| A variety of glycoproteins with abundant functions are widely distributed in biological bodies. Studies have shown that sugar chain plays an important role in stabilizing the tertiary structure of proteins, preventing protein hydrolysis, and enhancing immune adjustment, etc. It is also beneficial to cells recognition, tumor cells inhibiting, anti-oxidation, anti-aging etc. In addition, it has broad prospects in developing new special drugs and functional food. In recent years, much scientific research has been investigated in glycoprotein pharmacological effects and health care system, which has achieved a series of progress.Cordyceps(Cordyceps sinensis), known as one of the three treasures of traditional Chinese medicine, is a traditional tonic luxury Chinese herbe, which has important effects in regulating the immune system, anti-tumor, anti-fatigue, etc. With the development of modern medicine and popularity of traditional Chinese medicine in the world, more and more studies confirm that Cordyceps has vatriant immune activity. However, few reports have focused on the immune regulation systematically.Hirsutella sinensis, isolated from natural Cordyceps, is one of the Cordyceps sinensis, which has similar biological functions with Cordyceps. Hirsutella sinensis can be substitution of Cordyceps in case of lack in natural resources. This study contains three parts:separation and purification, structural analysis, and immune adjustment function of a novel extracellular glycoprotein (HS002-Ⅱ) from Hirsutella sinensis.First, we got the purified glycoprotein component of HS002-Ⅱfrom the fermentation products of Hirsutella sinensis by using DEAE and Sephacryl column chromatography.We found that the relative molecular weight of HS002-Ⅱis 44KDa by HPLC method, and the relative protein content is 42.1%, sugar content is 57.9%.Then its structure was analyzed by HPLC and IR method. We found that HS002-Ⅱcontains D-mannose, L-rhamnose, D-glucuronic acid, D-glucose, D-xylose and L-arabinose; HS002-Ⅱcontains Asp, Thr, Ser, Glu, Gly, Ala and other 10 amino acids.Finally, we investigated the effects of HS002-Ⅱon the macrophage functions and its related mechanisms. We selected the marine macrophage cell line RAW264.7 as cellular model to observe it. The effects of HS002-Ⅱon cell proliferation, NO release and cytokine production of RAW264.7 cell were inspected by MTT, Griess reagent, ELISA, respectively. Results showed that HS002-Ⅱsignificantly promoted the proliferation of RAW264.7 cell in the concentration of 50μg/ml. In addition, HS002-Ⅱremarkably increased both the secretion of NO and the production of cytokines TNF-a in RAW264.7 in the concentration-dependent manner. The mRNA expression of inflammatory factors and their signaling molecules in RAW264.7 cell were also analyzed by reverse transcription polymerase chain reaction (RT-PCR). HS002-Ⅱremarkably up-regulated the mRNA expression levels of inflammatory factors and NF-κB. Furthermore, the nuclear transcription factor NF-κB activation in RAW264.7 cells was analyzed by western-blotting. Results indicated that HS002-Ⅱdramatically reduced the the level of IκB-αand NF-κB p65 in cytoplasm of RAW264.7 cells, and significantly improved the level of NF-κB p65 in nucleus of RAW264.7 cells as well. In conclusion, this reasearch demonstrated that HS002-Ⅱpromoted macrophage function and activation of NF-κB.In the present work, the glycoprotein HS002-Ⅱof Hirsutella sinensis was extracted and purified. The structure and immunomodulation function of HS002-Ⅱwas then investigated. It provided a basis for further study of glycoproteins from Hirsutella Sinensis. |
PDF Full Text Request