Effects Of Bupleurum Polysaccharides On The Immunologic Function And TLR4Signaling Pathway Of Macrophages

Macrophages are originated in the bone marrow precursor cells and widely distributed in the body. They have tissue specificity, such as liver Kupffer cells, brain microglia, peritoneal macrophages, alveolar macrophages and osteoclast, which are located in different tissues. The phagocytosis of activated macrophages would be enhanced during inflammation, and they would kill pathgens directly through phagocytosis or indirectly by the secretion of inflammatory cytokines (eg. TNF-a, IL-6, and IL-1β), reactive oxygen species, NO and any other mediators against pathogenic microorganisms in order to effectively defense the invasion that would cause inflammation as well as tissue injury. As the first line of host defense system against infections, macrophages play critical roles in the innnate immune system. Macrophages target the structurally conserved pathogen-associated molecule patterns (PAMPs) through the pattern recognition receptors (PRRs) on their surface, thus effectively perceive the invasion of pathogenic microorganisms and induce the immune response.Lipopolysaccharide (LPS), the major cell wall constitute of Gram-negative bacteria, is one of most potent inducers of inflammatory reponse. Toll-like receptors (TLRs), which function as key innate immune receptors responsible for recognizing PAMPs, play essential roles in the host defense system. TLRs are mainly expressed in monocytes and dendritic cells, and each TLR identifies limited pathogens. Among TLRs, TLR4principally recognizes LPS and mediates the LPS signal transduction so as to regulate inflammatory response and clear the foreign bodies.Perennial herb Bupleurum (family Umbelliferae), known as’Chai-Hu’, is regarded as a traditional Chinese medicine. Radix Bupleuri (dried roots from Bupleurum chinense DC or Bupleurum smithii var. parvifolium) possesses anti-inflammatory and immuno-modulatory effects. Bupleurum smithii var. parvifolium is abundantly distributed in the northwest region of China and Bupleurum Polysaccharides (BPs) in this study was extracted from its roots. Previous research in our lab demonstrated that (1) treatment of BPs by oral gavage was able to elevate phagocytic activity of macrophages towards self-apoptosis cells, antibody-mediated sheep red blood cells (IgG-SRBC) and chicken red blood cells (CRBC) but suppress LPS-induced production of proinflammatory mediators such as NO, TNF-α, IL-6and IL-1β;(2) in animal model of acute lung injury (ALI) induced by intratracheal instillation of LPS, BPs could alleviate the pathological damage and inhibit over activation of complement and complement deposits in lung tissues.Based on our previous studies, in the present research, we observed the effects of BPs on phagocytosis and secretory function of murine peritoneal macrophages for the purpose of analysing the immunomodulatory effect of it. Additionally, in view of the important roles of TLR4signaling transduction in mononuclear macrophages, we investigated the effects of BPs on LPS-induced TLR4signaling pathway in order to thoroughly explore the specific mechanism of BPs to inhibit inflammatory response and to further understand the possible therapeutic target of BPs in ALI.1. Effects of Bupleurum Polysaccharides on Phagocytosis and Secretory Function of Murine Peritoneal MacrophagesAim:To determine the roles of BPs in phagocytosis and secretory functon of peritoneal macrophages and then to discover its immunomodulative effect on mononuclear macrophages.Methods:(1) The effect of BPs on phagocytosis of macrophages:Balb/c mice were randomly divided into six groups:control group, BPs (10,20,40,80μg/ml) treated groups, Dexamethasone (Dex,1μM) treated group. Macrophages were incubated with Dex (1μM) or different concentration of BPs for30min, followed by addition of the E. coli/Z.A-FITC fluorescent bioparticles to continue the cells cultivation for1-2h. Then the mean fluorescence intensity and phagocytic rate of macrophages were tested;(2) The effect of BPs on secretory functon of macrophages:Balb/c mice were randomly divided into fifteen groups:control group, BPs (10,20,40,80μg/ml) treated groups, LPS (1μg/ml) stimulated group, LPS combined with BPs (10,20,40,80μg/ml) treated groups, Polymyxin B (PMB,1μg/ml) treated group, BPs (10,20,40,80μg/ml) pretreated with PMB groups. Macrophages were treated for24h, and the level of nitric oxide (NO) and cytokines (TNF-a, IL-6, IL-1β, IL-12p40, IL-10, and IFN-β) was respectively quantified by Griess reaction and enzyme-linked immunosorbent assay (ELISA) to discover the effects of BPs on the secretory functon of macrophages.Results:(1) BPs20,40,80μg/ml drastically enhanced the mean fluorescence intensity and phagocytic rate of macrophages towards E.coli (P<0.05) but decreased that towards Z.A (P<0.05).(2) BPs40,80μg/ml obviously inhibited LPS-stimulated production of NO, TNF-a, IL-6, IL-1β, IL-12p40, and IFN-β(P<0.05). Nevertheless, BPs80μg/ml significantly promoted LPS-stimulated secretion of IL-10(P<0.05). The effects of BPs itself to the secretory function of macrophages were as follows:on the one hand, BPs40,80p,g/ml dramatically increased the production of TNF-a, IL-6, IL-12p40(P<0.01), and PMB pretreatment significantly lessened (P<0.05) but not removed this facilitation effect of BPs; on the other hand, BPs80μg/ml notably elevated the amount of IFN-P (P<0.001), but each concentration of BPs had almost no effect on NO, IL-1β, IL-10secretion, and PMB pretreatment did not affect these roles of BPs.Conclusions:BPs promoted the phagocytosis of macrophages towards E.coli but suppressed that towards Z.A, which could be related to the possibility that there was distinct mechanism and target when BPs engulfed different foreign bodies; BPs lessened LPS-induced production of inflammatory mediator NO and proinflammatory cytokines like TNF-α, IL-6, IL-1β, IL-12p40but increased the amount of anti-inflammatory cytokine IL-10. All these implied the fact that BPs can affact the activation of transcription factors which lead to the elevated production of inflammatory mediators during LPS/TLR4signaling transduction; Moreover, BPs inhibited LPS-induced secretion of IFN-β, a typical cytokine generated by the activation of IRF-3in MyD88-independent pathway of LPS/TLR4signaling transduction. All these results suggested BPs may exert anti-inflammatory roles by influencing TLR4signaling. Furthermore, this study threw light on the immunomodulative function of BPs, an effect that it could not only inhibit inflammatory response but also promote pathogen elimination.2. Effects of Bupleurum Polysaccharides on LPS-induced TLR4Signaling Pathway in Murine Peritoneal MacrophagesAim:To determine the mechanism of BPs to inhibit LPS-induced inflammatory response.Methods:Balb/c mice were randomly divided into six groups:control group, LPS (1μg/ml)-stimulated group, LPS combined with BPs (10,20,40,80μg/ml) treated groups. Immunofluorescence (IF) was utilized to examine the effects of BPs on LPS-induced cell surface TLR4and CD14expression; Western blot (WB) was applied to test the roles of BPs in LPS-induced expression of TLR4, CD14, TLR2, MyD88, IRAK4, TRAF6and phosphorylation of NF-κB p65, MAPKs (ERK, JNK, p38).Results:LPS significantly augmented expression of TLR4, CD14, TLR2, MyD88, IRAK4, TRAF6and phosphorylation of NF-κB p65as well as ERK, JNK, p38(P<0.05). BPs40,80μg/ml remarkably suppressed LPS-stimulated TLR4, CD14, TLR2, IRAK4, TRAF6activation and NF-κB p65, JNK phosphorylation (P<0.05). However, BPs had almost no effect on LPS-stimulated MyD88activation and ERK, p38phosphorylation.Conclusions:BPs was able to regulate and inhibit LPS-induced TLR4signaling pathway, which is mainly embodied in reducing LPS-induced high protein levels of TLR4, CD14, and p-NF-κB. All these results demonstrated the inhibitory effects of BPs on LPS-induced TLR4signaling, which might account for its anti-inflammatory action.