[Interventional effect and inflammatory regulation mechanism of dihydroartemisinin combined with pregabalin on neuropathic pain mice].

This study aims to clarify the effects of dihydroartemisinin(DHA) combined with pregabalin(PGB) on neuropathic pain(NP) in mice and explore the neuroinflammatory regulatory mechanism. NP mice model was established using spinal nerve ligation, whereas the sham group exposed the spinal nerve without ligation. The mice were randomly divided into sham group, model group, PGB groups of low, medium, and high doses(PGB-L, PGB-M, and PGB-H, with 22, 45, and 91 mg·kg~(-1)), DHA group(16 mg·kg~(-1)), and DHA combined with PGB groups of low, medium, and high doses(DHA + PGB-L, DHA + PGB-M, and DHA + PGB-H). Administration by gavage 18 days after modeling. Von Frey and cold plate were used to detect mechanical pain threshold and cold pain sensitivity in mice. The tail suspension test and forced swimming test were used to investigate depressive behavior, and the open field test was used to estimate anxiety behavior. The Morris water maze was used to evaluate cognitive function. Liquid suspension chip technology was used to quantitatively analyze immune inflammation-related factors. Immunofluorescence was used to detect the expression of CC chemokine ligand 3(CCL3) and transmembrane protein 119(TMEM119). The results showed that compared with the sham group, the mechanical pain and cold pain sensitivity thresholds of the model group were significantly reduced, and the struggle time was significantly increased in the tail suspension test and forced swimming test. The activity time in the central area was significantly reduced in the open field test. The residence time in the second/fourth quadrant was significantly longer than that in other quadrants, and the latency time of platform climbing significantly increased after platform withdrawal in the Morris water maze experiment. The expression of CCL3 was significantly increased; the number of TMEM119 positive cells and the cell body area were significantly increased. Compared with the model group, the DHA + PGB-M group showed a significant increase in mechanical pain and cold pain sensitivity thresholds, as well as a significant increase in struggle time in the tail suspension test and forced swimming test. The activity time in the central area of the open field test was significantly reduced. The residence time in the second/fourth quadrant was significantly shorter than that in other quadrants, and the latency time of platform climbing after platform withdrawal was significantly reduced. Compared with the PGB-M group, the mechanical pain threshold of D14-17 in the DHA + PGB-M group was significantly increased, and the struggle time during forced swimming was significantly increased. The residence time in the second/fourth quadrant of the Morris water maze was significantly shorter than that in other quadrants. Compared with the model group, the expression of CCL3, the number of TMEM119 positive cells, and the cell body area in the DHA + PGB-M group were significantly decreased. This study indicates that DHA + PGB can enhance the analgesic effect of PGB on NP mice, break through the limitations of PGB tolerance, and make up for the shortcomings of PGB in antidepressant and cognitive improvement. Its mechanism may be related to regulating neuroinflammation by inhibiting the activation of microglial cells and expression of CCL3.

[Simultaneous determination of seven artemisinin-related compounds in Artemisia annua by UPLC-QQQ-MS/MS].

A variety of compounds in Artemisia annua were simultaneously determined to evaluate the quality of A. annua from multiple perspectives. A method based on ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-QQQ-MS/MS) was established for the simultaneous determination of seven compounds: amorpha-4,11-diene, artemisinic aldehyde, dihydroartemisinic acid, artemisinic acid, artemisinin B, artemisitene, and artemisinin, in A. annua. The content of the seven compounds in different tissues(roots, stems, leaves, and lateral branches) of A. annua were compared. The roots, stems, leaves, and lateral branches of four-month-old A. annua were collected and the content of seven artemisinin-related compounds in different tissues was determined. A multi-reaction monitoring(MRM) acquisition mode of UPLC-QQQ-MS/MS was used, with a positive ion mode of atmospheric pressure chemical ion source(APCI). Chromatographic separation was achieved on an Eclipse Plus RRHD C_(18) column(2.1 mm×50 mm, 1.8 µm). The gradient elution was performed with the mobile phase consisted of formic acid(0.1%)-ammonium formate(5 mmol·L~(-1))(A) and the methanol(B) gradient program of 0-8 min, 55%-100% B, 8-11 min, 100% B, and equilibrium for 3 min, the flow rate of 0.6 mL·min~(-1), the column temperature of 40 ℃, the injection volume of 5 µL, and the detection time of 8 min. Through methodological investigation, a method based on UPLC-QQQ-MS/MS was established for the simultaneous quantitative determination of seven representative compounds involved in the biosynthesis of artemisinin. The content of artemisinin in A. annua was higher than that of artemisinin B, and the content of artemisinin and dihydroartemisinic acid were high in all the tissues of A. annua. The content of the seven compounds varied considerably in different tissues, with the highest levels in the leaves and neither artemisinene nor artemisinic aldehyde was detected in the roots. In this study, a quantitative method based on UPLC-QQQ-MS/MS for the simultaneous determination of seven representative compounds involved in the biosynthesis of artemisinin was established, which was accurate, sensitive, and highly efficient, and can be used for determining the content of artemisinin-related compounds in A. annua, breeding new varieties, and controlling the quality of Chinese medicinal materials.

The effect of an anti-malarial herbal remedy, Maytenus senegalensis, on electrocardiograms of healthy Tanzanian volunteers.

BACKGROUND: The emergence of resistance to artemisinin-based combination therapy necessitates the search for new, more potent antiplasmodial compounds, including herbal remedies. The whole extract of Maytenus senegalensis has been scientifically investigated for potential biological activities both in vitro and in vivo, demonstrating strong antimalarial activity. However, there is a lack of data on the electrocardiographic effects of M. senegalensis in humans, which is a crucial aspect in the investigation of malaria treatment. Assessing the electrocardiographic effects of M. senegalensis is essential, as many anti-malarial drugs can inadvertently prolong the QT interval on electrocardiograms. Therefore, the study's objective was to evaluate the electrocardiographic effects of M. senegalensis in healthy adult volunteers. METHODS: This study is a secondary analysis of an open-label single-arm dose escalation. Twelve healthy eligible Tanzanian males, aged 18 to 45, were enrolled in four study dose groups. A single 12-lead electrocardiogram (ECG) was performed at baseline and on days 3, 7, 14, 28, and 56. RESULTS: No QTcF adverse events occurred with any drug dose. Only one volunteer who received the highest dose (800 mg) of M. senegalensis experienced a moderate transient change (△QTcF > 30 ms; specifically, the value was 37 ms) from baseline on day 28. There was no difference in maximum QTcF and maximum △QTcF between volunteers in all four study dose groups. CONCLUSIONS: A four-day regimen of 800 mg every 8 h of M. senegalensis did not impact the electrocardiographic parameters in healthy volunteers. This study suggests that M. senegalensis could be a valuable addition to malaria treatment, providing a safer alternative and potentially aiding in the battle against artemisinin-resistant malaria. The results of this study support both the traditional use and the modern therapeutic potential of M. senegalensis. They also set the stage for future research involving larger and more diverse populations to explore the safety profile of M. senegalensis in different demographic groups. This is especially important considering the potential use of M. senegalensis as a therapeutic agent and its widespread utilization as traditional medicine. Trial registration ClinicalTrials.gov, NCT04944966. Registered 30 June 2021-Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT04944966?term=kamaka&draw=2&rank=1.

Cancer nutritional-immunotherapy with NIR-II laser-controlled ATP release based on material repurposing strategy.

Enlightened by the great success of the drug repurposing strategy in the pharmaceutical industry, in the current study, material repurposing is proposed where the performance of carbonyl iron powder (CIP), a nutritional intervention agent of iron supplement approved by the US FDA for iron deficiency anemia in clinic, was explored in anti-cancer treatment. Besides the abnormal iron metabolic characteristics of tumors, serving as potential targets for CIP-based cancer therapy under the repurposing paradigm, the efficacy of CIP as a catalyst in the Fenton reaction, activator for dihydroartemisinin (DHA), thus increasing the chemo-sensitivity of tumors, as well as a potent agent for NIR-II photothermal therapy (PTT) was fully evaluated in an injectable alginate hydrogel form. The CIP-ALG gel caused a rapid temperature rise in the tumor site under NIR-II laser irradiation, leading to complete ablation in the primary tumor. Further, this photothermal-ablation led to the significant release of ATP, and in the bilateral tumor model, both primary tumor ablation and inhibition of secondary tumor were observed simultaneously under the synergistic tumor treatment of nutritional-photothermal therapy (NT/PTT). Thus, material repurposing was confirmed by our pioneering trial and CIP-ALG-meditated NT/PTT/immunotherapy provides a new choice for safe and efficient tumor therapy.

Dihydroartemisinin alleviates erosive bone destruction by modifying local Treg cells in inflamed joints: A novel role in the treatment of rheumatoid arthritis.

Treg cell-based therapy has exhibited promising efficacy in combatting rheumatoid arthritis (RA). Dihydroartemisinin (DHA) exerts broad immunomodulatory effects across various diseases, with its recent spotlight on T-cell regulation in autoimmune conditions. The modulation of DHA on Treg cells and its therapeutic role in RA has yet to be fully elucidated. This study seeks to unveil the influence of DHA on Treg cells in RA and furnish innovative substantiation for the potential of DHA to ameliorate RA. To this end, we initially scrutinized the impact of DHA-modulated Treg cells on osteoclast (OC) formation in vitro using Treg cell-bone marrow-derived monocyte (BMM) coculture systems. Subsequently, employing the collagen-induced arthritis (CIA) rat model, we validated the efficacy of DHA and probed its influence on Treg cells in the spleen and popliteal lymph nodes (PLN). Finally, leveraging deep proteomic analysis with data-independent acquisition (DIA) and parallel accumulation-serial fragmentation (PASEF) technology, we found the alterations in the Treg cell proteome in PLN by proteomic analysis. Our findings indicate that DHA augmented suppressive Treg cells, thereby impeding OC formation in vitro. Consistently, DHA mitigated erosive joint destruction and osteoclastogenesis by replenishing splenic and joint-draining lymph node Treg cells in CIA rats. Notably, DHA induced alterations in the Treg cell proteome in PLN, manifesting distinct upregulation of alloantigen Col2a1 (Type II collagen alfa 1 chain) and CD8a (T-cell surface glycoprotein CD8 alpha chain) in Treg cells, signifying DHA's targeted modulation of Treg cells, rendering them more adept at sustaining immune tolerance and impeding bone erosion. These results unveil a novel facet of DHA in the treatment of RA.

A Biotinylated cpFIT-PNA Platform for the Facile Detection of Drug Resistance to Artemisinin in Plasmodium falciparum.

The evolution of drug resistance to many antimalarial drugs in the lethal strain of malaria (Plasmodium falciparum) has been a great concern over the past 50 years. Among these drugs, artemisinin has become less effective for treating malaria. Indeed, several P. falciparum variants have become resistant to this drug, as elucidated by specific mutations in the pfK13 gene. This study presents the development of a diagnostic kit for the detection of a common point mutation in the pfK13 gene of P. falciparum, namely, the C580Y point mutation. FIT-PNAs (forced-intercalation peptide nucleic acid) are DNA mimics that serve as RNA sensors that fluoresce upon hybridization to their complementary RNA. Herein, FIT-PNAs were designed to sense the C580Y single nucleotide polymorphism (SNP) and were conjugated to biotin in order to bind these molecules to streptavidin-coated plates. Initial studies with synthetic RNA were conducted to optimize the sensing system. In addition, cyclopentane-modified PNA monomers (cpPNAs) were introduced to improve FIT-PNA sensing. Lastly, total RNA was isolated from red blood cells infected with P. falciparum (WT strain - NF54-WT or mutant strain - NF54-C580Y). Streptavidin plates loaded with either FIT-PNA or cpFIT-PNA were incubated with the total RNA. A significant difference in fluorescence for mutant vs WT total RNA was found only for the cpFIT-PNA probe. In summary, this study paves the way for a simple diagnostic kit for monitoring artemisinin drug resistance that may be easily adapted to malaria endemic regions.

Malaria: biochemical, physiological, diagnostic, and therapeutic updates.

Background: Malaria has been appraised as a significant vector-borne parasitic disease with grave morbidity and high-rate mortality. Several challenges have been confronting the efficient diagnosis and treatment of malaria. Method: Google Scholar, PubMed, Web of Science, and the Egyptian Knowledge Bank (EKB) were all used to gather articles. Results: Diverse biochemical and physiological indices can mirror complicated malaria e.g., hypoglycemia, dyslipidemia, elevated renal and hepatic functions in addition to the lower antioxidant capacity that does not only destroy the parasite but also induces endothelial damage. Multiple trials have been conducted to improve recent points of care in malaria involving biosensors, lap on-chip, and microdevices technology. Regarding recent therapeutic trials, chemical falcipain inhibitors and plant extracts with anti-plasmodial activities are presented. Moreover, antimalaria nano-medicine and the emergence of nanocarrier (either active or passive) in drug transportation are promising. The combination therapeutic trials e.g., amodiaquine + artemether + lumefantrine are presented to safely counterbalance the emerging drug resistance in addition to the Tafenoquine as a new anti-relapse therapy. Conclusion: Recognizing the pathophysiology indices potentiate diagnosis of malaria. The new points of care can smartly manipulate the biochemical and hematological alterations for a more sensitive and specific diagnosis of malaria. Nano-medicine appeared promising. Chemical and plant extracts remain points of research.

A Cancer Nanovaccine Based on an FeAl-Layered Double Hydroxide Framework for Reactive Oxygen Species-Augmented Metalloimmunotherapy.

The complexity and heterogeneity of individual tumors have hindered the efficacy of existing therapeutic cancer vaccines, sparking intensive interest in the development of more effective in situ vaccines. Herein, we introduce a cancer nanovaccine for reactive oxygen species-augmented metalloimmunotherapy in which FeAl-layered double hydroxide (LDH) is used as a delivery vehicle with dihydroartemisinin (DHA) as cargo. The LDH framework is acid-labile and can be degraded in the tumor microenvironment, releasing iron ions, aluminum ions, and DHA. The iron ions contribute to aggravated intratumoral oxidative stress injury by the synergistic Fenton reaction and DHA activation, causing apoptosis, ferroptosis, and immunogenic cell death in cancer cells. The subsequently released tumor-associated antigens with the aluminum adjuvant form a cancer nanovaccine to generate robust and long-term immune responses against cancer recurrence and metastasis. Moreover, Fe ion-enabled T1-weighted magnetic resonance imaging can facilitate real-time tumor therapy monitoring. This cancer-nanovaccine-mediated metalloimmunotherapy strategy has the potential for revolutionizing the precision immunotherapy landscape.

Dihydroartemisinin abolishes cisplatin-induced nephrotoxicity in vivo.

Dihydroartemisinin (DHA), a derivative of artemisinin which is primarily used to treat malaria in clinic, also confers protective effect on lipopolysaccharide-induced nephrotoxicity. While, the activities of DHA in cisplatin (CDDP)-caused nephrotoxicity are elusive. To investigate the role and underlying mechanism of DHA in CDDP-induced nephrotoxicity. Mice were randomly separated into four groups: normal, CDDP, and DHA (25 and 50 mg/kg were orally injected 1 h before CDDP for consecutive 10 days). All mice except the normal were single injected intraperitoneally with CDDP (22 mg/kg) for once on the 7th day. Combined with quantitative proteomics and bioinformatics analysis, the impact of DHA on renal cell apoptosis, oxidative stress, biochemical indexes, and inflammation in mice were investigated. Moreover, a human hepatocellular carcinoma cells xenograft model was established to elucidate the impact of DHA on tumor-related effects of CDDP. DHA reduced the levels of creatinine (CREA) (p < 0.01) and blood urea nitrogen (BUN) (p < 0.01), reversed CDDP-induced oxidative, inflammatory, and apoptosis indexes (p < 0.01). Mechanistically, DHA attenuated CDDP-induced inflammation by inhibiting nuclear factor κB p65 (NFκB p65) expression, and suppressed CDDP-induced renal cell apoptosis by inhibiting p63-mediated endogenous and exogenous apoptosis pathways. Additionally, DHA alone significantly decreased the tumor weight and did not destroy the antitumor effect of CDDP, and did not impact AST and ALT. In conclusion, DHA prevents CDDP-triggered nephrotoxicity via reducing inflammation, oxidative stress, and apoptosis. The mechanisms refer to inhibiting NFκB p65-regulated inflammation and alleviating p63-mediated mitochondrial endogenous and Fas death receptor exogenous apoptosis pathway.

Bioprospecting of Artemisia genus: from artemisinin to other potentially bioactive compounds.

Species from genus Artemisia are widely distributed throughout temperate regions of the northern hemisphere and many cultures have a long-standing traditional use of these plants as herbal remedies, liquors, cosmetics, spices, etc. Nowadays, the discovery of new plant-derived products to be used as food supplements or drugs has been pushed by the exploitation of bioprospection approaches. Often driven by the knowledge derived from the ethnobotanical use of plants, bioprospection explores the existing biodiversity through integration of modern omics techniques with targeted bioactivity assays. In this work we set up a bioprospection plan to investigate the phytochemical diversity and the potential bioactivity of five Artemisia species with recognized ethnobotanical tradition (A. absinthium, A. alba, A. annua, A. verlotiorum and A. vulgaris), growing wild in the natural areas of the Verona province. We characterized the specialized metabolomes of the species (including sesquiterpenoids from the artemisinin biosynthesis pathway) through an LC-MS based untargeted approach and, in order to identify potential bioactive metabolites, we correlated their composition with the in vitro antioxidant activity. We propose as potential bioactive compounds several isomers of caffeoyl and feruloyl quinic acid esters (e.g. dicaffeoylquinic acids, feruloylquinic acids and caffeoylferuloylquinic acids), which strongly characterize the most antioxidant species A. verlotiorum and A. annua. Morevoer, in this study we report for the first time the occurrence of sesquiterpenoids from the artemisinin biosynthesis pathway in the species A. alba.

Drug-induced stress mediates Plasmodium falciparum ring-stage growth arrest and reduces in vitro parasite susceptibility to artemisinin.

During blood-stage infection, Plasmodium falciparum parasites are constantly exposed to a range of extracellular stimuli, including host molecules and drugs such as artemisinin derivatives, the mainstay of artemisinin-based combination therapies currently used as first-line treatment worldwide. Partial resistance of P. falciparum to artemisinin has been associated with mutations in the propeller domain of the Pfkelch13 gene, resulting in a fraction of ring stages that are able to survive exposure to artemisinin through a temporary growth arrest. Here, we investigated whether the growth arrest in ring-stage parasites reflects a general response to stress. We mimicked a stressful environment in vitro by exposing parasites to chloroquine or dihydroartemisinin (DHA). We observed that early ring-stage parasites pre-exposed to a stressed culture supernatant exhibited a temporary growth arrest and a reduced susceptibility to DHA, as assessed by the ring-stage survival assay, irrespective of their Pfkelch13 genotype. These data suggest that temporary growth arrest of early ring stages may be a constitutive, Pfkelch13-independent survival mechanism in P. falciparum.IMPORTANCEPlasmodium falciparum ring stages have the ability to sense the extracellular environment, regulate their growth, and enter a temporary growth arrest state in response to adverse conditions such as drug exposure. This temporary growth arrest results in reduced susceptibility to artemisinin in vitro. The signal responsible for this process is thought to be small molecules (less than 3 kDa) released by stressed mature-stage parasites. These data suggest that Pfkelch13-dependent artemisinin resistance and the growth arrest phenotype are two complementary but unrelated mechanisms of ring-stage survival in P. falciparum. This finding provides new insights into the field of P. falciparum antimalarial drug resistance by highlighting the extracellular compartment and cellular communication as an understudied mechanism.

Ferroptosis: A New Research Direction of Artemisinin and Its Derivatives in Anti-Cancer Treatment.

Ferroptosis, an iron-dependent cell death mechanism driven by an accumulation of lipid peroxides on cellular membranes, has emerged as a promising strategy to treat various diseases, including cancer. Ferroptosis inducers not only exhibit cytotoxic effects on multiple cancer cells, including drug-resistant cancer variants, but also hold potential as adjuncts to enhance the efficacy of other anti-cancer therapies, such as immunotherapy. In addition to synthetic inducers, natural compounds, such as artemisinin, can be considered ferroptosis inducers. Artemisinin, extracted from Artemisia annua L., is a poorly water-soluble antimalarial drug. For clinical applications, researchers have synthesized various water-soluble artemisinin derivatives such as dihydroartemisinin, artesunate, and artemether. Artemisinin and artemisinin derivatives (ARTEs) upregulate intracellular free iron levels and promote the accumulation of intracellular lipid peroxides to induce cancer cell ferroptosis, alleviating cancer development and resulting in strong anti-cancer effects in vitro and in vivo. In this review, we introduce the mechanisms of ferroptosis, summarize the research on ARTEs-induced ferroptosis in cancer cells, and discuss the clinical research progress and current challenges of ARTEs in anti-cancer treatment. This review deepens the current understanding of the relationship between ARTEs and ferroptosis and provides a theoretical basis for the clinical anti-cancer application of ARTEs in the future.

Multifunctional Deep Eutectic Solvent-Based Microemulsion for Transdermal Delivery of Artemisinin.

As a serious public health issue, malaria threatens the health of millions of people. Artemisinin, a gift from traditional Chinese medicine, has been used in the treatment of malaria and has shown good therapeutic efficiency. However, due to its low solubility, poor bioavailability, and short half-life time, some smart delivery strategies are still required. Herein, a multifunctional DES prepared from ibuprofen and menthol was prepared. This DES was shown to efficiently promote the solubility of artemisinin up to 400-fold. Then, it was further applied as the oil phase to construct an O/W microemulsion with the help of Tween-80 + Span-20 mixed surfactants. The prepared microemulsion displayed high efficiency in improving the permeability of artemisinin, which can be ascribed to the presence of the permeation enhancer menthol in DES and the microstructure of the O/W microemulsion. Moreover, the simultaneous permeation of artemisinin and ibuprofen further indicated the potential benefits of the presented formulation in the treatment of malaria. To sum up, the microemulsion based on multifunctional DES presented herein provided an effective method for transdermal delivery of artemisinin.

Genetics and metabolic responses of Artemisia annua L to the lake of phosphorus under the sparingly soluble phosphorus fertilizer: evidence from transcriptomics analysis.

The medicinal herb Artemisia annua L. is prized for its capacity to generate artemisinin, which is used to cure malaria. Potentially influencing the biomass and secondary metabolite synthesis of A. annua is plant nutrition, particularly phosphorus (P). However, most soil P exist as insoluble inorganic and organic phosphates, which results to low P availability limiting plant growth and development. Although plants have developed several adaptation strategies to low P levels, genetics and metabolic responses to P status remain largely unknown. In a controlled greenhouse experiment, the sparingly soluble P form, hydroxyapatite (Ca5OH(PO4)3/CaP) was used to simulate calcareous soils with low P availability. In contrast, the soluble P form KH2PO4/KP was used as a control. A. annua's morphological traits, growth, and artemisinin concentration were determined, and RNA sequencing was used to identify the differentially expressed genes (DEGs) under two different P forms. Total biomass, plant height, leaf number, and stem diameter, as well as leaf area, decreased by 64.83%, 27.49%, 30.47%, 38.70%, and 54.64% in CaP compared to KP; however, LC-MS tests showed an outstanding 37.97% rise in artemisinin content per unit biomass in CaP contrary to KP. Transcriptome analysis showed 2015 DEGs (1084 up-regulated and 931 down-regulated) between two P forms, including 39 transcription factor (TF) families. Further analysis showed that DEGs were mainly enriched in carbohydrate metabolism, secondary metabolites biosynthesis, enzyme catalytic activity, signal transduction, and so on, such as tricarboxylic acid (TCA) cycle, glycolysis, starch and sucrose metabolism, flavonoid biosynthesis, P metabolism, and plant hormone signal transduction. Meanwhile, several artemisinin biosynthesis genes were up-regulated, including DXS, GPPS, GGPS, MVD, and ALDH, potentially increasing artemisinin accumulation. Furthermore, 21 TF families, including WRKY, MYB, bHLH, and ERF, were up-regulated in reaction to CaP, confirming their importance in P absorption, internal P cycling, and artemisinin biosynthesis regulation. Our results will enable us to comprehend how low P availability impacts the parallel transcriptional control of plant development, growth, and artemisinin production in A. annua. This study could lay the groundwork for future research into the molecular mechanisms underlying A. annua's low P adaptation.

Arteannuin B, a sesquiterpene lactone from Artemisia annua, attenuates inflammatory response by inhibiting the ubiquitin-conjugating enzyme UBE2D3-mediated NF-κB activation.

BACKGROUND: Anomalous activation of NF-κB signaling is associated with many inflammatory disorders, such as ulcerative colitis (UC) and acute lung injury (ALI). NF-κB activation requires the ubiquitination of receptor-interacting protein 1 (RIP1) and NF-κB essential modulator (NEMO). Therefore, inhibition of ubiquitation of RIP1 and NEMO may serve as a potential approach for inhibiting NF-κB activation and alleviating inflammatory disorders. PURPOSE: Here, we identified arteannuin B (ATB), a sesquiterpene lactone found in the traditional Chinese medicine Artemisia annua that is used to treat malaria and inflammatory diseases, as a potent anti-inflammatory compound, and then characterized the putative mechanisms of its anti-inflammatory action. METHODS: Detections of inflammatory mediators and cytokines in LPS- or TNF-α-stimulated murine macrophages using RT-qPCR, ELISA, and western blotting, respectively. Western blotting, CETSA, DARTS, MST, gene knockdown, LC-MS/MS, and molecular docking were used to determine the potential target and molecular mechanism of ATB. The pharmacological effects of ATB were further evaluated in DSS-induced colitis and LPS-induced ALI in vivo. RESULTS: ATB effectively diminished the generation of NO and PGE2 by down-regulating iNOS and COX2 expression, and decreased the mRNA expression and release of IL-1ß, IL-6, and TNF-α in LPS-exposed RAW264.7 macrophages. The anti-inflammatory effect of ATB was further demonstrated in LPS-treated BMDMs and TNF-α-activated RAW264.7 cells. We further found that ATB obviously inhibited NF-κB activation induced by LPS or TNF-α in vitro. Moreover, compared with ATB, dihydroarteannuin B (DATB) which lost the unsaturated double bond, completely failed to repress LPS-induced NO release and NF-κB activation in vitro. Furthermore, UBE2D3, a ubiquitin-conjugating enzyme, was identified as the functional target of ATB, but not DATB. UBE2D3 knockdown significantly abolished ATB-mediated inhibition on LPS-induced NO production. Mechanistically, ATB could covalently bind to the catalytic cysteine 85 of UBE2D3, thereby inhibiting the function of UBE2D3 and preventing ubiquitination of RIP1 and NEMO. In vivo, ATB treatment exhibited robust protective effects against DSS-induced UC and LPS-induced ALI. CONCLUSION: Our findings first demonstrated that ATB exerted anti-inflammatory functions by repression of NF-κB pathway via covalently binding to UBE2D3, and raised the possibility that ATB could be effective in the treatment of inflammatory diseases and other diseases associated with abnormal NF-κB activation.

Discovery of Artemisinins as Microsomal Prostaglandins Synthase-2 Inhibitors for the Treatment of Colorectal Cancer via Chemoproteomics.

Colorectal cancer remains the second leading cause of cancer-related mortalities worldwide. While artemisinin (ART), a key active compound from the traditional Chinese medicinal herb Artemisia annua, has been recognized for its antiproliferative activity against colon cancer cells, its underlying molecular underpinnings remain elusive. Whereas promiscuity of heme-dependent alkylating of macromolecules, mainly proteins, has been seen pivotal as a universal and primary mode of action of ART in cancer cells, accumulating evidence suggests the existence of unique targets and mechanisms of actions contingent on cell or tissue specificities. Here, we employed photoaffinity probes to identify the specific targets responsible for ART's anti-colon cancer actions. Upon validation, microsomal prostaglandins synthase-2 emerged as a specific and reversible target of ART in HCT116 colorectal cancer cells, whose inhibition resulted in reduced cellular prostaglandin E2 biosynthesis and cell growth. Our discovery opens new opportunities for pharmacological treatment of colon cancer.

Artemisinins: Promising drug candidates for the treatment of autoimmune diseases.

Autoimmune diseases are characterized by the immune system's attack on one's own tissues which are highly diverse and diseases differ in severity, causing damage in virtually all human systems including connective tissue (e.g., rheumatoid arthritis), neurological system (e.g., multiple sclerosis) and digestive system (e.g., inflammatory bowel disease). Historically, treatments normally include pain-killing medication, anti-inflammatory drugs, corticosteroids, and immunosuppressant drugs. However, given the above characteristics, treatment of autoimmune diseases has always been a challenge. Artemisinin is a natural sesquiterpene lactone initially extracted and separated from Chinese medicine Artemisia annua L., which has a long history of curing malaria. Artemisinin's derivatives such as artesunate, dihydroartemisinin, artemether, artemisitene, and so forth, are a family of artemisinins with antimalarial activity. Over the past decades, accumulating evidence have indicated the promising therapeutic potential of artemisinins in autoimmune diseases. Herein, we systematically summarized the research regarding the immunoregulatory properties of artemisinins including artemisinin and its derivatives, discussing their potential therapeutic viability toward major autoimmune diseases and the underlying mechanisms. This review will provide new directions for basic research and clinical translational medicine of artemisinins.

Graphene enhances artemisinin production in the traditional medicinal plant Artemisia annua via dynamic physiological processes and miRNA regulation.

We investigated the effects of graphene on the model herb Artemisia annua, which is renowned for producing artemisinin, a widely used pharmacological compound. Seedling growth and biomass were promoted when A. annua was cultivated with low concentrations of graphene, an effect which was attributed to a 1.4-fold increase in nitrogen uptake, a 15%-22% increase in chlorophyll fluorescence, and greater abundance of carbon cycling-related bacteria. Exposure to 10 or 20 mg/L graphene resulted in a âˆ¼60% increase in H2O2, and graphene could act as a catalyst accelerator, leading to a 9-fold increase in catalase (CAT) activity in vitro and thereby maintaining reactive oxygen species (ROS) homeostasis. Importantly, graphene exposure led to an 80% increase in the density of glandular secreting trichomes (GSTs), in which artemisinin is biosynthesized and stored. This contributed to a 5% increase in artemisinin content in mature leaves. Interestingly, expression of miR828 was reduced by both graphene and H2O2 treatments, resulting in induction of its target gene AaMYB17, a positive regulator of GST initiation. Subsequent molecular and genetic assays showed that graphene-induced H2O2 inhibits micro-RNA (miRNA) biogenesis through Dicers and regulates the miR828-AaMYB17 module, thus affecting GST density. Our results suggest that graphene may contribute to yield improvement in A. annua via dynamic physiological processes together with miRNA regulation, and it may thus represent a new cultivation strategy for increasing yield capacity through nanobiotechnology.

Green synthesis and characterization of an orally bioactive artemisinin-zinc nanoparticle with enhanced bactericidal activity.

The occurrence of multidrug-resistant bacteria necessitates the development of new antibacterial agents. This study synthesized artemisinin-zinc nanoparticles (AZ NPs) using a simple green method and investigated their physicochemical properties, antibacterial activity, and oral biological activity. A spherical shape morphology of AZ NPs was observed by scanning and transmission electron microscopy, with a particle size of 73 ± 2.604 nm. Energy dispersive spectrometry analysis showed that the AZ NPs consisted mainly of Zn, C, N, and O elements. According to differential scanning calorimeter analysis, the AZ NPs were stable up to 450 °C. Fourier-transform infrared spectroscopy revealed that artemisinin successfully bound to zinc acetate. The AZ NPs showed antibacterial activity against Salmonella and Escherichia coli, with a minimum inhibitory concentration of 0.056 mg/mL for both and minimum bactericidal concentrations of 0.21 and 0.11 mg/mL, respectively. The mechanisms by which AZ NPs mediate membrane damage were revealed by the downregulation of gene expression, and potassium ion and protein leakage. In vivo safety trials of these drugs revealed low toxicity. After AZ NPs were administered to infected mice, the intestinal bacteria decreased significantly, liver and kidney function were restored, histopathological damage to the liver and spleen were reduced, and the expression of inflammatory cytokines decreased. Therefore, AZ NPs have the potential as an oral antibacterial agent and can be used in antibiotic development and in the pharmaceutical industry.

Phytochemical screening, antioxidant analyses, and in vitro and in vivo antimalarial activities of herbal medicinal plant - Rotheca serrata (L.) Steane & Mabb.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria is a major global health concern that is presently challenged by the emergence of Plasmodium falciparum (Pf) resistance to mainstay artemisinin-based combination therapies (ACTs). Hence, the discovery of novel and effective antimalarial drugs is pivotal to treating and controlling malaria. For many years, traditional plant-based herbal medicines have been employed in the treatment of various illnesses. Rotheca serrata (L.) Steane & Mabb. belongs to the Lamiaceae family that has been traditionally used to treat, cure, and prevent numerous diseases including malaria. AIM: The present investigation sought to assess the phytoconstituents, antioxidant, cytotoxicity, antimalarial activities of Rotheca serrata extract and its fractions. The in vitro antiplasmodial activity was assessed in chloroquine-sensitive Pf3D7 and artemisinin-resistant PfCam3.IR539T cultures, and the in vivo antimalarial activity was analyzed in Plasmodium berghei (Pb) ANKA strain-infected BALB/c mouse model. MATERIALS AND METHODS: The fresh leaves of Rotheca serrata were extracted in methanol (RsMeOH crude leaf extract). A portion of the extract was used to prepare successive solvent fractions using ethyl acetate (RsEA) and hexane (RsHex). The in vitro antiplasmodial activity was evaluated using [3H]-hypoxanthine incorporation assays against Pf3D7 and PfCam3.IR539T cultures. In vitro cytotoxicity study on HeLa, HEK-293T, and MCF-7 cell lines was carried out using MTT assay. The human red blood cells (RBCs) were used to perform the hemolysis assays. In vitro antioxidant studies and detailed phytochemical analysis were performed using GC-MS and FTIR. The four-day Rane's test was performed to evaluate the in vivo antimalarial activity against Pb ANKA strain-infected mice. RESULTS: Phytochemical quantification of Rotheca serrata extract (RsMeOH) and its fractions (RsEA and RsHex) revealed that RsMeOH crude extract and RsEA fraction had higher contents of total phenol and flavonoid than RsHex fraction. The RsEA fraction showed potent in vitro antiplasmodial activity against Pf3D7 and PfCam3.IR539T with IC50 values of 9.24 ± 0.52 µg/mL and 17.41 ± 0.43 µg/mL, respectively. The RsMeOH crude extract exhibited moderate antiplasmodial activity while the RsHex fraction showed the least antiplasmodial activity. The GC-MS and FTIR analysis of RsMeOH and RsEA revealed the presence of triterpenes, phenols, and hydrocarbons as major constituents. The RsMeOH crude extract was non-hemolytic and non-cytotoxic to HeLa, HEK-293T, and MCF-7 cell lines. The in vivo studies showed that a 1200 mg/kg dose of RsMeOH crude extract could significantly suppress parasitemia by ∼63% and prolong the survival of treated mice by ∼10 days. The in vivo antiplasmodial activity of RsMeOH was better than the RsEA fraction. CONCLUSION: The findings of this study demonstrated that traditionally used herbal medicinal plants like R. serrata provide a platform for the identification and isolation of potent bioactive phytochemicals that in turn can promote the antimalarial drug research. RsMeOH crude extract and RsEA fraction showed antiplasmodial, antimalarial and antioxidant activities. Chemical fingerprinting analysis suggested the presence of bioactive phytocompounds that are known for their antimalarial effects. Further detailed investigations on RsMeOH crude extract and RsEA fraction would be needed for the identification of the entire repertoire of the active antimalarial components with potent pharmaceutical and therapeutic values.