Protective Effect Of Metformin On Brain Slices Injury Induced By H₂O₂ In Mice

Objectives:To establish in vitro cerebral injury model,the brain slices were stimulated by hydrogen peroxide. Metformin was given to observe the protective effect PICK1(protein interacting with C kinase 1) signaling pathways mediated to resistance oxidative stress and cell apoptosis of brain slices.Methods:Preparation of cerebral injury models and grouping:before preparation of brain slices 160 Kunming mice were randomly divided into normal group,model group,low dose group and high dose group.Four groups were incubated in artificial cerebrospinal fluid at 37℃ for 30 min.Drug groups were incubated in ACSF at 37℃ with different concentrations of metformin for 30 min, the normal group and model group were incubated in normal artificial cerebrospinal fluid at 37℃ for 30 min.The model group and drug groups were incubated in artificial cerebrospinal fluid with hydrogen peroxide at 37℃ for 60 min,the normal group was incubated in normal artificial cerebrospinal fluid at 37℃for 60 min.After 120 min, the brain slices were prepared for further assays.The oxidative damage and concentration of formazan were detected by TTC staining.The activity of LDH(Lactate dehydrogenase),T-SOD(total-Superoxide Dismutase),MDA(malondialdehyde) and GSH-PX(glutathione peroxidase) were detected by LDH kit,T-SOD kit,MDA kit and GSH-PX kit.The expression of PICK1 m RNA was measured by q RT-PCR and apoptosis related protein Bcl-2/Bax(B-cell Leukemia/Lymphoma 2/Bcl-2 Associated X protein) were measured by Western Blot.Results:(1) Results show that compared with normal control group,the colors of TTC staining of model group and metformin groups were significantly lighter,and compared with model group,the metformin groups were obviously deepened,and the high dose group deeper than the low dose group.(2) Results show that compared with control group,the concentration of formazan significantly decreased(P<0.01).Compared with model group,low dose group increased(P<0.05) and high dose group significantly increased(P<0.01).(3) Results show that compared with normal control group,the activity of Lactate dehydrogenase(LDH) significantly increased(P<0.01).Compared with model group,low dose group(P<0.05) and high dose group(P<0.05) increased.But the difference between low dose group and high dose group was not obvious.(4) ?The activity of T-SOD of brain slices:Compared with control group,the activity of T-SOD of model group significantly decreased(P<0.01).Compared with model group,low dose group increased(P<0.05) and high dose group significantly increased(P<0.01).?The activity of MDA of brain slices:compared with control group,the activity of MDA of model group increased(P<0.05).Compared with model group,low dose group decreased(P<0.05) and high dose group significantly decreased(P<0.01).?The activity of GSH-PX of brain slices:Compared with control group,the activity of GSH-PX of model group decreased(P<0.05).Compared with model group, high dose group increased(P<0.05).However low dose group increased(P>0.05),but dose not have statistical significance.(5)The expression of PICK1 m RNA:compared with control group,the model group significantly increased(P<0.01);And metformin pre-treated slices(low dose group and high dose group) decreased compared with model group(P<0.05,P<0.05).High dose group reduced more than low dose group.(6)The expression of Bcl-2/Bax:The model group decreased obviously compared with control group(P<0.05).Compared with model group,low dose group(P<0.01) and high dose group(P<0.01) significantly increased.Conlusion:(1)Hydrogen peroxide can induce brain slices damage,however metformin have protective effect on brain slices induced by hydrogen peroxide.(2)The brain slices in oxidative stress can increased the expression of PICK1 m RNA,metformin have great protective effect on brain slices via decreasing the expression of PICK1 m RNA.(3)Metformin have protective effect on brain slices in oxidative stress induced by hydrogen peroxide via incresing the expression of Bcl-2/Bax.