- NLRP3 inflammasome: Meisoindigo inhibits NLRP3 inflammasome activation. [2]
- TLR4/NF-κB signaling pathway: Meisoindigo suppresses the TLR4/NF-κB signaling pathway. [2]

- Cell Growth Inhibition (Leukemia): Meisoindigo inhibited the growth of HL-60, NB4, and U937 AML cell lines in a dose- and time-dependent manner. The 24-hour IC50 values were approximately between 5 and 10 μM for these cell lines. [1]
- Apoptosis Induction (Leukemia): Treatment with 10 μM meisoindigo for 24 hours markedly increased the percentage of apoptotic cells in HL-60, NB4, and U937 cells, as shown by Annexin V/PI staining. The apoptosis was partially blocked by the pan-caspase inhibitor z-VAD-fmk (50 μM) and the caspase-3 inhibitor z-DEVD-fmk (20 μM), suggesting both caspase-dependent and -independent pathways. Meisoindigo increased cleaved caspase-3 and pro-apoptotic Bak, and decreased anti-apoptotic Bcl-2 and Bcl-xL levels in HL-60 cells. No change in CD95 (Fas) expression was observed, indicating apoptosis is likely mediated via the intrinsic mitochondrial pathway. [1]
- Cell Cycle Arrest (Leukemia): Meisoindigo treatment (5, 10 μM for 24 hours) caused cell-cycle arrest in HL-60, NB4, and U937 cells, with more cells in sub-G1 and G0/G1 phases and fewer cells in the S phase. This was accompanied by increased levels of p21 and p27 proteins. No significant changes in phospho-cdc2 (Tyr 15) were detected. [1]
- Myeloid Differentiation (Leukemia): Meisoindigo (5 μM for 48 hours) induced myeloid differentiation in HL-60 and NB4 cells, as shown by morphologic changes (cytoplasmic vacuolation, nuclear condensation/indentation), increased percentage of CD11b+ cells, and increased nitroblue tetrazolium (NBT) reduction activity. [1]
- hTERT Downregulation (Leukemia): Treatment with 5 μM meisoindigo for 24 hours significantly down-regulated hTERT promoter activity and hTERT mRNA levels in HL-60 and NB4 cells. Treatment for 3 days markedly decreased hTERT protein levels. [1]
- Chemotherapeutic Agent Synergy (Leukemia): Meisoindigo additively enhanced the cytotoxicity of cytarabine and idarubicin against HL-60 and NB4 cells, with a combination index (CI) value of approximately 1, indicating additivity. [1]
- Primary AML Cell Activity: Meisoindigo (5, 10 μM for 2 days) inhibited the growth of primary AML cells from eight patients. In samples from two patients, 5 μM meisoindigo for 24 hours induced marked apoptosis, increased p21, p27, and cleaved caspase-3 levels, and down-regulated Bcl-2 expression. No change in CD95 expression was observed. [1]
- Neuroprotection (OGD/R): In HT-22 (hippocampal neurons) and BV2 (microglia) cells, meisoindigo treatment (30 μM for BV2, 50 μM for HT-22) significantly improved cell viability following oxygen-glucose deprivation/reperfusion (OGD/R). Meisoindigo significantly decreased the expression of NLRP3, ASC, CL-caspase-1, and IL-1β in both cell lines after OGD/R. It also downregulated the M1 marker iNOS and upregulated the M2 marker Arg-1. Meisoindigo blocked the activation of the TLR4/NF-κB signaling pathway induced by OGD/R and LPS stimulation, suppressing TLR4, p-NF-κB p65, and downstream NLRP3 inflammasome proteins. This was accompanied by a shift from M1 to M2 microglia phenotype in BV2 cells. [2]

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Meisoindigo (Dian III; 5–20 M; for 24 hours) prevents AML cell lines from proliferating[1].
Meisoindigo (10 μM; for 24 hours) induces apoptosis of acute myeloid leukemia[1].
Meisoindigo (5-10 μM; for 24 hours) causes cell-cycle arrest[1].
Meisoindigo (5-10 μM; for 24 hours) elevates pro-apoptotic Bak and cleaved caspase-3 while lowering Bcl-2 and Bcl-xL levels in HL-60 cells[1].
Meisoindigo (10, 30, 50, 100, 150 μM; 24 hours) blocks NLRP3 inflammasome activation and M1/M2 polarization brought on by LPS (1 μg/mL) in HT-22 and BV2 cells by down-regulating TLR4 pathways after OGD/R[2].

- Anti-leukemic Activity (NOD/SCID Mice): In NOD/SCID mice injected intraperitoneally with HL-60 cells, daily intraperitoneal administration of meisoindigo (50, 100, or 150 mg/kg for 14 days, starting 7 days after cell injection) decreased spleen size in a dose-dependent manner, indicating moderate in vivo anti-AML activity. [1]
- Neuroprotection (MCAO Mice): In adult male C57BL/6J mice subjected to middle cerebral artery occlusion (MCAO) and reperfusion, post-treatment with meisoindigo (4, 8, 12 mg/kg i.p., given before MCAO and 2h after reperfusion) significantly reduced infarct volume, ameliorated neurological deficits, and reduced brain water content (edema) and AQP4 expression at 3 days post-stroke. The 8 mg/kg dose had the greatest protective effect. Meisoindigo reduced the number of NLRP3+ cells, MPO+ neutrophils, and iNOS+ M1 microglia/macrophages, while increasing YM1/2+ M2 microglia/macrophages in the ischemic penumbra. It suppressed the expression of NLRP3, ASC, CL-caspase-1, IL-18, TLR4, p-NF-κB p65, HMGB1, and IL-1β. The effects were similar to those of the specific NLRP3 inhibitor MCC950 (50 mg/kg) and the TLR4 inhibitor TAK-242 (3 mg/kg). [2]

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Meisoindigo (Dian III; 50–150 mg/kg; IP; daily; for 14 days) has anti-leukemic activity in vivo[1].
Meisoindigo significantly reduces infarct volume and improves neurological deficits 3 days after middle cerebral artery occlusion (MCAO) in Wild-type C57BL/6J mice (25-30 g) when administered intravenously (2, 4, 8, 12 mg/kg) prior to MCAO and 2 hours after reperfusion. Meisoindigo decreases brain edema and AQP4 expression[2].

- Cell Viability (MTT): AML cell lines (HL-60, NB4, U937) were plated in 96-well plates and treated with indicated concentrations of meisoindigo. At various time points, MTT was added, and after 3 hours of incubation, the formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. For primary AML cells, 10% 5637 bladder cancer cell-conditioned medium was included. [1]
- Cell Viability (CCK-8 for OGD/R): HT-22 and BV2 cells were seeded in 96-well plates and treated with different concentrations of meisoindigo during OGD. After OGD/R, CCK-8 solution was added, and absorbance was measured at 450 nm. [2]
- Apoptosis Assay (Annexin V/PI Flow Cytometry): Cells were treated with meisoindigo, then labeled with Annexin V-FITC and propidium iodide (5 μg/mL) for 30 minutes in the dark, followed by flow cytometric analysis. Caspase inhibitors (z-VAD-fmk 50 μM, z-DEVD-fmk 20 μM) were added to some groups. [1]
- Cell Cycle Analysis: Cells were treated with meisoindigo, then prepared using a hypotonic method. Data were acquired by flow cytometry and analyzed using ModFit LT software. [1]
- Myeloid Differentiation Assays: Differentiation was assessed by staining cells with CD11b antibody for flow cytometry, measuring NBT reduction activity (cells were treated with phorbol myristate acetate and NBT, and formazan deposits were counted), and examining Giemsa-Wright stained cytospin slides for morphological changes. [1]
- hTERT Promoter Activity Assay: HL-60 and NB4 cells were transfected with a luciferase reporter containing the hTERT promoter and a Renilla luciferase control plasmid. After meisoindigo treatment, luciferase activity was measured. [1]
- Real-Time RT-PCR: Total RNA was extracted, reverse transcribed to cDNA, and quantitative PCR was performed using SYBR Green to measure mRNA levels of hTERT, M1 markers (iNOS, CD32, CD16), M2 markers (CD206, YM1/2, Arg-1), and inflammatory cytokines (TNF-α, IL-1β). [1][2]
- Western Blot Analysis: Cells were lysed, and protein samples were separated by SDS-PAGE, transferred to PVDF membranes, and probed with specific primary antibodies (e.g., cleaved caspase-3, Bcl-2, Bak, p21, p27, NLRP3, ASC, CL-caspase-1, IL-1β, TLR4, p-NF-κB p65, iNOS, Arg-1). Protein bands were detected using chemiluminescence. [1][2]
- Immunofluorescence (in vitro): For in vitro OGD/R experiments, cells were stained with specific antibodies to assess protein expression. [2]

- Anti-leukemic Activity (NOD/SCID Mice): 6-8 week old NOD/SCID mice were injected intraperitoneally with 1 × 10⁶ HL-60 cells. Seven days after injection, meisoindigo (dissolved in DMSO then diluted in sterile saline) was administered once daily by intraperitoneal injection at doses of 50, 100, or 150 mg/kg body weight for 14 days. The vehicle control group received the solvent alone. Mice were sacrificed, and spleens were harvested and weighed. [1]
- Neuroprotection (MCAO Mice): Adult male C57BL/6J mice (25-30 g) were subjected to 1 hour of MCAO followed by reperfusion. Meisoindigo (dissolved in DMSO and diluted with sterile saline) was administered intraperitoneally before MCAO and 2 hours after reperfusion at doses of 2, 4, 8, and 12 mg/kg. MCC950 (50 mg/kg, a specific NLRP3 inhibitor) was administered i.p. 1 and 3 hours after occlusion. TAK-242 (3 mg/kg, a specific TLR4 inhibitor) was injected i.p. 1 hour after occlusion. Neurological deficit scores were evaluated 3 days after MCAO. Mice were then euthanized, and brains were collected for infarct volume measurement (TTC staining), brain water content (wet/dry method), immunofluorescence, and Western blot analysis. [2]

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HL-60, NB4, U937 leukemic cell lines
5, 10, 15, 20 μM
IP; daily; for 14 days

- General: Meisoindigo is a second-generation derivative of indirubin with general water solubility and is well-tolerated. It does not have the toxicity of indirubin. [2]
- Clinical Use: Meisoindigo has been used clinically in China for treating chronic myeloid leukemia (CML). [1][2]

[1]. Meisoindigo is a promising agent with in vitro and in vivo activity against human acute myeloid leukemia. Leuk Lymphoma. 2010 May;51(5):897-905.

[2]. Meisoindigo Protects Against Focal Cerebral Ischemia-Reperfusion Injury by Inhibiting NLRP3 Inflammasome Activation and Regulating Microglia/Macrophage Polarization via TLR4/NF-κB Signaling Pathway. Front Cell Neurosci. 2019 Dec 16;13:553.

- Background: Meisoindigo is a 3,3'-linked bisindole and a second-generation derivative of indirubin, an active ingredient of the traditional Chinese medicine Dangui Luhui Wan (which contains Qing Dai). It has been used in China for chronic myeloid leukemia (CML). The parent compound, indirubin, has poor water solubility and severe gastrointestinal side effects, which meisoindigo overcomes. [1][2]
- Mechanism in AML: Meisoindigo induces apoptosis in AML cells primarily through the intrinsic mitochondrial pathway (down-regulating Bcl-2/Bcl-xL, up-regulating Bak/Bax, activating caspase-3) and induces myeloid differentiation (up-regulating CD11b, increasing NBT reduction). It also down-regulates hTERT expression. It additively enhances the cytotoxicity of cytarabine and idarubicin. [1]
- Mechanism in Ischemic Stroke: Meisoindigo protects against cerebral ischemia-reperfusion injury by inhibiting the TLR4/NF-κB signaling pathway, which in turn suppresses NLRP3 inflammasome activation. This leads to a shift of microglia/macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, reducing neuroinflammation. The study also found that after stroke, NLRP3 is expressed more in neurons than in microglia/macrophages. [2]

C18H14N2O2

290.31

276.089

C, 74.47; H, 4.86; N, 9.65; O, 11.02

97207-47-1

97207-47-1

126274

Red solid powder

1.4±0.1 g/cm3

482.0±45.0 °C at 760 mmHg

236-237ºC

245.3±28.7 °C

0.0±1.2 mmHg at 25°C

1.698

1.53

1

2

1

21

614

0

O=C1C(=C2C3C(=CC=CC=3)N(C)C2=O)C2C(=CC=CC=2)N1

XZYXCQXTKOYHGK-UHFFFAOYSA-N

InChI=1S/C17H12N2O2/c1-19-13-9-5-3-7-11(13)15(17(19)21)14-10-6-2-4-8-12(10)18-16(14)20/h2-9,21H,1H3

3-(2-hydroxy-1-methylindol-3-yl)indol-2-one

Meisoindigo; N-Methylisoindigotin; Methyl isoindigotin

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: ~55 mg/mL (~199.1 mM)
Ethanol: ~1 mg/mL (~3.6 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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