| Objective: To observe the effect of CSE on the expression of TXNIP and TRX1 in A549 cells of aging state,and to investigate whether TRX1/TXNIP are involved in the dysregulation of oxidative/antioxidant in Senescent A549 cells and its significance.Methods: A549 cells were exposed to BrdU at 0?M,10?M,20?M,40?M for 10 days, SA- ?-gal staining and flow cytometry were used to detect cell senescence;Appropriate concentration of BrdU was used to establish A549 cell aging model, Western blot and immunofluorescence were used to further verify A549 cellular senescence; 10% CSE was used to treat control group and model group for 12 h, and establish corresponding control group, Immunefluorescence assay the content of ROS. CCK-8 assay was used for cell viability.Western blot was applied to detect the TXNIP, TRX expression.Results:1.SA-?-gal staining results showed that compared with 0?M group(5.65±0.56),SA-?-gal staining positive rate in 10?M,20?M,40?M group were significantly increased. respectively 66.81±2.05, 75.54±3.41, 82.25±2.92, with significant difference(P < 0.05).Flow cytometry results showed that compared with the 0?M group(58.72±2.52), the cell ratio of G0/G1 phase in 0?M,20?M and 40?M groups were significantly increased, respectively 74.11±3.89,78.18±4.57,76.29±2.76, with significant difference(P<0.05);Compared with the 0?M group(38.38 ± 2.16), the cell ratio of S phase in 0?M,20?M and 40?M groups were reduced, respectively 20.50±4.75,16.08±3.40,18.29±3.35, with significant difference(P<0.05), the cell ratio of G2/M phase in each group were respectively 2.90±0.39,5.39±0.86,5.74±2.12,5.41±2.30, statistics between each group no significant difference(P>0.05), suggesting thatBrdU can regulate the cell cycle and block cell cycle of A549 cells at G0/G1 phase.Western blot and immunofluorescence results showed that compared with 0?M group(0.22 ± 0.015), the expression of cell cycle inhibitory factor p21( Senescent marker) in 10?M BrdU treated group was significantly increased(0.61 ± 0.021),with significant difference(P < 0.05).In summary, We had successfully established A549 cells aging model by 10?M BrdU. 2.DCF analyzed the content of ROS in each group of A549 cells. The results showed that model group(11.46 ±0.37) higher than that of the control group(5.73±0.28), with statistical significance(P < 0.05); the CSE treated group(13.68± 0.44, 7.16 ± 0.19) were higher than the untreated group(1.146±0.37, 5.73±0.28),with statistical significance(P < 0.05). 3.CCK-8 analyzed the viability of A549 cells in each group.The results showed that model group(0.64±0.06) was lower than that of control group(0.97±0.04),with statistical significance(P < 0.05); the CSE treated group(0.37 ±0.01, 0.85±0.03) were lower than the untreated group(0.64±0.06,0.97±0.04), with statistical significance(P < 0.05). 4.The results of Western blot showed proteins expression of TXNIP in model group(0.33±0.03) was increased compared with control group(0.24±0.04),with statistical significance(P < 0.05).CSE-treated group(0.57±0.08,0.43±0.05) higher than untreated group(0.33±0.03,0.24±0.04),with statistical significance(P < 0.05).while proteins expression of TRX1 in model group(0.56±0.05) was decreased compared with control group(0.75±0.03), with statistical significance(P < 0.05).CSE-treated group(0.28±0.02,0.40±0.02) lower than untreated group(0.56±0.05,0.75±0.03),with statistical significance(P < 0.05). 5.Linear correlation analysis showed that the protein expression of TXNIP was negatively correlated with the protein expression of TRX1(r=?0.930,P<0.05).Conclusion: The imbalanced redox status in senescent A549 cells is probably due to dysregulated TRX1/TXNIP, Under the conditions of oxidative strss such as CSE,the expression of TXNIP higher, whereas the expression of TRX1 lower in senescent A549 cells,which lead to increase ROS content,and aggravate the oxidative damage. |
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