GSK-3β (IC50 = 0.6 μM ); CDK2/CyclinA (IC50 = 2.2 μM); CDK5/p35 (IC50 = 5.5 μM); CDK1/CyclinB (IC50 = 10 μM); CDK4/CyclinD1 (IC50 = 12 μM)
Epidermal Growth Factor Receptor (EGFR): IC₅₀ = 15 μM (inhibition of EGFR phosphorylation in epidermal keratinocytes) [2]
- Vascular Endothelial Growth Factor Receptor 2 (VEGFR2): IC₅₀ = 20 μM (inhibition of VEGFR2 kinase activity); Janus Kinase 2 (JAK2): IC₅₀ = 25 μM (inhibition of JAK2 phosphorylation in endothelial cells) [3]
- Tubulin: Ki = 12 μM (binding affinity to tubulin, competing with colchicine) [6]

Indirubin (Courupitine B) 显着降低 Td-EC 血管生成、侵袭、迁移和血管生成[1]。

---

1. 在DNCB致敏小鼠的脾淋巴细胞中，Indirubin（NSC 105327; Couroupitine B）（10 μM、20 μM、40 μM，处理48小时）呈剂量依赖性抑制Th1和Th17细胞分化：40 μM浓度下，Th1细胞因子IFN-γ分泌减少约60%，Th17细胞因子IL-17分泌减少约55%（ELISA检测）；同时，调节性T细胞（Treg）标志物Foxp3表达在40 μM浓度下增加约2.3倍（Western blot检测） [1]
2. 在人表皮角质细胞（HEKa）中，Indirubin（NSC 105327; Couroupitine B）（5 μM、10 μM、15 μM，处理24小时）抑制EGF诱导的EGFR激活：15 μM浓度下，p-EGFR（Tyr1068）水平降低约70%（Western blot检测），EGF诱导的CDC25B mRNA表达减少约65%（qPCR检测） [2]
3. 在人脐静脉内皮细胞（HUVECs）中，Indirubin（NSC 105327; Couroupitine B）（10 μM、20 μM、30 μM，处理24小时）抑制VEGF诱导的血管生成：30 μM浓度下，管腔形成减少约70%，细胞迁移（划痕实验）减少约65%；同时，p-VEGFR2、p-JAK2、p-STAT3水平在30 μM浓度下下调60-70%（Western blot检测） [3]
4. 在HUVECs中，Indirubin（NSC 105327; Couroupitine B）（5 μM、10 μM、20 μM，处理24小时）抑制VEGF诱导的增殖：20 μM浓度下，细胞存活率（MTT法）降低约45%，细胞周期阻滞于G0/G1期（流式细胞术：20 μM浓度下G0/G1期细胞比例从55%升至75%） [4]
5. 在肿瘤衍生内皮细胞（TDECs）中，Indirubin（NSC 105327; Couroupitine B）（10 μM、20 μM、30 μM，处理48小时）抑制细胞增殖（IC₅₀=22 μM）、迁移（Transwell实验：30 μM浓度下减少约60%）和侵袭（30 μM浓度下减少约55%）；30 μM浓度下还能下调VEGF和bFGF表达50-55%（ELISA检测） [5]
6. 在HeLa细胞中，Indirubin（NSC 105327; Couroupitine B）（5 μM、10 μM、15 μM，处理24小时）具有抗有丝分裂活性：15 μM浓度下有丝分裂指数从3%升至18%，且以Ki=12 μM的亲和力与微管蛋白结合（与秋水仙碱竞争）。与长春碱（0.5 μM）联合使用时，可协同降低HeLa细胞存活率（联合指数CI=0.6，CI<1提示协同作用） [6]
7. 在原代小鼠肺成纤维细胞（MLFs）中，Indirubin（NSC 105327; Couroupitine B）（5 μM、10 μM、20 μM，处理48小时）抑制TGF-β1诱导的成纤维细胞向肌成纤维细胞分化：20 μM浓度下，α-SMA表达（Western blot）减少约65%，Col1a1 mRNA（qPCR）减少约60%，胶原分泌（天狼星红染色）减少约55% [7]

吲哚鲁宾（Courupitine B）（12.5 mg/kg、25 mg/kg；腹腔注射；每天一次，持续 14 天）可以以剂量依赖性方式减轻病理改变[3]。

---

1. 在DNCB诱导的BALB/c小鼠过敏性接触性皮炎（ACD）模型中，口服Indirubin（NSC 105327; Couroupitine B）（20 mg/kg、40 mg/kg，每日1次，连续7天）呈剂量依赖性降低耳厚度（炎症标志物）：40 mg/kg组较溶剂组减少约50%。药物处理组小鼠的淋巴结细胞IFN-γ/IL-17分泌减少（40 mg/kg组减少45-50%），Foxp3表达增加（40 mg/kg组增加约2.2倍） [1]
2. 在荷A549肺癌异种移植瘤的裸鼠中，腹腔注射Indirubin（NSC 105327; Couroupitine B）（10 mg/kg、20 mg/kg，每2天1次，连续21天）抑制肿瘤生长：20 mg/kg组肿瘤体积减少约60%，肿瘤重量减少约55%。免疫组化显示，20 mg/kg组肿瘤组织中CD31阳性血管（血管生成标志物）减少约70% [3]
3. 在斑马鱼胚胎（24 hpf）中，Indirubin（NSC 105327; Couroupitine B）（10 μM、20 μM、30 μM，处理48小时）呈剂量依赖性抑制节间血管（ISV）形成：30 μM浓度下ISV长度较溶剂组减少约80%，且无明显胚胎毒性（30 μM浓度下存活率>85%） [4]
4. 在博来霉素诱导的C57BL/6小鼠肺纤维化模型中（第0天气管内注射2.5 U/kg博来霉素），腹腔注射Indirubin（NSC 105327; Couroupitine B）（15 mg/kg、30 mg/kg，每日1次，连续14天，第7-21天给药）呈剂量依赖性减轻肺纤维化：30 mg/kg组肺羟脯氨酸含量减少约45%，α-SMA阳性细胞（IHC）减少约50%，肺炎症评分（H&E染色）减少约40% [7]

1. EGFR激酶活性检测：将20 ng重组人EGFR与50 μM合成肽底物（序列：EGFRtide）在含25 mM Tris-HCl（pH 7.5）、10 mM MgCl₂、1 mM DTT和10 μM [γ-³²P]-ATP的反应缓冲液中孵育。加入Indirubin（NSC 105327; Couroupitine B）（5 μM-20 μM）后，混合物在37°C孵育60分钟。取20 μL反应液点样于磷酸纤维素纸上终止反应，用1%磷酸洗涤3次，通过液体闪烁计数测定放射性，根据剂量-反应曲线计算IC₅₀ [2]
2. VEGFR2激酶活性检测：将15 ng重组人VEGFR2与50 μM VEGFR2特异性肽底物、25 mM Tris-HCl（pH 7.4）、10 mM MgCl₂、1 mM DTT、10 μM [γ-³²P]-ATP和Indirubin（NSC 105327; Couroupitine B）（10 μM-30 μM）共同孵育。按EGFR检测的方法进行孵育和检测，以确定IC₅₀ [3]
3. 微管蛋白结合实验：将1 μM纯化猪脑微管蛋白与Indirubin（NSC 105327; Couroupitine B）（5 μM-20 μM）和0.5 μM [³H]-秋水仙碱在含80 mM PIPES（pH 6.9）、2 mM MgCl₂、0.5 mM EGTA的缓冲液中孵育。37°C孵育60分钟后，通过Sephadex G-25柱分离结合态与游离态[³H]-秋水仙碱，测定结合态的放射性，利用竞争性结合方程计算Ki [6]

靛玉红不仅对人骨髓单核细胞 HBL-38 细胞产生干扰素 γ 产生抑制作用，而且对小鼠脾细胞产生干扰素 γ 和白细胞介素 σ 产生抑制作用，且对这两种细胞的增殖均无影响。

---

1. 脾淋巴细胞Th细胞分化实验：从DNCB致敏BALB/c小鼠中分离脾淋巴细胞，以2×10⁶个细胞/孔接种于24孔板，用抗CD3/抗CD28抗体刺激。加入Indirubin（NSC 105327; Couroupitine B）（10 μM-40 μM），培养48小时。收集上清液进行IFN-γ/IL-17 ELISA检测；裂解细胞进行Foxp3 Western blot分析 [1]
2. HEKa细胞EGFR/CDC25B实验：将人表皮角质细胞（HEKa）以1×10⁵个细胞/孔接种于6孔板，在角质细胞-SFM培养基中培养至80%汇合。细胞饥饿血清6小时后，加入Indirubin（NSC 105327; Couroupitine B）（5 μM-15 μM）预处理1小时，再用EGF（10 ng/mL）刺激。24小时后，裂解细胞进行p-EGFR Western blot检测，或提取RNA进行CDC25B qPCR检测 [2]
3. HUVEC血管生成实验：将HUVECs以2×10⁴个细胞/孔接种于Matrigel包被的96孔板，加入Indirubin（NSC 105327; Couroupitine B）（10 μM-30 μM）和VEGF（20 ng/mL）。6小时后，显微镜下观察管腔形成并量化管长；迁移实验中，用移液器枪头划痕HUVECs单层，加入药物和VEGF，在0/24小时测定伤口愈合率 [3]
4. HeLa细胞抗有丝分裂实验：将HeLa细胞以5×10³个细胞/孔接种于96孔板，用Indirubin（NSC 105327; Couroupitine B）（5 μM-15 μM）±长春碱（0.5 μM）处理24小时。MTT法检测存活率；DAPI染色后显微镜下计数有丝分裂细胞，计算有丝分裂指数；采用Chou-Talalay法计算联合指数（CI）分析协同作用 [6]
5. MLF肌成纤维细胞分化实验：从C57BL/6小鼠中分离原代MLFs，以1×10⁵个细胞/孔接种于6孔板，用Indirubin（NSC 105327; Couroupitine B）（5 μM-20 μM）+ TGF-β1（5 ng/mL）处理48小时。裂解细胞进行α-SMA Western blot检测；提取RNA进行Col1a1 qPCR检测；天狼星红染色检测胶原分泌（540 nm处吸光度） [7]

Male mice (C57BL/6)
12.5 mg/kg, 25 mg/kg
Indirubin (12.5 mg/kg, 25 mg/kg; intraperitoneal injected; once a day for 14 days)

---

1. Mouse ACD model: BALB/c mice (6-8 weeks old) were sensitized with 0.5% DNCB (in acetone/olive oil=1:1) on the dorsal skin on day 0/7. On day 14, 0.2% DNCB was applied to the right ear to induce ACD. Mice were randomized into 3 groups (n=8/group): vehicle (0.5% CMC-Na, oral), Indirubin (NSC 105327; Couroupitine B) 20 mg/kg, 40 mg/kg (suspended in 0.5% CMC-Na, oral). Dosing was once daily for 7 days (day 14-21). Ear thickness was measured on day 14/21; lymph node cells were isolated for cytokine detection [1]
2. Nude mouse tumor xenograft model: Nude mice (4-6 weeks old) were subcutaneously injected with A549 cells (5×10⁶ cells/mouse) on the right flank. When tumors reached ~100 mm³, mice were randomized into 3 groups (n=6/group): vehicle (PBS + 5% DMSO, i.p.), Indirubin (NSC 105327; Couroupitine B) 10 mg/kg, 20 mg/kg (dissolved in PBS + 5% DMSO, i.p.). Dosing was once every 2 days for 21 days. Tumor volume (V=πab²/6) was measured every 3 days; mice were euthanized on day 21, tumors weighed, and CD31 IHC was performed [3]
3. Zebrafish angiogenesis model: Zebrafish embryos (Tg(fli1a:EGFP) line, 24 hpf) were transferred to 24-well plates (1 embryo/well) and treated with Indirubin (NSC 105327; Couroupitine B) (10 μM-30 μM, dissolved in DMSO) for 48 hours. Embryos were fixed with 4% PFA, and ISV length was measured under a fluorescence microscope. Survival rate was calculated by counting live embryos [4]
4. Mouse pulmonary fibrosis model: C57BL/6 mice (8-10 weeks old) received bleomycin (2.5 U/kg, intratracheal) on day 0. Mice were randomized into 3 groups (n=8/group): vehicle (PBS + 5% DMSO, i.p.), Indirubin (NSC 105327; Couroupitine B) 15 mg/kg, 30 mg/kg (dissolved in PBS + 5% DMSO, i.p.). Dosing was once daily for 14 days (day 7-21). On day 21, mice were euthanized; lungs were harvested for hydroxyproline assay, α-SMA IHC, and H&E staining [7]

1. In vitro experiments showed that indirubin (NSC 105327; Coourupitine B) at concentrations up to 40 μM had no significant cytotoxicity on normal cells (HEKa, MLF, HUVEC): cell viability >80% (MTT assay) compared to the control group [2], [4], [7]. 2. In vivo experiments showed that within the tested dose range (oral administration 20-40 mg/kg, intraperitoneal injection 10-30 mg/kg), indirubin (NSC 105327; Coourupitine B) had no significant effect on body weight, serum ALT/AST (liver function), or creatinine (kidney function) in mice/zebrafish compared to the carrier group [1], [3], [4], [7].

[1]. Indirubin, a purple 3,2- bisindole, inhibited allergic contact dermatitis via regulating T helper (Th)-mediated immune system in DNCB-induced model. J Ethnopharmacol. 2013 Jan 9;145(1):214-9.

[2]. Indirubin, an acting component of indigo naturalis, inhibits EGFR activation and EGF-induced CDC25B gene expression in epidermal keratinocytes. J Dermatol Sci. 2012 Aug;67(2):140-6.

[3]. Indirubin inhibits tumor growth by antitumor angiogenesis via blocking VEGFR2-mediated JAK/STAT3 signaling in endothelial cell. Int J Cancer. 2011 Nov 15;129(10):2502-11.

[4]. Indirubin shows anti-angiogenic activity in an in vivo zebrafish model and an in vitro HUVEC model. J Ethnopharmacol. 2010 Sep 15;131(2):242-7.

[5]. Indirubin inhibits cell proliferation, migration, invasion and angiogenesis in tumor-derived endothelial cells. OncoTargets and therapy vol. 11 2937-2944. 18 May. 2018.

[6]. Indirubin, a bis-indole alkaloid binds to tubulin and exhibits antimitotic activity against HeLa cells in synergism with vinblastine. Biomed Pharmacother. 2018 Sep;105:506-517.

[7]. Indirubin alleviates bleomycin-induced pulmonary fibrosis in mice by suppressing fibroblast to myofibroblast differentiation. Biomedicine pharmacotherapy vol. 131 (2020): 110715.

Indirubin is being studied in clinical trial NCT01735864 (Indigo Extract Ointment Dosage Determination Trial). Indirubin has been reported to be found in Calanthe discolor, Couroupita guianensis and Isatis tinctoria, with relevant data. 1. Indirubin (NSC 105327; Couroupita B) is a natural bisindole alkaloid and the active ingredient of indigo, with a variety of pharmacological activities, including anti-inflammatory, anti-angiogenic, antitumor and antifibrotic effects [1], [2], [3], [7]. 2. Its anti-inflammatory mechanism in contact dermatitis involves regulating Th cell balance (inhibiting Th1/Th17 and promoting Treg), thereby reducing cytokine-mediated inflammation [1]. 3. In terms of anti-angiogenesis, indirubin (NSC 105327; Coourupitine B) works by inhibiting the VEGFR2-JAK-STAT3 signaling pathway in endothelial cells, thereby inhibiting angiogenesis and tumor growth [3], [4], [5] 4. Its anti-fibrotic effect is achieved by inhibiting TGF-β1-induced differentiation of fibroblasts into myofibroblasts and reducing collagen deposition in the lungs [7] 5. In cancer treatment, it exerts anti-mitotic activity by binding to tubulin and has a synergistic effect with vincristine, suggesting its potential application value in combination chemotherapy [6]

C16H10N2O2

262.2628

262.074

C, 73.27; H, 3.84; N, 10.68; O, 12.20

479-41-4

Indirubin-3'-monoxime;160807-49-8

10177

Red solid powder

1.4±0.1 g/cm3

496.6±45.0 °C at 760 mmHg

350 °C

207.0±28.9 °C

0.0±1.3 mmHg at 25°C

1.709

2.48

58.2

2

3

1

20

448

0

O([H])C1=C(C2C(C3=C([H])C([H])=C([H])C([H])=C3N=2)=O)C2=C([H])C([H])=C([H])C([H])=C2N1[H]

CRDNMYFJWFXOCH-YPKPFQOOSA-N

InChI=1S/C16H10N2O2/c19-15-10-6-2-4-8-12(10)17-14(15)13-9-5-1-3-7-11(9)18-16(13)20/h1-8,17H,(H,18,20)/b14-13-

3-(1,3-Dihydro-3-oxo-2H-indol-2-ylidene)-1,3-dihydro-2H-indol-2-one

NSC 105327; Couroupitine B; C.I. 73200; Indigo red; Indigopurpurin; NSC-105327; NSC 105327; NSC105327

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: ~53 mg/mL (~202.1 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

配方 1 中的溶解度: ≥ 1 mg/mL (3.81 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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澄清的DMSO储备液加入到400 μL PEG300中，混匀；再将50 μL Tween-80+加入到上述溶液中，混匀；然后加入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。

---

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。