Digitoxin regulates autophagy and apoptosis of thyroid follicular epithelial cells via inhibiting activation of Akt/mTOR signaling pathway
Objective It is to investigate the effects of digitoxin on apoptosis and autophagy of human thyroid follicular epithelial cell line Nthy-roi3-1 stimulated by lipopolysaccharide(LPS),and to detect its mechanism.Methods ①Nthy roi3-1 cells were divided into control group,LPS group,LPS + digitoxin low dose(digitoxin-L)group,LPS + digitoxin medium dose(digitoxin-M)group and LPS +digitoxin high dose(digitoxin-H)group.After corresponding treatment,the viability of Nthy-roi3-1 cells in each group was detected by CCK-8 method,the expression of microtubule-associated protein 1 light chain 3(LC3)in Nthy-roi3-1 cells was observed by immunofluorescence staining,the apoptosis rate of Nthy-roi3-1 cells of each group was detected by flow cytometry,the expressions of LC3Ⅱ/LC3Ⅰ and Beclin1 protein,phosphorylated protein kinase B(p-Akt),protein kinase B(Akt),phosphorylated mammalian target of rapamycin(p-mTOR),mammali-an target of rapamycin(mTOR)and its downstream factor p70S6K protein in Nthy-roi3-1 cells of each group were detected by Western blot.②Nthy-roi3-1 cells were divided into control group,LPS group,LPS +digitoxin group,LPS +digitoxin + autophagy inhibitor group.After corresponding treatment,the apoptosis rate of each group was detected by flow cytometry,and the protein expressions of LC3Ⅱ/LC3Ⅰ,Beclin1,p-Akt,Akt,p mTOR,mTOR and p70S6K were detected by West-ern blot.Results Compared with LPS group,the cell viability of each LPS + digitoxin group were significantly increased(all P<0.05);the fluorescence staining intensity of LC3 was gradually increased with the increase of dose of digitoxin,the apoptosis rates were all significantly decreased(all P<0.05);the relative protein expressions of LC3Ⅱ/LC3Ⅰ and Beclin1 in the cells were significantly up-regulated(all P<0.05),and the relative protein expressions of p-Akt/Akt,p-mTOR/mTOR and p70S6K in the cells were significantly down-regulated(all P<0.05).②Compared with LPS group,the apoptosis rate of LPS +digitoxin +group was significantly decreased(P<0.05);the relative protein expressions of LC3Ⅱ/LC3Ⅰ and Beclin1 in the cells were significantly up-regulated(all P<0.05),meanwhile,the relative protein expressions of p-Akt/Akt,p-mTOR/mTOR and p70S6K in the cells were significantly down-regulated(all P<0.05).Compared with LPS +digitoxin +group,the apoptosis rate of LPS +digitoxin +autophagy inhibitor group was significantly increased(P<0.05);the relative protein expressions of LC3Ⅱ/LC3Ⅰ and Beclin1 in the cells were significantly down-regulated(all P<0.05),meanwhile,the relative protein expressions of p-Akt/Akt,p-mTOR/mTOR and p70S6K in the cells were signifi-cantly up-regulated(all P<0.05).Conclusion Digitoxin can promote autophagy and inhibit apoptosis of Nthy-Roi 3-1 cells stimulated by LPS,the mechanism may be related to inhibiting the activation of Akt/mTOR signaling pathway.
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维普
