Anti-inflammatory, immunomodulatory, and heme oxygenase-1 inhibitory activities of ravan napas, a formulation of uighur traditional medicine, in a rat model of allergic asthma

Abstract

Ravan Napas (RN) is a traditional formula used to treat pulmonary symptoms and diseases such as coughing, breathing difficulty, and asthma in traditional Uighur medicine. The purpose of this study was to investigate the anti-inflammatory, and immuno-modulatory activity of RN in a well-characterized animal model of allergic asthma. Rats were sensitized with intraperitoneal (ip) ovalbumin (OVA) and alum, and then challenged with OVA aerosols. The asthma model rats were treated with RN; saline- and dexamethasone- (DXM-) treated rats served as normal and model controls. The bronchoalveolar lavage fluid (BALF) cellular differential and the concentrations of sICAM-1, IL-4, IL-5, TNF-α, INF-γ, and IgE in serum were measured. Lung sections underwent histological analysis. The immunohistochemistry S-P method was used to measure the expression of ICAM-1 and HO-1 in the lung. RN significantly reduced the number of inflammatory cells in BALF and lung tissues, decreased sICAM-1, IL-4, IL-5, TNF-α, and IgE in serum, and increased serum INF-γ. There was a marked suppression of ICAM-1 and HO-1 expression in the lung. Our results suggest that RN may have an anti-inflammatory and immuneregulatory effect on allergic bronchial asthma by modulating the balance between Th1/Th2 cytokines.

Figure 1

Experimental protocol. Rats were immunized with intraperitoneal injection of a suspension containing 40 mg ovalbumin (OVA) and 2 mg aluminium hydroxide. 15 days after the immunization, rats were challenged by exposure to an aerosol of 1% OVA in PBS for 20 minutes once daily for 8 consecutive days (days 15 to 22). Three groups were treated for 22 days after sensitization, with saline (controls), RN1, and RN2 (RN orally at 0.25 g/kg or 0.5 g/kg per day). DXM group was treated with dexamethasone 10 mg/kg on days 20, 21, and 22.

Figure 2

Effect of Ravan Napas (RN) on the recruitment of inflammatory cells in BALF obtained from OVA-induced rat model of asthma. Rat Bronchoalveolar Lavage Fluid (BALF) was harvested during the 24 h after last OVA challenge. All rats were sensitized with OVA: (a) total leucocytes, (b) lymphocytes and neutrophils, and (c) eosinophils. Normal: rats sensitized with OVA and challenged with saline. Control: rats sensitized and challenged with OVA. DXM: OVA-sensitized and challenged rats treated with Dexamethasone (10 mg/kg). RN-1: OVA-sensitized and challenged rats treated with RN (0.25 g/kg/day). RN-2: OVA-sensitized and challenged rats treated with RN (0.5 g/kg/day). Data are expressed as mean ± S.E.M., n = 10 rats per treatment group. ^▲▲▲ P < .01 versus Normal; ***P < .01 versus Control.

Figure 3

Effect of Ravan Napas (RN) on pulmonary inflammation in OVA-induced rat model of asthma. Lung tissues were obtained on the day after the last OVA challenge. Tissues were stained with hematoxylin and eosin (H&E, 400x) (a). The inflammatory cell infiltration in the lung tissues was scored as described in the method section (b). Normal: rats sensitized with OVA and challenged with saline. Control: rats sensitized and challenged with OVA. DXM: OVA-sensitized and challenged rats treated with Dexamethasone (10 mg/kg). RN-1: OVA-sensitized and challenged rats treated with RN (0.25 g/kg/day). RN-2: OVA-sensitized and challenged rats treated with RN (0.5 g/kg/day). Data are expressed as mean ± S.E.M., n = 10 rats per treatment group. ^▲▲▲ P < .01 versus Normal; ***P < .01 versus Control.

Figure 4

Effect of Ravan Napas (RN) on sICAM-1 and total IgE concentration in serum of OVA-induced rat model of asthma. Blood was sampled and serum obtained 24 h after last OVA challenge. sICAM-1 and total IgE were measured by ELISA as described in the materials and methods section. (a): sICAM-1 concentrations; (b): IgE concentrations; Normal: rats sensitized with OVA and challenged with saline. Control: rats sensitized and challenged with OVA. DXM: OVA-sensitized and challenged rats treated with Dexamethasone (10 mg/kg). RN-1: OVA-sensitized and challenged rats treated with RN (0.25 g/kg/day). RN-2: OVA-sensitized and challenged rats treated with RN (0.5 g/kg/day). Data are expressed as mean ± S.E.M., n = 10 rats per group.  ^▲▲▲ P < .01 versus Normal; ***P < .01 versus Control.

Figure 5

Effect of Ravan Napas (RN) on IL-4, IL-5, INF-α, and INF-γ in serum of OVA-induced rat model of asthma. Blood was sampled and serum obtained 24 h after last OVA challenge. IL-4, IL-5, TNF-α, and INF-γ were analyzed by ELISA as described in the materials and methods section. Normal: rats sensitized with OVA and challenged with saline. Control: rats sensitized and challenged with OVA. DXM: OVA-sensitized and challenged rats treated with Dexamethasone (10 mg/kg). RN-1: OVA-sensitized and challenged rats treated with RN (0.25 g/kg/day). RN-2: OVA-sensitized and challenged rats treated with RN (0.5 g/kg/day). Data are expressed as mean ± S.E.M., n = 10 rats per group. ^▲▲▲ P < .01 versus Normal; ***P < .01 versus Control.

Figure 6

Effect of RN on the expression of ICAM-1 and HO-1 in lung tissue of OVA-induced rat model of asthma. Wistar rat lung tissue was obtained 24 hours after last Ova challenge. Immunohistochemistry was performed as described in materials and methods section. Expression of ICAM-1 and HO-1 in the lung tissues was scored as described in Section 2. Normal: rats sensitized with OVA and challenged with saline. Control: rats sensitized and challenged with OVA. DXM: OVA-sensitized and challenged rats treated with Dexamethasone (10 mg/kg). RN-1: OVA-sensitized and challenged rats treated with RN (0.25 g/kg/day). RN-2: OVA-sensitized and challenged rats treated with RN (0.5 g/kg/day). Data are expressed as mean ± S.E.M., n = 10 rats per group. ^▲▲▲ P < .01 versus Normal; ***P < .01 versus Control.

Figure 7

Main anti-inflammatory and immunoregulatory targets of Ravan Napas (RN) in allergic asthma. This figure illustrates the cascade of activations and inactivations resulting from exposure to allergen after sensitization in asthma, from exposure of Antigen-presenting cells (APC) to activation of mast cells and neutrophils, activation of Th2 cells liberating ICAM, interleukins, and TNFα, inactivation of Th1 cells releasing INFγ, and so forth. From the results reported, RN, as indicated, acts on various pathways implicated in the bronchial response to antigens: stimulation of Th1, inactivation of Th2, decreased ICAM and HO-1 expression, decreased IgE production, and decreased recruitment of neutrophils and other inflammatory cells.