| Objective To observe the effect of Bufei Yishen Recipe on differentiation and expression of inflammatory factors of Dendritic cells(DCs) induced by lipopolysaccharide(LPS), to explore the mechanisms of Bufei Yishen adjusting DCs for treatment of chronic obstructive pulmonary disease(COPD). Methods 1 Culture of rat bone marrow derived DCs in vitro: SPF grade SD rats’ femur and tibia were separated aseptically, the bone marrow mononuclear cells were obtained from the rat femur, tibia bone marrow through spalled RBC by Tris-NH4 Cl, added to the RPMI 1640 culture medium with rat recombinant granulocyte- macrophage colony stimulating factor(GM-CSF) and rat recombinant interleukin- 4(IL-4), by the methods of screening DCs colony, cultured for 8 d in vitro, collect article 8d loosely adherent and suspended cells, which is derived from rat bone marrow DCs. DCs were divided into blank group, Bufei Yishen group, dexamethasone group, LPS group, LPS + Bufei Yishen group and LPS + dexamethasone group. 2 The changes of growth and morphology of DCs were observed by inverted microscope. 3 Phenotype and phagocytosis of DCs were determined by flow cytometry, such as OX-62, MHC-II, CD80, CD86 and phagocytosis. 4 Levels of DCs cell secreted cytokines, such as IL-6, IL-10, IL-12P40, IL-12P70, TNF- alpha, IFN- gamma, were measured by using enzyme-linked immuno sorbent assay(ELISA) kits. 5 Gene expressions of toll like receptor(TLR) 2 and TLR4 in DCs cells were determined by using fluorescence quantitative polymerase chain reaction(PCR) technology.Results 1 Successful established the culture method of rat bone marrow derived DCs in vitro. Each 1×107 rat bone marrow cells can harvest about 1×106 DCs, the cell purity can reach more than 50%, and has the typical morphology of DCs. 2 Compared with the control group, dexamethasone group and Bufei Yishen group expressed OX-62, MHC-II and CD80 and CD86 decreased(P<0.01); compared with the dexamethasone group, the Bufei Yishen group’s expression was lower, but the difference was not significantly(P >0.05); LPS can up regulate the expression of OX-62, MHC-II, CD80 and CD86 of DCs, and the expression of control group was more higher(P<0.01); compared with the LPS group, LPS+dexamethasone group and LPS+Bufei Yishen group can significantly reduce the expression of OX-62, MHC-II, CD80 and CD86(P<0.01); and LPS+Bufei Yishen group decreased more significantly effect than LPS+dexamethasone(P<0.05 or P<0.01). 3 Compared with the control group, dexamethasone group and Bufei Yishen group phagocytic function of DCs were increased(P<0.01); and Bufei Yishen group was significantly higher than that in dexamethasone group(P<0.05); LPS group’s expression of phagocytic function was significantly decreased compared with the control group(P<0.01); compared with the LPS group, the function of phagocytic of LPS+dexamethasone groupand LPS+Bufei Yishen group were significantly increased(P<0.05); and the expression of LPS+Bufei Yishen group increased more than that in LPS+dexamethasone group(P<0.05). 4 Compared with the control group, dexamethasone group and Bufei Yishen group the secretion of IL-6 and IL-10 were significantly increased(P<0.01), the secretion of TNF-alpha, IFN-gamma, IL-12P40 and IL-12P70 were significantly decreased(P<0.01); but there was no significant difference between two groups(P> 0.05); the secretion of IL-6, TNF-alpha, IFN-gamma, IL-12P40 and IL-12P70 in LPS group were significantly increased than control group(P<0.01), and decreased of IL-10(P<0.01); compared with the LPS group, LPS+dexamethasone group and the LPS+Bufei Yishen group the secretion of IL-6 and IL-10 were increased(P<0.05 or P<0.01), the expression of TNF-alpha, IFN-gamma, IL-12P40 and IL-12P70 were decreased significantly(P<0.05 or P<0.01); and the chages of LPS+Bufei Yishen group was particularly significant(P<0.05 or P<0.01). 5 Compared with the control group, dexamethasone group and Bufei Yishen group can significantly increase the expression level of TLR2, TLR4(P<0.01); but there was no significant difference between two groups(P > 0.05); a low level of TLR2, TLR4 expressed in LPS group, which was lower than the control group(P<0.01); compared with the LPS group, LPS+dexamethasone group and LPS+Bufei Yishen group the expression of TLR2, TLR4 was significantly increased(P<0.01); and LPS+Bufei Yishen group’s expression level was significantly increased than that in LPS+dexamethasone group(P<0.01). Conclusion 1 Bufei Yishen recipe inhibited the differentiation and maturation of LPS induced DCs, by regulating morphology, cytokine secretion, gene and cell phenotype expression. 2 Bufei Yishen recipe for the treatment of stable COPD, might be related to the inhibition of LPS induced DCs maturation, and the reduction of inflammation. |
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