Dihydromyricetin Inhibits The Proliferation Of Retinoblastoma Cell Line Y79 Through JAK2/STAT3/Autophagy Pathway

[Objective] To observe the effect of dihydromyricetin(DMY)on the proliferation of retinoblastoma cell line Y79 cells and explore its possible mechanism.[Method] 1.The experiment was divided into the following groups:the control group: Y79 cells were cultured in RPMI-1640 medium with10% calf serum for 24 h.DMY group: Y79 cells were treated with DMY(20,40,60 and 80 mg/L)for 12,24 and 48 h,respectively.3-MA(3-methyladenine)group: Y79 cells were cultured in RPMI-1640 medium containing 3-MA(5 mmol/L)for 24 h.3-MA + DMY group: the cells were treated with RPMI-1640 medium containing 3-MA(5 mmol/L)for 0.5 h,then DMY(100 μg/m L)was added and cells were co-treated for24 h.WP1066 group: cells were cultured in RPMI-1640 medium containing 3-MA(5 mmol/L)for 24 h.WP1066 + DMY group: Y79 cells were treated with RPMI-1640 medium containing JAK2/STAT3 signal pathway inhibitor wp1066(10 μ mol/L)for 0.5 h,then DMY(100 μg/m L)was added and cells were co-treated for 24 h.2.MTT assay was used to detect the cell viability.3.The ultrastructure of the cells was observed by transmission electron microscope.4.The expressions of autophagy related proteins beclin-1,LC-Ⅱ and p62,and the expressions of total JAK2(t-JAK2),phosphorylated JAK2(p-JAK2),total STAT3(t-STAT3)and phosphorylated STAT3(p-STAT3)were detected by Western blot.[Results] 1.After treated with DMY(20,40,60 and 80 mg/L)for 12,24 and 48 h,the proliferation of Y79 cells was significantly decreased in a dose and time-dependent manner(all P<0.05).2.After treated with DMY(20,40,60 and 80 mg / L)for 12,24 and48 h,the proliferation inhibition rates of Y79 cells in each group were significantly higher than that in the control group,and the proliferation inhibition rates were gradually increased with the increase of treatment time and concentration of DMY(all P<0.05).3.Compared with the control group,the expressions of autophagy related proteins beclin-1,LC-Ⅱ and the ratio of LC-Ⅱ to LC-Ⅰ(LC-Ⅱ/LC-Ⅰ)in DMY(60 mg/L)group were significantly increased(all P<0.05),while the expression of p62 was significantly decreased(P<0.05).4.The results of electron microscopy showed that compared with the control group,the number of autophagy bodies in DMY treatment group was significantly increased.Compared with the control group,the percentage of autophagic vesicles in the total cytoplasmic area of DMY(60 mg/L)group was significantly increased(P<0.05).5.Compared with DMY group,the proliferation level of Y79 cells in DMY(60 μmol/L)+ 3-MA group was significantly increased(P<0.05).6.Compared with the control group,the protein expressions of p-JAK2 and p-STAT3 and the ratio of p-JAK2/t-JAK2 and p-STAT3/t-STAT3 in DMY(60 mg/L)group were significantly increased(all P<0.05).Compared with the control group,the expression of t-JAK2 protein in DMY treatment group had no significant difference(P>0.05).7.Compared with DMY group,the proliferation level of Y79 cells in DMY(60 μmol/L)+ WP1066 group was significantly increased(P<0.05).[Conclusion] Dihydromyricetin inhibits the proliferation of human retinoblastoma cell line Y79 cells,which the mechanism may be related to the activation of JAK2/STAT3 signaling pathway and the promotion of autophagy induced by DMY.