Dihydroartemisinin(DHA) Induces PC12 Differentiation Through Activation Of Autophagy Signaling Interactive Pathway

Objective: Dihydroartemisinin(DHA)neurotoxicity experiments showed that after DHA intervene PC12,the synaptic extension of cells and increased vitality after DHA intervention.We investigated the mechanism by which DHA induces PC12 differentiation.It is expected to develop effective and safe drugs for the treatment of neurodegenerative diseases.1.Method:The optimum concentration screening of DHA was detected by CCK8,respectively.The optimal concentration of DHA with 0.01,0.1,1,10,and 25 mg/L was treated PC12.2.The count of morphological changes of PC12 cells after DHA intervene and the growth of cell synapses was detected by microscope.3.Different concentrations of DHA was treated PC12 and cytometry(FACS)analysis ROS in PC12 cells.4.Neuronal markers MAP2 was detected by immunofluorescence staining after DHA intervention with PC12.5.The expression of Beclin,LC-3,ATG5,and MAPK was detected by RT-PCR after DHA intervention with PC12.6.Beclin,LC-3,ATG5,MAPK and p38 MAPK Protein Levels was Detected Western Blotting after DHA intervention with PC12Results:Different concentrations of DHA(0.01,0.1,1,10,25 μg/m L)treated with PC-12 cells for24 h.The results of CCK8 showed that the cytotoxicity was increased and the cell growth was inhibited in the concentration 10 and 25 μg/m L.The concentration of DHA(0.01,0.1,and 1 μg/m L)treated with PC-12 cell for 24 h.There was no significant difference between the cell viability and the normal group(p>0.05).We Selected concentrations of DHA(0.01,0.1,and 1 μg/m L),respectively,to treat with PC-12 cells,after12,24,48,and 72 h detected cell viability.The results showed that when DHA treated PC-12 cells at 24 h,the viability of the cells was significantly decreased at concentrations of 0.1 and 1 μg/m L(p<0.05 or p<0.001);after 72 h,the viability of the cells at concentrations of 0.01,0.1,and 1 μg/m L was significantly reduced.(p<0.001 or p<0.05 or p<0.001);Treated of PC-12 cells after 48 h,the cell viability in the 0.01,0.1,and 1 μg/m L groups had no difference compared with the normal group(p>0.05),we selected concentration was 0.01,0.1 and 1 μg/m L DHA for 48 h treatment of PC-12 cells,was used as a follow-up study condition.Count the number of synaptic proliferations of PC-12 cells treated with different concentrations.The results showed that concentrations of DHA(0.01,0.1,and 1 μg/m L)significantly promoted synaptic proliferation(p<0.01).Fluorescence microscopy was used to observe the number of MAP2 positive cells treated with different concentrations.The results showed that concentrations of DHA(0.01,0.1,and 1 μg/m L)the expression of MAP2 protein increased in PC-12 cells,and 0.1 μg/m L of DHA had the greatest number of MAP2 positive cells in PC-12 cells.Flow cytometry was used to detect the level of ROS production in PC-12 cells treated with different concentrations of DHA.The results showed that 0.01,0.1,and 1 μg/m L of DHA all increased ROS in PC-12 cells.The largest number of MAP2 positive cells was observed in PC-12 cells treated with 0.1μg/m L DHA.we selected DHA concentration of 0.1μg/m L intervention PC-12 cells after48 h,atg5 and beclin m RNA expression levels were significantly increased.Western Blotting was used to detect the expression of Beclin,LC-3,ATG5,MAPK and p38 MAPK in PC-12 cells treated with DHA.The results showed that Beclin and ATG5 protein levels were significantly increased,and LC-3 protein levels were increased.The p38 MAPK was significantly elevated at the protein level.conclusions: 1.High concentration of DHA significantly inhibited the growth of PC-12 cells.Low concentrations have no apparent toxicity.2.After low concentration of DHA treated PC-12 cell,the viability of PC12 cells increased,the synaptic elongation increased,and the expression of MAP2 proteins is increased,and cell differentiation is enhanced.