Cloning and functional characterization of the legumin A gene (EuLEGA) from Eucommia ulmoides Oliver.

Legumin A is a seed storage protein that provides nutrients for seed germination. The purpose of this study was to describe the structure and expression pattern of the EuLEGA gene in Eucommia ulmoides Oliver (E. ulmoides) and to infer its functional role. The 1287 bp coding sequence of the EuLEGA CDS of the EuLEGA gene, encoding a protein containing 428 amino acid residues, was cloned. The structure predicted that the protein belonged to the RmlC (deoxythymidine diphosphates, dTDP)-4-dehydrorhamnose 3,5-epimerase)-like cupin conserved domain family, which contains both RmlC, a key enzyme for the synthesis of rhamnose and legumin A. The overexpression (OE) vector of the EuLEGA gene was constructed and genetically transformed into tobacco and E. ulmoides; the RNA interference (RNAi) vector of the EuLEGA gene was constructed and genetically transformed into E. ulmoides; and the contents of legumin A and rhamnose were detected. The results showed that the EuLEGA gene could significantly increase the content of legumin A in transgenic tobacco leaves and transgenic E. ulmoides regenerative buds, and the OE of this gene in E. ulmoides could promote an increase in rhamnose content. RNAi caused a significant decrease in the legumin A content in the regenerated buds of E. ulmoides. These was a significant increase in legumin A in the transgenic tobacco seeds, and these results indicate that the expression of the EuLEGA gene is closely related to the accumulation of legumin A. Subcellular localization studies revealed that EuLEGA is localized to the cytoplasm with the vacuolar membrane. Analysis of the EuLEGA gene expression data revealed that the expression level of the EuLEGA gene in the samaras was significantly greater than that in the leaves and stems. In addition, the study also demonstrated that GA3 can upregulate the expression levels of the EuLEGA gene, while ABA and MeJA can downregulate its expression levels.

EuHDZ25 positively affects rubber biosynthesis by targeting EuFPS1 in Eucommia leaves.

Eucommia ulmoides is a temperate gum source plant that produces trans-polyisoprene (TPI), also known as Eucommia rubber. The structural configuration and function of TPI offer a new material with important potential for industrial development. In this study, we detected the TPI content in the leaves of diploid and triploid E. ulmoides plants. The average TPI content in the leaves of triploid E. ulmoides was significantly higher than that of diploid. Transcriptome data and weighted gene co-expression network analyses identified a significant positive correlation between the EuFPS1 gene and TPI content. Overexpression of EuFPS1 increased the density of rubber particles and TPI content, indicating its crucial role in TPI biosynthesis. In addition, the expression of EuHDZ25 in E. ulmoides was significantly positively correlated with EuFPS1 expression. Yeast one-hybrid and dual-luciferase assays demonstrated that EuHDZ25 mainly promotes TPI biosynthesis through positive regulation of EuFPS1 expression. The significantly up-regulated expression of EuHDZ25 and its consequent upregulation of EuFPS1 during the biosynthesis of TPI may partially explain the increased TPI content of triploids. This study provides an important theoretical foundation for further exploring the molecular mechanism of secondary metabolites content variation in polyploids and can help to promote the development and utilization of rubber resources.

Cloning and functional characterization of the peptide deformylase encoding gene EuPDF1B from Eucommia ulmoides Oliv.

Peptide deformylase can catalyse the removal of formyl groups from the N-terminal formyl methionine of the primary polypeptide chain. The peptide deformylase genes of a few herbaceous plants have been studied to some extent, but the peptide deformylase genes of woody plants have not been studied. In this study, we isolated EuPDF1B from Eucommia ulmoides Oliv. The full-length sequence of EuPDF1B is 1176 bp long with a poly-A tail and contains an open reading frame of 831 bp that encodes a protein of 276 amino acids. EuPDF1B was localized to the chloroplast. qRT‒PCR analysis revealed that this gene was expressed in almost all tissues tested but mainly in mature leaves. Moreover, the expression of EuPDF1B was enhanced by ABA, MeJA and GA and inhibited by shading treatment. The expression pattern of EuPDF1B was further confirmed in EuPDF1Bp: GUS transgenic tobacco plants. Among all the transgenic tobacco plants, EuPDF1Bp-3 showed the highest GUS histochemical staining and activity in different tissues. This difference may be related to the presence of enhancer elements in the region from - 891 bp to - 236 bp of the EuPDF1B promoter. In addition, the expression of the chloroplast gene psbA and the net photosynthetic rate, fresh weight and height of tobacco plants overexpressing EuPDF1B were greater than those of the wild-type tobacco plants, suggesting that EuPDF1B may promote the growth of transgenic tobacco plants. This is the first time that PDF and its promoter have been cloned from woody plants, laying a foundation for further analysis of the function of PDF and the regulation of its expression.

Establishment of an Efficient Protoplast Isolation and Transfection Method for Eucommia ulmoides Oliver.

BACKGROUND: Eucommia ulmoides Oliver is a unique high-quality natural rubber tree species and rare medicinal tree species in China. The rapid characterization of E. ulmoides gene function has been severely hampered by the limitations of genetic transformation methods and breeding cycles. The polyethylene glycol (PEG)-mediated protoplast transformation system is a multifunctional and rapid tool for the analysis of functional genes in vivo, but it has not been established in E. ulmoides. METHODS: In this study, a large number of highly active protoplasts were isolated from the stems of E. ulmoides seedlings by enzymatic digestion, and green fluorescent protein expression was facilitated using a PEG-mediated method. RESULTS: Optimal enzymatic digestion occurred when the enzyme was digested for 10 h in an enzymatic solution containing 2.5% Cellulase R-10 (w/v), 0.6% Macerozyme R-10 (w/v), 2.5% pectinase (w/v), 0.5% hemicellulase (w/v), and 0.6 mol/L mannitol. The active protoplast yield under this condition was 1.13 × 106 protoplasts/g fresh weight, and the protoplast activity was as high as 94.84%. CONCLUSIONS: This study established the first protoplasm isolation and transient transformation system in hard rubber wood, which lays the foundation for subsequent functional studies of E. ulmoides genes to achieve high-throughput analysis, and provides a reference for future gene function studies of medicinal and woody plants.

Positive regulation of the Eucommia rubber biosynthesis-related gene EuFPS1 by EuWRKY30 in Eucommia ulmoides.

Eucommia rubber is a secondary metabolite from Eucommia ulmoides that has attracted much attention because of its unique properties and enormous potential for application. However, the transcriptional mechanism regulating its biosynthesis has not yet been determined. Farnesyl pyrophosphate synthase is a key enzyme in the Eucommia rubber biosynthesis. In this study, the promoter of EuFPS1 was used as bait, EuWRKY30 was screened from the cDNA library of EuFPS1 via a yeast one-hybrid system. EuWRKY30 belongs to the WRKY IIa subfamily and contains a WRKY domain and a C2H2 zinc finger motif, and the expressed protein is located in the nucleus. EuWRKY30 and EuFPS1 exhibited similar tissue expression patterns, and yeast one-hybrid and dual-luciferase experiments confirmed that EuWRKY30 directly binds to the W-box element in the EuFPS1 promoter and activates its expression. Moreover, the overexpression of EuWRKY30 significantly upregulated the expression level of EuFPS1, further increasing the density of the rubber particles and Eucommia rubber content. The results of this study indicated that EuWRKY30 positively regulates EuFPS1, which plays a critical role in the synthesis of Eucommia rubber, provided a basis for further analysis of the underlying transcriptional regulatory mechanisms.

Physiology and transcriptome of Eucommia ulmoides seeds at different germination stages.

E. ulmoides (Eucommia ulmoides) has significant industrial and medicinal value and high market demand. E. ulmoides grows seedlings through sowing. According to previous studies, plant hormones have been shown to regulate seed germination. To understand the relationship between hormones and E. ulmoides seed germination, we focused on examining the changes in various indicators during the germination stage of E. ulmoides seeds. We measured the levels of physiological and hormone indicators in E. ulmoides seeds at different germination stages and found that the levels of abscisic acid (ABA), gibberellin (GA), and indole acetic acid (IAA) significantly varied as the seeds germinated. Furthermore, we confirmed that ABA, GA, and IAA are essential hormones in the germination of E. ulmoides seeds using Gene Ontology and Kyoto Encyclopedia of Genes and Genomics enrichment analyses of the transcriptome. The discovery of hormone-related synthesis pathways in the control group of Eucommia seeds at different germination stages further confirmed this conclusion. This study provides a basis for further research into the regulatory mechanisms of E. ulmoides seeds at different germination stages and the relationship between other seed germination and plant hormones.

EuTGA1, a bZIP transcription factor, positively regulates EuFPS1 expression in Eucommia ulmoides.

Eucommia ulmoides (E. ulmoides) is widely cultivated and exhibits remarkable adaptability in China. It is the most promising rubber source plant in the temperate zone. E. ulmoides gum (EUG) is a trans-polyisoprene with a unique "rubber-plastic duality", and is widely used in advanced materials and biomedical fields. The transcription of Farnesyl pyrophosphate synthase (FPS), the rate-limiting enzyme of EUG biosynthesis, is controlled by regulatory mechanisms that remain poorly elucidated. In this research, 12 TGA transcription factors (TFs) in E. ulmoides were identified. Promoter prediction results revealed that the EuFPS1 promoter had binding sites for EuTGAs. Subsequently, the EuTGA1 was obtained by screening the E. ulmoides cDNA library using the EuFPS1 promoter as a bait. The individual yeast one­hybrid and dual-luciferase assays confirmed that in the tobacco plant, EuTGA1 interacted with the EuFPS1 promoter, resulting in a more than threefold increase in the activity of the EuFPS1. Subcellular localization study further revealed that EuTGA1 is localized in the nucleus and acts as a TF to regulate EuFPS1 expression. In addition, qRT-PCR analysis demonstrated that the expression trend of EuFPS1 and EuTGA1 was the same at different time of the year. Notably, low temperature and MeJA treatments down-regulated EuTGA1 expression. Additionally, the transient transformation of EuTGA1 enhanced NtFPS1 expression in tobacco plants. Overall, this study identified a TF that interacted with EuFPS1 promoter to positively regulate EuFPS1 expression. The findings of this study provide a theoretical basis for further research on the expression regulation of EuFPS1.

Cloning and functional analysis prohibitins protein-coding gene EuPHB1 in Eucommia ulmoides Oliver.

As multifunctional proteins, prohibitins(PHBs) participate in many cellular processes and play essential roles in organisms. In this study, using rapid amplification of cDNA end (RACE) technology, EuPHB1 was cloned from Eucommia ulmoides Oliver (E. ulmoides). A subcellular localization assay preliminarily located EuPHB1 in mitochondria. Then EuPHB1 was transformed into tobacco, and phenotype analyses showed that overexpression of EuPHB1 caused leaves to become chlorotic and shrivel. Furthermore, genes related to hormone and auxin signal transduction, auxin binding, and transport, such as ethylene-responsive transcription factor CRF4-like and ABC transporter B family member 11-like, were significantly inhibited in response to EuPHB1 overexpression. Its overexpression disturbs the original signal transduction pathway, thus causing the corresponding phenotypic changes in transgenic tobacco. Indeed, such overexpression caused fading of palisade tissue and an increase in the number of certain mesophyll cells. It also increased adenosine triphosphate (ATP) synthase activity, mitochondrial membrane potential, ATP content, and reactive oxygen species (ROS) levels in cells. Our results suggest that EuPHB1 expression promotes cellular energy metabolism by accelerating the oxidative phosphorylation of the mitochondrial respiratory chain. Elevated levels of EuPHB1 in the mitochondria, which helps supply the extra energy required to support rapid rates of cell division.

Eucommia lignans alleviate the progression of diabetic nephropathy through mediating the AR/Nrf2/HO-1/AMPK axis in vivo and in vitro.

Lignans derived from Eucommia ulmoides Oliver (Eucommia lignans) inhibit the progression of inflammatory diseases, while their effect on the progression of diabetic nephropathy (DN) remained unclear. This work was designed to assess the function of Eucommia lignans in DN. The major constituents of Eucommia lignans were analyzed by UPLC-Q-TOF-MS/MS. The binding between Eucommia lignans and aldose reductase (AR) was predicted by molecular docking. Eucommia lignans (200, 100, and 50 mg·kg-1) were used in model animals to evaluate their renal function changes. Rat glomerular mesangial cells (HBZY-1) were transfected with sh-AR, sh-AMPK, and oe-AR in the presence of high glucose (HG) or HG combined with Eucommia lignans to evaluate whether Eucommia lignans affected HG-induced cell injury and mitochondrial dysfunction through the AR/Nrf2/HO-1/AMPK axis. Eucommia lignans significantly attenuated the progression of DN in vivo. Eucommia lignans notably reversed HG-induced upregulation of inflammatory cytokines and mitochondrial injury, while downregulating the levels of Cyto c, caspase 9, AR, and NOX4 in HBZY-1 cells. In contrast, HG-induced downregulation of Nrf2, HO-1 and p-AMPKα levels were abolished by Eucommia lignans. Meanwhile, knockdown of AR exerted similar therapeutic effect of Eucommia lignans on DN progression, and AR overexpression reversed the effect of Eucommia lignans. Eucommia lignans alleviated renal injury through the AR/Nrf2/HO-1/AMPK axis. Thus, these findings might provide evidence for the use of Eucommia lignans in treating DN.

Identification of a Coprinellus strain and its application in Eucommia ulmoides gum extraction by fermenting leaves.

White rot fungi is a kind of filamentous fungi which can degrade lignin, hemicellulose and cellulose effectively. In this study, a wild white rot fungi collected from Pingba Town, Bijie City of China was identified as Coprinellus disseminatus (fruiting body) based on morphological and molecular identification. The mycelium of C. disseminatus cultured in the medium supplemented xylan as carbon showed the higher xylanase (XLE) and cellulase (CLE) activity. Further, the activities of tissue degradation-related enzymes including XLE, CLE, acetyl xylanesterase (AXE) and α-L-arabinofuran glycosidase (α-L-AF) were determined after fermenting Eucommia ulmoides leaves by inoculating C. disseminatus mycelium. The results showed that the activities of XLE, CLE, AXE and α-L-AF of mycelium cultured in xylan-contained medium reached the maximum at 5 d after inoculation, which were 777.606 ± 4.248 U mL-1, 9.594 ± 0.008 U mL-1, 4.567 ± 0.026 U mL-1 and 3.497 ± 0.10 U mL-1 respectively. Also, the activities of AXE and α-L-AF both reached the maximum in C. disseminatus mycelium cultured in glucose-contained medium. By comparing the yield of E. ulmoides gum under different fermentation treatments, the extraction yield of E. ulmoides gum were 2.156 ± 0.031% and 2.142 ± 0.044% at 7 d and 14 d after fermentation with mycelium supplemented xylan as carbon source, which were significantly higher than other groups. This study provides a theoretical reference for the preparation of E. ulmoides gum by large-scale fermentation of E. ulmoides leaves with C. disseminatus.

Nocardiopsis changdeensis sp. nov., an endophytic actinomycete isolated from the roots of Eucommia ulmoides Oliv.

Strain Mg02T was isolated from roots of Eucommia ulmoides Oliv. collected from Changde City, Hunan Province, China. Strain Mg02T, which exhibited distinct chemotaxonomic characteristics of the genus Nocardiopsis: cell-wall chemotype III/C, i.e., meso-diaminopimelic acid as diagnostic amino acid in whole-cell hydrolysates and menaquinone MK-10 with variable degrees of saturation in the side chain as the predominant isoprenoid quinone, was investigated by a polyphasic approach to determine their taxonomic position. Sequence analysis of the 16S rRNA gene indicated that strain Mg02T is affiliated to the genus Nocardiopsis, having highest sequence similarity to Nocardiopsis flavescens CGMCC 4.5723T (99.1%) and <98.7% to other species of the genus Nocardiopsis with validly published names. Phylogenetic analysis of 16S rRNA gene indicated strain Mg02T formed a separate evolutionary clade, suggesting that it could be a novel Nocardiopsis species. Phylogenomic analysis showed that strain Mg02T was closely related to N. flavescens CGMCC 4.5723T and distinct from the latter according to the clustering patterns. The Average Nucleotide Identity and digital DNA-DNA hybridization values between strain Mg02T and N. flavescens CGMCC 4.5723T were far below the species-level thresholds. Based on phenotypic, phylogenetic and chemotaxonomic characteristics, we think that strain Mg02T should represent a novel Nocardiopsis species, for which the name Nocardiopsis changdeensis sp. nov. is proposed. The type strain is Mg02T (=MCCC 1K06174T = JCM 34709T).

Quercetin-3-O-α-L-arabinopyranosyl-(1→2)-ß-D-glucopyranoside Isolated from Eucommia ulmoides Leaf Relieves Insulin Resistance in HepG2 Cells via the IRS-1/PI3K/Akt/GSK-3ß Pathway.

For nearly 2000 years, Eucommia ulmoides Oliver (EUO) has been utilized in traditional Chinese medicine (TCM) throughout China. Flavonoids present in bark and leaves of EUO are responsible for their antioxidant, anti-inflammatory, antitumor, anti-osteoporosis, hypoglycemic, hypolipidemic, antibacterial, and antiviral properties, but the main bioactive compound has not been established yet. In this study, we isolated and identified quercetin glycoside (QAG) from EUO leaves (EUOL) and preliminarily explored its molecular mechanism in improving insulin resistance (IR). The results showed that QAG increased uptake of glucose as well as glycogen production in the palmitic acid (PA)-induced HepG2 cells in a dose-dependent way. Further, we observed that QAG increases glucose transporters 2 and 4 (GLUT2 and GLUT4) expression and suppresses the phosphorylation of insulin receptor substrate (IRS)-1 at serine612, thus promoting the expression of phosphatidylinositol-3-kinase (PI3K) at tyrosine458 and tyrosine199, as well as protein kinase B (Akt) and glycogen synthase kinase (GSK)-3ß at serine473 and serine9, respectively. The influence posed by QAG on the improvement of uptake of glucose was significantly inhibited by LY294002, a PI3K inhibitor. In addition, the molecular docking result showed that QAG could bind to insulin receptors. In summary, our data established that QAG improved IR as demonstrated by the increased uptake of glucose and glycogen production through a signaling pathway called IRS-1/PI3K/Akt/GSK-3ß.

Central and Enteric Neuroprotective Effects by Eucommia ulmoides Extracts on Neurodegeneration in Rotenone-induced Parkinsonian Mouse.

Parkinson's disease (PD) is a progressive neurodegenerative disease of both the central and peripheral / enteric nervous systems. Oxidative stress and neuroinflammation are associated with the pathogenesis of PD, suggesting that anti-oxidative and anti-inflammatory compounds could be neuroprotective agents for PD. Eucommia ulmoides (EU) is a traditional herbal medicine which exerts neuroprotective effects by anti-inflammatory and anti-oxidative properties. Our previous study showed that treatment with chlorogenic acid, a component of EU, protected against neurodegeneration in the central and enteric nervous systems in a PD model. In this study, we examined the effects of EU extract (EUE) administration on dopaminergic neurodegeneration, glial response and α-synuclein expression in the substantia nigra pars compacta (SNpc), and intestinal enteric neurodegeneration in low-dose rotenone-induced PD model mice. Daily oral administration of EUE ameliorated dopaminergic neurodegeneration and α-synuclein accumulation in the SNpc. EUE treatment inhibited rotenone-induced decreases in the number of total astrocytes and in those expressing the antioxidant molecule metallothionein. EUE also prevented rotenone-induced microglial activation. Furthermore, EUE treatment exerted protective effects against intestinal neuronal loss in the PD model. These results suggest that EU exerts neuroprotective effects in the central and enteric nervous systems of rotenone-induced parkinsonism mice, in part by glial modification.

Transcriptomic and biochemical analyses revealed the improved growth, lipid metabolism, and flesh quality of grass carp (Ctenopharyngodon idellus) by dietary Eucommia ulmoides bark and leaf supplementation.

The objective of this study was to investigate the dietary effects of Eucommia ulmoides bark and leaf (EB, EL) supplementation on the growth, lipid metabolism, flesh quality, and transcriptome of grass carp (Ctenopharyngodon idellus). EB and EL were individually added to the basal diet (control) at concentrations of 20 g/kg and 40 g/kg, respectively, and then the three diets were fed to grass carp (59.7 ±â€…0.3 g) for 60 d. The results showed that the weight gain was improved, and the feed conversion ratio was decreased by supplementation with EB and EL (P < 0.05). Compared to the control, the EB and EL groups showed higher flesh hardness; water-holding capacity; and collagen, docosahexaenoic acid (DHA), and n-3 polyunsaturated fatty acids (n-3PUFAs) contents and lower mesenteric lipid and muscle crude lipid contents (P < 0.05). Moreover, dietary EB and EL supplementation increased the activities of superoxide dismutase and glutathione peroxidase and decreased the contents of malondialdehyde and protein carbonyl in flesh (P < 0.05). In muscle transcriptome profiling, a total of 979, 1980 differentially expressed genes (DEGs) were identified, and 29, 199 Gene Ontology (GO) terms and 13, 39 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in the EB and EL groups, respectively. Some key pathways and genes involved in promoting growth, lipid metabolism and flesh quality were obtained, including mTOR and PPAR signaling pathways, muscle cytoskeleton- and extracellular matrix-related genes (myosin and collagen), etc. Overall, dietary EB and EL supplementation improved the growth, lipid metabolism, and flesh quality of grass carp, and several potential pathways and genes were identified behind the improvement mechanism of EB and EL supplementation.

As a traditional herb, Eucommia ulmoides (E. ulmoides) has been utilized in East Asia for at least 2 000 years. In recent years, E. ulmoides has been applied in the culture of fish for its functions of promoting growth, lipid metabolism, and flesh quality. However, the underlying molecular mechanism of improving growth, lipid metabolism, and flesh quality is not well understood. Our study showed that the improvement of flesh quality is the combined effect of antioxidant capacity, muscle texture, water-holding capacity, and nutritional composition. Additionally, several potential pathways and differentially expressed genes were identified through RNA sequencing to further study the improvement mechanism of dietary E. ulmoides bark and leaf supplementation on growth, lipid metabolism, and flesh quality in fish.

[Eucommia ulmoides Oliv polysaccharide reduces the injury of IL-1ß-induced chondrocyte by inhibiting NF-κB pathway].

OBJECTIVE: To investigate the effects of Eucommia ulmoides Oliv polysaccharide on the injury of interleukin-1ß(IL-1ß)-induced chondrocyte and its possible mechanism. METHODS: ATDC5 was treated with 10 µg/ml IL-1ß to establish osteoarthritis chondrocyte inflammation model, mouse chondrocyte ATDC5 were cultured in vitro and randomly divided into blank group, model group, model+Eucommia ulmoides Oliv polysaccharide low concentration group, model+Eucommia ulmoides Oliv polysaccharide medium concentration group and model+Eucommia ulmoides Oliv polysaccharide high concentration group. The cells in the blank group were cultured with conventional medium;the cells in the model group cells were cultured with a medium containing 10 ?g/ml IL-1ß, and the cells in the model+Eucommia ulmoides Oliv polysaccharide low concentration group, model+Eucommia ulmoides Oliv polysaccharide medium concentration group and model+Eucommia ulmoides Oliv polysaccharide high concentration group were co-cultured with medium containing 100, 200, 400 µg/ml Eucommia ulmoides Oliv polysaccharide and 10 µg/ml IL-1ß. After the cells of each group were cultured for 24 h, 48 h and 72 h, CCK-8 method was used to detect cell viability. After the cells of each group were cultured for 48 h, flow cytometry and DAPI staining were used to detect cell apoptosis;ELISA method was used to detect the expression of TNF-α, NO, IFN-γ and IL-6 in cells; DCFH-DA method was used to detect the content of ROS in cells;Western blot was used to detect the protein expression of TIMP-1, MMP-13 and NF-κB signaling pathway-related P65 and p-P65;Immunofluorescence staining was used to observe the localization of NF-κB P65 cells. RESULTS: Compared with the blank group, the ATDC5 cell viability and the protein expression of TIMP-1 in the model group reduced (P<0.05), while apoptosis rate, the levels of TNF-α, NO, IFN-γ and IL-6, the content of ROS, the protein expression of MMP-13 and p-P65, and the number of P65+ in the nucleus increased(P<0.05). Compared with the model group, the ATDC5 cell viability and the protein expression of TIMP-1 in the model+Eucommia ulmoides Oliv polysaccharide low concentration group, model+Eucommia ulmoides Oliv polysaccharide medium concentration group and model+Eucommia ulmoides Oliv polysaccharide high concentration group increased (P<0.05), while apoptosis rate, the levels of TNF-α, NO, IFN-γ and IL-6, the content of ROS, the protein expression of MMP-13 and p-P65, and the number of P65+ in the nucleus reduced (P<0.05). CONCLUSION: The results showed that Eucommia ulmoides Oliv polysaccharide could promote proliferation of IL-1ß-induced chondrocyte ATDC5 and inhibit its apoptosis, inflammatory response and matrix degradation. Its mechanism may be related to the inhibition of the activation of NF-κB pathway.

In vitro effects of Eucommia ulmoides and its active components on the growth, lipid metabolism and collagen metabolism of grass carp (Ctenopharyngodon idellus) hepatocyte and intramuscular fibroblast.

Two experiments were conducted to investigate the in vitro effects of Eucommia ulmoides (E. ulmoides) and its active components on the growth, lipid metabolism and collagen metabolism of grass carp's (Ctenopharyngodon idellus) hepatocytes and intramuscular fibroblasts. In experiments 1 and 2 (Expt. 1, 2), hepatocytes and intramuscular fibroblasts were treated with 2.5, 5, 10, 20, 40 and 80 µg ml-1 of Eucommia bark extract (EBE), Eucommia leaf extract (ELE), pinoresinol diglucoside (PDG), chlorogenic acid (CGA), quercetin (QC) and aucubin (AU) for 24 h, respectively, then the cell growth, lipid and collagen metabolism-related gene expressions were evaluated. The results showed that the cell proliferation rate of hepatocytes and intramuscular fibroblasts was significantly improved by the supplementation of EBE, ELE, CGA, QC and AU. Moreover, triglyceride concentration of hepatocytes was significantly decreased by the EBE, ELE, CGA and QC supplementations compared to the control. Meanwhile, EBE, ELE, CGA, QC and AU supplementations significantly upregulated the relative gene expressions of insulin-like growth factor-1 (igf1), protein kinase B (akt), target of rapamycin (tor) and eukaryotic initiation factor 4E binding protein 1 (4ebp1) in hepatocytes, and ribosomal protein S6 kinase 1 (s6k1) transcription was significantly activated by ELE, CGA and QC supplementations. Nonetheless, phosphatidylinositol 3-kinase (pi3k) was unaffected by any of the supplements. In addition, the mRNA expressions of genes associated with lipid metabolism (peroxisome proliferator activated receptor α pparα, carnitine palmitoyltransferase 1 cpt1, adipose triglyceride lipase atgl, hormone-sensitive lipase hsl, peroxisome proliferator activated receptor γ pparγ) were significantly upregulated by EBE, ELE, CGA and QC. In intramuscular fibroblasts, the EBE, ELE, CGA, QC and AU supplementations significantly increased in vitro hydroxyproline concentrations, promoted the relative expressions of transforming growth factor-ß1 (tgfß1), connective tissue growth factor (ctgf), collagen type I alpha 1/2 chain (col1a1, col1a2), lysine oxidase (lox) and tissue inhibitor of matrix metalloproteinase-2 (timp2), and decreased matrix metalloproteinase-2 (mmp2) gene expression. Also, the gene expressions of drosophila mothers against decapentaplegic protein 2/4 (smad2, smad4) and proline hydroxylase (phd) were significantly upregulated by ELE, CGA, QC and AU supplementations. Based on the present in vitro results of grass carp, EBE, ELE, CGA, QC and AU improved the growth and lipid metabolism (except AU) in hepatocytes, and promoted the collagen deposition in intramuscular fibroblast, which is partly attributed to the signalling pathways of AKT/TOR, PPARα and TGF-ß/Smads/CTGF.

UPLC-MS analysis and network pharmacology-based investigation into the active ingredients and molecular mechanisms of anti-fatigue of male flowers with Eucommia ulmoides Oliv.

The male flowers of Eucommia ulmoides Oliv. (MFEU) was a natural product that could alleviate fatigue and accelerate fatigue alleviation. Nonetheless, the active ingredients and underlying pharmacological mechanisms remain unknown. This study aimed to decode the active ingredients and potential action mechanisms of MFEU in the therapy of anti-fatigue using an integrated UPLC-MS analysis, network pharmacology approach, and cell experiments. Characterizations of chemical constituents of MFEU extract were identified by UPLC-Q-TOF-MS. The corresponding drug targets were retrieved from the drug target database and used to construct the "composite-target-pathway" network. The Cytoscape was used to identify potential protein targets of these MFEU components, indicating that 24 anti-fatigue compounds in MFEU regulate 18 anti-fatigue-related targets in 10 signaling pathways. The 16 components of MFEU were verified at the cellular level. The results of cell experiments showed that MFEU extract (0.361 µg/ml), Caffeic acid, Deacetylasperulosidic acid, Naringenin, Acanthoside B, Geniposidic acid, Rutin, and Quercetin could promote testosterone secretion on Leydig cells at 50 µM. The MFEU extract and seven compounds in MFEU might play a role in anti-fatigue by participating in the regulation of testosterone secretion. Finally, the results of PCR analysis showed that MFEU promotes the secretion of testosterone, which is related to CYPIIa1 and 17ß-HSD, STAR in the signal pathway of testosterone synthesis. This study provides a basis for further exploring the anti-fatigue mechanism of MFEU, adopting the method of multi-compound and multi-target.

Influences of dietary Eucommia ulmoides leaf extract on the hepatic lipid metabolism, inflammation response, intestinal antioxidant capacity, intestinal microbiota, and disease resistance of the channel catfish (Ictalurus punctatus).

The purpose of the study was to investigate the effects of Eucommia ulmoides leaf extract (ELE) on the common occurrence of liver steatosis, chronic inflammation, oxidative stress, disturbance of gut microbiota, and disease susceptibility in high-fat diet-fed channel catfish. Channel catfish fed three diets, including a high-fat diet (11% crude fat) and ELE-supplemented diets containing 1‰ or 2‰ ELE for 4 weeks. The results showed the contents of liver triacylglycerol of 1‰ and 2‰ ELE groups were reduced, and ELE treatments decreased the expression of lipogenesis related genes (srebp-1c, pparγ, and acc-1), and increased the expression of lipolysis related genes (pparα). In addition, the supplementation of ELE improved the inflammatory response of the liver and intestine. ELE could improve the destruction of intestinal morphology structure and increase the expression level of hif-1a and tight junction proteins (Occludin, Claudin2, Claudin15). 2‰ ELE significantly enhanced the antioxidant capacity of intestine by increasing the activity of SOD enzyme. Moreover, the supplement of ELE significantly increased the abundance of Cetobacterium and Romboutsia (p < 0.05). Compared with the control group, the expression of immune factor nf-κb had a significant decrease, and il-1ß showed a tendency to decrease in the ELE supplement groups after pathogenic bacteria challenge. In conclusion, the ELE alleviated fatty liver disease and inflammation response, improved the oxidative capacity and physiological structure of intestine, and improved the structure of intestinal microbiota and disease resistance in HFD-fed channel catfish.

Effects of dietary eucommia ulmoides leaf extract on growth performance, expression of feeding-related genes, activities of digestive enzymes, antioxidant capacity, immunity and cytokines expression of large yellow croaker (Larimichthys crocea) larvae.

A 30-d feeding trial was conducted to investigate effects of dietary eucommia ulmoides leaf extract (ELE) on growth performance, activities of digestive enzymes, antioxidant capacity, immunity, expression of inflammatory factors and feeding-related genes of large yellow croaker larvae. Five micro-diets were formulated with supplementation of 0 g kg-1 (the control), 5 g kg-1 (0·5 %), 10 g kg-1 (1·0 %) and 20 g kg-1 (2·0 %) of ELE, respectively. Results showed that the best growth performance was found in larvae fed the diet with 1·0 % ELE. Furthermore, ELE supplementation significantly increased the npy expression at 1·0 % dosage, while increased ghrelin in larvae at 0·5 % dosages. The activity of leucine aminopeptidase in larvae fed the diet with 1·0 % ELE was significantly higher than the control, while alkaline phosphatase was significantly upregulated in larvae fed the diet with 2·0 % ELE. A clear increase in total antioxidant capacity in larvae fed the diet with 1·0 % ELE was observed, whereas catalase activity was significantly higher in 1·0 % and 2·0 % ELE supplementation compared with the control. Larvae fed the diet with 1·0 % ELE had a significantly higher activities of lysozyme, total nitric oxide synthase and nitric oxide content than the control. Moreover, transcriptional levels of cox-2, il-1ß and il-6 were remarkably downregulated by the supplementation of 0·5-1·0 % ELE. This study demonstrated that the supplementation of 1·0 % ELE in diet could increase the growth performance of large yellow croaker larvae probably by promoting expression of feeding-related genes, enhancing antioxidant capacity and immunity and inhibiting expression of inflammatory factors.

Chemical Constituents from Staminate Flowers of Eucommia ulmoides Oliver and Their Anti-Inflammation Activity in Vitro.

Three new monoterpenoids, named eucomylides A-C (1-3), along with six known compounds (4-9) were isolated from the staminate flowers of Eucommia ulmoides Oliver. The structures were elucidated by extensive analyses of spectroscopic methods, and their absolute configurations were established by time-dependent density functional theory electronic circular dichroism (TDDFT ECD) calculation. All the compounds along with previously isolated components (10-14) were tested for their anti-inflammatory effects. Two iridoid glycosides (11 and 12) and a flavonoid glycoside (14) showed potent suppressive effects on nitric oxide (NO) production in RAW 264.7 cells, with IC50 values ranging from 17.11 to 22.26 µM.