Inhibitory Effects And Mechanism Of Activin A On LPS-activated BV2 Cells

Activin A is a multifunctional growth and differentiation factor,which belongs to the transforming growth factor-beta(TGF-?)superfamily.It exerts different biological effects in different tissues and different stages of the body development.It plays an important role in the induction of early embryo development,promoting the secretion of gonadotropins from the anterior pituitary gland cells,inhibition of inflammatory cytokines secreted by activated macrophages,induction of B lymphoma cells apoptosis and so on.Activin A can be generated by a variety of cells of the immune system,and in turn,regulates the function of immune cells,including macrophages,monocytes,T lymphocytes and dendritic cells Microglia are widely distributed in the brain and spinal cord,which is considered as macrophages to be settled in the central nervous system.Many studies show that activin A can affect the function of macrophages originating in somatic cell,but the mechanism of activin A in regulation of microglia activation has not been elucidated clearly.Therefore,effects and mechanism of activin A on LPS-activated BV2 cells was explore in this study.1.Inhibiting effects of activin A on LPS-induced BV2 cellsIn this study,the expressions of activin A and its receptors,signal transduction proteins of Smads in BV2 cells were first detected by RT-PCR.The results showed that activin A,activin type IIA receptor(Act R?A),activin type IIB receptor(Act R?B),Smad2,3,4 m RNA were all expressed in BV2 cells,suggesting that activin A may exert an effect on BV2 cells.Effects of activin A and LPS on morphologic changes of BV2 cells were then examined by Giemsa staining.The results showed that LPS could induce morphologic changes of BV2 cells,and activin A significantly inhibited the effect of LPS on morphologic changes of BV2 cells.There was no effect of activin A on the NO secretion and i NOS expression of BV2 cells,but activin A could inhibit the secretion of IL-6,IL-1? and TNF-? significantly.LPS,as a classical macrophage activating factor,could promote the secretion of NO and IL-6,IL-1?,TNF-? and increase the expression of i NOS,IL-6,IL-1 beta,TNF-?m RNA in BV2 cells,but activin A inhibited LPS-induced NO and inflammatory factors secretion,as well as the expression of inflammatory factors m RNA.In order to further explore the mechanism of activin A on activation of BV2 cells induced by LPS,the expressions of activin signal transduction related molecules were detected by RT-PCR,The results revealed that activin A and LPS could induce the expressions of Act RIIA,Act RIIB and Smad2,Smad3 m RNA,respectively,but there was no significant effect on the expression of Smad4 m RNA.Interestingly,activin A can inhibit the effects of LPS promoting the expressions of Act RIIA,Act RIIB,Smad2 and Smad3 m RNA.The results of flow cytometry showed that LPS significantly promoted the expression of TLR4 in BV2 cells.Although activin A had no significant effects on the expression of TLR4,it can inhibit the up-regulation of TLR4 induced by LPS.Both activin A and LPS could promote the expression of TLR2 on BV2 cells,and there was a much stronger effect when BV2 cells were treated with activin A and LPS together.These results suggest that activin A may inhibit the expression of TLR4 to inhibit the activation of BV2 cells induced by LPS.2.Studies on the mechanism of activin A mediated by Smad3 in BV2 cellsSmad3 is a classic signal transduction protein of activin.The expression of Smad3 m RNA increased in BV2 cells after activin A treatment.Therefore,BV2 cells transfected with Smad3 by transient transfection were used to further confirm the effects of activin A and LPS on the secretion of inflammatory factors.The results showed that after Smad3 overexpressed in BV2 cells,the expressions of i NOS,IL-6,IL-1? and TNF-? m RNA were all increased compared with pc DNA3 empty plasmid control group,and the secretion and expression of inflammatory factors treated by LPS were significantly higher than those of pc DNA3 group.At the same time,the expressions of Act RIIA and Act RIIB were also increased obviously in Smad3 overexpression BV2 cells,but the expression of Smad4 was significantly inhibited.The results of flow cytometry showed that the expression of TLR2 in Smad3 overexpression BV2 cells was not changed after LPS and activin A treatment.But after overexpression of Smad3,LPS significantly enhanced the expression of TLR4,and activin A could inhibit the effects on the expression of TLR4 induced by LPS.In conclusion,activin A significantly inhibited the activation of BV2 cells induced by LPS,which wasclosely related to Smad3 signaling pathway and TLR4 expression.