Metabolites,immunomodulatory Effects And Mechanism Of Tolypocladium Inflatum F 56,a Fungus Isolated From Cordyceps Sinensis

Objective:Tolypocladium inflatum F 56(TIF 56)is a medicinal fungus isolated from Cordyceps.Preliminary studies have found that the fungal extracts of this genus have similar components as Cordyceps sinensis,and have definite activities in immune regulation,antiradiation damage,as well as anti-tumor,and promises good development prospects.So far,research on the main component,pharmacological activity and mechanism of action concerning TIF 56 fermented mycelium is not sufficient,which limits the development and utilization of this medicinal fungus.Our investigaion focuses on this fungus TIF56 as the research object,analyzes its chemical components,and at the same time conducts experimental research on its anti-immune damage effects and immune response molecular mechanism,so as to provide evidence for in-depth development of Cordyceps alternative resources.Research Methods and Results:1.Component analysisUPLC-QTOF-MS/MS method was used to qualitatively analyze the effective components of TIF 56 fermented Cordyceps mycelia.Results revealed that TIF 56 contained 11 types of nucleotides,17 types of amino acids,11 types of cyclic peptides,6 types of monosaccharides and oligosaccharides,as well as 20 types of fatty acids,alkanes,esters and more than 20 types of other components.Using chromatographic methods to separate chemical components,this thesis obtained 18 kinds of small molecules,among which cyclic peptides were reported for the first time in this species.2.Protective effects of TIF 56 on immune lesionExperimental study was conducted on the repair effects against immune injury.Whole body irradiation mice with 8 Gy dosage of 60 Co y-ray were used as a classic immune injury model.The anti-injury effects of TIE(with a dosage of 100,200,400 mg/kg)were studied on the whole body animal,including the main immune organs(spleen and thymus),reproductive organs(testis),digestive organs(small intestine),and histopathological,morphological and hematological effects were evaluated as well.The results showed that TIE could improve the spleen index,peripheral blood and sperm quality of the irradiated mice.Histopathological evaluation indicated that TIE could effectively protect the hematopoietic function,lymphocyte regeneration,complete morphology of testicular tissue and intestinal mucosal barrier of mice,and consequently could produce broad-spectrum antiradiation effects on hematopoiesis,immunity,digestive tract and reproductive system.Our study demonstrated that it could achieve good immune regulation and repair effects on immune lesion induced by high dose radiation.Chemokines were measured using primary mouse macrophages.The results showed that the chemokines secreted by macrophages after TIE treatment include C5/C5a,CCL11,CXCL1,CCL2,CCL17,TIMP-1,TNF-α,significantly improved;this chemokine combination can significantly promote the phosphorylation level of ERK1/2 in endothelial cells;long-term histological and hematological results show that doses as high as 200 mg/kg have significant effects on mouse tissue and blood,indicating no significant impact.This shows that it has good immune dysfunction modulation and repair effect on animal radiation-induced tissue damage and shows good biological safety.3.Immune regulation mechanism of TIE3.1 Proteomic study of TIE on immune responsePrimary mouse peritoneal macrophages without polarization were treated with TIE(witha dosage of 1 mg/mL)in vitro.Proteomic techniques were used to analyze the protein expression levels of TIE-treatment murine macrophages.By DSEq2 method,as compared with those of the control group,132 up-regulated proteins and 63 down-regulated proteins were identified,of which the up-regulation levels of the inflammatory response-related proteins IFIT1,IFIH1 and RSAD2 were significantly regulated.The results of STRING protein-protein interaction network analyses showed that the 10 key genes,such as ISG15,RSAD2,IFIH1,IFIT1,OASL,STAT1,DDX58,IFI35 and OAS3 were in the key position in response to TIE treatment.GO and Metascape signaling pathway enrichment analyses indicated that TIE treatment mainly promoted immune response-related signaling pathways,such as innate immune response.GSEA results showed that TIE intervention activated RIGⅠ signaling pathway and STING-cGAS signaling pathway in mouse macrophages.3.2 The molecular mechanism of TIE and its polysaccharide component TIP in immune regulationUndifferentiated RAW264.7 cells were used as research carriers to construct lipopolysaccharide LPS and interleukin IL-4-induced inflammation model.CCK8 assay showed that TIE had few effect on the proliferation of mouse macrophage RAW264.7.RTPCR experiments demonstrated that TIE and TIP could regulate the mRNA expression levels of key chemokines in IL-4-induced M1 and LPS-induced M2 macrophages.In LPS-induced M1 pro-inflammatory polarization state,TIE and TIP could significantly inhibit the expression levels of INF-α,INF-β,TNF-α,IL-6,ISG15 and other genes,suggesting that TIE and TIP regulated the expression levels of macrophage-related factors in pro-inflammatory polarization state,but differences existed in the two.ELISA assay was used to explore the effects of TIE on the secretion of different chemokines by M1 and M2 macrophages.Finally,the WB method was used to verify that TIE treatment significantly up-regulated the expression and phosphorylation levels of related proteins in the cGAS-STING-TBK-IRF3 signaling pathway in macrophages RAW264.7 with TIE treatment.These results show that TIE and TIP play an important role in the regulation of M1 or M2 macrophages.3.3 Application of TIE in rescuing nervous system inflammation caused by LPSThe CCK8 experiment was used to analyze the effects of various concentrations of TIE on the proliferation of mouse microglia BV2,varifyin the biological safety of TIE;RT-PCR,NO kits and other methods were used to examine that TIE inhibits the release of proinflammatory mediator factors in BV2 cells caused by LPS;using transcriptome sequencing and bioinformatics analysis,it is speculated that TIE may respond to external cellular stress through the mitochondrial energy metabolism pathway;using the Seahorse Mitochondrial Stress Kit,it is clear that TIE can modulating the oxygen consumption rate of LPS-induced BV2 cells;using cell transmission electron microscopy,it is clear that TIE treatment has no effect on the mitochondrial morphology of BV2 cells;through metabolome analysis,it is inferred that TIE may promote energy by affecting the β-oxidation of long-chain fatty acids in BV2 cells Metabolism;using the LPS-induced nervous system inflammation model,it was verified that TIE can rescue the LPS-induced nervous system inflammatory response and neural activity at the animal level.ConclusionIn summary,this thesis first performed the biological identification of Cordyceps fungus TIF 56,analyzed the chemical components of its extracts,and then studied its multiorgan protective effects on high-dose radiation-damaged animals,indicating that it displays good immune protection.Then,proteomics analyses showed that TIE intervention triggered immune response by activating RIG-I signaling pathway and STING-cGAS signaling pathway in mouse macrophages.In addition,the expression levels of related genes,such as the levels of various extracellular factors and the expression levels of key transcription factors in the signaling pathway were verified in the polarized state of macrophages treated with TIE.Finally,the network pharmacology technology and proteomics technology were used to study the interaction network of cyclic peptide chemical components-disease targets.In general,TIF 56 extracts displayed the potential to regulate the immune status of organisms in both directions.Its polysaccharide component TIP and cyclic peptide components may play a synergistic role in integration,consequently it has a similar role of "correction and righting" as Cordyceps.This study provides a scientific evidence for furher development and utilization of Cordyceps medicinal fungi.