RARγ/retinoic acid receptor-γ

Dectin-1 and pro-inflammatory cytokines are not expressed when BMS961 is present [1].The expression of RARγ was upregulated after stimulation with A. fumigatus. RARγ mRNA began to rise at 4h and peaked at 8h (P<0.001). The protein of RARγ reached to the peak at 16h (P<0.001). Pretreated with BMS961 before A. fumigatus hyphae stimulation, expression of Dectin-1, TNF-α and IL-6 decreased dramatically at mRNA and protein levels. Conclusion: HCECs can express RARγ and A. fumigatus hyphae infection can increase RARγ expression. BMS961 can inhibit the expression of Dectin-1 and pro-inflammatory cytokines, and play an anti-inflammatory role in innate immune responses against A. fumigatus.

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BMS961 Modulates the Dectin-1 Expression Induced by Aspergillus fumigatus in Human Corneal Epithelial Cells [1]
In order to know whether Dectin-1 expression are modulated in response to the activation of RARγ. HCECs were pretreated with or without BMS961 (1 µg/mL) for 30min, before incubated with hyphae for 8h. The mRNA and protein levels of Dectin-1 were measured by qRT-PCR and Western blot respectively. The mRNA expression of Dectin-1 were increased in HCECs after hyphae stimulation for 8h, but the levels were down-regulated by BMS961 pretreatment (P<0.001; Figure 2A). Similarly, compared with HCECs exposed to hyphae, the protein levels of Dectin-1 were also inhibited by BMS961 pretreatment (P<0.001; Figure 2B).

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BMS961 Pretreatment Inhibited Pro-inflammatory Cytokines Production Induced by Aspergillus fumigatus [1]
To investigate whether RARγ can modulate the innate immune response to A. fumigatus in HCECs, HCECs were pretreated with BMS961 (1 µg/mL) for 30min, followed by hyphae for 8h or 16h. The mRNA and protein levels of TNF-α and IL-6 were measured by qRT-PCR and ELISA, respectively. Figure 3 showed that the expressions of TNF-α and IL-6 increased at 8h post-infection with A. fumigatus in HCECs compared with the control group. Agitated of RARγ by BMS961 suppressed the production of these cytokines compared with the infection group (P<0.01; Figure 3A). Meanwhile, the expressions of TNF-α and IL-6 in the supernatant were also down-regulated, which were consistent with the change of mRNA levels (P<0.01; Figure 3B).

Human Corneal Epithelial Cells Culture and Stimulation [1]
HCECs were cultured and maintained in HCECs growth medium in a humidified 5% CO2 incubator at 37°C. HCECs growth medium contains 1:1 DMEM/HamF-12 supplemented with 5% fetal bovine serum (FBS), 10 ng/mL human epidermal growth factor (EGF), 5 mg/mL insulin, and 50 mg/mL penicillin and streptomycin. For stimulation, HCECs were treated with A. fumigatus hyphae (5×107/mL) in different times. And HCECs pretreated with or without BMS961 (1 µg/mL) for 0.5h were stimulated by A. fumigatus hyphae. Total RNA, protein and supernatant were collected for qRT-PCR, Western blot and ELISA. BMS961 was dissolved in DMSO, preliminary experiments showed no obvious difference between the DMSO group and normal group.
The HCECs were stimulated with A. fumigatus hyphae for 0, 2, 4, 8, 12 and 16h. RARγ mRNA and protein levels were tested by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Then HCECs were pretreated with or without BMS961 (RARγ agonist, 1 µg/mL). The mRNA and protein expression of Dectin-1 and the downstream cytokines (TNF-α and IL-6) were determined by qRT-PCR, Western blot and enzyme-linked immunosorbent assay (ELISA).

[1]. Effects of retinoic acid receptor-γ on the Aspergillus fumigatus induced innate immunity response in human corneal epithelial cells. Int J Ophthalmol. 2016 Dec 18;9(12):1713-1718.

BMS961 is a selective retinoic acid receptor γ (RARγ) agonist. To further investigate the effects of RARγ on the immune response of corneal epithelial cells infected with Aspergillus fumigatus, we used the RARγ agonist BMS961 to observe changes in Dectin-1 and pro-inflammatory cytokines. Interestingly, in our study, BMS961 pretreatment almost completely suppressed the expression and secretion of Dectin-1-mediated TNF-α and IL-6 in HCECs. Further investigation is warranted to determine whether this significant effect of BMS961 is related to the nature of the immune challenge. This is consistent with findings from studies on other cell types where the RAR signaling pathway is crucial for enhancing Raldh2 expression. Raldh2 suppresses pro-inflammatory cytokines in dendritic cells (DCs) by inducing Socs3, a known regulator of pro-inflammatory responses. Our previous research has shown that Dectin-1 plays an important role in the defense against fungal infections. It can trigger a series of cellular responses, including respiratory burst, ligand endocytosis and phagocytosis, dendritic cell maturation, and the production of cytokines and chemokines (including TNF-α, IL-6, IL-10, IL-2, GM-CSF, and G-CSF). Studies have shown that Dectin-1 expression is upregulated in fungal-infected corneas, and compared to control mice, infected Dectin-1-/- mice exhibit reduced cellular infiltration and fungal clearance in the cornea. Under these conditions, the regulatory effect of RARγ is similar to that observed in Aspergillus fumigatus experiments. Our data suggest that RARγ can regulate the expression and activation of Dectin-1, which may be the mechanism by which it exerts its anti-inflammatory effect. Other mechanisms may also contribute to the anti-inflammatory effect of RARγ, as Manicassamy et al. found that the RA signaling pathway can attenuate p38 MAPK activation.
It is well known that corneal inflammation is a double-edged sword. Appropriate inflammation can stimulate an effective host defense response without causing significant tissue damage; however, excessive inflammation can severely impair visual function. From the host's perspective, a balanced inflammatory response ensures immune defense while avoiding collateral damage caused by excessive inflammation. Therefore, an appropriate inflammatory response is crucial for clearing fungal inflammation, while excessive inflammation can severely impair visual function. In the early stages of the inflammatory response, an increase in pro-inflammatory cytokines is needed to initiate the response. However, especially in the later stages, downregulating the inflammatory response can reduce corneal tissue damage. When excessive inflammation occurs, timely and appropriate use of immunosuppressants can reduce damage and scarring, and promote future healing. In summary, RARγ plays an indispensable role in FK's fight against Aspergillus fumigatus-induced keratitis. This study opens new avenues for exploring the role of RARγ in fungal infections and further elucidates the molecular mechanisms of its immunomodulatory function. This study also raises awareness of the clinical importance of antifungal drugs and highlights the need for long-term research on the antifungal activity of RARγ in the cornea. Therefore, RARγ's ability to inhibit the expression and activation of Dectin-1 and pro-inflammatory cytokines provides a novel treatment approach for corneal diseases caused by Dectin-1-induced fungal inflammation leading to tissue damage. RARγ can reduce damage caused by pro-inflammatory cytokines and promote ulcer healing. [1]

C23H26FNO4

399.455250263214

399.185

C, 69.16; H, 6.56; F, 4.76; N, 3.51; O, 16.02

185629-22-5

2418

White to off-white solid powder

5.194

3

5

4

29

634

0

FC1=C(NC(=O)C(O)C2C=CC3=C(C(C)(C)CCC3(C)C)C=2)C=CC(C(O)=O)=C1

AANFHDFOMFRLLR-UHFFFAOYSA-N

InChI=1S/C23H26FNO4/c1-22(2)9-10-23(3,4)16-11-13(5-7-15(16)22)19(26)20(27)25-18-8-6-14(21(28)29)12-17(18)24/h5-8,11-12,19,26H,9-10H2,1-4H3,(H,25,27)(H,28,29)

3-fluoro-4-[[2-hydroxy-2-(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2-yl)acetyl]amino]benzoic acid

BMS 961; BMS-189961; 185629-22-5; BMS189961; BMS-961; 3-fluoro-4-[[2-hydroxy-2-(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2-yl)acetyl]amino]benzoic acid; BMS961; BMS 189961; 3-Fluoro-4-[[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8,-tetrahydro-2-naphthalenyl)acetyl]amino]-benzoic acid; BENZOIC ACID, 3-FLUORO-4-[[(2R)-HYDROXY(5,6,7,8-TETRAHYDRO-5,5,8,8-TETRAMETHYL-2-NAPHTHALENYL)ACETYL]AMINO]-;

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.

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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.)