Seselin targeted Janus kinase 2 (Jak2) to block its activity and downstream signal transducer and activator of transcription 1 (STAT1) signaling pathway, thereby suppressing the proinflammatory phenotype of macrophages [3].

P-388 and HT-29 cells are hazardous to selenium, with ED50 values of 8.66 and 9.94 μg/mL, respectively [1]. The cytokine output of LPS- and IFN-γ-stimulated macrophages is inhibited by selvin (5–20 μM; 0.5–24 h) [3]. The expression of pro-inflammatory macrophage markers (iNOS, phagocytosis, and CD11c) in BMDMs is inhibited by selvin (5–20 μM; 12 h) [3]. The STAT1 signaling pathway is blocked by selvin (5–20 μM; 0.5–6 h) [3].

---

Seselin (20 μM) suppressed the mRNA levels of proinflammatory cytokines (IL-1β, IL-6, TNF-α, IL-23) and chemokines (Ccl3, Ccl7, Cxcl9, Cxcl11) in bone marrow-derived macrophages (BMDMs) stimulated with LPS and IFN-γ in a concentration-dependent manner. [3]
Seselin (10, 20, 40 μM) reduced secreted IL-6 and TNF-α protein levels in BMDM supernatants stimulated with LPS and IFN-γ. [3]
Seselin (10, 20, 40 μM) markedly suppressed IL-1β and TNF-α secretion in BMDMs stimulated with LPS followed by ATP. [3]
Seselin (10 μM) suppressed the mRNA levels of IL-1β, IL-6, and TNF-α in BMDMs stimulated with LPS and IFN-γ in a time-dependent manner (peaking at 3-6 h). [3]
Seselin (10, 20, 40 μM) significantly down-regulated iNOS expression, reduced phagocytosis (uptake of fluorescent red latex beads), and decreased the percentage of CD11c-positive cells in BMDMs stimulated with LPS and IFN-γ. [3]
Seselin (10, 20, 40 μM) suppressed the phosphorylation levels of STAT1 (p-STAT1) and p65 (p-p65) in BMDMs stimulated with LPS and IFN-γ in both concentration- and time-dependent manners, as shown by Western blot. [3]
Seselin (10 μM) inhibited the nuclear accumulation of STAT1 and p65 in RAW 264.7 cells stimulated with LPS and IFN-γ, as shown by immunofluorescence assay. [3]
Seselin (10, 20, 40 μM) reduced the levels of p-p65 and cleaved caspase-1 (p10 and p20) in BMDMs stimulated with LPS and ATP. [3]
Seselin (20 μM) decreased the interaction between Jak2 and IFN-γR, as well as the interaction between Jak2 and STAT1, as determined by co-immunoprecipitation (co-IP). [3]
Seselin enhanced the thermal stability of Jak2 protein in RAW 264.7 cells at higher temperatures in a concentration-dependent manner, as shown by cellular thermal shift assay (CETSA). [3]
Seselin showed very little cytotoxicity on normal, unstimulated BMDMs or those stimulated with LPS and IFN-γ or IL-4, even at a high concentration (80 μM), as measured by MTT assay. [3]
Seselin did not affect the proliferation of RAW 264.7 cells stimulated with LPS and IFN-γ or IL-4, as assessed by CFSE staining. [3]
Seselin (20 μM) slightly inhibited the expression of CD14 (a marker of THP-1 differentiation) in THP-1 cells stimulated with PMA. [3]
Seselin (10, 20, 40 μM) significantly suppressed the mRNA levels of proinflammatory cytokines (IL-6 and TNF-α) in THP-1 derived macrophages. [3]

In mice, selenium (0.5–40.5 mg/kg; sc; once) has antinociceptive and peripheral anti-inflammatory effects [2]. Mice with sepsis brought on by cecal ligation and puncture respond better to sevelin (3–30 mg/kg; ig; once) [3].

---

Seselin (3, 10, 30 mg/kg, intragastrically) administered 2 h before cecal ligation and puncture (CLP) surgery significantly improved the survival rate of septic mice. [3]
Seselin (10, 30 mg/kg) suppressed systemic levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in the serum of CLP-induced septic mice. [3]
Seselin (10, 30 mg/kg) significantly decreased proinflammatory cytokines (IL-1β, IL-6, TNF-α) and total infiltrated cell numbers in bronchoalveolar lavage fluid (BALF) of CLP mice. [3]
Seselin (10, 30 mg/kg) decreased the mRNA levels of IL-1β, IL-6, and TNF-α in lung tissue of CLP mice. [3]
Seselin (30 mg/kg) clearly ameliorated lung damage (leukocyte infiltration, interstitial and intraalveolar oedema) induced by CLP, as shown by H&E staining. [3]
Seselin (10, 30 mg/kg) significantly mitigated infiltration of immune cells (CD11b-positive) and decreased CD11c expression (marker of proinflammatory macrophages) in lung tissue of CLP mice, as shown by immunofluorescence and immunohistochemistry. [3]
Seselin (30 mg/kg) inhibited the levels of p-Jak2, p-STAT1, and p-p65 in lung tissue of CLP mice. [3]
Seselin (30 mg/kg) significantly down-regulated the proportion and total number of F4/80+CD11c+ alveolar macrophages in BALF of CLP mice. [3]
Seselin (30 mg/kg) significantly decreased the proportion of CD11b+Ly6C+ monocytes in BALF and lung tissue, but only partially reduced CD11b+Ly6G+ neutrophils. [3]
Seselin (20 μM) pretreated proinflammatory phenotypic BMDMs (stimulated with LPS and IFN-γ) were adoptively transferred to macrophage-depleted mice. These mice showed improved survival rate and reduced serum levels of IL-6 and TNF-α after LPS challenge (10 mg/kg, i.p.). [3]
Seselin (0.5, 4.5, 40.5 mg/kg, s.c.) injected 30 min prior to acetic acid inhibited the mice writhing response in a dose-dependent manner by 19.5%, 26.2%, and 41.4%, respectively. [2]
Seselin (4.5, 40.5 mg/kg, s.c.) injected 30 min prior to formalin reduced the licking time during the first phase (neurogenic) by 34.4% and 66.9%, respectively. [2]
Seselin (0.5, 4.5, 40.5 mg/kg, s.c.) injected 30 min prior to formalin elicited a significant inhibition of formalin response during the second phase (inflammatory) by 90.3%, 97.8%, and 95.3%, respectively. [2]
Seselin (40.5 mg/kg, s.c.) produced no significant antinociceptive effect in the hot plate test (reaction time) during 90 min of observation. [2]

RT-PCR[3]
Cell Types: Bone marrow-derived macrophages (BMDMs)
Tested Concentrations: 5, 10 and 20 μM
Incubation Duration: 6 h
Experimental Results: decreased the mRNA for cytokines (IL-1β, IL-6, Tnf-α and IL-23) and chemokines (Ccl3, Ccl7, Cxcl9 and Cxcl11) concentration-dependently in BMDMs.

---

Western Blot Analysis[3]
Cell Types: BMDMs
Tested Concentrations: 5, 10 and 20 μM
Incubation Duration: 0.5, 1.5, 3 and 6 h
Experimental Results: Suppressed expression of p-STAT1 and p-p65 both concentration and time dependently.

---

Seselin cytotoxicity and macrophage phenotype assay: Bone marrow-derived macrophages (BMDMs) were established by flushing femurs from mice with PBS. Harvested cells were grown in RPMI 1640 containing 10% FBS and 10 ng/mL macrophage-colony stimulating factor (M-CSF) for 7 days. Adherent macrophages were stimulated with 10 ng/mL LPS and 10 ng/mL IFN-γ or 20 ng/mL IL-4. MTT assay: Cells were seeded in 96-well plates and incubated with various concentrations of Seselin, with or without LPS and IFN-γ or IL-4 for 24 h. MTT (20 μL of 4 mg/mL) was added and incubated for 4 h. The plate was centrifuged, supernatant discarded, DMSO (200 μL) added, and absorbance at 570 nm measured. [3]
Seselin cell proliferation assay (CFSE): RAW 264.7 cells were incubated with 2 μM CFSE solution for 30 min at 37°C. Labelled cells were incubated with various doses of Seselin in the presence of LPS and IFN-γ or IL-4 for 24 h. Cell proliferation was assessed by flow cytometry. [3]
Seselin quantitative PCR: Total RNA was extracted from cells or tissues and reverse transcribed to cDNA. Quantitative PCR was performed with a detection system. The primer sequences for mouse and human genes (e.g., IL-1β, IL-6, TNF-α, IL-23, Ccl3, Ccl7, Cxcl11, Fizz1, Ym1, Arg-1, Ccl22, Cdl163, β-actin) were used. [3]
Seselin Western blot: Collected cells were lysed in lysis buffer, separated by 10% SDS-PAGE, and transferred onto PVDF membranes. Membranes were blocked with 3% BSA, probed with primary antibodies (e.g., p-STAT1, STAT1, p-p65, p65, p-Jak2, Jak2, TLR4, p-IKKa/β, IKK, caspase1, IFNγR1, tubulin, actin) overnight at 4°C, then incubated with HRP-coupled secondary antibody. Protein bands were visualized. [3]
Seselin flow cytometry for activated BMDMs: Cultured cells were harvested, washed with cold PBS, and stained with specific antibodies (e.g., PE-anti-iNOS, PE Rat IgG2a isotype control) for 30 min at 4°C in the dark and analysed. Phagocytosis assays were conducted using fluorescent red latex beads (1 μm diameter). Opsonized beads were added to Seselin-treated cells at a ratio of 10:1 (beads:cells) and incubated at 37°C for 2 h. Cells were harvested, washed, and analysed by flow cytometry. [3]
Seselin confocal microscopy: Cells were washed, fixed in Lyse/fix buffer for 30 min, permeabilized with 0.5% Triton X-100, and blocked with 3% BSA. Cells were stained with anti-STAT1 or anti-p65 overnight at 4°C and then with specific fluorescent-coupled secondary antibody. Coverslips were counterstained with DAPI and imaged. [3]
Seselin co-immunoprecipitation (co-IP): RAW 264.7 cells were pretreated with Seselin in the presence of LPS and IFN-γ. Cell lysates were incubated with 2 μg appropriate antibody at 4°C overnight and precipitated with protein A/G-agarose beads for 4 h at 4°C. Beads were washed, and immunoprecipitated proteins were analysed by Western blot. [3]
Seselin cellular thermal shift assay (CETSA): Cultured cells were incubated with 20 μM Seselin or DMSO for 2 h. Harvested cells were equally divided and lysed using three cycles of freeze-thawing with liquid nitrogen, then heated at various temperatures with a PCR instrument, centrifuged to separate soluble fractions from precipitates. Supernatants were analysed by Western blot. [3]
Seselin molecular docking: Molecular docking analysis was used to investigate possible binding mode of Seselin with the crystal structures of Jak2 (31-516aa) (PDB ID: 4Z32) and Jak2 (840-1132aa) (PDB ID: 3UGC). [3]
Seselin THP-1 differentiation: THP-1 cells were stimulated with 500 mM PMA for 24 hours in the presence of various doses of Seselin. CD14 expression was detected by flow cytometry. [3]

Animal/Disease Models: Male Swiss mice[2]
Doses: 0.5, 4.5 or 40.5 mg/kg
Route of Administration: subcutaneous (sc)injection; once
Experimental Results: Inhibited the writhing response induced by acetic acid in a significant and dose-dependent manner, by 19.5%, 26.2% and 41.4% at dose of 0.5, 4.5 or 40.5 mg/kg, respectively. Elicited a significant inhibition of formalin response during the second phase (inflammatory), by 90.3%, 97.8% and 95.3%, respectively.

---

Animal/Disease Models: C57BL/6 mice, caecal ligation and puncture (CLP) induced sepsis model[3]
Doses: 3, 10 and 30 mg/kg
Route of Administration: intragastric (po)administration, once
Experimental Results: Ameliorated lung injury and diminished JAK2 phosphorylation level in lung tissue during sepsis. diminished the immune cell counts in BALF induced by CLP.

---

Seselin in CLP-induced sepsis model: Female C57BL/6 mice (6-8 weeks old, 18-22 g) were used. Seselin was dissolved in olive oil and administered intragastrically at doses of 3, 10, or 30 mg/kg (100 μL per 20 g body weight) 2 h prior to CLP surgery. CLP surgery: Mice were anaesthetized with i.p. injection of 2% sodium pentobarbital (150 μL per 20 g). The caecum was ligated tightly with a 3-0 silk suture at the middle and punctured through-and-through twice with a 20-gauge needle. Prewarmed (37°C) normal saline (1 mL) was injected immediately after the operation. For survival studies, mice were given Seselin or vehicle 2 h before CLP, and survival was recorded for 60 h. For sample collection, blood and lung tissues were collected 4 h after CLP. [3]
Seselin in LPS-induced sepsis model with macrophage depletion and adoptive transfer: Systemic macrophages were depleted by i.p. administration of clodronate liposome (100 μL per 20 g). BMDMs were polarized to proinflammatory phenotype with 10 ng/mL LPS and 10 ng/mL IFN-γ and treated with 20 μM Seselin or DMSO for 6 h. Pretreated cells (1×10^6 cells per 20 g) were transferred i.v. to macrophage-depleted mice 2 h before LPS challenge. Mice were injected with LPS (i.p., 10 mg/kg) and survival was monitored. Blood samples were collected 4 h after LPS challenge. [3]
Seselin in acetic acid-induced writhing test: Male Swiss mice (25-30 g) were pretreated s.c. with Seselin (0.5, 4.5, or 40.5 mg/kg), morphine (5 mg/kg), or indomethacin (5 mg/kg). After 30 min, 0.6% (v/v) solution of acetic acid was injected i.p. (10 mL/kg). The number of writhes occurring between 0 and 30 min after acetic acid injection was recorded. [2]
Seselin in formalin test: Mice were pretreated s.c. with Seselin (0.5, 4.5, or 40.5 mg/kg), morphine (5 mg/kg), or indomethacin (5 mg/kg). After 30 min, 1% aqueous formalin (20 μL) was administered by intraplantar route into the right hind paw. The time spent licking the injected paw was measured during the first 5 min (Phase 1, neurogenic) and 15-30 min after formalin injection (Phase 2, inflammatory). [2]
Seselin in hot plate test: Mice were dropped twice on a heated plate (51 ± 1°C), separated by a 30 min interval. Groups of six mice each received s.c. saline, Seselin (40.5 mg/kg), morphine (5 mg/kg), or indomethacin (2 mg/kg) in a volume of 10 mL/kg. Reaction times (licking paw or jumping) were measured at time zero (0 time) and 30, 60, and 90 min after compound administration, with a cut-off time of 40 s. [2]

Seselin showed very little cytotoxicity on normal, unstimulated BMDMs or those stimulated with LPS and IFN-γ or IL-4, even at a high concentration (80 μM), as measured by MTT assay. [3]
Seselin did not affect the proliferation of RAW 264.7 cells stimulated with LPS and IFN-γ or IL-4, as assessed by CFSE staining. [3]

[1]. Cytotoxic and anti-platelet aggregation constituents from the root wood of Melicope semecarpifolia. Planta Med. 2005 Nov;71(11):1078-81.

[2]. Antinociceptive activity of the pyranocoumarin seselin in mice. Fitoterapia. 2006 Dec;77(7-8):574-8.

[3]. Seselin ameliorates inflammation via targeting Jak2 to suppress the proinflammatory phenotype of macrophages. Br J Pharmacol. 2019 Jan;176(2):317-333.

Seselin is a coumarin compound and a metabolite. It has been reported to exist in angelica, banyan trees, and other organisms with relevant data.

---

Seselin is an angular pyranocoumarin isolated from some Rutaceae spp. (e.g., Sigmatanthus trifoliatus Huber ex Emmerich) and has been reported to have antifungal, antibacterial, anti-inflammatory, and antinociceptive properties. [2][3]
Seselin has been suggested to possess anti-inflammatory activity in the carrageenin-induced inflammation assay in rats. [2]
Seselin inhibits indole acetic acid oxidase and peroxidase enzyme systems in plants. [3]
Seselin is effective against blowfly larvae and selectively activates some yeast strains. [3]
The anti-inflammatory activity of Seselin is mediated by targeting Jak2 to block the proinflammatory phenotype of macrophages, suggesting its possible application in treating inflammatory diseases such as sepsis. [3]

C14H12O3

228.24

228.079

523-59-1

68229

White to off-white solid powder

1.222g/cm3

403ºC at 760 mmHg

119 - 120 °C

170.5ºC

1.583

2.977

0

3

0

17

394

0

QUVCQYQEIOLHFZ-UHFFFAOYSA-N

InChI=1S/C14H12O3/c1-14(2)8-7-10-11(17-14)5-3-9-4-6-12(15)16-13(9)10/h3-8H,1-2H3

8,8-dimethylpyrano[2,3-f]chromen-2-one

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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