| Isaria cicadae Miquel,as an edible and medicinal chinese medicine with thousands of years in folk,was widely distributed in province of Jiangsu,Zhejiang and Southwest of China.Because of its wide biological activities and abundant resources,it was of great significance to develop and utilize the Isaria cicadae Miquel rationally.Polysaccharide was main active constituent of this medicinal material.Although there have been some reports about the characterization and immunomodulatory activity of polysaccharide extracted from sari a cicadae Miquel,it was still not systematic and comprehensive enough.In our previous study,a homogeneous polysaccharide named as JCH-1 was separated from Isaria cicadae Miquel.In this paper,RAW264.7,a mouse macrophage,was used as a target cell to evaluate its immunomodulatory activity and the immunomodulatory mechanism of JCH-1 was further researched.Besides that,the characterization of JCH-1 was aimed to clarify.Our research aims to lay the foundation for the development of medicinal resources and health products of entomogenous fungi such as Isaria cicadae Miquel.Objective:1.The contents of total sugar,protein,sulfate group,glucuronic acid and monosaccharide composition of JCH-1 were determined to elucidate its characterization.2.The effects of JCH-1 on the morphology,proliferation,phagocytosis and the release amount of NO and cytokines(TNF-a and IL-6)of RAW264.7 were explored to evaluate its immunoregulatory activity.3.The effects of JCH-1 on the expression of proteins related to TLR4-MAPK-NF-?B signal pathway were measured to explore its immunomodulatory mechanism.Methods:1.Characterization of JCH-1The contents of total sugar,protein,glucuronic acid and sulfate group of JCH-1 were determined by the methods of phenol-sulfuric acid,coomassie brilliant blue,sulfuric acid-carbazole and barium chloride-gelatin respectively.The monosaccharide composition of JCH-1 were determined by sucronitrile ester derivatization.The sample was scanned and analyzed by ultraviolet and infrared spectroscopy.2.Immunomodulatory activity of JCH-1 on RAW264.7To explore the influence of JCH-1 on RAW264.7,the morphological changes of RAW264.7 were observed and photographed,the proliferation of cells was detected by MTT method,and the phagocytosis was detected by neutral red method.Besides that,the levels of NO,TNF-a and IL-6 were measured by Griess or ELISA method.3.Immunomodulatory mechanism of JCH-1 on RAW264.7.The method of RT-PCR was used to measure the mRNA expression of iNOS,TNF-a and IL-6 before and after treating RAW264.7 with different concentrations of JCH-1;The RAW264.7 was pretreated with inhibitors of TLR4(C34),MAPK(SP600125?PD98059 and SB203580)and NF-K B(Bayll-7082)signal pathway for some minutes respectively,then the intervention of JCH-1 was performance for 24 h.After that,the contains of NO,TNF-?and IL-6 were measured;The effects of JCH-1 on the expression of proteins related to TLR4-MAPK-NF-K B signal pathway were measured by the method of Western Blotting;The method of Western Blotting was used to detected the influence of inhibitors of TLR4(C34),MAPK(SP600125?PD98059 and SB203580)and NF-K B(Bayll-7082)signal pathway on the the expression of proteins about TLR4-MAPK-NF-?B signal pathway activated by JCH-1.Results:1.The content of total sugar,glucuronic acid and sulfate group in JCH-1 are 90.15%,1.15%and 8.65%respectively.But the protein in JCH-1 was not detected.JCH-1 was consisted of glucose,mannose and galactose,and the molar ratio of them were detected as 1.70:1.37:1.00.2.JCH-1 could activate the function of RAW264.7 and make them change from resting state to active state.Beside that,JCH-1 could promote the proliferation of RAW264.7 and enhance its phagocytosis ability.In addition,JCH-1 could promote the secretion of NO,TNF-a and IL-6 of RAW264.7 in a dose-dependent manner.3.The mRNA expression level of iNOS,TNF-a and IL-6 in RAW264.7 were up-regulated after being treated with JCH-1.4.After being pretreated with inhibitors of TLR4(C34),MAPK(SP600125?PD98059 and SB203580)and NF-K B(Bayll-7082)signal pathway,the ability of RAW264.7 to secrete NO,TNF-? and IL-6 promoted by JCH-1 were decreased.5.JCH-1 could improve phosphorylation and nuclear entry of P65,promote phosphorylation and degradation of I?B-?.Besides that,JCH-1 could increased phosphorylation level of P38,JNK and ERK.6.The ability of JCH-1 to improve phosphorylation and nuclear entry of P65,promote phosphorylation and degradation of I?B-? and increased phosphorylation level of P38,JNK and ERK were suppressed after the RAW264.7 was pretreated with inhibitor of TLR4(C34).Besides that,the ability of JCH-1 to improve phosphorylation and nuclear entry of P65 and promote phosphorylation and degradation of I?B-? were decreased after the RAW264.7 was pretreated with inhibitorsof MAPK(SP600125?PD98059 and SB203580)signal pathway,However,the inhibitor of NF-?B(Bayll-7082)have no influences on the ability of JCH-1 to increased phosphorylation level of P38,JNK and ERK.Conclusion:1.The content of total sugar,glucuronic acid and sulfate group in JCH-1 were 90.15%,1.15%and 8.65%respectively.There was no protein in JCH-1 detected.JCH-1 was consisted of glucose,mannose and galactose,and the molar ratio of them were detected as 1.70:1.37:1.00.2.JCH-1 could enhance the proliferation and phagocytosis of macrophages,promote the release of NO and cytokines(TNF-a,IL-6),thus improve the immune function of organisms.3.JCH-1 could activate TLR4-MAPK-NF-?B signaling pathway of macrophages,thereby improving the immune function of organism. |
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