Protective Effects Of Safflor Yellow On Alzheimer’s Disease Induced By Tau Phosphorylation In Vitro

Safflower,a dry tubular flower of Carthamus tinctorius L.is a traditional Chinese medicine.Modern pharmacological studies have reported that the water-soluble safflower yellow pigements including hydroxy safflor yellow A(HSYA),Safflower yellow A(SYA)and Safflower yellow B(SYB)are the main effective components of safflower.In this study,SYB was purified from safflower yellow pigments;Human neuroblastocytes(SH-SY5Y)cells were cultured to model the Alzheimer’s Disease(AD)in vitro.To investigate the protective effects and mechanisms of HSYA、SYA and SYB on the synaptic damage and the abnormal phosphorylation of Tau protein in SH-SY5 Y cells induced by OA,and provide experimental basis for its clinical applications.The main contents and results are as follows:(1)After alcohol precipitation purifying,the safflower products were eluted by AB-8resin column chromatography to obtain safflower yellow B crude,then the crude were going through Sephadex LH-20 chromatography to get the pure safflower yellow B,and its purity was over 98%.The preparing method that can obtain the higher purity products without any impurities is simple,cheap and safe.(2)Establish an AD cell model via OA-induced synaptic atrophy and abnormal phosphorylation of Tau protein in differentiated mature neurons in vitro.From the observation of cell morphology,the measurement and calculation the ratio of synapses and cell body,and the detection of Tau Ser-262 protein phosphorylation by western blot,we found that the synapses increased significantly(P < 0.01),and the cell cycle stagnated at G0/G1 phase,and the cells showed typical differentiated neuronal characteristics compared with normal cells after inducement of 5 μ mol/L ATRA for 4 days.When different concentrations of OA(0、10、20、30、40 nmol/L)were used to deal with differentiated mature neurons at different times(3、6、9、12、18 h),cell synapses were shrinking and their morphology changed significantly.After the 40 nmol/L OA challenge,the synaptic atrophy of the cells was obvious in a time dependent manner,and the phosphorylation of the Ser-262 site of Tau protein increased first and then decreased overthe time changing.The final cell model conditions were defined as follows: the SH-SY5 Y cells can differentiate into mature neurons at 5 μmol/L ATRA for 4 days;it can be caused synapse atrophy and Tau protein abnormal phosphorylation after the treatment of 40nmol/L OA for 6 h.(3)After co-treatment of the 40 nmol/L OA and 0.2-1 μmol/L HSYA、SYA、SYB respectively for 6 hours,the cell morphology were observed by Giemsa staining and the phosphorylation of the Ser-262 site of Tau protein were detected by Western blot.The synergistic atrophy of neurons induced by OA was inhibited by HSYA、SYA and SYB resulting in the cells spread well and tend to normal cells,and the phosphorylation of tau protein Ser262 site was decreased.Moreover,individual compounds can inhibit the abnormal phosphorylation of Tau protein in a dose dependent manner.(4)To evaluate the function of mitochondria,the Flow cytometry was used to detect the level of reactive oxygen species in the cells using DCFH-DA fluorescence probes,and the changes in mitochondrial membrane potential using JC-1 fluorescent probe staining,the changes in mitochondrial reactive oxygen species and mitochondria mass using Mito SOX fluorescence probes and MitoTracker? Green FM probes respectively;The ATP content detection kit was used to detect the changes in intracellular ATP content.The results showed that the intracellular reactive oxygen species and mitochondrial reactive oxygen species was decreased,mitochondrial membrane potential,the number of mitochondria and ATP synthesis was increased.Conclusion: The study demonstrates that SY has multiple protective effects in differentiated SH-SY5 Y neuroblastoma cells,it could effectively inhibit OA-induced synaptic atrophy,increase the level of synaptophysin expression,meanwhile the level of the phosphorylation of Tau protein at serine262 markedly decreased.The likely mechanism might be linked to mitochondrial function.