| Objective To study the effect of Qi-Boosting Toxin-Resolving Formulae(QBTRF)on autophagy and apoptosis of human nasopharyngeal carcinoma CNE-2Z cells,and to explore the relationship between autophagy and apoptosis of CNE-2Z cells.Methods1.Detection of apoptosis of CNE-2Z cells induced by QBTRF MTT assay was used to detect the effect of different concentrations of QBTRF on the proliferation of CNE-2Z cells after 24 h and 48 h of nasopharyngeal carcinoma and the half inhibitory concentration of 48 h was calculated as the follow-up drug intervention concentration.The changes of apoptosis rate after QBTRF intervened CNE-2Z cells for 12 h,24h,36 h and 48 h and the effect of Z-VAD-fmk(Inhibitor of apoptosis)on the apoptosis rate were detected by Annexin V-FITC/PI.The expressions of Cleaved caspase-3,Bax,Bcl-2 and Cleaved PARP after QBTRF intervened CNE-2Z cells for12 h,24h,36 h and 48 h and the effect of Z-VAD-fmk on Cleaved caspase-3,Bax,Bcl-2 and Cleaved PARP protein expression were detected by Western Blot.2.Detection of autophagy of CNE-2Z cells induced by QBTRF MDC was used to observe the formation of autophagosomes after QBTRF intervened CNE-2Z cells for 12 h,24h,36 h and 48 h.Western blot was used to detect the protein expression of LC3 and Beclin1 after QBTRF intervened CNE-2Z cells for 12 h,24h,36 h and 48 h and 3-MA(Autophagy inhibitor)intervened CNE-2Z cells.In addition,western blot was used to detect the protein expression of PI3K/Akt/mTOR signaling pathway.3.Study on the relationship between autophagy and apoptosis The autophagy and apoptosis of QBTRF were observed under transmission electron microscope.Annexin V-FITC/PI was used to detect the change of apoptotic rate of CNE-2Z cells after 3-MA.The expression of LC3 and Beclin1 protein after Z-VAD-fmk was detected by Western Blot.Results1.Effect of QBTRF on CNE-2Z cells proliferation MTT results showed that QBTRF significantly inhibited the proliferation of CNE-2Z cells compared with the Control group,and the results were extremely significant(p<0.01),and the inhibitory effect was concentration dependent and time-dependent.2.Effects of QBTRF on apoptosis of CNE-2Z Cells(1)Annexin V-FITC/PI results showed that the apoptotic rates of CNE-2Z cells treated with QBTRF for 12 h,24h,36 h and 48 h were(38.69±3.22)%,(54.30±6.93)%,(69.53±4.14)% and(80.87±4.15)% respectively,compared with Control group(1.11±0.83)%,the difference was statistically significant(p <0.01).10μM Z-VAD-fmk pretreatment significantly reduced the rate of apoptosis induced by QBTRF,the apoptosis rate of Z-VAD-fmk pretreatment +QBTRF group was(14.30±3.07)%,and the apoptosis rate of QBTRF group was(58.66±5.66)%,the difference was statistically significant.There was no significant difference between the Z-VAD-fmk group(2.02±0.50%)and the Control group(1.39±0.58)%(p(29)0.05).(2)Western Blot results showed that the expression of Cleaved caspase-3,Bax and Cleaved PARP protein was up-regulated and the expression of Bcl-2 protein was decreased after QBTRF intervention in CNE-2Z cells(12h、24h、36h 和 48h).The difference was statistically significant(p all <0.01).The expression of Cleaved caspase-3,Bax and Cleaved PARP decreased and the expression of Bcl-2 was up-regulated in Z-VAD-fmk pretreatment+ QBTRF group,which was significantly different from that in QBTRF group(p <0.05 and p <0.01).3.Effects of QBTRF on autophagy of CNE-2Z Cells(1)MDC results showed that the fluorescence intensity of CNE-2Z cells treated withQBTRF for 12 h,24h,36 h and 48 h were(0.044 ± 0.009),(0.078 ± 0.010),(0.053 ±0.010)and(0.030 ± 0.007)respectively,compared with Control group(0.011 ± 0.008),the difference was statistically significant(p <0.05 and p <0.01).(2)Western Blot results showed that QBTRF intervention CNE-2Z cells 12h、24h、36h and 48 h,LC3-II and Beclin1 enhanced expression compared with Control group,with statistically significant difference(p <0.05 and p <0.01),at 24 h,the protein expression of LC3-II and Beclin1 increased the peak;In addition,when intervention24 h,the protein expression of PI3 K,p-AKT and p-mTOR in the QBTRF group were lower than that in the Control group(p <0.05 and p <0.01);While,the expression of PI3 K,p-AKT and p-mTOR protein was up-regulated(p <0.05 and p <0.01)in 3-MA pretreatment + QBTRF group.4.Study on the correlation between autophagy and apoptosis of CNE-2Z cells(1)The results of transmission electron microscopy showed that QBTRF interference in CNE-2Z cells 12 h after the emergence of a small number of cells in the cell,the cell structure is clear;After the intervention of 24 h,the number of autophagy in the cells increased significantly,and the nuclei began to shrink;After 36 h intervention,the relative reduction in the number of autophagosomes,nuclear pyknosis obviously increased,intracytoplasmic vacuoles;48h after the intervention,reduce autophagy in cells,nuclear pyknosis into blocks,cell volume reduction and by sprouting.(2)Annexin V-FITC/PI showed that 3-MA significantly inhibited the apoptosis of CNE-2Z cells.The apoptosis rate of 3-MA group was(1.66±0.76)%,and group Control(1.97±0.68)%,the difference was not statistically significant(p=0.594);3-MA pretreatment group + QBTRF apoptosis rate was(19.40±2.59)%,and the apoptosis rate of QBTRF group(54.79±4.06)%,there was statistical significance between the two the difference(p=0.000).(3)Western Blot showed that autophagy inhibitor 3-MA pretreatment group+QBTRF Cleaved caspase-3 and Cleaved PARP apoptosis protein and the expression level of Bax decreased,the expression level of the inhibitor of apoptosis protein Bcl-2is up-regulated,the differences were statistically significant(p <0.01),compared withQBTRF group;Compared with QBTRF group,Z-VAD-fmk pretreatment group+QBTRF apoptosis inhibitor of autophagy related protein LC3 and Beclin1 protein expression decreased significantly,there were statistically significant differences(p<0.05).Conclusions QBTRF inhibited the proliferation of human nasopharyngeal carcinoma CNE-2Z cells and induced apoptosis and autophagy.QBTRF induced autophagy is associated with activation of the PI3K/AKT/mTOR pathway.QBTRF-induced autophagy could promote apoptosis. |
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