SGLT2 Inhibitors Empagliflozin and Canagliflozin Ameliorate Allergic Asthma Responses in Mice

Abstract

Inhibitors of sodium/glucose cotransporter 2 (SGLT2), such as empagliflozin and canagliflozin, have been widely used to block glucose reabsorption in the proximal tubules of kidneys in patients with diabetes. A meta-analysis suggested that SGLT2 inhibitors are associated with a decreased risk of asthma development. Therefore, we investigated whether SGLT2 inhibitors could suppress allergic asthma. Empagliflozin and canagliflozin suppressed the in vitro degranulation reaction induced by antigens in a concentration-dependent manner in RBL-2H3 mast cells. Empagliflozin and canagliflozin were administered to BALB/c mice sensitized to ovalbumin (OVA). The administration of empagliflozin or canagliflozin significantly suppressed OVA-induced airway hyper-responsiveness and increased the number of immune cells and pro-inflammatory cytokine mRNA expression levels in bronchoalveolar lavage fluid. The administration of empagliflozin and canagliflozin also suppressed OVA-induced histopathological changes in the lungs. Empagliflozin and canagliflozin also suppressed serum IgE levels. These results suggested that empagliflozin and canagliflozin may be applicable for the treatment of allergic asthma by suppressing immune responses.

Keywords: SGLT2; allergy; asthma; canagliflozin; pulmonary pharmacology.

Figure 1

Effects of empagliflozin and canagliflozin on the degranulation of RBL-2H3 mast cells. RBL-2H3 cells were stimulated with dinitrophenyl-human serum albumin (DNP-HSA) after sensitization with anti-dinitrophenyl immunoglobulin E (DNP-IgE) for 18 h. Empagliflozin (A) and canagliflozin (B) were added at the concentrations indicated 30 min before DNP-HSA stimulation. There was basal degranulation observed in negative control samples without DNP-HSA and DNP-IgE treatment, and the degranulation seen with DNP-HAS and DNP-IgE was used as a positive control. The results are shown as means ± the standard error of the mean (SEM, n = 3). *** p < 0.001 vs. the HSA-untreated group. # p < 0.05, ## p < 0.01 vs. the HSA-treated group.

Figure 2

Effects of empagliflozin and canagliflozin on airway hyper-responsiveness in an OVA-induced murine asthma model. We determined Penh (enhanced pause) values as airway hyper-responsiveness in empagliflozin (1 or 3 mg/kg)-, canagliflozin (5 or 15 mg/kg)-, or PBS-treated mice by rising the methacholine concentrations. PBS: phosphate-buffered saline (PBS)-treated mice, OVA: ovalbumin (OVA)-challenged mice, OVA + empagliflozin (1 mg/kg), OVA + empagliflozin (3 mg/kg), OVA + canagliflozin (5 mg/kg), and OVA + canagliflozin (15 mg/kg). The results are shown as means ± the SEM (n = 5). ** p < 0.01 vs. the PBS-treated group, ## p < 0.01, # p < 0.05 vs. the OVA-treated group.

Figure 3

Effects of empagliflozin and canagliflozin on the rises in immune cell numbers induced by OVA in BALF. (A) Mice sensitized with OVA twice by intraperitoneal injection at day 0 and 14 were subsequently exposed to OVA nebulized on D28, D29, and D30. Empagliflozin (1 and 3 mg/kg) or canagliflozin (5 and 15 mg/kg) was intraperitoneally injected 30 min before the challenge of OVA. We stained and counted the cells in the bronchoalveolar lavage fluid (BALF) using the May–Grünwald stain. Lined areas on upper panels are enlarged in lower panels. Red arrows indicate macrophages, green arrows indicate eosinophils, and yellow arrows indicate lymphocytes. (B) Numbers of lymphocyte, eosinophil, macrophage, and total cells in the BALF are shown. The cell count results are shown as means ± the SEM (n = 5). *** p < 0.001 vs. the PBS-treated one, ### p < 0.001, # p < 0.05, ## p < 0.01 vs. the OVA-treated one.

Figure 4

Effects of empagliflozin and canagliflozin on the expression levels of cytokines mRNAs in BALF cells. The levels of the Th2 cytokines Il-4 and Il-13, the Th1 cytokine Ifn-γ, and the Th17 cytokine Il-17a mRNA expression of in BALF cells were determined. (A) Ifn-γ, (B) Il-13, (C) Il-4, and (D) Il-17a. The levels of cytokines mRNAs were expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene. Values are shown as means ± the SEM (n = 5). ** p < 0.01, *** p < 0.001 vs. the PBS-treated one, ### p < 0.001, # p < 0.05, ## p < 0.01 vs. the OVA-treated one.

Figure 5

Effects of empagliflozin and canagliflozin on airway inflammation and mucin production. (A) Hematoxylin and eosin (H&E)-stained sections of lung tissues from the PBS-, OVA-, empagliflozin (1 or 3 mg/kg)-, and canagliflozin (5 or 15 mg/kg)-treated groups. The small navy-blue dots around the bronchioles are eosinophils. In the PBS-treated group, we rarely observed eosinophils. In the OVA-treated group, we found extensively accumulated eosinophils around the bronchioles (green arrows). (B) A histogram of the inflammatory scores in H&E-stained slides. (C) Periodic acid–Schiff (PAS)/hematoxylin-stained sections of lung tissues from the PBS-, OVA-, empagliflozin (1 or 3 mg/kg)-, and canagliflozin (5 or 15 mg/kg)-treated groups. Mucin is stained purple with PAS. In the OVA-treated group, a darker and thicker purple color was observed surrounding the bronchioles compared with that in the PBS-treated group (red arrows). (D) A histogram of the numbers of PAS-stained cells in the slides. Values represent means ± the SEM (n = 5). *** p < 0.001 vs. the PBS-treated one, ### p < 0.001, # p < 0.05, ## p < 0.01 vs. the OVA-treated one.

Figure 6

Effects of empagliflozin and canagliflozin on the expression levels of cytokines mRNAs in the lungs. The expression of the Th1 cytokine Ifn-γ, the Th2 cytokines Il-4 and Il-13, and the Th17 cytokine Il-17a mRNAs in the lung tissues were determined. (A) Ifn-γ, (B) Il-13, (C) Il-4, and (D) Il-17a. The levels of cytokines mRNAs were expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene. Values are shown as means ± the SEM (n = 5). *** p < 0.001, ** p < 0.01 vs. the PBS-treated one, # p < 0.05, ## p < 0.01, vs. the OVA-treated one.

Figure 7

Effects of empagliflozin and canagliflozin on IL-13 levels in BALF and IgE levels in serum. IL-13 levels in BALF (A) and IgE protein levels in serum (B) were measured by enzyme-linked immunosorbent assays. The results are shown as means ± the SEM (n = 5). *** p < 0.001, * p < 0.05 vs. the PBS-treated one, ## p < 0.01 vs. the OVA-treated one.