| Part 1 Effects of High Glucose on Papillary Thyroid Cancer K1 Cells' Proliferation and PI3K/Akt/Nrf2 Signaling PathwayObjective To observe the effect of different concentrations of glucose on the proliferation of papillary thyroid cancer K1 cells.To investigate the effect of high glucose on the expression and nuclear translocation of Nrf2 in K1 cells,at the same time,to study its possible pathway.Methods1.K1 cells were treated with(5.5?10?25?50)mmol/L glucose,MTT analysis was used to detected K1 cells proliferation at 12,24,36and 48 h.The same osmotic pressure group was used as control.2.According to the results of MTT,K1 cells were divided into normal glucose group(5.5mmol/l glucose),high glucose group(25mmol/l glucose)and hypertonic group(25mmol/l mannitol).K1 cells were extracted from each group at 0,12,24,48 h.Then,Western blot method was used to detect the protein expressions of Nrf2,phosphoinositide 3-kinase(PI3K),protein kinase B(Akt)and phosphorylated-Akt(p-Akt).Meanwhile,using immunefluorence staining for determination of distribution of Nrf2 in K1 cells.Spearman correlation analysis was used to test the correlation of Nrf2,PI3 K and p-Akt/Akt with the proliferation of K1 cells treated with high glucose.Results1.The proliferation of K1 cells have altered in depend on elevated glucose concentration,and it reached the peak at 25mmol/L glucose concentration(148.76±5.19)%,however,it got the lowest point(84.72±3.26)% at 50mmol/L glucose concentration,compared with5.5mmol/l glucose concentration,each group of the proliferation rate of K1 cells were statistically different(p<0.01).2.At the same concentration of glucose,K1 cells proliferation changed as time goes on,it start to increase at 12 h and reached the peak at 24 h,then declined.3.The expressions of Nrf2,PI3 K,p-Akt/Akt and the distribution of Nrf2 in the nucleus of K1 affected by 25mmol/l glucose after 24 h were(Nrf2:0.869 ±0.075?PI3K:0.783±0.020 ?p-Akt/Akt:0.480±0.048?proportion of Nrf2 at nucleus:91.2%,260/285),all of them reached maxima.The alteration of expressions of these proteins and proportion of Nrf2 at nucleus synchronized with MTT results profoundly.4.Spearman correlation analysis showed that each expression of Nrf2,PI3 K,p-Akt/Akt was positively correlated with the proliferation of K1 cells in the high glucose group,and the correlation coefficient r was0.908,0.910,0.916,respectively,p<0.00.5.There was no significant difference in the the expression of Nrf2,PI3 K,p-Akt/Akt and the proliferation of K1 cells(p>0.05).Conclusions1.Glucose concentration-and time-dependently influenced theproliferation of K1 cells.Moreover,the proliferation of K1 cells could end up on top by treating with 25mmol/L glucose concentration 24 hours.2.High glucose may promote the proliferation of papillary thyroid cancer K1 cells by promoting Nrf2 expression and nuclear translocation,which may be related to the up regulation of PI3K/Akt signaling pathway in K1 cells.Part 2 Inhibitory Effect of Berberine on the Proliferation of K1 Cells Induced by High Glucose and Its Possible MechanismObjective To observe the inhibitory effect of berberine on the proliferation of high glucose-induced papillary thyroid cancer K1 cells.To investigate the effect of berberine on high glucose induced the protein expression and nuclear translocation of Nrf2 in K1 cells,and to explore its possible signaling pathway.In hope to provide new ideas and methods for the treatment of early diabetic patients with papillary thyroid cancer.Methods 1.High glucose(25mmol/L)and different concentration of berberine(0?10?40?80?mol/L)were added to coculture K1 cells for24 hours.The proliferation of K1 cells in each group were detected by using MTT.Setting normal glucose group(5.5mmol/l)as control.2.Western blot was used to measure the expression levels of Nrf2,PI3 K,Akt and p-Akt.Meanwhile,the distribution of Nrf2 in K1 cells were determined using immunofluorence staining.All experiments were setted normal glucose group(5.5mmol/L)as control.Results1.Compared with normal glucose group,the proliferation rate of K1 cells,the expression levels of PI3 K,p-Akt/Akt,Nrf2 as well as the proportion of Nrf2 in the nuclear distribution in high glucose group were significantly elevated,respectively.(proliferation rate:(126.64±5.41)% vs(87.31±3.67)%;PI3K:(0.425±0.019)vs(0.272±0.039);p-Akt/Akt:(0.446±0.021)vs(0.168±0.035);Nrf2:(0.597±0.014)vs(0.308±0.026);proportion of Nrf2 at nucleus:(93.0%,265/285)vs(23.1%,45/195),p<0.01 for all.2.Compared with high glucose group,all the proliferation rate of K1 cells in high glucose added with 10,40,80?mol/L berberine groups((111.76±4.10)%,(70.03±2.18)%,(32.41±3.76)%)were lowered.Meanwhile,the inhibitory effect was the strongest in80?mol/L berberine group,and which was more effective in 40 ?mol/L berberine group than in 10 ?mol/L berberine group.3.The expression levels of PI3 K,p-Akt/Akt,Nrf2 as well as the proportion of Nrf2 in the nuclear distribution were down-regulated obviously(p<0.05),when compared with high glucose group.And these changes were more significant with the concentration of berberine increasing.Conclusions Berberine concentration-dependently inhibited the proliferation of high glucose-induced K1 cells,which may be related to that berberine down-regulated the expression of Nrf2 as well as nuclear translocation of Nrf2 through stressing the activation of PI3K/Akt signaling pathway. |
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