Previous studies demonstrated the dependence of cancer on nerve. Recently, a growing number of studies reveal that cancer cells share the property and regulatory network with neural stem/progenitor cells. However, relationship between the property of neural stemness and cell tumorigenicity is unknown.Monosiga brevicollis, a unicellular species that is a closest known relative of metazoans, are biased toward neural cells.We show that neural stem/progenitor cells, but not non-neural embryonic or somatic stem/progenitor cell types, exhibit tumorigenicity and the potential for differentiation into tissue types of all germ layers when they are placed in non-native environment by transplantation into immunodeficient nude mice. Likewise, cancer cells capable of tumor initiation have the property of neural stemness because of their abilities in neurosphere formation in neural stem cell-specific serum-free medium and in differentiation potential, in addition to their neuronal differentiation potential that was characterized previously. Moreover, loss of a pro-differentiation factor in myoblasts, which have no tumorigenicity, lead to the loss of myoblast identity, and gain of the property of neural stemness, tumorigenicity and potential for re-differentiation. By contrast, loss of neural stemness via differentiation results in the loss of tumorigenicity. These suggest that the property of neural stemness contributes to cell tumorigenicity, and tumor phenotypic heterogeneity might be an effect of differentiation potential of neural stemness. Bioinformatic analysis reveals that neural genes in general are correlated with embryonic development and cancer, in addition to their role in neural development; whereas non-neural genes are not. Most of neural specific genes emerged in typical species representing transition from unicellularity to multicellularity during evolution. Genes in We suggest that the property of neural stemness is the source of cell tumorigenicity. This is due to that neural biased unicellular state is the ground state for multicellularity and hence cell type diversification or differentiation during evolution, and tumorigenesis is a process of restoration of neural ground state in somatic cells along a default route that is pre-determined by an evolutionary advantage of neural state. Cancer is a complex disease characterized by inter- and intra-tumor heterogeneity and regulatory mechanisms that include thousands of cancer related genes, tens of millions of cancer related mutations, and numerous intertwined signaling pathways. There is not a unified explanation for cancer due to the complexity. In recent years, a growing number of studies revealed that cancer cells share properties of neural cells. Dependence of cancer on nerve has been observed since 1946 , 3. ThisWe showed previously that cells of different cancer types were characteristic of neural stem/progenitor cells (NSCs/NPCs) because (1) cancer cells exhibit neuronal differentiation potential when endogenous cancer-promoting factors are inhibited, and (2) expression of most  cancer promoting genes or genes being upregulated/activated in cancer cells is neural specific or at least enriched in embryonic neural cells , 11. In Among embryonic and adult cell types, ESCs and the induced pluripotent stem cells (iPS) are known to be tumorigenic . Howeverl-glutamine , 1 × MEM nonessential amino acids . A375, C2C12WT, C2C12Myod1−/−, U-118 MG, HCT 116, HepG2 and mouse embryonic fibroblasts (MEFs) from E13.5 embryos were cultured in DMEM. NE-4C cells were cultured in MEM  containing 1% glutamax  and 1% MEM non-essential amino acids. NCI-H460 was culture in PRMI-1640 , U-2OS in McCoy’s 5A , and SW480 was cultured in Leibovitz’s L-15 medium . Mesenchymal stem cells (MSCs) from rat bone marrow was cultured in MSC complete medium (Cellbank of Chinese Academy of Sciences. Cat. No.: #SCSP-615). All culture media except MSCs were supplemented with 10% fetal bovine serum  (15% FBS for mESCs) and with 50 U/ml penicillin and 50 μg/ml streptomycin. Cells were cultured at 37 °C with 5% CO2. mESCs were cultured in petri dishes coated with 0.1% gelatin, and NE-4C was cultured in dishes coated with 10 μg/ml poly-d-lysine . Besides culturing cells in regular media, cells were also cultured in a serum-free medium, which is described below.C57BL/6 mESCs were cultured in Dulbecco's Modified Eagle's Medium , 1 ng/ml human LIF , 100 μM β-mercaptoethanol, 2 mM A375 (Cat. No.: #SCSP-533), HCT 116 (Cat. No.: #TCHu 99), U-118 MG (Cat. No.: #TCHu216), SW480 (Cat. No.: #TChu172), NCI-H460 (Cat. No.: #TCHu205), U-2OS (Cat. No.: #TCHu 88), HepG2 (Cat. No.: SCSP-510), NE-4C (Cat. No.: #SCSP-1501) and MSCs (Cat. No.: #SCSP-402) were purchased from the Cellbank of Chinese Academy of Sciences . C2C12 was a gift from Dr. Minsheng Zhu, and mESCs were a gift from Dr. Jinzhong Qin at the Model Animal Research Center of Nanjing University. All cells were detected free of mycoplasma contamination with PCR. Cancer cell lines were authenticated with short tandem repeat profiling, which was performed in September 2019 and May 2020, respectively. Cells with fewer than eight passages were used for experiments.2, which was used for differentiation of ESCs into neuroectodermal cells [WT, C2C12Myod1−/−, NE-4C, A375, HCT 116, U-118 MG, NCI-H460, SW480, U-2OS and MSCs were also cultured in this medium to test their ability in neurosphere formation. All cells were plated at a density of 1 × 105/cm2. Except SW480, U-2OS, MSCs, MEFs and C2C12WT that could not form a spherical structure in the medium, other cell types began to form a spherical structure after usually 4 days of culture. The spherical structures gradually grew larger and started to float freely  with the extension of culture time. For injection of mESC-derived primNSCs into immunodeficient nude mice, neurospheres were dissociated by trituration.Primitive NSCs (primNSCs) were differentiated from mESCs by culturing mESCs in defined serum-free medium Ndiff 227  at 37 °C with 5% COal cells . primNSCMyod1−/− cells were treated with retinoic acid (RA) at 1 μM for 72 h, while cells treated with DMSO were used as a control. After treatment, cells were used for assays of immunoblotting, soft agar colony formation, and subcutaneous injection into nude mice.To determine cell tumorigenicity after differentiation, NE-4C and C2C12All experiments with animals (including xenograft tumor assays below) in the study  were approved by and in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) at the Model Animal Research Center of Nanjing University. For isolation of embryonic cortical cells, C57BL/6 mice with gestation on day 13.5 were sacrificed and embryos were resected. Brains were excised from embryos with forceps, and then cortical tissues were separated after removal of meninges and the ganglionic eminences. Cortical tissues were washed with ice-cold DMEM medium thrice. Afterwards the tissues were placed in 1 ml of 0.25% trypsin–EDTA and incubated in a water bath at 37 °C for 10 min. Trypsinization was terminated by addition of three volumes of DMEM containing 10% FBS. Cortical cells were then collected by centrifugation at 1000 rpm for 5 min at room temperature, washed twice with PBS, and re-suspended in PBS. Cortical cells were cultured in serum-free medium for neurosphere formation and immunofluorescence. Part of the cells were also homogenized in TRIzol  and stored at − 80 °C for later use for total RNA extraction.For isolation of MEFs, the head regions, limbs and visceral organs were removed from E13.5 embryos. The remaining trunk regions were minced into fine pieces, which were suspended in 1 ml of 0.25% trypsin–EDTA, and incubated in a 37 °C water bath for 15 min. Afterwards, three volumes of DMEM containing 10% FBS were used to stop trypsin activity. Cells were collected by centrifugation, resuspended and plated into petri dishes for culture in complete MEF medium. MEFs were used for injection into nude mice, or used for culture in serum-free medium as a control for neurosphere formation assay.nu mice with an age of five to six weeks were purchased from the National Resource Center for Mutant Mice  and maintained in a specific-pathogen-free facility. Different numbers of cells, depending on cell types as listed in Additional file t-test. Tumor volume was calculated using the formula: length × width2/2. The significance of difference in tumor volumes between two groups was calculated using two-way ANOVA followed by Bonferroni/Dunn (ANOVA-Bonferroni/Dunn) tests.Immunodeficiency nude Foxn1After measurement, tumor tissues were cut into two parts. One part was homogenized in TRIzol for total RNA extraction; the other part was fixed with 4% paraformaldehyde and embedded in paraffin, and used for histological examination with hematoxylin and eosin (HE) staining and immunohistochemistry analysis. Sections were viewed and photographed with a microscope (Olympus BX53).6 cells were suspended in 100 μl of sterile PBS and injected into mice via tail vein. Mice were sacrificed in one to three months after injection, depending on the progression of tumor growth.NE-4C cells were also injected via tail vein of nude mice to evaluate their tumorigenicity in different regions of animal body. Each 1 × 10Whole cell lysates (WCL) were prepared as described . IB dete2O for 5 min each. Antigen unmasking was performed by steaming slides in 0.01 M sodium citrate buffer (pH 6.0) for 25 min. Afterwards, sections were washed in dH2O for three times, followed by incubation in 3% H2O2, and washed again in dH2O. After blocking, sections were incubated with primary antibodies diluted in antibody diluent overnight at 4 °C. Primary antibodies were removed and sections were washed, and then incubated with biotinylated secondary antibody (1:500). DAB substrate was used for signal visualization. Cell nuclei were counter-stained with hematoxylin. Primary antibodies were: ACP5 , ACTA2 , AFP , BGLAP , CTSK , KRT5 , MAP2 , PAX6 , SOX1 , SOX9 , and anti-human nuclei antibody . Negative controls were performed in parallel in which primary antibodies were not used.Proteins marking different tissue differentiation in tumors were detected with immunohistochemistry using conventional method. Briefly, sections were deparaffinized and rehydrated with three washes of xylene for 5 min each, two washes of 100% ethanol for 10 min each, two washes of 95% ethanol for 10 min each, and two washes in dHIF detection of marker proteins in cells, myotubes, neurospheres or neurosphere-like structures derived from cancer cells was performed exactly as described . Primary-Act/β-ACT was detected as a loading control. Significance in changes of gene expression was calculated based on experiments in triplicate using two-tailed Student’s t-test. Final results were presented as histograms with relative units of transcription levels. Primers for RT-qPCR were listed in Additional file Cells or tumor tissues were homogenized in TRIzol. Total RNAs were extracted following the protocol provided by the manufacturer. cDNAs were synthesized from the total RNAs using the HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) , which contains reagent for removal of genomic DNA contamination. qPCR was performed on a LightCycler® 96 system (Roche). Amplification parameters were as follows: one cycle of pre-denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing and extension at 60 °C for 30 s, and an additional cycle for melting curve. Transcription level of βMyod1 gene using an online design tool (crispr.mit.edu). gRNA target oligos were cloned to the BsmBI site of lentiCRISPRv2 vector. Lentivirus packaging was performed as described [Myod1 gene was PCR-amplified and assayed with agarose gel electrophoresis. Clones with an extra smaller DNA band were considered as potentially mutated clones and their amplified Myod1 gene was further sequenced. Amplified PCR products with two bands also showed two heterozygous peaks in sequencing electropherograms, meaning possible deletion in one allele of Myod1 gene. Cell clones with heterozygous deletions were passaged, picked again for single cells. These single cells were cultured, DNA extracted and gene sequenced again, until a cell clone with homozygous gene deletion was identified. Two pairs of primers were used for genotyping: F0: ACTCCTCTGACAGGACAGGA, R0: TCTCGAAGGCCTCATTCACT; F1: TACTGTTGGGGTTCCGGAGT, R1: GCACATGCTCATCCTCACGA.gRNAs were designed from the first exon of mouse escribed . C2C12 cCells were collected by trypsinization and washed in PBS for twice. Each 300 μl of lysis buffer (10 mM Tris–HCl (pH 8.0), 0.4 M NaCl, 35 mM SDS, 2 mM EDTA) was added to re-suspend cells, followed by addition of 5 μl of proteinase K at 20 μg/ml. The mixture was incubated in waterbath at 55 °C for 4 h. Flocculent genomic DNA was precipitated by addition of 700 μl of absolute ethanol.Myod1 knockout C2C12 cells were analyzed with RNA-seq. Total RNAs of C2C12WT and C2C12Myod1−/− cells, each in triplicate, were prepared with TRIzol. Briefly, total RNAs were quantified and rRNAs were removed. After fragmentation of the remaining total RNAs, sequencing libraries were constructed with NEBNext® Ultra™ RNA Library Prep Kit for Illumina® , and sequenced with an Illumina NovaSeq platform. Raw data were processed for quality control and cleaned up to generate clean data, which were mapped against the mouse reference genome GRCm38 with Hisat2/Tophat software. Transcript sequence reads were assembled and quantified using Stringtie software. Differentially expressed genes (DEGs) were calculated with limma package. A gene was considered as differentially expressed if |log2FC|≥ 1 and meanwhile p-Value ≤ 0.05. Enrichment analyses were made using software KOBAS (http://kobas.cbi.pku.edu.cn), which covers seven pathway databases , five human disease databases , and the Gene Ontology database. Sequencing, signal processing and data analyses were performed by Beijing CapitalBio Technology Co., Ltd. . RNA-seq data were deposited to the Gene Expression Omnibus (GEO) under accession number GSE137507.Transcriptomic profiles of wild type and WT and C2C12Myod1−/− cells were cultured in DMEM containing 2% horse serum . For detecting neuron differentiation potential, C2C12WT and C2C12Myod1−/− cells were first cultured in Ndiff 227 medium for 6 days. After dissociation of C2C12Myod1−/− cells, both C2C12WT and C2C12Myod1−/− cells were treated with 2 μM retinoic acid (RA) for 24 h.For detecting muscle differentiation potential, C2C12Myod1−/− phenotype by introducing extraneous cDNA encoding MYOD1 into the cells, human MYOD1 cDNA was PCR-amplified from the vector pDONER223-hMYOD1  and subcloned to the sites ClaI/XbaI of pCS2 + eGFPmcs vector using primers Forward: ccgg atcgat CAG GAT ATG GAG CTA CTG TCG C, and Reverse: ccgg tctaga GAG CAC CTG GTA TAT CGG GTT G. The resulting plasmid encodes a fusion protein MYOD1-eGFP. To introduce the plasmids into C2C12Myod1−/− cells, 4 μg of either empty vector or MYOD1-eGFP plasmid was mixed with 1 × 106 cells in 200 μl of electroporation buffer in the Cell Line Nucleofector™ Kit V (Lonza. Cat. No.: #VCA-1003). Cuvette containing the mixture was placed in a Nucleofector™ 2b device (Lonza. Cat. No.: #AAB-1001) and electroporation was performed with program B-032. Afterwards, cells were re-suspended in DMEM medium plus 10% FBS. Twenty-four hours after electroporation, cells were cultured in DMEM containing 2% horse serum  for 5 days to detect the rescuing effect in C2C12Myod1−/− cells. Moreover, C2C12Myod1−/− cells that were replenished with MYOD1 were also tested for their tumorigenicity using soft agar colony formation and xenograft assays.In order to rescue C2C12A series of genes were analyzed for their conservativeness and expression patterns in tissues, in particular embryonic tissues.Homo sapiens’  and zebrafish ZFIN databases (https://zfin.org). Tissue expression in mouse was mainly searched from mouse database MGI (http://www.informatics.jax.org/). When expression pattern for a gene is not available in these databases, gene expression in mouse tissues was also determined according to description in the database Emage (http://www.emouseatlas.org/emage/home.php). In very rare cases, gene expression patterns in chicken were determined based on description in literatures. Expression only in neural precursor tissues, including neural ectoderm, neural plate, neural folds, neural tube or neural crest during early embryonic development, or later in central nervous system (CNS) or sensory nervous systems  was considered as neural specific. Expression was mainly in the neural tissues above but with weak expression in other tissues, for example, a gene with strong expression in the CNS and weak expression in somites in Xenopus tailbud embryos, was considered as enriched neural expression. Embryonic tissue expression of genes for the about 2600 human neuronal proteins , Amphimedon queenslandica (A. queenslandica) and Trichoplax adhaerens (T. adhaerens), whose genomes have been sequenced, using BLAST  sequence similarity searches against the non-redundant (nr) protein databases. The top hits of output sequences of these three lower species with significant similarity  were vice versa BLASTed against human and mouse protein databases. If the original input of human, mouse, Xenopus or zebrafish proteins whose genes with neural expression (either specific or non-specific) matched the BLAST output of human/mouse proteins using M. brevicollis, A. queenslandica or T. adhaerens sequences, then these M. brevicollis, A. queenslandica or T. adhaerens proteins were considered as proteins homologous to neural related proteins in vertebrates.To determine how these neural related genes are conserved during animal evolution, we compared the protein sequences of these genes with protein or peptide sequences of M. brevicollis are biased towards certain cell types in vertebrates, we analyzed gene homology between M. brevicollis and mammals, and analyzed the expression of conserved genes in vertebrate embryonic tissues. Gene homology was determined by reciprocal sequence comparison between all 9275 protein (peptide) sequences of M. brevicollis and human or mouse non-redundant (nr) protein databases using protein BLAST program, as described above. M. brevicollis protein (peptide) sequences, their entry numbers and entry names were downloaded from Uniprot (http://www.uniprot.org)  with M. brevicollis proteins. However, some M. brevicollis proteins have highly conserved domains or motifs, for example, those in protein families. It has been difficult to ascertain the homologs based on the conserved motifs or domains. Thus, these proteins were not included as vertebrate homologs. Subsequently, the homologous genes were searched for their expression in mainly embryonic tissues of mouse, Xenopus or zebrafish, as described above.In order to examine whether the genes in Besides these genes above, we also collected about 7000 genes that are expressed in non-neural tissues or whose expression patterns are unknown . 2 × 104 cells suspended in 200 μl of serum-free culture medium were added to the upper compartment, and 500 μl of culture medium containing 10% FBS was added in the lower compartment. Plates were then incubated at 37 °C for a time period, as indicated in the text. Afterwards, cells were fixed with 37% formaldehyde, followed by staining with 0.5% crystal violet for 10 min. Cells that did not migrate were removed. Migrated cells were washed with PBS and photographed.Experiments were performed as described . C2C12WT5 cells were added to Matrigel, which was submerged in the culture medium in the lower compartment. Plates were incubated at 37 °C for time periods as indicated in the text. Later on, cells were treated using the same manner as in the migration assay.For cell invasion assay, each 80 μl of Matrigel  was diluted in PBS (1:8) and distributed uniformly onto a 24-well transwell insert. 1 × 10t-test.Colony formation assay was performed as described . The top6 cells were injected, both mESCs and primNSCs formed tumor in all injected nude mice , endodermal  and ectodermal  differentiation were generally activated in tumor  differentiated into neuroectodermal cells, the primNSCs , 25, 28 Acta2, T, Desmin, Kdr) and endodermal tissue  differentiation  and endodermal  tissue differentiation  and more genes for neural stemness  were reduced  and neural stemness genes   were activated in tumor  Fig. a. Pluripted Fig. a. Actualnd mouse  besides MAP2 and SYN1, neural stemness genes CDH2, MSI1, NCAM1, SOX2/9, VIM and ZEB2   in wild type gene , a reagent inducing neuronal differentiation of NSCs/NPCs, did not alter the myotube phenotype  and neural stemness genes  in tumors than in C2C12Myod1−/− cells  were identified in these organisms  Tumors formed by mESCs and primNSCs in nude mice, which were injected at the same number (1×106 cells each mouse). (B-C) Difference in volumes (B) and weight (C) between tumors formed by mESCs and primNSCs. In (B) and (C), data are shown as mean±SEM. Significance of difference in tumor volume between two groups was calculated using two-way ANOVA-Bonferroni/Dunn test, and significance of difference in weight was calculated using two-tailed Student’s t-test. *p<0.05, **p<0.01. Figure S2. Tumor formation in nude mice by NE-4C cells injected via tail vein. (A) Tumor formation occurred in different parts of the body, as indicated by arrows. v, ventral view; d, dorsal view. (B) An HE stained section of a tumor dissected from the hind leg, showing the tumor was surrounded by muscle tissue. Objective magnification: 2×. Figure S3. Detection of tumorigenicity of MSCs via subcutaneous injection of cells into nude mice. No tumor formation could be observed in the mice in 114 days after injection. Figure S4. Detection of tissue origin in xenograft tumors using an antibody specific for human cell nuclei (HuNu). (A) IHC detection of HuNu in sections of tumors derived from different cancer cells. IHC without addition of HuNu antibody was used as negative controls. (B) IHC detection of HuNu in a section of the tumor derived from mouse NE-4C cells. Objective magnification: 4×; insets: 20×. Figure S5. Myod1 gene knockout in C2C12 cells using CRISPR/Cas9 and genotyping. (A) Diagram showing mouse Myod1 gene structure, sgRNA, and primer pairs used for genotyping. Numbers indicate position of bases, with the first base of transcription start site being assigned as +1. (B-C) PCR amplification of genomic DNA from a selected clone No. 132 and wild type (WT) C2C12 cells using primer pairs F0/R0 (B) and F1/R1 (C). M: DNA molecular size marker. (D) Sequencing electropherogram showing a deletion of 209 bp in Myod1 gene in the cells of selected clone, which led to a frameshift and premature translational stop. Underlined in the deleted region is the sequence matching sgRNA. (E) Conceptual translation of the truncated Myod1 gene and comparison with the wild type Myod1. (F) RT-PCR detection of Myod1 transcription in wild type and knockout cells using primer pair F0/R0. β-Act was detected as a loading control. RT-: Reverse transcription without transcriptase. M: DNA molecular size marker. (G) IF detection of Myod1 in wild type and knockout cells. Nuclei were counterstained with DAPI. (H) Morphological difference between wild type and knockout cells in C2C12 normal culture medium. Figure S6. Rescue of KO phenotype and characterization of neuronal differentiation potential of KO cells. (A) Rescuing effect of electroporation of a plasmid coding for MYOD1-eGFP fusion protein into KO cells. Electroporation of the empty vector pCS2+eGFPmcs was used as a control. Expression of MYOD1 and muscle cell markers was detected with IF. (B) Phenotypic difference of WT and KO cells cultured in serum-free medium for 6 days followed by RA treatment. (C-E) IF assays of muscle-specific (C) and neuron-specific marker (D and E) expression in WT and KO cells, as treated in (B). In (A) and (C-E), nuclei were counterstained with DAPI. Figure S7. The effect of re-introduction of MYOD1 on tumorigenicity of C2C12 KO cells. (A) IB detection of forced expression of MYOD1 in C2C12 KO cells. (B and C) The effect of forced expression of MYOD1 on the ability of colony formation in soft agar in a 10-day period. Significance in difference of colony formation (C) was calculated based on experiments in triplicate using two-tailed Student’s t-test. ***p<0.01. Colonies larger than 25 μm in diameter were counted. (E-G) shows the difference in tumor formation between control C2C12 KO cells and cells with forced MYOD1 expression in nude mice. (E) shows tumors in five of five mice injected with control cells but no tumor formation by cells with forced MYOD1 expression, and (F) and (G) show difference in tumor volume (F) and weight (G) between the two groups. In (F), significance of difference in tumor volume between two groups of mice was calculated using two-way ANOVA-Bonferroni/Dunn test. In (G), significance of difference in tumor weight was calculated using two-tailed Student’s t-test. Data are shown as mean±SEM. ***p<0.001. Figure S8. Association between the genes that are not expressed in vertebrate neural tissues with GO (A), pathway (B) and human diseases (C). Cancer is highlighted with a triangle. Figure S9. Average gene length of three groups of human genes. Table S1. Xenograft analysis on different types of cells. Table S2. Comparison of tumorigenicity between mESCs and primNSCs by injecting different numbers of cells in mice, as measured 36 days after injection. Table S3. Primers for RT-qPCR.Additional file 2: Table S4. 5283 neural specific genes or genes enriched in neural tissues, the evidence for expression patterns, and conservativeness of these genes.Additional file 3: Table S5. 738 genes with neural expression but not specifically or not mainly expressed in neural tissues, the evidence for expression patterns, and conservativeness of these genes.Additional file 4: Table S6. Human neuronal genes retrieved from GO database.Additional file 5: Table S7. Complete list of 9275 proteins in M. brevicollis, and homology of these proteins with mammalian proteins, and genes of homologous proteins with neural expression.Additional file 6: Table S8. 7238 genes that are not expressed in neural tissues or for which the expression patterns are unknown.Additional file 7: Table S9. Transcript lengths of 5283 neural genes, 738 non-specific genes and 7238 non-neural genes.Additional file 8: Table S10. 1801 downregulated genes and 1400 upregulated genes in C2C12Myod1−/− cells compared with C2C12WT


In contrast, odontoblastic differentiation of DPCs was suppressed by RORα knockdown. Moreover, we found that melatonin elevated the expression of odontogenic markers, which was accompanied by the upregulation of RORα (p < 0.001). Utilising small interfering RNA, we further demonstrated that RORα inhibition attenuated melatonin-induced odontogenic gene expression, ALP activity and matrix mineralisation (p < 0.01). Collectively, these results provide the first evidence that RORα can promote odontoblastic differentiation of DPCs and mediate the pro-odontogenic effect of melatonin.Dental papilla cells (DPCs), precursors of odontoblasts, are considered promising seed cells for tissue engineering. Emerging evidence suggests that melatonin promotes odontoblastic differentiation of DPCs and affects tooth development, although the precise mechanisms remain unknown. Retinoid acid receptor-related orphan receptor α (RORα) is a nuclear receptor for melatonin that plays a critical role in cell differentiation and embryonic development. This study aimed to explore the role of RORα in odontoblastic differentiation and determine whether melatonin exerts its pro-odontogenic effect via RORα. Herein, we observed that RORα was expressed in DPCs and was significantly increased during odontoblastic differentiation in vitro and in vivo. The overexpression of RORα upregulated the expression of odontogenic markers, alkaline phosphatase (ALP) activity and mineralized nodules formation ( Oral diseases such as dental caries, pulp and periodontal diseases, dental trauma and tooth loss are major public health problems worldwide, affecting the general health and quality of life of individuals . CurrentMelatonin (N-acetyl-5-methoxytryptamine) is mainly synthesised in and secreted by the pineal gland in a circadian rhythm. It has diverse physiological and pharmacological functions, including free radical scavenging, antiaging, immune regulation and reproduction control ,16,17,18The retinoid acid receptor-related orphan receptors (RORs), which are members of the steroid hormone receptor superfamily, comprise three distinct subtypes, RORα, RORβ, and RORγ, which exhibit a typical structure containing four functional domains—a highly variable N-terminal domain, a highly conserved DNA-binding domain, a C-terminal ligand-binding domain and a hinge domain . The molBoth dentin and bone are hard tissues, and their formation has many similarities. Moreover, in previous studies, we found that the pro-odontogenic effect of melatonin is not mediated by membrane receptors . Based oTo detect whether nuclear receptor RORs are expressed in rat DPCs (rDPCs), we performed reverse transcription polymerase chain reaction (RT-PCR) and agarose gel electrophoresis assay using specific primers for RORα, RORβ, and RORγ. RT-PCR data revealed that RORα and RORβ, but not RORγ, were expressed in rDPCs; moreover, the brightness of band of RORα was higher than that of RORβ a. Immunop < 0.05), which implied that RORα may be involved in odontoblastic differentiation  for 3 or 7 days and then the mRNA and protein levels of RORα and odontoblastic markers were detected by quantitative RT-PCR (qRT-PCR) and western blotting, respectively. After 3- and 7-day induction, the mRNA levels of odontoblast-related genes, including dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), and alkaline phosphatase (ALP), were remarkably elevated a. Consisntiation a,b.To further verify that RORα was increased during odontoblastic differentiation, we detected the protein expression pattern of RORα during odontoblastic differentiation in the first lower molar of 1-day postnatal Sprague-Dawley rats. Immunochemical analysis showed all stages of the differentiation of DPCs into odontoblasts a. RORα wTo determine the functional role of RORα in the regulation of odontoblastic differentiation, we overexpressed RORα in rDPCs by transfecting them with pcDNA3.1-RORα. Cells transfected with pcDNA3.1-NC were used as the negative control. The efficiency of RORα overexpression was confirmed by qRT–PCR and western blotting, which demonstrated a marked upregulation of RORα at both the mRNA and protein levels in the RORα overexpression group when compared with the negative control a. p < 0.001) (p < 0.001), whereas only DSPP and ALP were upregulated in the maintained medium group (p < 0.01)  were significantly increased in the OS group (< 0.001) b. When c < 0.01) b. Consis < 0.01) c. Moreov < 0.01) d. To detTo confirm the effect of RORα on odontoblastic differentiation, we knocked down RORα using small interfering RNA (si RORα) in rDPCs. Cells in the negative control group were transfected with si NC. After transient transfection, both the mRNA and protein levels of RORα were efficiently downregulated when compared with those of the negative control group a. si RORp < 0.001), whereas in the OS group, their transcriptional levels were significantly attenuated in response to RORα knockdown (p < 0.05). Similarly, under odontogenic induction, the protein levels of DSPP and DMP1 were increased on day 7, but they were downregulated in the RORα knockdown group when compared with the negative control group (p > 0.05). Accordingly, RORα acted as a positive regulator of the odontoblastic differentiation of DPCs.As shown in ol group c. ALP acol group d. Moreovol group e. Howeve−8 mol/L melatonin displaying the optimal stimulative effect [−8 mol/L melatonin was used for our subsequent studies. To determine whether RORα mediates the pro-odontogenic effect of melatonin on rDPCs, we first examined whether melatonin induces RORα expression. We treated rDPCs with OS medium in the presence or absence of 10−8 mol/L melatonin for 7 days. As shown in p < 0.001), along with the apparent upregulation of DSPP, DMP1, and ALP (p < 0.05). Previous studies of our group have suggested that under odontogenic induction, melatonin promotes the odontoblastic differentiation of rDPCs and matrix mineralisation in a dose-dependent manner, with 10e effect ,32. Thus−8 mol/L melatonin for 7 days. Compared with the negative control group, the mRNA levels of DSPP, DMP1, and ALP were notably downregulated in the RORα knockdown group (p < 0.001)  e. Furthe< 0.001) f. These In this study, we explored the role of the nuclear receptor RORα in odontoblastic differentiation. First, we demonstrated that endogenous RORα was expressed in rDPCs and was significantly upregulated during odontoblastic differentiation both in vitro and in vivo. Second, RORα overexpression was shown to promote the odontoblastic differentiation of rDPCs, whereas RORα knockdown inhibited it. Finally, our results revealed that RORα mediated the pro-odontogenic effect of melatonin. Collectively, our findings provide the first evidence that the nuclear receptor RORα is a novel positive regulator of the odontoblastic differentiation of DPCs and is a critical mediator in melatonin-induced odontoblastic differentiation.Nuclear receptors, as DNA-binding transcription factors, can regulate the expression of specific target genes at the transcriptional level. These genes play fundamental roles in cell proliferation, differentiation, and embryonic development . MultiplOdontoblastic differentiation is a critical process of tooth development. During this process, DPCs are first induced to become preodontoblasts, followed by their elongation and polarisation to become immature odontoblasts, finally differentiating into mature odontoblasts. Differentiated odontoblasts, including immature and mature odontoblasts, synthesise and secrete extracellular matrix proteins to produce dentin . The proMounting evidence has shown the key role of RORα in both cellular differentiation and bone metabolism. Previous functional studies have demonstrated that RORα overexpression can improve the expression of osteogenic markers, including ALP, bone sialoprotein (BSP), DMP1, osteocalcin, and collagen type I, and promote the formation of mineralized nodules during osteogenesis, whereas RORα suppression inhibits these responses ,46,67. M−8 mol/L, promotes the differentiation of DPCs into odontoblasts, although, the precise mechanism remains unknown [−8 mol/L melatonin enhanced the expression of DSPP, DMP1, and ALP, which is consistent with the findings of previous studies [Initially, RORα was considered an orphan nuclear receptor without endogenous ligands; however, after a series of studies, melatonin is now regarded as a moderate-affinity ligand for RORα ,71. Alth unknown ,32,78. M unknown ,20,21. H unknown . Next, w studies ,32. Nota studies ,32. Give studies ,80,81, fIn conclusion, the present study provides the first evidence that the nuclear receptor RORα is a positive regulator of odontoblastic differentiation and an important mediator of melatonin-induced DPC differentiation into odontoblasts. These findings not only elucidate the downstream mechanism of the pro-odontogenic effect of melatonin but also provide a potential target for dental tissue regeneration.3 in size). The tissue pieces were then seeded on 10-cm culture dishes and maintained in α-minimal essential medium  containing 20% fetal bovine serum , 100 U/mL penicillin, and 100 μg/mL streptomycin  at 37 °C in 5% CO2 humidified air. Upon reaching 80–90% confluence, cells were passaged using TrypLE  and purified by a distinct digestion method. Cells within 3–4 passages were used for subsequent experiments.All animal experiments were approved by the Ethics Committee of Zhongshan School of Medicine, Sun Yat-sen University, China (No.2017-218). The dental papilla was gently isolated from the first molar of 1-day postnatal Sprague-Dawley rats  under a stereomicroscope  and minced into small pieces . The induction medium was changed every 2 days. The cells were harvested for qRT-PCR, western blotting, ALP activity assays, and alizarin red staining at different time points.4 cells per well. Upon reaching 30–50% confluence, small interfering RNA (siRNA) targeting rat RORα  or negative control siRNA  were transfected into DPCs at a final concentration of 100 nM with RNAFit transfection reagent  for 8 h according to the manufacturer’s protocol. To overexpress RORα expression, DPCs were seeded in 12-well plates at a density of 1.5 × 105 cells per well. The next day, when cells reached 60–80% confluence, they were transfected with 1 μg expression vector encoding rat RORα  or 1 μg empty vector  per well using NeofectTM DNA transfection reagent  according to the manufacturer’s instructions. Twenty-four hours after transfection, the serum-free medium was replaced with maintained medium or OS medium.To knockdown RORα expression, DPCs were seeded in 12-well plates at a density of 7.5 × 10DPCs seeded on 35 mm glass bottom dishes were fixed with 100% cold methanol (−20 °C) for 5 min at room temperature and were blocked in 5% bovine serum albumin (BSA) for 1 h. Subsequently, DPCs were incubated with anti-RORα primary antibody  at 4 °C overnight, followed by incubation with Dylight 488-conjugated secondary antibody  for 1 h at room temperature in the dark. The nuclei were stained using 4-6-diamidino-2-phenylindole  for 5 min in the dark. The cells were then visualised under a laser scanning confocal microscope .The mandibles were dissected from 1-day postnatal Sprague-Dawley rats , fixed in 10% buffered paraformaldehyde for 48 h, and decalcified with 10% ethylenediaminetetraacetic acid (pH 7.4) for 5 days. After dehydration and paraffin embedding, the tissues were cut into 5-μm sagittal sections. Tissue sections were deparaffinized, rehydrated, and heat-retrieved in 0.01 mol/L citrate buffer (pH 6.0) for 15 min at 100 °C followed by cooling at room temperature. Subsequently, they were incubated in 3% hydrogen peroxide for 15 min at room temperature, blocked with 5% BSA for 1 h, and then incubated with anti-RORα primary antibody  at 4 °C overnight. After washing with PBS, the specimens were incubated in Polymer Helper  and horseradish peroxidase (HRP)- anti-rabbit IgG  at 37 °C for 20 min and then visualised using diaminobenzidine in the dark and counterstained with haematoxylin. Finally, the slides were dehydrated, transparentised with dimethylbenzene, and sealed using a mounting medium. The slides were observed using a digital pathology slide scanner .Total RNA was isolated from DPCs using an RNA-Quick purification kit  according to the manufacturer’s instructions. For qRT-PCR, first-strand cDNA was synthesised using PrimeScriptTM RT Master Mix Kit  and then amplified with SYBR Green I Master Mix  in the LightCycler 480 Real-Time PCR System . The relative mRNA expression of target genes was normalised to that of glyceraldehyde-3-phosphate dehydrogenase. For the agarose gel electrophoresis assay, amplified RT-PCR products were synthesised using the HiScript II One Step RT-PCR Kit  according to the manufacturer’s protocol, then separated on a 2% agarose gel and visualised with a gel imaging system . The specific primers used in this study are listed in Total protein was extracted from the cells using a radio-immunoprecipitation assay lysis buffer (RIPA) supplemented with protease and phosphatase inhibitors  and measured with a bicinchoninic acid (BCA) protein assay kit . Equal amount of protein lysates (20–30 μg/lane) were separated by 4–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes . Then, the membranes were blocked with 5% non-fat milk for 2 h at room temperature. Subsequently, they were incubated overnight at 4 °C with the following primary antibodies: polyclonal rabbit anti-RORα antibody , monoclonal mouse anti-DSPP antibody , polyclonal rabbit anti-DMP1 antibody , and monoclonal mouse anti-β-actin . After washing with tris-buffered saline and tween 20 (TBST), the membranes were incubated with the corresponding HRP-conjugated secondary antibodies  at room temperature for 1 h. The immunoreactive bands were detected using chemiluminescence detection reagents  and visualised using an ImageQuant LAS 4000 mini system . The intensities of the bands were quantified using ImageJ 1.36b .DPCs were cultured in OS medium for 7 days. ALP activity in cellular lysates was measured using an ALP activity detection kit  according to the manufacturer’s instructions. The protein concentration was quantified using a BCA protein assay kit . One unit of ALP activity was defined as the amount that liberated 1 mol p-nitrophenol per mg protein.DPCs were seeded into 12-well plates and cultured in OS medium for 7 days. After fixation in 4% paraformaldehyde for 30 min, DPCs were stained with 1% alizarin red staining solution  for 5 min at room temperature and were then rinsed with distilled water. The matrix calcium deposition was scanned using an inverted phase-contrast microscope . To quantify the mineralized nodules, the stained cells were incubated in 0.1M hexadecylpyridinium chloride monohydrate  for 30 min. The absorbance of the supernatant was then measured at a wavelength of 562 nm.t-test. Multiple group comparisons were conducted by one-way analysis of variance followed by Fisher’s least significant difference post hoc test. Statistical analysis was performed using SPSS 22.0 software  and p < 0.05 was considered statistically significant.All data are presented as the mean ± standard deviation of triplicate independent experiments. Two-group comparisons were assessed using Student’s two-tailed


Melodinus henryi is a good source of terpenoid indole alkaloids, and traditionally used as a folk medicine in the treatment of meningitis and fracture. In order to further exploit their potential uses, its anti-inflammatory and immunosuppressive activities, safety evaluations and chemical profiles have been illustrated. Compared to the crude methanol extract from M. henryi and its non-alkaloidal fraction, the total alkaloidal fraction (MHTA) had the strongest anti-inflammatory and immunosuppressive activities. In the acute oral toxicity assay, the half lethal dose (LD50) of MHTA was more than 2000 mg/kg. The sub-acute toxicity assay for consecutive 28 days exhibited MHTA at a lower concentrations of less than 500 mg/kg might be regarded as safe, and might damage spleen, liver, kidney, and heart when the dose is higher than 1000 mg/kg. In addition, a phytochemical investigation on MHTA led to the isolation of 15 monoterpenoid indole alkaloids. Thus, in regard with the potent side effects of MHTA, it should be used with caution in the development of phytomedicine. Medicinal plants have a long history in the prevention and treatment of human various diseases due to their health promoting potentials . The natMelodinus (Apocynaceae) are rich in terpenoid indole alkaloids (TIAs) and widely distributed in the tropical and sub-tropical regions [M. henryi is a perennial vine mainly distributed in China, Thailand, and Burma [M. henryi present a series of structurally novel TIAs in the meric and dimeric forms, such as melodinines A-G [Plants of the genus  regions . Among tnd Burma  and has nd Burma . Phytochines A-G  and V [1ines A-G , melodinines A-G  and W1-Wines A-G , melohenines A-G , melodinines A-G , 16. Somines A-G , 11, 17.M. henryi were evaluated their biological activities. Furthermore, the toxicological evaluations of MHTA were performed by assessing acute and sub-acute toxicities in vivo. The chemical constituents of MHTA were carried out by column chromatographic methods and identified by a combination with NMR and MS analysis.Previous studies of this plant are mainly focused on the discovery of TIAs with novel structures, and significant cytotoxic activity. However, the information of bioactive components responsible for the treatment of inflammatory diseases remains unclear. In order to further exploit its potential uses, it is necessary to illustrate the bioactive extract, evaluate its toxicity, investigate its chemical constituents. Thus, the obtained total alkaloidal extract (MHTA) and non-alkaloidal extract (MHNA) from M. henryi. Among them, the MHTA showed the highest inhibition of nitric oxide release  as a positive control. As showed in Fig. p > 0.05) when compared to those of the control group . The food and water intakes and weight gain decreased slightly with no significant difference (p < 0.05), whereas a similar phenomenon was observed at 500 mg/kg group but was not significant (p > 0.05) .The organs of all animals were collected and weighted after the experiment, and the relative organ coefficients were calculated as shown in Table p < 0.05). Besides, the LYM% and MON in females treated with 500 mg/kg MHTA as well as males and females that received 1000 mg/kg MHTA were obviously increased (p < 0.05) than those in mice of the control group, The GRA% and RBC significantly decreased in mice of 1000 mg/kg (both sexes) and RBC also significantly decreased in females of 500 mg/kg (p < 0.05). The MCV levels in 1000 mg/kg MHTA significantly increased than the control group. PLT levels significantly increased in females at dose of 500 mg/kg and all animals at dose of 1000 mg/kg, while PCT% significantly increased in the 1000 mg/kg MHTA group (p < 0.05). In addition, no significant changes were observed in other biochemical parameters between the control and MHTA treated mice.The hematological parameters in animals are shown in Table p < 0.05). Meanwhile, ALB levels were significantly decreased in males treated with 500 and 1000 mg/kg MHTA (p < 0.05), and that in females were also decreased with no statistical significance. The levels of AST in 1000 mg/kg MHTA group and ALT, BUN and CRE in 500 and 1000 mg/kg MHTA groups significantly increased (p < 0.05) compared to the control group. Na concentrations in females treated with 1000 mg/kg MHTA statistically decreased (p < 0.05), while K values markedly increased  (p < 0.05) in comparison with the control ones. Moreover, the parameters such asTBIL, TG, TC, GLU and Cl were found in a normal range with no significant difference to the control.In sub-acute toxicity, the serum biochemical parameters are summarized in Table The histopathological studies of the vital organs  of the control group, 125 and 1000 mg/kg MHTA groups were performed. The photomicrograph results of those vital organs were as follows: for liver parenchyma, normal morphology of hepatic lobules were observed in the control Fig. A1 and lo1H NMR, 13C NMR and HRESIMS spectroscopic data (13C NMR data in Table 1–3) [4) [5) [6) [7) [8) [9) [10) [O-methylepivincanol (11) [12) [13) [14) [15) [In order to provide the information of chemical investigations, a phytochemistry investigation of MHTA were performed. A total of 15 TIAs were isolated by a series of column chromatographic separations and purification via MPLC, Pre-HPLC, and sephadex LH-20 Fig. . Their sble 1–3) , taberso [4) [5) , 11-meth [5) [6) , lochner [6) [7) , kopsini [7) [8) , tubotai [8) [9) , ( −)-eb[9) [10) , O-methynol (11) , scandin11) [12) , 10-hydr12) [13) , melaxil13) [14) , melosca14) [15) .Fig. 4ChM. henryi has been proved to be a good source of TIAs and has been used as a folk medicine for the treatment of some inflammatory diseases. Previous investigations on M. henryi are mainly focused on the isolation of TIAs with novel structures, evaluation of their cytotoxic activities on the human cancer cell lines. However, the researches on the anti-inflammatory potentials of the bioactive extract, and their phytochemicals have not been exploited. In this paper, M. henryi was extracted by methanol to obtain the crude extract (CE), and then processed to yield the non-alkaloidal extract (MHNA) and total alkaloidal extract (MHTA). We firstly reported the anti-inflammatory and immunosuppressive activities of M. henryi. The result showed that MHTA had the strongest anti-inflammatory and immunosuppressive activity than CE and MHNA.People also depend on traditional herbal medicine to treat diseases in developing countries . Plants 50 of the MHTA can be considered greater than 2000 mg/kg, which was regarded as practically low toxic.In order to provide the information of MHTA for the treatment of disease, the preclinical safety assessment is necessary to performed. The acute toxicity test was initially performed in mice of females due to the higher sensitivity than males, as it provides data to guide the other toxicity tests . There wp < 0.05). The body weight of all animals in the 1000 mg/kg group was significantly lower compared to the control (p < 0.05), meaning that high dose of MHTA may be toxic to mice. The organ coefficient is a crucial detection index in the evaluation of drug safety, and provides a quantitative reference for drug evaluation. When the relative organ weights significantly changes, some underlying disease or damage  may happen [p < 0.05), suggesting that doses of 1000 mg/kg MHTA might induce liver damage. The liver played a key role in the biotransformation, detoxification, and synthesis of serum proteins [In the sub-acute toxicity experiments, the decreases of food consumption and water intake were observed in the dose of 1000 mg/kg during the experiment period (y happen . In thisproteins . To furtp < 0.05) in 500 and 1000 mg/kg groups were most likely induced by the body inflammation, which could be partly confirmed by the significant increases of LYM and MON. RBC and HGB levels were also significantly decreased at high doses of MHTA, and was in agreement with the previously reported damages in spleen [The hematological parameters could provide direct evidence for toxicity assessment in humans and animals . The sign spleen . In addin spleen . Pervioun spleen . Therefon spleen . Moreoven spleen . Daily op < 0.05), suggesting that the ability of protein synthesis may be reduced. ALB shows a downward trend, suggesting the rise of GLB in the 500 and 1000 mg/kg groups. The increase of GLB is a sign of activation of the immune system, indicating the occurrence of inflammation in the body [In the analysis of biochemical indicators, the TP, composed of ALB and GLB, in the groups of 500 and 1000 mg/kg decreased significantly , indicating that high doses of MHTA might damage the liver. The observed increase in the relative liver organ weight and reduction of RBC confirmed this assumption, which was consistent with the reported result [p < 0.05). Meanwhile, the content of sodium ions significantly decreased, while the content of potassium ions significantly increased in the highest dose group, indicating that the kidney was damaged and glomerular filtration function injury resulted in the impairment of ion balance in the blood [The liver is the main organ responsible for natural products metabolism and increased indicative activities of serum enzymes AST and ALT are considered sensitive markers of hepatocellular damage . Higher d result . The kidd result . The levhe blood . This fiBiochemical data of high concentrations of MHTA revealed side effects to vital organs in mice, the histopathological studies provide a supportive evidence for hematological and biochemical analysis. The histopathology changes mainly occurred in the liver, kidney, spleen, and heart at high doses of MHTA (1000 mg/kg). Meanwhile, the severity of the dose is related to increased treatment duration, suggesting accumulative toxic effect of the alkaloids.M. henryi so far [1–3), aspidosperma-type alkaloids (4–8), condylocarpan-type alkaloid (9), eburna-type alkaloids (10–11), and quinoline-type alkaloids (12–15).Furthermore, there is an urgent need for phytochemical investigation on bioactive ingredients of this plant to understand the chemical components of MHTA. Phytochemical research showed that about 66 alkaloids of different structures have been isolated from i so far –14, 19. 4, 5, 6 and 8 had significant cytotoxic activities on human cancer cell lines, while other types of compounds have no obvious cytotoxic activity [Melodinus plants, such as 15-dihydroscandine, 15β-hydroxy-14,15-dihydroscandine, melaxillines A and B, melodinine T, scandine, 11-hydroxytabersonine, and O-methyl-Δ14-vincanol-3-one exhibited significant anti-inflammatory activity [4) could attenuate LPS-induced acute lung injury and inhibited LPS-mediated macrophage activation in vitro [5) showed inhibitory effect on NO production [9) showed good inhibitions on COX-1 and COX-2 in anti-inflammatory assay in vitro [14), scandine (12), and meloscandonine (15) significantly inhibited in release of β-glucuronidase [Previous reports on the cytotoxic activities of the compounds showed that aspidosperma-type alkaloids activity , 19. Theactivity , 44, 45.in vitro , 11-hydroduction , tubotaiin vitro , and melronidase , suggestAs we all known, the combined effects  in the mixture of compounds are exist in bioactivity evaluation , and theM. henryi to fill its gap in the traditional applications. MHTA showed the strongest anti-inflammatory and immunosuppressive activities than CE and MHNA. In order to obtain the safety information of MHTA in the treatment of inflammatory diseases, the acute and sub-acute toxicity evaluations were conducted in animal models. It did not display any abnormal behaviors and mortality when administered in a single dose of 2000 mg/kg. However, the repeated administration of sub-acute toxicity experiment, MHTA (greater than 500 mg/kg) might selectively damage the organ tissues, such as kidney, heart, liver, and spleen, suggesting caution is required for the use. In addition, 15 pure TIAs were also isolated and further performed as standards for the control of the MHTA quality. Therefore, the present work provides deeper knowledge about the species M. henryi, expands the applications of the MHTA on the inflammatory diseases under a safe dose as a quality controllable drug candidate.In the present study, we firstly performed the anti-inflammatory and immunosuppressive activities of M. henryi (10 kg) was collected from Jinghong of China (GPS: 21°32′N/100°28′E) in October 2017, and identified by Dr. Y.P. Liu, Kumming Institute of Botany, CAS. A voucher specimen (No. Cheng20171008-03) was stored in the cool and dry environment of the faculty of agriculture and food, Kunming University of science and technology.The aerial part of 3OH for three times (48 h at a time). The extract solvent was collected and then concentrated under vacuum to obtain a crude extract (CE). The residue was dissolved in acidic aqueous solvent including 0.3% hydrochloric acid (v/v) and partitioned with EtOAc  for three times. The acidic aqueous solution was adjusted with 5% ammonia to have a pH value about 9–10. The alkaline aqueous solution was then partitioned with EtOAc  for three times. After collection and concentration of EtOAc solvent, the total alkaloidal extract was obtained (MHTA). The remaining part was evaporated under reduced pressure at 50 °C to obtain the non-alkaloid extract (MHNA).The sample was cut into pieces, powdered and extracted with 90% aqueous CH2 at 37 °C.RAW264.7 macrophages were purchased from Kunming cell bank, CAS, and were cultured in DMEM with 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) under a humidified atmosphere containing 5% COCytotoxicity of CE, MHTA and MHNA on RAW264.7 macrophage cells was performed by MTT assay . Then th6 cell/mL. Then cells were exposed to the test samples in the presence of concanavalin A (Con A) and lipopolysaccharides (LPS), respectively, using the Con A-/LPS-treated splenocytes as the experimental control, dexamethasone (DXM) as positive control. After incubation for 44 h, 10 μL of CCK-8 was added and incubated for another 4 h and the absorbance was measured at 450 nm by using a microplate reader . Finally, the inhibitory effects of different sample were analyzed.A previously reported CCK-8 method was used to carry out the immunosuppressive activity . In brieSixty mice  weighing between 18 and 22 g provided by Kunming Medical University  were used. The animals were acclimated in a specificpathogen free (SPF) laboratory , and fed a basic animal feed  for 7 days. The toxicological evaluations were conducted according to the recommendations from the National Institutes of Health Guide for Laboratory Animal Care and Use.50) was assessed under the principle of “Acute Toxic Class Method”.Twenty female mice were used to evaluate the acute toxicity study due to the OECD423 guideline . The aniThe sub-acute toxicity experiment was performed in mice  according to the OECD guideline . The micThe hematological parameters, including percentage of lymphocytes (LYM%), lymphocyte (LYM), red blood cell (RBC), red blood cell volume (HCT), white blood cell (WBC), monocytes (MON), mean corpuscular hemoglobin (MCH), percentage of granulocytes (GRA%), mean platelet volume (MPV), mean corpuscular volume (MCV), platelet (PLT), hemoglobin (HGB), and platelet pressure (PCT) were detected by an automatic blood cell analyzer HF-3800 Plus .The blood samples were centrifuged at 4000 rpm for 10 min at 4 °C for biochemical analysis. The serum biochemical parameters, including aminotransferase (ALT), aspartate transaminase (AST), total protein (TP), albumin (ALB), triglycerides (TG), total cholesterol (TC), total bilirubin (TBIL), glucose (GLU), alanine blood urea nitrogen (BUN), creatinine (CRE), potassium (K), sodium (Na), and chloridne (Cl) were analyzed by using commercial kits .The tissues  were collected, and weighed to calculate organ coefficients . All tis18MPLC column with a gradient solvent MeOH/H2O (from 20:80 to 100:0) to afford six fractions (A-F). Fraction A (2.6 g) was isolated on a silica gel column (30:1) with petroleum ether-acetone  and then purified by Sephadex LH-20 (MeOH) column and a preparative HPLC (CH3CN/H2O: 50:50) to yield compound 4 (9.2 mg) and 5 (28.7 mg) 7 (4 mg), 8 (13.6 mg), and 10 (28.4 mg). Fr.B (9.7 g) was chromatographed by MPLC  to yield three subfractions (B1–B3). Compounds 2 (33 mg) and 6 (19.6 mg) were obtained from sbufraction B-3 (203 mg) by a preparative HPLC purification . Fr.C (7.7 g) was separated by MPLC  and further purified by silica gel column using a petroleum ether-acetone (3:1) to give compounds 1 (15 mg), 13 (17 mg) and 11 (29 mg). Fr.D (2.6 g) was separated by MPLC with CH3OH/H2O (30:70–50:50) and further resolved by HPLC withCH3OH/H2O (50:50) to yield compounds 3 (28 mg), and 14 (3.6 mg). Fr.E (6.3 g) was separated by MPLC with CH3OH/H2O (20:80–60:40) to get compound 9 (2.9 mg) and 12 (21.2 mg). Fr.F (30 g) was separated by MPLC with CH3OH/H2O (20:80–50:50) and purified by a silica gel column  to afford compound 15 (8.8 mg).The crude alkaloidal extract (98.0 g) was separated on a RP-Cp < 0.05 was considered significant.All tests were carried out in triplicate and data were expressed as mean ± SD (standard deviation). Statistical analysis was performed with one-way analysis of variance (ANOVA). Statistical significance of differences between the groups was assessed using the Student's t-test. The


Tetracentron sinense is an endemic and endangered deciduous tree. It belongs to the Trochodendrales, one of four early diverging lineages of eudicots known for having vesselless secondary wood. Sequencing and resequencing of the T. sinense genome will help us understand eudicot evolution, the genetic basis of tracheary element development, and the genetic diversity of this relict species.T. sinense genome. We assemble the 1.07 Gb genome sequence into 24 chromosomes and annotate 32,690 protein-coding genes. Phylogenomic analyses verify that the Trochodendrales and core eudicots are sister lineages and showed that two whole-genome duplications occurred in the Trochodendrales approximately 82 and 59 million years ago. Synteny analyses suggest that the γ event, resulting in paleohexaploidy, may have only happened in core eudicots. Interestingly, we find that vessel elements are present in T. sinense, which has two orthologs of AtVND7, the master regulator of vessel formation. T. sinense also has several key genes regulated by or regulating TsVND7.2 and their regulatory relationship resembles that in Arabidopsis thaliana. Resequencing and population genomics reveals high levels of genetic diversity of T. sinense and identifies four refugia in China.Here, we report a chromosome-scale assembly of the T. sinense genome provides a unique reference for inferring the early evolution of eudicots and the mechanisms underlying vessel element formation. Population genomics analysis of T. sinense reveals its genetic diversity and geographic structure with implications for conservation.The The online version contains supplementary material available at 10.1186/s13059-020-02198-7. Eudicots are the largest and most diverse group of angiosperms, containing 75% of all angiosperm species . Recent Tetracentron sinense and Trochodendron aralioides). Although there has been controversy about the phylogenetic position of the Trochodendrales  were cut with razor blades and then softened in 2% ethylenediamine at 60 °C for 2 days. Fifteen-micrometer-thick transverse, radial, and tangential sections were cut with a sliding microtome. Thereafter, sections were stained sequentially with 1% aqueous safranin solution, rinsed, and finally mounted beneath cover slides. Images were captured by a light microscope .Sectioning blocks of T. sinense were collected from the upper slope of Fenshuiling, Yunnan province, China. Stem samples were boiled in water and sectioned on a sliding microtome, then cut into pieces about 1 cm in length and fixed overnight in 2.5% glutaraldehyde, followed by washing with 0.1 M PBS (pH 7.2) three times. After sequential dehydration in 30%, 60%, 80%, 90%, 95%, and 100% ethyl alcohol (15 min each), the samples were incubated in liquid carbon dioxide at 0 °C and 20 °C sequentially (20 min each), then subjected to critical point drying at 35 °C for 15 min using a Tousimis autosamdri-825 critical point dryer. Vacuum coating was performed using a HITACHI MC 10003 Sputter Coater under 10 Pa and 1000 V for 2 min. The images were taken with an accelerating voltage of 5–10 kV using a Hitachi SU80202 SEM.For scanning electron microscope (SEM) analysis, the stem materials from T. sinense were imaged by X-ray microtomography using a Bruker Skyscan microCT system 1172  with a 20–100 kV, 10 W X-ray source. The microCT device was set to operate at a voltage of 33 kV and power of 7 W. The sample was scanned at a voxel size resolution of 1.792 μm. The software Skyscan’s NRecon program  was used to perform the reconstruction of the projection images, resulting in a volume of 3304 × 3304 × 1806 μm from 1032 slice images with a resolution of 1888 × 1888 × 1086 pixels each. Imaris volume rendering and analysis software  was used to render images from the three-dimensional data set.Wood blocks of AtVND7, AtLBD18/AtLBD30, AtMYB46/AtMYB83, AtCESA4, AtCESA8, and AtXCP1/AtXCP2 are key genes involved in the development of vessel elements in A. thaliana [. These genes belong to the VNS, LBD, R2R3-MYB, CesA, and PLCP gene families [T. sinense genes orthologs, genes of VNS, LBD, CesA, and PLCP gene families from 22 representatives of major plant lineages, including 19 species of angiosperms used in the phylogenetic analysis of species, 3 gymnosperms , 1 lycophyte (Selaginella moellendorffii), and 1 moss (Physcomitrella patens/ Physcomitrium patens), were obtained using the hmmsearch command in the HMMER package [R2R3-MYB transcription factor homologs were obtained by a BLASTp search with e-value 1e−5 (Extended Data Information). Then, identical and defective sequences were identified and manually eliminated in BioEdit. Protein sequences or nucleotide sequences were aligned using MAFFT [thaliana , 49. Thefamilies –48, resp package  with theng MAFFT  and the ng MAFFT .AtVND6, AtVND7, TsVND6.1, TsVND6.2, and TsVND7.2 fusions with GFP. To this end, the AtVND6, AtVND7, TsVND6.1, TsVND6.2, and TsVND7.2 coding region fragments were amplified from wild-type (WT) complementary DNA (cDNA) of A. thaliana and T. sinense by using PCR with gene-specific primer sets . Images were captured using a Zeiss LSM 880 Airyscan  for confocal microscopy. An argon laser (488-nm line) was used for GFP excitation and the emission was captured at 510–550 nm (green).To investigate the subcellular localization of the VNDs, we created transgenic plants expressing TsVND6.1, TsVND7.1, TsVND7.2 or TsLBD30a transcription factors and the promoters of TsVND7.2, TsMYB, TsCesA4, TsCesA8, TsXCPa, TsXCPb, and TsXCPc (about 500 bp in length from the translation start site). All the promoters and transcription factor genes were amplified by PCR using TransStart FastPfu DNA polymerase . Using BamHI/EcoRI or EcoRI/SacI digestion and T4 ligation reactions, the transcription factor genes were inserted into the AD vector  and the promoters were inserted into the BD vector . Then, the vectors were transformed into yeast strain Y187 and the transformed yeast was cultured for 2–3 days on SD/-Trp/-His double deficiency medium. Single clones harboring different vector combinations were transferred to SD/-THL medium containing 10, 20, 30, 40, 50, and 60 mM 3-AT and incubated at 30 °C for 0.5–8 h, and the color changes in the yeast cells were observed. All primers are shown in Table S5 in Additional file Yeast one-hybrid analysis was performed to investigate the interactions between TsVND6.1, TsVND7.1, TsVND7.2, TsLBD30a, and TsLBD30b to the TsVND7.1, TsVND7.2, TsMYB, TsCesA4, TsCesA8, TsXCPa, TsXCPb, and TsXCPc promoters, the pGreenII 0800-LUC reporter vector and pGreenII 62-SK effector vector (Biovector Science Lab) were used. The TsVND7.1, TsVND7.2, TsMYB, TsCesA4, TsCesA8, TsXCPa, TsXCPb, and TsXCPc promoters were cloned into pGreenII 0800 at the HindIII and BamHI sites to fuse them with the LUC reporter gene . The coding sequences for TsVND6.1, TsVND7.1, TsVND7.2, TsLBD30a, and TsLBD30b were cloned into the pGreenII 62-SK vector at the EcoRI and HindIII sites. The recombinant vectors were co-transformed into A. tumefaciens GV3101. After centrifugation, the bacteria were collected and resuspended at A600 = 0.6 in infection solution  for infiltration. The prepared suspensions were infiltrated into N. benthamiana leaves and grown in the dark for 48 h. The leaves were then sprayed with luciferin and kept in the dark for 10 min to quench the fluorescence. A CCD imaging apparatus  was used for image capture.To assay the activating activity of T. sinense trees, was extracted and sequenced using Illumina technology. Raw data were filtered using Fastq [T. sinense genome using BWA-MEM [DNA of 55 individuals from 6 populations, which represent most of the known distribution of ng Fastq . Clean r BWA-MEM . Duplica BWA-MEM . FreeBay BWA-MEM  was usedK (K = 1 to 10). We performed 5-fold cross-validation to determine the most likely number of clusters. The GCTA tool [T. aralioides was used as an outgroup.Ancestry estimation of individuals was inferred using ADMIXTURE  with difCTA tool  was usedCTA tool  based onPopulation genetic parameters, including nucleotide diversity (π)  and the Additional file 1. Supplementary Notes, Supplementary Figs. S1–S14, and Supplementary Tables S1–S5.Additional file 2. Review history.


Syzygium cumini is a flavonoid-rich medicinal plant that contains myricetin and gallic acid. Little is known about the potential antiplatelet properties of S. cumini and its constituent flavonoids.Flavonoids have been characterized as a prominent class of compounds to treat thrombotic diseases through the inhibition of thiol isomerases. S. cumini leaf and its most prevalent polyphenols, myricetin and gallic acid.To evaluate the antiplatelet effects and mechanism of action of a polyphenol-rich extract (PESc) from In vitro platelet adhesion and thrombus formation as well as in vivo bleeding time were performed. Finally, myricetin was incubated with recombinant thiol isomerases to assess its potential to bind and inhibit these, while molecular docking studies predicted possible binding sites.PESc, myricetin, and gallic acid were incubated with platelet-rich plasma and washed platelets to assess platelet aggregation and activation. in vitro without affecting hemostasis in vivo. Fluorescence quenching studies suggested myricetin binds to different thiol isomerases with similar affinity, despite inhibiting only protein disulfide isomerase (PDI) and ERp5 reductase activities. Finally, molecular docking studies suggested myricetin formed non-covalent bonds with PDI and ERp5.PESc decreased platelet activation and aggregation induced by different agonists. Myricetin exerted potent antiplatelet effects, whereas gallic acid did not. Myricetin reduced the ability of platelets to spread on collagen, form thrombi PESc and its most abundant flavonoid myricetin strongly inhibit platelet function. Additionally, myricetin is a novel inhibitor of ERp5 and PDI, unveiling a new therapeutic perspective for the treatment of thrombotic disorders. In platadhesion . Besidesadhesion . Particutivation . Therefotivation  and natutivation . Accorditivation .Syzygium cumini (L.) Skeels (Myrtaceae) is a worldwide cultivated medicinal plant, popularly known as jamun, black plum, jambolan, or jambolão (S. cumini has been proposed as a prominent source of bioactive compounds against cardiometabolic disorders (S. cumini has been shown to inhibit the hyperactivation of platelets from diabetic patients (S. cumini (PESc) leaf, which consisted of gallic acid, quercetin, myricetin, and its derivatives myricetin-3-α-arabinopyranoside and myricetin deoxyhexoside  were purchased from Sigma-aldrich . PAPA-NONOate was purchased from Tocris . PE/Cy5 anti human CD62P and PAC-1 FITC antibodies were purchased from BD Biosciences . FITC-conjugated fibrinogen was purchased from Agilent . Collagen was purchased from Nycomed  whereas Collagen-Related Peptide (CRP) was obtained from Prof Richard Farndale . Anti-phospho-vasodilator-stimulated phospho-protein (VASP) (Ser239) was purchased from Cell Signalling , anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Proteintech , and Alexa-488 conjugated phalloidin secondary antibody was bought from Life Technologies Myricetin, gallic acid, ADP, thrombin, phorbol-12-myristate-13-acetate (PMA), Thrombin Receptor Activator Peptide 6 (TRAP-6), human fibrinogen, and 1,4-Dithiothreitol (DTT) and 3,3′-Dihexyloxacarbocyanine iodide (DIOCS. cumini leaves were collected from different trees at the campus (2°33´11.7´´S 44°18´22.7´´W) of the Federal University of Maranhão (UFMA) in São Luís, Maranhão, Brazil. Samples were identified and catalogued at the Herbarium MAR of the Department of Biology of the same institution, where a voucher specimen was deposited under n° 4573.The extract was prepared according to As we have previously characterized the phytochemical composition of PESc , confirmg for 10 min at 22°C to obtain PRP. To obtain washed platelets (WP), PRP was centrifuged twice  in the presence of 1.25 μg/ml prostacyclin. The final platelet pellet was resuspended in modified Tyrode’s-HEPES buffer,  and rested for 30 min at 30°C before experiments. All protocols using human blood samples were approved by the Research Ethics Committees of the Federal University of Maranhão and the University of Reading.Healthy volunteers who did not use antiplatelet drugs and had previously provided informed consent had their blood samples collected in tubes containing 1:5 v/v acid citrate dextrose  or 3.8% (w/v) sodium citrate for platelet aggregation experiments using platelet-rich plasma (PRP). Whole blood was centrifuged at 250 × 8 platelets/ml) were incubated for 25 min at 37°C with 10, 100, or 1,000 μg/ml of PESc prior to the addition of ADP (2.5 or 5 µM), thrombin (0.01 or 0.02 U/ml), or PMA (100 nM). For experiments using myricetin and gallic acid, these were incubated in PRP  or WP  for 10 min at 37°C followed by the addition of agonists collagen (1 µg/ml) or TRAP-6 (10 µM). Aggregation traces from at least three different donors were recorded for 5 min.PRP and WP aggregation assays were performed in a four-channel aggregometer . PRP samples (2–3 × 108 platelets/ml) were incubated with PESc at the same concentrations and conditions used for platelet aggregation experiments. Then WP were incubated for 10 min with thrombin (0.02 U/ml). FITC-conjugated PAC-1 antibody was added for 10 min in the dark and fluorescence read using a flow cytometer FACS Calibur . For experiments using myricetin and gallic acid, these were incubated with WP for 10 min at 37°C followed by the addition of agonists CRP (1 µg/ml) or TRAP-6 (10 µM). FITC-conjugated fibrinogen and PE/Cy5-conjugated anti-human CD62P were incubated for 30 min, then platelets were read after a 25-fold dilution with Tyrodes-HEPES buffer.WP (2–3 × 107 platelets/ml) were incubated in absence or presence of myricetin  for 10 min at 37°C, then 300 µl of the solution was dispensed onto a fibrinogen or collagen (100 µg/ml)-coated coverslip for 45 min at 37°C. Non-adhered platelets were removed and the coverslip washed three times with PBS. Adhered platelets were fixed using 0.2% paraformaldehyde for 10 min. This solution was then removed and coverslips washed three times with PBS before the addition of 0.1% (v/v) Triton-X to permeabilize the cells. After removal of Triton-X and further washes using PBS, platelets were stained with Alexa Fluor 488 or 647-conjugated phalloidin  for 1 h in the dark at room temperature. Coverslips were mounted onto microscope glass slides and imaged using a 100× oil immersion lens on a Nikon A1-R Confocal microscope.WP (2 × 106 (5 µM) for 30 min at 30°C, whilst Vena8 bio-chips  were coated with collagen (100 µg/ml) for 60 min at 37°C. Prior to experiments, blood was pre-treated with myricetin (30 µM) or vehicle control for 10 min at 37°C and Vena8 bio-chips were washed with Tyrode’s-HEPES buffer. Whole blood was then perfused at a shear rate of 45 dyn/cm2 and images recorded every 4 s for 10 min using a 20× air lens on a Nikon A1-R Confocal microscope exciting at 488 nm and detecting emission at 500 to 520 nm. Fluorescence intensity was calculated using NIS Elements Software .Whole blood was pre-incubated with DIOCmus musculus) with 10–12 weeks of age and 30–35 grams were acquired from the Animal Facility House of the Federal University of Maranhão (UFMA), São Luís–MA. Animals were kept under a 12 h light cycle, controlled temperature (22–24°C) and food and water ad libitum. Mice were given myricetin at 25 mg/kg or 50 mg/kg or vehicle control for three consecutive days through oral gavage. One hour after the last dose, animals were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and 5 mm of the tail was amputated using sharp scissors. The bleeding tail was then placed in filtered PBS buffer at 37°C and time to cessation of bleeding recorded for up to 20 min, after which mice were terminated. All procedures were performed in alignment with the National Council for the Control of Animal Experimentation  and approved by the local Animal Care and Welfare Committee of UFMA, under ruling number 23115.018725/2017-19.Healthy female Swiss mice (Escherichia coli to generate a glutathione s-transferase (GST)-tagged fusion protein. The fusion protein was then purified by affinity chromatography using glutathione agarose and the GST cleaved using PreScision protease as per manufacturer instructions . Finally, the proteins were submitted to a gel filtration on Superdex 75 purification resin  to remove contaminants.The generation of recombinant thiol isomerases was performed as previously described . BrieflyMyricetin (0.01–10 µM) or vehicle (1:400 v/v DMSO : PBS) were incubated with recombinant ERp5, ERp57, ERp72, or PDI (2 µM) in PBS buffer containing ethylenediaminetetracetic acid  for 10 min at 25°C in a black 96-well plate. Fluorescence intensity was read using a Flexstation 3 fluorimeter , with 280 nm excitation and emission scanned from 300 to 420 nm in 5 nm slits, at a speed of 0.17 s per well. Appropriate blanks in which no protein was added were also acquired to establish that myricetin had no autofluorescence at the specified excitation/emission wavelengths. Data obtained are the means of at least three independent experiments run at least in duplicate.KSV), peak fluorescence intensity at 330 nm was used and constant determined from the following equation:To calculate the Stern-Volmer quenching constant  was determined according to the equation of The apparent binding constant /F] versus log [1/([L] − n (F0 − F)[P]/F0)] was plotted and n determined as the slope of the regression, as described by n was substituted back into the equation and Kb determined for the highest concentration tested. Finally, the dissociation constant (Kd) was calculated as Kd = 1/Kb.In which The reductase activity of the thiol isomerases was determined through the fluorescent probe dieosin glutathione disulfide . Di-E-GssG was synthesized and used as previously described . MyricetThe predicted poses of interaction between myricetin and different thiol isomerases were assessed using AutoDock 4.2 package, similar to previously described . The 3D 8 platelets/ml), were incubated with myricetin  or the nitric oxide donor PAPA-NONOate (100 µM), lysed in reducing Laemmli sample buffer  and heated for 5 min. Samples were loaded into a 10% Mini-PROTEAN TGX precast protein gel submerged in 1X Tris/Glycine/SDS buffer , then submitted to vertical transfer in a tetra vertical electrophoresis cell  using constant voltage of 150V for 45 min. After protein separation, semi-dry transfer was performed at 15V for 2 h using a BioRad Trans-blot semidry blotter. Membranes were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies against VASP (Ser239) or GAPDH at 1:1,000 v/v dilution overnight. After washing the primary antibody off, Alexa-488 conjugated phalloidin secondary antibody was incubated for 1 h at room temperature at 1:4,000 v/v dilution. Membranes were visualized using a Typhoon imaging system .WP . Quantitative results were expressed as mean ± SEM and individual points for all bar graphs, in order to improve transparency on the variability of data. Sample size varied from three to six independent repeats. Statistical analysis was performed through paired one-way ANOVA and Tukey as post-test with level of significance of 5%.The initial approach focused on whether PESc would inhibit platelet aggregation. Therefore, different concentrations of PESc were incubated with PRP and platelets were activated with various agonists. The composition of the batch of PESc used in this study is consistent with previously reported   and gallic acid (second most abundant compound) on platelet aggregation. Both PRP Figure 2Further studies were conducted to assess the effect of both myricetin and gallic acid in platelet function through flow cytometry. Results in Upon vascular injury, platelets start to adhere to components of the sub-endothelium, such as collagen and fibrinogen . In ordeGiven the antiplatelet effects and inhibition of adhesion to collagen exerted by myricetin, we hypothesized this flavonoid could impact thrombus formation. Therefore, blood was perfused under physiological arterial shear rate into collagen-coated Vena8 biochips as shown in After establishing antiplatelet and anti-thrombotic properties for myricetin, we then tested its impact on hemostasis. Healthy mice (10–12 weeks of age) were given myricetin (25 or 50 mg/kg) orally for 3 consecutive days, upon which bleeding time was measured after tail tip removal. Results are shown in 3 function located at the end of the platelet activation pathway, 3) that myricetin inhibited platelet spreading on collagen but not on fibrinogen, similar to a PDI knockout model  myricetin inhibited platelet aggregation induced by different agonists, 2) that PDI is a key modulator of integrin αIIbβut model  4) that ut model , and 5) ut model , we deciKSV, the quencher coefficient Kq, the binding constant Kb, and the dissociation constant Kd for each protein studied. Values for KSV and Kq were within the same order of magnitude for all of the thiol isomerases tested, indicating a similar binding affinity between these proteins and myricetin , which suggests some compounds were able to cross the platelet cell membrane, probably targeting PKC or downstream molecules, i.e. signaling that occurs at the end of the platelet activation pathway. Moreover, the inhibition of platelet function herein described for PESc is in accordance with reports showing that a green tea flavonoid-rich extract reduced platelet aggregation and integrin αIIbβcollagen . Given tin vivo.Myricetin inhibited platelet aggregation and activation induced by agonists of the collagen and thrombin pathways, whereas gallic acid showed little to no effect even at 10× higher concentrations. This is in agreement with previous reports showing that myricetin strongly inhibited collagen-  and aracThe effect of myricetin on platelet activation is also compatible with that previously shown for quercetin, a flavonoid of similar structure. 239 (2 (TxA2) receptor (TPR) and this could also be a potential mechanism of action for myricetin. However, previous literature has shown that SQ-29548, a specific TPR inhibitor, displayed little effect on CRP-induced platelet activation  and the local Animal Care and Welfare Committee of the Federal University of Maranhao, under ruling number 23115.018725/2017-19.RG designed the study, performed experiments, analyzed data, and drafted the manuscript. SS, JS, JF, HS, and VC performed experiments and analyzed data. SA generated recombinant proteins used in experiments. AT supervised experiments, discussed data, and reviewed the manuscript. JG discussed data and reviewed the manuscript. AP designed the study, discussed the data, drafted and reviewed the manuscript.This study was funded by the British Heart Foundation (RG/15/2/31224), Medical Research Council (MR/J002666/1), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001, and Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão, FAPEMA . AT was supported by CSIC grupos I+D 2014 and 2018 (536) and FAPEMA (PVI-05558/15). AP was supported by FAPEMA (BEEP-02511/18).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer [MC] declared a past co-authorship with one of the authors [JG] to the handling editor.


Ovarian cancer (OC) is difficult to diagnose at an early stage and leads to the high mortality rate reported in the United States. Standard treatment for OC includes maximal cytoreductive surgery followed by platinum-based chemotherapy. However, relapse due to chemoresistance is common in advanced OC patients. Therefore, it is necessary to develop new anticancer drugs to suppress OC progression. Recently, the anticancer effects of laminarin, a beta-1,3-glucan derived from brown algae, have been reported in hepatocellular carcinoma, colon cancer, leukemia, and melanoma. However, its effects in OC are not reported. We confirmed that laminarin decreases cell growth and cell cycle progression of OC cells through the regulation of intracellular signaling. Moreover, laminarin induced cell death through DNA fragmentation, reactive oxygen species generation, induction of apoptotic signals and endoplasmic reticulum (ER) stress, regulation of calcium levels, and alteration of the ER-mitochondria axis. Laminarin was not cytotoxic in a zebrafish model, while in a zebrafish xenograft model, it inhibited OC cell growth. These results suggest that laminarin may be successfully used as a novel OC suppressor. In the United States, approximately 22,240 human ovarian cancer (OC) cases with 14,070 deaths were reported in 2018. OC is difficult to diagnose and is, therefore, associated with a high mortality rate. The risk of a woman developing OC was 1 in 78 with a diagnosis rate of 11.5 per 100,000 people in the United States between 2010 and 2014 . Thus, md-glucose polysaccharides, which occur in cell walls of bacteria, fungi, yeast, cereals, and seaweeds [d-glucopyranose residues with 6-O branching part in main and β--interstrand linkages [β-Glucan consists of a group of β-seaweeds . The physeaweeds . β-Glucaseaweeds . Additioseaweeds . β-Glucaseaweeds . Dietaryseaweeds . One of seaweeds . Laminarlinkages . Howeverlinkages ,18, melalinkages , and brelinkages . HoweverTherefore, we investigated the effects of laminarin specifically in terms of (i) apoptosis in vitro (ES2 and OV90 cells) and in vivo (zebrafish), (ii) cell cycle progression and reactive oxygen species (ROS) production in vitro, (iii) cytosolic or mitochondrial calcium concentrations and mitochondrial membrane potential (MMP) in vitro, and (iv) intracellular signaling pathways in vitro.p < 0.05) and OV90  cells in a dose-dependent manner. Cell cycle assays ) with some β- interstrand linkages and branch point A. We dete assays D,E reveap < 0.001; OV90: up to 0.3-fold, p < 0.01), JNK , and p38  in both OC cell types compared with non-treated cells  related to cell proliferation  revealeded cells E–G.Pretreatment with pathway inhibitors, LY294002 or U0126, almost completely blocked the laminarin-induced decrease in the phosphorylation of AKT and S6 proteins in OC cells A,C. Althp < 0.01; OV90: up to 11.5 times, p < 0.01) compared with vehicle-treated control. Collectively, these results suggest that laminarin induces cell apoptosis by increasing DNA fragmentation and apoptosis-related proteins in OC cells.The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay revealed abundant DNA fragmentation in the nuclei of laminarin-treated ES2 cells and some DNA fragmentation in OV90 cells, but no apoptotic damage in vehicle-treated cells A,B, indip < 0.001; p < 0.001; p < 0.001; p < 0.001; p < 0.001; p < 0.001; Flow cytometry analysis of human OC cells incubated with fluo-4 or rhod-2 following treatment with laminarin showed a dose-dependent increase in intracellular calcium levels  in ES2 cells and 217.3% (p < 0.05) in OV90 cells compared with the control, but co-incubation with the calcium chelators prevented the laminarin-induced increase in calcium levels (p < 0.001) in ES2 cells and 522.9% (p < 0.01) in OV90 cells by laminarin treatment alone, but co-treatment prevented any change in mitochondrial calcium levels (p < 0.01) and OV90 cells , but co-incubation with calcium chelators repressed the laminarin-mediated MMP loss  increased late apoptotic cells (ES2: 642.9% and OV90: 565.2%), but co-treatment with either of the three calcium chelators—2-APB, BAPTA, and ruthenium red—showed a decrease in late apoptotic cells , ATF6α , p-PERK , GADD153 , p-eIF2α , and GRP78  in both OC cell types compared with the control  and 48.5% (p < 0.001) for ES2 cells (p < 0.001) and 45.6% (p < 0.001) for OV90 cells, respectively  for 24 h did not show any change in viability or development A. When OS2 cells B, and byectively C.Laminaria japonica, Ecklonia kurome, and Eisenia bicyclis [Laminarin is a polysaccharide present in brown algae, such as bicyclis . While ibicyclis ,22, hepabicyclis , colon cbicyclis ,18,19, abicyclis , its effLaminarin repressed cell proliferation and PI3K/MAPK intracellular signaling pathways and increased the number of late apoptotic cells, subG1 cell population, MMP, apoptotic proteins, ER stress sensor proteins, and ER-mitochondrial tethering proteins in ES2 and OV90 cells. It also suppressed tumor formation in a zebrafish xenograft model.In the present study, we first confirmed that laminarin repressed OC cell viability. According to previous reports, laminarin suppresses proliferation, colony formation, and migration of human melanoma cells by inhibiting matrix metalloproteinase-2 (MMP-2), MMP-9, and the p-ERK1/2 signaling cascade . MoreoveMitochondrial-dependent cell death is associated with mitochondrial calcium concentration overload and mitochondrial membrane permeabilization as well as cleavage of caspases, the release of cytochrome c, and activation of pro-apoptotic proteins . In partIntracellular signal transduction is important in cancer progression, metastasis, and chemotherapy resistance. The PI3K/MAPK pathway serves as a therapeutic target for controlling cancer cell development . LaminarLaminaria digitata  was purchased from Sigma-Aldrich  and dissolved in sterile water before use. The antibodies used are described previously [Laminarin extracted from eviously .ES2 (ovarian clear cell carcinoma cells) and OV90 (papillary serous adenocarcinoma cells) were purchased from American Type Culture Collection  and incubated as described previously .Proliferation assays were performed using the Cell Proliferation ELISA, BrdU Kit . Briefly, both cell lines were incubated in a 96-well plate and serum-starved for 24 h in MaCoy’s 5A medium. Cells were then treated with laminarin for 48 h. BrdU was added to the cell culture medium, and the cells were incubated for an additional 2 h at 37 °C. BrdU was detected with an anti-BrdU-POD antibody and a color developed after the addition of the substrate.Both cell lines were incubated in confocal dishes and starved for 24 h in FBS-free conditions. Cells were incubated with laminarin for 48 h, air-dried, and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h at room temperature. The cells were briefly rinsed with PBS and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. Subsequently, the cells were subjected to a TUNEL staining mixture using the In Situ Cell Death Detection kit with tetramethylrhodamine (TMR) red (Roche) for 1 h at 37 °C in the dark. Cells were then washed with PBS and overlaid with DAPI. Fluorescence was detected using an LSM710  confocal microscope.5 cells) were seeded in 6-well plates and treated with laminarin for 48  h. The supernatants were discarded and the adherent cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA). The cells were collected by centrifugation, washed with PBS, and resuspended using 1× binding buffer at a dilution of 1  ×  106 cells/mL. Then, 100 μL of the cell suspension (1  ×  106 cells) was transferred to a 5 mL culture tube and incubated with 5  μL FITC Annexin V and 5  μL PI for 15  min at room temperature in the dark. After this, 400 μL of 1× binding buffer was added to the 5 mL culture tube. Fluorescence intensity was determined using a FACS Calibur (BD Biosciences).The induction of apoptosis in OC cells by laminarin was analyzed using the FITC Annexin V Apoptosis Detection Kit I . The cells  was transferred to a 5 mL culture tube and incubated with 5  μL of RNase A and 5  μL of PI for 30  min at room temperature in the dark. After this, 300 μL of 1× binding buffer was added to the 5 mL tube. The intensity of the fluorescence was determined using a FACS Calibur flow cytometer (BD Biosciences).The cells were seeded in 6-well plates and treated with various doses in laminarin for 48 h at 37 °C in a CO2 incubator. The treated cells were then washed with PBS again. Fluorescent DCF intensity was analyzed using a flow cytometer (BD Biosciences).Intracellular ROS production was estimated using 2′,7′-dichlorofluorescein diacetate , which is converted to fluorescent 2′,7′-dichlorofluorescein (DCF) in the presence of peroxides. Cells were detached with trypsin-EDTA, collected by centrifugation, and washed with PBS. The cells were treated with 10 μM DCFH-DA for 30 min at 37 °C, washed twice with PBS, and treated with different doses of laminarin for 1 h at 37 °C in a CO2 incubator. Supernatants were discarded, and adherent cells were detached with trypsin-EDTA. The cells were collected by centrifugation, resuspended in 3 μM fluo-4 acetoxymethyl ester , and incubated at 37 °C in a CO2 incubator for 20 min. The stained cells were washed with PBS, and the fluorescence intensity was analyzed using a flow cytometer (BD Biosciences).ES2 and OV90 cells were seeded in 6-well plates and incubated for 24 h in serum-free medium when the cells reached 70% to 80% confluency. The cells were then treated with different concentrations of laminarin for 48 h at 37 °C in a CO2 incubator. Supernatants were discarded, and adherent cells were detached with trypsin-EDTA. The cells were collected by centrifugation, resuspended in 3 μM rhod-2, and incubated at 37 °C in a CO2 incubator for 20  min. The stained cells were washed with PBS and the fluorescence intensity was analyzed using a flow cytometer (BD Biosciences).Both cell lines were seeded in 6-well plates and incubated for 24 h in serum-free medium when cells reached 70–80% confluency. The cells were then treated with different concentrations of laminarin for 48 h at 37 °C in a CO2 incubator. Supernatants were discarded, and adherent cells were detached with trypsin-EDTA. The cells were collected by centrifugation, resuspended in a staining solution, which included 200 × JC-1 and 1 × staining buffer, and incubated at 37 °C in a CO2 incubator for 20 min. The stained cells were collected by centrifugation and washed once with 1 × JC-1 staining buffer. The cells were then centrifuged once more and resuspended in 1 mL staining buffer. Fluorescence intensity was analyzed using a FACSCalibur (BD Biosciences).Changes in the JC-1 MMP were determined using a Mitochondria Staining Kit . The cells were seeded in 6-well plates and incubated for 24 h in serum-free medium until they reached 70% confluency. Then, cells were treated with laminarin in a dose-dependent manner for 48 h at 37 °C in a COThe protein concentrations in whole-cell extracts were determined using the Bradford Protein Assay  with BSA as the standard. Proteins were denatured, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes. Blots were developed using enhanced chemiluminescence detection and quantified by measuring the intensity of light emitted from correctly sized bands under ultraviolet light using a ChemiDoc EQ system and Quantity One software (Bio-Rad). Immunoreactive proteins were detected using goat anti-rabbit polyclonal antibodies against phosphorylated and total proteins at a 1:1000 dilution and separated by 10% SDS-PAGE. As a loading control, total protein and α-tubulin (TUBA) were used to normalize results for the detection of target proteins. Multiple exposures of each western blot were used to ensure the linearity of chemiluminescent signals.To confirm laminarin toxicity, embryos were exposed to 0.003% phenylthiourea (PTU) for 14 h to suppress pigmentation, the embryo’s shell was removed, and the embryo was treated with various concentrations of laminarin in 24-well plates. After 24 h, embryo viability and development were observed under light microscopy (Carl Zeiss Stereo microscope DV4). Pictures were taken by fixing zebrafish embryos onto a glass slide with 3% methylcellulose (Sigma-Aldrich).Xenograft studies were performed according to previous experiments  with modifications. Fertilized eggs were treated with Danieau’s solution containing 0.003% PTU at 28.5 °C for 48 h to suppress pigmentation. Micropipettes were created from a 1.0 mm glass capillary  by using a micropipette puller  for injection and anesthesia. At 48 h after fertilization, zebrafish were anesthetized in 0.02% tricaine (Sigma) and immobilized on an agar plate. Cells were treated with laminarin in a dose-dependent manner for 22 h and stained for an additional 2 h with 4 μM of CellTracker CM-Dil Dye (Invitrogen). Cells (100-200) were injected into the yolk sac by microinjection . Subsequently, zebrafish were incubated in 24-well plates containing Danieau’s solution with 0.003% PTU at 28.5 °C for 72 h. Fish were immobilized in a drop of 3% methylcellulose in Danieau’s solution on a glass slide. Pictures were taken using fluorescence microscopy . Fluorescent tumors were quantified by ImageJ software .p-value less than 0.05 was considered significant. Data are presented as least-square means with standard errors.All quantitative data were subjected to a least-squares analysis of variance using the General Linear Models procedure in the Statistical Analysis System program . Western blot data were corrected for differences in sample loading using total protein or TUBA data as a covariate. All tests of significance were performed using the appropriate error terms according to the expectation of the mean squares for the error. A In summary, our results provide the first evidence that laminarin has anticancer effects in human OC cells. Laminarin increased cytosolic and mitochondrial calcium levels in human OC cells through the regulation of ER-mitochondrial proteins. Laminarin also decreased PI3K and MAPK signaling in OC cells, whereas it activated ER stress, leading to OC cell death. Zebrafish xenograft model results showed that laminarin prevents tumor formation within the embryo yolks. Our results suggest that laminarin may serve as a novel therapeutic for the treatment of OC.