The main findings of the present study are that artesunate significantly suppressed CS-induced airway inflammation as well as airway remodeling in vivo and in vitro. The effect of artesunate was associated with the CS-induced airway remodeling by targeting PPAR-γ/TGF-β1/Smad2/3 signaling.
Experimental studies have already confirmed that CS exposure directly contributed to the changes in structural cells seen in the lung tissue and small airways caused by airway inflammation and oxidative stress . Thus, to some extent, inhibiting the inflammatory response, oxidative stress, as well as airway remodeling may offer viable choices for the therapy of COPD. First, we established a 12-week CS-exposure rat model to evaluate the therapeutic effects of artesunate, and we found that artesunate treatment reduced the levels of IL-6, IL-8, TNF-α, ICAM-1, ROS and GSH in the BALF of CS-exposed rats in a dose-dependent manner. These results are consistent with another study showing that artesunate could modulate multiple inflammatory and oxidative stress mediators in CS or ovalbumin exposure mice model .
Previous studies on both animals and humans have shown that CS induces the production of pro-inflammatory cytokines such as IL-6, IL-8, TNF-α, and ICAM-1 as well as ROS and GSH, which amplify the inflammatory process and play an integral role in the coordination and persistence of inflammatory process that occur in airway remodeling of COPD patients [29, 30]. This pathological changes in the lungs exposed to CS were significantly ameliorated by artesunate treatment. Importantly, isometric force increase and lung function decline, including PIF, PEF, IP and IP-sloPe, as representatives of airway hyperresponsiveness and small airway resistance, respectively, were also ameliorated. Besides its anti-malaria property, artesunate has also been reported to show a variety of pharmacological activities. For example, artesunate was reported to reduce lung damage in CS-induced mice , which is consistent with our results. Artesunate ameliorated CS-induced airway inflammation, inhibited the PI3Kδ/Akt pathway, restored HDAC2 activity, consequently reversing CSE-induced glucocorticoid insensitivity . All these findings have prompted us to further characterize the underlying mechanisms in detail. Therefore, artesunate, as a multi-target drug, provides a novel use for the repurposing of ‘old’ drugs in airway remodeling, a key feature of COPD and asthma . To further validate the animal experiment results, cellular experiments were performed in HBSM cells. It was widely accepted that CSE has a significant effect on airway abnormal contractility and proliferation in HBSM cells . Consistently, our results were consistent with these findings and further demonstrated that artesunate inhibited HBSM cells proliferation and reduced levels of the proliferation marker cyclin D1 as well as α-SMA, a marker of myofibroblast in vivo and in vitro. However, previous studies have reported that artesunate suppressed the proliferation of human leukemic cells proliferation by regulating c-Myb and cyclin D2 expression . Moreover, artesunate significantly inhibited the proliferation of hepatoma cell line via STAT3 inhibition and DR4 augmentation . In our study, we observed an increase in HBSM cells proliferation caused by CSE can be inhibited by artesunate, accompanied with the recoveries of cyclin D1 and α-SMA expresisons, revealing the different mechanisms of artesunate in inhibiting cell proliferation in various cell systems.
It has been reported that PPAR-γ activation reduces lipopolysaccharide-induced inflammation in mice model, suggesting that an activator of PPAR-γ may have a beneficial effect the inflammatory response of COPD . In fact, several evidences have suggested the reduced level and activity of PPAR-γ in the lungs of both CS-exposed mice lungs as well as in the lung of smokers and COPD patients [38, 39]. Consistently, our data demonstrated that PPAR-γ expression in both rat lungs and HBSM cells were significantly inhibited in response to CS, which is in agreement with previous reports that CS can inhibit PPAR-γ activation . Whereas treatment with artesunate significantly enhanced PPAR-γ activation in vivo and in vitro, which is in agreement with previous reports that PPAR-γ agonists reversed CS-induced airway injury in bronchial epithelial cells . Thus, we have reason to speculate that the activation of PPAR-γ may involve the protection of artesunate for CS-induced airway inflammation and remodeling.
It has been reported that TGF-β1-Smad signaling pathway coud be activated by CS in bronchial rat explants, which is identified as the key signaling pathway in EMT and airway remodeling [11, 42]. It has been reported that TGF-β1/Smad2 pathway was significantly activated in bronchial smooth muscle cells exposed to CS . More important, the inhibition of the TGF-β1 gene by PPAR-γ activation can be applied for treating TGF-β1-induced pathophysiologic disorders such as fibrosis . We further investigated in our this study whether artesunate’s effect involve the TGF-β1 signaling via activation of PPAR-γ. We found that PPAR-γ knockdown and the inactivation of TGF-β1/Smad signaling pathway attenuated the effect of artesunate on CS-induced cell proliferation in vitro, which is consistent with previous study on the activation of PPAR-γ in various cells and diseases . For instance, activation of PPAR-γ in myeloid cells could promote the progression of epithelial lung tumors through the regulation of TGF-β1 signaling pathway. PPAR-γ expression was increased in NSCLC cell lines, and knockdown of PPAR-γ inhibited EMT . Therefore, our results further confirmed that the effect of artesunate by ameliorating CS-induced bronchial remodeling involved in PPAR-γ/TGF-β1/Smad2/3 signaling pathway.