Fig 1.
The effect of ivabradine (IVA) on the carrageenan-induced 50% paw withdrawal threshold (pain threshold) in rats.
Carrageenan at 1% was subcutaneously injected into the hindpaw, causing acute inflammation. IVA at different concentrations was subcutaneously injected into the hindpaw, combined with carrageenan. (A) The time-course of the pain threshold until 8 hours after the injection of test solutions (CA: only carrageenan; CA + IVA10: carrageenan + IVA at 10 μM; CA + IVA20: carrageenan + IVA at 20 μM; CA + IVA50: carrageenan + IVA at 50 μM). (B) The pain threshold 2 hours after the injection of test solutions. *P< 0.05, ****P< 0.0001 compared with CA. Data represent the mean ± SD (n = 6 for each).
Fig 2.
The effect of lamotrigine (LTG) to reverse the action of ivabradine (IVA) on the carrageenan-induced 50% paw withdrawal threshold (pain threshold) in rats.
Carrageenan at 1% was subcutaneously injected into the hindpaw, causing acute inflammation. IVA at 20 μM and/or LTG at 10 μM was injected into the hindpaw combined with carrageenan. (A) The time-course of the pain threshold until 8 hours after the injection of test solutions (CA: only carrageenan; CA + IVA20: carrageenan + IVA at 20 μM; CA + IVA20 + LTG: carrageenan + IVA at 20 μM + LTG at 10 μM; CA + LTG: carrageenan + LTG at 10 μM). (B) The pain threshold 2 hours after the injection of test solutions. **P< 0.01, ****P< 0.0001 compared with CA. # P< 0.05 compared with CA + IVA20. Data represent the mean ± SD (n = 6 for each).
Fig 3.
The effect of forskolin (FSK) to reverse the action of ivabradine (IVA) on the carrageenan-induced 50% paw withdrawal threshold (pain threshold) in rats.
Carrageenan at 1% was subcutaneously injected into the hindpaw, causing acute inflammation. IVA at 20 μM and/or FSK at 10 μM was injected into the hindpaw combined with carrageenan. (A) The time-course of the pain threshold until 8 hours after the injection of test solutions (CA: only carrageenan; CA + IVA20: carrageenan + IVA at 20 μM; CA + IVA20 + FSK: carrageenan + IVA at 20 μM + FSK at 10 μM; CA + FSK: carrageenan + FSK at 10 μM). (B) The pain threshold 2 hours after the injection of test solutions. **P< 0.01, ****P< 0.0001 compared with CA # P< 0.05 compared with CA+ IVA20. Data represent the mean ± SD (n = 6 for each).
Fig 4.
The effect of ZD7288 on the carrageenan-induced 50% paw withdrawal threshold (pain threshold) in rats.
Carrageenan at 1% was subcutaneously injected into the hindpaw, causing acute inflammation. ZD7288 at 50 μM was subcutaneously injected into the hindpaw combined with carrageenan. The figure shows the time-course of the pain threshold until 8 hours after the injection of test solutions (CA: only carrageenan; CA + ZD: carrageenan + ZD7288 at 50 μM). *P< 0.05, ****P< 0.0001 compared with only carrageenan. Data represent the mean ± SD (n = 6 for each).
Fig 5.
Hematoxylin-eosin staining findings in the hindpaw area of rats (2 hours after injection).
The following test solutions were subcutaneously injected into the hindpaw: physiological saline (A), 1% carrageenan (B), 1% carrageenan + ivabradine (IVA) at 20 μM (C), 1% carrageenan + IVA at 20 μM + forskolin at 10 μM (FSK) (D), and 1% carrageenan + IVA at 50 μM (E). Scale bar represents 200 μm.
Fig 6.
The effect of ivabradine (IVA) on carrageenan-induced accumulation of leukocytes in the hindpaw of rats.
The following test solutions were subcutaneously injected into the hindpaw: physiological saline (PS), 1% carrageenan (CA), CA + IVA at 20 μM (IVA20), CA + IVA20 + forskolin at 10 μM (FSK), and CA + IVA at 50 μM (IVA50). *P< 0.05, ****P< 0.0001 compared with physiological saline. # P< 0.05, ### P< 0.001, #### P< 0.0001 compared with CA. Data represent the mean ± SD (n = 6 for each).
Fig 7.
Immunohistochemical expression of TNF-alpha in the hindpaw area of rats (2 hours after injection).
The following test solutions were subcutaneously injected into the hindpaw: physiological saline (A, E), 1% carrageenan (B, F), 1% carrageenan + ivabradine at 20 μM (IVA20) (C, G), and 1% carrageenan + IVA20 + forskolin at 10 μM (FSK) (D, H). A-D are shown at low magnification, and black scale bars represent 200 μm. E-H are shown at a higher magnification, and blue scale bars represent 100 μm.
Fig 8.
The effect of ivabradine (IVA) on TNF-alpha production in mouse macrophage-like cells.
The cells were incubated with LPS at 10 ng/mL. TNF-alpha levels in the supernatants of the cells following 2-, 4-, and 6-hour incubation with only LPS, LPS + IVA at 10, 20, and 50 μM were measured. *P< 0.05, ***P< 0.001, ****P< 0.0001 compared with only LPS. Data represent the mean ± SD (n = 5 for each).
Fig 9.
The effect of ZD7288 (ZD) on TNF production in mouse macrophage-like cells.
The cells were incubated with LPS at 10 ng/mL. TNF-alpha levels in the supernatants of the cells following 2-, 4-, and 6-hour incubation with only LPS and LPS + ZD at 10 and 50 μM were measured. **P< 0.01, ****P< 0.0001 compared with only LPS. Data represent the mean ± SD (n = 5 for each).
Fig 10.
The effect of forskolin (FSK) to reverse the action of ivabradine (IVA) on LPS-stimulated TNF production in mouse macrophage-like cells.
The cells were incubated with LPS at 10 ng/mL. TNF-alpha levels in the supernatants of the cells following 2-hour incubation with only LPS, LPS + IVA at 20 μM, LPS + FSK at 10 μM, and LPS + IVA at 20 μM + FSK at 10 μM were measured. ***P< 0.001, ****P< 0.0001 compared with only LPS. Data represent the mean ± SD (n = 5 for each).