| Fuzhuan brick tea is a kind of dark tea with a history of more than 400 years.It has many beneficial effects on human health,such as anti-obesity,protecting liver,immunomodulatory,hypolipidemic and hypoglycemic effect.As a kind of post-fermented tea products,it is characterized by its unique fermentation process called “the golden flower blossoming”.Aspergillus cristatus,termed the “Golden Flower Fungus”,is the dominant fungus involved in the fermentation of Fuzhuan brick tea.It has great influence on the organoleptic qualities and health properties of tea,and its amount is considered as an important indicator of Fuzhuan brick tea quality.There is limited information available on the exopolysaccharides(EPSs)and intracellular polysaccharides(IPSs)from A.cristatus,as possible important bioactive substances present in Fuzhuan brick tea.However,it is limited to some preliminary studies on extraction optimization,physicochemical properties and antioxidant,hypolipidemic and anti-tumor activities.However,the influences of EPSs and IPSs from A.cristatus on the immune system and gut microbiota in vivo have not been reported.Current knowledge about the interactions among EPSs/IPSs,immune system and gut microbiota is still unclear.Therefore,EPSs and IPSs were isolated and purified,and their physicochemical properties,preliminary structure and immunomodulatory activities in vitro were analyzed briefly in this study.Then,we focused on the regulating effects of EPSs and IPSs from A.cristatus on immune function and gut microbiota in immunosuppressive mice.In addition,the alleviative effect of EPSs and IPSs on colitis and the regulation of intestinal barrier function were further studied.The role in modulating immune responses and in maintaining gut homeostasis was highlighted in this study.The main results are as follows:1.Isolation and purification,physicochemical properties,preliminary structure and immunomodulatory activity in vitro of EPSs and IPSsIn this study,the crude extra-and intra-cellular polysaccharides(CEPSs and CIPSs)were extracted from the fermentation broth and mycelium of A.cristatus by water extractalcohol precipitation and ultrasonic-assisted extraction method,respectively.After removing protein and various small molecule impurities by Sevage method and dialysis,EPSs-2 and IPSs-2,two mainly purified fractions,were purified by a DEAE-Fast Flow column.EPSs and IPSs have higher total carbohydrates,which is mainly neutral polysaccharide.EPSs contained a higher content of uronic acid than IPSs,while IPSs-2 contained almost no uronic acid.After purification,the total carbohydrates of EPSs-2 and IPSs-2 was remarkably increased,while the protein and polyphenol contents were significantly decreased.Both EPSs and IPSs are heteropolysaccharides composed mainly of mannose and galactose.The monosaccharide composition of EPSs-2 was mannose,ribose,glucuronic acid,glucose,galactose at a molar ratio of 4.3 : 0.5 : 0.4 : 1.2 : 3.6.IPSs-2 were composed of mannose,ribose,glucose,galactose at a molar ratio of 4.4 : 0.4 : 1.1 : 4.1.The average molecular weights of EPSs-2 and IPSs-2 were 5.60 and 17.98 k Da,respectively.Both EPSs and IPSs have good thermal stability.In addition,the immunomodulatory activity of EPSs-2 in vitro was evaluated with murine macrophage RAW264.7 cell model.It was found that EPSs-2 had no apparently cytotoxic effect to RAW264.7 cells and significantly improved the secretion of NO,TNF-α and IL-6 in a dose-dependent manner,indicating that EPSs-2 exhibited excellent immunomodulatory effect on RAW264.7 cells.2.The regulation of immune function and gut microbiota of EPSs and IPSsThe modulation of EPSs and IPSs on immune function and gut microbiota was evaluated by cyclophosphamide(Cy)induced immunosuppressive mice model.The results showed that EPSs-2,CIPSs and IPSs-2 had strong immunomodulatory effects on Cy-induced immunosuppressed mice.EPSs-2,as the main purified fraction,showed more effective activity than CEPSs,while there were no significant differences between CIPSs and IPSs-2.It was manifested in the recovery of body weight and daily intake,the improvement of immune organ indexes(thymus and spleen indexes),the significant increase of cytokines(TNF-α,IL-1β,IFN-γ)and immunoglobulin(Ig A)levels in serum.It was found that EPSs and IPSs attenuated the damages of jejunum and colon tissues and significantly increased the villi length of jejunal and the ratio of villus length to crypt depth(V/C)by the morphology pathological analysis.Furthermore,the expressions of tight junction proteins(ZO-1,Occludin,Claudin-1)and mucin(MUC2)were promoted to reinforce the intestinal barrier function and mucosal immunity.Meanwhile,EPSs and IPSs intervention significantly increased the production of different kinds of short chain fatty acids(SCFAs).EPSs-2 and IPSs-2 significantly promoted the expression of SCFAs receptors(GPR41,GPR43)and cytokines(TNF-α,IL-6,IFN-γ)in colon.In addition,EPSs and IPSs intervention balanced the dysbiosis of gut microbiota induced by Cy in mice.Thereinto,EPSs modulated the microbial composition by increasing the growth of Muribaculaceae,Prevotellaceae＿UCG-001,Bacteroides,Parabacteroides,Tidjanibacter while decreasing the relative abundances of Helicobacter,Bilophila,Mucispirillum,Lachnospiraceae,Ruminococcaceae and Clostridiales.IPSs maintained the homeostasis of the microbial ecology through elevating the growth of beneficial bacteria positively correlated with immune parameters(Muribaculaceae,Muribaculum,Duncaniella＿muris,Bacteroides acidifaciens,Parabacteroides,Barnesiella)while reducing the abundance of pathobiont negatively correlated with immune parameters(Lachnospiraceae,Ruminococcaceae,Clostridiales,Bilophila,Helicobacter,Mucispirillum＿schaedleri).3.EPSs and IPSs alleviated colitis and regulated intestinal barrier function in miceThe alleviating effects of EPSs and IPSs on colitis and the regulation of intestinal barrier function was investigated by the establishment of dextran sulphate sodium(DSS)induced colitis mice model.The results showed that EPSs-2,CIPSs and IPSs-2 had strong effects on the improvement of colitis in mice,and IPSs-2 especially exhibited more effective activity.It was manifested in the recovery of body weight,the morphology,color and weight of the solid feces of the mice,the improvement of colonic morphology and inhibiting the symptoms of colonic atrophy.Besides,the level of Lcn-2 in feces,and the secretion of TNF-α,IL-1β,IFN-γ,IL-6 and LPS in serum were significantly inhibited.The expressions of antiinflammatory cytokine IL-10 in colon of mice was up-regulated and the inflammation-related genes TNF-α,IL-1β,i NOS and TLR4 were down-regulated.In addition,the alleviating effects of EPSs and IPSs on colitis in mice were closely related to the regulation of intestinal barrier function.EPSs-2 and IPSs significantly alleviated the pathological damage of the colon,and improved the DSS-induced reduction of goblet cells and mucus production defects.In addition,the expression of ZO-1,Occludin,Claudin-1 and MUC2 at m RNA level was enhanced,and then the production of tight junction proteins and mucin was promoted to enhance intestinal barrier function and intestinal mucosal immunity.Meanwhile,EPSs-2 and IPSs significantly promoted the production of SCFAs,and up-regulated the m RNA expressions of the major SCFAs receptors GPR43,GPR41 and GPR109 A,which could play an important role in maintaining intestinal barrier function and alleviating colitis. |
PDF Full Text Request