Phytotherapeutic BS012 and Its Active Component Ameliorate Allergic Asthma via Inhibition of Th2-Mediated Immune Response and Apoptosis

Abstract

Asthma is a chronic inflammatory disorder of the lungs that results in airway inflammation and narrowing. BS012 is an herbal remedy containing Asarum sieboldii, Platycodon grandiflorum, and Cinnamomum cassia extracts. To elucidate the anti-asthma effect of BS012, this study analyzed the immune response, respiratory protection, and changes in metabolic mechanisms in an ovalbumin-induced allergic asthma mouse model. Female BALB/c mice were exposed to ovalbumin to induce allergic asthma. Bronchoalveolar lavage fluid and plasma were analyzed for interleukin and immunoglobulin E levels. Histological analyses of the lungs were performed to measure morphological changes. Apoptosis-related mediators were assayed by western blotting. Plasma and lung tissue metabolomic analyses were performed to investigate the metabolic changes. A T-helper-2-like differentiated cell model was used to identify the active components of BS012. BS012 treatment improved inflammatory cell infiltration, mucus production, and goblet cell hyperplasia in lung tissues. BS012 also significantly downregulated ovalbumin-specific immunoglobulin E in plasma and T-helper-2-specific cytokines, interleukin-4 and -5, in bronchoalveolar lavage fluid. The lungs of ovalbumin-inhaled mice exhibited nerve growth factor-mediated apoptotic protein expression, which was significantly attenuated by BS012 treatment. Ovalbumin-induced abnormalities in amino acid and lipid metabolism were improved by BS012 in correlation with its anti-inflammatory properties and normalization of energy metabolism. Additionally, the differentiated cell model revealed that N-isobutyl-dodecatetraenamide is an active component that contributes to the anti-allergic properties of BS012. The current findings demonstrate the anti-allergic and respiratory protective functions of BS012 against allergic asthma, which can be considered a therapeutic candidate.

Keywords: Apoptosis; Asthma; Inflammation; Metabolomics; Ovalbumin.

Fig. 1

Scheme of the experimental procedure (A) and effect of BS012 on the levels of ovalbumin-specific IgE and Th2-specific cytokines production in ovalbumin-induced asthma mice. The level of (B) OVA-specific IgE in plasma, (C) BALF IL-4, and (D) BALF IL-5 was measured. All the data are expressed as the mean ± SEM (n=5 per group). *** Significantly different (p<0.001) from Control group. ^##, ^### Significantly different (p<0.01, 0.001) from OVA group.

Fig. 2

Assessment of effect of BS012 on histological examination of lung tissues by H&E staining. (A) H&E-stained sections of lung tissues (original magnification ×200). (B) Measured the thickness of bronchial epithelium. Data are presented as mean ± SEM (n=5 per group). *** Significantly different (p<0.001) from Control group. ^#, ^## Significantly different (p<0.05, 0.01) from OVA group. Assessment of effect of BS012 on goblet cell hyperplasia in lung tissue by PAS staining. (C) PAS staining results of the lung tissues (original magnification ×200), mucus is stained as a purple color. (D) Quantitative analyses of PAS positive cells in bronchial. Data are presented as mean ± SEM (n=5 per group). *** Significantly different (p<0.001) from Control group. ^### Significantly different (p<0.001) from OVA group.

Fig. 3

Effect of BS012 on OVA-induced activation of NGF-mediated JNK signaling pathway in the lungs. The protein expression levels of (A) NGF, (B) p75NTR, (C) P-JNK/JNK, and (D) P-c-Jun/c-Jun activation in the lung tissues were determined via western blot analysis. Quantitative analysis was performed by densitometric analysis. All the data are expressed as the mean ± SEM (n=5 per group). *** Significantly different (p<0.001) from Control group. ^#, ^##, ^### Significantly different (p<0.05, 0.01, 0.001) from OVA group.

Fig. 4

Effect of BS012 on regulating (A-C) Bcl-2 family related apoptosis proteins and (D-F) cytochrome c-initiated caspase activation pathway in ovalbumin-induced asthma mice lung tissues. Expression levels of (A) P-Bad/Bad, (B) Bax, and (C) Bcl-xl, (D) cytochrome C, (E) cleaved-caspase 9/caspase-9, and (F) cleaved-caspase 3/caspase-3 proteins in mice lung tissues were detected by western blot analysis. Quantified protein expression levels were performed by densitometric analysis. Data are presented as mean ± SEM (n=5 per group). **, *** Significantly different (p<0.01, 0.001) from Control group. ^#, ^##, ^### Significantly different (p<0.05, 0.01, 0.001) from ovalbumin group.

Fig. 5

Changes in plasma metabolome in OVA-induced asthma mice administered with BS012 at 100, 200 mg/kg, and dexamethasone 1.5 mg/kg. The partial least squares discriminant analysis (PLS-DA) score plot derived from (A) positive and (B) negative ionization mode. (C) Heatmap summarizing the relative levels of metabolites significantly changed in the OVA-induced group compared to the control group or BS012 treated group compared to the OVA-induced group. Relative levels represent the standardized intensity values by auto scaling (the value divided by the mean center and the standard deviation of each variable).

Fig. 6

Changes in lung tissue metabolome in OVA-induced asthma mice administered with BS012 at 100, 200 mg/kg, and dexamethasone 1.5 mg/kg. The partial least squares discriminant analysis (PLS-DA) score plot derived from (A) positive and (B) negative ionization mode. (C) Heatmap summarizing the relative levels of metabolites significantly changed in the OVA-induced group compared to the control group or BS012 treated group compared to the OVA-induced group. Relative levels represent the standardized intensity values by auto scaling (the value divided by the mean center and the standard deviation of each variable).

Fig. 7

Overall metabolic pathways altered in systemic (plasma) and lung tissue of OVA-induced asthma mice after BS012 administration. Red arrows indicate significant changes between the OVA-induced group and the control group, while blue arrows denote significant changes between the BS012 treated group and the OVA-induced group.

Fig. 8

Effect of N-isobutyl-dodecatetraenamide on cytokine production in CD4⁺ T cells under Th2-skewing condition. Bone marrow cells were obtained from the femur and tibia of naïve mice. Dendritic cells (DC) were prepared for co-culture by performing CD11c MACS enrichment in bone marrow derived dendritic cells. CD4⁺ T cells were enriched by collecting and pooling spleens and peripheral lymph nodes from 6-week male C57BL/6N mice, followed by CD4 MACS enrichment. The enriched samples were identified and flow-sorted for naïve CD4⁺ gated as CD4⁺CD44^lowCD25-CD62L^hi using FACSAria III cell sorter. For DC - CD4⁺ T cells co-culture assays, cells were co-cultured in complete media with soluble anti-CD3ε Ab (clone 145-2C11), anti-IFNγ (clone XMG1.2), IL-2, IL-4 in the presence of N-isobutyl-dodecatetraenamide for 3 days. Intracellular cytokines were analyzed in CD4⁺ T cells under Th2-skewing condition targeting (A) IL-4 and (B) IL-13. From the CD4⁺ T cells co-cultured with dendritic cells, (C) IL-13 production was analyzed. All the data are expressed as the mean ± SEM (n=5 per group). *, **, *** Significantly different (p<0.05, 0.01, 0.001) from Control group.