Application of 4-methylumbelliferone in preparation of anti-allergic drugs
A methylumbelliferone and anti-allergic technology, which is applied in the field of biopharmaceuticals, can solve the problems of large side effects and poor anti-allergic drug effects, and achieve the effect of reducing degranulation and alleviating the symptoms of local and systemic allergic reactions in the body
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Embodiment 1
[0020] Inhibitory effect of 4-MU on rat basophil (RBL-2H3) degranulation:
[0021] Mouse basophils are widely used in the diagnosis and immunotherapy of allergies. RBL-2H3 is easy to culture and is widely used in degranulation and signaling pathways, mast cell stabilizers, physicochemical properties of FcεR and its cytoskeleton Interaction studies etc. RBL-2H3 has high-affinity IgE receptors, which can be activated to secrete histamine and other transmitters by accumulating these receptors or synergizing with calcium ionophores.
[0022] In order to verify the inhibitory effect of 4-MU on the degranulation of rat basophils (RBL-2H3), its specific implementation is as follows:
[0023] Cell culture: Rat basophilic leukemia-2h3 cell lines (RBLs) were purchased from Cellcook Biotechnology Company (Guangzhou, China) and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 4.0 mM l-glutamine Amide, 1.0mM sodium pyruvate, 100U / ml penicillin, 100μg / ml streptomycin, non-es...
Embodiment 2
[0028] 4-MU inhibits degranulation of bone marrow-derived mast cells (BMMCs):
[0029] Cell culture: BMMCs were isolated from Balb / c mice (purchased from Guangdong Medical Experimental Animal Center), cultured in RPMI (Hyclone), 10% FBS (Gibico), 1% double antibody, 10ng / ml IL-3 and SCF4 -6 weeks.
[0030] CCK8 detects the toxicity of cells to 4-MU: BMMC (1×104 / well) were seeded in a 96-well plate, T-5224 was added after 24 hours, and incubation was continued for 24 hours. Then add 10ul CCK8 reagent, incubate for 1 hour, and finally measure the absorbance (OD value) with a microplate reader at a wavelength of 450nm.
[0031] Release of β-aminoglycosidase: Inoculate BMMC in a 24-well plate, incubate overnight with anti-DNP IgE for sensitization, incubate with 4-MU for 1 hour, stimulate with DNP-HSA (sigma) for 30 minutes, take 50ul supernatant and 50ul The substrate was reacted at 37°C for 90mins. The absorbance was measured at 405nm. After the supernatant was discarded, 500...
Embodiment 3
[0035] Effects of 4-MU on passive skin hypersensitivity in mice:
[0036] Breeding of BALB / c mice: Female mice (4-5 weeks old BALB / C) were purchased from the Guangdong Medical Laboratory Animal Center (Foshan, Guangdong) and placed in a temperature (24±1°C) and humidity (55±10 %) in a relatively stable SPF environment for 1 week. Mice were used to isolate bone marrow-derived mast cells (BMMC) and to perform a passive cutaneous anaphylaxis (PCA) model and an active systemic anaphylaxis (ASA) model.
[0037] Mice were injected with anti-DNP-IgE 500ng / ear. 24 hours later, 4-MU (50 mg / kg) or ketotifen (50 mg / kg, positive control group) was intraperitoneally injected (i.p.) into the ear (N=5 / group). One hour later, the mice were injected with 200 μg of DNP-HSA PBS containing 0.5% Evans blue dye into the tail vein. Mice were euthanized 1 h after DNP-HSA injection. Ears were taken and placed in 700 μl formamide for extraction at 65°C for 12 hours. Dye absorbance was measured at ...
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