The Effect Of Sparassis Latifolia Polysaccharides On Immune Receptors TLR4、TLR2 And Signal Transduction Pathways

[Objective] To isolate and purify the neutral polysaccharides of Sparassis latifolia,study the macrophage RAW264.7 immune receptors TLR4 and TLR2 of neutral polysaccharides and acid polysaccharides of Sparassis latifolia with a molecular weight of 6456 Da,and explore receptor-mediated signal transduction pathways.It will provide a theoretical basis for further exploring the immunoregulatory mechanism of Sparassis latifolia polysaccharides and its development as a raw material for immune function.[Methods](1)The crude polysaccharide of Sparassis latifolia was extracted by the traditional water extraction and alcohol precipitation method.Sparassis latifolia polysaccharide were decolored by HZ-830 macroporous resin and isolated by DEAE-52 with distilled water and collected the eluent components.Then the neutral polysaccharide components was purified by Sepharose CL-6B and collected.The components of neutral polysaccharides of Sparassis latifolia were analyzed using high-performance gel permeation chromatography,fourier infrared chromatography,ion chromatography,etc.(2)The immune receptors TLR4 and TLR2 of neutral polysaccharides and acid polysaccharides of Sparassis latifolia were studied.The effect of polysaccharide concentrations of two components in the range of 0~4000 μg/m L on the proliferation activity of macrophage RAW264.7 was determined by MTT method,and screened the concentration with the strongest ability to promote macrophage proliferation.Macrophage RAW264.7 were treated with the two polysaccharide components at the concentrations selected;TLR4antibody and TLR2 antibody were applied to macrophage RAW264.7 for 1 h,and then cultured with cell culture medium containing two polysaccharide components.The cell culture supernatant and cells were collected separately,and detected the secretion of NO,IL-6,TNF-α,and IFN-β in the cell culture supernatant;Total intracellular RNA was extracted,and the mRNA expression of macrophage immunomodulatory receptors was determined by RT-PCR.The total protein of macrophages was extracted and the protein expression of immunoregulatory receptors were determined by western blot,and determined the immune recepters of two polysaccharide components of Sparassis latifolia.(3)For the identified immune receptors,the effect of two polysaccharide components on immunoreceptor-mediated signal transduction pathways were studied.Macrophage RAW264.7 was treated with two polysaccharide components,and then the cells were collected separately.Total RNA was extracted from the cells.The relative mRNA expression of TRAF6,IRF3,and MAPKs(JNK,ERK,p38)were determined by RT-PCR.[Results](1)Sparassis latifolia polysaccharide was obtained by HZ-830 macroporous resin and separated DEAE-52.After purification by Sepharose CL-6B,two neutral polysaccharides SLP1 and SLP2 were obtained.Both SLP1 and SLP2 had characteristic structures of polysaccharides.SLP1 was consisted of arabinose: galactose: glucose: xylose: mannose with a molar ratio of 4:10:12:3:2.SLP2 was consisted of arabinose: galactose: glucose: xylose: mannose with a molar ratio of 6:12:63:10:5.From the highperformance gel permeation chromatogram of SLP1,there were two peaks,and the molecular weight of the main peak component was 6.9 × 106 Da,but the amount was small.The molecular mass of SLP2 is 3.2 × 105 Da,and the high-performance gel permeation chromatogram showed a single symmetrical peak with good separation effect and high purity.It was a homogeneous polysaccharide.Therefore,SLP2 was selected for subsequent research on immune receptors and signal transduction pathways.(2)Both the neutral polysaccharide SLP2 and the acid polysaccharide of Sparassis latifolia could promote the proliferation of RAW264.7 macrophages,increased the secretion of NO,TNF-α,IL-6 and IFN-β,and there was a trend of first increase and then decrease with the increase of polysaccharide concentration.When the concentrations were 250 μg/m L and 31.25 μg/m L,the ability to promote cell proliferation were the strongest.After the addition of TLR4 antibody,the secretion of NO,IL-6,TNF-α,and IFN-β was significantly reduced,and the difference was extremely significant(p<0.01).With the addition of TLR2 antibody,the secretion of NO,IL-6,TNF-α,and IFN-β decreased,but there was no statistically significant difference.After SLP2 and acid polysaccharides of Sparassis latifolia treated cells,the mRNA and protein expression of TLR4 receptors increased significantly and the difference was extremely significant(p<0.01).This indicates that TLR4 was a receptor for the neutral polysaccharide SLP2 and acid polysaccharides of Sparassis latifolia,and TLR2 may not be their receptor.(3)Neutral polysaccharides SLP2 and acid polysaccharides of Sparassis latifolia could increase macrophage RAW264.7 surface immunomodulatory receptor TLR4-mediated signal transduction pathway related genes TRAF6,IRF3,JNK,ERK,p38 relative mRNA expression.When the concentration of neutral polysaccharide SLP2 was 250 μg/m L and the concentration of acid polysaccharide of Sparassis latifolia was31.25 μg/m L,the relative expression of each gene was the largest.This showed that TRAF6,IRF3,JNK,ERK,and p38 were the molecular targets of the downstream pathways of these two polysaccharides.[Conclution] Neutral polysaccharide SLP2 and acid polysaccharide of Sparassis latifolia have immunomodulatory effects.TLR4 is the receptors of neutral polysaccharide SLP2 and acid polysaccharide of Sparassis latifolia,and through the TLR4 receptor-mediated signal transduction pathway to regulate the immune function of macrophage RAW264.7.