The Research Of The Inhibitory Effect Of CI Combined With PDTC On Inflammation Improved The Proliferation Of A549 Cells And The Relevant Mechanism

Objective: This experiment concentrated on the inhibitory effect of cinobufacini injection(CI)combined with pyrrolidine dithiocarbama(PDTC)on propagation of non-small cell lung cancer cell A549 stimulated by inflammation and the inflammatory factors from RAW264.7 cells induced by lipopolysaccharide,and inquire into the relevant mechanism about NF-?B signaling pathway.It could provide experimental basis for the prevention and treatment of inflammation improved the deterioration of lung cancer via CI combined with PDTC.Methods: 1.The inhibitory effect of CI combined with PDTC on the proliferation of A549 cells: A549 cells were seeded and incubated for attachment.The control group,PDTC group,CI group and union group were seting.After 24 h or 48 hours with additting dr?gs,CCK-8 assay was used to detect the cell survival rate.2.The effect of CI combined with PDTC on the viability of RAW264.7 cells stimulated by LPS: RAW264.7 cells were seeded and incubated for attachment.The control group,LPS stimulated group,LPS stimulated PDTC group,LPS stimulated CI group and LPS stimulated union group were seting.The treatment groups were incubated with LPS for 2h following by adding drugs for 24 h.CCK-8 assay was used to detect the cell survival rate.3.The inhibitory effect of CI combined with PDTC on inflammatory factors: RAW264.7 cells were seeded in 12-well plates and grouped after LPS induction for 2h and reagents treatment for 24 h.The NO productions from supernatant were detected by NO kit.The TNF-?,IL-6 and IL-1? productions from supernatant were tested by ELISA kit respectively.4.The inhibitory effect of CI combined with PDTC on the proliferation of A549 cells stimulated by inflammation: A549 cells were seeded and incubated for attachment.The control group,inflammation stimulated group,inflammation stimulated PDTC group,inflammation stimulated CI group and inflammation stimulated union group were seting.The noninflammation stimulated groups were setting for contrast.The inflammation stimulated group was added the supernatant medium from RAW264.7 cells induce by LPS.The drug groups were added reagents confected by the supernatant medium or normal medium.After incubated for 48 h,the cell survival rate was tested by CCK-8 assay.5.The colocalization of NF-?B p65 and the nucleus: RAW264.7 cells were seeded in confocal capsules and grouped after reagents treatment for 24 h.Laser Scanning Confocal Microscope was used to observe the colocalization of NF-?B p65 and the nucleus.6.CI combined with PDTC inhibited the degradation of I?B-?: RAW264.7 cells were seeded in 6-well plates and grouped after reagents treatment for 24 h.The cells were collected and the proteins were extracted.Western Blot was applied to test the expression of I?B-?.Results: The results of CCK-8 show that both CI and PDTC had inhibitory effect on proliferation of A549 cells.The effect was promotional when CI cooperated with PDTC(P<0.05).There is no influence on the viability of RAW264.7 treated by LPS,CI and PDTC(P > 0.05).Anti-inflammatory effection exhibited that the contents of inflammatory factors were increased obviously after induced by LPS on RAW264.7 cells(P<0.05).CI and PDTC had different degrees of inhibition effect on NO,TNF-?,IL-6 and IL-1?.When CI combined with PDTC,the effect of higher then that in union groups in total.The cell survival rate was increasing stimulated by inflammation(P<0.05).The inhibitory effect of CI combined with PDTC on the proliferation of A549 cells stimulated by inflammation was stronger than single group(P<0.05).The pictures taken by Laser Scanning Confocal Microscope indicated that NF-?B p65 was increased obviously in nucleus after stimulated by LPS in RAW264.7 cells.Both CI and PDTC could inhibite the nucleus displacement of NF-?B p65 and it is more effective in the combined utilization.The results of Western Blot declared that LPS could improve the degradation of I?B-?(P<0.05).The degradation of I?B-? in RAW264.7 cells was decreased by CI or PDTC.The inhibitory effect of degradation of I?B-? was advanced by CI combined with PDTC(P<0.05).Conclusion:(1)CI combined with PDTC could effectively inhibited the secretion of NO,TNF-?,IL-6 and IL-1? in RAW264.7 cells induced by LPS and it's stronger than that in union group in total.(2)The proliferation of A549 cells were improved by inhibitory stimulation.CI combined with PDTC could effectively inhibited the proliferation of A549 cells with or without inflammatory stimulation and the inhibitory effect was more higher than the single drug groups.(3)The mechanism of inhibiting inflammation improved the proli feration of A549 cells using CI combined with PDTC might be suppressing the nuclear translocation of NF-?B via decreasing the degradation of I?B-? in RAW264.7 cells resulting in NF-?B could not come into cell nucleus to bind and transcribe the related genes of NO,TNF-?,IL-6 and IL-1?,consequently reduced the secretion of these pro-inflammatory factors,futher to inhibite the accelerated effection of inflammatory stimulation on A549 cells.