into 3 groups: Met+MCAO grou、Met+Veh+MCAO group and CC+Met +MCAO group. Intraperitoneal injection were administered using CC before MCAO model were established. Western blot was used to detect AMPK levels found 7) To test autophagy levels and lactic acid levels after ischemic-reperfusion 24 h.C57BL / 6 mice were used. Mice were randomly divided into 3 groups: Met+MCAO group、Met+Veh+MCAO group and CC+Met+MCAO group. Intraperitoneal injection were administered using CC before MCAO model were established. western blot was used to analysis the LC3, beclin1 and P62. Lactic kit was used to test lactic lever.Conclusion: After ischemia-reperfusion 24 h with chronic metformin preconditioning, autophagy was increased and played a neuroprotective role,which also involved in the regulation of the level of Hsp70.Summary:It was found that chronic metformin preconditioning can reduce the level of AMPK after early schemic-reperfusion 6h, reducing the level of lactic acid and apoptosis cells. Meanwhile with chronic metformin preconditioning, autophagy was increased and played a neuroprotective role,which involved in the regulation of the level of Hsp70.