Effect And Mechanism Of The JNK Signaling Pathway In The Apoptosis Of NCI-H292 Cells Induced By Poly-L-Arginine

Objectives To investigate the effect and molecular mechanism of c-Jun N-terminal kinase(JNK)signaling pathway in the apoptosis of NCI-H292 cells induced by poly-L-arginine(PLA).Materials and methods 1.Cell culture:NCI-H292 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum(FBS),100 mg/l penicillin,100 mg/l streptomycin,and 12mmol/l L-glutamine at 37℃ in an incubator with 5% CO2 and subcultured with medium change every two days.2.Detection of protein expression in the JNK signaling pathway by Western blot:NCI-H292,an airway epithelial cell line,was stimulated by PLA to induce cell apoptosis.PAL at concentrations of 0mg/L,10 mg/L,20 mg/L,40 mg/L,and 60 mg/L was applied to stimulate NCI-H292 cells for 24 hours.Total proteins of each group were extracted and the differences in JUK and P-JUK were detected among groups by Western blot.3.Determination of cell apoptosis rate by Annexin V-FITC/PI double staining : NCI-H292 cells were divided into the control group,the PLA group,the SP600125 group,and the PLA+ SP600125 group.According to the protocol,NCI-H292 Cells were pre-treated with SP600125,an inhibitor specifically against the JNK signaling pathway,for 1 hour,followed by PLA treatment for 24 hours.Cells in each group were collected and stained by Annexin V-PI double staining.Apoptotic NCI-H292 cells were stained and detected by flow cytometry within 4 hours.Apoptosis of NCI-H292 cells was observed before and after the treatment of SP600125(30μM).4.Measurement of the expression of apoptosis related proteins,Bax,Bcl-2 and Casepase-3 by Western blot:The expressions of Bax,Bcl-2 and Casepase-3 as well as JNK and P-JNK in the JNK signaling pathway in NCI-H292 cells of the blank control group,the PLA group,the SP600125 group and the PLA+ SP600125 group were detected by Western blot.5.Statistical analysis : The data were presented as mean± standard deviation and analyzed by the SPSS17.0 statistical software.The one-way ANOVA was used to compare data among multiple groups and the LSD-t test was used to compare the difference between two groups when the variance was homogeneous and the Dunnett ’s T3 test otherwise.Differences were considered statistically significant at p<0.05.Results 1.No statistically significant difference in the phosphorylation level of JNK was observed between NCI-H292 cells treated with 10mg/l PLA and those with 0mg/l PLA(P=0.179).The phosphorylation levels of JNK in the groups treated with 20 mg/l,40mg/l,and 60mg/l PLA were increased compared with that of the group treated with 0mg/l PLA in a concentration dependent manner,and the difference was statistically significant(F=4.239,all p <0.05).2.Apoptosis rates of NCI-H292 cells in the blank control group,the PLA group,the SP600125 group,and the PLA+SP600125 group were 5.13±1.12%,22.62±1.66%,5.69±0.18%,and 8.99±1.74 %,respectively,with statistical difference among groups(F=113.961,p<0.001);the apoptosis rate of the PLA group was significantly increased compared with that of the blank control group(p<0.001);and the apoptosis rate of the PLA+SP600125 group was significantly decreased compared with that of the PLA group(p<0.001).3.Compared with the blank control group,the ratio of P-JNK/JNK in the NCI-H292 cells treated with 40mg/l PLA was significantly increased(F=31.778,p<0.001),the ratio of Bcl-2/Bax(apoptosis related proteins)was decreased(F=21.077,p<0.01),and the expression of Caspase-3 was significantly increased(F=31.768,p<0.01).4.After the intervention of JNK pathway-specific inhibitor SP600125,the ratio of P-JNK/JNK was significantly decreased(p<0.001),that of Bcl-2/Bax was significantly increased(p<0.001),and the expression of Caspase-3 was significantly reduced(p<0.001)in NCI-H292 cells of the PLA+SP600125 group compared with that of the PLA group,respectively.Conclusion 1.The JNK signaling pathway in the airway epithelial cell NCI-H292 is activated by PLA.2.PLA upregulates the expression of proapoptotic proteins,Bax and Caspase-3,and downregulates the expression of anti-apoptotic protein,Bcl-2,in NCI-H292 cells.3.The JNK signaling pathway mediates PLA induced apoptosis in NCI-H292 cells by promoting Bax and Caspase-3 expression and inhibiting Bcl-2 expression.