Immunomodulatory Activity And Molecular Mechanism Of Cyclocarya Paliurus Polysaccharide Based On Sulfated Modification

The immune system plays a vital role in the host’s defense against microorganisms and foreign pathogens.However,the dysfunction of the immune system can cause the body to generate inflammation,infection and even cancer.Therefore,the search for natural active substances with low toxicity and no side effects to enhance the immunity of the body is crucial for health.It has been found that sulfated polysaccharides have potential immunomodulatory activity and can exert immunomodulatory effects by stimulating the immune system or controlling the activity of immune cells,which is a potentially valuable bioactive substance for use as an adjuvant in the treatment of clinical immune system diseases.Cyclocarya paliurus polysaccharide（CP）,as one of the main active components of Cyclocarya paliurus,shows a variety of biological activities.As an effective method to improve the bioactivity of CP,sulfated modification can enhance the antioxidant,anti-inflammatory and anti-tumor bioactivities of CP.At present,most of the immunomodulatory activities of sulfated Cyclocarya paliurus polysaccharide have been studied in vivo or in vitro as a single aspect.In addition,its immunomodulatory mechanism remains unclear.Therefore,in this work,sulfated Cyclocarya paliurus polysaccharide（SCP3）was prepared by the chlorosulfate-pyridine method.The immunomodulatory activity and molecular mechanism of SCP3 were comprehensively investigated in vivo and in vitro by macrophages RAW264.7 and cyclophosphamide（CTX）-induced immunosuppression mice.Moreover,the immunomodulatory mechanism of SCP3 was analyzed from multiple perspectives,including intestinal flora and metabolomics.The major research contents and results of the study are as follows:（1）The sulfated Cyclocarya paliurus polysaccharide（SCP3）with a substitution degree（DS）of 0.75 was prepared by the chlorosulfate-pyridine method（chlorosulfonic acid:pyridine,v/v,1:3）.The physicochemical analysis showed that SCP3 contained 33.29%total sugars,14.67%uronic acid and 10.36%protein with a molecular weight of 2.19×10⁵Da.Analysis of the monosaccharide composition analysis revealed that SCP3 consisted of six monosaccharides and two alduronic acid,which were rhamnose,arabinose,galactose,glucose,xylose,mannose,galacturonic acid and glucuronic acid with molar ratios of 0.26:1.00:1.36:1.20:0.21:0.46:1.47:0.27.FT-IR spectra showed the presence of characteristic absorption peaks of the polysaccharide and the sulfate groups in SCP3,which indicated the sulfated group was successful bound to the polysaccharide.The UV spectra showed that SCP3 was a heteropolysaccharide containing protein.Scanning electron microscopy showed that the morphology of the polysaccharide showed fragmented and striped structures with rougher surfaces and larger voids after sulfated modification.（2）The immunomodulatory activity and molecular mechanism of SCP3 were studied using macrophages RAW264.7 as an in vitro cell model.The results showed that SCP3 could increase the viability of macrophages,enhance phagocytosis,promote intracellular ROS content and activate downstream signaling pathways.Meanwhile,SCP3 could increase the phosphorylation expression of ERK1/2,JNK,p38 and NF-κB p65,activating cellular MAPK and NF-κB signaling pathways.In addition,TLR4 receptor inhibitors were able to inhibit the ability of SCP3 to promote the secretion of NO and TNF-α,as well as block the expression of phosphorylated proteins of the downstream MAPK signaling pathway,indicating that TLR4 is a membrane receptor for SCP3 activation of the downstream signaling pathway.Finally,validation using specific inhibitors of MAPK and NF-κB signaling pathways.The results demonstrate that SCP3 could activate macrophages through TLR4-mediated MAPK and NF-κB signaling pathways to promote the production of cytokines TNF-αand NO,which in turn regulate macrophages immunity.（3）The in vivo immunomodulatory activity and mechanism of SCP3 were investigated using splenic lymphocytes and cyclophosphamide（CTX）-induced immunosuppressed mice as models.Results showed that SCP3 could enhance the proliferatione of splenic lymphocytes in normal mice,and could synergistically promote the proliferative activity of T and B lymphocytes with Con A and LPS.By cytokine assay,it was found that CP significantly increased the secretion of TNF-α,IL-2 and IFN-γand activated the differentiation of Th cells into Th1 type,while SCP3significantly increased the secretion of TNF-α,IL-2,IFN-γand IL-4 and activated the differentiation of Th cells into Th1 and Th2 type,indicating that the sulfated modification of SCP3 could mediate both cellular immunity and humoral immunity,and improved the immunomodulatory ability of the polysaccharide on splenic lymphocytes.The immunomodulatory activity of SCP3 on CTX-induced immunosuppressed mice and its possible molecular mechanism were investigated.The results showed that SCP3 could improve the body weight of immunosuppressed mice and alleviate the atrophy of immune organs and the abnormal blood parameters caused by CTX.Moreover,SCP3 increased the proliferation of T and B lymphocytes,promoted the secretion of TNF-α,IL-10,and NO from splenic lymphocytes,as well as up-regulated the ratio of CD3⁺,CD4⁺,and CD8⁺T lymphocytes.Meanwhile,SCP3ameliorated oxidative stress damage in the liver by increasing T-AOC capacity,antioxidant enzyme（SOD,CAT,GSH-Px）activity and reducing malondialdehyde（MDA）content.In addition,SCP3 at a dose of 50 mg/kg significantly promoted the expression of My D88,phosphorylation of JNK,ERK 1/2,p38 in MAPKs signaling pathway,as well as increased Akt,NF-κB（p65）,IκB-αphosphorylation and promoted Ik B-αdegradation.In conclusion,SCP3 was able to regulate immune activity in immunosuppressed mice through the My D88-dependent MAPK/NF-κB/PI3K-Akt signaling pathway.（4）The intestinal mucosal immunomodulatory activity of SCP3 in immunosuppressed mice was investigated from the perspective of intestinal mucosal immunity.The results showed that SCP3 increased the villi length（V）and crypt depth（C）,improved the ratio of V/C in small intestine,and reduced swelling and cellular infiltration in the intestinal lumen.Moreover,SCP3 increased the number of goblet cells（GCs）and mucin content,improved the intestinal barrier,restored the morphological integrity of the intestine,and reduced the serum levels of lipopolysaccharide binding protein（LBP）and endotoxin（ET）.Moreover,SCP3 was able to increase the expression of Ig A-secreting cells and the number of intestinal CD4⁺and CD8⁺T cells,promote the increase of intestinal TNF-αand IL-1βand regulated intestinal immunity by improving the antioxidant capacity of intestine.In addition,SCP3 also increased the expression of tight junction proteins ZO-1,Occludin and Claudin-1 to promote cellular tight junctions,enhance intestinal barrier,attenuate intestinal permeability,and prevent the invasion of bacterial endotoxins and pathogenic microorganisms.（5）An in vitro model of oxidative damage to intestinal epithelial cells was used to investigate the protective effect of SCP3 against oxidative damage of IEC-6 using RNA-seq sequencing.RNA-seq sequencing results showed that there were 2630differential genes up-regulated and 1082 differential genes down-regulated in the blank control group compared with the model group（B vs M）;393 differential genes up-regulated and 859 differential genes down-regulated in the model group compared with the CP group（M vs CP）;and 870 differential genes up-regulated and 1982differential genes down-regulated in the model group compared with SCP3（M vs SCP3）.KEGG enrichment analysis showed that the protection of SCP3 against oxidative damage in IEC-6 cells might be related to MAPK and PI3K-Akt signaling pathways.The expression of key proteins in MAPK and PI3K-Akt signaling pathways was verified by Western blot based on the information related to genomic changes.The results showed that H₂O₂significantly increased the phosphorylation of JNK,ERK and p38 of MAPK signaling pathway and the phosphorylation of Akt of PI3K-Akt signaling pathway in IEC-6 cells,while the pretreatment of cells by SCP3significantly attenuated the activation of this pathway by H₂O₂and reduced the expression of phosphorylated proteins.Thus,SCP3 could protect the intestinal cells from oxidative stress damage through MAPK/PI3K-Akt signaling pathway.（6）The mechanism of immunomodulatory effect of SCP3 on immunosuppressed mice was studied based on intestinal flora.Bacterial 16S r RNA sequencing results showed that SCP3 was able to altered the structure of intestinal microbes of immunosuppressed mice,increasing the diversity of the intestinal flora and modulating the relative abundance of specific bacteria,including increasing the abundance of Bacteroidetes,Firmicutes,Tenericutes,Oscillospira,and Akkermansia,and decreasing the abundance of Proteobacteria and Verrucomicrobia.The results of species differences and biomarker bacteria analysis indicated that g＿Lactobacillus,g＿Acidovorax,c＿Chloroplast,g＿Gemella,o＿Streptophyta,f＿Gemellaceae,o＿Gemellales,g＿Acinetobacter,and f＿Moraxellaceae may be associated with the pathogenesis of CTX-induced immunosuppression.However,after CP intervention,the biomarker bacteria included g＿Pediococcus,o＿Bacteroidales,f＿Prevotellaceae,g＿[Prevotella],f＿Erysipelotricaceae and the marker species of SCP3 include g＿Clostridium,g＿[Ruminococcus],f＿Dehalobacterium,g＿AF12,g＿Butyricimonas and f＿（Odoribacteraceae）.In addition,SCP3 significantly increased the content of total short-chain fatty acid（SCFAs）,acetic acid,propionic acid and butyric acid in the colonic contents compared to CP.Functional prediction of intestinal colonization revealed that the effects of CP and SCP3 on metabolic pathways in immunosuppressed mice may be related to amino acid metabolism,carbohydrate metabolism and fatty acid synthesis and metabolic pathways.（7）The mechanism of immunomodulatory effects of SCP3 on immunosuppressed mice was investigated by UPLC-Q-TOF/MS metabolomics.PCA and OPLS-DA analysis revealed that the treatment of CTX affected the metabolic profile of mouse serum,while the intervention of CP and SCP3 had the ability to modulate the alteration of endogenous small molecule substances.A total of 31differential metabolites were identified between the NC and MC groups,29differential metabolites were identified between the MC and CP groups,33differential metabolites were identified between the MC and SCP3 groups,and 29differential metabolites were identified between the CP and SCP3 groups.Moreover,it was found that CTX mainly affected aminoacyl-t RNA biosynthesis,phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,ubiquinone and other terpenoid-quinone biosynthesis,α-linolenic acid metabolism and tyrosine metabolism these six metabolic pathways.However,CP and SCP3 were able to regulate serum metabolites in immunosuppressed mice from different metabolic pathways,respectively.Among them,the intervention of CP mainly regulated fatty acid metabolism,includingα-Linolenic acid metabolism and Arachidonic acid metabolism,while the intervention of SCP3 could regulate not only fatty acid metabolism（linoleic acid metabolism）but also amino acid metabolism,including phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,aminoacyl t-RNA biosynthesis,etc.Compared to CP,SCP3 regulates more metabolic pathways for serum metabolites and the regulation of metabolites tends to be more toward normal levels.Correlation analysis by key metabolites with immune indicators in mice.The results revealed that six metabolites,valine,isoleucine,phenylalanine,tyrosine,（9Z,12Z,15Z）-octadecatrienoic acid,and arachidonic acid,were significantly correlated with mouse immune indexes and could be used as possible biomarkers of polysaccharide-modulated CTX immunocompromised mice.