In the present study, we provide evidence that RESV can suppress CSE- induced endothelial apoptosis via autophagy induction. RESV treatment also resulted in an increased expression of active Notch1 in HUVECs. DAPT significantly blocked Notch-1 activation and increased apoptotic protein expression, which can be partially reversed by RESV. Furthermore, the protective autophagy induced by RESV in HUVECs depends on a Notch1 signaling. Taken together, these data suggest that restoration of Notch1 signaling and induction of autophagy play critical roles in the protective effect of resveratrol on CSE- induced endothelial apoptosis.
Apoptosis of pulmonary structural cells including pulmonary endothelial cells is significantly elevated in human smokers with COPD [3, 9], and mice with emphysema caused by CS exposure [4, 5]. Our previous studies also demonstrated that CSE triggers apoptosis of endothelial cells in vitro [6–8, 10]. Consistent with previous studies, the present study further confirmed the promoting effect of CSE on the apoptosis of HUVECs. It has been shown that direct induction of pulmonary endothelial cell apoptosis using an endothelial cell-specific peptide results in emphysematous lung destruction. Furthermore, inhibition of endothelial apoptosis effectively prevented the emphysematous changes [4, 5]. These results highlight a crucial role of pulmonary endothelial cell apoptosis in the pathogenesis of CS related COPD. Therefore, preventing CS-induced pulmonary endothelial cell apoptosis and strengthening the pulmonary endothelial barrier may be an effective therapeutic strategy for COPD.
RESV is a polyphenol naturally found in a variety of plant species such as grapes, apples, berries, and peanuts. It is well known to have beneficial effects, including anti-inflammatory, anti-oxidative stress and anti-apoptosis [11, 18]. The anti-apoptotic effects of RESV have been widely investigated in multiple organs, including lungs. Ceramide was reported to be up-regulated in the lungs of COPD patients and CS exposed mice, and was shown to be associated with the increased apoptosis of bronchial epithelial cells . RESV protects bronchial epithelial cells from CS-induced apoptosis in vitro and in mouse models through attenuating the accumulation of ceramide . As a natural activator of SIRT1, RESV is capable of protecting bronchial epithelial cells against CSE-induced apoptosis through activating SIRT1 . The results indicated the antiapoptotic function of RES in bronchial epithelial cells in the condition of CS exposure. However, few studies investigated whether it has a similar protective effect on CSE-induced endothelial cell apoptosis. In the present study, we have strong evidence that that resveratrol attenuates CSE- induced HUVEC apoptosis. Pretreatment with RESV remarkably attenuated the expression of cleaved caspase-3, an indicator for cell apoptosis, in the presence of CSE. Moreover, its protective effect against HUVEC apoptosis under CSE is associated with autophagy. Autophagy is an essential intracellular process responsible for the selective degradation of pathogens, damaged organelles and proteins that cannot be degraded by the proteasome, to support protein and organellar homeostasis, as well as recycling of biomolecular resources, to maintain energy homeostasis and cell survival . Emerging evidence indicates that RESV attenuates endothelial oxidative injury in HUVECs by inducing autophagy[13, 24]. Here, in the present study we found that blocking autophagy with 3-MA aggravated the apoptosis by upregulating caspase3 expression, while inducing autophagy with rapamycin relieved the apoptosis, indicating that autophagy confers the ability to protect endothelial cells from CSE- induced apoptosis. Moreover, we observed a promoting effect of RESV on autophagy by inhibiting mTOR signaling pathway. Cells treated with RESV showed a decrease in the expression of mTOR protein, resulting in an upregulation in Beclin1 and LC3b-II expression, with a decrease in p62 levels compared to those without RESV treatment. However, the exact underlying mechanism remains unclear and requires further investigation.
Nocth1 receptor protein is one of the four single-pass transmembrane receptors in Notch system, which controls cell proliferation, differentiation, and apoptosis. Notch signal activation involves the ligand-receptor interaction from the surface of two neighbouring cells, which triggers two successive proteolytic cleavages and release of the NICD from the full-length heteromeric receptor. Then, NICD travels to the nucleus where it interacts with the DNA-binding protein and converts it into a potent transcriptional activator . Our previous studies have demonstrated that Notch-1 was downregulated in lung tissue of COPD compared to those of non-smokers . Furthermore, overexpressing Notch-1 has been shown to alleviate CSE mediated endothelial apoptosis [6, 7]. Recently, several studies have shown that Notch1 is involved in the autophagic process [17, 26]. We speculate that autophagy may be related to the protective effect of Notch1 against endothelial apoptosis. We found that Notch1 expression was decreased after CSE exposure in consistent with the previous studies. Notch1 inhibition leads to an increase in the expression of mTOR and a decrease Beclin1 and LC3b-II expression accompanied with increased p62 levels, as well as an increase in endothelial apoptosis. Overexpression of Notch1 induced autophagy by inhibiting mTOR signaling, meanwhile attenuated the HUVECs apoptosis in endothelial cells. Furthermore, rapamycin reversed the cell apoptosis resulting from the inhibition of Notch1, while 3-MA blocked the anti-apoptotic effect of Notch1 signaling. These results suggested that Notch1 heavily inhibits cell apoptosis of HUVECs by promoting mTOR-mediated autophagy.
Recent years, RESV has received increasing attention for its role in regulating the Notch signaling. However, conflicting results of RESV effects on Notch1 signaling were reported that RESV can enhance [14, 27] or suppress  Notch1 in a cell type dependent manner. In this work, we examined what role RESV plays in HUVECs in the condition of CSE exposure. We observed that RESV treatment significantly restored the Notch1 protein levels suppressed by CSE. Furthermore, supplementation with RESV improves the cell apoptosis in HUVECs resulted from the inhibition of Notch1 signaling. Taken together, the results from the present study indicated that RESV elicits its protective effect against CSE-induced apoptosis through regulating Notch1-mediated autophagy.