Study On The Mechanism Of Artesunate Regulating Autophagy And Inhibiting OGD/R Induced PC12 Cell Injury

Ischemic stroke is one of the diseases in which cerebral vascular disease causes a rapid decrease in blood supply to brain tissue,resulting in insufficient blood supply,leading to metabolic disorders and death of brain cells,and ultimately irreversible injury to brain tissue.Artesunate(ART)can penetrate the blood brain barrier and maintain a high concentration in the brain,and has very slight neurotoxicity.To study the effect of ART on PC12 oxygen-glucose deprivation/ reperfusion(OGD/R)induced excessive autophagy,as well as the important role of AMPK/mTOR signaling pathway in it,and explore new methods and drugs for the prevention and treatment of ischemic stroke,to explore the potential role of ART in ischemic stroke.In this study,PC12 cells were used as the research object.First,the OGD/R model was established using the 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromination method(MTT method).Then,MTT assay was used to detect the effect of different concentrations of ART on cell activity,and to screen the effective working concentration of ART to inhibit OGD/R induced cell injury.The changes of cell morphology and growth status in the control group,OGD/R group,OGD/R+ART group,and OGD/R+ autophagy inhibitor Bafilomycin A1(BAF-A1)group were observed through inverted microscope.Hoechst 33342 staining was used to observe the protective effects of OGD/R and ART on OGD/R induced cell injury.Flow cytometry was used to observe the changes of apoptosis in each group.Immunocytochemical staining was used to detect the expression of related autophagic proteins p62,Beclin-1,LC3-Ⅱ/LC3-Ⅰin each group.Finally,Western blotting was used to detect the expression of autophagy related marker proteins p62,Beclin-1,LC3-Ⅱ/LC3-Ⅰ in cells of each group,and the effect on the expression of related proteins AMPK,p-AMPK,mTOR,and p-mTOR in the AMPK/mTOR signal pathway.The results showed that PC12 cells were cultured in vitro,and the proliferation inhibition rates of cells at different OGD/R time periods were calculated,and a dose-effect curve was drawn.It was concluded that the half inhibition time of OGD/R on cells was 6 hours of oxygen glucose deprivation/24 hours of reperfusion.According to the MTT results,the ART concentration is below 25μM has no effect on cell survival.After 6hours of OGD,the cells were co-cultured with different concentrations of ART reoxygenated glucose for 24 hours,and the survival rate was measured by MTT.The experiment was divided into five groups:(1)control group,(2)OGD/R group,(3)OGD/R+ART low concentration group(1μM),(4)OGD/R+ART high concentration group(25μM),(5)OGD/R+BAF-A1 group.The inverted microscope observed that the cells in the control group had multiple protrusions,with different numbers of protrusions,the synapses are long and resemble neuronal axons.The cells in the OGD/R group were mostly circular,without protrusions,or had shorter protrusions.The cells in the OGD/R+ART group gradually recovered to longer synapses as the concentration increased.The morphology of cells in the BAF-A1 group was similar to that in the OGD/R+ART group.Hoechst33342 staining experiment observed that compared with the control group,the nucleus of OGD/R group showed a bright blue color,indicating a significant increase in cell apoptosis.The number of bright blue cells in the nucleus of OGD/R+ART group decreased as the concentration of ART increased.The trend of OGD/R+BAF-A1 group was similar to that of OGD/R+ART group.The results of flow cytometry were consistent with the above experimental results.The OGD/R group showed significant apoptosis,and ART could reduce the OGD/R induced apoptosis.The results of immunocytochemical staining and Western blotting showed that after treatment with different concentrations of ART on OGD/R induced cells,compared with the control group,the expression of autophagy related protein p62 in OGD/R group was significantly decreased(P<0.01),and the expression of Beclin-1 was significantly increased(P<0.01).The proportion of LC3-Ⅱ/LC3-Ⅰ was increased.Both ART and BAF-A1 co-cultures significantly the expression of p62,reduce the expression of Beclin-1,and enhance the proportion of LC3-Ⅱ/LC3-Ⅰ(P<0.05).The results of Western blotting showed that the expression of the classical autophagic AMPK/mTOR signaling pathway related protein p-AMPK/AMPK was increased and the expression level of p-mTOR/mTOR was significantly decreased in OGD/R group(P<0.01);In the OGD/R+ART group,the expression of p-AMPK/AMPK decreased and the expression of p-mTOR/mTOR increased with the increase of ART concentration(P<0.01).In conclusion,ART can inhibit OGD/R induced PC12 cell injury,which may be related to regulating the AMPK/mTOR signaling pathway to inhibit excessive autophagy.Therefore,Therefore,ART may be a potential drug for the treatment of ischemic stroke.