Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Artemisitene is identified as a novel Nrf2 activator. It acts by decreasing Nrf2 ubiquitination, which increases its stability and protein half-life. [1]

Artemisitene selectively induces DNA double-strand breaks (DSBs) and apoptosis in a variety of human cancer cells by inhibiting the expression of topoisomerase in human cancer cells. Artemisinin enhances c-Myc ubiquitination by preferentially activating c-Myc E3 ligase NEDD4, hence selectively destabilizing c-Myc in human cancer cells [2].

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Artemisitene activates the antioxidant responsive element (ARE) in a dose-dependent manner. In MDA-MB-231 cells transfected with an ARE-luciferase reporter, it induced a 2-fold increase in luciferase activity at 0.5 μM and a maximum 3.5-fold increase at 2.5 μM. [1]
Treatment of MDA-MB-231 cells with artemisitene (2 μM for 16 h) increased Nrf2 protein levels without changing Keap1 levels. It also increased the mRNA expression of Nrf2 target genes, including NQO1 and Mrp2. [1]
In mouse macrophage-like RAW264.7 cells, artemisitene activated the Nrf2-dependent response more potently. Treatment with 1 μM AT strongly induced Nrf2 protein, and 0.5 μM AT induced NQO1 and HO-1 mRNA expression by up to 10-fold and 2-fold, respectively. [1]
Artemisitene protects cells from oxidative stress. In MDA-MB-231 cells, pretreatment with 2 μM AT for 8 h reduced H₂O₂-induced cellular ROS levels. Furthermore, AT pretreatment (2 μM for 12 h) significantly increased cell viability against 1 mM H₂O₂ challenge, with a survival rate of 0.965 compared to 0.499 in the control group. [1]
Mechanistically, artemisitene increases the half-life of Nrf2 protein. In cycloheximide chase assays, the half-life of Nrf2 increased from 20.8 minutes in untreated MDA-MB-231 cells to 34.5 minutes in AT-treated cells. [1]
An in vivo ubiquitination assay showed that artemisitene (2 μM), similar to the positive control tBHQ, decreases the ubiquitination of Nrf2. [1]
The activation of Nrf2 by artemisitene is dependent on Keap1. In A549 cells, which harbor a dysfunctional Keap1, treatment with AT (1-4 μM) failed to increase Nrf2 protein levels. [1]

In Nrf2 wild-type mice, systemic administration of artemisinin (5-10 mg/kg; i.p.) substantially reduced bleomycin-induced lung damage [1].

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Systemic administration of artemisitene (10 mg/kg, i.p.) in B6 mice effectively activated the Nrf2-dependent response in lung tissue. At 48 hours post-injection, pulmonary Nrf2 protein levels were upregulated, and mRNA levels of its downstream genes NQO1 and HO-1 were increased. [1]
In a bleomycin (BLM)-induced lung injury mouse model, artemisitene treatment (10 mg/kg, i.p., given 48h before each BLM injection) significantly reduced BLM-induced pulmonary pathological alterations, as assessed by H&E staining. [1]
Artemisitene attenuated BLM-induced lung fibrosis. Masson's trichrome staining showed less collagen deposition, and hydroxyproline content (an index of collagen) was significantly lower in the AT+BLM group compared to the BLM-only group. It also inhibited the BLM-induced upregulation of fibrosis markers α-SMA and TGF-β mRNA in lung tissue. [1]
Artemisitene reduced BLM-induced inflammatory response. Bronchoalveolar lavage fluid analysis showed that AT treatment decreased the BLM-induced increase in total inflammatory cells, macrophages, and neutrophils. It also inhibited the BLM-induced upregulation of pro-inflammatory cytokines IL-6, IL-4, TGF-β, and MCP-1, and restored the levels of IL-2 and IFN-γ in lung tissue. [1]

ARE-luciferase Reporter Assay: A stable MDA-MB-231 cell line expressing an ARE-luciferase construct was established. Cells were seeded, grown to 80% confluence, and treated with test compounds (e.g., artemisitene at 1 μM, tBHQ at 50 μM) for 24 hours. Cells were then lysed, and luciferase activity was measured. For a dual-luciferase assay, MDA-MB-231 cells were co-transfected with an ARE-firefly luciferase plasmid and a Renilla luciferase control plasmid. Transfected cells were treated with increasing doses of artemisitene (0-2.5 μM) or 5 μM SF for 24 hours. Both firefly and Renilla luciferase activities were measured, with firefly activity normalized to Renilla activity. [1]
ROS Detection and Cell Viability: MDA-MB-231 cells were pretreated with 2 μM artemisitene or 5 μM SF for 8 hours, then challenged with 0.2 mM H₂O₂ for another 8 hours. Cellular ROS levels were detected by dichlorofluorescein staining and flow cytometry. For cell viability, cells in a 96-well plate were pretreated with DMSO or 2 μM AT for 12 hours, then treated with 1 mM H₂O₂ for 24 hours. MTS reagent was added, incubated for 4 hours, and absorbance at 490 nm was recorded to calculate the survival rate. [1]
Nrf2 Protein Half-Life Measurement: MDA-MB-231 cells were left untreated or treated with 2 μM artemisitene for 4 hours. Protein synthesis was then blocked by adding 50 μM cycloheximide. Total cell lysates were collected at different time points (0, 15, 30, 45, 60 min) and subjected to immunoblot analysis with an anti-Nrf2 antibody. Band intensities were quantified, and the half-life was calculated. [1]
In Vivo Ubiquitination Assay: MDA-MB-231 cells were co-transfected with expression vectors for Nrf2, Keap1, and hemagglutinin-ubiquitin. Transfected cells were left untreated or treated with 50 μM tBHQ or 2 μM artemisitene along with 10 μM MG132 for 4 hours. Cells were lysed, and Nrf2 was immunoprecipitated with an anti-Nrf2 antibody. The immunoprecipitated proteins were then analyzed by immunoblot with an anti-ubiquitin antibody to detect ubiquitin-conjugated Nrf2. [1]
qRT-PCR for Gene Expression: Total mRNA was extracted from cells or tissues using TRIZOL reagent and reverse-transcribed to cDNA. Real-time PCR was performed using Taqman probes or SYBR Green with specific primers for human and mouse genes (NQO1, Mrp2, HO-1, IL-2, IL-4, IL-6, IFN-γ, MCP-1, TGF-β, α-SMA, and housekeeping genes β-actin or 18s rRNA). Relative mRNA levels were calculated using the comparative Ct method. [1]

Pilot Nrf2 Activation Study:** Eight-week-old B6 mice were injected intraperitoneally with PBS or 10 mg/kg artemisitene. Mice were sacrificed 48 hours post-injection, and lung tissues were isolated for immunoblot analysis of Nrf2 and qRT-PCR analysis of NQO1 and HO-1 mRNA. [1]
* **BLM-Induced Lung Injury Model (Fibrosis/Pathology):** B6 mice were randomly divided into 4 groups (n=5): Control (PBS), AT only (10 mg/kg), BLM only, and AT+BLM. Mice received an i.p. injection of PBS or 10 mg/kg AT. 48 hours later, mice in the BLM and AT+BLM groups were injected i.p. with 2 mg BLM. This BLM injection was repeated once a week for 3 weeks. Seven days after the third BLM injection, mice were sacrificed, and lung tissues were collected for H&E staining, Masson's trichrome staining, hydroxyproline assay, and qRT-PCR analysis of fibrosis and inflammation markers. [1]
* **BLM-Induced Acute Inflammation Study:** B6 mice were pre-injected i.p. with PBS or 5 mg/kg AT. 24 hours later, they were injected with 2 mg BLM. On the seventh day after BLM injection, mice were sacrificed, and bronchoalveolar lavage was performed to analyze total and differential inflammatory cell counts (neutrophils, macrophages, lymphocytes). [1]

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Pilot Nrf2 Activation Study: Eight-week-old B6 mice were injected intraperitoneally with PBS or 10 mg/kg artemisitene. Mice were sacrificed 48 hours post-injection, and lung tissues were isolated for immunoblot analysis of Nrf2 and qRT-PCR analysis of NQO1 and HO-1 mRNA. [1]
BLM-Induced Lung Injury Model (Fibrosis/Pathology): B6 mice were randomly divided into 4 groups (n=5): Control (PBS), AT only (10 mg/kg), BLM only, and AT+BLM. Mice received an i.p. injection of PBS or 10 mg/kg AT. 48 hours later, mice in the BLM and AT+BLM groups were injected i.p. with 2 mg BLM. This BLM injection was repeated once a week for 3 weeks. Seven days after the third BLM injection, mice were sacrificed, and lung tissues were collected for H&E staining, Masson's trichrome staining, hydroxyproline assay, and qRT-PCR analysis of fibrosis and inflammation markers. [1]
BLM-Induced Acute Inflammation Study: B6 mice were pre-injected i.p. with PBS or 5 mg/kg AT. 24 hours later, they were injected with 2 mg BLM. On the seventh day after BLM injection, mice were sacrificed, and bronchoalveolar lavage was performed to analyze total and differential inflammatory cell counts (neutrophils, macrophages, lymphocytes). [1]

In the in vivo studies, artemisitene alone at a dose of 10 mg/kg showed no obvious toxicity. Histological examination of lung, liver, and kidney tissues from mice treated with AT alone revealed no pathological alterations. [1]

[1]. Artemisitene activates the Nrf2-dependent antioxidant response and protects against bleomycin-induced lung injury. FASEB J. 2016 Jul;30(7):2500-10.

[2]. Artemisitene suppresses tumorigenesis by inducing DNA damage through deregulating c-Myc-topoisomerase pathway. Oncogene. 2018 Sep;37(37):5079-5087.

Artemisinin is a terpene lactone. It has been reported that dehydroartemisinin is found in Artemisia annua, Artemisia argyi, and Artemisia umbellata; relevant data are available for reference.

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Artemisitene is a derivative of the antimalarial agent artemisinin and a natural component of the sweet wormwood herb (Artemisia annua). Unlike artemisinin, it possesses Nrf2-activating properties. [1]
It acts as a potent Nrf2 activator by inhibiting the Keap1-dependent ubiquitination of Nrf2, leading to its stabilization and enhanced transcriptional activity of antioxidant response element-driven genes. [1]
By activating the Nrf2 pathway, artemisitene provides cytoprotection against oxidative stress (H₂O₂) in vitro and against bleomycin-induced lung injury, inflammation, and fibrosis in vivo. [1]
The study suggests that artemisitene-based therapeutic approaches targeting Nrf2 may offer antioxidant protection against tissue damage caused by environmental insults and toxic chemicals. [1]
It was observed that artemisitene activates the Nrf2 pathway to different extents in different cell types, being more potent in RAW264.7 macrophage-like cells than in MDA-MB-231 breast cancer cells. [1]

C15H20O5

280.3163

280.131

C, 64.27; H, 7.19; O, 28.54

101020-89-7

11000442

White to off-white solid powder

160-162°C (lit.)

3.52E-07mmHg at 25°C

2.315

0

5

0

20

489

6

O=C1O[C@H]2[C@@]34OO[C@](O2)(C)CC[C@H]3[C@@H](CC[C@H]4C1=C)C

IGEBZMMCKFUABB-KPHNHPKPSA-N

InChI=1S/C15H20O5/c1-8-4-5-11-9(2)12(16)17-13-15(11)10(8)6-7-14(3,18-13)19-20-15/h8,10-11,13H,2,4-7H2,1,3H3/t8-,10+,11+,13-,14-,15-/m1/s1

(1R,4S,5R,8S,12S,13R)-1,5-dimethyl-9-methylidene-11,14,15,16-tetraoxatetracyclo[10.3.1.04,13.08,13]hexadecan-10-one

(+)-Artemisitene; Artemisitene; Dehydroqinghaosu; Methenyl-artemisinin; ATT.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 100 mg/mL (~356.74 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)