Shikonin alleviates asthma phenotypes in mice via an airway epithelial STAT3-dependent mechanism

Abstract

Background: Asthma is an inflammatory disease where the balance between Th1/Th2 and Th17/Treg plays a crucial role in its pathogenesis. Shikonin is used to treat a variety of autoimmune diseases due to its good anti-inflammatory activity. However, the effect and mechanism of shikonin on asthma remain unknown.

Method: Mice were sensitized with ovalbumin (OVA)/house dust mite (HDM) and treated with shikonin. Lung inflammation was assessed histologically and via flow cytometry. Bronchoalveolar lavage fluid (BALF) was analyzed for cell counts and cytokines. Shikonin's impact on p-STAT3 was studied in vivo and in vitro.

Results: Shikonin inhibited OVA or HDM-induced inflammation and airway hyperresponsiveness. Upon treatment, a restoration of the Th1/Th2 and Th17/Treg balance was observed, evidenced by a reduction in IL-4 and IL-17A levels in BALF, alongside an elevation in interferon-gamma and IL-10. Furthermore, shikonin impeded the infiltration of eosinophils, neutrophils, macrophages, and lymphocytes into lung tissue. The observed decrease in STAT3 phosphorylation and diminished nuclear translocation of p-STAT3 confirmed that shikonin promotes the balance of Th1/Th2 and Th17/Treg by regulating airway epithelial STAT3.

Conclusion: Shikonin mitigates asthma symptoms through a STAT3-dependent mechanism, indicating its potential as an anti-asthmatic therapeutic agent.

Keywords: STAT3; Th1/Th2; Th17/Treg; asthma; shikonin.

Figure 1

Shikonin alleviates the phenotypes of OVA- and HDM-induced asthma mice. (a) HE staining was used to detect the inflammatory infiltration of the central airway in the lung sections of mice. (b) HE staining was used to detect peripheral airway inflammatory infiltration in the lung sections of mice. (c) Central airway inflammation score. (d) Peripheral airway inflammation score. (e) Semi-quantitative score of inflammatory infiltration in the lung sections of mice. (f) The effect of shikonin on airway reactivity in asthma mice. Compared with the control group, **p < 0.01; compared with the OVA group, ^## p < 0.01; compared with the HDM group, ^△△ p < 0.01, n = 3.

Figure 2

Shikonin alleviates the imbalance of Th1/Th2 and Th17/Treg in OVA- and HDM-induced asthma mice. Flow cytometry was used to evaluate the percentages of CD4⁺ IFN-γ⁺ Th1 cells (a), CD25⁺ FOXP3⁺ Treg cells (b), CD4⁺ IL-4⁺ Th2 cells (c), and CD4⁺ IL-17⁺ Th17 cells (d) in lung tissues. Compared with the control group, ^** p < 0.01; compared with the OVA group, ^## p < 0.01; compared with the HDM group, ^△△ p < 0.01, n = 3.

Figure 3

Shikonin alleviates immune cell infiltration and regulates the expression of Th cell-related cytokines. (a) The number of inflammatory cells, eosinophils (Eos), neutrophils (Neu), macrophages (Mac), and lymphocytes (Lym) in bronchoalveolar lavage fluid (BALF). (b) The concentration of Th cell-related inflammatory factors in BALF was detected by ELISA. Compared with the control group, ^** p < 0.01; compared with the OVA group, ^# p < 0.05; compared with the OVA group, ^## p < 0.01; compared with the HDM group, ^△△ p < 0.01, n = 3.

Figure 4

Shikonin suppresses the expression and localization of p-STAT3 in airway epithelial cells. (a) Western blot analysis was conducted to examine the phosphorylation level of STAT3 in mouse airway epithelial cells. (b) The expression pattern of p-STAT3 in airway epithelial cells was detected by immunofluorescence staining. Compared with the control group, ^** p < 0.01; compared with the OVA group, ^## p < 0.01; compared with the HDM group, ^△△ p < 0.01, n = 3.

Figure 5

Activation of STAT3 can reverse the inhibitory effect of shikonin on the function of MLE-12 cells. (a) Western blot analysis was performed to measure the expression levels of STAT3 and p-STAT3 in mouse epithelial cell line MLE-12. (b) The localization of p-STAT3 in mouse bronchial epithelial cell line MLE-12 was detected by immunofluorescence staining. (c) Western blot was applied to measure the expression levels of HIF-α, IFN-γ, IL-1β, and MMP9. (d) ELISA was used to detect the concentrations of inflammatory factors (IL-1β and IFN-γ) in the MLE-12 cell medium. Compared with the control group, ^** p < 0.01; compared with the model group, ^## p < 0.01; compared with the shikonin group, ^△ p < 0.05; compared with the shikonin group, ^△△ p < 0.01, n = 3.