Pharmacology and immunological actions of a herbal medicine ASHMI on allergic asthma

Abstract

Allergic asthma is a chronic and progressive inflammatory disease for which there is no satisfactory treatment. Studies reported tolerability and efficacy of an anti-asthma herbal medicine intervention (ASHMI) for asthma patients, developed from traditional Chinese medicine. To investigate the pharmacological actions of ASHMI on early- and late-phase airway responses (EAR and LAR), Ovalbumin (OVA)-sensitized mice received 6 weeks of ASHMI treatment beginning 24 h following the first intratracheal OVA challenge. EAR were determined 30 min following the fourth challenge and LAR 48 h following the last challenge. ASHMI effects on cytokine secretion, murine tracheal ring contraction and human bronchial smooth muscle cell prostaglandin (PG) production were also determined.ASHMI abolished EAR, which was associated with significantly reduced histamine, leukotriene C4, and OVA-specific IgE levels, as well as LAR, which was associated with significantly reduced bronchoalveolar lavage fluid (BALF) eosinophils, decreased airway remodeling, and lower Th2 cytokine levels in BALF and splenocyte cultures. Furthermore, ASHMI inhibited contraction of murine tracheal rings and increased production of the potent smooth muscle relaxer PGI(2). ASHMI abrogation of allergic airway responses is associated with broad effects on asthma pathological mechanisms.

Figure 1. 3D-HPLC-DAD fingerprint of the ASHMI extract

Alkaloids, flavonoids, and triterpenoids in ASHMI extract are shown by labels. Peak analysis and assignment were performed using standard samples and/or LC-MS method. Matrine, sophocarpine, oxymatrine, kushenol O, trifolirhizin correspond to the compounds from Radix Sophorae Flavescentis. Ganoderic acid B, D, A, H, and F and ganolucidic acid D correspond to Ganoderma lucidum; Liquiritin, liquiritigenin, 7, 4’dihydroxyflavone, Isoononin, 3’7-Dihydroxy-4’-methoxy-isoflavone, and glycyrrhizic acid correspond to compounds from Radix glycyrrhiza.

Figure 2. Early ASHMI treatment abolished EAR and reduced histamine, LT C4 and Ag-specific IgE levels

(A) Mice were sensitized, challenged, and treated with ASHMI as described in the protocol. (B) PEF values were measured 30 min following the 4th challenge. Plasma samples were obtained immediately following PEF measurement. Histamine (C) and LTC4 (D) levels were measured by ELISA. In separate experiments mice were sacrificed immediately following PEF measurement and BALF were obtained from mice in each group and pooled. BALF histamine levels (D) and BALF LT C4 levels (E) were determined. (F) Plasma OVA-specific IgE levels were determined by ELISA. Results of B, C, D and G are expressed as means ± SEM for each group of mice (n = 10–15/group from 2–3 sets of experiments. *, p < 0.05 vs. sham; **, p < 0.01 vs. sham; *** p < 0.001 vs. sham; # p < 0.05 vs. naïve; # # #, p < 0.001 vs. naïve. Results of E and F are means ± SEM of triplicates from pooled BALF of 4 mice. Sham, OVA sensitized/challenged mice received water as sham treatment. ASHMI, OVA sensitized/challenged mice received ASHMI treatment. Naïve, not sensitized/challenged, nor treated.

Figure 3. ASHMI treatment eliminated LAR and reduced airway inflammation and remodeling

(A) Mice were sensitized, challenged, and treated with ASHMI as described in the protocol. (B) APTI following ACh provocation was measured 2 days after the 5th OVA challenge. (C) Percent eosinophils in BALF immediately following APTI was determined. (D) Total lung collagen content was measured in homogenized lung supernatants. Lung histology analysis (E) shows numerous goblet cells (PAS positive cells, purple color) in airway from a sham-treated mouse, markedly reduced mucus goblet cells in airways of an ASHMI-treated mouse and the absence of goblet cells in airways of a naïve mouse. Results of B and C are expressed as means ± SEM for each group of mice (n = 10–15/per group from 2 sets of experiments. ***, p < 0.001 vs. sham; ###, p < 0.001 vs. naïve. Results of (D) are Mean ± SEM of 5–6 mice from each group. *, p < 0.05; vs. sham; #, p < 0.05 vs. naïve.

Figure 4. Immunomodulatory effect of ASHMI on T cell cytokine profiles

In a separate experiment, mice were sensitized, challenged, and treated with ASHMI as in Fig 3A. Mice were sacrificed 24 h following the 5th challenge and BALF was collected from each group of mice (n = 4) and pooled. Cytokine levels were determined by ELISA. Results are expressed as means ± SEM of triplicates.

Figure 5. ASHMI modulation of airway smooth muscle contractility

(A) Effect of ASHMI on tracheal ring contractility. Tracheal rings from OVA sensitized/challenged (OVA) or naive mice were pretreated with or without ASHMI for 30 min. Contractile responses to increasing doses of ACh were measured. Results are expressed as mean ± SEM of rings. # #, p < 0.01 and # # # p < 0.001 vs. OVA. ***, p < 0.001 vs. OVA + ASHMI. (B) Effect of β2-adrenergic receptor antagonist on ASHMI mediated effect on tracheal rings. Tracheal rings from OVA mice were pretreated with ASHMI in the presence or absence of β2-adrenergic receptor antagonist ICI 11855 (50 µM) for 30 min. Contractile responses to increasing doses of ACh were measured. Results are expressed as mean ± SEM of 4–5 rings. ***, p < 0.001 vs. OVA + ASHMI, # # #, p < 0.001 vs. OVA + ASHMI + ICI118,551. OVA, tracheal rings from OVA sensitized and challenge mice; Naive, tracheal rings from naive mice; OVA+ASHMI, tracheal rings from OVA sensitized and challenge mice pretreated with ASHMI; Naïve + ASHMI, tracheal rings from naive mice pretreated with ASHMI.

Figure 6. ASHMI induced the release of PGI2 production by human tracheal smooth muscle cells (HTSMCs)

HTSMC were cultured for 30 min and 6 h with or without ASHMI at different concentrations as indicated. Supernatants were then harvested and PGI2 levels (A) and (B) and PGD2 levels (C) and (D) were determined by measuring levels of PGI2 metabolite 2,3-dinor-6-keto PGF1α and PGD2 metabolite 11β-PGF2α. Data are mean ± SEM of triplicate ELISA. *p < 0.05 vs. untreated. The Y axis of PGI2 (A) and (B) and PGD2 (C) and (D) are not on the same scale.