In recent years, increasing attention is being paid to asthma treatments due to the increasing incidence of asthma. Various polysaccharides are now considered as functional food or potential medicines to affect the development of asthma 36–37. Our experiment firstly found that E. sibiricum bulb polysaccharides (E1P and ESBP) could improved the pulmonary pathological states, decreased the infiltration of inflammatory of lung cells and prevented airway remodelling in asthmatic mice. The action mechanisms of E1P and ESBP were also explored based on the Th1/Th2 and Th17/Treg balances.
Based on evidence, asthma is a kind of chronic disease with typical symptoms of airway inflammation, hyper-responsiveness and airway remodelling 38. Airway inflammation involves multitudinous activated cells, such as eosinophils, neutrophils, monocytes and basophils, and eosinophils have been implicated in eosinophilic asthma with the exacerbation of illness 39. For this reason, many therapeutics that aim to limit the numbers of eosinophils or deplete them are currently widely used to treat asthma. Pathologic mucus is challenging to clear due to its high viscosity and elasticity, thereby efficiently contributing to airway blockage 40–41. As a biomarker of Th2-induced inflammation, elevated serum IgE has been considered to induce the exacerbation of asthma 42. In the present study, the results of counting inflammatory cells and HE staining suggested that E1P and ESBP could reduce pulmonary inflammation in asthma. The data of PAS and HE indicated that the accumulation of mucus and airway remodelling in asthmatic mice were prevented by the two polysaccharides. The decreased serum IgE level of asthmatic mice reconfirmed the anti-asthmatic effect of E. sibiricum bulb polysaccharides.
The Th1/Th2 balance is critical for developing asthma. Asthma presents with an increase in Th2 cytokines and a decrease in Th1 cytokines. As the essential Th2 cytokines, IL-4 and IL-13 participate in the pathogenesis of asthma. An increase in IL-4 level can facilitate allergic sensitisation, switching to IgE and eosinophilia 43. IL-13 also influences IgE production and eosinophil chemotaxis, and participates in smooth muscle contractility and goblet cell hyperplasia in different ways from IL-4 44. The Th1 cytokine IFNγ has the effects of suppressing fibroblast activity and antagonising the functions of Th2 cytokines 45. Given that the differentiation of Th1 and Th2 cells was achieved by specific transcription factors, the abundance of related transcription factors could affect the balance between Th1 and Th2 cells. Gata3 is crucial to the differentiation of Th cells into Th2, and T bet is a master determinant of Th1 cells because the deficiency of T bet leads to the lack of Th1 immune responses 46. Mutual restrain also exists between T bet and Gata3. Therefore, the imbalance between T bet and Gata3 changes the Th1/Th2 balance 47. We also examined the mRNA abundance and the levels of protein expression of T bet and Gata3 in the lung. Our experimental data indicated that, in cytokines level, the imbalance of Th1/Th2 caused by asthma was reversed by ESBP and E1P. The difference was that ESBP increased the Th1 cytokine IFNγ and simultaneously decreased the Th2 cytokines IL-4 and IL-13, but E1P only reduced Th2 cytokines in asthma. Meanwhile, ESBP and E1P could reduce the elevated quantity of Gata3 mRNA in the OVA-challenged group, and raised the transcription level of T bet that reduced by the OVA challenge. The experimental data of protein expression levels were consistent with the RT-PCR findings, confirming that ESBP and E1P can regulate the Th1/Th2 balance by simultaneously reducing Gata3 and increasing T bet expression in asthmatic reactions. Therefore, E. sibiricum bulb polysaccharides treated asthma by balancing Th1/Th2.
Along with the development of mechanism research of asthma, the Th17/Treg balance gradually came into prominence. In the cytokine level, the decreased cytokine production of Treg cells on asthma and the overproduction of Th17 cytokine exacerbate asthma 48. As a typical cytokine of Th17 cells, IL-17 could promote neutrophilia in the lungs and mucus accumulation, thereby contributing to goblet cell hyperplasia and airway remodelling 49. IL-10, a representative cytokine of Treg cells, affects asthma by diverse mechanisms in different situations because IL-10 could suppress Th2 and Th17 immune responses 50. At the protein transcriptional level, TGFβ not only affects the differentiation of Th17 and Treg cells but also airway epithelia into myofibroblasts 7, thereby confirming that TGFβ is pivotal to airway remodelling and inflammation of asthma. Th17 cells are believed to help induce the inflammation of asthma, and their crucial molecule RORγt implicates the differentiation and maturation of Th17 cells 51. Conversely, the functions of Foxp3 include inhibition of Th17 differentiation and driving Treg differentiation 52. In the present study, ESBP reversed the cytokine levels of Treg and Th17 cells induced by OVA challenge while E1P only decreased the level of IL-17. The treatment of E1P and ESBP suppressed TGFβ and RORγt expression but significantly increased Foxp3 expression in the lung tissue. These finding suggested the effects of E. sibiricum bulb polysaccharides on suppression of Th17 cells’ function and the enhancement of Treg cells.
The improvement of polysaccharides solubility is beneficial to enhance absorption and bioavailability in vivo, which can confer polysaccharides with more diversified functions and wider application 53. Therefore, from the perspective of solubility, E1P might have further development potential over ESBP. However, regard to the differences in pharmacological action between E1P and ESBP, the result differs from the reports suggesting that enzyme-assisted extraction could enhance the bioactivity of polysaccharides 54–56. Compared with ESBP, E1P extracted with enzyme assistance was deficient in the effects of increasing IFNγ and IL-10 cytokines, RORγt mRNA and T bet protein. Different structures and physicochemical properties of polysaccharides contribute to changes in their bioactivities 57. The amylase used to extract E1P might destroy the 1, 4-glycosidic bonds of polysaccharides, and more amylopectin or pectic polysaccharides can be released 58. Thus, the structure and composition of E1P might be changed and different from ESBP. The structural differences and structure–effect relationship between E1P and ESBP deserve further study.