Structure of the Human cGAS-DNA Complex Reveals Enhanced Control of Immune Surveillance.

Cyclic GMP-AMP synthase (cGAS) recognition of cytosolic DNA is critical for immune responses to pathogen replication, cellular stress, and cancer. Existing structures of the mouse cGAS-DNA complex provide a model for enzyme activation but do not explain why human cGAS exhibits severely reduced levels of cyclic GMP-AMP (cGAMP) synthesis compared to other mammals. Here, we discover that enhanced DNA-length specificity restrains human cGAS activation. Using reconstitution of cGAMP signaling in bacteria, we mapped the determinant of human cGAS regulation to two amino acid substitutions in the DNA-binding surface. Human-specific substitutions are necessary and sufficient to direct preferential detection of long DNA. Crystal structures reveal why removal of human substitutions relaxes DNA-length specificity and explain how human-specific DNA interactions favor cGAS oligomerization. These results define how DNA-sensing in humans adapted for enhanced specificity and provide a model of the active human cGAS-DNA complex to enable structure-guided design of cGAS therapeutics.

The Translation Inhibitor Rocaglamide Targets a Bimolecular Cavity between eIF4A and Polypurine RNA.

A class of translation inhibitors, exemplified by the natural product rocaglamide A (RocA), isolated from Aglaia genus plants, exhibits antitumor activity by clamping eukaryotic translation initiation factor 4A (eIF4A) onto polypurine sequences in mRNAs. This unusual inhibitory mechanism raises the question of how the drug imposes sequence selectivity onto a general translation factor. Here, we determined the crystal structure of the human eIF4A1⋅ATP analog⋅RocA⋅polypurine RNA complex. RocA targets the "bi-molecular cavity" formed characteristically by eIF4A1 and a sharply bent pair of consecutive purines in the RNA. Natural amino acid substitutions found in Aglaia eIF4As changed the cavity shape, leading to RocA resistance. This study provides an example of an RNA-sequence-selective interfacial inhibitor fitting into the space shaped cooperatively by protein and RNA with specific sequences.

Pharmacodynamic study of chitosan in combination with salvianolic acid B in the treatment of CC14 -induced liver fibrosis in mice.

Liver fibrosis is a chronic liver lesion caused by excessive deposition of the extracellular matrix after liver damage, resulting in fibrous scarring of liver tissue. The progression of liver fibrosis is partially influenced by the gut microbiota. Chitosan can play a therapeutic role in liver fibrosis by regulating the gut microbiota based on the 'gut-liver axis' theory. Salvianolic acid B can inhibit the development of liver fibrosis by inhibiting the activation of hepatic stellate cells and reducing the production of extracellular matrix. In this study, the therapeutic effect of chitosan in combination with salvianolic acid B on liver fibrosis was investigated in a mouse liver fibrosis model. The results showed that the combination of chitosan and salvianolic acid B was better than the drug alone, improving AST/ALT levels and reducing the expression of α-SAM, COL I, IL-6 and other related genes. It improved the structure of gut microbiota and increased the abundance of beneficial bacteria such as Lactobacillus. The above results could provide new ideas for the clinical treatment of liver fibrosis.

Divergent reactivity of usnic acid and evaluation of its derivatives for antiproliferative activity against cancer cells.

Natural products continue to be an inspiration for new drugs to treat debilitating diseases such as cancer. Usnic acid is a secondary metabolite isolated predominately from lichen species and has been shown to exhibit antiproliferative properties, however its application is limited by poor drug-like properties and low specificity. We report our work on investigating the reactivity of usnic acid for incorporating heterocyclic rings and the divergent reactivity that can be obtained by simply altering the reaction solvent and temperature. The synthesised derivatives were then tested against HeLa cancer cells for their antiproliferative properties. A number of promising compounds were obtained including 4, 5 and 9 that showed an IC50 of 878, 311 and 116 nM, respectively, against HeLa cancer cells after 48 h of treatment.

Transfer of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs) from oral exposure into cow's milk - part II: toxicokinetic predictive models for risk assessment.

Understanding the transfer of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) as well as polychlorinated biphenyls (PCBs) from oral exposure into cow's milk is not purely an experimental endeavour, as it has produced a large corpus of theoretical work. This work consists of a variety of predictive toxicokinetic models in the realms of health and environmental risk assessment and risk management. Their purpose is to provide mathematical predictive tools to organise and integrate knowledge on the absorption, distribution, metabolism and excretion processes. Toxicokinetic models are based on more than 50 years of transfer studies summarised in part I of this review series. Here in part II, several of these models are described and systematically classified with a focus on their applicability to risk analysis as well as their limitations. This part of the review highlights the opportunities and challenges along the way towards accurate, congener-specific predictive models applicable to changing animal breeds and husbandry conditions.

Salvianolic Acid B Attenuates Hypertrophic Scar Formation In Vivo and In Vitro.

BACKGROUND: Hypertrophic scars (HTSs) are a fibroproliferative disorder that occur following skin injuries. Salvianolic acid B (Sal-B) is an extractant from Salvia miltiorrhiza that has been reported to ameliorate fibrosis in multiple organs. However, the antifibrotic effect on HTSs remains unclear. This study aimed to determine the antifibrotic effect of Sal-B in vitro and in vivo. METHODS: In vitro, hypertrophic scar-derived fibroblasts (HSFs) were isolated from human HTSs and cultured. HSFs were treated with (0, 10, 50, 100 µmol/L) Sal-B. Cell proliferation and migration were evaluated by EdU, wound healing, and transwell assays. The protein and mRNA levels of TGFßI, Smad2, Smad3, α-SMA, COL1, and COL3 were detected by Western blots and real-time PCR. In vivo, tension stretching devices were fixed on incisions for HTS formation. The induced scars were treated with 100 µL of Sal-B/PBS per day according to the concentration of the group and followed up for 7 or 14 days. The scar condition, collagen deposition, and α-SMA expression were analyzed by gross visual examination, H&E, Masson, picrosirius red staining, and immunofluorescence. RESULTS: In vitro, Sal-B inhibited HSF proliferation, migration, and downregulated the expression of TGFßI, Smad2, Smad3, α-SMA, COL1, and COL3 in HSFs. In vivo, 50 and 100 µmol/L Sal-B significantly reduced scar size in gross and cross-sectional observations, with decreased α-SMA expression and collagen deposition in the tension-induced HTS model. CONCLUSIONS: Our study demonstrated that Sal-B inhibits HSFs proliferation, migration, fibrotic marker expression and attenuates HTS formation in a tension-induced HTS model in vivo. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Herpetin Promotes Bone Marrow Mesenchymal Stem Cells to Alleviate Carbon Tetrachloride-Induced Acute Liver Injury in Mice.

Herpetin, an active compound derived from the seeds of Herpetospermum caudigerum Wall., is a traditional Tibetan herbal medicine that is used for the treatment of hepatobiliary diseases. The aim of this study was to evaluate the stimulant effect of herpetin on bone marrow mesenchymal stem cells (BMSCs) to improve acute liver injury (ALI). In vitro results showed that herpetin treatment enhanced expression of the liver-specific proteins alpha-fetoprotein, albumin, and cytokeratin 18; increased cytochrome P450 family 3 subfamily a member 4 activity; and increased the glycogen-storage capacity of BMSCs. Mice with ALI induced by carbon tetrachloride (CCl4) were treated with a combination of BMSCs by tail-vein injection and herpetin by intraperitoneal injection. Hematoxylin and eosin staining and serum biochemical index detection showed that the liver function of ALI mice improved after administration of herpetin combined with BMSCs. Western blotting results suggested that the stromal cell-derived factor-1/C-X-C motif chemokine receptor 4 axis and the Wnt/ß-catenin pathway in the liver tissue were activated after treatment with herpetin and BMSCs. Therefore, herpetin is a promising BMSC induction agent, and coadministration of herpetin and BMSCs may affect the treatment of ALI.

A high-resolution mass spectrometric method for identification and characterization of the in vitro metabolites of senkyunolide H.

RATIONALE: Ligusticum chuanxiong Hort is a well-known herb medicine that has been widely prescribed to treat cardiovascular diseases in China for hundreds of years. Senkyunolide H (SNH) is one of the major bioactive ingredients extracted from L. chuanxiong, and it displayed neuroprotective effects. To fully understand its mechanism of action, the metabolism needs to be investigated. METHODS: In vitro studies were conducted by incubating SNH with rat and human hepatocytes, and the metabolites were identified and characterized using liquid chromatography in combination with hybrid quadrupole Orbitrap mass spectrometry (LC-Orbitrap-MS). The structures of the metabolites were proposed by accurate mass analysis of respective precursor ions, indicative product ions, and elemental compositions. RESULTS: Under the current conditions, a total of 10 metabolites were identified, and among these metabolites, M3 and M4 were the most abundant metabolites both in rat and human hepatocytes. Our results demonstrated that hydroxylation, hydration, glucuronidation, and GSH conjugation were the primary metabolic pathways of SNH. CONCLUSIONS: The present study provides new information on the metabolism of SNH, which would help prospects of the disposition of SNH.

Transcription Factor SmSPL2 Inhibits the Accumulation of Salvianolic Acid B and Influences Root Architecture.

The SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factor play vital roles in plant growth and development. Although 15 SPL family genes have been recognized in the model medical plant Salvia miltiorrhiza Bunge, most of them have not been functionally characterized to date. Here, we performed a careful characterization of SmSPL2, which was expressed in almost all tissues of S. miltiorrhiza and had the highest transcriptional level in the calyx. Meanwhile, SmSPL2 has strong transcriptional activation activity and resides in the nucleus. We obtained overexpression lines of SmSPL2 and rSmSPL2 (miR156-resistant SmSPL2). Morphological changes in roots, including longer length, fewer adventitious roots, decreased lateral root density, and increased fresh weight, were observed in all of these transgenic lines. Two rSmSPL2-overexpressed lines were subjected to transcriptome analysis. Overexpression of rSmSPL2 changed root architectures by inhibiting biosynthesis and signal transduction of auxin, while triggering that of cytokinin. The salvianolic acid B (SalB) concentration was significantly decreased in rSmSPL2-overexpressed lines. Further analysis revealed that SmSPL2 binds directly to the promoters of Sm4CL9, SmTAT1, and SmPAL1 and inhibits their expression. In conclusion, SmSPL2 is a potential gene that efficiently manipulate both root architecture and SalB concentration in S. miltiorrhiza.

[124I]IBETA: A New Aß Plaque Positron Emission Tomography Imaging Agent for Alzheimer's Disease.

Several fluorine-18-labeled PET ß-amyloid (Aß) plaque radiotracers for Alzheimer's disease (AD) are in clinical use. However, no radioiodinated imaging agent for Aß plaques has been successfully moved forward for either single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. Radioiodinated pyridyl benzofuran derivatives for the SPECT imaging of Aß plaques using iodine-123 and iodine-125 are being pursued. In this study, we assess the iodine-124 radioiodinated pyridyl benzofuran derivative 5-(5-[124I]iodobenzofuran-2-yl)-N,N-dimethylpyridin-2-amine ([124I]IBETA) (Ki = 2.36 nM) for utilization in PET imaging for Aß plaques. We report our findings on the radioiododestannylation reaction used to prepare [124/125I]IBETA and evaluate its binding to Aß plaques in a 5 × FAD mouse model and postmortem human AD brain. Both [125I]IBETA and [124I]IBETA are produced in >25% radiochemical yield and >85% radiochemical purity. The in vitro binding of [125I]IBETA and [124I]IBETA in transgenic 5 × FAD mouse model for Aß plaques was high in the frontal cortex, anterior cingulate, thalamus, and hippocampus, which are regions of high Aß accumulation, with very little binding in the cerebellum (ratio of brain regions to cerebellum was >5). The in vitro binding of [125I]IBETA and [124I]IBETA in postmortem human AD brains was higher in gray matter containing Aß plaques compared to white matter (ratio of gray to white matter was >5). Anti-Aß immunostaining strongly correlated with [124/125I]IBETA regional binding in both the 5 × FAD mouse and postmortem AD human brains. The binding of [124/125I]IBETA in 5 × FAD mouse and postmortem human AD brains was displaced by the known Aß plaque imaging agent, Flotaza. Preliminary PET/CT studies of [124I]IBETA in the 5 × FAD mouse model suggested [124I]IBETA was relatively stable in vivo with a greater localization of [124I]IBETA in the brain regions with a high concentration of Aß plaques. Some deiodination was observed at later time points. Therefore, [124I]IBETA may potentially be a useful PET radioligand for Aß plaques in brain studies.

Repurposing of FDA-approved drugs against active site and potential allosteric drug-binding sites of COVID-19 main protease.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still has serious negative effects on health, social life, and economics. Recently, vaccines from various companies have been urgently approved to control SARS-CoV-2 infections. However, any specific antiviral drug has not been confirmed so far for regular treatment. An important target is the main protease (Mpro ), which plays a major role in replication of the virus. In this study, Gaussian and residue network models are employed to reveal two distinct potential allosteric sites on Mpro that can be evaluated as drug targets besides the active site. Then, Food and Drug Administration (FDA)-approved drugs are docked to three distinct sites with flexible docking using AutoDock Vina to identify potential drug candidates. Fourteen best molecule hits for the active site of Mpro are determined. Six of these also exhibit high docking scores for the potential allosteric regions. Full-atom molecular dynamics simulations with MM-GBSA method indicate that compounds docked to active and potential allosteric sites form stable interactions with high binding free energy (∆Gbind ) values. ∆Gbind values reach -52.06 kcal/mol for the active site, -51.08 kcal/mol for the potential allosteric site 1, and - 42.93 kcal/mol for the potential allosteric site 2. Energy decomposition calculations per residue elucidate key binding residues stabilizing the ligands that can further serve to design pharmacophores. This systematic and efficient computational analysis successfully determines ivermectine, diosmin, and selinexor currently subjected to clinical trials, and further proposes bromocriptine, elbasvir as Mpro inhibitor candidates to be evaluated against SARS-CoV-2 infections.

Separate Upper Pathway Ring Cleavage Dioxygenases Are Required for Growth of Sphingomonas wittichii Strain RW1 on Dibenzofuran and Dibenzo-p-Dioxin.

Sphingomonas wittichii RW1 is one of a few strains known to grow on the related compounds dibenzofuran (DBF) and dibenzo-p-dioxin (DXN) as the sole source of carbon. Previous work by others (B. Happe, L. D. Eltis, H. Poth, R. Hedderich, and K. N. Timmis, J Bacteriol 175:7313-7320, 1993, https://doi.org/10.1128/jb.175.22.7313-7320.1993) showed that purified DbfB had significant ring cleavage activity against the DBF metabolite trihydroxybiphenyl but little activity against the DXN metabolite trihydroxybiphenylether. We took a physiological approach to positively identify ring cleavage enzymes involved in the DBF and DXN pathways. Knockout of dbfB on the RW1 megaplasmid pSWIT02 results in a strain that grows slowly on DBF but normally on DXN, confirming that DbfB is not involved in DXN degradation. Knockout of SWIT3046 on the RW1 chromosome results in a strain that grows normally on DBF but that does not grow on DXN, demonstrating that SWIT3046 is required for DXN degradation. A double-knockout strain does not grow on either DBF or DXN, demonstrating that these are the only ring cleavage enzymes involved in RW1 DBF and DXN degradation. The replacement of dbfB by SWIT3046 results in a strain that grows normally (equal to the wild type) on both DBF and DXN, showing that promoter strength is important for SWIT3046 to take the place of DbfB in DBF degradation. Thus, both dbfB- and SWIT3046-encoded enzymes are involved in DBF degradation, but only the SWIT3046-encoded enzyme is involved in DXN degradation.IMPORTANCES. wittichii RW1 has been the subject of numerous investigations, because it is one of only a few strains known to grow on DXN as the sole carbon and energy source. However, while the genome has been sequenced and several DBF pathway enzymes have been purified, there has been very little research using physiological techniques to precisely identify the genes and enzymes involved in the RW1 DBF and DXN catabolic pathways. Using knockout and gene replacement mutagenesis, our work identifies separate upper pathway ring cleavage enzymes involved in the related catabolic pathways for DBF and DXN degradation. The identification of a new enzyme involved in DXN biodegradation explains why the pathway of DBF degradation on the RW1 megaplasmid pSWIT02 is inefficient for DXN degradation. In addition, our work demonstrates that both plasmid- and chromosomally encoded enzymes are necessary for DXN degradation, suggesting that the DXN pathway has only recently evolved.

Discovery of 5-(N-hydroxycarbamimidoyl) benzofuran derivatives as novel indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) are rate-limiting enzymes in the kynurenine pathway (KP) of l-tryptophan (l-Trp) metabolism and are becoming key drug targets in the combination therapy of checkpoint inhibitors in immunoncology. To discover a selective and potent IDO1 inhibitor, a structure-activity relationship (SAR) study of N-hydroxybenzofuran-5-carboximidamide as a novel scaffold was investigated in a systematic manner. Among the synthesized compounds, the N-3-bromophenyl derivative 19 showed the most potent inhibition, with an IC50 value of 0.44 µM for the enzyme and 1.1 µM in HeLa cells. The molecular modeling of 19 with the X-ray crystal structure of IDO1 indicated that dipole-ionic interactions with heme iron, halogen bonding with Cys129 and the two hydrophobic interactions were important for the high potency of 19.

Synthesis and in vitro study of nitro- and methoxy-2-phenylbenzofurans as human monoamine oxidase inhibitors.

A new series of 2-phenylbenzofuran derivatives were designed and synthesized to determine relevant structural features for the MAO inhibitory activity and selectivity. Methoxy substituents were introduced in the 2-phenyl ring, whereas the benzofuran moiety was not substituted or substituted at the positions 5 or 7 with a nitro group. Substitution patterns on both the phenyl ring and the benzofuran moiety determine the affinity for MAO-A or MAO-B. The 2-(3-methoxyphenyl)-5-nitrobenzofuran 9 was the most potent MAO-B inhibitor (IC50 = 0.024 µM) identified in this series, whereas 7-nitro-2-phenylbenzofuran 7 was the most potent MAO-A inhibitor (IC50 = 0.168 µM), both acting as reversible inhibitors. The number and position of the methoxyl groups on the 2-phenyl ring, have an important influence on the inhibitory activity. Molecular docking studies confirmed the experimental results and highlighted the importance of key residues in enzyme inhibition.

Chuanxiongdiolides R4 and R5, phthalide dimers with a complex polycyclic skeleton from the aerial parts of Ligusticum chuanxiong and their vasodilator activity.

Chuanxiongdiolides R4-R6 (1-3), three novel phthalide dimers featuring two classes of unreported monomeric units (ligustilide/senkyunolide A and ligustilide/neocnidilide) with an unprecedented linkage style (3a,7'/7a,7'a), were isolated from the aerial parts of Ligusticum chuanxiong, together with three pairs of enantiomeric phthalide dimers [(-)/(+)-4a/4b, 5a/5b, and 6a/6b]. The bioassays revealed that compounds 1, 3, 4, 5, and 6 showed significant vasodilation effects, and the mechanism may be attributed to Cav1.2 activation blockade. Based on the established compounds library, the structure activity relationship of the phthalides was proposed. Our findings afford possible leads for developing new vasodilator against cardiovascular and cerebrovascular diseases such as hypertension and ischemic stroke.

Analytical method development and percutaneous absorption of propylidene phthalide, a cosmetic ingredient.

Propylidene phthalide (PP) is a cosmetic ingredient used in the fragrance industry and regulated for the limited content of 0.01% in cosmetic products in Korea. The aim of this study was to determine PP dermal absorption rate according to the Korea Ministry of Food and Drug Safety (MFDS) guidelines using in vitro Franz diffusion system. An analytical method in assessing PP was developed through method validation using LC-MS/MS. Linearity, precision, and accuracy were acceptable based upon MFDS guidelines. The stability of PP in receptor fluid (50% ethanol) at 32°C was sufficient up to 24 hr. Cream formulation (o/w) was topically applied to excised rat skin at a dose of 113 mg/cm2 containing 0.7% PP. The time points for receptor fluid collection were set at 0, 1, 2, 4, 8, 12, and 24 hr. After 24 hr, the remaining formulation on the skin and stratum corneum (SC) were collected through swabbing with an alcohol cotton and tape stripping, respectively. The collected samples (swabbed-remained formulation, SC, and skin) were extracted using acetonitrile for 24 hr. Total dermal absorption rate of PP was approximately 24% in cream formulation. These findings may be used for further exposure evaluation of PP in human consumers.

Exploring the extremes: applying high concentration of yeast extract leads to drastic morphological changes and elimination of (+)-geodin and asterric acid production in Aspergillus terreus submerged cultures.

OBJECTIVE: Evaluation of morphology and secondary metabolites production in Aspergillus terreus ATCC 20542 cultures over a wide range of lactose and yeast extract concentrations from 0.2 up to an extremely high level of 200 g l-l. RESULTS: The morphological differences of mycelial objects were quantified with the use of morphological parameters calculated by applying the tools of digital image analysis. At 200 g l-l of yeast extract clumps and loose hyphae were recorded instead of pellets commonly observed in submerged cultures of A. terreus. Under these conditions the biosynthesis of (+)-geodin and asterric acid was totally blocked, lovastatin formation was found to be at a relatively low level and biomass production turned out to be greater than in the remaining variants, where the pelleted growth was observed. At 200 g l-l of lactose the production of lovastatin, (+)-geodin and asterric acid was visibly stimulated compared to the media containing 0.2, 2 and 20 g l-l of the sugar substrate, but at the same time no traces of butyrolactone I could be detected in the broth. Lactose at the extremely high concentration of 200 g l-l did not induce the drastic morphological changes observed in the case of 200 g l-1 of yeast extract. It was proved that at the C/N values as low as 4 and as high as 374 A. terreus not only continued to display growth but also exhibited the production of secondary metabolites. The use of cultivation media representing the equivalent C/N ratios led to different metabolic and morphological outcomes depending on the concentration of lactose and yeast extract that contributed to the given C/N value. CONCLUSION: The extremely high concentration of yeast extract leads to marked morphological changes of A. terreus and the elimination of (+)-geodin and asterric production, while applying the excess of lactose is stimulatory in terms of lovastatin production.

Simultaneous determination of 3-n-butylphthalide and its metabolite 10-hydroxy-butylphthalide in rat plasma using liquid chromatography-tandem mass spectrometry and application to a pharmacokinetic study.

3-n-Butylphthalide (NBP) is a potent drug for the treatment of ischemic stroke. The aim of this study was to develop a simple and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method for the simultaneous determination of NBP and its circulating metabolite 10-hydroxy-NBP in rat plasma using senkyunolide I as the internal standard (IS). The analytes and IS were extracted from the plasma by ethyl acetate-ethyl ether (1:5, v/v) and then separated on an ACQUITY BEH C18 column (2.1 × 50 mm, 1.7 µm). The mobile phase consisted of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid, which was delivered at a flow rate of 0.3 mL/min with gradient elution. MS detection was achieved under selective reaction monitoring mode with precursor-to-product transitions at m/z 191.1 > 145.1 for NBP, m/z 207.1 > 171.1 for 10-hydroxy-NBP and m/z 207.1 > 161.1 for IS, respectively. The assay showed excellent linearity over the concentration range of 0.5-1000 ng/mL for both analytes, with correlation coefficient greater than 0.998. The other validation parameters were all within the required limits. The validated UPLC-MS/MS method has been further applied to the pharmacokinetic study of NBP and 10-hydroxy-NBP in rats after they were orally administered with NBP (30 mg/kg).

R2R3-MYB Transcription Factor SmMYB52 Positively Regulates Biosynthesis of Salvianolic Acid B and Inhibits Root Growth in Salvia miltiorrhiza.

The dried root of Salvia miltiorrhiza is a renowned traditional Chinese medicine that was used for over 1000 years in China. Salvianolic acid B (SalB) is the main natural bioactive product of S. miltiorrhiza. Although many publications described the regulation mechanism of SalB biosynthesis, few reports simultaneously focused on S. miltiorrhiza root development. For this study, an R2R3-MYB transcription factor gene (SmMYB52) was overexpressed and silenced, respectively, in S. miltiorrhiza sterile seedlings. We found that SmMYB52 significantly inhibited root growth and indole-3-acetic acid (IAA) accumulation, whereas it activated phenolic acid biosynthesis and the jasmonate acid (JA) signaling pathway. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed that SmMYB52 suppressed the transcription levels of key enzyme-encoding genes involved in the IAA biosynthetic pathway and activated key enzyme-encoding genes involved in the JA and phenolic acid biosynthesis pathways. In addition, yeast one-hybrid (Y1H) and dual-luciferase assay showed that SmMYB52 directly binds to and activates the promoters of several key enzyme genes for SalB biosynthesis, including SmTAT1, Sm4CL9, SmC4H1, and SmHPPR1, to promote the accumulation of SalB. This is the first report of a regulator that simultaneously affects root growth and the production of phenolic acids in S. miltiorrhiza.

MiR408-SmLAC3 Module Participates in Salvianolic Acid B Synthesis in Salvia miltiorrhiza.

MicroRNAs (miRNAs) are important regulators of gene expression involved in plant development and abiotic stress responses. Recently, miRNAs have also been reported to be engaged in the regulation of secondary plant metabolism. However, there are few functional studies of miRNAs in medicinal plants. For this study, we obtained Sm-miR408 interference lines to investigate the function of Sm-miR408 in a medicinal model plant (Salvia miltiorrhiza). It was found that inhibiting the expression of Sm-miR408 could increase the content of salvianolic acid B and rosmarinic acid in the roots. The SmLAC3 and Sm-miR408 expression patterns were analyzed by qRT-PCR. A 5' RLM-RACE assay confirmed that Sm-miR408 targets and negatively regulates SmLAC3. Moreover, the overexpression of SmLAC3 in S. miltiorrhiza promoted the accumulation of salvianolic acids in the roots. Furthermore, the lignin content of the roots in overexpressed SmLAC3 lines was decreased. Taken together, these findings indicated that Sm-miR408 modulates the accumulation of phenolic acids in S. miltiorrhiza by targeting SmLAC3 expression levels.