Uncovering Druggable Targets in Aortic Dissection: An Association Study Integrating Mendelian Randomization, pQTL, and Protein-Protein Interaction Network.

Aortic dissection (AD) is a life-threatening acute aortic syndrome. There are limitations and challenges in the discovery and application of biomarkers and drug targets for AD. Mendelian randomization (MR) analysis is a reliable analytical method to identify effective therapeutic targets. We aimed to identify novel therapeutic targets for AD and investigate their potential side-effects based on MR analysis. Data from protein quantitative trait loci (pQTLs) were used for MR analyses to identify potential therapeutic targets. We probed druggable proteins involved in the pathogenesis of aortic dissection from deCODE. In this study, a two-sample MR analysis was conducted, with druggable proteins as the exposure factor and data on genome-wide association studies (GWAS) of AD as the outcome. After conducting a two-sample MR, summary data-based Mendelian randomization (SMR) analysis and colocalization analysis were performed. A protein-protein interaction (PPI) network was also constructed to delve into the interactions between identified proteins. After MR analysis and the Steiger test, we identified five proteins as potential therapeutic targets for AD. SMR analysis and colocalization analysis also confirmed our findings. Finally, we identified ASPN (OR = 1.36, 95% CI: 1.20, 1.54, p = 4.22 × 10-5) and SPOCK2 (OR = 0.57, 95% CI: 0.41, 0.78, p = 4.52 × 10-4) as the core therapeutic targets. Through PPI network analysis, we identified six druggable targets, enabling the subsequent identification of six promising drugs from DrugBank for treating AD. This discovery of specific proteins as novel therapeutic targets represents a significant advancement in AD treatment. These findings provide more effective treatment options for AD.

Genomics and resequencing of Fagopyrum dibotrys from different geographic regions reveals species evolution and genetic diversity.

Fagopyrum dibotrys, belonging to the family Polygonaceae and genus Fagopyrum, is used in traditional Chinese medicine and is rich in beneficial components, such as flavonoids. As its abundant medicinal value has become increasingly recognized, its excessive development poses a considerable challenge to wild germplasm resources, necessitating artificial cultivation and domestication. Considering these factors, a high-quality genome of F. dibotrys was assembled and the evolutionary relationships within Caryophyllales were compared, based on which 58 individual samples of F. dibotrys were re-sequenced. We found that the samples could be categorized into three purebred populations and regions distributed at distinct elevations. Our varieties were cultivated from the parental populations of the subpopulation in central Yunnan. F. dibotrys is speculated to have originated in the high-altitude Tibetan Plateau region, and that its combination with flavonoids can protect plants against ultraviolet radiation; this infers a subpopulation with a high accumulation of flavonoids. This study assembled a high-quality genome and provided a theoretical foundation for the future introduction, domestication, and development of cultivated varieties of F. dibotrys.

Emerging roles of FOXK2 in cancers and metabolic disorders.

FOXK2, a member of the Forkhead box K (FOXK) transcription factor family, is widely expressed in various tissues and organs throughout the body. FOXK2 plays crucial roles in cell proliferation, differentiation, autophagy, de novo nucleotide biosynthesis, DNA damage response, and aerobic glycolysis. Although FOXK2 is recognized as an oncogene in colorectal cancer and hepatocellular carcinoma, it acts as a tumor suppressor in breast cancer, cervical cancer, and non-small cell lung cancer (NSCLC). This review provides an overview of the recent progress in understanding the regulatory mechanisms of FOXK2 and its downstream targets, highlights the significant impact of FOXK2 dysregulation on cancer etiology, and discusses the potential of targeting FOXK2 for cancer treatment.

Deacylases-structure, function, and relationship to diseases.

Reversible S-acylation plays a pivotal role in various biological processes, modulating protein functions such as subcellular localization, protein stability/activity, and protein-protein interactions. These modifications are mediated by acyltransferases and deacylases, among which the most abundant modification is S-palmitoylation. Growing evidence has shown that this rivalrous pair of modifications, occurring in a reversible cycle, is essential for various biological functions. Aberrations in this process have been associated with various diseases, including cancer, neurological disorders, and immune diseases. This underscores the importance of studying enzymes involved in acylation and deacylation to gain further insights into disease pathogenesis and provide novel strategies for disease treatment. In this Review, we summarize our current understanding of the structure and physiological function of deacylases, highlighting their pivotal roles in pathology. Our aim is to provide insights for further clinical applications.

ZDHHC5-mediated NLRP3 palmitoylation promotes NLRP3-NEK7 interaction and inflammasome activation.

The nucleotide-binding domain (NBD), leucine-rich repeat (LRR), and pyrin domain (PYD)-containing protein 3 (NLRP3) inflammasome is a critical mediator of the innate immune response. How NLRP3 responds to stimuli and initiates the assembly of the NLRP3 inflammasome is not fully understood. Here, we found that a cellular metabolite, palmitate, facilitates NLRP3 activation by enhancing its S-palmitoylation, in synergy with lipopolysaccharide stimulation. NLRP3 is post-translationally palmitoylated by zinc-finger and aspartate-histidine-histidine-cysteine 5 (ZDHHC5) at the LRR domain, which promotes NLRP3 inflammasome assembly and activation. Silencing ZDHHC5 blocks NLRP3 oligomerization, NLRP3-NEK7 interaction, and formation of large intracellular ASC aggregates, leading to abrogation of caspase-1 activation, IL-1ß/18 release, and GSDMD cleavage, both in human cells and in mice. ABHD17A depalmitoylates NLRP3, and one human-heritable disease-associated mutation in NLRP3 was found to be associated with defective ABHD17A binding and hyper-palmitoylation. Furthermore, Zdhhc5-/- mice showed defective NLRP3 inflammasome activation in vivo. Taken together, our data reveal an endogenous mechanism of inflammasome assembly and activation and suggest NLRP3 palmitoylation as a potential target for the treatment of NLRP3 inflammasome-driven diseases.

Glut10 restrains neointima formation by promoting SMCs mtDNA demethylation and improving mitochondrial function.

Neointimal hyperplasia is a major clinical complication of coronary artery bypass graft and percutaneous coronary intervention. Smooth muscle cells (SMCs) play a vital roles in neointimal hyperplasia development and undergo complex phenotype switching. Previous studies have linked glucose transporter member 10(Glut10) to the phenotypic transformation of SMCs. In this research, we reported that Glut10 helps maintain the contractile phenotype of SMCs. The Glut10-TET2/3 signaling axis can arrest neointimal hyperplasia progression by improving mitochondrial function via promotion of mtDNA demethylation in SMCs. Glut10 is significantly downregulated in both human and mouse restenotic arteries. Global Glut10 deletion or SMC-specific Glut10 ablation in the carotid artery of mice accelerated neointimal hyperplasia, while Glut10 overexpression in the carotid artery triggered the opposite effects. All of these changes were accompanied by a significant increase in vascular SMCs migration and proliferation. Mechanistically, Glut10 is expressed primarily in the mitochondria after platelet-derived growth factor-BB (PDGF-BB) treatment. Glut10 ablation induced a reduction in ascorbic acid (VitC) concentrations in mitochondria and mitochondrial DNA (mtDNA) hypermethylation by decreasing the activity and expression of the Ten-eleven translocation (TET) protein family. We also observed that Glut10 deficiency aggravated mitochondrial dysfunction and decreased the adenosinetriphosphate (ATP) content and the oxygen consumption rate, which also caused SMCs to switch their phenotype from contractile to synthetic phenotype. Furthermore, mitochondria-specific TET family inhibition partially reversed these effects. These results suggested that Glut10 helps maintain the contractile phenotype of SMCs. The Glut10-TET2/3 signaling axis can arrest neointimal hyperplasia progression by improving mitochondrial function via the promotion of mtDNA demethylation in SMCs.

Lymph-Node-Targeted Drug Delivery for Effective Immunomodulation to Prolong the Long-Term Survival After Heart Transplantation.

The chronic rejection responses and side effects of the systematic administration of immunosuppressants are the main obstacles to heart allograft and patient survival. The development of xenotransplantation also urgently requires more efficient immune regulation strategies. Herein, it is demonstrated that lymph-node (LN)-targeted drug delivery can realize LN-specific immunomodulation with attenuated immune suppression on distant peripheral immune organs to effectively prolong long-term survival after heart transplantation in a chronic murine heart transplantation model. A chemokine C-C motif ligand 21 (CCL21) specific aptamer for LN targeting is decorated onto the surface of the hybrid nanoparticular delivery vector mainly composed of CaCO3 /CaP/heparin. The targeting delivery system can dramatically enhance accumulation of the loaded immunosuppressant, fingolimod hydrochloride (FTY720), in draining lymph nodes (dLNs) for inducing powerful immune suppression. By promoting the generation of endogenous regulatory T cells (Tregs ) and decreasing the proportion of effector T cells (Teffs ) in dLNs after heart transplantation, the LN-targeting strategy can effectively regulate local immune responses instead of systemic immunity, which reduces the incidence of long-term complications. This study provides an efficient strategy to improve the survival rate after organ transplantation by precise and localized immunoregulation with minimized side effects of immunosuppression.

Advances in pharmacology, biosynthesis, and metabolic engineering of Scutellaria-specialized metabolites.

Scutellaria Linn., which belongs to the family Lamiaceae, is a commonly used medicinal plant for heat clearing and detoxification. In particular, the roots of S. baicalensis and the entire herb of S. barbata have been widely used in traditional medicine for thousands of years. The main active components of Scutellaria, including: baicalein, wogonin, norwogonin, scutellarein, and their glycosides have potential or existing drug usage. However, the wild resources of Scutellaria plants have been overexploited, and degenerated germplasm resources cannot fulfill the requirements of chemical extraction and clinical usage. Metabolic engineering and green production via microorganisms provide alternative strategies for greater efficiency in the production of natural products. Here, we review the progress of: pharmacological investigations, multi-omics, biosynthetic pathways, and metabolic engineering of various Scutellaria species and their active compounds. In addition, based on multi-omics data, we systematically analyze the phylogenetic relationships of Scutellaria and predict candidate transcription factors related to the regulation of active flavonoids. Finally, we propose the prospects of directed evolution of core enzymes and genome-assisted breeding to alleviate the shortage of plant resources of Scutellaria. This review provides important insights into the sustainable utilization and development of Scutellaria resources.

The Combination of Rhodosin and MMF Prolongs Cardiac Allograft Survival by Inhibiting DC Maturation by Promoting Mitochondrial Fusion.

Despite being the gold-standard treatment for end-stage heart disease, heart transplantation is associated with acute cardiac rejection within 1 year of transplantation. The continuous application of immunosuppressants may cause side effects such as hepatic and renal toxicity, infection, and malignancy. Developing new pharmaceutical strategies to alleviate acute rejection after heart transplantation effectively and safely is of critical importance. In this study, we performed a murine model of MHC-full mismatch cardiac transplantation and showed that the combination of Rhodosin (Rho) and mycophenolate mofetil (MMF) could prevent acute rejection and oxidative stress injury and prolong the survival time of murine heart transplants. The use of Rho plus MMF in allografts improved the balance of Tregs/Teff cells, which had a protective effect on allotransplantation. We also isolated bone marrow-derived dendritic cells (BMDCs) and determined that Rho inhibited DC maturation by promoting mitochondrial fusion mainly through the mitochondrial fusion-related protein MFN1. Herein, we demonstrated that Rho, an active ingredient isolated from the plant Rhodiola rosea with antioxidant and anti-inflammatory activities, could efficiently alleviate acute rejection and significantly prolong murine heart allograft survival when used with a low dose of MMF. More importantly, we found that Rho restrained DC maturation by promoting mitochondrial fusion and decreasing reactive oxygen species (ROS) levels, which then alleviated acute rejection in murine cardiac transplantation. Interestingly, as a novel immunosuppressant, Rho has almost no side effects compared with other traditional immunosuppressants. Taken together, these results suggest that Rho has good clinical auxiliary applications as an effective immunosuppressant and antioxidant, and this study provides an efficient strategy to overcome the side effects of immunosuppressive agents that are currently used in organ transplantation.

Iron deficiency exacerbates aortic medial degeneration by inducing excessive mitochondrial fission.

Iron deficiency (ID) is a global nutritional deficiency that was shown to be involved in the pathogenesis of aortic aneurysm and dissection (AAD) in our previous studies. Some studies suggested that mitochondrial dynamics was involved in the apoptosis and phenotypic transformation of vascular smooth muscle cells (VSMCs). However, little is known about the role of mitochondrial dynamics in aortic medial degeneration (AMD) promoted by an iron deficient diet. The present study investigated the effect of ID on the phenotypic transformation of VSMCs, the progression of AMD, and the underlying mechanism. The expression of p-Drp1 (Ser616) and Fis1 was markedly upregulated in the aortic media of AAD patients and ApoE-/- mice with subcutaneous AngII osmotic pumps. ID facilitated the formation of mitochondria-associated endoplasmic reticulum membranes (MAMs), which triggered excessive mitochondrial fission, induced the phenotypic transformation of VSMCs, and ultimately accelerated the progression of AMD. Furthermore, the present study indicated that an inhibitor of Drp1 could partially reverse this process. Maintaining iron balance in the human body may prevent the development of AAD.

miR-223: An Immune Regulator in Infectious Disorders.

MicroRNAs (miRNAs) are diminutive noncoding RNAs that can influence disease development and progression by post-transcriptionally regulating gene expression. The anti-inflammatory miRNA, miR-223, was first identified as a regulator of myelopoietic differentiation in 2003. This miR-223 exhibits multiple regulatory functions in the immune response, and abnormal expression of miR-223 is shown to be associated with multiple infectious diseases, including viral hepatitis, human immunodeficiency virus type 1 (HIV-1), and tuberculosis (TB) by influencing neutrophil infiltration, macrophage function, dendritic cell (DC) maturation and inflammasome activation. This review summarizes the current understanding of miR-223 physiopathology and highlights the molecular mechanism by which miR-223 regulates immune responses to infectious diseases and how it may be targeted for diagnosis and treatment.

Mangiferin Inhibits PDGF-BB-Induced Proliferation and Migration of Rat Vascular Smooth Muscle Cells and Alleviates Neointimal Formation in Mice through the AMPK/Drp1 Axis.

Mangiferin is a naturally occurring xanthone C-glycoside that is widely found in various plants. Previous studies have reported that mangiferin inhibits tumor cell proliferation and migration. Excessive proliferation and migration of vascular smooth muscle cells (SMCs) is associated with neointimal hyperplasia in coronary arteries. However, the role and mechanism of mangiferin action in neointimal hyperplasia is still unknown. In this study, a mouse carotid artery ligation model was established, and primary rat smooth muscle cells were isolated and used for mechanistic assays. We found that mangiferin alleviated neointimal hyperplasia, inhibited proliferation and migration of SMCs, and promoted platelets derive growth factors-BB- (PDGF-BB-) induced contractile phenotype in SMCs. Moreover, mangiferin attenuated neointimal formation by inhibiting mitochondrial fission through the AMPK/Drp1 signaling pathway. These findings suggest that mangiferin has the potential to maintain vascular homeostasis and inhibit neointimal hyperplasia.

Metabolic Modifications, Inflammation, and Cancer Immunotherapy.

Cancer immunotherapy has accomplished significant progresses on treatment of various cancers in the past decade; however, recent studies revealed more and more heterogeneity in tumor microenvironment which cause unneglectable therapy resistance. A central phenomenon in tumor malignancy is metabolic dysfunctionality; it reprograms metabolic homeostasis in tumor and stromal cells thus affecting metabolic modifications on specific proteins. These posttranslational modifications include glycosylation and palmitoylation, which usually alter the protein localization, stability, and function. Many of these proteins participate in acute or chronic inflammation and play critical roles in tumorigenesis and progression. Therefore, targeting these metabolic modifications in immune checkpoints and inflammation provides an attractive therapeutic strategy for certain cancers. In this review, we summarize the recent progresses on metabolic modifications in this field, focus on the mechanisms on how glycosylation and palmitoylation regulate innate immune and inflammation, and we further discuss designing new immunotherapy targeting metabolic modifications. We aim to improve immunotherapy or targeted-therapy response and achieve more accurate individual therapy.

Combination of matrine and tacrolimus alleviates acute rejection in murine heart transplantation by inhibiting DCs maturation through ROS/ERK/NF-κB pathway.

Matrine, an alkaloid derived from traditional Chinese herbs, has been confirmed to regulate immunity and exert anti-inflammatory effects. Matrine injection has been widely used in clinic therapy for anti-tumor and anti-inflammatory diseases. Heart transplantation(HT) is the only solution for the end-stage heart failure, but it is restricted by the cardiac allograft rejection. One of the important pathophysiological processes of post-transplantation rejection is inflammatory cell infiltration. Matrine has been shown to exert a positive protective effect against oxidative stress injury and inflammation, which likely benefits allograft survival. However, it remains unclear whether matrine inhibits alloimmunity or allograft rejection. In this study, we established the heart transplantation model in mouse and extracted bone marrow-derived dendritic cells (BMDCs) to explore the function and mechanism of matrine in heart transplantation. Moreover, combination treatment with matrine and tacrolimus(FK506) had a synergistic effect in preventing acute rejection of heart transplants. Here we found that matrine can prolong the survival of post-transplant and inhibit inflammatory cell infiltration in transplanted hearts of mice. At the same time, matrine increased Treg ratio and decreased CD4+/CD8 + ratio in mice. More importantly, matrine inhibited DCs maturation in mice and reduced oxidative damage and apoptosis in allograft hearts. Furthermore, matrine also downregulated NF-κB pathway and upregulated ERK1/2 signaling pathway. Overall, our study reveals a novel immunosuppressive agent that has the potential to reduce the side effects of existing immunosuppressive agents when used in combination with them.

Artesunate Restrains Maturation of Dendritic Cells and Ameliorates Heart Transplantation-Induced Acute Rejection in Mice through the PERK/ATF4/CHOP Signaling Pathway.

BACKGROUND: Heart transplantation (HT) is the only effective treatment for end-stage heart failure because it can effectively improve the survival rate and quality of life of patients with heart failure. Artesunate (ART) is an artemisinin derivative, with good water solubility and higher oral bioavailability. The main aim of this study was to determine the role of ART in HT mice. METHODS: In animal experiments, mice were divided into the control group, HT group, low ART+HT group, and high ART+HT group. Next, inflammatory cell infiltration, oxidative stress injury, and myocardial cell apoptosis were determined in heart tissue. The proportion of multiple lymphocytes in spleen and lymph nodes was then determined using flow cytometry. In addition, cell experiments were conducted to determine the changes in expression of surface maturation markers of BMDC and changes in intracellular reactive oxygen species after LPS stimulation. Finally, western blot analysis was performed to determine the levels of endoplasmic reticulum stress-related proteins (CHOP/ATF4/PERK). RESULTS: The survival time of mice in the ART treatment group was significantly prolonged and was positively correlated with the dose. In animal experiments, ART significantly reduced inflammatory cell infiltration in heart tissue and the proportion of CD4+CD8+ T cells in spleens and lymph nodes. Moreover, ART treatment lowered the 8-OHdg in hearts and myocardial apoptosis. In cell experiments, ART treatment slowed down the development and maturation of BMDCs by inhibiting the expression of endoplasmic reticulum stress-related proteins. Furthermore, the treatment alleviated the oxidative stress damage of BMDCs. CONCLUSION: ART can inhibit maturation of dendritic cells through the endoplasmic reticulum stress signaling pathway, thereby alleviating acute rejection in mice after heart transplantation.

Comparative Genome Analysis of Scutellaria baicalensis and Scutellaria barbata Reveals the Evolution of Active Flavonoid Biosynthesis.

Scutellaria baicalensis (S. baicalensis) and Scutellaria barbata (S. barbata) are common medicinal plants of the Lamiaceae family. Both produce specific flavonoid compounds, including baicalein, scutellarein, norwogonin, and wogonin, as well as their glycosides, which exhibit antioxidant and antitumor activities. Here, we report chromosome-level genome assemblies of S. baicalensis and S. barbata with quantitative chromosomal variation (2n = 18 and 2n = 26, respectively). The divergence of S. baicalensis and S. barbata occurred far earlier than previously reported, and a whole-genome duplication (WGD) event was identified. The insertion of long terminal repeat elements after speciation might be responsible for the observed chromosomal expansion and rearrangement. Comparative genome analysis of the congeneric species revealed the species-specific evolution of chrysin and apigenin biosynthetic genes, such as the S. baicalensis-specific tandem duplication of genes encoding phenylalanine ammonia lyase and chalcone synthase, and the S. barbata-specific duplication of genes encoding 4-CoA ligase. In addition, the paralogous duplication, colinearity, and expression diversity of CYP82D subfamily members revealed the functional divergence of genes encoding flavone hydroxylase between S. baicalensis and S. barbata. Analyzing these Scutellaria genomes reveals the common and species-specific evolution of flavone biosynthetic genes. Thus, these findings would facilitate the development of molecular breeding and studies of biosynthesis and regulation of bioactive compounds.

Bre1-dependent H2B ubiquitination promotes homologous recombination by stimulating histone eviction at DNA breaks.

Repair of DNA double-strand breaks (DSBs) requires eviction of the histones around DNA breaks to allow the loading of numerous repair and checkpoint proteins. However, the mechanism and regulation of this process remain poorly understood. Here, we show that histone H2B ubiquitination (uH2B) promotes histone eviction at DSBs independent of resection or ATP-dependent chromatin remodelers. Cells lacking uH2B or its E3 ubiquitin ligase Bre1 exhibit hyper-resection due to the loss of H3K79 methylation that recruits Rad9, a known negative regulator of resection. Unexpectedly, despite excessive single-strand DNA being produced, bre1Δ cells show defective RPA and Rad51 recruitment and impaired repair by homologous recombination and response to DNA damage. The HR defect in bre1Δ cells correlates with impaired histone loss at DSBs and can be largely rescued by depletion of CAF-1, a histone chaperone depositing histones H3-H4. Overexpression of Rad51 stimulates histone eviction and partially suppresses the recombination defects of bre1Δ mutant. Thus, we propose that Bre1 mediated-uH2B promotes DSB repair through facilitating histone eviction and subsequent loading of repair proteins.

Rtt105 functions as a chaperone for replication protein A to preserve genome stability.

Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA-ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA-importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA-ssDNA complex. Single-molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.

Chemical and genetic diversity of Astragalus mongholicus grown in different eco-climatic regions.

Astragalus mongholicus Bunge (Fabaceae) is an important plant source of the herbal drug known as Radix Astragali, which is used worldwide as a medicinal ingredient and a component of food supplement. Russian Federation, Mongolia, Kazakhstan, and China are the main natural distribution areas of A. mongholicus in the world. However, the quality of medicinal plant varies among different locations. As for A. mongholicus, limited literature focused on its biodiversity mechanism. Here, we combined the chemometric analysis of chemical components with genetic variation, as well as climatic and edaphic traits, to reveal the biodiversity mechanism of A. mongholicus. Results showed that the detected chemical, genetic and climatic traits comprehensively contributed to the quality diversity of A. mongholicus. The eight main chemical components, as well as the inorganic elements of P, B and Na were all significant chemical factors. The precipitation and sunshine duration were the main distinguishing climatic factors. The inorganic elements As, Mn, P, Se and Pb were the distinguishing edaphic factors. The systematic method was firstly established for this medicinal plant in order to illustrate the formation of diversity in terms of quality, and provide scientific evidence for geographic indications and climatic adaptation in production and in the clinical application of herbal medicinal plants.

Three-Dimensional Evaluation on Ecotypic Diversity of Traditional Chinese Medicine: A Case Study of Artemisia annua L.

Artemisinin is the first-line drug for anti-malaria recommended by the World Health Organization (WHO). As the sole natural plant source of artemisinin, ecotypes of Artemisia annua L. vary widely in artemisinin content between nations, and China is the main producing area of A. annua. Here we present a three-dimensional evaluation on ecotypic diversity of A. annua from 12 main producing areas in China using high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD) method, DNA barcoding and ecological analyses. The results indicated that A. annua exhibited high ecotypic diversity. A. annua grown in the South of the Qinling Mountains-Huaihe River Line had a high artemisinin content, whereas the northern ones were low. Similar pattern was noted in the genetic diversity. The southern A. annua had high intraspecific variation in contrast to the northern A. annua. In terms of ecological analyses, humidity and sunshine time could be the major limiting ecological factors that affect the accumulation of artemisinin. This is the first reported three-dimensional evaluation integrating chemical, molecular and ecological analyses of the ecotypic diversity of A. annua. The work will facilitate exploring the genetic basis of chemical variations and developing strategies for the breeding and cultivation of high quality A. annua.