| Cordyceps militaris is a traditional Chinese medicinal fungus with natural medicinal value and nutritional value.Cordyceps militaris has a significant role in regulating the body’s immunity.One of its main active ingredients is Cordyceps militaris polysaccharides(CMP).Studies have shown that Cordyceps militaris polysaccharides have anti-inflammatory,antioxidant,anti-tumor,hypoglycemic and other activities.At present,CMP is mainly extracted from the fruiting bodies of Cordyceps militaris,which is limited,and the extraction rate of existing methods is not high.As the residue of the medium after the cultivation of edible fungi,the spent mushroom substrate also contains a large amount of mycelium components produced during the cultivation of edible fungi.Usually,after the cultivation of edible fungi,the spent mushroom substrate is used as organic fertilizer or feed,resulting in the waste of the residual mycelium.Therefore,in this study,Cordyceps militaris fungus chaff was used as raw material to extract Cordyceps militaris crude polysaccharides,and the response surface optimization experiment was carried out to obtain the best extraction technology of it.Based on the fact that CMP has many pharmacological activities such as anti-inflammatory and improving immune ability,an in vitro cellular anti-inflammatory model was established by using mouse mononuclear macrophage RAW264.7 as experimental material.The effect of CMP on macrophage polarization was detected by adding exogenous CMP,and the anti-inflammatory activity was studied.The mechanism of macrophage polarization induced by CMP was analyzed from the point of view of LncRNAs.The main results are as follows.1.Cordyceps militaris crude polysaccharides was extracted by water extraction and alcohol precipitation method from Cordyceps militaris bran.The extraction conditions of Cordyceps militaris crude polysaccharides were optimized.The extraction rate was 13.395 in this condition: the temperature was 55.01 ℃,the ratio of liquid to material was 28.2,and the extraction time was 0.5005 h.Through the analysis of the components of Cordyceps militaris crude polysaccharides by liquid chromatography,it is proved that the crude polysaccharides contain the main components of CMP such as glucose,mannose and galactose,as well as ribose and other components,which are more complex.2.3.13mg/mL was used as the best concentration to stimulate mouse mononuclear macrophag.The gene expression levels of M1 polarization markers TNF-α,IL-6,iNOS and IL-12,and M2 polarization markers MR and FIZZL were detected after stimulation.ELISA method was used to detect the changes of inflammatory cytokines TNF-α and IL-12 secreted into the culture medium after stimulation.The results showed that the stimulation of CMP could promote the M1 polarization of macrophages,and the polarization process activated the activation of NF-κB pathway involved in inflammatory immune response.3.More than 100 differentially expressed LncRNAs were screened by RNA-seq,and four differentially expressed LncRNAs(MALAT1,GAS5,H19 and HOTAIR)were detected by RT-qPCR.The RNA-seq results were verified,indicating that LncRNAs are involved in the regulation of M1 polarization of macrophages stimulated by CMP.In this study,the extraction method of Cordyceps militaris crude polysaccharides was optimized,which provided a technical support for making full use of edible fungus resources and reducing waste,and was expected to provide reference for the extraction of polysaccharides from other edible fungi.Through cell experiments and RNA-seq techniques,the mechanism of CMP stimulating macrophage polarization was preliminarily revealed.To provide a theoretical basis for further understanding the regulation of immunity by CMP.However,there is a lack of research on the receptor of macrophage response to CMP and the signal pathway regulated by differentially expressed LncRNAs.These deficiencies are expected to be discussed in the follow-up experiments. |
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