Protective Effect And Mechanism Of Exopolysaccharides From Genistein-induced Monascus On Intestinal Health

Exopolysaccharides（EPS）from Monascus,as an important class of metabolites,has been shown to have a variety of biological functions such as anti-tumor,antibacterial,and immune enhancement.However,the current research on the immunomodulation of polysaccharides from Monascus was mainly focused on the determination of some simple indicators,such as cytokines and phagocytosis,and the immunomodulatory mechanism of polysaccharides from Monascus,especially the study of intestinal immunity,was less reported,which seriously limits the development and utilization of the endemic resources of Monascus in our country.Therefore,the present thesis was based on the study of EPS-producing Monascus,and its liquid fermentation conditions were optimized,and then the exopolysaccharides from genistein-stimulated Monascus（G-EMP）was studied,and the structure of G-EMP was analyzed to evaluate its in vitro immunomodulatory effect and toxicity.Meanwhile,the protective effects of G-EMP on intestinal health were evaluated by constructing an intestinal injury model with cyclophosphamide（Cy）and dextran sulfate sodium（DSS）,and its possible mechanisms were further investigated in terms of gut immunity,gut microbiota,gut metabolism,and signaling pathways.The main results of the thesis are as follows:（1）Optimization of liquid fermentation conditions for EPS production by Monascus and comparative transcriptomics analyses were performed to understand the effect of liquid fermentation conditions on EPS synthesis.The results suggested that the optimal liquid medium composition was:40 g/L sucrose,4 g/L yeast powder,1 g/L Mg SO₄·7H₂O,0.8 g/L KH₂PO₄,1.6 g/L K₂HPO₄·3H₂O,2 m L/L Tween 80,and 3 g/L genistein;the optimal liquid culture conditions were:inoculum amount of 7%,culture temperature of 30℃,initial p H 6.0,rotational speed of 180 rpm,and culture time of 4d.A total of 8095 unigenes were obtained from transcriptomics analysis,and 17 key enzymes for EPS synthesis were identified.Interestingly,12 carbohydrate metabolism subcategories were enriched in the group with 4 days of fermentation compared to 2days,with most of the differentially expressed genes（DEGs）being upregulated,but only nine carbohydrate metabolism subcategories were enriched with longer fermentation time,with all DEGs being downregulated.Furthermore,genistein not only facilitated the production of EPS by regulating cell membrane permeability,enhancing cellular respiratory metabolism and monosaccharide precursor synthesis pathways,and enhancing antioxidant enzyme activity to reduce oxidative stress damage,but also affected the structure of polysaccharides by influencing the activity of enzymes in the monosaccharide synthesis pathway and the activity of enzymes encoding polysaccharide spatial structure.（2）The effect of the genistein on the structure of EPS was explored.The results revealed that the addition of genistein could lead to significant changes in the chemical composition,solubility,molecular weight,zavg,zeta potential,molar ratio of monosaccharide composition,surface morphology,spatial structure and thermal stability of EPS.Meanwhile,EMP_（0.1）is mainly composed of→4)-β-D-Galp-（1→、→2）-β-D-Glcp-（1→and a small amount of→6）-α-D-Glcp-（1→、→2,6）-α-D-Glcp-（1→、→4）-α-D-Manp-(1→connected to each other to form a backbone andα-D-Manp-（1→connected to the O-2 position in→2,6）-α-D-Glcp-(1→at the O-2 position to form a branched chain;whereas G-EMP_（0.1）is mainly composed of→4)-α-D-Galp-（1→、→2）-β-D-Manp-（1→and a small number of→4）-α-D-Galp-（1→、→2,6）-α-D-Glcp-(1→interconnected to form a backbone,withα-D-Manp-（1→attached to the O-2 position of→2,6）-α-D-Glcp-(1→to form a multibranch chain.（3）The classical mouse macrophage cell line RAW264.7 was adopted to investigate the immunostimulatory activity of G-EMP.The results revealed that the G-EMP improved the immunomodulatory activity of RAW264.7 macrophages by increasing the secretion of reactive oxygen species,nitric oxide and cytokines.Moreover,phospho-Jun N-terminal kinase（p-JNK）,phospho-p38（p-p38）,phospho-extracellular regulated protein kinase（p-ERK）mitogen-activated protein kinase（MAPK）and nuclear factor-kappa B（NF-κB）phospho-p65（p65）proteins were remarkably upregulated by G-EMP stimulation,blocking Toll-like receptor 4（TLR4）that dramatically reduced the pinocytic and phagocytic capacities.More importantly,G-EMP exhibited stronger immunomodulatory activity compared to EMP,which result was closely related to the higher solubility,monosaccharide constituent,branching degree,andα-D-1,4-Gal glycosidic bond of G-EMP.（4）Cy was adopted to construct an immunosuppressed mouse model to explore the interactions of G-EMP in intestinal immune response,intestinal injury and gut microbiota in mice.The results suggested that G-EMP effectively alleviated the signs of weight reduction and diet reduction caused by Cy,increased fecal water content and splenic index,and decreased the oxidative stress of the liver.Simultaneously,G-EMP boosted the levels of cytokines,CD4⁺and CD8⁺T cells in the mouse colon and markedly enhanced immunomodulation via the activation of PI3K/AKT-MAPKs/NF-κB signal pathways.It also improved Cy-induced intestinal injury by restoring villus length,increasing the number of cupped cells,upregulating the expression of mucin and tight junction proteins,and downregulating the ratio of apoptotic proteins（Bax/Bcl-2）.Furthermore,G-EMP intervention displayed a positive association with most immunological indexes by elevating the levels of short-chain fatty acids,varying gut microbiota composition,and enhancing beneficial bacteria（Lactobacillaceae,Prevotellaceae,and S24-7）.（5）A mouse model of colitis intestinal injury was constructed using DSS targeting to estimate the mitigating properties of G-EMP on colitis mice in terms of pathology,biochemistry,inflammatory response,and intestinal barrier.The results suggested that G-EMP intervention reduced body weight loss,degree of colon injury and shortening,Disease Activity Index score and histopathological score,decreased MPO activity,restored the number of goblet cells,and effectively regulated the balance of the number of immune cells regulating the immune response in spleen and colon and the Th1/Th2/Th17 cytokine axis to regulate the immune response to protect against intestinal injury induced by DSS in mice.Additionally,G-EMP not only effectively regulated the hepatic and colonic oxidative stress in DSS-induced intestinal injury mice and improved the intestinal barrier and intestinal integrity;it also activated the Nrf2/HO-1 signaling pathway by increasing the expression of mucin and tight junction proteins,thus exerting the protective effect of G-EMP against DSS-induced intestinal injury.（6）Metabolomics techniques were combined with 16S r RNA sequencing to explore the potential mechanisms of G-EMP on host metabolism and intestinal microbiota of DSS-treated colitis intestinal injury mice.The results suggested that G-EMP intervention effectively reversed the reduction of SCFAs in DSS-induced intestinal injury mice.Meanwhile,16S r RNA sequencing revealed that G-EMP intervention increased the abundances of gut microbiota of DSS-treated colitis intestinal injury mice.At the phylum level,G-EMP was able to downregulate the abundances of Bacteroidota and increase the abundances of Firmicutes.At the genus level,G-EMP was able to increase the abundances of Akkermansia,Lactobacillus and Allobaculum,while decreasing the abundances of Bacteroides,Helicobacter and Clostridium.At the family level,G-EMP enriched Lactobacillaceae.Furthermore,serum metabolic analyses based on UHPLC-QE-MS revealed that 100 differential metabolites were identified for NC vs MC,77 differential metabolites for MC vs EMP,95 differential metabolites for MC vs G-EMP,and 23 differential metabolites for EMP vs G-EMP.The metabolic pathway results showed that G-EMP mitigated the effects of DSS-induced intestinal damage mainly through the maintenance of intestinal immune homeostasis metabolic pathways,such as the biosynthesis of phenylalanine,tyrosine,and tryptophan,the metabolism of phenylalanine,the biosynthesis of aminoacyl-t RNA,and the biosynthesis of ubiquinone and other terpene quinones.（7）Based on RNA-seq analysis,bioinformatics,PCR and western blot,the potential mechanism of G-EMP to alleviate DSS-induced colonic injury was explored in depth.The results showed that compared with the control group,the DSS group expressed 408 DEGs,of which 169 were elevated and 239 were decreased.Compared with the DSS group,the G-EMP group had 2670 DEGs,of which 959 were elevated and 1711 were decreased.Compared with the control group,the G-EMP group had a total of 2422 DEGs,of which 946 were elevated and 1476 were decreased.Furthermore,both GO analysis and KEGG enrichment analysis indicated that G-EMP could alleviate DSS-induced colonic intestinal injury by regulating multiple pathways such as cytokines,apoptosis,TLR4/MAPK/NF-κB,and ERS.Meanwhile,bioinformatics,PCR and western blot results further confirmed the RNA-seq sequencing results.（8）A mouse model of DSS-induced intestinal injury was constructed,and specific inhibitors（4-PBA）and activators（2-DG）borrowed from ERS were employed to deeply investigate the effects of G-EMP-regulated ERS/UPR on alleviating DSS-induced intestinal injury.The results suggested that G-EMP intervention alleviated the over-activation of PERK-e IF2a-ATF4-CHOP and IREla-XBP1 signaling pathways,as well as ERS-related apoptotic pathways,resulting in an improved intestinal barrier.Meanwhile,the co-intervention of 4-PBA and 2-DG with G-EMP revealed that ERS was involved in the regulation of intestinal immune cells and cytokines by G-EMP,and that G-EMP could effectively regulate the intestinal immune TLR4/MAPK/NF-κB signaling pathway through the inhibition of ERS stress,which would in turn exert a positive effect on the intestinal injury caused by DSS.Furthermore,the co-intervention of 4-PBA and 2-DG with G-EMP revealed the involvement of ERS in the positive regulatory effect of G-EMP on the metabolic disorders of intestinal SCFAs induced by DSS.