Mechanism of Cordyceps Cicadae in Treating Diabetic Nephropathy Based on Network Pharmacology and Molecular Docking Analysis

Objective. To systematically study the mechanism of cordyceps cicadae in the treatment of diabetic nephropathy (DN) with the method of network pharmacology and molecular docking analysis, so as to provide theoretical basis for the development of new drugs for the treatment of DN. Methods. TCMSP, Symmap, PubChem, PubMed, and CTD database were used to predict and screen the active components and therapeutic targets for DN. The network of active components and targets was drawn by Cytoscape 3.6.0, the protein-protein interaction (PPI) was analyzed by the STRING database, and the DAVID database was used for the enrichment analysis of intersection targets. Molecular docking studies were finished by Discovery Studio 3.5. Results. A total of 36 active compounds, including myriocin, guanosine, and inosine, and 378 potential targets of cordyceps cicadae were obtained. PPI network analysis showed that AKT1, MAPK8, and TP53 and other targets were related to both cordyceps cicadae and DN. GO and KEGG pathway analysis showed that these targets were mostly involved in R-HSA-450341, 157.14-3-3 cell cycle, and PDGF pathways. Docking studies suggested that myriocin can fit in the binding pocket of two target proteins (AKT1 and MAPK8). Conclusion. Active ingredients of cordyceps cicadae such as myriocin may act on DN through different targets such as AKT1, MAPK8, and TP53 and other targets, which can help to develop innovative drugs for effective treatment of DN.

The transcriptome analysis on urea response mechanism in the process of ergosterol synthesis by Cordyceps cicadae

Nitrogen source is required for the growth of Cordyceps cicadae and involved in the regulation of metabolite synthesis. In order to further investigate the regulatory effects of nitrogen sources on the ergosterol synthesis by C. cicadae. We first confirmed that urea could significantly increase the ergosterol synthesis. The transcriptome analysis showed that compared with biomass cultured in the control fermentation medium (CFM), 1340 differentially expressed genes (DEGs) were obtained by Gene Ontology (GO) annotation, and 312 DEGs were obtained by Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation from the biomass cultured in CFM + CO(NH2)2. Urea up-regulated D-3-phosphoglycerate dehydrogenase gene transcription level and down-regulated enolase and L-serine/L-threonine ammonialyase gene transcription level, increased serine synthesis, allosterically activate pyruvate kinase, to promote the synthesis of pyruvate and CH3CO ~ SCOA, the primer of ergosterol; Urea increase the genes transcription related with ergosterol synthesis by up-regulating the steroid regulatory element binding protein gene transcription levels. The transcriptome results were provided by those of qRT-PCR. Collectively, our finding provided valuable insights into the regulatory effect of nitrogen source on the ergosterol synthesis by C. cicadae.

Different Cultivation Environments Affect the Yield, Bacterial Community and Metabolites of Cordyceps cicadae

Cordyceps cicadae is an entomogenous fungus with important uses in traditional Chinese medicine. However, its wild resources have not met consumers’ demand due to excessive harvesting practices. Artificial cultivation is therefore an important alternative, but research on cultivating C. cicadae in natural habitats has not been reported. In this study, we aimed to explore the viability of cultivating C. cicadae in a natural habitat, in the soil of Pinus massoniana forest. We assessed and compared the yield, metabolite contents and bacterial community composition of C. cicadae grown in the Antheraea pernyi pupae at different growth stages, and under different cultivation conditions, in the soil of a natural habitat and in sterile glass bottles. Our results showed that cultivating C. cicadae in a natural habitat is feasible, with up to 95% of pupae producing C. cicadae fruiting bodies. The content of nitrogen compounds (amino acids) in C. cicadae cultivated in a natural habitat was significantly higher than in glass bottles, while the yield and carbon compound (mannitol and polysaccharide) and nucleoside (cordycepin and adenosine) contents were lower. Different bacterial genera were enriched in C. cicadae at different growth stages and cultivation environments, and these bacterial genera were closely related to metabolites contents during growth. This study demonstrated the viability of a novel cultivation method of C. cicadae, which could be used as an alternative to wild stocks of this fungus. These findings provided new insights into the growth mechanism of C. cicadae and its interaction with soil microorganisms.

Functional and In Vitro Antioxidant Properties of Wild and Insect-Cultured Cordyceps Cicadae Polysaccharides

Cordyceps cicadae is an entomogenous fungus, which is used as both medicine and food due to the presence of rich bioactive substances. Currently, most studies focus on the pharmacological effects of the bioactive components of C. cicadae, but the research on their processing is still in its infancy. In this study, polysaccharides were extracted from insect-cultivated C. cicadae (tussah pupae C. cicadae and domestic silkworm pupae C. cicadae) and wild C. cicadae. The functional properties of C. cicadae polysaccharides were evaluated. At the same time, the in vitro antioxidant activity of the insect-cultivated C. cicadae was determined and compared with the wild C. cicadae. The results showed that the cultivated C. cicadae polysaccharides and the wild one had similar structures, and both had appreciable water solubility, oilholding capacity, thermal stability, and emulsifying property compared to foaming property and foaming stability. Further, the C. cicadae polysaccharides cultured on silkworm pupae and tussah pupae showed comparable antioxidant and reducing power to the wild C. cicadae. Our data indicates that the C. cicadae polysaccharides have the potential as a food additive, among which the domestic silkworm pupae C. cicadae showed the best outcome, and the C. cicadae polysaccharides cultured by bionic method have the same antioxidant activity as the wild one.

Potential Protection Effect of ER Homeostasis of N6-(2-Hydroxyethyl)adenosine Isolated from Cordyceps cicadae in Nonsteroidal Anti-Inflammatory Drug-Stimulated Human Proximal Tubular Cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) belong to a class of universally and commonly used anti-inflammatory analgesics worldwide. A diversity of drawbacks of NSAIDs have been reported including cellular oxidative stress, which in turn triggers the accumulation of unfolded proteins, enhancing endoplasmic reticulum stress, and finally resulting in renal cell damage. Cordyceps cicadae (CC) has been used as a traditional medicine for improving renal function via its anti-inflammatory effects. N6-(2-hydroxyethyl)adenosine (HEA), a physiologically active compound, has been reported from CC mycelia (CCM) with anti-inflammatory effects. We hypothesize that HEA could protect human proximal tubular cells (HK–2) from NSAID-mediated effects on differential gene expression at the mRNA and protein levels. To verify this, we first isolated HEA from CCM using Sephadex® LH–20 column chromatography. The MTT assay revealed HEA to be nontoxic up to 100 µM toward HK–2 cells. The HK–2 cells were pretreated with HEA (10–20 µM) and then insulted with the NSAIDs diclofenac (DCF, 200 µM) and meloxicam (MXC, 400 µM) for 24 h. HEA (20 µM) effectively prevented ER stress by attenuating ROS production (p < 0.001) and gene expression of ATF–6, PERK, IRE1α, CDCFHOP, IL1β, and NFκB within 24 h. Moreover, HEA reversed the increase of GRP78 and CHOP protein expression levels induced by DCF and MXC, and restored the ER homeostasis. These results demonstrated that HEA treatments effectively protect against DCF- and MXC-induced ER stress damage in human proximal tubular cells through regulation of the GRP78/ATF6/PERK/IRE1α/CHOP pathway.

Molecular record for the first authentication of Isaria cicadae from Vietnam

The entomopathogenic fungus T011, parasitizing on nymph of Cicada, collected in the coffee garden in Dak Lak Province, Vietnam, was preliminarily morphologically identified as Isaria cicadae, belonged to order Hypocreales and family Clavicipitaceae. To ensure the authenticity of T011, phylogenetic analysis of the concatenated set of multiple genes including ITS, nrLSU, nrSSU, Rpb1, and Tef1 was applied to support the identification. Genomic DNA was isolated from dried sample T011. The PCR assay sequencing was applied to amplify ITS, nrLSU, nrSSU, Rpb1, and Tef1 gene. For phylogenetic analysis, the concatenated data of both target gens were constructed with MEGAX with a 1,000 replicate bootstrap based on the neighbor-joining, maximum likelihood, maximum parsimony method. As the result, the concatenated data containing 62 sequences belonged to order Hypocreales, families Clavicipitaceae, and 2 outgroup sequences belonged to order Hypocreales, genus Verticillium. The phylogenetic analysis results indicated that T011 was accepted at subclade Cordyceps and significantly formed the monophyletic group with referent Cordyceps cicadae (Telemorph of Isaria cicadae) with high bootstrap value. The phylogenetically analyzed result was strongly supported by our morphological analysis described as the Isaria cicadae. In summary, phylogenetic analyses based on the concatenated dataset were successfully applied to strengthen the identification of T011 as Isaria cicadae.