Effects Of TXNIP On Proliferation Of INS-1 Islet β Cells

Background:With the rapid increase in the incidence rate,diabetes has become one of the most serious diseases that endanger human health in today’s society.The decrease in the number of pancreatic islet β cells is the main cause of diabetes.Therefore,inhibiting apoptosis and promoting proliferation of pancreatic β-cells is one of the key methods to treat diabetes.Currently,Thioredoxin-interacting protein(TXNIP)is the only known substence that binds to reduced Thioredoxin(Trx)activity via its cysteine at position 247.TXNIP combined with Trx can inhibits its activity and then mediates oxidative stress,induce cell apoptosis and other effects.Studies have confirmed that the expression of TXNIP is significantly up-regulated during the development of diabetes and can induce apoptosis of islet β cells.Studies have shown that the expression of TXNIP is significantly decreased in many tumor tissues,and increased TXNIP expression can inhibit the growth of tumors,suggesting that TXNIP is also closely related to cell proliferation,but its effect on the proliferation of pancreatic islet beta cells remains unclear.Combining with Trx is the main way that TXNIP works.However,studies have also shown that TXNIP can inhibit glucose uptake in skin fibroblasts,and this effect is independent of Trx.In adipocytes,the use of the cysteine-mutated TXNIP at position247 also inhibits glucose uptake,demonstrating that this effect has nothing to do with its ability to inhibit Trx,suggesting that TXNIP has a mechanism that is not dependent on the Trx system.According to these research backgrounds,we designed this experiment.First,We selected two main methods of adenovirus infection of cells,and compared which is more suitable for constructing a TXNIP overexpression model in INS-1 cells.After confirming the adenovirus infection method,We explored the effects and possiblemechanisms of Trx-dependent and Trx-independent pathways on INS-1 pancreaticβ-cell proliferation by constructing different adenoviral vectors and setting up control groups.Part I: Comparison of Two Adenovirus Infection Methods to Construct TXNIP Overexpression Model in INS-1 Islet β Cells Objective:In rat INS-1 pancreatic β-cells,a more stable and efficient TXNIP overexpression model was sought by comparing different adenovirus infection methods.Methods:1.Construction of empty adenovirus vector(Ad-GFP)and TXNIP overexpression adenovirus vector(Ad-TXNIP-GFP),both vectors contain green fluorescent protein(GFP).2.Based on the multiplicity of infection(MOI)of 50,different groups of viruses were counted and then infected with INS-1 cells under normal culture conditions.The normal group in Method I was directly added with 1 mL of 1640 medium,Ad-GFP and Ad-TXNIP-GFP groups were added with 1 mL of a mixture of adenovirus and1640 medium.Method I was supplemented with 3 mL of 1640 complete medium 4hours after adenovirus infection,cultured to 24 hours followed by change of medium,and then continued to culture to 48 hours;In Method Ⅱ,Normal group was added with 1 mL 1640 medium,Ad-GFP group and Ad-TXNIP-GFP group were added with1 mL of a mixture of adenovirus and 1640 medium After 4h of adenovirus infection,1640 medium containing adenovirus was washed away with PBS,and 4m L complete medium was changed to 48 h without any treatment.3.The expression of GFP in each group was observed under a fluorescence microscope.4.Flow cytometry was used to detect GFP positive rate and average fluorescence intensity in each group.5.CCK-8 test INS-1 cell survival rate in each group.6.Real-time PCR was used to detect of TXNIP expression in mRNA levels.7.Western blot was used to detect of TXNIP expression in protein levels.Results:1.Method Ⅱ adenovirus infects INS-1 cells more efficiently than method I.Fluorescence microscopy showed that the expression of GFP in method I was significantly lower than that in method Ⅱ(P <0.05)at 48 h.Flow cytometry showed that the GFP-positive rate and average fluorescence intensity of the method I Ad-GFP group and Ad-TXNIP-GFP group were lower than that of the method Ⅱ.2.The survival rate of INS-1 cells of method Ⅱ is higher than that of method I.The results of CCK-8 showed that the cell viability of Ad-GFP group was increased by 26%(n = 6,P<0.05)compared with method I;the cell viability of Ad-TXNIP-GFP group was increased by 13%(n = 6,P <0.05),the survival rate of Normal group was close(n = 6,P> 0.05).3.The expression of TXNIP in INS-1 cells in method Ⅱ was higher than that of method I.The results of Real-time PCR detection showed that the expression level of TXNIP in Ad-TXNIP-GFP group of Ⅱ was significantly higher than that of method I(n=6,P<0.05).Western blot analysis showed that the expression of TXNIP gene in Ad-TXNIP-GFP group of Ⅱ was significantly higher than that in method I(n=6,P<0.05).Conclusions:1.Method Ⅱ infection is more efficient and has less negative impact on cellsurvival.2.The target gene TXNIP in method Ⅱ is expressed more highly in cells.The establishment of TXNIP overexpression model is more stable and efficient,which is more conducive to the follow-up experiments.Part Ⅱ: Effects of TXNIP overexpression on proliferation of INS-1 islet β cells by adenovirus vector Objective:1.To investigate the effect of TXNIP overexpression on the proliferation of INS-1 cells and its possible mechanism under normal culture conditions.2.To explore the effect of TXNIP-independent Trx pathway on INS-1 islet cell proliferation and its possible mechanism after TXNIP overexpression.Methods:1.Experimental grouping: INS-1 cells under normal culture conditions were divided into four groups: Normal group,Ad-GFP group,Ad-TXNIP-GFP group,Ad-TXNIPc247s-GFP.2.Using adenovirus infection technique.The calculated adenovirus in each group was mixed with 1 mL of 1640 medium and added into the culture flask of INS-1 cells under normal culture conditions.The normal group was added with 1 m L of virus-free 1640 medium and the other three groups were added 1 m L of 1640 medium containing the corresponding virus mixture,and then placed in an incubator to culture 24 h,wash the medium with PBS,and then replace 4 mL1640 complete medium,continue to culture to 48 h.3.After adenovirus infected cells,the positive rate and the average fluorescence intensity of GFP and the proliferation of INS-1 cells after EDU and Ki67 labeling were detected by flow cytometry.4.Real-time PCR detected the expression of TXNIP at the level of mRNA afterthe adenovirus infected cells.5.Western blot was used to detect the expression of TXNIP,AKT,P-AKT,ERK,P-ERK,beta-Catenin,P-beta-Catenin,Ki67 at protein levels.Results:1.Determine the best conditions for infection of adenovirus.Flow cytometry was used to detect the positive rate of GFP and the average fluorescence intensity of GFP under the condition of different adenovirus infection time: 24 h,48h,72 h and the different multiplicity of infection(MOI): 25,50,75,100.The results showed that when the infection time was 48 h and the MOI was 75,the positive rate of GFP and the average fluorescence intensity were the highest.Therefore,we follow the best conditions for adenovirus infection for follow-up experiments.2.The expression of TXNIP in adenovirus infected INS-1 cells was significantly increased.The expression of TXNIP at mRNA level was detected by Real-time PCR.The expression of TXNIP in Ad-TXNIP-GFP group and Ad-TXNIPc247s-GFP group was significantly higher than that in Normal group(n = 6,P<0.01).The expression of TXNIP at Western blot showed that in Ad-TXNIP-GFP group and Ad-TXNIPc247s-GFP group was significantly higher than that in Normal group(n =6,P<0.05).The results showed that adenovirus vector was successfully constructed and TXNIP overexpression model was established to meet the experimental requirements.3.After overexpression of TXNIP,INS-1 cell proliferation can be inhibited from both Trx-dependent and Trx-independent pathways.The results of EdU showed that proliferation of Ad-GFP group was not significantly different from that of Normal group.Ad-TXNIP-GFP group and Ad-TXNIPc247s-GFP group significantly inhibited cell proliferation(n = 6,P <0.05)Ad-TXNIP-GFP group inhibited cells more than Ad-TXNIPc247s-GFP group(n = 6,P <0.05).According to the Ki67 test results,Normal group and Ad-GFP group had no significant difference(n=6,P>0.05),compared with the Normal group,Ad-TXNIP-GFP group and Ad-TXNIPc247s-GFP group significantly inhibited cell proliferation(n=6,P<0.05),and inhibition of Ad-TXNIP-GFP group of cells are still higher than that of Ad-TXNIPc247s-GFP group(n=6,P<0.05).It is concluded that TXNIP overexpression can inhibit the proliferation of INS-1 cells by Trx pathway and Trx independent pathway,and the inhibitory effect of Trx dependent pathway on cell proliferation is higher than that of Trx independent pathway.4.Trx-independent pathways can inhibit the phosphorylation of AKT and ERK.The results of Western blot showed that Ad-TXNIP-GFP group and Ad-TXNIPc247s-GFP group inhibited the phosphorylation of AKT and ERK(P =0.05,P <0.05)).β-catenin and P-β-catenin showed no significant difference between the four groups(n = 6,P> 0.05).The results showed that TXNIP could inhibit the phosphorylation of AKT and ERK by Trx-independent pathways after TXNIP overexpression,while β-catenin did not participate in the regulation of TXNIP on cell proliferation.5.Inhibition of AKT and ERK phosphorylation can inhibit INS-1 cell proliferation.We added 10 μmol / L AKT phosphorylation inhibitor Perifosine and 20 μmol / L ERK phosphorylation inhibitor PD98059 into Normal group and Ad-GFP group respectively.According to Western blot results.There was no significant difference in Ad-GFP group compared with Normal group.The expression of P-AKT and Ki67 in Normal + Perifosine group,Ad-GFP + Perifosine group,Ad-TXNIP-GFP group and Ad-TXNIPc247s-GFP group were significantly inhibited(n = 6,P <0.01).The inhibition of Ki67 in Ad-TXNIP-GFP group was higher than that of Ad-TXNIPc247s-GFP group.The expression of P-ERK and Ki67 in Normal +PD98059 group,Ad-GFP + PD98059 group,Ad-TXNIP-GFP group and Ad-TXNIPc247s-GFP group were also significantly inhibited compared with Normal group(n = 6,P < 0.01).Conclusions:1.TXNIP can inhibit the proliferation of INS-1 islet β cells through the Trx-dependent and Trx-independent pathways,and the inhibition of Trx-dependent pathway is higher.2.Trx-independent pathway can inhibit the proliferation of INS-1 cells by inhibiting the phosphorylation of AKT and ERK.