Alleviatory Role of Panax Notoginseng Saponins in Modulating Inflammation and Pulmonary Vascular Remodeling in Chronic Obstructive Pulmonary Disease: mechanisms and Implications.

COPD is an inflammatory lung disease that limits airflow and remodels the pulmonary vascular system. This study delves into the therapeutic potential and mechanistic underpinnings of Panax notoginseng Saponins (PNS) in alleviating inflammation and pulmonary vascular remodeling in a COPD rat model. Symmap and ETCM databases provided Panax notoginseng-related target genes, and the CTD and DisGeNET databases provided COPD-related genes. Intersection genes were subjected to protein-protein interaction analysis and pathway enrichment to identify downstream pathways. A COPD rat model was established, with groups receiving varying doses of PNS and a Roxithromycin control. The pathological changes in lung tissue and vasculature were examined using histological staining, while molecular alterations were explored through ELISA, RT-PCR, and Western blot. Network pharmacology research suggested PNS may affect the TLR4/NF-κB pathway linked to COPD development. The study revealed that, in contrast to the control group, the COPD model exhibited a significant increase in inflammatory markers and pathway components such as TLR4, NF-κB, HIF-1α, VEGF, ICAM-1, SELE mRNA, and serum TNF-α, IL-8, and IL-1ß. Treatment with PNS notably decreased these markers and mitigated inflammation around the bronchi and vessels. Taken together, the study underscores the potential of PNS in reducing lung inflammation and vascular remodeling in COPD rats, primarily via modulation of the TLR4/NF-κB/HIF-1α/VEGF pathway. This research offers valuable insights for developing new therapeutic strategies for managing and preventing COPD.

Comprehensive genomic analysis of Burkholderia arboris PN-1 reveals its biocontrol potential against Fusarium solani-induced root rot in Panax notoginseng.

Panax notoginseng (Burkill) F.H. Chen, a valuable traditional Chinese medicine, faces significant yield and quality challenges stemming from root rot primarily caused by Fusarium solani. Burkholderia arboris PN-1, isolated from the rhizosphere soil of P. notoginseng, demonstrated a remarkable ability to inhibit the growth of F. solani. This study integrates phenotypic, phylogenetic, and genomic analyses to enhance our understanding of the biocontrol mechanisms employed by B. arboris PN-1. Phenotype analysis reveals that B. arboris PN-1 effectively suppresses P. notoginseng root rot both in vitro and in vivo. The genome of B. arboris PN-1 comprises three circular chromosomes (contig 1: 3,651,544 bp, contig 2: 1,355,460 bp, and contig 3: 3,471,056 bp), with a 66.81% GC content, housing 7,550 protein-coding genes. Notably, no plasmids were detected. Phylogenetic analysis places PN-1 in close relation to B. arboris AU14372, B. arboris LMG24066, and B. arboris MEC_B345. Average nucleotide identity (ANI) values confirm the PN-1 classification as B. arboris. Comparative analysis with seven other B. arboris strains identified 4,628 core genes in B. arboris PN-1. The pan-genome of B. arboris appears open but may approach closure. Whole-genome sequencing revealed 265 carbohydrate-active enzymes and identified 9 gene clusters encoding secondary metabolites. This comprehensive investigation enhances our understanding of B. arboris genomes, paving the way for their potential as effective biocontrol agents against fungal plant pathogens in the future.

Identification of candidate genes and residues for improving nitrogen use efficiency in the N-sensitive medicinal plant Panax notoginseng.

BACKGROUND: Nitrogen (N) metabolism-related key genes and conserved amino acid sites in key enzymes play a crucial role in improving N use efficiency (NUE) under N stress. However, it is not clearly known about the molecular mechanism of N deficiency-induced improvement of NUE in the N-sensitive rhizomatous medicinal plant Panax notoginseng (Burk.) F. H. Chen. To explore the potential regulatory mechanism, the transcriptome and proteome were analyzed and the three-dimensional (3D) information and molecular docking models of key genes were compared in the roots of P. notoginseng grown under N regimes. RESULTS: Total N uptake and the proportion of N distribution to roots were significantly reduced, but the NUE, N use efficiency in biomass production (NUEb), the recovery of N fertilizer (RNF) and the proportion of N distribution to shoot were increased in the N0-treated (without N addition) plants. The expression of N uptake- and transport-related genes NPF1.2, NRT2.4, NPF8.1, NPF4.6, AVP, proteins AMT and NRT2 were obviously up-regulated in the N0-grown plants. Meanwhile, the expression of CIPK23, PLC2, NLP6, TCP20, and BT1 related to the nitrate signal-sensing and transduction were up-regulated under the N0 condition. Glutamine synthetase (GS) activity was decreased in the N-deficient plants, while the activity of glutamate dehydrogenase (GDH) increased. The expression of genes GS1-1 and GDH1, and proteins GDH1 and GDH2 were up-regulated in the N0-grown plants, there was a significantly positive correlation between the expression of protein GDH1 and of gene GDH1. Glu192, Glu199 and Glu400 in PnGS1 and PnGDH1were the key amino acid residues that affect the NUE and lead to the differences in GDH enzyme activity. The 3D structure, docking model, and residues of Solanum tuberosum and P. notoginseng was similar. CONCLUSIONS: N deficiency might promote the expression of key genes for N uptake (genes NPF8.1, NPF4.6, AMT, AVP and NRT2), transport (NPF1.2 and NRT2.4), assimilation (proteins GS1 and GDH1), signaling and transduction (genes CIPK23, PLC2, NLP6, TCP20, and BT1) to enhance NUE in the rhizomatous species. N deficiency might induce Glu192, Glu199 and Glu400 to improve the biological activity of GS1 and GDH, this has been hypothesized to be the main reason for the enhanced ability of N assimilation in N-deficient rhizomatous species. The key genes and residues involved in improving NUE provide excellent candidates for the breeding of medicinal plants.

PnNAC2 promotes the biosynthesis of Panax notoginseng saponins and induces early flowering.

KEY MESSAGE: PnNAC2 positively regulates saponin biosynthesis by binding the promoters of key biosynthetic genes, including PnSS, PnSE, and PnDS. PnNAC2 accelerates flowering through directly associating with the promoters of FT genes. NAC transcription factors play an important regulatory role in both terpenoid biosynthesis and flowering. Saponins with multiple pharmacological activities are recognized as the major active components of Panax notoginseng. The P. notoginseng flower is crucial for growth and used for medicinal and food purposes. However, the precise function of the P. notoginseng NAC transcription factor in the regulation of saponin biosynthesis and flowering remains largely unknown. Here, we conducted a comprehensive characterization of a specific NAC transcription factor, designated as PnNAC2, from P. notoginseng. PnNAC2 was identified as a nuclear-localized protein with transcription activator activity. The expression profile of PnNAC2 across various tissues mirrored the accumulation pattern of total saponins. Knockdown experiments of PnNAC2 in P. notoginseng calli revealed a significant reduction in saponin content and the expression level of pivotal saponin biosynthetic genes, including PnSS, PnSE, and PnDS. Subsequently, Y1H assays, dual-LUC assays, and electrophoretic mobility shift assays (EMSAs) demonstrated that PnNAC2 exhibits binding affinity to the promoters of PnSS, PnSE and PnDS, thereby activating their transcription. Additionally, an overexpression assay of PnNAC2 in Arabidopsis thaliana witnessed the acceleration of flowering and the induction of the FLOWERING LOCUS T (FT) gene expression. Furthermore, PnNAC2 demonstrated the ability to bind to the promoters of AtFT and PnFT genes, further activating their transcription. In summary, these results revealed that PnNAC2 acts as a multifunctional regulator, intricately involved in the modulation of triterpenoid saponin biosynthesis and flowering processes.

Integrated metabolome and transcriptome analysis reveals the regulatory mechanism of low nitrogen-driven biosynthesis of saponins and flavonoids in Panax notoginseng.

BACKGROUND: Nitrogen (N) is an important macronutrient involved in the biosynthesis of primary and secondary metabolites in plants. However, the metabolic regulatory mechanism of low-N-induced triterpenoid saponin and flavonoid accumulation in rhizomatous medicinal Panax notoginseng (Burk.) F. H. Chen remains unclear. METHODS: To explore the potential regulatory mechanism and metabolic basis controlling the response of P. notoginseng to N deficiency, the transcriptome and metabolome were analysed in the roots. RESULTS: The N content was significantly reduced in roots of N0-treated P. notoginseng (0 kg·N·667 m-2). The C/N ratio was enhanced in the N-deficient P. notoginseng. N deficiency promotes the accumulation of amino acids (L-proline, L-leucine, L-isoleucine, L-norleucine, L-arginine, and L-citrulline) and sugar (arabinose, xylose, glucose, fructose, and mannose), thus providing precursor metabolites for the biosynthesis of flavonoids and triterpenoid saponins. Downregulation of key structural genes (PAL, PAL3, ACC1, CHS2, PPO, CHI3, F3H, DFR, and FGT), in particular with the key genes of F3H, involved in the flavonoid biosynthesis pathway possibly induced the decrease in flavonoid content with increased N supply. Notoginsenoside R1, ginsenoside Re, Rg1, Rd, F1, R1 + Rg1 + Rb1 and total triterpenoid saponins were enhanced in the N0 groups than in the N15 (15 kg·N·667 m-2) plants. Higher phosphoenolpyruvate (an intermediate of glycolyticwith pathway metabolism) and serine (an intermediate of photorespiration) levels induced by N deficiency possibly promote saponin biosynthesis through mevalonic acid (MVA) and methylerythritol (MEP) pathways. Genes (MVD2, HMGS, HMGR1, HMGR2, DXR, and HMGR1) encoding the primary enzymes HMGS, HMGR, DXR, and MVD in the MVA and MEP pathways were significantly upregulated in the N0-treated P. notoginseng. The saponin biosynthesis genes DDS, DDS, CYP716A52, CYP716A47, UGT74AE2, and FPS were upregulated in the N-deficient plants. Upregulation of genes involved in saponin biosynthesis promotes the accumulation of triterpenoid saponins in the N0-grown P. notoginseng. CONCLUSIONS: N deficiency enhances primary metabolisms, such as amino acids and sugar accumulation, laying the foundation for the synthesis of flavonoids and triterpenoid saponins in P. notoginseng. F3H, DDS, FPS, HMGR, HMGS and UGT74AE2 can be considered as candidates for functional characterisation of the N-regulated accumulation of triterpenoid saponins and flavonoids in future.

Cucumber mosaic virus impairs the physiological homeostasis of Panax notoginseng and induces saponin-mediated resistance.

As an important medicinal plant, Panax notoginseng often suffers from various abiotic and biotic stresses during its growth, such as drought, heavy metals, fungi, bacteria and viruses. In this study, the symptom and physiological parameters of cucumber mosaic virus (CMV)-infected P. notoginseng were analyzed and the RNA-seq was performed. The results showed that CMV infection affected the photosynthesis of P. notoginseng, caused serious oxidative damage to P. notoginseng and increased the activity of several antioxidant enzymes. Results of transcriptome analysis and corresponding verification showed that CMV infection changed the expression of genes related to plant defense and promoted the synthesis of P. notoginseng saponins to a certain extent, which may be defensive ways of P. notoginseng against CMV infection. Furthermore, pretreatment plants with saponins reduced the accumulation of CMV. Thus, our results provide new insights into the role of saponins in P. notoginseng response to virus infection.

Physiological and metabolomic analyses reveal that Fe3O4 nanoparticles ameliorate cadmium and arsenic toxicity in Panax notoginseng.

Heavy metal(loid)-contaminated available arable land seriously affects crop development and growth. Engineered nanomaterials have great potential in mitigating toxic metal(loid) stress in plants. However, there are few details of nanoparticles (NPs) involved in Panax notoginseng response to cadmium (Cd) and arsenic (As). Herein, integrating physiological and metabolomic analyses, we investigated the effects of Fe3O4 NPs on plant growth and Cd/As responses in P. notoginseng. Cd/As treatment caused severe growth inhibition. However, foliar application of Fe3O4 NPs increased beneficial elements in the roots and/or leaves, decreased Cd/As content by 10.38% and 20.41% in the roots, reduced membrane damage and regulated antioxidant enzyme activity, thereby alleviating Cd/As-induced growth inhibition, as indicated by increased shoot fresh weight (FW), the rootlet length and root FW by 40.14%, 15.74%, and 46.70% under Cd stress and promoted the shoot FW by 27.00% under As toxicity. Metabolomic analysis showed that 227 and 295 differentially accumulated metabolites (DAMs) were identified, and their accumulation patterns were classified into 8 and 6 clusters in the roots and leaves, respectively. Fe3O4 NPs altered metabolites significantly involved in key pathways, including amino sugar and nucleotide sugar metabolism, flavonoid biosynthesis and phenylalanine metabolism, thus mediating the trade-off between plant growth and defense under stress. Interestingly, Fe3O4 NPs recovered more Cd/As-induced DAMs to normal levels, further supporting that Fe3O4 NPs positively affected seedling growth under metal(loid)s stress. In addition, Fe3O4 NPs altered terpenoids when the seedlings were subjected to Cd/As stress, thus affecting their potential medicinal value. This study provides insights into using nanoparticles to improve potential active ingredients of medicinal plants in metal(loid)-contaminated areas.

Effects of borneol combined with astragaloside IV and Panax notoginseng saponins regulation of microglia polarization to promote neurogenesis after cerebral ischaemia.

OBJECTIVE: To study the effect of borneol combined with astragaloside IV and Panax notoginseng saponins (BAP) on promoting neurogenesis by regulating microglia polarization after cerebral ischaemia-reperfusion(CI/R) in rats. METHODS: A focal CI/R injury model was established. Evaluated the effects of BAP on ischaemic brain injury, on promoting neurogenesis, on inhibiting Inflammatory microenvironment and TLR4/MyD88/NFκB signalling pathway. A microglia oxygen-glucose deprivation reoxygenation (OGD/R) model was established that evaluated the effects of BAP on regulating the polarization of microglia and inflammatory microenvironment. RESULTS: BAP can inhibit the expression of TLR4, MyD88 and NFκB proteins, reduce IL-1ß and increase IL-10, reduce M1 type microglia and increase M2 microglia. The proliferation of neural stem cells increased, synaptic gap decreased, synaptic interface curvature increased, expression of SYN and PSD95 proteins increased, which improved the neurological dysfunction and reduced the volume of cerebellar infarction and nerve cell injury. CONCLUSION: BAP can reduce CI/R injury and promote neurogenesis, the effect is related to inhibition of the activation of TLR4/MyD88/NFκB, regulating the polarization of microglia from M1 type to M2 type and inhibition of inflammatory response.

Genome-wide identification and characterization of members of the LEA gene family in Panax notoginseng and their transcriptional responses to dehydration of recalcitrant seeds.

BACKGROUND: Late embryogenesis abundant (LEA) proteins play an important role in dehydration process of seed maturation. The seeds of Panax notoginseng (Burkill) F. H. Chen are typically characterized with the recalcitrance and are highly sensitive to dehydration. However, it is not very well known about the role of LEA proteins in response to dehydration stress in P. notoginseng seeds. We will perform a genome-wide analysis of the LEA gene family and their transcriptional responses to dehydration stress in recalcitrant P. notoginseng seeds. RESULTS: In this study, 61 LEA genes were identified from the P. notoginseng genome, and they were renamed as PnoLEA. The PnoLEA genes were classified into seven subfamilies based on the phylogenetic relationships, gene structure and conserved domains. The PnoLEA genes family showed relatively few introns and was highly conserved. Unexpectedly, the LEA_6 subfamily was not found, and the LEA_2 subfamily contained 46 (75.4%) members. Within 19 pairs of fragment duplication events, among them 17 pairs were LEA_2 subfamily. In addition, the expression of the PnoLEA genes was obviously induced under dehydration stress, but the germination rate of P. notoginseng seeds decreased as the dehydration time prolonged. CONCLUSIONS: We found that the lack of the LEA_6 subfamily, the expansion of the LEA_2 subfamily and low transcriptional levels of most PnoLEA genes might be implicated in the recalcitrant formation of P. notoginseng seeds. LEA proteins are essential in the response to dehydration stress in recalcitrant seeds, but the protective effect of LEA protein is not efficient. These results could improve our understanding of the function of LEA proteins in the response of dehydration stress and their contributions to the formation of seed recalcitrance.

The combination of astragaloside IV and Panax notoginseng saponins attenuates cerebral ischaemia-reperfusion injury in rats through ferroptosis and inflammation inhibition via activating Nrf2.

OBJECTIVES: This study aimed to observe the effect of the combination of astragaloside IV (AST IV) and Panax notoginseng saponins (PNS) on cerebral ischaemia-reperfusion injury (CIRI) and explore the specific mechanism of the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated combination of AST IV and PNS against CIRI based on ferroptosis and inflammatory response. METHODS: The therapeutic effect and mechanism of AST IV and PNS were evaluated by constructing a Sprague-Dawley rat middle cerebral artery ischaemia-occlusion-reperfusion model. The specific mechanism of the combination of AST IV and PNS against CIRI was revealed through the combined intervention of the Nrf2-specific inhibitor brusatol. KEY FINDINGS: After AST IV and PNS treatment, the cerebral infarction area of the rats was reduced; behavioural performance was improved; Fe2+, malondialdehyde, lipid peroxidation, interleukin-6, interleukin-1ß, tumour necrosis factor-α and myeloperoxidase levels were reduced; and glutathione and glutathione peroxidase 4 levels were increased. In addition, the expression of Nrf2 was significantly increased, the combined treatment was more effective than the single treatment, and the Nrf2 inhibitor brusatol could reverse the effects of the combined intervention of AST IV and PNS. CONCLUSIONS: The findings of this study suggest that combining AST IV and PNS attenuates CIRI by activating Nrf2 to inhibit ferroptosis and inflammatory responses.

Engineered Glycosidase for Significantly Improved Production of Naturally Rare Vina-Ginsenoside R7.

Ginsenosides are the main bioactive ingredients in plants of the genus Panax. Vina-ginsenoside R7 (VG-R7) is one of the rare high-value ginsenosides with health benefits. The only reported method for preparing VG-R7 involves inefficient and low-yield isolation from highly valuable natural resources. Notoginsenoside Fc (NG-Fc) isolated in the leaves and stems of Panax notoginseng is a suitable substrate for the preparation of VG-R7 via specific hydrolysis of the outside xylose at the C-20 position. Here, we first screened putative enzymes belonging to the glycoside hydrolase (GH) families 1, 3, and 43 and found that KfGH01 can specifically hydrolyze the ß-d-xylopyranosyl-(1 → 6)-ß-d-glucopyranoside linkage of NG-Fc to form VG-R7. The I248F/Y410R variant of KfGH01 obtained by protein engineering displayed a kcat/KM value (305.3 min-1 mM-1) for the reaction enhanced by approximately 270-fold compared with wild-type KfGH01. A change in the shape of the substrate binding pockets in the mutant allows the substrate to sit closer to the catalytic residues which may explain the enhanced catalytic efficiency of the engineered enzyme. This study identifies the first glycosidase for bioconversion of a ginsenoside with more than four sugar units, and it will inspire efforts to investigate other promising enzymes to obtain valuable natural products.

High nitrogen inhibits biomass and saponins accumulation in a medicinal plant Panax notoginseng.

Nitrogen (N) is an important macronutrient and is comprehensively involved in the synthesis of secondary metabolites. However, the interaction between N supply and crop yield and the accumulation of effective constituents in an N-sensitive medicinal plant Panax notoginseng (Burkill) F. H. Chen is not completely known. Morphological traits, N use and allocation, photosynthetic capacity and saponins accumulation were evaluated in two- and three-year-old P. notoginseng grown under different N regimes. The number and length of fibrous root, total root length and root volume were reduced with the increase of N supply. The accumulation of leaf and stem biomass (above-ground) were enhanced with increasing N supply, and LN-grown plants had the lowest root biomass. Above-ground biomass was closely correlated with N content, and the relationship between root biomass and N content was negatives in P. notoginseng (r = -0.92). N use efficiency-related parameters, NUE (N use efficiency, etc.), NC (N content in carboxylation system component) and P n (the net photosynthetic rate) were reduced in HN-grown P. notoginseng. SLN (specific leaf N), Chl (chlorophyll), NL (N content in light capture component) increased with an increase in N application. Interestingly, root biomass was positively correlated with NUE, yield and P n. Above-ground biomass was close negatively correlated with photosynthetic N use efficiency (PNUE). Saponins content was positively correlated with NUE and P n. Additionally, HN improved the root yield of per plant compared with LN, but reduced the accumulation of saponins, and the lowest yield of saponins per unit area (35.71 kg·hm-2) was recorded in HN-grown plants. HN-grown medicinal plants could inhibit the accumulation of root biomass by reducing N use and photosynthetic capacity, and HN-induced decrease in the accumulation of saponins (C-containing metabolites) might be closely related to the decline in N efficiency and photosynthetic capacity. Overall, N excess reduces the yield of root and C-containing secondary metabolites (active ingredient) in N-sensitive medicinal species such as P. notoginseng.

Genome-wide identification and expression analysis of MYB gene family under nitrogen stress in Panax notoginseng.

The myeloblastosis (MYB) gene family, involved in regulating many important physiological and biochemical processes, is one of the largest transcript factor superfamilies in plants. Since the identification of genome sequencing of Panax notoginseng has been completed, there was little known about the whole genome of its specific MYB gene family and the response to abiotic stresses, in consideration of the excessive application of nitrogen fertilizers in P. notoginseng. In this study, 123 PnMYB genes (MYB genes of P. notoginseng) have been identified and divided into 3 subfamilies by the phylogenetic analysis. These PnMYB genes were unevenly located on 12 chromosomes. Meanwhile, the gene structure and protein conserved domain were established by MEME Suite. The analysis of collinear relationships reflected that there were 121 homologous genes between P. notoginseng and Arabidopsis and 30 between P. notoginseng and rice. Moreover, cis-acting elements of PnMYB gene promoters were predicted which indicated that PnMYBs are involved in biotic, abiotic stress, and hormone induction. The expressions of PnMYB transcription factors in its roots, flowers, and leaves were detected by qRT-PCR and they had tissue-specific expressions and related to the growth of different tissues. Under nitrogen stress, MYB transcription factors had great feedback. Ten R2R3-MYB subfamily genes were significantly induced and indicated the possible function of protecting P. notoginseng from excess nitrogen. With further knowledge on identification of PnMYB gene related to tissue selectivity and abiotic stresses, this study laid the foundation for the functional development of PnMYB gene family and improved the cultivation of P. notoginseng.

New Glycosyltransferases in Panax notoginseng Perfect Main Ginsenosides Biosynthetic Pathways.

Ginsenosides, the main bioactive ingredients of the Panax genus, are dammarane or oleanane triterpenoids with glycosylated modifications at C3/C6/C20 hydroxyls or C28 carboxyl, and their diverse glycosylation pattern has attracted great attention. However, the biosynthesis of some important saponins is still unclear. In this study, six UGTs were characterized, two of which were novel. PnUGT71A3 catalyzes not only the C6 hydroxyl glycosylation of protopanaxatriol (PPT) and F1 to form Rh1 and Rg1, respectively, but also the C20 hydroxyl glycosylation of protopanaxadiol (PPD)-type Rg3 to generate Rd. Especially, PnUGT94M1 is UDP-ß-l-rhamnose (UDP-Rha)-dependent, regioselectively catalyzing the C2' hydroxyl rhamnosylation of C6 glucose of the PPT-type ginsenosides Rg1 and Rh1 to generate ginsenosides Re and Rg2, respectively. Site-directed mutagenesis showed that His21, Asp120, Ser363, and Pro372 are key residues, and the triple mutant (G344S/G345S/L346T) highly improved the activity toward Rg1 and Rh1. The findings in this study, perfect main ginsenosides biosynthetic pathways in the Panax genus, expand the biocatalyst toolbox for ginsenoside production and show that the PSPG motif is one of the options to modify UGTs to improve their activities.

Multi-algorithm cooperation research of WRKY genes under nitrogen stress in Panax notoginseng.

WRKY transcription factors play an important role in the immune system and the innate defense response of plants. WRKY transcription factors have great feedback on nitrogen stress. In this study, bioinformatics was used to detect the WRKYs of Panax notoginseng (PnWRKYs). The response of PnWRKYs under nitrogen stress was also well studied. PnWRKYs were distributed on 11 chromosomes. According to PnWRKY and Arabidopsis thaliana WRKY (AtWRKY) domains, these PnWRKY proteins were divided into three groups by phylogenetic analysis. MEME analysis showed that almost every member contained motif 1 and motif 2. PlantCARE online predicted the cis-acting elements of the promoter. PnWRKY gene family members obtained 22 pairs of repeat fragments by collinearity analysis. The expression levels of PnWRKYs in different parts (roots, flowers, and leafs) were analyzed by the gene expression pattern. They reflected tissue-specific expressions. The qRT-PCR experiments were used to detect 74 PnWRKYs under nitrogen stress. The results showed that the expression levels of 8 PnWRKYs were significantly induced. The PnWRKY gene family may be involved in biotic/abiotic stresses and hormone induction. This study will not only lay the foundation to explore the functions of PnWRKYs but also provide candidate genes for the future improvement of P. notoginseng.

Ginsenoside Rb1, a principal effective ingredient of Panax notoginseng, produces pain antihypersensitivity by spinal microglial dynorphin A expression.

Panax notoginseng (Chinese ginseng, Sanqi), one of the major ginseng species, has been traditionally used to alleviate different types of chronic pain. The raw P. notoginseng powder is commonly available in China as a non-prescription drug to treat various aliments including arthritic pain. However, strong scientific evidence is needed to illustrate its pain antihypersensitive effects, effective ingredients and mechanism of action. The oral P. notoginseng powder dose-dependently alleviated formalin-induced tonic hyperalgesia, and its total ginsenosides remarkably inhibited neuropathic pain hypersensitivity. Ginsenoside Rb1, the most abundant ginsenoside of P. notoginseng, dose-dependently produced neuropathic pain antihypersensitivity. Conversely, ginsenosides Rg1, Re and notoginseng R1, the other major saponins from P. notoginseng, failed to inhibit formalin-induced tonic pain or mechanical allodynia in neuropathic pain. Ginsenoside Rb1 metabolites ginsenosides Rg3, Compound-K and protopanaxadiol also had similar antineuropathic pain efficacy to ginsenoside Rb1. Additionally, intrathecal ginsenoside Rb1 specifically stimulated dynorphin A expression which was colocalized with microglia but not neurons or astrocytes in the spinal dorsal horn and primary cultured cells. Pretreatment with microglial metabolic inhibitor minocycline, dynorphin A antiserum and specific κ-opioid receptor antagonist GNTI completely blocked Rb1-induced mechanical antiallodynia in neuropathic pain. Furthermore, the specific glucocorticoid receptor (GR) antagonist Dex-21-mesylate (but not GPR30 estrogen receptor antagonist G15) also entirely attenuated ginsenoside Rb1-related antineuropathic pain effects. All these results, for the first time, show that P. notoginseng alleviates neuropathic pain and ginsenoside Rb1 is its principal effective ingredient. Furthermore, ginsenoside Rb1 inhibits neuropathic pain by stimulation of spinal microglial dynorphin A expression following GR activation.

Components of Salvia miltiorrhiza and Panax notoginseng Protect Pericytes Against OGD/R-Induced Injury via Regulating the PI3K/AKT/mTOR and JNK/ERK/P38 Signaling Pathways.

Salvia miltiorrhiza (SAL) and Panax notoginseng (PNS) are widely used in treating of ischemic stroke. However, it is unknown which components of SAL and PNS protect brain microvascular pericytes after an ischemic stroke. We evaluated the protective effects and mechanisms of SAL and PNS components in pericytes subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Pericytes were subjected to OGD/R. Cell Counting Kit-8 (CCK-8) was used to evaluate cell viability. ROS and SOD kits were used to detect oxidative stress. Flow cytometry was performed to analyze cell apoptosis. To evaluate cell migration, a scratch assay was performed. Expression of cleaved caspase-3, Bcl-2, Bax, VEGF, Ang-1, PDGFR-ß, PI3K/AKT/mTOR, and JNK/ERK/P38 signaling pathways were identified using western blot. The results revealed that salvianolic acid B (Sal B), salvianolic acid D (Sal D), notoginsenoside R1 (R1), ginsenoside Rb1 (Rb1), and ginsenoside Rg1 (Rg1) increased the cell viability of pericytes subjected to OGD/R, reduced the level of ROS, and increased the expression of SOD. The components reduced cell apoptosis, increased the protein level of Bcl-2/Bax, reduced the level of cleaved caspase-3/caspase-3, increased cell migration, and enhanced the levels of Ang-1, PDGFR-ß, and VEGF. The components could activate PI3K/AKT/mTOR pathway while inhibiting the JNK/ERK/P38 pathway. Studies found that Sal B, Sal D, R1, Rb1, and Rg1 inhibited oxidative stress and apoptosis while increasing the release of pro-angiogenic regulators of pericytes related to the PI3K/AKT/mTOR and JNK/ERK/P38 signaling pathways. This provides a potential foundation for developing monomeric drugs for treating ischemic stroke.

Panaxydol Derived from Panax notoginseng Promotes Nerve Regeneration after Sciatic Nerve Transection in Rats.

BACKGROUND: Peripheral nerve regeneration is a coordinated process of Schwann cell (SC) reprogramming and intrinsic neuronal growth program activation. Panaxydol (PND) is a strong biologically active traditional Chinese medicine monomer extracted from Panax notoginseng rhizomes. In vitro, PND protects neurons and SCs from injury and stimulates the expression and secretion of neurotrophic factors (NTFs) by SCs. We hypothesized that PND may also promote peripheral nerve regeneration in adult animals. METHODS: PND (10 mg/kg body weight) was injected intraperitoneally into the Sprague-Dawley (SD) rats for two consecutive weeks after sciatic nerve transection. The morphology of the repaired sciatic nerve was evaluated after 16 weeks, and sensory and motor function recovery was evaluated using functional and behavioral techniques. RESULTS: PND was biologically safe at an injection dose of 10 mg/kg/day. After 14 days, it significantly increased the myelination of regenerated nerve fibers, and promoted sensory and motor function recovery. In the early stage of injury, PND significantly upregulated the mRNA expression of brain-derived neurotrophic factor (BDNF) and its receptors in distal injured nerves, which may represent a possible mechanism by which PND promotes nerve regeneration in vivo. CONCLUSIONS: Our study demonstrated that PND leads to sensory and motor recovery in a sciatic nerve transection model rat. Furthermore, we showed that BDNF mRNA level was significantly increased in the injured distal nerve, potentially contributing to the functional recovery. Further research is warrantied to examine whether direct injection is a more efficient method to increase BDNF expression compared to an exogenous BDNF administration.

Multi-algorithm cooperation comprehensive research of bZIP genes under nitrogen stress in Panax notoginseng.

Basic leucine zipper (bZIP) transcription factors play an irreplaceable position in the regulation of plant secondary metabolism, growth and development, and resistance to abiotic stress. Panax notoginseng is a traditional medicinal plant in China, but the systematic identification and the resistance of Panax notoginseng bZIP (PnbZIP) family under nitrogen stress have not been reported before, considering the excessive application of N fertilizers. In this study, we conducted a genome-wide identification of the PnbZIP family and analyzed its phylogeny, tissue selectivity, and abiotic resistence. 74 PnbZIPs were distributed on 12 chromosomes and 8 were not successfully located. Through phylogenetic analysis of Arabidopsis and Panax notoginseng, we divided them into 14 subgroups. In the same subgroup, bZIPs had similiar intron/exon structure and conserved motifs. In the analysis of chromosome structure, two PnbZIP genes were duplicated in tandem on chromosome 3. Intraspecific collinearity analysis showed that 28 PnbZIPs participated in segmental replication. Each PnbZIP promoter contained at least one stress response element or stress-related hormone response element. RNA-seq and qRT-PCR methods were used to analyze the expression patterns of the PnbZIP gene in different tissues (roots, flowers, and leaves) and under different nitrogen stresses. The results showed that the PnbZIP gene had the highest expression level in flowers and reflected tissue-specific expressions. Meanwhile, under the stress of ammonium nitrogen fertilizer and nitrate nitrogen fertilizer, PnbZIPs in roots were differently expressed. 10 PnbZIP stress-responsive genes were screened for significant expression, among which PnbZIP46 was significantly up-regulated, which could be a candidate gene for resistance to Nitrogen stress. This study laid the foundation for functional identification of PnbZIPs and improved the cultivation of Panax notoginseng.

Key Glycosyltransferase Genes of Panax notoginseng: Identification and Engineering Yeast Construction of Rare Ginsenosides.

Panax notoginseng is one of the most famous valuable medical plants in China, and its broad application in clinical treatment has an inseparable relationship with the active molecules, ginsenosides. Ginsenosides are glycoside compounds that have varied structures for the diverse sugar chain. Although extensive work has been done, there are still unknown steps in the biosynthetic pathway of ginsenosides. Here, we screened candidate glycosyltransferase genes based on the previous genome and transcriptome data of P. notoginseng and cloned the full length of 27 UGT genes successfully. Among them, we found that PnUGT33 could catalyze different ginsenoside substrates to produce higher polarity rare ginsenosides by extending the sugar chain. We further analyzed the enzymatic kinetics and predicted the catalytic mechanism of PnUGT33 by simulating molecular docking. After that, we reconstructed the biosynthetic pathway of rare ginsenoside Rg3 and gypenoside LXXV in yeast. By combining the Golden Gate method and overexpressing the UDPG biosynthetic genes, we further improved the yield of engineering yeast strain. Finally, the shake-flask culture yield of Rg3 reached 51 mg/L and the fed-batch fermentation yield of gypenoside LXXV reached 94.5 mg/L, which was the first and highest record.