Evaluation of reference genes for quantitative expression analysis in Mylabris sibirica (Coleoptera, Meloidae).

Mylabris sibirica is a hypermetamorphic insect whose adults feed on oilseed rape. However, due to a shortage of effective and appropriate endogenous references, studies on molecular functional genes in Mylabris sibirica, have been tremendously limited. In this study, ten internal reference genes (ACT, ARF1, AK, EF1α, GAPDH, α-TUB, RPL6, RPL13, RPS3 and RPS18) were tested and assessed under four selected treatments including adult ages, adult tissues, temperatures, and sex by RT-qPCR based on five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). Our findings showed that RPL6 and RPL13 were the most optimal internal reference gene combination for gene expression during various adult ages and under diverse temperatures; The combination of RPL6 and RPS18 was recommended to test gene transcription levels under different adult tissues. AK and RPL6 were the best reference genes in male and female adults. RPL6 and RPL13 were the most appropriate reference gene pair to estimate gene expression levels under four different tested backgrounds. The relative transcript levels of a uridine diphosphate (UDP)-N-acetylglucosamine-pyrophosphorylase (MsUAP), varied greatly according to normalization with the two most- and least-suited reference genes. This study will lay the basis for further molecular physiology and biochemistry studies in M. sibirica, such as development, reproduction, sex differentiation, cold and heat resistance.

The first chromosome-level genome assembly and transcriptome sequencing provide insights into cantharidin production of the blister beetles.

Blister beetles (Coleoptera: Meloidae) produce a natural defensive toxin cantharidin (CTD), which has been used for various cancer treatments and other diseases. Currently, the lack of chromosome-level reference genomes in Meloidae limits further understanding of the mechanism of CTD biosynthesis and environmental adaptation. In this study, the chromosome-level genome assembly of Mylabris phalerata was generated based on PacBio and Hi-C sequencing. This reference genome was about 136.68 Mb in size with contig N50 of 9.17 Mb and composed of 12 chromosomes. In comparison to six other Coleoptera insects, M. phalerata exhibited multiple expanded gene families enriched in juvenile hormone (JH) biosynthetic process pathway, farnesol dehydrogenase activity, and cytochrome P450, which may be related to CTD biosynthesis. Consistently, the transcriptomic analysis suggested the "terpenoid backbone biosynthesis" pathway and "the juvenile hormone" as putative core pathways of CTD biosynthesis and presented eight up-regulated differential expression genes in male adults as candidate genes. It is possible that the restricted feeding niche and lifestyle of M. phalerata were the cause of the gene family's contraction of odorant binding proteins. The ABC transporters (ABCs) related to exporting bound toxins out of the cell and the resistance to the self-secreted toxins (e.g. CTD) were also contracted, possibly due to other self-protection strategies in M. phalerata. A foundation of understanding CTD biosynthesis and environmental adaptation of blister beetles will be established by our reference genome and discoveries.

Five New Cantharidin Derivatives from the Insect Mylabris cichorii L. and Their Potential against Kidney Fibrosis In Vitro.

Five new monoterpenoids including three 1-hydroxymethyl-2-methyl cantharimide-type derivatives (1, 2, and 5) and two 1,2-dimethyl cantharimide-type derivatives (3 and 4), together with three known compounds (6-8) were isolated from the insect Mylabris cichorii Linnaeus. The structures of these new compounds, including their absolute configurations, were characterized by detailed analysis of NMR, chemical derivatization, and quantum chemical ECD calculations. All of the compounds were tested for their biological activity against kidney fibrosis. The results revealed that compounds 2, 4, and 7 could inhibit kidney fibrosis in vitro at 40 µM by inhibiting the expression of fibronectin and collagen I in TGF-ß1-induced NRK-52e cells.

Quality Evaluation of Traditional Chinese Medicine Mylabris Based on Heavy Metal Risk Assessment and Analysis of Pharmacological Component Contents.

As a highly representative traditional Chinese anti-tumor medicinal material, the biomass of Mylabris is collected from the wild. However, the living environments of Mylabris is differ, so Mylabris may be contaminated by heavy metal pollution depending on the environment. These environments may also affect the amount of biosynthesis of its medicinal ingredient, cantharidin, there by affecting the quality of Mylabris. In this study, we determined the heavy metal content in Mylabris from different origins by using ICP-MS, evaluated the risk posed by these heavy metals, and recommended theoretical maximum limits of heavy metals in medicinal Mylabris. The results show that the Cu content in Mylabris is substantially higher than that in Cr, As, Pb, Cd, and Hg. A quantitative risk assessment showed that Mylabris poses no noncarcinogenic risks. The results of the total carcinogenic risk value showed that origins S12 and S13 pose carcinogenic risk by Cr and As, and the rest of the origins were in the human-tolerable carcinogenic risk range. We found large differences in the cantharidin content in Mylabris from different origins. In general, the Mylabris from origins S2, S3 and S4 had a higher in vivo cantharidin content, which proved that the quality of the medicinal materials was higher here than in other production areas. Finally, we providing a reference for the quality evaluation of medicinal Mylabris materials.

The mitochondrial genome of Mylabris mongolica Dokhtouroff, 1887 (Coleoptera: Meloidae).

Mylabris mongolica Dokhtouroff, 1887 is a traditional medicine material and an important predator in China. The mitochondrial genome of M. mongolica is presented for the first time in this study. The mitogenome is 15,034 bp in length and comprises 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and partial control region. The nucleotide composition of M. mongolica was 36.7% of A, 18.1% of C, 11.1% of G, and 34.1% of T. The phylogenetic results divide all Meloidae species into two clades. The genus Mylabris was retrieved as a paraphyletic group, with Mylabris having a closer relationship with Hycleus than other genera within Meloidae. This study provides useful genetic data for future studies on the phylogeny and evolution of Meloidae species.

Pharmacological mechanism of mylabris in the treatment of leukemia based on bioinformatics and systematic pharmacology.

Leukemia is a common blood cancer, whose treatment usually necessitates chemo/radiotherapy and bone marrow transplant. Hence, safer and more effective options are urgently needed. Mylabris, the dried body of blister beetles, has been used extensively in traditional Chinese medicine. This study applied bioinformatics and systematic pharmacology to investigate the mechanism of action of mylabris in the treatment of leukemia. Five effective components and 35 corresponding target proteins were identified by screening the TCMSP database; whereas 776 genes related to leukemia were selected using OMIM, GeneCards, and the Therapeutic Target Database. Eight genes common to mylabris and leukemia were identified. Protein-protein interaction network analysis and a component-target-pathway diagram identified TP53 and PTEN as key gene targets of mylabris in the treatment of leukemia. GO enrichment analysis pointed to DNA damage and cell cycle disorder caused by p53 signaling as the most significant processes; whereas KEGG enrichment pointed to the p53 signaling pathway. In summary, mylabris may exert a therapeutic effect on leukemia by triggering DNA damage, inducing apoptosis, as well as inhibiting the growth and proliferation of tumor cells through the regulation of TP53 and PTEN. These findings provide a mechanistic rationale for the treatment of leukemia with traditional Chinese medicine.

Identification of the molecular targets and mechanisms of compound mylabris capsules for hepatocellular carcinoma treatment through network pharmacology and bioinformatics analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese herbal formulas have been proven to exert an inhibitory effect on tumor. Compound mylabris capsules (CMC) has been used for treating cancer, especially hepatocellular carcinoma (HCC), for years in China. However, its therapeutic mechanisms on HCC remain unclear. AIM OF THE STUDY: This research aimed to elucidate the molecular targets and mechanisms of CMC for treating HCC. MATERIALS AND METHODS: First, the bioactive ingredients and potential targets of CMC, as well as HCC-related targets were retrieved from publicly available databases. Next, the overlapped genes between potential targets of CMC and HCC-related targets were determined using bioinformatics analysis. Then, networks of ingredient-target and gene-pathway were constructed. Finally, cell experiments were carried out to examine the effects of CMC-medicated serum on HCC and validate the core molecular targets. RESULTS: In total, 151 bioactive ingredients and 255 potential targets of CMC were selected, 982 differentially expressed genes of HCC were identified. Among them, 34 overlapped genes were finally selected. In addition, 20 pathways and 429 GO terms were significantly enriched. Protein-protein interaction and gene-pathway networks indicated that Cyclin B1(CCNB1) and Cyclin Dependent Kinase 1(CDK1) were the core gene targets for the treatment of CMC on HCC. Moreover, in vitro studies showed that CMC-medicated serum significantly inhibited the viability of HepG2 cells. Furthermore, CMC downregulated CCNB1 and CDK1 expressions and induced G2/M phase cell cycle arrest. CONCLUSIONS: CMC plays a therapeutic role in HCC via multi-component, -target and -pathway mechanisms, in which CCNB1 and CDK1 may be the core molecular targets. This study indicates that the integration of network pharmacology and bioinformatics analysis, followed by experimental validation, can serves as an effective tool for studying the therapeutic mechanisms of traditional Chinese medicine.

Pharmacokinetics and tissue distribution of cantharidin after oral administration of aqueous extracts from Mylabris in rats.

A sensitive gas chromatography-mass spectroscopy method was established for the determination of cantharidin (CTD) in rat plasma and liver homogenates. During the experiment, rats were randomly divided into two groups (low, high) and were administered aqueous extract of Mylabris compound for 7 days. Then, plasma and tissue samples were taken at different time points to study the pharmacokinetics and tissue distribution of CTD in rats. The selected reaction monitoring transitions for CTD and clofibrate (internal standard) were m/z 128 → 85 and m/z 169 → 141, respectively. The calibration curve ranged from 10.26 to 3,078 ng/ml for plasma and from 10.26 to 246.24 ng/ml for liver homogenates. The lower limits of quantification were 10.26 ng/ml for both plasma and liver. The intra- and inter-day precision and accuracy were <20% for both plasma and liver homogenates. Extraction recovery ranged from 89.21 to 103.61% for CTD in rat plasma and liver and from 83.79 to 102.74% for IS in rat plasma and liver. Matrix effects ranged from 93.06 to 110.44% for CTD and from 91.65 to 110.80% for IS.

Rapid profiling of cantharidin analogs in Mylabris phalerata Pallas by ultra-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry.

We evaluated the protective effect and toxicity of extracts from Mylabris phalerata Pallas by measuring the activated partial thromboplastin time, prothrombin time, venous thrombosis and acute toxicity in rats. Results showed the petroleum ether and water fractions of M. phalerata inhibited thrombosis but hardly prolonged the activated partial thromboplastin time and prothrombin time in rats. The trichloromethane fraction had obvious toxicity with an LD50 of 0.2 g/kg in vivo, and contained many cantharidin analogs (CAs) by ultra-performance liquid chromatography-quadrupole ion trap-tandem mass spectrometry (UPLC-QTRAP-MS/MS). CAs are the major potential bioactivity constituent in M. phalerata. An effective and reliable UPLC-QTRAP-MS/MS method was successfully developed to separate and identify CAs. The fragmentation patterns of five purified compounds were applied to elucidate the structure of their analogs. Thirty-four CAs were characterized or tentatively identified, eight of which are proposed to be novel compounds (13-17, 20, 21, 23), and their fragmentation patterns were investigated for the first time. Most importantly, a rapid and reliable UPLC-MS method was developed to identify the CAs of M. phalerata. This method has contributed to the discovery of most of these unknown analogs or their metabolites in M. phalerata effectively and quickly, and does not rely on limited chemical structural diversity libraries.

Phylogenetic relationship and characterization of the complete mitochondrial genome of Mylabris calida (Coleoptera:Meloidae).

Beetle genus Mylabris (Meloidae) was described by Fabricius (1775) and had been well known due to its relevance to traditional medicine (e.g., cantharidin production). Here, we sequenced and annotated the mitochondrial genome (mitogenome) of Mylabris calida, one of species within Mylabris. This mitogenome was 15,149 bp long and encoded 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs) and two ribosomal RNA unit genes (rRNAs). All 13 PCGs were initiated by the ATN (ATG, ATT and ATA) codon. All PCGs terminated with the stop codon TAA or TAG except for cox1, cox2, nad5 and nad4 which end with the incomplete codon T-. Phylogenetic analysis showed that M. calida got together with the same genus species Mylabris sp., M. aulica and three Hycleus species (H. cichorii, H. phaleratus and H. marcipoli), indicating Mylabris has a closer relationship with Hycleus than other genera within Meloidae.

Historical evolution in processing and some problems in modern research of Mylabris / 中草药

Mylabris is a traditional Chinese medicine and ethnic medicine, which has significant effects on treatment of malignant tumors and dermatophytosis and so on. It has been recorded in most historical Chinese Pharmacopoeias. However, because of its hypertoxicity in the crud forms, Mylabris needs to be furtherly processed if it is taken orally. There are various methods about how to process it in previous Materia Medica Classics, whereas the most modern approaches are mainly using cleansing technology and stir-baked with rice technology. Moreover, the technologies stipulated by local processing regulations are different from the stipulations of pharmacopoeias. This research combs through the history evolution systematically in the processing of Mylabris, and analyzed the existential issues in modern research on it, so as to provide references for the processing and the quality control of Mylabris.

Draft Genome of a Blister Beetle Mylabris aulica.

Mylabris aulica is a widely distributed blister beetle of the Meloidae family. It has the ability to synthesize a potent defensive secretion that includes cantharidin, a toxic compound used to treat many major illnesses. However, owing to the lack of genetic studies on cantharidin biosynthesis in M. aulica, the commercial use of this species is less extensive than that of other blister beetle species in China. This study reports a draft assembly and possible genes and pathways related to cantharidin biosynthesis for the M. aulica blister beetle using nanopore sequencing data. The draft genome assembly size was 288.5 Mb with a 467.8 Kb N50, and a repeat content of 50.62%. An integrated gene finding pipeline performed for assembly obtained 16,500 protein coding genes. Benchmarking universal single-copy orthologs assessment showed that this gene set included 94.4% complete Insecta universal single-copy orthologs. Over 99% of these genes were assigned functional annotations in the gene ontology, Kyoto Encyclopedia of Genes and Genomes, or Genbank non-redundant databases. Comparative genomic analysis showed that the completeness and continuity of our assembly was better than those of Hycleus cichorii and Hycleus phaleratus blister beetle genomes. The analysis of homologous orthologous genes and inference from evolutionary history imply that the Mylabris and Hycleus genera are genetically close, have a similar genetic background, and have differentiated within one million years. This M. aulica genome assembly provides a valuable resource for future blister beetle studies and will contribute to cantharidin biosynthesis.

Research progress on derivative cantharidin bioactivities and toxic regulations / 中草药

Cantharidin is the main active component of traditional Chinese medicine Mylabris. Cantharidins received increasing attentions for having bioactivities such as anticancer, anti-inflammatory and enhancing leukocytes, meanwhile, many researches were reported about cantharidin bioactivities, structure modifications, and synthesis methods. In this review, structures of derivative cantharidins were summarized and bioactivity and toxic regulations were developed, which can provide reference for cantharidin compound researches and modernizations of Mylabris.

Pharmacognostical study of Chinese medicine Mylabris / 中草药

Objective: To provide the scientific evidence for the identification of Mylabris phalerata and Mylabri scichorii by observing the characteristics and microscopic characteristics of the two species, which may be of relevance to the compilation of 2020 edition of Chinese Pharmacopoeia. Methods: The pharmacognosy of six batches of M. phalerata and four batches of M. scichorii were studied by character identification, micro-morphological identification, conventional microscopic identification, polarized light microscopic identification and microsublimation methods. Results: Colored holographic image of the micro-morphological characteristics (body length, antenna, elytron and hind-wing), microscopic characteristics (bristle, body wall fragment, elytron debris, hind-wing debris, muscle fiber, debris of gas pipeline and undigested plant tissue) and crystalline sublimate characteristics were obtained for the first time. Conclusion: The results of micro-morphological identification complement the fine structural characteristics of traditional character identification. The microscopic and microsublimation methods for identification can be used as specific markers for identifying Mylabris.

Unique Responses of Hepatocellular Carcinoma and Cholangiocarcinoma Cell Lines toward Cantharidin and Norcantharidin.

The present study aimed to investigate whether cell lines from human gastric and liver cancers respond differently toward cantharidin (CTD) and norcantharidin (NCTD) than other types of cancer cells. We first established the half maximal inhibitory concentrations (IC50s) of CTD for a large panel of cancer cell lines representing the 12 major types of human cancers and the mode of cell death induced by the two compounds. We next compared the growth inhibitory effects as well as the corresponding modes of action of CTD and NCTD. The IncuCyte ZOOM system was used as a semi-high throughput means to define IC50s and 90% inhibitory doses (IC90s) as a reference for the maximal tolerable doses (MTDs) for the two compounds in 72 cancer cell lines. Classical clonogenic survival assay was used to assess the anti-proliferative effect of CTD on selected cell lines of interest. In addition, DNA content-based flow was used to interrogate the modes of cell death following CTD or NCTD exposure. The results of these experiments led to several findings. 1). Cell lines representing hepatocellular carcinomas (HCCs) and cholangiocarcinomas (CCs) were among the most sensitive toward CTD, consistent with the previous clinical study of this compound and its source of origin, Mylabris. 2). Among the individual cell lines of a given cancer types, the sensitivity trends for CTD and NCTD did not exhibit a good correlation. 3) CTD and NCTD caused distinctive cytotoxic effects on HepG2 cells. Specifically, while a cytostatic effect is the primary cause of growth inhibition of CTD, cytotoxic effect is the main contributing factor for the growth inhibition of NTCD. These results indicate that liver cancer cell lines are among the most sensitive to CTD and that CTD and NCTD exhibit their effects through distinct mechanisms.

[Molecular mechanism of Mylabris for colorectal cancer based on integrative pharmacology].

At present, the disjointing situation is generally present between "systemic"and "local", "macro" and "micro", "in vivo process" and "activity evaluation" in the study of traditional Chinese medicine (TCM). An urgent task for the modernization of TCM is to establish new strategies and methods which can reflect the overall characteristics of TCM. The introduction of integrative pharmacology provided a feasible approach to solve the problem of the fragmentation of TCM. Internet-based computation platform method was adopted in this study to explore the active molecular mechanism of Mylabris in the treatment of colorectal cancer. Based on the analysis of the functional integration of internet-based computation platform V1.0 version software, the "core components-key target- main pathway" multidimensional network of Mylabris in treatment of colorectal cancer disease was constructed to explore the potential molecular mechanism of Mylabris in treatment of colorectal cancer from multiple perspectives. The results showed that Mylabris can treat the colorectal cancer and the mechanism might be associated with amino acid metabolism, NF-κB signaling pathway, immune system, endocrine system, nervous system, and chemokine signal transduction pathway, epithelial cell signaling in helicobacter pylori infection, T cell receptor signaling pathway, B cell receptor signaling pathway and so on.

Molecular mechanism of Mylabris for colorectal cancer based on integrative pharmacology / 中国中药杂志

At present, the disjointing situation is generally present between "systemic"and "local", "macro" and "micro", " process" and "activity evaluation" in the study of traditional Chinese medicine (TCM). An urgent task for the modernization of TCM is to establish new strategies and methods which can reflect the overall characteristics of TCM. The introduction of integrative pharmacology provided a feasible approach to solve the problem of the fragmentation of TCM. Internet-based computation platform method was adopted in this study to explore the active molecular mechanism of Mylabris in the treatment of colorectal cancer. Based on the analysis of the functional integration of internet-based computation platform V1.0 version software, the "core components-key target- main pathway" multidimensional network of Mylabris in treatment of colorectal cancer disease was constructed to explore the potential molecular mechanism of Mylabris in treatment of colorectal cancer from multiple perspectives. The results showed that Mylabris can treat the colorectal cancer and the mechanism might be associated with amino acid metabolism, NF-κB signaling pathway, immune system, endocrine system, nervous system, and chemokine signal transduction pathway, epithelial cell signaling in helicobacter pylori infection, T cell receptor signaling pathway, B cell receptor signaling pathway and so on.

Cloning and functional analysis of farnesyl pyrophosphate synthase (FPPS) gene from Mylabris cichorii.

Cantharidin, a defensive terpene compound synthesized by the meloid beetle (Coleoptera, Meloidae), is an important anticancer agent. However, there has been little study done on how this compound synthesized by the beetle. In this paper, a farnesyl pyrophosphate synthase (FPPS) gene, designated McFPPS, was isolated from Mylabris cichorii by reverse transcription PCR based on conserved domains in other organisms. Multiple alignment analysis showed that the deduced amino acids shared >70% homology with FPPSs from other species and contained typically seven conservative regions. Gene expression profile analysis revealed that McFPPS was expressed throughout the tested growth stages of M. cichorii adults, whereas the transcripts accumulated to the highest level at 20 days in male adults while the highest expression level appeared at 15 days in females. Tissue expression pattern analysis showed that McFPPS was expressed constitutively in all tested tissues and a relatively higher expression level in the alimentary canal of males, but no significant tissue difference in the females. For the first time, a RNA interference strategy was employed to induce a greater suppression of McFPPS mRNA, and thus a sharp decrease in the expression levels of downstream genes and the concentration of product. All these results indicated that McFPPS may be directly involved or play an essential role in the biosynthesis of cantharidin.

Cantharis by photosynthetic bacteria biotransformation: Reduced toxicity and improved antitumor efficacy.

ETHNOPHARMACOLOGICAL RELEVANCE: The blister beetle, also known as Mylabris cichorii, is not only widely used in clinical applications in Chinese anticancer medicine, but is also one of the main ingredients in a variety of traditional Chinese medicinal preparations with anticancer activity. However, the strong toxicity exhibited by this beetle species limits its clinical applicability, with the photosynthetic bacteria featuring a strong biological conversion function. Therefore, in this study we explore the use of photosynthetic bacteria for bioconversion of the blister beetle in order to reduce the toxicity and in effort to enhance the overall antitumor effect. METHODS AND RESULTS: In the first set of experiments, we utilized an orthogonal experimental design to optimize the culture medium of photosynthetic bacteria. Concurrently, the growth curve of photosynthetic bacteria was used to determine the inoculation amount of the photosynthetic bacteria and the safe concentration of cantharis. Through antitumor activity experiments conducted in vitro we found that the inhibition rate increased through cantharis by PSB biotransformation of HepG2, A549 and BEL-7406 cells. Furthermore, through acute toxicity tests in mice it was found that the blister beetle water extraction liquid exhibits a LD50 value of 1383mg/kg, while the blister beetle transformation liquid exhibits a LD50 value of 206mg/kg. The LD50 value of the blister beetle water extract was found to be 6.7 times higher than the transformation liquid, thus demonstrating that the toxicity of cantharis by PSB biotransformation may be decreased. More strikingly, decreased toxicity was observed in the mouse liver and in pathological sections of the kidneys after transformation. CONCLUSIONS: In this paper we demonstrate for the first time that PSB bioconversion of the blister beetle is able to reduce the toxicity of a common method used in anticancer treatments as part of the principles in traditional Chinese medicine and may therefore improve antitumor activity in vitro.

[Chemical constituents from Mylabris phalerata and their cytotoxic activity in vitro].

Ten compounds were isolated from Mylabris phalerata by using preparative HPLC and column chromatography over MCI gel. On the basis of physical-chemical properties, NMR and MS data analysis, the compounds were identified as 5'-[(1 R,2 R,3 S,6R)-1-hydroxymethyl-2-methyl-3,6-epoxycyclohexane-1,2-dicarboximide]- ethyl-2'-methyl-2'-butenoate (1),cantharidin (2), cyclo-(L-Pro-L-Ala) (3), cyclo-(R-Pro-R-Leu) (4), cyclo-(S-Pro-R-Leu) (5), cyclo-(D-Pro-L-Tyr) (6), indole-3-aldehyde (7), 3-indoleacetic acid (8), valerolactam (9), and 4-hydroxyphthalid (10).Compound 1 was a new compound, and compounds 2-10 were obtained from this genus for the first time. Compounds 1-9 were subjected to cytotoxic activity on HCT-116, HepG2, BGC-823, NCI-H1650, A2780 cell lines, and only compound 2 showed inhibitory effect on all cancer cell lines.