| Cordyceps genus is a kind of important entomogenous fungi,which their life cycle and infection mechanism are mainly similar,and the difference lies only in the host insects and growth conditions.Cordyceps kyushuensis Kob,which was found in Meng Mountain of Shandong Province and the eastern mountain area of Shenyang,Liaoning Province,is the only species of Cordyceps growing on the larvae of Clanis bilineata Walker.It was verified as a new species in 2000 in China,and its agamotype was identified as Verticillium kyushuensis.Studies have shown that there are a variety of active ingredients in Cordyceps kyushuensis,such as cordycepin,pentastatin,cordycepin,adenosine,flavonoids,polysaccharides,amino acids and so on.Pharmacological experiments in vivo and vitro showed that Cordyceps kyushuensis has many pharmacological activities,for example,cordycepin exerts anti-tumor effects by inducing apoptosis of non-small cell lung cancer cells,specifically inhibiting the transcription of human cervical cancer cells,and it could also play immunoregulatory function by inducing mature dendritic cells to regulate T cell proliferation;Combination of cordycepin and pentastatin could not only be effective in the treatment of mice infected with African trypanosomiasis,but also that OncoVista corporation in USA used both cordycepin and pentostatin to treat terminal deoxynucleotide positive leukemia cells(TdT+)with approval of FDA.Although the pharmacological effects of some single active ingredients of Cordyceps kyushuensis have been proved,the pharmacological activities brought by many ingredients are not simple additivity,which makes it face the bottleneck of "diversified chemical ingredients,unclear mechanism of action.unspecified scientific theory" in clinical application.It is not only Cordyceps kyushuensis,but also the application of traditional Chinese medicine is restricted.With the development and progress of modern biotechnologies,microbiome technology which is represented by genomics,transcriptomics and proteomics.and systems pharmacology whose core is "understanding organisms as a whole and exploring the network relationships of life systems at different levels" have emerged,and they provide a new thinking for the modern development of traditional Chinese medicine and the development of newtype drugs from Chinese medicine sources.Combining various tools of omics to obtain complete biological information at different molecular levels can help to elucidate the changes of genes and proteins in different developmental stages and under diseases or health states.The thinking concept of "multiple drugs correspond to multiple targets and multiple diseases" in systems pharmacology coincides with the dialectical thinking of traditional Chinese medicine.The interaction between drugs and human genes or proteins(targets)constructs a complex and robust network,which treats diseases by regulating the targets in vivo and participating in many biological processes of organisms.In this multi-component and multi-target synergistic system,we study drugs through theoretical calculation and molecular biology pathway,and with omics technology to provide practical and effective methods for rapid study,clarify and verify the therapeutic effect of traditional Chinese medicine at the molecular level,to make contribution and promotion to the development of traditional Chinese medicine by applying new concepts of pharmacology.In this paper,the object of study is C.kyushuensis.Transcriptomics and proteomics were used to study the differential expressed genes and proteins in different developmental stages of C.kyushuensis.Complete mitochondrial genome of C.kyushuensis was sequenced and phylogenetic tree was completed to classify and verify the evolutionary status of C.kyushuensis.Under the premise of summarizing the active components of C.kvushuensis,we used systematic pharmacological techniques and analytical methods to predict potential pharmacological activity.Based on the predicted results,the hypoglycemic and hypolipidemic effects of water extract of C.kyushuensis were verified with zebrafish diseases model.Meanwhile,the changes of autophagic expression under high sugar/drug administration status were analyzed,and verify the pharmacological activities of C.kyushuensis theoretically and practically.The main contents and conclusions of this paper are as follows:1.The transcriptome of C.kyushuensis at four developmental stages(mycelium stage CKK1,coloring stage CKK2,stromata-forming initial stage CKK3,fruiting body stage CKK4)and proteome of C.kyushuensis at twodevelopmental stages(CKK1 and CKK4)were sequenced and analyzed for the first time,and key enzymes during fruiting body stage were identified.The results showed that there were 21351(CKK1),17858(CKK2),17869(CKK3)and 17088(CKK4)unigenes transcribed at four developmental stages.RNA-Seq data was used to identify genes showing significant changes in expression during the four stages.CKK1-VS-CKK2,CKK1-VS-CKK3.CKK1-VS-CKK3.CKK2-VS-CKK3.CKK2-VS-CKK4.CKK3-VS-CKK4 were the six groups to analysis the differential expression genes between them.There were 878 DEGs and 552 up-regulated genes(CKK1-VS-CKK2),811 DEGs and 536 up-regulated genes(CKK1-VS-CKK3).718 DEGs and 479 up-regulated genes(CKK1-VS-CKK4),1057 DEGs and 566 up-regulated genes(CKK2-VS-CKK3),912 DEGs and 493 up-regulated senes(CKK2-VS-CKK4),907 DEGs and 453 up-regulated genes(CKK3-VS-CKK4).GO,KEGG and KOG databases were carried out to further analysis the DEGs obtained above among the six groups,and key enzymes in fruiting body stage were identified.There were 42 unigenes participated in fruiting body stage.Among them,12 unigenes belonged to NADH dehydrogenase subunit,2 unigenes belonged to NADPH oxidase,2 unigenes belonged to pheromone receptor,9 unigenes belonged to G protein subunit and 1 unigene belonged to adenylyl cyclase and 2 unigenes belonged to MAPK pathway,etc.KEGG pathways analysis showed that MAPK pathway is one of the top twenty annotated pathways,which suggested that the development of fruiting body maybe more dependent on the MAPK sigaling pathway.In proteome sequence,there were 4185 proteins identified in two developmental stages,and according to FC>1.5 or FC<0.67,445 differential expressed proteins were screened,then KEGG.GO and KOG annotation were analyzed.2.On the basis of transcriptome and proteome databases,the synthetic gene cluster of cordycepin and pentastatin was identified by Blast.As the "protector" of cordycepin in vivo,pentostatin could protect cordycepin from degradation by adenosine deaminase,thereby enhances its pharmacological activity.It has been identified that pentostatin and pentostatin share the same synthetic gene cluster in C.kyushuensis.The synthetic gene cluster contains four sgenes,named ckl-ck4.respectively.Among them,the conserved region of ckl gene is redox/dehydrogenase region,the conserved region of ck2 gene is metal-dependent phosphatase HDc family,the conserved region of ck3 gene is N-terminal nucleotide kinase and C-terminal HisG region,and ck4 is annotated by blast as a potential ATP-binding cassette transporter.3.The whole mitochondrial genome of C.kyushuensis was sequenced and analyzed,and phylogenetic tree was constructed to analyze the evolutionary process.The length of mitochondrial genome of C.kyushuensis was 37520 bp.and GC content was 26.55%.There were 22 coding genes(3 ATP synthase F0 subunits,1 deheme cytochrome b gene.3 cytochrome oxidase subunits.5 NADH dehydrogenase subunits.1 NADH-quinone oxidoreductase and 9 open reading frames)and 21 non-coding genes(19 tRNA.1 rrnL and 1 rrnS).The phylogenetic tree showed that C.kuyshuensis and Ophiocordyeps sinensis separated very early and had a distant relationship,but it is close with with C.militaris.The evolutionary results are consistent with the re-classification of Cordyceps fungi in recent years.O.sinensis was classified as Ophiocordvceps of Ophiocordycipitaceae.while C.militaris and C.kyushuensis still belong to Cordyceps genus of Cordycipitaceae.4.C.kyushuensis was cultured in different beans culture medium.The contents of cordycepin were detected on 7,20 and 30 days respectively,and the high-yield culture medium of cordycepin was screened.The increase of cordycepin content was in time manner.Among the five kind of bean culture mediums,black bean culture medium had the highest cordycepin content,followed by soybean culture medium.Rice medium had the lowest cordycepin content.Considering of the yield of cordycepin,black bean and soybean medium were the optimal culture medium.In addition to screening high-yield culture medium for cordycepin.the antioxidant activity of different beans culture media fermentated by C.kyushuensis were also studied.The results showed that the antioxidant activity(ABTS free radical scavening rate,DPPH free radical scavenging rate and Fe2+ reduction antioxidant capacity)of beans culture media were enhanced after fermentated by C.kyushuensis.5.Based on systems pharmacology to predict the pharmacological activities of C.kyushuensis.Taking oral bioavailability(?30%)as the criterion for screening the pharmacological active compounds of C.kyushuensis,and 42 candidate compounds were obtained,corresponding to 301 human targets.According to our previous research and the need of following research,five diseases(non-small cell lung cancer,tissue lymphoma,leukemia,hyperglycemia and hyperlipidemia)were selected to construct "compound-target-disease network" and "protein-protein interaction network".The topological property of networks showed that prostaglandin G/H synthase 2(PTGS2),prostaglandin G/H synthase 1(PTGS1),tumor necrosis factor(TNF)and other targets play an important role in the treatment of tumor,in which PTGS2 is highly expressed in both benign and malignant tumors;Adrenergic receptor 1(ADRB1)and adrenergic receptor 2(ADRB2)are potential targets in both hyperglycemia and hyperlipidemia,and insulin resistance and adipocytokine signaling pathways are both enriched in hyperglycemia and hyperlipidemia,which suggested that there are inner links between glucose metabolism and lipid metabolism in human body.By analyzing the potential relationships among pharmacological compounds,target proteins and diseases,the potential molecular biological mechanism for C.kyushuensis to treat diseases was revealed.6.Based on the pharmacological activities predicted previously,studied the hypoglycemic and hypolipidemic effects of C.kyushuensis and the changes of autophagy caused by hyperglycemia.Adult zebrafish were immersed in 20 g/L glucose solution to establish hyperglycemia model and zebrafish larve were fed with 1%yolk water to establish hyperlipidemia model.Then,different concentrations of aqueous extracts of C.kvushuensis were used to treat the zebrafish model and related blood sugar indices(blood glucose level,fructosamine level)and blood lipid(SOD activity,malondialdehyde content)were detected.The results showed that the aqueous extract of C.kyushuensis could significantly reduce blood glucose and blood lipid of zebrafish,and they were in a dose-dependent manner.Besides phenotypic observation,transcriptome of three zebrafish groups(control group,10 mg/L group and hyperglycemia)were sequenced and analyzed.There was big difference in differential expressed genes between "control group virus 10 mg/L group" and"control group virus hyperglycemia",while 6300 and 6521 genes up-regulated,4632 and 5768 genes down-regulated respectively.There were 941 up-regulated genes and 1673 down-regulated genes in lOmg/L group and high-glucose group.respectively,which indicated that although after administration the blood glucose level of hyperglycemia zebrafish returned to normall.the changes of related genes caused by long-term high-glucose level in vivo could not be restored in a short time.In combination with the actual situation,diabetes patients still have a series of complications which occurred after controlling blood glucose stability,which is the puzzle of diabetes as a comprehensive metabolic disease,indicating the chronicity and difficulty in therapeutic process.Considering that the long-term high glucose level in vivo may have changed the expression of some genes and pathways in cells,the changes of autophagic expression in different target cells are still uncertain at high glucose level.In this study,the genes related to glucose metabolism(ins,pckl,pdx1,gck,g6pca.2)and autophagy related genes(atg3,atg4b,atg5,foxo3)were detected and analyzed.The results showed that the autophagy level in zebrafish cells was inhibited by high concentration of glucose.after administration with C.kyushuensis,the autophagy ability recovered.but it was still weaker than control group.This provides a inspiration for the following work:under the premise of stable blood glucose level,continue to treat hyperglycemia zebrafish with C.kyushuensis for a long time,observe and detect whether the expression of related genes return to normal level,and further explore the pathogenesis and curative effect of diabetes-related complicationsIn summary,modern microbiome technology was used to analyze the medicinal research of C.kyushuensis in gene and protein level.Then,the whole discrimination theory of traditional Chinese medicine was corresponded to the micro-expression,that is."multiple active compounds of C.kyushuensis act on multiple targets to construct complex networks".Systems pharmacology was used to predict and analyze the pharmacological activity of Ckyushuensis.All of these showed the new application of modern biotechnology and analytical methods in the development of traditional Chinese medicine,provided guidance for the modern application of traditional Chinese medicine,increased the scientificity of traditional Chinese medicine,and provided new ideas for the new use of old medicine,the development of new drugs and the treatment of complex human diseases. |
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