Immunomodulatory Effects of Mulberry Leaf Polysaccharide on Porcine Alveolar Macrophages
[Objective]The purpose of this study was to investigate the effects of mulberry leaf polysaccharide(MLP)on the immune function of porcine alveolar macrophages and its mechanism.[Method]The experiment took porcine alveolar macrophages as the study object,CCK-8 method was used to detect the proliferation rate of porcine alveolar macrophages treated with different concentrations of MLP,and screen the appropriate concentration of MLP.The cells were divided into blank control group(BC group),positive control group(LPS group)and MLP groups with different concentrations.The release of nitric oxide(NO)in porcine alveolar macrophages was detected by Griess method.The phagocytosis ability of porcine alveolar macrophages was detected by neutral red phagocytosis assay.The contents of interleukin-10(IL-10),IL-6 and tumor necrosis factor-α(TNF-α)in porcine alveolar macrophages were determined by ELISA.The mRNA expression levels of TNF-α,Toll-like receptor 4(TLR4),IL-6,IL-10 and nuclear transcription factor κB(NF-κB)were detected by Real-time quantitative PCR.The expression levels of TLR4 and p65 proteins were detected by Western blotting.[Result]Compared with 0 μg/mL MLP group,the proliferation rate of porcine alveolar macrophages in 20,40 and 80 μg/mL MLP groups was extremely significantly increased(P＜0.01),and showed a dose-dependent effect.Therefore,20,40 and 80 μg/mL MLP were selected for follow-up tests.Compared with BC group,the phagocytosis rate and NO release of porcine alveolar macrophages in different concentration of MLP groups were extremely significantly increased(P＜0.01).ELISA results showed that the contents of IL-6 and TNF-α in porcine alveolar macrophages were significantly or extremely significantly higher than those in BC group when MLP concentration was 80 μg/mL(P＜0.05 or P＜0.01).Real-time quantitative PCR results showed that the mRNA expressions of TNF-α,IL-6 and TLR4 genes in porcine alveolar macrophages at MLP concentrations of 40 and 80 μg/mL were extremely significantly higher than those in BC group(P＜0.01).The results of Western blotting showed that when MLP concentration was 80 μg/mL,the protein expressions of p65 and TLR4 were extremely significantly higher than those in BC group(P＜0.01).[Conclusion]MLP could activate porcine alveolar macrophages,significantly promote the release of NO and the secretion of IL-6 and TNF-α,and had good immunomodulatory activities,and its immunomodulatory mechanism was related to the activation of TLR4 on the surface of macrophages.
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