Tuo-Min-Ding-Chuan Decoction Alleviate Ovalbumin-Induced Allergic Asthma by Inhibiting Mast Cell Degranulation and Down-Regulating the Differential Expression Proteins

Abstract

Allergic asthma is a stubborn chronic inflammatory disease, and is considered a co-result of various immune cells, especially mast cells, eosinophils and T lymphocytes. At present, the treatment methods of allergic asthma are limited and the side effects are obvious. Traditional Chinese medicine has been used to treat diseases for thousands of years in China. One such example is the treatment of allergic asthma, which take the characteristics of less adverse reactions and obvious curative effect. Tuo-Min-Ding-Chuan Decoction (TMDCD) is a traditional Chinese medicine compound for the treatment of allergic asthma optimized from Ma-Xing-Gan-Shi Decoction (MXGSD), which was put forward in Treatise on Febrile Diseases by Zhang Zhongjing in the Eastern Han Dynasty. The compound shows a significant clinical effect, but the mechanism of its influence on the immune system is still unclear. The purpose of this study was to observe whether TMDCD could alleviate the symptoms of ovalbumin (OVA) challenged allergic asthma mice, and to explore its immune regulatory mechanism, especially on mast cell (MC) degranulation. The results showed TMDCD could not only reduce the airway hyperresponsiveness (AHR), inflammatory cell infiltration and mucus secretion in the lung tissue of OVA challenged mice, but also decrease the levels of total IgE, OVA-specific IgE, histamine and LTC4 in serum. We found that TMDCD can downregulate the expression of Fractalkine, Tryptase ε, IL-25, CCL19, MCP-1, OX40L, Axl, CCL22, CD30, G-CSF, E-selectin, OPN, CCL5, P-selectin, Gas6, TSLP in OVA challenged mice serum by using mouse cytokines antibody array. It has been reported in some literatures that these differentially expressed proteins are related to the occurrence of allergic asthma, such as tryptase ε, MCP-1, CCL5, etc. can be released by MC. And the results of in vitro experiments showed that TMDCD inhibited the degranulation of RBL-2H3 cells stimulated by DNP-IgE/BSA. Taken together, we made the conclusion that TMDCD could reduce the infiltration of inflammatory cells in lung tissue and alleviate airway remodeling in mice with allergic asthma, showed the effects of anti-inflammatory and antiasthmatic. TMDCD could also reduce the levels of IgE, histamine, LTC4, Tryptase ε, and other MC related proteins in the serum of allergic asthma mice, and the in vitro experiments showed that TMDCD could inhibit IgE mediated degranulation and histamine release of RBL-2H3 cells, proved its anti allergic effect.

Keywords: Chinese herbal compound; TMDCD; allergic asthma; differential expression proteins; mast cells.

FIGURE 1

Schematic diagram of allergic asthma model establishment. Sensitization: The mixture of OVA and aluminum hydroxide gel was given to BALB/c mice (i.p.) at day 0 and day 14. Challenge: 1% OVA was given by aerosol inhalation to mice in all the groups except NC group (given PBS, instead) from day 21 to day 25, continuously. Treatment: The TMDCD group mice were given TMDCD by gavage after day 14, altogether for 12 days. From day 21 to day 25, 1 h before OVA challenge, gave dexamethasone to the mice in OVA + Dex group by gavage. The mice were sacrificed on day 26.

FIGURE 2

Detection of AHR. (A, B), after stimulation with MeCh, the difference and overall trend of Penh value among the groups. (C, D), after stimulation with MeCh, the difference and overall trend of Pause value among the groups. (E, F), the difference and overall trend of R_L values among groups after MeCh stimulation. (G, H), after stimulation with MeCh, the difference and overall trend of Cdyn values among groups. The results were expressed as Mean ± SEM, compared with NC group, *p < 0.05, *p < 0.01, *p < 0.001. Compared with OVA group, #p < 0.05, #p < 0.01, #p < 0.001. (WBPs: n = 5–6 mice/group, RCs: n = 3–5 mice/group. The results were from two independent experiments).

FIGURE 3

The number of eosinophils and IgE level in BALF. (A) Eosinophils count. (B) The level of IgE was detected by ELISA. The results were expressed as Mean ± SEM. Compared with NC group, **p < 0.01, ***p < 0.001, compared with OVA group, ##p < 0.01, ###p < 0.001 (n = 5 mice/group).

FIGURE 4

H&E and PAS staining of mice lung tissue. (A) Histopathology: infiltration of inflammatory cells around the bronchus (H&E staining, magnification×100). (B) Histopathology: secretion of mucus around bronchus (PAS staining, magnification×100). The results were expressed as Mean ± SEM. Compared with NC group, *p < 0.05, **p < 0.01, compared with OVA group, #p < 0.05 (n = 5 mice/group).

FIGURE 5

Total IgE, OVA specific IgE, histamine and LTC4 in serum were detected by ELISA. The results were expressed as Mean ± SEM. Compared with NC group, *p < 0.05, ***p < 0.001, compared with OVA group, ##p < 0.01, ###p < 0.001 (n = 5 mice/group).

FIGURE 6

The regulation of TMDCD on serum differential expressed proteins. The results were expressed as Mean ± SEM. Compared with NC group, *p < 0.05, **p < 0.01, ***p < 0.001, compared with OVA group, #p < 0.5, ##p < 0.01, ###p < 0.001 (n = 4–5 mice/group).

FIGURE 7

(A) Principal component analysis of differential expressed proteins. (B) Clustering analysis of differential expression proteins, A: NC group, B: OVA group, C: OVA + DEX group, D: OVA + TMDCD group. The color key indicates the protein expression level: red represents high-level expression, blue represents low-level expression. (C) GO pathway enrichment analysis of CC. (D) GO pathway enrichment analysis of MF (top 10). (E) GO pathway enrichment analysis of BP (top 10). (F) KEGG pathway enrichment analysis.

FIGURE 8

Effect of TMDCD on degranulation of mast cells. (A) Effect of TMDCD on the viability of RBL-2H3 cells. (B) Effect of TMDCD on the release of β-hexosaminidase. The results were expressed as Mean ± SEM. (C) Effect of TMDCD on histamine release. (D) Effect of TMDCD on morphology of RBL-2H3 cells sensitized by DNP-IgE/BSA. a. Blank group; b. Model group; c. Dexamethasone group; d. TMDCD 20 μg/ml group; e. TMDCD 40 μg/ml group; f. TMDCD 80 μg/ml group (toluidine blue staining, original magnification ×100). Compared with the control group, ***p < 0.001, compared with the model group, #p < 0.05, ##p < 0.01 and ###p < 0.001.