Immune Regulatory Functions Of Biologically Active Proteins From Cordyceps Militaris

The current research on active ingredients of edible mushrooms is mainly focused on the activity characterization and structural identification of polysaccharides.There are fewer studies on the structural analysis and immune activity of edible mushroom proteins,and there is a lack of comprehensive evaluation of the immunomodulatory activity of different edible mushroom proteins.In this study,the immunomodulatory effects of edible mushroom proteins were comprehensively evaluated through isolation and purification,activity screening,structural analysis,and activity evaluation,and the immunomodulatory proteins with good immunomodulatory activity were selected for further purification,activity evaluation,and structural characterization.The main results of this paper are as follows:1、Edible mushroom proteins have a variety of biological activities.Here,twenty-two edible mushrooms are selected,namely Ophiocordyceps sinensis,Cordyceps militaris,Agrocybe aegerita,Coriolus versicolor,Phellinus igniarius,Ganoderma lucidum,Grifola frondose,Morchella esculenta,Inonotus obliquus,Boletus,Tricholoma matsutake(S.Ito&S.Imai)Singer,Russula alutacea,Termitornyces albuminosus,Tuber melanosporum,Pleurotus eryngii,Flammulina velutipes,Volvariella volvacea,Hericium erinaceus,Pleurotus ostreatus,Lentinula edodes,Agaricus bisporus,Hypsizygus marmoreus.The edible mushroom proteins were extracted from thirteen rare edible mushrooms and nine common edible mushrooms and their purity reached more than 60%.We evaluated the immunomodulatory activity of the edible mushroom proteins using a macrophage immunomodulation model and analyzed their constituents.The results showed that all22 edible mushroom crude proteins increased the proliferation and phagocytosis of mouse macrophages and we found that these edible mushroom proteins had an effect on the secretion of reactive oxygen species and nitric oxide by mouse macrophages.Further studies on the secretion of cytokines from mouse macrophages showed a significant increase in pro-inflammatory cytokines,demonstrating that the edible mushroom proteins promoted the polarization of macrophages to the classical M1type macrophages,further demonstrating that edible mushroom proteins could enhance immunity.The immunomodulatory activity of rare edible mushroom proteins was found to be significantly higher than that of bulk edible mushroom proteins by homogenization of the modal length method,with the highest being C.militaris and the lowest being the H.marmoreus.Among the thirteen rare edible mushrooms,the maximum modal length|X||~2was 9.98±1.31 at a concentration of 200μg/m L for C.militaris protein,followed by C.versicolor,which at 200μg/m L.Therefore,the immunomodulatory activity of C.militaris proteins was significantly higher compared to other edible mushrooms.2、Through the evaluation of immunomodulatory activity of edible mushroom proteins,the highest immunomodulatory activity of C.militaris proteins was selected for further study.The crude protein of C.militaris was extracted by alkaline solubilization and acid precipitation,and purified by anion-exchange chromatography and dextran gel chromatography,and the RAW 264.7 immunomodulatory model was established to evaluate the activity of different fractions,and a fraction with the best immunomodulatory activity CMP 2b was isolated.The relative molecular mass was determined by matrix-assisted laser desorption ionization mass spectrometry technique,and the relative molecular mass was 22460 Da.3、By establishing a macrophage immunomodulation model,it was found that CMP 2b significantly increased the proliferation and phagocytosis of macrophages,with the highest cell proliferation rate reaching 152.91±5.55%and phagocytosis increasing by 23.34%of the blank control group;and promoted the secretion of NO,ROS,IL-1β,IL-6,IFN-γand TNF-αby macrophages.Among them,NO secretion level reached 2.31 times of that of the blank control group,ROS secretion level reached 82.33%of that of the positive control LPS,and TNF-α,IL-1β,IFN-γand IL-6 secretion were 5.51,17.42,4.18 and 26.75 times of that of the blank control group,respectively,which significantly enhanced the immunomodulatory ability of macrophages.Pretreatment with anti-SR,anti-NLRP1,anti-NLRP3,anti-NLRC4,anti-TLR1,anti-TLR2 and anti-TLR4 before stimulation with CMP 2b.RAW 264.7indicates that TLR1 and NLRP3 are membrane receptors for CMP 2b recognition by mouse macrophages.