Researches On The Antitumor Mechanisms Of Active Components From Cordyceps Cicadae And Artificial Cultivation With Bombyx Mori Pupa As A Substitute Host

Cordyceps cicadae,a kind of traditional Chinese medicine,is composed of dried Cicada flammata larvae that have died and stiffened from infection with Paecilomyces cicadas.It is rich in protein,amino acid,cellulose,fatty acid,vitamins and trace elements.And its bioactive components mainly include nucleoside,polysaccharide,ergosterol and its peroxide,wormholic acid,polyglobin.C.cicadae exerts anti-tumor,immunomodulation,nerve protection,improvement of renal function,hypoglycemia,and antibacterial effects.As an important pharmacological action,the antitumor function of C.cicadae is lack of systematic research.At the same time,the natural resources of C.cicadae is exhausted,it can not meet the market demand.Based on the above issues,we isolated and identified the active components from the C.cicadae,and explored their antitumor effects and mechanisms in this paper.1.MTT assay was used to detect the antitumor effects of WEC(water extract of C.cicadae)and EEC(ethanolic extract of C.cicadae).It was found that EEC had stronger antitumor effect than WEC.And EEC had cytotoxicity to SGC-7901,H1 299,HeLa,HepG2 and A549 cells at high concentration,and EEC has optimal antitumor activity against SGC-7901 cells.2.The antitumor effect of EEC were studied by cell apoptosis detection,cell cycle detection,MMP detection,intracellular Ca2+level and western blotting.They showed that EEC could induce SGC-7901 cell apoptosis,S phase arrest,MMP depolarization and Ca2+overload,which may be the effective ways for EEC to inhibit the growth of SGC-7901 cells.In addition,EEC can regulate the expression levels of apoptosis,cell cycle and endoplasmic reticulum(ER)stress related proteins in SGC-7901 cells.EEC inhibited the proliferation of SGC-7901 cells by inducing caspase-dependent apoptosis,arresting the cell cycle in the S phase and increasing ER stress.This study revealed that C.cicadae is a potential natural source of anticancer drugs3.The active components in EEC were determined by phenol-sulfuric acid assay,BCA protein assay kit and HPLC(High Performance Liquid Chromatography,高效液相色谱)assay.The results of phenol-sulfuric acid assay showed that the total sugar content of EEC was 211.6 μg/mg.The results of BCA assay showed that the protein content of EEC was 336.5 μg/mg.The results of the HPLC assay suggest that EEC contained 1.841± 0.007,6.015 ±0.018,1.774± 0.030 and 5.388 ± 0.019 μg/mg adenine,uridine,adenosine and N6-(2-Hydroxyethyl)-adenosine,respectively.MTT assay showed that adenine,uridine,adenosine and N6-(2-hydroxyethyl)-adenosine(HEA)inhibited the growth of SGC-7901 cells,and HEA had optimal antitumor activity.4.HEA is a natural adenosine derivative with a variety of biological and pharmacological activities.The antitumor effect of HEA and its mechanisms were studied by CCK-8 assay,cell apoptosis detection,cell cycle detection,reactive oxygen species(ROS)detection,MMP detection,intracellular Ca2+level and western blotting.The results showed that HEA exerted cytotoxic effects against gastric carcinoma cells(SGC-7901 and AGS).Additionally,HEA induced ROS production and MMP depolarization,triggered caspase-dependent apoptosis,promoted intracellular Ca2+-related ER stress and autophagy.This study demonstrated that HEA is a potential chemotherapeutic agent for gastric carcinoma.5.Gene expression profiling showed that HEA treatment induced differentially expressed genes(DEGs)of SGC-7901 cells,which were involved in apoptosis,cell cycle arrest,ER stress,and autophagy.GO analysis and KEGG pathway analysis showed that the up-regulated or down-regulated genes of HEA were mainly involved in a variety of signaling pathways of cancer occurrence and development.6.In order to study the antitumor effect and mechanism of HEA in vivo,subcutaneous xenograft models of SGC-7901 cells were established.HEA could inhibit the growth of transplanted tumors,cause tumor tissues cells apoptosis,and achieve antitumor effect in vivo.7.Paecilomyces cicadae was isolated from the C.cicadae by tissue separation method.The strain was inoculated into Bombyx mori pupa by spraying.By simulating the ecological environment conditions of natural C.cicadae,"Jincanhua" was cultivated,and the suitable artificial cultivation conditions were explored.In this paper,we studied the antitumor effect and mechanism of EEC,and we found that HEA is one of the main effective components of EEC.Therefore,we further explored the antitumor effect and mechanism of HEA in vivo and in vitro.Those results provided theoretical basis for medicinal and health use of C.cicadae and laid a foundation for its further development and utilization.