Immunomodulatary Effect And Regulation Mechanism Of Plantagoasiatica Polysaccharide On Mouse Macrophages

Plantago asiatica is one of the commonly used Chinese herbal medicines included in “the Pharmacopoeia of People's Republic of China”.Previous studies suggested that Plantago asiatica Polysaccharide(PLP),a class of Polysaccharides,extracted from Plantago asiatica,has various biological activities including scavenging free radicals and regulating macrophage autophagy.Our previous studies showed that PLP could improve the titer of Newcastle disease vaccine immune and transformation rate of lymphocyte of chicken.Some studies have found a variety of Chinese medicine polysaccharide could promote secretion of cytokine,NO and improve the phagocytosis ability of macrophage.Macrophage can identify polysaccharide and activate the downstream signal pathways to regulate the immune function,through its surface receptors such as Toll like receptors(TLR),Mannose receptor,(MR),Complement receptor 3(CR3)and beta glucan receptor(Dectin-1).Some studies showed that the Bupleurum Polysaccharides,Yupingfeng Polysaccharide and Polysaccharides of Boschniakiarossica et al can exert their functions on cells through nuclear transcription factor kappa B(NF-?B)signal pathway;Agaricus blazei polysaccharide,Saussurea Tridactyla Polysaccharide and Astragalus Polysaccharide may have effects on cells through activating mitogen protein kinase(MAPK)signaling pathway.However,little was known about its effects on innate immunity and the mechanism.In the current study,we employ cell and molecular biological techniques to analysis the role of PLP in the regulatory of immune function and the mechanism of immune regulation in mouse macrophage.The main results and conclusions are as follows:1.The effects of Plantago asiatica Polysaccharide on immune function in macrophagesIn this study,mouse macrophages were divided into 8 groups,PLP groups(20,40,60,80,100,120 and 160 mg/m L),lipopolysaccharide(LPS)group(1 mg/m L)and control group(cells without drugs).Macrophages were cultured with PLP or LPS for 24 h,and then the cell culture medium was collected to detect the expression of NO,IL-6 and TNF-?.At the same time,the cells were collected to extract the total RNA,followed by detecting the expression of i NOS m RNA.Phagocytic activity of macrophages was detected using neutral red.Macrophages proliferation ability was detected by MTS.The results showed that PLP(20,40,60,80,100 and 120 ?g/m L)and LPS(1 ?g/m L)could significantly promote the phagocytosis rate,secretion of NO and the expression of i NOS m RNA(P <0.01)of macrophages.PLP(20,40,80 and 160 ?g/m L)could significantly increase the secretion of IL-6(P < 0.01).PLP(20,40,80 and 160?g/m L)could enhance the secretion of TNF-?,and the TNF-? of each group were significantly higher than control group(P < 0.01)except the 20 ?g/m L group.PLP(20,40,60,80,100 and 120?g/m L)could improve the proliferation rate(%)of mouse macrophages(P < 0.01),and the highest proliferation rate appeared at 100 ?g/m L group,which was133.2 ±7.7,then it decreased.These results showed that PLP could improve the phagocytosis,secretory and proliferation function of mouse macrophages,suggesting that PLP could regulate the immune function.2.The mechanism of Plantago asiatica Polysaccharide on macrophage immune regulationIn this study,we tried to find the main receptors of macrophages toward PLP and determine the effect of PLP on NF-kappa B and MAPK signaling pathway.Total RNA,total protein and culture medium from PLP treated cells were collected at different time points,then quantitative RT-PCR and Western blot were performed to detect the expression change of TLR-NF-?B and MAPK signaling pathway gene m RNA and protein.TLR2 antibody(anti-TLR2)and P-ERK blocker(U120)were used to block the TLR2 and P-ERK,then changes of m RNA and protein expression of TLR2 and P-ERK were detected,the TNF-? secretion quantity change of macrophages was detected at the same time.The results showed that the TLR2 and TLR4 m RNA expression were significantly increased in PLP stimulated macrophages after 5h(P < 0.01),and the expression decreased with the passage of time.Blocking TLR2 with TLR2 antibody,the expression of TLR2/4 m RNA in anti-TLR2+PLP group was 1.88 ± 0.18 and 0.91 ± 0.23,which were significantly lower than PLP group.TNF-? concentration of anti-TLR2(10 ?g/m L)+PLP and anti-TLR2(25 ?g/m L)+ PLP groups were significantly decreased(P < 0.01),PLP could not enhance the TNF-? concentration of macrophages when incubated with anti-TLR2(10 or 25?g/m L).NF-?B and My D88 m RNA expression of PLP groups were significantly higher than control group(P < 0.01)when TLR2 was blocked.The NF-?B and My D88 m RNA expression were significantly reduced compared with PLP group(P<0.01).The expression of P65 protein was significantly up-regulated after PLP stimulated for 15 min or 30 min,and reduced to normal level after 1 h.The expression levels of ERK1,JNK1 and P38 m RNA were significantly increased after PLP stimulation(P < 0.05),and the expression levels of ERK2 and JNK2 m RNA had no significant change compared with control group(P > 0.05).PLP could not increase m RNA expression levels of three channel subunits of MAPK when macrophages were incubated with anti-TLR2.ERK1 and P-ERK1/2 protein expression levels were significantly increased compared with the control group,while the protein expression level of ERK2 had no significant change and they were decreased with the pass of time.After macrophages were incubated with U-120,PLP could not increase the phosphorylation level of P-ERK1/2 and the m RNA expression of My D88,so did the protein expression level of NF-?B P65,and they were significantly different compared with PLP group(P < 0.01).When incubated with anti-TLR2 and U120,the m RNA expression of i NOS was significantly reduced compared with PLP group(P <0.01),while it was significantly higher than the control group(P < 0.01).The results suggested that the major receptor for recognizing PLP was TLR2 in macrophages.One of the pathways that PLP regulated immunologic function of macrophages was TLR2-My D88-MAPK-NF-?B signaling pathway.The MAPK pathway might be involved in the regulation of i NOS m RNA expression.The results above showed that Plantago asiatica Polysaccharide could regulate the immune function macrophages through TLR2-My D88-MAPK-NF-?B signaling pathway.Plantago asiatica Polysaccharide could increase the protein and m RNA expression of ERK1,JNK1,P38 and NF-?B,suggesting that ERK1,JNK1,P38 and NF-?B were pharmacological targets of Plantago asiatica Polysaccharide.