Ligustilide Promotes Autophagy Through LKB1-AMPK-mTOR Signaling Pathway To Protect OGD/R Induced PC12 Apoptosis

Objective: Nervous system diseases have become one of the most important diseases that seriously threaten human health.The oxygen and glucose deprivation(OGD)model of nerve cells is mostly used to simulate ischemic stroke at the cellular level,and it is mainly used to quickly recover oxygen and glucose supply after oxygen and glucose deprivation,so as to simulate cerebral ischemia reperfusion injury for cell self-repair and drug research.Ligustilide(LIG)is a kind of phthalide in chemical structure,which mainly exists in the volatile oil of angelica sinensis and ligusticum chuanxiong.In this study,we investigated whether LIG could fight PC12 cell apoptosis induced by OGD/R through autophagy.This provides a theoretical basis for the protective effect of LIG on nerve injury in acute ischemic cerebrovascular disease.Methods: 1.PC12 cells were cultured and passaged when the cell growth reached 90% and the fusion was achieved,the cells were passed at a ratio of 1:3,the culture conditions was 37℃ and 5%CO2.2.MTS method was used to detect the effects of OGD/R at different times(3h,12 h,24h,48h)on the proliferation activity of PC12 cells.3.Inverted microscope was used to observe the effects of different concentrations of LIG(10-5-10-9M)on the morphology of PC12 cells at different time(3h,12 h,24h).4.Flow cytometry was used to detect the effects of OGD/R on PC12 cell apoptosis at different time(3h,12 h,24h,48h).5.The effect of autophagy pathway inhibitor 3-MA and AMPK inhibitor Dorsomorphin on cell apoptosis was detected by flow cytometry.6.Immunofluorescence was used to detect the expression of autophagy-related protein LC3-Ⅱ in PC12 cells.7.Electron transmission microscopy was used to detect the expression of autophagosomes in PC12 cells.8.Western blot was used to detect the expression of apoptosis-related proteins Bcl-2 and Bax after LIG(10-6M)treatment.9.Western blotting was used to detect the protein expression of LKB1,p-LKB1,AMPK,p-AMPK,m TOR and p-m TOR in the cell signaling pathway after LIG(10-6M).10.Detect the effect of addition pathway inhibition of 3-MA on the expression of LKB1,p-LKB1,AMPK,p-AMPK,m TOR and p-m TOR proteins;Results: 1.PC12 cells were cultured in DMEM-F12 culture medium(10% fetal bovine serum,1% penicillin and streptomycin)for 3 days,reaching 90%,and were spread on the bottom of the bottle for passage in a ratio of 1:3.2.After the LIG concentration of 0,10-5 ~ 10-9M was used for 0,3,12,and 24 hours,it was found that the LIG concentration of 10-5,10-6,and 10-7M significantly increased the proliferation activity of cells after 3 hours(compared with the dexamethasone group,P < 0.05).Therefore,we chose LIG pretreatment(10-6M,3h)as the concentration and time entry point for subsequent tests.3.PC12 cell apoptosis was detected by flow cytometry.Under the OGD/R condition,PC12 cell apoptosis was induced at different time(3h,12 h,24h).LIG significantly reduced the apoptosis rate of PC12 cells induced by OGD/R.4.PC12 cell apoptosis induced by OGD/R was detected by flow cytometry,and the apoptosis rate was significantly increased after autophagy inhibition by 3-MA.After the addition of AMPK inhibitor Dorsomorphin,the apoptosis rate of Dor-group increased,and the protective effect of LIG was weakened after the application of autophagy and pathway inhibitors.These results again demonstrated that autophagy was involved in the neuroprotective effect of LIG,which attenuated the apoptosis induced by OGD/R.5.After OGD/R(12h)treatment,a small amount of autophagy-related protein LC3-Ⅱ accumulated around the nucleus,and the expression and distribution of LC3-Ⅱ were significantly enhanced after LIG treatment.Autophagy inhibitor 3-MA can partially reverse this process,indicating that LIG can improve the autophagy level of PC12 cells.6.Electron transmission microscopy was used to detect autophagosomes in PC12 cells,and LIG was up-regulated in PC12 cells.7.Western blot analysis showed that LIG increased the protein expression of Bcl-2 in OGD/R treated PC12 cells and decreased the protein expression of Bax.8.LIG+OGD/R group significantly increased AMPK activation(phosphorylation)and AMPK upstream kinase LKB1(phosphorylation),indicating that LIG had the ability to up-regulate LKB1 and AMPK activities.In this group,m TOR phosphorylation was reduced,and LIG may induce autophagy level acceleration through the LKB1-AMPK-m TOR signaling pathway.Conclusions: 1.The pretreatment of 10-6M LIG for 3h can significantly inhibit OGD/ Rinduced PC12 cell apoptosis.2.LIG inhibited PC12 cell apoptosis by up-regulating Bcl-2 protein expression and down-regulating Bax protein expression.3.LIG can inhibit apoptosis by upregulating autophagy and play a neuroprotective role.4.LIG inhibits OGD/R-induced PC12 cell apoptosis by increasing autophagy and may be achieved by activation of the LKB1-AMPK-m TOR autophagy signaling pathway.