Transcriptome Analysis Provides Insights into Catalpol Biosynthesis in the Medicinal Plant Rehmannia glutinosa and the Functional Characterization of RgGES Genes.

Rehmannia glutinosa, a member of the Scrophulariaceae family, has been widely used in traditional Chinese medicine since ancient times. The main bioactive component of R. glutinosa is catalpol. However, the biogenesis of catalpol, especially its downstream pathway, remains unclear. To identify candidate genes involved in the biosynthesis of catalpol, transcriptomes were constructed from R. glutinosa using the young leaves of three cultivars, Beijing No. 3, Huaifeng, and Jin No. 9, as well as the tuberous roots and adventitious roots of the Jin No. 9 cultivar. As a result, 71,142 unigenes with functional annotations were generated. A comparative analysis of the R. glutinosa transcriptomes identified over 200 unigenes of 13 enzymes potentially involved in the downstream steps of catalpol formation, including 9 genes encoding UGTs, 13 for aldehyde dehydrogenases, 70 for oxidoreductases, 44 for CYP450s, 22 for dehydratases, 30 for decarboxylases, 19 for hydroxylases, and 10 for epoxidases. Moreover, two novel genes encoding geraniol synthase (RgGES), which is the first committed enzyme in catalpol production, were cloned from R. glutinosa. The purified recombinant proteins of RgGESs effectively converted GPP to geraniol. This study is the first to discover putative genes coding the tailoring enzymes mentioned above in catalpol biosynthesis, and functionally characterize the enzyme-coding gene in this pathway in R. glutinosa. The results enrich genetic resources for engineering the biosynthetic pathway of catalpol and iridoids.

Neuroprotective effects of Rehmannia glutinosa polysaccharide on chronic constant light (CCL)-induced oxidative stress and autophagic cell death via the AKT/mTOR pathway in mouse hippocampus and HT-22 cells.

Rehmannia glutinosa polysaccharide (RGP) has been reported to exhibit anti-anxiety effects, yet the underlying mechanism remains unclear. Chronic constant light (CCL) induced cognitive dysfunction associated with oxidative stress in mice has been reported. Here, the neuroprotective effect of RGP on hippocampal neuron damage in CCL-treated mice was investigated. In vivo study, mice were subjected to CCL for 4 weeks and/or oral administration of 100, 200 and 400 mg/kg RGP every other day. In vitro experiment, hippocampal neuron cells (HT-22) was exposed to LED light and/or supplemented with 62.5, 125 and 250 µg/mL RGP. Mice exposed to CCL showed impaired cognitive and depressive-like behavior in the hippocampus, which were reversed by RGP. Meanwhile, RGP reversed light-induced oxidative stress and autophagy both in mice and hippocampal neuron cells (HT-22). Furthermore, compared with Light-exposed group, RGP treatment activated the AKT/mTOR pathway. Importantly, the AKT inhibitor Perifosine significantly weakened the neuroprotective of RGP on Light-induced oxidative stress and autophagy in HT-22 cells by inhibiting AKT/mTOR pathway and increasing the content of autophagy-related protein. Our data demonstrated, for the first time, that oxidative stress and the AKT/mTOR pathway plays a critical role in Light-induced apoptosis and autophagic cell death in mice and HT-22 cells.

Effects of Catalpol from Rehmannia glutinosa Extract on Skin Flaps.

BACKGROUND: Flaps are commonly used for repairing tissues and wounds in surgery. However, various factors can cause postoperative necrosis in these flaps. Catalpol is a bioactive component in extracts from Rehmannia glutinosa , which has pharmacologic characteristics that may improve flap survival. METHODS: The experiments were performed in 36 male Sprague-Dawley rats divided into three groups: control, low-dose catalpol, and high-dose catalpol. The flap survival rate, neutrophil density, microvessel density, superoxide dismutase, and malondialdehyde levels were measured; histopathologic analysis was performed 7 days after surgery. Blood flow was measured by laser Doppler flowmetry and lead oxide-gelatin angiography. The levels of vascular endothelial growth factor, toll-like receptor 4, nuclear factor-kappa B, tumor necrosis factor-α, interleukin (IL)-6, nod-like receptor 3, cysteinyl aspartate specific proteinase-1 (caspase-1), IL-1ß, and IL-18 were determined by immunohistochemistry. RESULTS: Catalpol treatment increased flap survival, reduced neutrophil recruitment and release, decreased malondialdehyde levels, and increased superoxide dismutase levels; thus, it effectively reduced oxidative stress, up-regulated the expression of vascular endothelial growth factor, and increased microvessel density. Laser Doppler flowmetry and lead oxide-gelatin angiography showed that catalpol treatment improved angiogenesis. Immunohistochemical analyses showed that catalpol inhibited the production of inflammatory factors, such as tumor necrosis factor-α and IL-6, by down-regulating toll-like receptor 4 and nuclear factor-κB. Furthermore, catalpol reduced cell pyroptosis by inhibiting the production of nod-like receptor 3 inflammasomes, thereby down-regulating the release of IL-1ß and IL-18. CONCLUSION: Catalpol can improve the rate of flap survival. CLINICAL RELEVANCE STATEMENT: The research verified that the Rehmannia extract catalpol, through angiogenesis, inflammatory response, ischemia-reperfusion injury, and pyroptosis-related pathways, effectively improved the flap survival rate, which will provide new ideas for clinical medication.

Omics analyses of Rehmannia glutinosa dedifferentiated and cambial meristematic cells reveal mechanisms of catalpol and indole alkaloid biosynthesis.

BACKGROUND: Rehmannia glutinosa is a rich source of terpenoids with a high medicinal reputation. The present study compared dedifferentiated cells (DDCs) and cambial meristematic cells (CMCs) cell cultures of R. glutinosa for terpenoid (catalpol) and indole alkaloid (IA) biosynthesis. In this regard, we used widely targeted metabolomics and transcriptome sequencing approaches together with the comparison of cell morphology, cell death (%), and catalpol production at different time points. RESULTS: We were able to identify CMCs based on their morphology and hypersensitivity to zeocin. CMCs showed higher dry weight content and better catalpol production compared to DDCs. The metabolome analysis revealed higher concentrations of IA, terpenoids, and catalpol in CMCs compared to DDCs. The transcriptome sequencing analysis showed that a total of 27,201 genes enriched in 139 pathways were differentially expressed. The higher catalpol concentration in CMCs is related to the expression changes in genes involved in acetyl-CoA and geranyl-PP biosynthesis, which are precursors for monoterpenoid biosynthesis. Moreover, the expressions of the four primary genes involved in monoterpenoid biosynthesis (NMD, CYP76A26, UGT6, and CYP76F14), along with a squalene monooxygenase, exhibit a strong association with the distinct catalpol biosynthesis. Contrarily, expression changes in AADC, STR, and RBG genes were consistent with the IA biosynthesis. Finally, we discussed the phytohormone signaling and transcription factors in relation to observed changes in metabolome. CONCLUSIONS: Overall, our study provides novel data for improving the catalpol and IA biosynthesis in R. glutinosa.

A Rehmannia glutinosa caffeic acid O-methyltransferase functional identification: Reconstitution of the ferulic acid biosynthetic pathway in Saccharomyces cerevisiae using Rehmannia glutinosa enzymes.

Rehmannia glutinosa produces many pharmacological natural components, including ferulic acid (FA) which is also an important precursor of some medicinal ingredients, so it is very significant to explore FA biosynthesis for enhancing the production of FA and its derivations. This study aimed to determine and reconstitute the R. glutinosa FA biosynthetic pathway from phenylalanine (Phe) metabolism in Saccharomyces cerevisiae as a safe host for the biosynthesis of plant-derived products. Although plant caffeic acid O-methyltransferases (COMTs) are thought to be a vital catalytic enzyme in FA biosynthesis pathways, to date, none of the RgCOMTs in R. glutinosa has been characterized. This study identified an RgCOMT and revealed its protein enzymatic activity for FA production in vitro. The RgCOMT overexpression in R. glutinosa significantly increased FA yield, suggesting that its molecular function is involved in FA biosynthesis. Heterologous expression of the RgCOMT and reported R. glutinosa genes, RgPAL2 (encoding phenylalanine ammonia-lyase [PAL] protein), RgC4H (cinnamate 4-hydroxylase [C4H]), and RgC3H (p-coumarate-3-hydroxylase [C3H]), in S. cerevisiae confirmed their catalytic abilities in the reaction steps for the FA biosynthesis. Importantly, in this study, these genes were introduced into S. cerevisiae and coexpressed to reconstitute the R. glutinosa FA biosynthetic pathway from Phe metabolism, thus obtaining an engineered strain that produced an FA titer of 148.34 mg L-1 . This study identified the functional activity of RgCOMT and clarified the R. glutinosa FA biosynthesis pathway in S. cerevisiae, paving the way for the efficient production of FA and its derivatives.

Purple Rehmannia: Investigation of the activation of R2R3-MYB transcription factors involved in anthocyanin biosynthesis.

Engineering anthocyanin biosynthesis in herbs could provide health-promoting foods for improving human health. Rehmannia glutinosa is a popular medicinal herb in Asia, and was a health food for the emperors of the Han Dynasty (59 B.C.). In this study, we revealed the differences in anthocyanin composition and content between three Rehmannia species. On the 250, 235 and 206 identified MYBs in the respective species, six could regulate anthocyanin biosynthesis by activating the ANTHOCYANIDIN SYNTHASE (ANS) gene expression. Permanent overexpression of the Rehmannia MYB genes in tobacco strongly promoted anthocyanin content and expression levels of NtANS and other genes. A red appearance of leaves and tuberous/roots was observed, and the total anthocyanin content and the cyanidin-3-O-glucoside content were significantly higher in the lines overexpressing RgMYB41, RgMYB42, and RgMYB43 from R. glutinosa, as well as RcMYB1 and RcMYB3 in R. chingii and RhMYB1 from R. henryi plants. Knocking out of RcMYB3 by CRISPR/Cas9 gene editing resulted in the discoloration of the R. chingii corolla lobes, and decreased the content of anthocyanin. R. glutinosa overexpressing RcMYB3 displayed a distinct purple color in the whole plants, and the antioxidant activity of the transgenic plants was significantly enhanced compared to WT. These results indicate that Rehmannia MYBs can be used to engineer anthocyanin biosynthesis in herbs to improve their additional value, such as increased antioxidant contents.

Genome-wide identification and characterization of the HD-Zip gene family and expression analysis in response to stress in Rehmannia glutinosa Libosch.

The HD-Zip family of transcription factors is unique to the plant kingdom, and play roles in modulation of plant growth and response to environmental stresses. R. glutinosa is an important Chinese medicinal material. Its yield and quality are susceptible to various stresses. The HD-Zip transcription factors is unique to the plant, and roles in modulation of plant growth and response to environmental stresses. However, there is no relevant research on the HD-ZIP of R. glutinosa. In this study, 92 HD-Zip transcription factors were identified in R. glutinosa, and denominated as RgHDZ1-RgHDZ92. Members of RgHDZ were classified into four groups (HD-ZipI-IV) based on the phylogenetic relationship of Arabidopsis HD-Zip proteins, and each group contains 38, 18, 17, and 19 members, respectively. Expression analyses of RgHDZ genes based on transcriptome data showed that the expression of these genes could be induced by the endophytic fungus of R. glutinosa. Additionally, we showed that RgHDZ genes were differentially expressed in response to drought, waterlogging, temperature, and salinity treatments. This study provides important information for different expression patterns of stress-responsive HD-Zip and may contribute to the better understanding of the different responses of plants to biotic and abiotic stresses, and provide a molecular basis for the cultivation of resistant varieties of R. glutinosa.

Co-Expression Analysis Reveals Differential Expression of Homologous Genes Associated with Specific Terpenoid Biosynthesis in Rehmannia glutinosa.

Terpenoids are naturally occurring compounds involved in respiration, photosynthesis, membrane fluidity, and pathogen interactions and are classified according to the structure of their carbon skeleton. Although most terpenoids possess pharmacological activity, knowledge about terpenoid metabolism in medicinal plants is insufficient. Rehmannia glutinosa (R. glutinosa) is a traditional herb that is widely used in East Asia and has been reported to contain various terpenoids. In this study, we performed a comprehensive transcriptome analysis of terpenoid metabolism in R. glutinosa using two RNA sequencing platforms: Illumina and PacBio. The results show that the sterol, saponin, iridoid, and carotenoid pathways are active in R. glutinosa. Sterol and saponin biosynthesis were mevalonate pathway dependent, whereas iridoid and carotenoid biosynthesis were methylerythritol 4-phosphate pathway dependent. In addition, we found that the homologous genes of key enzymes involved in terpenoid metabolism were expressed differentially and that the differential expression of these genes was associated with specific terpenoid biosynthesis. The different expression of homologous genes encoding acetyl-CoA acetyltransferase, 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, mevalonate diphosphate decarboxylase, farnesyl pyrophosphate synthase, squalene synthase, and squalene epoxidase was associated with sterol and saponin biosynthesis. Homologous genes encoding 1-deoxy-D-xylulose 5-phosphate synthase were also differentially expressed and were associated with carotenoid and iridoid biosynthesis. These results suggest that the biosynthesis of specific terpenoids can be regulated by the homologous of key enzymes involved in plant terpenoid metabolism.

Efficient CRISPR/Cas9-mediated genome editing in Rehmannia glutinosa.

KEY MESSAGE: Here, we cloned a phytoene desaturase (PDS) gene from Rehmannia glutinosa, and realized RgPDS1 knock out in R. glutinosa resulted in the generation of albino plants. Rehmannia glutinosa is a highly important traditional Chinese medicine (TCM) with specific pharmacology and economic value. R. glutinosa is a tetraploid plant, to date, no report has been published on gene editing of R. glutinosa. In this study, we combined the transcriptome database of R. glutinosa and the reported phytoene desaturase (PDS) gene sequences to obtain the PDS gene of R. glutinosa. Then, the PDS gene was used as a marker gene to verify the applicability and gene editing efficiency of the CRISPR/Cas9 system in R. glutinosa. The constructed CRISPR/Cas9 system was mediated by Agrobacterium to genetically transform into R. glutinosa, and successfully regenerated fully albino and chimeric albino plants. The next-generation sequencing (NGS) confirmed that the albino phenotype was indeed caused by RgPDS gene target site editing, and it was found that base deletion was more common than insertion or replacement. Our results revealed that zCas9 has a high editing efficiency on the R. glutinosa genome. This research lays a foundation for further use of gene editing technology to study the molecular functions of genes, create excellent germplasm, accelerate domestication, and improve the yield and quality of R. glutinosa.

Transcriptome-based identification and expression characterization of RgABCC transporters in Rehmannia glutinosa.

ABCC multidrug resistance-associated proteins (ABCCs/MRPs), a subfamily of ABC transporters, are involved in multiple physiological processes. Although these proteins have been characterized in some plants, limited efforts have been made to address their possible roles in Rehmannia glutinosa, a medicinal plant. Here, we scanned R. glutinosa transcriptome sequences and identified 18 RgABCC genes by in silico analysis. Sequence alignment revealed that the RgABCCs were closely phylogenetically related and highly conserved with other plant ABCCs/MRPs. Subcellular localization revealed that most of the RgABCCs were deposited in vacuoles and a few in plasma membranes. Tissue-specific expression of the RgABCCs indicated significant specific accumulation patterns, implicating their roles in the respective tissues. Differential temporal expression patterns of the RgABCCs exhibited their potential roles during root development. Various abiotic stress and hormone treatment experiments indicated that some RgABCCs could be transcriptionally regulated in roots. Furthermore, the transcription of several RgABCCs in roots was strongly activated by cadmium (Cd), suggesting possible roles under heavy metal stresses. Functional analysis of RgABCC1 heterologous expression revealed that it may increase the tolerance to Cd in yeast, implying its Cd transport activity. Our study provides a detailed inventory and molecular characterization of the RgABCCs and valuable information for exploring their functions in R. glutinosa.

Molecular and Biochemical Pathways of Catalpol in Alleviating Diabetes Mellitus and Its Complications.

Catalpol isolated from Rehmannia glutinosa is a potent antioxidant and investigated against many disorders. This review appraises the key molecular pathways of catalpol against diabetes mellitus and its complications. Multiple search engines including Google Scholar, PubMed, and Science Direct were used to retrieve publications containing the keywords "Catalpol", "Type 1 diabetes mellitus", "Type 2 diabetes mellitus", and "diabetic complications". Catalpol promotes IRS-1/PI3K/AKT/GLUT2 activity and suppresses Phosphoenolpyruvate carboxykinase (PEPCK) and Glucose 6-phosphatase (G6Pase) expression in the liver. Catalpol induces myogenesis by increasing MyoD/MyoG/MHC expression and improves mitochondria function through the AMPK/PGC-1α/PPAR-γ and TFAM signaling in skeletal muscles. Catalpol downregulates the pro-inflammatory markers and upregulates the anti-inflammatory markers in adipose tissues. Catalpol exerts antioxidant properties through increasing superoxide dismutase (sod), catalase (cat), and glutathione peroxidase (gsh-px) activity in the pancreas and liver. Catalpol has been shown to have anti-oxidative, anti-inflammatory, anti-apoptosis, and anti-fibrosis properties that in turn bring beneficial effects in diabetic complications. Its nephroprotective effect is related to the modulation of the AGE/RAGE/NF-κB and TGF-ß/smad2/3 pathways. Catalpol produces a neuroprotective effect by increasing the expression of protein Kinase-C (PKC) and Cav-1. Furthermore, catalpol exhibits a cardioprotective effect through the apelin/APJ and ROS/NF-κB/Neat1 pathway. Catalpol stimulates proliferation and differentiation of osteoblast cells in high glucose condition. Lastly, catalpol shows its potential in preventing neurodegeneration in the retina with NF-κB downregulation. Overall, catalpol exhibits numerous beneficial effects on diabetes mellitus and diabetic complications.

Overexpression of RgPAL family genes involved in phenolic biosynthesis promotes the replanting disease development in Rehmannia glutinosa.

Rehmannia glutinosa production is affected by the replanting disease, which involves autotoxic harm mediated by specific endogenous allelochemicals in root exudates. Many phenolics that act as allelochemical agents are mostly phenylpropanoid products of secondary metabolism in plants. Phenylalanine ammonia-lyase (PAL) is the first enzyme that catalyses the deamination of l-phenylalanine for entrance into the phenylpropanoid pathway. PAL family genes have been isolated and functionally characterized in many plant species. However, PAL family genes involved in phenolic biosynthesis remain largely uncharacterized in R. glutinosa. Here, we identified and characterized four PAL family genes (RgPAL2 to RgPAL5) in the species whose sequences exhibited highly conserved domains of PALs according to in silico analysis, implying their potential function in phenolic biosynthesis. Overexpression of RgPALs in R. glutinosa enhanced phenolic production, verifying that RgPAL family genes participate in phenolic biosynthesis pathways. Moreover, we found that the release of several allelopathic phenolics from the roots of RgPAL-overexpressing transgenic R. glutinosa increased, implying that the RgPALs positively promote their release. Importantly, under continuous monoculture stress, we found that the RgPAL transgenic plants exhibited more significant autotoxic harm than did non-transgenic (WT) plants by activating the phenolics/phenylpropanoid pathway, indicating that RgPAL family genes function as positive regulators of the replanting disease development in R. glutinosa. This study revealed that RgPAL family genes are involved in the biosynthesis and release of several phenolics and positively control the replanting disease development in R. glutinosa, laying a foundation for further clarification of the molecular mechanisms underlying the disease formation.

Biogenic Synthesis of Silver Nanoparticles with High Antimicrobial and Catalytic Activities using Sheng Di Huang (Rehmannia glutinosa).

Silver nanoparticles (AgNPs) are widely sought after for a variety of biomedical and environmental applications due to their antimicrobial and catalytic properties. We present here a green and simple synthesis of AgNPs utilizing traditional Chinese medicinal herbs. The screening of 20 aqueous herb extracts shows that Sheng Di Huang (Rehmannia glutinosa) had the most promising potential in producing AgNPs of 30±6 nm, with narrow size distribution and high crystallinity. The antimicrobial activities of these AgNPs conducted on E. coli cells were found to be superior in comparison to poly(vinylpyrrolidone)-capped AgNPs synthesized using common chemical method. Additionally, the AgNPs obtained possess excellent catalytic performance in the reduction of 4-nitrophenol to 4-aminophenol. We compared the phytochemical and FTIR spectral analyses of the herb extract before and after synthesis, in order to elucidate the phytochemicals responsible for the reduction of Ag+ ions and the capping of the AgNPs produced.

Green synthesized zinc oxide nanoparticles regulates the apoptotic expression in bone cancer cells MG-63 cells.

Management of degenerative spine pathologies frequently leads to the need for bone growth. Rehmanniae Radix (RR), a Chinese herbal formulation was found to exhibit numerous therapeutic properties including its potent effect against cancer cell lines. However, the underlying mechanism through which the Zinc oxide nanoparticles (ZnONPs) synthesized from Rehmanniae Radix exerts its anti-cancer activity against osteosarcoma cell line MG-63 needs to be explored. Therefore, the study was performed to evaluate the anticancer, cytotoxicity and apoptotic effectiveness of ZnONPs from RR against MG-63 cells. Characterization studies such UV-vis spectroscopy, FTIR, TEM and XRD analysis were performed. Cytotoxicity assay, mitochondrial membrane potential (MMP), morphological examination of cells and formation of reactive oxygen species (ROS), and apoptosis inducing ability of RR were evaluated by various procedures. Western blot analysis of apoptotic markers such as Bax, caspase-3 and caspase-9 were also performed. RR was found to inhibit growth of MG-63 cells at increasing dose. AO/EB staining confirmed the apoptotic efficacy of ZnONPs induced by RR in MG-63 cells. ZnONPs was also found to initiate increased generation of ROS and decreased MMP. Decreased MMP has resulted in increased levels of apoptotic proteins Bax, caspase-3 and caspase-9 and induction of apoptosis was substantiated by western blot analysis. The outcomes of the work propose that ZnONPs from RR exhibits strong anticancer action and inducing apoptosis on MG-63 cells via stimulating increased generation of ROS. Thus, ZnONPs from RR might be used as a hopeful drug target against several types of cancer cell lines.

Molecular Regulation of Catalpol and Acteoside Accumulation in Radial Striation and non-Radial Striation of Rehmannia glutinosa Tuberous Root.

Rehmannia glutinosa L., a perennial plant of Scrophulariaceae, is one of the most commonly used herbs in traditional Chinese medicine (TCM) that have been widely cultivated in China. However, to date, the biosynthetic pathway of its two quality-control components, catalpol and acteoside, are only partially elucidated and the mechanism for their tissue-specific accumulation remains unknown. To facilitate the basic understanding of the key genes and transcriptional regulators involved in the biosynthesis of catalpol and acteoside, transcriptome sequencing of radial striation (RS) and non-radial striation (nRS) from four R. glutinosa cultivars was performed. A total of 715,158,202 (~107.27 Gb) high quality reads obtained using paired-end Illumina sequencing were de novo assembled into 150,405 transcripts. Functional annotation with multiple public databases identified 155 and 223 unigenes involved in catalpol and acteoside biosynthesis, together with 325 UGTs, and important transcription factor (TF) families. Comparative analysis of the transcriptomes identified 362 unigenes, found to be differentially expressed in all RS vs. nRS comparisons, with 143 upregulated unigenes, including those encoding enzymes of the catalpol and acteoside biosynthetic pathway, such as geranyl diphosphate synthase (RgGPPS), geraniol 8-hydroxylase (RgG10H), and phenylalanine ammonia-lyase (RgPAL). Other differentially expressed unigenes predicted to be related to catalpol and acteoside biosynthesis fall into UDP-dependent glycosyltransferases (UGTs), as well as transcription factors. In addition, 16 differentially expressed genes were selectively confirmed by real-time PCR. In conclusion, a large unigene dataset of R. glutinosa generated in the current study will serve as a resource for the identification of potential candidate genes for investigation of the tuberous root development and biosynthesis of active components.

Metabolite accumulation and metabolic network in developing roots of Rehmannia glutinosa reveals its root developmental mechanism and quality.

Rehmannia glutinosa root contains many compounds with important medicinal properties and nutritional benefits, but only more than 140 compounds have been reported so far. Many other compounds and their accumulation and metabolic networks during its development remain unclear. In order to clarify them, its metabolic profiles at three different developmental stages were analyzed using untargeted LC-MS analysis. Multivariate analysis revealed that 434 metabolites differently accumulated in its different stages, suggesting different change trends. The metabolites having the same trend share common metabolic pathways, the metabolites showing increasing contents during its development have medical and nutritional values, and some mature root-specific metabolites may be better candidates for its quality control; 434 metabolites were mapped onto 111 KEGG pathways including 62 enzymes, whose increasing and decreasing patterns were shown during its development. Some metabolites complicatedly interacted with some enzymes and the top-10 pathways enriched from 111 KEGG pathways in network analysis. These findings extended the dataset of its identified compounds, and revealed that its development and quality were associated with the accumulation of different metabolites. Our work will lay the foundation for the better understanding of its chemical constituents, quality and developmental mechanism.

Rehmannia glutinosa polysaccharide promoted activation of human dendritic cells.

In our previous study, we showed that Rehmannia glutinosa polysaccharide (RGP) treatment induced maturation of dendritic cells (DCs) and that it had an anticancer effect in mice. The effect of RGP has not been studied in human DCs, including monocyte-derived DCs (MDDCs) and peripheral blood DCs (PBDCs). In this study, we examined DC activation by RGP in human cells. The dendritic morphology of RGP-treated MDDCs was substantially altered as compared with that of phosphate-buffered saline (PBS)-treated control cells. Moreover, RGP treatment markedly decreased phagocytic activity and increased expression levels of co-stimulatory molecules in MDDCs. In addition, RGP treatment elevated the production of proinflammatory cytokines. Furthermore, RGP-induced activation of MDDCs was dependent on the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). RGP-treated MDDCs promoted upregulation of T-cell activation, including proliferation and interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) production. Analysis of the effect of RGP in PBDC subsets revealed that it induced upregulation of co-stimulatory molecule expression and proinflammatory cytokine production. These data suggest that RGP may function as an immune stimulatory molecule in humans.

Differential proteomic analysis of replanted Rehmannia glutinosa roots by iTRAQ reveals molecular mechanisms for formation of replant disease.

BACKGROUND: The normal growth of Rehmannia glutinosa, a widely used medicinal plant in China, is severely disturbed by replant disease. The formation of replant disease commonly involves interactions among plants, allelochemicals and microbes; however, these relationships remain largely unclear. As a result, no effective measures are currently available to treat replant disease. RESULTS: In this study, an integrated R. glutinosa transcriptome was constructed, from which an R. glutinosa protein library was obtained. iTRAQ technology was then used to investigate changes in the proteins in replanted R. glutinosa roots, and the proteins that were expressed in response to replant disease were identified. An integrated R. glutinosa transcriptome from different developmental stages of replanted and normal-growth R. glutinosa produced 65,659 transcripts, which were accurately translated into 47,818 proteins. Using this resource, a set of 189 proteins was found to be significantly differentially expressed between normal-growth and replanted R. glutinosa. Of the proteins that were significantly upregulated in replanted R. glutinosa, most were related to metabolism, immune responses, ROS generation, programmed cell death, ER stress, and lignin synthesis. CONCLUSIONS: By integrating these key events and the results of previous studies on replant disease formation, a new picture of the damaging mechanisms that cause replant disease stress emerged. Replant disease altered the metabolic balance of R. glutinosa, activated immune defence systems, increased levels of ROS and antioxidant enzymes, and initiated the processes of cell death and senescence in replanted R. glutinosa. Additionally, lignin deposition in R. glutinosa roots that was caused by replanting significantly inhibited tuberous root formation. These key processes provide important insights into the underlying mechanisms leading to the formation of replant disease and also for the subsequent development of new control measures to improve production and quality of replanted plants.

Lyophilized Powder of Catalpol and Puerarin Protected Cerebral Vessels from Ischemia by Its Anti-apoptosis on Endothelial Cells.

Catalpol and puerarin are two monomers of Rehmannia glutinosa and Lobed Kudzuvine Root, which are two herbs commonly used together in ancient prescriptions of traditional Chinese medicine for cerebral ischemia. Our previous study shows that the lyophilized powder of the two monomers improved the outcome of cerebral ischemia excellently in rodents. However, if it protects vessels from ischemia is unknown. The present research studied the protection of lyophilized powder of catalpol and puerarin (CP) on endothelial cells and the relative mechanism in vivo and in vitro. Middle cerebral artery occlusion (MCAO) rats were used to study the improvement of CP on neurological deficiency, regional cerebral blood flow (rCBF), and infarct volume. The morphology of vessels and the apoptosis of brain vascular endothelial cells (BVECs) were observed and detected by immunohistochemistry approaches. To study how CP protected primary BVECs (pBVECs) from ischemic penumbra, oxygen glucose deprivation (OGD)-damaged pBVECs were cultured in the condition of insufficient nutrition and low oxygen which recapitulate the low perfusion of ischemic penumbra. Using the cell model, the mechanism by which CP protected pBVECs was studied by shRNA and pathway inhibitors. CP at the dose of 65.4 mg/kg increased regional cerebral blood flow (rCBF), reduced infarct volume, protected vessel integrity and inhibited endothelial cell apoptosis in vivo. But it only improved rCBF, vessel integrity and BVECs apoptosis at the dose of 32.7 mg/kg. In vitro, the protection of CP on pBVECs was proved to be ERK/HIF-1a- and PI3K/AKT/mTOR/HIF-1a-dependent. This study indicates a possibility of CP being a new drug for cerebral ischemia. Besides, this research provides an alternative cell model for penumbra ECs study.

Integrating targeted glycomics and untargeted metabolomics to investigate the processing chemistry of herbal medicines, a case study on Rehmanniae Radix.

It is well-recognized that multiple components, the majority of which are secondary metabolites and carbohydrates, collectively contribute to the therapeutic effects of herbal medicines. The chemical characterization of herbal medicines has focused extensively on secondary metabolites but has largely overlooked carbohydrates. Here, we proposed an integrated chromatographic technique based targeted glycomics and untargeted metabolomics strategy simultaneously determining carbohydrates and secondary metabolites for the overall chemical profiling of herbal medicines; this strategy was successfully exemplified in an investigation of processing chemistry of Rehmanniae Radix (RR), a Chinese medicinal herb. It was demonstrated that the integrated strategy holistically illuminated the variations in the glycome and metabolome of RR samples processed by the traditionally-adopted nine cycles of steaming and drying, and further elucidated the processing-induced chemical transformation mechanisms of carbohydrates and secondary metabolites, and thereby revealed the inherent chemical connections between carbohydrates and secondary metabolites. The result suggested that the proposed strategy meets the technical demands for the overall chemical characterization of herbal medicines, and therefore could serve as a powerful tool for deciphering the scientific basis of herbal medicines.