| The use of chemotherapy in clinical often causes immune suppression,even hematopoietic dysfunction by modifying the genome of normal cells,and resulting in serious side effects.At present,the methods to alleviate immunosuppression or hematopoietic dysfunction mainly include surgery,chemosynthetic drug therapy,and cytokine therapy.However,these methods have some limitations,which increase the risk of infection,and their pharmacological effects are not obvious enough.Edible and medicinal fungi are of natural origin,with rich pharmacological activities and low toxic effects.Polysaccharide components in fungi are often used in the preparation of functional foods or drugs because of rich configuration and multiple targets.Russula griseocarnosa is a kind of ectomycorrhizal fungus,mainly distributed in Fujian Province of China.The previous studies showed that polysaccharide in Russula griseocarnosa had good immunoregulatory ability in in vitro,but the structure analysis of the polysaccharide was not deep enough,and its immunoregulatory activity and deeper hematopoietic improvement function have not yet been reported in in vivo.Therefore,this study regards Russula griseocarnosa as the research object,separated and purified its polysaccharides,explored the differences in the structure and composition of different polysaccharide components,and analyzed the mechanism of Russula griseocarnosa polysaccharide in alleviating hemopoietic dysfunction caused by chemotherapy in combination with immunocompromised mouse model and hemopoietic dysfunction mouse model.First of all,this study analyzed the main components of Russula griseocarnosa,and found that it contained 44.6%total dietary fiber,30.9%total protein,27.1%total sugar,13.45%mannitol,6.9%total ash,2.8%crude fat,0.74%total alkaloids,0.51%total polyphenols,0.43%total triterpenoids,0.4%total saponins,0.35%soluble protein,and 0.3%total sterols.And it was also found that it contained 17 amino acids such as glutamic acid,6 fatty acids such as C15:0,and 8 mineral elements such as manganese,and the content of heavy metals was lower than the national limit of heavy metals in fungus,which proved that Russula griseocarnosa is a kind of edible and medicinal fungus with rich material composition,high polysaccharide content,and safety.Combined with the single factor experiment and response surface design,the optimal conditions for hot water extraction of Russula griseocarnosa polysaccharide were determined:the ratio of liquid to material was 30:1,the temperature was 80℃,and the time was 3 h.The crude polysaccharide components were obtained by Sevag method and ethanol precipitation method,and the different polysaccharide components were separated and purified by chromatography.Finally,two main polysaccharides RGP1 and RGP2 were obtained.Further combine molecular weight detection,monosaccharide composition analysis,chromatography combined mass spectrometry,and nuclear magnetic resonance analysis to determine the difference in structure and composition of the two polysaccharides:the molecular weight of RGP1 was 31.4 k Da,it was mainly composed of five monosaccharides,and the molar ratio was galactose(Gal):3-O-methyl-galactose(3-O-Me-Gal):glucose(Glu):mannose(Man):fucose(Fuc)=25.77:8.37:2.51:1.52:1.41.RGP1 was a galactan composed of 1,6-α-Galp as the main chain,partially methylated at O-3 of→6)-α-Galp-(1→,and partially substituted at O-2 ofα-Fucp or a branched chain composed of→6)-α-Galp-(1→or→6)-α-O-Me-Galp-(1→withα-Fucp;The molecular weight of RGP2 was 11.8 k Da,it was mainly composed of four monosaccharides,and the molar ratio was Glc:Gal:Man:Glucuronic acid(Glc A)=30.62:3.43:1.90:1.41.RGP2 was a glucan composed of→6)-β-D-Glcp-(1→repeat connection or with→6)-α-D-Galp-(1→connection as the main chain,and existedβ-D-Glcp-(1→substitution at O-3 of→6)-β-D-Glcp-(1→.The results of structural analysis showed that there were significant differences in the structural composition between RGP1 and RGP2,which may lead to differences in their pharmacological activities.Next,screened the immunoregulatory activity of RGP1 and RGP2 in the immunocompromised mouse model constructed by 75 mg/kg cyclophosphamide.The results showed that both RGP1 and RGP2 decreased the increase of spleen index,relieved the expansion of germinal center of spleen nodules and the infiltration of neutrophils,restored the cytotoxicity of NK cells and the transformation ability of T lymphocytes in spleens.However,the results of cytokines detection showed that compared with RGP2,RGP1 significantly increased the level of immunoglobulin(Ig)A,Ig G,Ig M,interleukin(IL)-4,IL-6,interferonγ,tumor necrosis factorαin spleens.It was found that RGP1 had a better immunoregulation,therefore,the possible mechanism of RGP1 immunoregulation was further analyzed through intestinal flora and serum metabolomics.The results of intestinal flora analysis showed that RGP1significantly regulated the abundance of Lactobacillus,Faecalibacterium,Odoribacter and other genera between groups,and the metabolic pathway mediated by intestinal microorganisms was directly enriched in immune regulation.In addition,the results of metabolomics showed that RGP1 significantly changed the level of 20 metabolites in the model group,such as L-kynurenine,Uridine,Acetylcholine,and there was a significant correlation between the intestinal flora with significantly changed abundance and serum metabolites.Deeply exploring the mechanism,this study found that the immunoregulatory activity of RGP1 mainly lies in the influence of T cells differentiation.Furthermore,the hematopoietic dysfunction mouse model constructed with 100mg/kg cyclophosphamide was used to explore the hematopoietic activity and related mechanism of RGP1.The results showed that RGP1 alleviated the decrease of body weight and the increase of spleen index,and tissue lesions such as bone marrow vacuolation and sparse arrangement,increased the number of long-term hematopoietic stem cells,activated T cells and cluster of differentiation(CD)4~+T cells,regulated the level of peripheral blood cells in mice with hematopoietic dysfunction,but failed to affect the number of natural killer cells and B cells in bone marrow.These results preliminarily proved the improvement of RGP1 on hematopoietic function.Proteomics was used to further explore the mechanism,it was found that the pathways of significantly changed protein enrichment mainly included CD4 receptor binding,T cell activation and hematopoietic regulation,which was consistent with the previous results.The results of cytokine detection confirmed that RGP1 promoted the secretion of hematopoietic cytokines related to CD4~+T cells(IL-2,IL-3,IL-4,IL-5,IL-6,IL-10,and others),and these factors further activated the Janus kinase(JAK)/signal transduction and activator of transcription(STAT)3 signal pathway,further induced the expression of downstream target protein c-Myc.In conclusion,RGP1 promoted hematopoiesis by stimulating the differentiation of CD4~+T cells and activation of JAK/STAT3 pathway.In summary,the results of this study showed that:(1)Russula griseocarnosa was rich in many nutrients and active components,and its polysaccharide content was high,which can be used for the extraction and activity research of polysaccharide;(2)Russula griseocarnosa contained two main polysaccharide components,and their molecular weight and structural composition were quite different;(3)Compared with RGP2,RGP1 showed better immunoregulatory activity,and the related mechanism may be to regulate T cell differentiation;(4)RGP1 alleviated the hematopoietic dysfunction of mice caused by chemotherapy,and the related mechanism may be to promote the differentiation of CD4~+T cells and activate JAK/STAT3 pathway.The results of this study provide data support for the development of functional food or clinical drugs with the main component of Russula griseocarnosa polysaccharide,which may alleviate chemotherapy-induced hematopoietic dysfunction in clinical. |
