Female balb c mice

BALB/c female mice (6 weeks of age) were purchased from Orientbio Inc. (Seoul, Korea) and acclimated in an animal-care facility for 1 week. The mice were housed in environmentally controlled and specific pathogen-free conditions (22°C, 12 h light/12 h dark cycle) and were provided with water and standard chow ad libitum. Mice were randomly divided into five groups (eight animals/group): NC (normal control group: phosphate buffered saline (PBS)-sensitization/challenge + oral gavage of PBS), OVA (OVA-sensitization/challenge group + oral gavage of PBS), Mon (OVA-sensitization/challenge group + oral gavage of montelukast at 30 mg/kg), GGTA-100 (OVA-sensitization/challenge group + oral gavage of GGTA at 100 mg/kg), and GGTA-200 (OVA-sensitization/challenge group + oral gavage of GGTA at 200 mg/kg). All experimental procedures were carried out in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Korea Institute of Oriental Medicine. Animal handling was carried out in accordance with the dictates of the National Animal Welfare Law of Korea.

All animal studies were approved by Ewha Womans University’s Institutional Animal Care and Use Committee (IACUC, approval ID: 16-023). All methods and experimental procedures were conducted according to the guidelines of the Ewha Womans University’s IACUC. The animals were housed under pathogen-free conditions with a 12-h light/12-h dark cycle, and were fed with standard diet and water ad libitum. 5 weeks old BALB/c female mice (Orientbio) were sensitized with OVA by i.p. injection of 1.3 mg of aluminum hydroxide (Sigma) and 100 µg of ovalbumin (Sigma) twice a week, followed by challenge with intranasal instillation of OVA on day 14, 16, and 18. Ten minutes before challenge, PBS, dexamethasone, and synthetic peptides were intraperitoneally injected. On days 15, 17, and 19, i.p administration was performed without antigen administration. On day 19, the animals were sacrificed 2 hours after the i.p injection, followed by BALF and lung tissue preparation.

BALB/c female mice (6 weeks old) were purchased from Orient Bio Inc. (Seongnam, Korea) and acclimated for 1 week prior to beginning the experiment. To induce asthma, mice were anesthetized and then sensitized with 75 μg of ovalbumin (OVA; Sigma-Aldrich Corporation) and 10 μg of polyinosinic-polycytidylic acid [poly(I:C); Calbiochem-Merck KGaA, Darmstadt, Germany] via intranasal administration on days 0, 1, 2, 3, and 7; they were then intranasally challenged with 50 μg of OVA with 10 μg of poly(I:C) on days 14, 21, 22, and 23. To verify the treatment effect of hUCB-MSCs or CoCl₂-MSCs, mice were intravenously injected into the tail vein on day 15 with hUCB-MSCs or CoCl₂-MSCs (1 × 10⁵ cells/100 μL/mouse). As the positive control group, several mice were administered equal volumes of PBS. All mice were sacrificed on day 24, and bronchoalveolar lavage fluid (BALF) was obtained from the left lung by lavaging three times with 1 mL of saline via trachea cannula, while the right lung was resected. Then, BALF was centrifuged, precipitated cells were resuspended in 1 mL of PBS, and the number of cells was counted under a biological microscope (Olympus Corporation, Tokyo, Japan). Isolated lungs were fixed with 4% paraformaldehyde, embedded in paraffin, and then cut into sections at a thickness of 3–4 μm, which were stained with hematoxylin and eosin.