| Sarcodon imbricatus?SI?,belonging to the family of Basidiomycotina and Aphyllophorales,is an edible and medicinal fungus with rich nutritional value and various pharmacological activities,widely distributed in Central Europe,North America and Asia.Large-scale artificial cultivation has been achieved in Sichuan,Yunnan,Tibet,Gansu and Xinjiang with the highest yield.SI has been used for thousands of years as a traditional medicine and healthy food.Traditional Chinese medicine pointed that it has the functions of chasing wind and dispelling cold,relaxing tendons and activating collaterals,lowering blood lipids,detoxifying qi,curing wind and breaking blood.Modern medicine indicated that SI,an ideal health food,has antitumor,detoxification and relieving hepatocirrhosis effection.Research about SI at home and aboard was not very extensive,mainly focusing on chemical composition,polysaccharide structure and fungi liquid fermentation culture optimization,antibacterial and antitumor activity,lacking of in-depth explore on pharmacological effects and mechanisms.Recently,immunoregulation and hematopoietic activity of SI have not been reported.In the present study,activity ingredient composition and content of SI dried products were systematically analyzed.On this basis,taking Sarcodon imbricatus polysaccharides?SIPS?as the research object,to explore the immunomodulation and hematopoietic recovery ability using cell and molecular biology techniques,combined with in vivo and in vitro experiments,and the corresponding pharmacological mechanisms of SIPS were preliminarily elucidated.Conclusions were summarized as follows:The total sugar content of SI accounted for 35.22%of dry weight,followed by crude fiber content 23.28%,total protein 18.33%,mannitol content 9.41%,total ash content 9.30%,total sterol content 3.16%,Vitamins 0.44%and fat accounted for only3.02%,total flavonoids(2.05×10-2%),total polyphenols?0.41%?and total triterpenes(4.12×10-2%)were less.Among the mineral elements,potassium was the highest element,about 39.57×10-2 mg/100 g dry weight,followed by iron?78.42 mg/100 g dry weight?and calcium?68.04 mg/100 g dry weight?,and heavy metals were not detected.Additionly,SI was composed of 17 amino acids?12.44%?and 24 fatty acids,including 8 essential amino acids,accounting for 4.35%of the total amino acids.In this paper,crude polysaccharides were isolated from SI by decolorization,hot water extraction,protein removal and alcohol precipitation,and fractional purification by DEAE-52 ion exchange column chromatography to obtain SIPS with purity of86.8%.High performance liquid chromatography?HPLC?showed that SIPS was a heterogeneous polysaccharide with molecular weight from 56331.8 to 93963.6 Da.Fourier infrared spectroscopy?FT-IR?showed that SIPS has O-H stretching vibration absorption peak at 3365.8 cm-1,C-H stretching vibration at 2922.2 cm-1,stronger sugar ring absorption peak at 1597.1 cm-1,C-H angular vibration absorption peak at1398.4 cm-1,C-O bending vibration at 1028.4 cm-1 caused by C-O-H and C-O-C stretching,and three weak absorption peaks between 1200 and 1000 cm-1 indicated the presence of pyranoid ring skeleton structure,which were typical characteristics of polysaccharidesWe investigated the effect of SIPS on the immune system function in cyclophosphamide?CTX?-induced immunosuppression mice.After 28 days of SIPS administration,body weight,spleen/thymus index,NK cell cytotoxicity,T lymphocyte transformation capacity and immune-related cytokines in CTX-induced immunosuppression mice were detected,and the expression of Nrf2 and downstream antioxidant enzymes were analyzed to interrogate the molecular mechanisms that SIPS exerted immunoregulation.The results demonstrated that body weight and spleen index returned to normal,NK cell cytotoxicity and lymphocyte transformation capacity were increased significantly compared with the model group.Moreover,SIPS promoted the secretion of immunoglobulin A?IgA?,IgG,IgM,IL-2,IL-6,IL-10,IL-12 and IFN-gamma?IFN-??in serum and spleen of immunosuppression mice.Notably,SIPS could reduce the accumulation of reactive oxygen species?ROS?in the spleen and thymus of immunosuppression mice,increase the total antioxidant capacity?SOD,GSH-Px and CAT?,and promote the synthesis of acid phosphatase?ACP?and lysozyme?LZM?in the thymus.Histopathological analysis of spleen revealed the protective effect of SIPS against CTX-induced immunosuppression.Western-blot showed that SIPS upregulated the expression of nuclear factor-erythroid 2-related factor 2?Nrf2?and its downstream target genes encoding antioxidant enzymes:SOD1,SOD2,haem oxygenase-1?HO-1?,CAT and quinone oxidoreductase 1?NQO1?,indicated that SIPS reversed CTX-induced immunosuppression might through Nrf2-mediated oxidative stress pathway,accompany with potential antioxidant activity.In order to explore the hematopoietic function of SIPS,effects of SIPS on cell viability,differentiation and apoptosis activity of two hematopoietic cell?K562 and CHRF?were investigated by XTT method,benzidine staining and flow cytometry?FCM?in vitro.The results showed that SIPS significantly increased hematopoietic cells proliferation,promoted K562 differentiation into erythroid and megakaryocyte lines,and upregulated the expression of RSK1p90,c-Myc and ELK1 in both hematopoietic cells,and without cytotoxicity,suggesting that SIPS promoted the proliferation and differentiation of hematopoietic cells might via activating the expression of transcription factors.Subsequently,we explored the effect of SIPS on bone marrow hematopoietic system function,and the underlying mechanisms of SIPS reconstructing hematopoietic function was revealed in CTX-induced myelosuppression mice model in vivo.After 28 days of administration,SIPS enhanced the body weight and thymus index,alleviated enlargement of the spleen and liver,and contributed to the recovery of lymphocyte?LY?,neutrophil?NE?and hemoglobin?HGB?in peripheral blood to normal levels.Moreover,SIPS improved the morphological structure of femur and organs,promoted the production of B lymphocyte and the proliferation capability of hematopoietic stem/progenitor cells?HSCs/HPCs?significantly in bone marrow.The results of antibody chip screening and ELISA validation analysis showed that SIPS significantly increased the levels of IL-2,IL-3,IL-4,IL-5,IL-6,granulocyte colony-stimulating factor?G-CSF?and macrophage colony-stimulating factor?M-CSF?in serum and spleen of myelosuppression mice,and inhibited the secretion of TNF-alpha?TNF-??,IFN-?,C-C motif chemokine-1?CCL1?and monocyte chemotactic protein-1?MCP1?.Meanwhile,SIPS upregulated the protein expression levels of G-CSF,granulocyte colony stimulating factor receptor?G-CSFR?,M-CSF,phosphotyrosine kinase?JAK2?and phosphorylation signal transduction and transcription activator?STAT3?in the spleen of myelosuppression mice,and the results were further confirmed in CHRF,K562,and bone marrow mononuclear cells?BMMNCs?cultured in vitro,suggesting that JAK2/STAT3 signal transduction pathway regulated by G-CSF might be the main mechanism of SIPS promoting hematopoietic function repair in mice with chemotherapy-induced injury.In conclusion,the above experimental results manifested that:?1?SIPS can significantly promote the immune function of CTX-induced immunosuppression mice,and its mechanism might be related to Nrf2-mediated oxidative stress pathway;?2?SIPS can significantly enhance the hematopoietic function of CTX-induced myelosuppression mice,and its mechanism might be related to G-CSF-mediated JAK2/STAT3 pathway.The proposal of this research topic will help to promote the transformation of SI from health food to biomedical field.And will also provide new ideas and new directions for screening and developing candidate drugs that can alleviate bone marrow immunosuppression induced by long-term chemotherapy. |
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