
Allergic asthma is a chronic inflammatory disease of the airways of the lungs and is characterized by chest tightness, coughing, shortness of breath, and wheezing with large heterogeneity of clinical phenotypes (Park and Im, 2019). More than 300 million people are patients of asthma worldwide (Park and Im, 2019). Environmental factors such as air pollution and allergens can cause asthma in addition to genetic factors (Aoki
Previously, carnosol has been reported to have anti-inflammatory effects. Carnosol activated the anti-inflammatory peroxisome proliferator-activated receptor-γ at the transcriptional level (Rau
Carnosol and other materials were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Rat RBL-2H3 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured at 37°C in a 5% CO2-humidified incubator as described previously (Huang
Female five-week-old BALB/c mice were purchased from Daehan Biolink (Seoul, Korea). They were housed in the laboratory animal facility at Pusan National University and fed water and food
Six-week-old BALB/c mice (22 g) were randomly divided into three groups (
Using a Cellspin® centrifuge (Hanil Electric, Seoul, Korea), the immune cells in BALF were adhered to a glass slide and fixed in methanol for 30 s. Cells on slides were stained with May-Grünwald solution for 8 min and subsequently by Giemsa solution for 12 min.
RT-PCR was performed to analyze the expression levels of Th2 and Th17 cells. Total RNA was isolated using TRIzol™ reagent (Invitrogen, Waltham, MA, USA), and used for cDNA synthesis. Promega GoTaq® DNA polymerase (Promega Corporation, Madison, WI, USA), primers for each gene, and the synthesized cDNA were reacted to amplify the specific genes. Primers sequences and PCR conditions for specific genes are described in a previous report (Park
Tissue sections of lungs from mice of each group were prepared. Periodic acid-Schiff (PAS) staining and hematoxylin and Eosin (H&E) staining were performed to identify mucus-secreting goblet cells and eosinophil infiltration, respectively. For PAS staining, the Schiff’s regent was used, and for H&E staining, hematoxylin and eosin regents were used (Heo and Im, 2019; Kim and Im, 2019).
The degree of lung inflammation was evaluated using a subjective scale of 0-3 by a treatment-blind observer (Heo and Im, 2019; Kim and Im, 2019). Mucin-secreting cells stained with PAS in the airways were counted from two lung sections per mouse. The length of the bronchi basal lamina using was also simultaneously measured ImageJ software (National Institute of Health, MD, USA). Mucous production was expressed by the number of PAS-positive cells per mm of bronchiole.
IL-4 levels in BALF were quantitated using an ELISA kit. A capture antibody and a biotinylated detection antibody specific for IL-4 were obtained from eBioscience (cat. 14-7041-68, and cat. 33-7042-68C, San Diego, CA, USA). Avidin-horseradish peroxidase was used and the absorbance was measured at 450 nm (Lee
The results are expressed as means ± standard errors (SEs). For statistical significance, analysis of variance (ANOVA) was used, followed by Turkey’s post hoc test using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA).
RBL-2H3 mast cells were used to ascertain if carnosol inhibits antigen-induced degranulation. β-Hexosaminidase activity was measured as degranulation activity in the medium after antigen exposure (Heo and Im, 2019; Kim and Im, 2019). Carnosol inhibited the release of β-hexosaminidase in a dose-dependent manner and also significantly inhibited degranulation at concentrations higher than 3 μM (Fig. 1).
An OVA-induced allergic asthma model in female BALB/c mice was utilized to verify the inhibitory effects of carnosol
We measured the mRNA levels of Th2 cytokines in the lungs and immune cells of BALF. We also measured the mRNA levels of IL-17A, as Th17 cells were recently recognized as regulators of allergic asthma (Wakashin
The lungs were histologically examined on D32. PAS staining was utilized to detect the presence of mucins. Stored or secreted mucins appear as violet in color in the PAS-stained sections. Dark purple-stained cells surrounded the bronchioles in the OVA-treated group, and carnosol treatment reduced mucin-stained cells (Fig. 4A). The eosinophils were rarely observed as small, navy blue dots in the control group, and in the OVA group, eosinophils were found to be densely surrounding the bronchioles by using H&E staining (Fig. 4B). Semi-quantitative analysis was performed by counting the PAS-stained cells per mm of the bronchioles (Fig. 4C) (Heo and Im, 2019; Kim and Im, 2019). PAS-stained cells were rarely found in the control group. Approximately 110 PAS-stained cells were counted per mm in the OVA group, and carnosol treatment significantly inhibited the PAS-stained cells (Fig. 4C). Also semi-quantitative evaluation using a subjective scale of 0-3 was applied to assess lung inflammation from H&E staining (Heo and Im, 2019; Kim and Im, 2019). The inflammation score in the OVA group was approximately 2.5, while carnosol treatment significantly inhibited the score (Fig. 4D).
The protein levels of IL-4 and IL-13 in BALF were measured by using ELISA to verify the effect of carnosol on Th2 cytokine expression. The IL-4 levels were increased in the OVA-induced group compared to the vehicle-treated control group, and the increase in IL-4 levels was significantly suppressed by carnosol treatment (Fig. 5). IL-13 levels in the samples were out of detectable ranges.
Although the anti-inflammatory, anti-oxidant, and anti-tumor effects of carnosol have been previously reported (Singletary, 1996; Johnson, 2011; Kashyap
Carnosol treatment significantly inhibited the allergic features in BALF and the lungs of mice in the OVA-induced murine allergic asthma model. The responses of Th2 cytokines such as IL-4 and IL-13 are very important in the development of asthma (Fish
The anti-allergic effect of carnosol has also been observed in other Th2 cell-related diseases. In skin pharmacology, topical application of carnosol has been reported in PMA-induced ear edema, phthalic anhydride-induced atopic dermatitis, and UVB-induced inflammation (Mengoni
Based on literature, the anti-asthma effects of carnosol could result from several mechanistic modulations in inflammation-related proteins: (i) carnosol activation of anti-inflammatory peroxisome proliferator-activated receptor-γ transcriptionally (Rau
In summary, we report that carnosol induces suppressive effects on degranulation of mast cells
This research was supported by the Basic Science Research Program of the Korean National Research Foundation, funded by the Korean Ministry of Education, Science and Technology (NRF-2019R1A2C1005523).
Authors declare that there is no conflict of interest.
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