|
Objective To observe the insulin-like growth factor -1 (IGF-1) expression in PC12 cells cystathionine-beta-synthase (CBS) and hydrogen sulphide (H2S) and possible mechanisms of cell signaling pathways. With different doses of IGF-1 (20, 40 and 80 ng / ml) were cultured in vitro rat pheochromocytoma (PC12) cells. After 24 hours, respectively, with Western blot detection CBS protein expression quantitative PCR CBS gene expression, sensitive sulfur electrode detect hydrogen sulfide gas levels. In the study of cell signaling mechanisms, select join the ERK upstream signaling molecule MEK inhibitor PD98059 PC12 cells the synchronization experiments select the optimal dose IGF-1: 80 ng / ml role in the 30 minutes before the Western blot detection of extracellular signal-regulated kinase / mitogen-activated protein kinase (ERK / MAPK)-related protein, and repeat the test. Results (1) IGF-1 20ng/mL, 40 ng / mL, 80 ng / mL group CBS gene expression than the control group raised (1 ± 0.185; 2.422 ± 0.185; 3.504 ± 0.185; 4.881 ± 0.185, all P ≤ 0.05 ), and expression gradually increased with the concentration. 80 ng / mL (optimal dose) group than 20ng/ml and 40 ng / ml group CBS gene expression raised more obvious. Western detect the CBS protein changes in gene changes (0.294 ± 0.123; 0.463 ± 0.123; 0.577 ± 0.123; 0.913 ± 0.123, all P ≤ 0.05). (2) IGF-1 20, 40, 80 ng / ml group H2S concentration higher than those in the control group (3.380 ± 0.282; 6.408 ± 0.282; 11.468 ± 0.282; 21.711 ± 0.282, all P ≤ 0.05), and 80ng/ml group than 20ng/ml and 40ng/ml group increased significantly, P ≤ 0.05). (3) IGF-1 80ng/ml group than the control group increase the Perk protein level (1.074 ± 0.0180; 0.738 ± 0.0180, P ≤ 0.05), ERK inhibitor PD98059 different doses (25, 50 uM) than IGF-1 80ng / ml group lowered the Perk protein expression (0.563 ± 0.0180; 0.260 ± 0.0180; 1.074 ± 0.0180, P <0.05), PD98059 50uM group than 25um group pERK down more significantly (0.260 ± 0.0180; 0.563 ± 0.0180, P <0.05), each group total ERK was no significant difference (F = 0.105, P = 0.955, P among the three groups were gt; 0.05) (4) the expression of ERK inhibitor PD98059 different doses (25um, 50uM) than IGF-1 80ng/ml group can be significantly lowered CBS (0.244 ± 0.009; 0.149 ± 0.009; 0.980 ± 0.009, P <0.05) and H2S concentration ( 4.979 ± 0.341; 2.614 ± 0.341; 24.342 ± 0.341, P ≤ 0.05), PD98059 50uM down than 25um CBS expression (0.149 ± 0.009; 0.244 ± 0.009, P <0.05), H2S change is consistent with CBS (2.614 ± 0.341; 4.979 ± 0.341, P ≤ 0.05). Conclusion IGF-1 can increase the CBS expression and H2S concentration in PC12 cells in vitro, and its mechanism may be realized through the activation of MAPK / ERK signaling pathway.
|