Screening Of The Active Parts Of Cicada Chinensis Immunomodulatory And Preliminary Study On Its Mechanism Of Action

Cordyceps cicadae Shing is a dry complex formed by C.cicadae Shing fungus parasitic on Cicada flammata Dist larvae.It is sweet in taste and cold in nature,and it is considered non-toxic.According to traditional Chinese medicine theory,its drug action is attributed to the lung and liver channels of the human body.It has the effects of evacuating wind and heat,calming shock and relieving spasmodic.Modern studies have shown that C.cicadae Shing contains polysaccharides,nucleosides,ergosterols,cordyceps acid and other chemical components,which have immunomodulative effect,antioxidant effect,anti-aging effect,anti-tumor effect and other pharmacological effects.Previously,our research group conducted a preliminary study on the pharmacological activities of C.cicadae Shing,and found that adenosine and n-butanol extracts from C.cicadae Shing could protect PC12 cells from glutamate-induced oxidative damage.Polysaccharides from C.cicadae Shing could prolong the lifespan of Drosophila under normal physiological condition and hydrogen peroxide stress condition.Polysaccharides from C.cicadae Shing CPA-1 and CPB-1 could promote the expression of dendritic cell surface molecules and induce dendritic cell maturation;but there was a lack of systematic research on the immunomodulatory activity of C.cicadae Shing.Therefore,RAW264.7 macrophage model was used to screen out the better part with immunomodulatory activity of C.cicadae Shing,the immunomodulatory activity of the better part on RAW264.7 cells was further studied,and the mechanism of the immunomodulatory activity was also preliminarily explored in this paper,which will promote the development of C.cicadae Shing medicinal resources,at the same time provide a reference for the development of new immunomodulatory drugs.Main research contents and results are as follows:Screening of the better part with immunomodulatory activity of C.cicadae Shing.Using RAW264.7 macrophage model,effects of total water extracts and different solvent extracts of C.cicadae Shing on proliferation,phagocytosis and cytokine NO secretion of RAW264.7 cells were compared,and the better part with immunomodulatory activity of C.cicadae Shing was screened out.The results showed that when concentrations were 25 50 100200 μg/m L,proliferation activities of RAW264.7 cells were increased after polysaccharides and supernatant extracts treatments.Phagocytic activities of RAW264.7 cells were increased after polysaccharides and residual water extracts treatments,but the effect of polysaccharides was better than that of residual water extracts.In addition,NO secretion of RAW264.7 cells was promoted after total water extracts and different solvent extracts of C.cicadae Shing treatments,and the effect of polysaccharides from C.cicadae Shing was better than that of other extracts.Therefore,polysaccharides from C.cicadae Shing were selected as the better part of C.cicadae Shing with immunomodulatory activity for further study.Study on the immunomodulatory activity of polysaccharides from C.cicadae Shing on macrophages.Proliferation activity of RAW264.7 cells was detected by MTT assay,phagocytosis activity of RAW264.7 cells was detected by neutral red assay,content of NO was determined by Griess assay,and the contents of TNF-α,IL-1β and IL-6 cytokines were determined by ELISA kits.The m RNA expression levels of i NOS,TNF-α,IL-1β and IL-6 genes were detected by RT-PCR,and the immunomodulatory activity of polysaccharides from C.cicadae Shing was further studied.The results showed that polysaccharides from C.cicadae Shing could promote proliferation of RAW264.7 cells and improve phagocytic activities of RAW264.7 cells in the concentration range of 3.125-400 μg/m L.Polysaccharides from C.cicadae Shing could change morphology of RAW264.7 cells in normal resting state,make cell volume larger,and some cells grow pseudopods.Polysaccharides from C.cicadae Shing could induce secretion of NO,TNF-α,IL-1β,IL-6 cytokines in RAW264.7 cells,and could also up-regulate the m RNA expression of i NOS,TNF-α,IL-1β,IL-6 genes.Study on the mechanism of polysaccharides from C.cicadae Shing on immunomodulatory activity of macrophages.Dectin-1 receptor inhibitor laminarin pretreated RAW264.7 cells,and then intervened with polysaccharides from C.cicadae Shing.The supernatant was taken,the content of NO in the supernatant was determined by Griess method,and the contents of TNF-α and IL-6 cytokines in the supernatant were determined by ELISA kits.The m RNA expression levels of i NOS,TNF-α and IL-6 genes were detected by RT-PCR.Protein-immunoimprinting assay was used to detect the effects of polysaccharides from C.cicadae Shing on Dectin-1/Syk/NF-κB signaling pathway related proteins in RAW264.7 cells.The results showed that compared with the single polysaccharide treatment group,the release of NO,TNF-α and IL-6 cytokines in the inhibitor treatment group was decreased,and the m RNA expression levels of the related genes i NOS,TNF-α and IL-6 were also down-regulated.After the cells were pretreated with Dectin-1 receptor inhibitors,the effects of polysaccharides from C.cicadae Shing promoted the expression of Syk kinase,the phosphorylation of IKKα/β protein in NF-κB pathway,and the degradation of IκBα protein in NF-κB pathway were weakened.Finally,it was also found that after treatment of RAW264.7 cells with polysaccharides from C.cicadae Shing,p65 subunits in the cells had nuclear translocation and the NF-κB signaling pathway was activated.These results suggest that polysaccharides from C.cicadae Shing could regulate the immunomodulatory activity of RAW264.7 cells through the downstream Syk/NF-κB signaling pathway mediated by Dectin-1 receptor on cell surface.