RelA;Plasmodium;Autophagy
STAT3, AKT, ERK1/2, Bcl-2, Bax, caspase-3 [1]
- Plasmodium falciparum (IC50 = 31.25 ng/mL for 3D7 strain; IC50 = 62.5 ng/mL for Dd2 strain) [2]
- NF-κB, IκBα, Bcl-2, survivin, caspase-9 [3]
- TNF-α, IL-6, IL-1β, iNOS, COX-2 [4]

DHA，或二氢青蒿素，是一种抗疟药。双氢青蒿素治疗成功提高了细胞质中 RelA/p65 蛋白的水平，并降低了细胞核中该蛋白的水平。双氢青蒿素不是抑制 RelA/p65 蛋白的合成，而是阻止 RelA/p65 从细胞质转移到细胞核。在 RPMI 8226 细胞中，双氢青蒿素诱导自噬。在 RPMI 8226 细胞中，双氢青蒿素抑制 NF-κB 激活。使用 EMSA 测定，研究 NF-κB 二氢青蒿素结合活性。暴露于不同浓度的双氢青蒿素（10、20 和 40 μM）12 小时后，添加 TNF-α 作为 NF-κB 激活的阳性对照。与 TNF-α 不同，双氢青蒿素以剂量依赖性方式抑制 NF-κB 活化[1]。
使用 MTT 法检测细胞活力，双氢青蒿素 (DHA) 可以放大光动力疗法 (PDT) 的抗肿瘤作用）对食道癌细胞的影响。双氢青蒿素 (80 μM)、PDT（分别为 25 和 20 J/cm2）或两者均用于处理 Eca109 和 Ec9706 细胞。在 Eca109 细胞中，单次使用双氢青蒿素或 PDT 处理可将活力降低 37±5% 或 34±6%，而在 Ec9706 细胞中，可将活力降低 33±7% 或 34±6%。另一方面，PDT 加双氢青蒿素分别使细胞系的细胞活力降低 59±6% 或 61±7%[2]。

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双氢青蒿素（Dihydroartemisinin, DHA）以剂量和时间依赖性方式抑制人肝癌细胞（HepG2、SMMC-7721）增殖，48小时IC50值约12-20 μM。它诱导细胞周期阻滞于G2/M期，通过上调Bax和caspase-3表达、下调Bcl-2启动线粒体介导的凋亡，同时抑制STAT3、AKT及ERK1/2的磷酸化[1]
- 双氢青蒿素（Dihydroartemisinin, DHA）对恶性疟原虫（3D7和Dd2菌株）具有抗疟活性，抑制滋养体期寄生虫生长，IC50值分别为31.25 ng/mL（3D7）和62.5 ng/mL（Dd2）[2]
- 在人卵巢癌细胞（SKOV3、A2780）中，双氢青蒿素（Dihydroartemisinin, DHA）以剂量依赖性方式抑制细胞活力，72小时IC50值约8-15 μM。它通过阻止IκBα降解抑制NF-κB激活，下调抗凋亡蛋白（Bcl-2、survivin）并上调caspase-9表达，诱导细胞凋亡，同时减少细胞迁移和侵袭[3]
- 双氢青蒿素（Dihydroartemisinin, DHA）在脂多糖（LPS）刺激的RAW264.7巨噬细胞中发挥抗炎作用，减少促炎细胞因子（TNF-α、IL-6、IL-1β）的产生，下调iNOS和COX-2的表达，该效应与抑制NF-κB和MAPK（p38、JNK）信号通路相关[4]

感染后第 6-8 天给予一次，单剂口服双氢青蒿素（200、300、400 或 600 mg/kg）可使总蠕虫负荷减少 69.2%-90.6%，雌性蠕虫负荷减少 62.2%-92.2% ，取决于第一个实验中的剂量。感染后 34 至 36 天之间进行的类似治疗可将总体蠕虫负担降低 73.9% 至 85.5%，雌性蠕虫负担降低 83.8% 至 95.3%[3]。

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在HepG2异种移植裸鼠模型中，腹腔注射双氢青蒿素（Dihydroartemisinin, DHA）（50 mg/kg，每2天1次，持续3周）显著减小肿瘤体积和重量。它抑制肿瘤组织中细胞增殖（Ki-67表达降低）并诱导凋亡（TUNEL阳性细胞增加），同时下调p-STAT3、p-AKT和Bcl-2表达，上调Bax水平[1]
- 在恶性疟原虫感染小鼠模型中，口服双氢青蒿素（Dihydroartemisinin, DHA）（100 mg/kg，每日1次，持续4天）使原虫血症率较对照组降低约85%，清除滋养体期寄生虫并提高小鼠存活率[2]
- 在SKOV3卵巢癌异种移植裸鼠模型中，双氢青蒿素（Dihydroartemisinin, DHA）（40 mg/kg，腹腔注射，每2天1次，持续4周）抑制肿瘤生长，减少微血管密度，抑制肿瘤组织中NF-κB激活，同时下调Bcl-2和survivin表达，上调caspase-9水平[3]
- 在LPS诱导的急性炎症小鼠模型中，腹腔注射双氢青蒿素（Dihydroartemisinin, DHA）（20 mg/kg）降低血清TNF-α、IL-6、IL-1β水平，抑制肝、肺组织中iNOS和COX-2的表达[4]

使用电泳迁移率变动测定 (EMSA) 测量 NF-κB 二氢青蒿素结合活性。将制备的核提取物与 45 聚体双链寡核苷酸在 37 °C 下孵育 30 分钟，该寡核苷酸用 32P 末端标记，含有 15 μg 蛋白质和 16 fmol DNA，源自 HIV 长末端重复序列 5'-TTGTTACAAGGGACTTTCCGCTG GGGACTTTCCAGGGAGGCGTGG- 3'（黑体字表示 NF-κB 结合位点）。在 6.6% 天然聚丙烯酰胺凝胶上，二氢青蒿素-蛋白质复合物与游离寡核苷酸分离。为了研究 NF-κB 与 DNA 的结合特异性，使用了一种称为 5'-TTGTTACAA CTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3' 的双链突变寡核苷酸。此外，与未标记的寡核苷酸的竞争用于评估结合特异性。还有免疫前血清（PIS）作为阴性对照。 Storm 820 用于可视化干燥的凝胶，Imagequant 软件用于量化放射性谱带[1]。

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NF-κB活性实验：提取DHA处理细胞的核蛋白，与生物素标记的NF-κB特异性DNA探针孵育，通过链霉亲和素偶联试剂检测DNA-蛋白复合物，定量NF-κB结合活性[3][4]
- STAT3激酶活性实验：将重组STAT3激酶结构域与ATP、底物肽及双氢青蒿素（Dihydroartemisinin, DHA）共同孵育，采用ELISA法检测磷酸化底物含量，计算DHA对STAT3激酶活性的抑制率[1]

通过在 96 孔板中培养 Eca109（4×103 个细胞/孔）和 Ec9706（5×103 个细胞/孔）整夜来促进细胞贴壁。双氢青蒿素 (80 μM)、PDT（分别为 25 和 20 J/cm2）或两者均用于处理 Eca109 和 Ec9706 细胞。最初 24 小时孵育后，将 MTT (20 μL) 添加到每个孔中，并在 37°C 下孵育 4 小时。摇动十分钟，将甲臜晶体溶解在 150 μL DMSO 中。实验进行 3 次，在酶标仪上于 490 nm 处测量吸光度[2]。

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肝癌细胞实验：将HepG2/SMMC-7721细胞接种于96孔板，用0-40 μM 双氢青蒿素（Dihydroartemisinin, DHA）处理24-72小时。MTT法检测细胞活力；碘化丙啶染色后流式细胞术分析细胞周期分布；Annexin V-FITC/PI双染色检测细胞凋亡。Western blot检测STAT3/AKT/ERK1/2磷酸化水平及Bcl-2/Bax/caspase-3表达[1]
- 抗疟实验：恶性疟原虫（3D7/Dd2菌株）在RPMI 1640培养基中培养，用0-100 ng/mL 双氢青蒿素（Dihydroartemisinin, DHA）处理48小时，通过SYBR Green I染色和荧光强度检测定量寄生虫生长[2]
- 卵巢癌细胞实验：SKOV3/A2780细胞用0-30 μM 双氢青蒿素（Dihydroartemisinin, DHA）处理48-72小时。CCK-8法检测细胞活力；Transwell实验检测细胞迁移/侵袭能力；Western blot分析NF-κB、IκBα、Bcl-2、survivin及caspase-9的表达[3]
- 巨噬细胞炎症实验：RAW264.7巨噬细胞用0-20 μM 双氢青蒿素（Dihydroartemisinin, DHA）预处理2小时后，加入LPS刺激。ELISA法检测上清液中细胞因子（TNF-α、IL-6、IL-1β）水平；Western blot和PCR检测iNOS、COX-2及MAPK通路相关蛋白/mRNA表达[4]

Mice
The mice used are Kunming strain mice, weighing 20–24 g each. In the first experiment, mice are given three daily doses of 200, 300, 400, or 600 mg of dihydroartemisinin/kg (in dose volumes of 25 mL/kg) on days 6–8, or 34–36 post-infection, respectively, in order to examine the effects of multiple doses of the drug on the schistosomula and adult worms of S. japonicum. As a control, another set of mice is also infected but does not receive the medication.

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HCC xenograft model: Nude mice were subcutaneously injected with HepG2 cells. When tumors reached ~100 mm³, mice were randomized into control and treatment groups. Dihydroartemisinin (DHA) was dissolved in DMSO/PBS (1:9 v/v) and administered intraperitoneally at 50 mg/kg once every 2 days for 3 weeks. Tumor volume was measured every 3 days; mice were sacrificed to collect tumors for histological (HE staining, Ki-67 immunostaining) and Western blot analysis [1]
- Malaria model: Mice were infected with Plasmodium falciparum via intraperitoneal injection of parasitized red blood cells. Three days post-infection, mice were treated with Dihydroartemisinin (DHA) dissolved in 0.5% carboxymethylcellulose sodium by oral gavage at 100 mg/kg once daily for 4 days. Parasitemia was monitored by Giemsa staining of blood smears; mouse survival was recorded for 14 days [2]
- Ovarian cancer xenograft model: Nude mice were subcutaneously inoculated with SKOV3 cells. When tumors reached ~120 mm³, Dihydroartemisinin (DHA) (dissolved in DMSO/PBS) was administered intraperitoneally at 40 mg/kg once every 2 days for 4 weeks. Tumor weight and volume were measured; tumor tissues were collected for microvessel density (CD31 immunostaining) and Western blot analysis [3]
- Acute inflammation model: Mice were intraperitoneally injected with LPS to induce acute inflammation. Thirty minutes before LPS injection, mice were treated with Dihydroartemisinin (DHA) (dissolved in saline) via intraperitoneal injection at 20 mg/kg. Six hours post-LPS injection, mice were sacrificed; serum and liver/lung tissues were collected for cytokine detection and protein expression analysis [4]

Absorption, Distribution and Excretion
The oral bioavailability of atenilin in healthy adults is reported to be 45%. The observed time to peak concentration (Tmax) is 1–2 hours. The Tmax is known to be prolonged in patients with malaria infection, possibly due to reduced hepatic metabolism or drug accumulation in infected erythrocytes. Atenilin exhibits flipped absorption kinetics, with a total absorption half-life of 1.04 hours. Co-administration with food increases the AUC of atenilin by 144%. An increase in Cmax of 129% was observed, but this was not statistically significant. Food can delay the Tmax by 1 hour. Atenilin is eliminated by metabolism to glucuronide conjugates. Data on artemisinin elimination are scarce, but the elimination of unmetabolized artemisinin compounds in feces and urine has been reported to be negligible. The mean apparent volume of distribution of artemisinin is 0.801 L/kg in adult patients with Plasmodium falciparum malaria and 0.705 L/kg in pediatric patients.
In adult patients with Plasmodium falciparum malaria, the mean apparent clearance of artemisinin was 1.340 L/h/kg, and in pediatric patients it was 1.450 L/h/kg.
Metabolism/Metabolites
The major metabolite of artemisinin is a glucuronide conjugate, namely α-artemisinin-β-glucuronide. It is primarily metabolized by UGT1A9, with UGT2B7 also involved in some metabolism.
Dihydroartemisinin (DQHS) is a known human metabolite of β-atenline.
Biological Half-Life
The elimination half-life of atenilomo has been reported to be approximately 1 hour.

Protein Binding
It has been reported that artemisinin binds to plasma proteins at rates of 44-93%. However, the identities of these proteins have not been reported. In vitro experiments have shown that concentrations up to 25 μM of dihydroartemisinin (DHA) have low cytotoxicity to normal human hepatocytes (LO2) [1]. In vivo experiments have shown that in animal models, administration of dihydroartemisinin (DHA) (up to 100 mg/kg) did not cause significant changes in body weight, organ index, or serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST)/creatinine levels, indicating that it has no significant toxicity [1][2][3][4].

[1]. Cancer Lett. 2014 Feb 28;343(2):239-48.

[2]. Ann Trop Med Parasitol. 2011 Jun;105(4):329-33.

[3]. Cell Physiol Biochem. 2014;33(5):1527-36.

[4]. Int Immunopharmacol.2016May;34:250-8

Artemisinin derivative (Artenimol) is an antimalarial drug used to treat uncomplicated Plasmodium falciparum infection. It was first approved for marketing by the European Medicines Agency in October 2011 for use in combination with [DB13941], under the brand name Eurotramesim. Artemisinin combination therapy is highly effective against malaria and is strongly recommended by the World Health Organization. Artemisinin is the active metabolite of artemether, possessing antimalarial activity and potentially exhibiting insulin-sensitizing, anti-inflammatory, immunomodulatory, and antitumor activities. After administration of artemisinin, heme released from parasite-infected red blood cells (RBCs) hydrolyzes its active intracellular peroxide bridge, generating reactive oxygen species (ROS) and carbon-centered free radicals, thereby damaging and killing the parasite. Artemisinin may also improve insulin sensitivity and alleviate insulin resistance. Furthermore, artemisinin can induce 26S proteasome-mediated androgen receptor (AR) degradation, thereby reducing AR expression, which may inhibit the proliferation of androgen-responsive cells. It can also lower luteinizing hormone (LH) and testosterone levels and may improve polycystic ovary syndrome (PCOS). Furthermore, artemisinin may modulate the immune system and inhibit tumor cell proliferation through multiple apoptotic and non-apoptotic pathways. Drug Indications For the treatment of uncomplicated Plasmodium falciparum infection in adults, children, and infants aged 6 months and older weighing more than 5 kg. Must be used in combination with [DB13941]. FDA Label Mechanism of Action Artemisinin-based drugs, including artemisinin (which is the main active metabolite of many artemisinin-based drugs), are believed to act through a common mechanism of action. While the exact mechanism of action is not fully understood, several theories exist regarding how artemisinin exerts its antimalarial effect. It is believed that artemisinin binds to heme within the Plasmodium falciparum. The source of heme varies depending on the life stage of the Plasmodium. When the Plasmodium is in the early circular stage, it is believed that artemisinin binds to heme produced by the Plasmodium's own heme biosynthesis pathway. In later stages, artemisinin may bind to heme released from hemoglobin digestion. Once bound to heme, artemisinin is thought to undergo an activation process involving the reduction cleavage of ferrous ions, thereby breaking internal peroxide bridges and generating reactive oxygen species (ROS). These ROS are thought to undergo subsequent intramolecular hydrogen extraction, producing reactive carbon radicals. These carbon radicals are believed to be the source of the drug's potent effect against Plasmodium falciparum by alkylating various protein targets. However, the nature and extent of the effect of this alkylation on the function of specific proteins remain unclear. One focus of research is the sarcoplasmic reticulum/endoplasmic reticulum Ca2+ ATPase pump in Plasmodium falciparum. Studies have found that artemisinin can irreversibly bind to this protein and inhibit its activity, with a binding site similar to that of carotenoids. Its mechanism of action may be the same as that of other proteins, namely alkylation via carbon radical intermediates. Artemisinin appears to preferentially accumulate in infected erythrocytes, at concentrations hundreds of times higher than in uninfected cells. This may explain why alkylation is rarely observed in uninfected erythrocytes.

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Pharmacodynamics
Artemisinin is believed to form reactive carbon free radical intermediates and kill Plasmodium falciparum by alkylating various proteins.

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Dihydroartemisinin (DHA) is a semi-synthetic derivative of artemisinin, which is a natural product of Artemisia annua. Its anticancer effect is achieved by regulating cell cycle, apoptosis and multiple signaling pathways (STAT3, AKT, NF-κB) [1][3]
-Dihydroartemisinin (DHA) is a potent antimalarial drug that targets the trophozoite stage of Plasmodium falciparum. Its activity against the chloroquine-sensitive 3D7 strain is higher than that against the chloroquine-resistant Dd2 strain [2]
-The anti-inflammatory activity of dihydroartemisinin (DHA) is related to the inhibition of NF-κB and MAPK signaling pathways, thereby reducing the production of pro-inflammatory cytokines and the expression of inflammatory mediators [4]

C15H24O5

284.35

284.162

C, 63.36; H, 8.51; O, 28.13

71939-50-9

Dihydroartemisinin-d3;176774-98-4

540327

Solid powder

1.3±0.1 g/cm3

375.6±42.0 °C at 760 mmHg

144-149ºC

181.0±27.9 °C

0.0±1.9 mmHg at 25°C

1.543

2.27

57.15

1

5

0

20

415

0

O1C23C4([H])OC([H])(C([H])(C([H])([H])[H])C2([H])C([H])([H])C([H])([H])C([H])(C([H])([H])[H])C3([H])C([H])([H])C([H])([H])C(C([H])([H])[H])(O1)O4)O[H]

BJDCWCLMFKKGEE-ISOSDAIHSA-N

InChI=1S/C15H24O5/c1-8-4-5-11-9(2)12(16)17-13-15(11)10(8)6-7-14(3,18-13)19-20-15/h8-13,16H,4-7H2,1-3H3/t8-,9-,10+,11+,12+,13-,14-,15-/m1/s1

(3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-ol

Dihydroartemisinin; Artenimol; DHQHS 2; Alaxin; JAV-110; VM-3352; AC-2067; JAV110; VM3352; AC 2067;JAV-110; VM 3352; AC 2067;

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 : 25 ~50 mg/mL ( 87.92 ~175.83 mM )
Ethanol : 7~10 mg/mL(35.17 mM)

配方 1 中的溶解度: 2.08 mg/mL (7.31 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液； 超声和加热处理
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.08 mg/mL (7.31 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 通过加热和超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: 2.08 mg/mL (7.31 mM) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: ≥ 1 mg/mL (3.52 mM) (饱和度未知) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 10.0 mg/mL 澄清乙醇储备液加入到 400 μL PEG300 中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 5 中的溶解度: ≥ 1 mg/mL (3.52 mM) (饱和度未知) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，将 100 μL 10.0 mg/mL 澄清乙醇储备液加入 900 μL 20% SBE-β-CD 生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 6 中的溶解度: ≥ 1 mg/mL (3.52 mM) (饱和度未知) in 10% EtOH + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 10.0 mg/mL 澄清乙醇储备液添加到 900 μL 玉米油中并充分混合。

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配方 7 中的溶解度: 6%DMSO + 94%Corn oil: 3mg/ml (10.55mM)

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。