| Objective:To investigate the effects of curcumin on proliferation,apoptosis,ROS generation,endoplasmic reticulum stress and autophagy in human neuroblastoma cell lines SK-N-SH and SH-SY5Y cells,and to preliminarily investigation of the molecular mechanisms of curcumin on SK-N-SH and SH-SY5Y cell proliferation,apoptosis and endoplasmic reticulum stress and autophagic activity via ROS.Methods:(1)SK-N-SH and SH-SY5Y cells were selected for the study,and different concentrations of curcumin(0,1.25,2.5,5,10 and 15 μmol/L)were applied to the cells,and the inhibition rate and half inhibition concentration(IC50)of curcumin on SK-N-SH and SH-SY5Y cells were detected by CCK-8 method.Subsequently,experimental groups were set up:control group,curcumin 1/2 IC50,IC50,2 IC50 groups,different drug concentrations were applied to SK-N-SH and SH-SY5Y cells,and the inhibition rates of different concentrations of curcumin on cells were detected by CCK-8 method;the effects of different concentrations of curcumin on apoptosis,cell cycle and ROS fluorescence intensity were detected by flow cytometry;Reagent kit to detect MDA activity;Real-time qPCR assay(RT-qPCR)to detect cellular Grp78 and CHOP mRNA expression;Western blot to detect cellular Caspase-3,cleaved Caspase-3,cyclinA2,p21,Grp78,CHOP,cleaved caspase-12,Beclin-1,LC3II,and LC3 I protein expression;And the co-localization of p62 and LC3 was observed by immunofluorescence technique.(2)SK-N-SH and SH-SY5Y cells were selected for the study,and the experiments were grouped into:control group,curcumin group(IC50),NAS(ROS scavenger)group(5mmol/L),and curcumin+NAC(IC50+5mmol/L).CCK-8 was applied to detect the cell inhibition rate;flow cytometry was applied to detect the apoptosis rate;Western blot was applied to detect the cell Grp78,CHOP,cleaved caspase-12,Beclin-1,LC3II,LC3 I protein expression.Results:(1)CCK-8 results showed that cell proliferation was inhibited after different concentrations of curcumin(0,1.25,2.5,5,10,15 μmol/L)on SK-N-SH and SH-SY5Y cells,and the inhibitory effect was concentration-dependent,and the IC50 values of curcumin on SK-N-SH and SH-SY5Y cells were 13.251 and 12.387,respectively.After the effect of NAC,curcumin group and curcumin+NAC on SK-N-SH and SH-SY5Y cells,the inhibition rate of SK-N-SH and SH-SY5Y cells in curcumin+NAC group was significantly increased compared with NAC group(P<0.01).(2)Flow cytometric and MDA activity results demonstrated that after curcumin 1/2 IC50,IC50,2IC50 action on SK-N-SH and SH-SY5Y cells,compared with the control group,the apoptosis rate of SK-N-SH and SH-SY5Y cells in curcumin 1/2 IC50 group,IC50 group and 2IC50 group was significantly increased(P<0.01).The G2/M phase cells of SK-N-SH cells were significantly increased in the curcumin 2IC50 group(P<0.01).Significant increase in ROS fluorescence intensity and decrease in MDA activity in SK-N-SH and SH-SY5Y cells in curcumin 1/2IC50,IC50 and 2IC50 groups(P<0.01).After NAC,curcumin group and curcumin+NAC action on SK-N-SH and SH-SY5Y cells,the apoptosis rate of SK-N-SH and SH-SY5Y cells was significantly increased in curcumin+NAC group compared with NAC group(P<0.01).(3)The results of qRT-PCR assay suggested that after curcumin 1/2 IC50,IC50 and 21C50 action on SK-N-SH and SH-SY5Y cells,the expression of Grp78 and CHOP mRNA in SK-N-SH and SH-SY5Y cells in curcumin IC50 and 2IC50 groups were significantly increased compared with the control group(P<0.05).(4)Western blot assay results showed that after curcumin 1/2 IC50,IC50,2IC50 action on SK-N-SH,SH-SY5Y cells.Compared with control group,the expression of cleaved Caspase-3,p21,Grp78,CHOP,cleaved caspase-12,Beclin-1,LC3Ⅱ,LC3Ⅱ/LC3Ⅰ protein was significantly increased in SK-N-SH,SH-SY5Y cells in curcumin IC50 group and 2IC50 group(P<0.05),the expression of cyclinA2 and LC3Ⅰ protein was significantly decreased in SK-N-SH and SH-SY5Y cells in curcumin IC50 group and 2IC50 group(P<0.05).After the action of NAC,curcumin group and curcumin+NAC on SK-N-SH and SH-SY5Y cells,Compared with the NAC group,the expression of CHOP protein was significantly increased in SK-N-SH cells and the expression of Grp78 and CHOP protein was significantly increased in SH-SY5Y cells in curcumin+NAC group(P<0.05).The expression of cleaved caspase-12,Beclin-1,LC3Ⅱ,and LC3Ⅱ/LC3Ⅰ protein was significantly increased in SK-N-SH and SH-SY5Y cells(P<0.05),and the difference in LC3Ⅰ protein expression was not statistically significant(P>0.05).(5)Immunofluorescence results demonstrated that after curcumin 1/2 IC50,IC50,2IC50 action on SK-N-SH,SH-SY5Y cells,compared with the control group,LC3 expression of SK-N-SH,SH-SY5Y cells in curcumin IC50,2IC50 group was significantly upregulated(P<0.05),and curcumin 1/2 IC50,IC50,2IC50 groups SH-SY5Y cells p62 expression was significantly decreased(P<0.05).Conclusion:(1)Curcumin inhibited the proliferation of human neuroblastoma SK-N-SH and SH-SY5Y cells in a concentration-dependent manner,induced apoptosis in SK-N-SH and SH-SY5Y cells,down-regulated cyclinA2 protein expression in SK-N-SH and SH-SY5Y cells and up-regulated p21 protein expression in SK-N-SH and SH-SY5Y cells.After curcumin(IC50,2IC50)treatment,ROS fluorescence intensity of SK-N-SH and SH-SY5Y cells was significantly increased,which could promote cellular Grp78,CHOP,cleaved caspase-12 and Beclin-1,LC3Ⅱ protein expression.(2)Curcumin inhibits SK-N-SH and SH-SY5Y cell proliferation,induces SK-N-SH and SH-SY5Y cell apoptosis,and promotes endoplasmic reticulum stress and autophagy in SK-N-SH and SH-SY5Y cells through upregulation of ROS production. |
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