| Objective To observe the influence of Bufei Yishen granule in MRC-5 cells induced by cigarette smoke extract(CSE)on cell proliferation,apoptosis,cytokine secretion,Matrix metalloproteinase secretion,extracellular matrix secretion,transformation and the expression of related gene and protein.To study the mechanism that Bufei Yishen Granule through NF-?B signal transduction pathway intervene MRC-5 cells inflammatory reaction induced by CSE.Methods MRC-5 cells can be divided into Control,BY,Am,CSE,CSE+BY,CSE+Am,si RNA,si RNA+BY,si RNA+Am,si RNA+CSE,si RNA+CSE+BY and si RNA+CSE+Am group.Each group MRC-5 cells' s proliferation and apoptosis was measured by flow cytometry after respectively tag the Carboxyflu orescein discrete succinimide ester(CFSE)and the Annexin V-FITC and PI.The secretion level of IL-1?,IL-8,TNF-?,TGF-?1,FN,LN,COL-I,COL-III and COL-IV of each group cell culture supernatant were detected by ELISA method.The alpha smooth muscle actin(?-SMA)expression of each group MRC-5 cells was tested by the cell immunofluorescence technique.The m RNA expression of IL-1?,TNF-?,TGF-?1,NF-?B p65 and I-?B? were determined by fluorescence quantitative PCR technology to detection;the protein expression of NF-?B p65,I-?B? were measured by the Western-BlotingResults 1 Cell proliferation Compare with Control group,cell proliferation rate was increased in BY group and Am group(P<0.05),while reduced significantly in CSE group(P<0.05);Compare with CSE group,cell proliferation rate was increased slightly in CSE+BY group and CSE+Am group(P<0.05);there was no statistical significance between CSE+BY and CSE+Am group(P>0.05);compare with si RNA group,cell proliferation rate was increased in si RNA+BY group and si RNA+Am group(P<0.05),while reduced in si RNA+CSE group(P<0.05);compare with si RNA+CSE group,cell proliferation rate was increased slightly in si RNA+CSE+BY group and si RNA+CSE+Am group(P<0.05),there was no statistical significance between si RNA+CSE+BY group and si RNA+CSE+Am group(P>0.05);NF-?B p65 si RNA groups compare with non-NF-?B p65 groups,there was no statistical significance in si RNA+CSE+BY group(P>0.05),cell proliferation rate in other groups were increased(P<0.05).2 Cell apoptosis Compare with Control group,cell apoptosis rate was reduced in BY group(P<0.05)and increased significantly in CSE group(P<0.05)but no statistical significance in Am group(P>0.05);compare with CSE group,cell apoptosis rate was reduced slightly in CSE+BY group and CSE+Am group(P<0.05);cell apoptosis rate reduce more significantly in CSE+BY group than CSE+Am group(P<0.05);compare with si RNA group,there was no statistical significance in si RNA+BY group and si RNA+Am group(P>0.05),while was increased in si RNA+CSE group(P<0.05);compare with si RNA+CSE group,there was no statistical significance in si RNA+CSE+BY group and si RNA+CSE+Am group(P>0.05);NF-?B p65 si RNA groups compare with non-NF-?B p65 groups,cell apoptosis rate in all the groups were decreased(P<0.05).3 IL-1?,IL-8,TNF-? and TGF-?1 Compare with the Control group,IL-1?,IL-8,TNF-? and TGF-?1 were increased significantly in BY group,Am group,CSE group(P<0.05);compare with CSE group,IL-1?,IL-8,TNF-? and TGF-?1 were reduced slightly in CSE+BY group and CSE+Am group(P<0.05);CSE+BY group compare with CSE+Am group,IL-1? and IL-8 were reduced slightly(P<0.05),TNF-? and TGF-?1 expression had no statistical significance(P>0.05);compare with si RNA group,IL-1?,IL-8,TNF-? and TGF-?1 were increased significantly in si RNA+BY group,si RNA+Am group and si RNA+CSE group(P<0.05);compare with si RNA+CSE group,IL-1?,IL-8,TNF-? and TGF-?1 were reduced slightly in si RNA+CSE+BY group and si RNA+CSE+Am group(P<0.05);si RNA+CSE+BY group compare with si RNA+CSE+Am group,IL-1?,IL-8 and TNF-? reduce slightly(P<0.05),TGF-?1 expression had no statistical significance(P>0.05);IL-1?,IL-8,TNF-? and TGF-?1 in NF-?B p65 si RNA groups decrese more than non-NF-?B p65 groups(P<0.05).4 MMP-9 and TIMP-1 Compare with Control group,MMP-9 and TIMP-1 were increased significantly in CSE group(P<0.05),there was no statistical significance in BY group and Am group group(P>0.05);compare with CSE group,MMP-9 and TIMP-1 were reduced slightly in CSE+BY group and CSE+Am group(P<0.05);there was no statistical significance of MMP-9 and TIMP-1 between in CSE+BY group and CSE+Am group(P>0.05);compare with si RNA group,there was no statistical significance in si RNA+BY group and si RNA+Am group(P>0.05),while increased significantly in si RNA+CSE group(P<0.05);compare with si RNA+CSE group,MMP-9 and TIMP-1 were reduced slightly in si RNA+CSE+BY group,si RNA+CSE+Am group(P<0.05);there was no statistical significance between in si RNA+CSE+BY group and si RNA+CSE+Am group(P>0.05);MMP-9 was reduced in NF-?B p65 si RNA groups more than non-NF-?B p65 groups(P<0.05),but TIMP-1 had no statistical significance(P>0.05).5 ECM Compare with Control group,FN,LN,COL-I,COL-III and COL-IV expression were no statistical significance in BY group and Am group(P>0.05),while was increased significantly in CSE group(P<0.05);compare with CSE group,FN,LN,COL-I,COL-III and COL-IV were reduced in CSE+BY group(P<0.05),the COL-I,COL-III and COL-IV expression were reduced slightly in CSE+Am group(P<0.05)but FN and LN has no statistical significance in CSE+Am group(P>0.05);CSE+BY group compare with the CSE+Am group,FN and LN were reduced slightly(P<0.05),COL-I,COL-III and COL-IV expression had no statistical significance(P>0.05);compare with si RNA group,there was no statistical significance in si RNA+BY group and si RNA+Am group(P>0.05),there was increased significantly in si RNA+CSE group(P<0.05);compare with si RNA+CSE group,there was reduced slightly in si RNA+CSE+BY group(P<0.05),COL-I,COL-III and COL-IV expression of the si RNA+CSE+Am group was reduced slightly(P<0.05)but FN,LN had no statistical significance(P>0.05);si RNA+CSE+BY group compare with si RNA+CSE+Am group,the FN and LN were reduced slightly(P<0.05),the COL-I,COL-III and COL-IV expression had no statistical significance(P>0.05);FN,LN,COL-I,COL-III and COL-IV expression in NF-?B p65 si RNA groups were decreased more than non-NF-?B p65 groups(P<0.05). 6 ?-SMA Compare with Control group,?-SMA expression was no statistical significance in BY group and Am group(P>0.05),there was increased significantly in CSE group(P<0.05);compare with CSE group,there was reduced slightly in CSE+BY group and CSE+Am group(P<0.05),there was reduced slightly in CSE+Am group more than CSE+BY group(P<0.05);compare with si RNA group,there was no statistical significance in si RNA+BY group and si RNA+Am group(P>0.05),while there was increased in si RNA+CSE group(P<0.05);compare with si RNA+CSE group,there was reduced slightly in si RNA+CSE+BY group and si RNA+CSE+Am group(P<0.05),but there was no statistical significance between the si RNA+CSE+BY group and si RNA+CSE+Am group(P>0.05);?-SMA expression in NF-?B p65 si RNA groups compare with non-NF-?B p65 groups,si RNA+CSE group,si RNA+CSE+BY group and si RNA+CSE+Am group were reduced(P<0.05),other groups had no statistical significance(P>0.05).7 IL-1?,TNF-? and TGF-?1 m RNA Compare with the Control group,the IL-1?,TNF-? and TGF-?1 m RNA expression of the BY group and Am group increase significantly(P<0.05),the CSE group increase significantly(P<0.05);compare with the CSE group,the IL-1?,TNF-? and TGF-?1 m RNA expression of the CSE+BY group and CSE+Am group reduce slightly(P<0.05);the CSE+BY group compare with the CSE+Am group,the IL-1? m RNA expression reduce significantly(P<0.05)and the TNF-? and TGF-?1 m RNA expression has no statistical significance(P>0.05);compare with the si RNA group,the si RNA+BY group and si RNA+Am group increase significantly(P<0.05),the si RNA+CSE group increase significantly(P<0.05);compare with the si RNA+CSE group,the si RNA+CSE+BY group and si RNA+CSE+Am group reduce slightly(P<0.05);the si RNA+CSE+BY group compare with the si RNA+CSE+Am group,the IL-1? and TNF-? m RNA expression reduce significantly(P<0.05)and the TGF-?1 m RNA expression has no statistical significance(P>0.05);NF-?B p65 si RNA groups compare with non-NF-?B p65 groups,I-?B? m RNA has no statistical significance(P>0.05)and other groups reduce(P<0.05).8 NF-?B p65 and I-?B? m RNA Compare with the Control group,the NF-?B p65 and I-?B? m RNA expression were increased significantly in CSE group(P<0.05),but there were no statistical significance in BY group and Am group(P>0.05);compare with CSE group,there were reduced slightly in CSE+BY group and CSE+Am group(P<0.05);CSE+BY group compare with the CSE+Am group,NF-?B p65 and I-?B? m RNA expression were reduced significantly(P<0.05);compare with si RNA group,there were no statistical significance in si RNA+BY group and si RNA+Am group(P>0.05);compare with si RNA+CSE group,there were reduced slightly in si RNA+CSE+BY group and si RNA+CSE+Am group(P<0.05);si RNA+CSE+BY group compare with si RNA+CSE+Am group,there was reduced significantly of NF-?B p65 and I-?B? m RNA(P<0.05);NF-?B p65 si RNA groups compare with non-NF-?B p65 groups,I-?B? m RNA had no statistical significance(P>0.05)and other groups were reduced(P<0.05).9 NF-?B p65 and I-?B? protein Compare with the Control group,NF-?B p65 and I-?B? protein expression in BY group and Am group had no statistical significance(P>0.05),but there was increased significantly in CSE group(P<0.05);compare with CSE group,there were reduced slightly in CSE+BY group and CSE+Am group(P<0.05),while NF-?B p65 and I-?B? protein expression were reduced significantly in CSE+BY more than CSE+Am group(P<0.05);compare with si RNA group,there was no statistical significance in si RNA+BY group and si RNA+Am group(P>0.05),while there was increased in si RNA+CSE group(P<0.05);compare with si RNA+CSE group,there were reduced in si RNA+CSE+BY group and si RNA+CSE+Am group(P<0.05);NF-?B p65 and I-?B? protein expression in si RNA+CSE+BY group were reduced significantly more than si RNA+CSE+Am group(P<0.05);NF-?B p65 p65 si RNA groups compare with non-NF-?B p65 p65 groups,I-?B? protein expression had no statistical significance(P>0.05)and other groups were reduced(P<0.05).10 Correlation analysis of indicators After analysis we found that the highly correlation degree or above have the following: NF-?B p65 and IL-1?(r=0.934,P=0.000),IL-8(r=0.942,P=0.000),TNF-?(r=0.926,P=0.000),TGF-?1(r=0.938,P=0.000),MMP-9(r=0.932,P=0.000),FN(r=0.903,P=0.000),LN(r=0.877,P=0.000),COL-I(r=0.928,P=0.000),COL-III(r=0.980,P=0.000),COL-IV(r=0.915,P=0.000),?-SMA(r=0.861,P=0.000)and NF-?B p65 m RNA(r=0.950,P=0.000);MMP-9 and TIMP-1(r=0.857,P=0.000),FN(r=0.823,P=0.001),LN(r=0.842,P=0.001),COL-I(r=0.986,P=0.000),COL-III(r=0.963,P=0.000),COL-IV(r=0.949,P=0.000);TIMP-1 and LN(r=0.893,P=0.000),COL-I(r=0.914,P=0.000),COL-III(r=0.806,P=0.002),COL-IV(r=0.937,P=0.000); IL-1? and IL-1? m RNA(r=0.935,P=0.000);TNF-? and TNF-? m RNA(r=0.910,P=0.000);TGF-?1 and TGF-?1 m RNA(r=0.823,P=0.001);I-?B? and I-?B? m RNA(r=0.971,P=0.000).Conclusion 1 Through transfection NF-?B p65 si RNA for MRC-5 cells,there have obvious inhibitory effect on cell apoptosis,inflammatory cytokines,matrix metalloproteinase and the expression of extracellular matrix.2 Bufei Yishen granule can effectively facilitate the MRC-5 cells induced by 5% CSE proliferation,inhibit the apoptosis,decrease the secretion of inflammatory cytokines,matrix metalloproteinase and extracellular matrix,reduce cell transformation down-regulate,Its mechanism may be through down-regulated NF-?B pathway to suppress the MRC-5 cell inflammatory response,reduce the process of airway chronic inflammation and then delay COPD airway remodeling.3 Cell experiment by single factor to stimulate can't completely simulate in vivo pathological situation,we can add different CSE concentrations or cell co-culture for further research. |
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