Effect Of LPS And NNK On The Proliferation And Related Gene Expression Of Raw264.7 Cells

Objective To observe the effect of LPS and NNK on the proliferation and C-myc gene and other related gene(NF-κB、i NOS、Arginase、TNFα、IL-1α、IL-6)expression in mouse macrophage cell line(Raw264.7 cells).Methods Raw264.7 cells were cultured in vitro and the C-myc m RNA expression in Raw264.7 cells treated with different concentration of LPS and NNK were detected by of Real time-PCR in order to determine the best drug concentration for further study. Then the C-myc 、NF-κB、Arginase、i NOS gene expression and the proliferation of Raw264.7 cells stimulated by LPS, NNK and LPS combined with NNK respectively were detected by Real time-PCR and cell counting instrument; the protein expression of C-myc was detected by western blot; the protein expression of TNF-α、IL-6 were detected by ELISA quantitative analysis. the protein expression of i NOS were detected by immunocytochemistry technology.the protein expression of TNF-α、IL-6 were detected by ELISA quantitative analysis.Results(1)The results showed that 10 ng/ml、250ng/ml、1μg /ml and 4μg /ml LPS could increase the C-myc m RNA relative expression of Raw264.7 cells,and LPS at 1μg /ml there was significant difference compared with the other groups(P<0.05).(2)NNK alone did not influence the C-myc m RNA expression of Raw264.7 cell, but could decrease the up-regulation of LPS to a certain extent at 24h、36h(<0.05).(3)LPS and LPS + NNK could decrease the proliferation activity of Raw264.7 cells; the proliferation activity of Raw264.7 cells treated with LPS and NNK was lower than LPS.(4) LPS and LPS and NNK could increase the expression of NF-κB、Arginase、i NOS m RNA at 24h、36h、48h point. The expression of NF-κB(24h、36h)、Arginase(36h) m RNA treated with LPS and NNK was higher than LPS alone(P <0.05).(5) Compared with control, LPS and LPS and NNK could significantly increase the expression of IL-6、IL-1a m RNA(P<0.01). The expression degree of IL-1a m RNA in LPS group was lower than LPS and NNK group(P<0.05).(6) The C-myc protein expression of Raw264.7 cell in NNK、LPS、LPS and NNK group were no significant difference compared with control group.(7)Compared with control, LPS、LPS and NNK could increase the protein expression of IL-6 and TNF-α at 24-48h(P<0.05). The protein expression degree of IL-6 in LPS group was lower than LPS+ NNK group(P<0.01). The protein expression degree of TNF-a in LPS group was higher than LPS and NNK grounp(P<0.05).Conclusion NNK could not effect the proliferation activity of macrophage cells、transformation into M1 macrophage、transcription factor expression. LPS could steep induce the C-myc 、NF-κB 、i NOS 、Arginase 、IL-1a m RNA, decrease the proliferation activity of Raw264.7 cells, but enhance Raw264.7 cells transformation into M1 macrophage. LPS plus NNK could induce the NF-κB、IL-1α up-regulation stimulated by LPS,but weaken Arginase gene expression; promote Raw264.7 cells activaty to M1 macrophage, then induce M1 macrophage release IL-1α, increase i NOS gene expression, therefore the acceleration of NO synthesis will lead to DNA damages and impede DNA repair, induce gene mutation, finally lung cancer occurred.