Mitophagy is a special type of autophagy, which plays a cellular protective role by clearing damaged mitochondria and reducing reactive oxygen species [28,29]. Mitophagy-mediated elimination of mitochondria is involved in many cellular processes including early embryonic development, cell differentiation, and apoptosis [30]. Accumulating evidence indicated that mitophagy functions and maintains health throughout life. Mitophagy defects are associated with various pathological processes such as neurodegeneration, heart failure, cancer, and aging [31]. Currently, there are limited reports about the relationship between mitophagy and LADC. Chang et al. have reported that high PINK1 expression (a marker of mitophagy) is an independent prognostic factor of LADC, which is correlated with poor response to chemotherapy [32]. In this study, we reported that mitophagy inhibition by elaiophylin could induce intracellular and mitochondrial ROS and suppress cell viability in in vitro and in vivo models of LADC. In addition, elaiophylin showed a slightly cytotoxic effect on human lung fibroblasts (MRC5), which were often used to evaluate the safety of anticarcinogen [33,34,35].The Sirtuins family is a class of NAD+-dependent deacetylases that participates in a large number of important redox reactions, such as gluconeogenesis, glycolysis, tricarboxylic acid cycle, oxidative respiratory chain, etc. [36]. The dysregulation of the Sirtuins family plays an important role in various human prevalent diseases such as tumors, cardiovascular diseases, neuronal diseases, liver diseases, inflammation, and aging [32,37]. Sirtuins are known to control autophagy and mitophagy in cancers by acting on transcription factors or proteins related to autophagy and mitophagy mechanisms [38]. It is shown that increased ROS production occurs when cells are under stress. Additionally, the co-enzyme NAD activates sirtuins and regulates the activity of antioxidant reactive elements (AREs), which in turn regulates transcription of pro- and antioxidant genes to maintain redox cascades [39,40]. As for the previous literature, sirtuins have been linked to the control of autophagy and mitophagy by modulating transcription of autophagy and mitophagy genes, by post-translational modification of proteins belonging to the autophagy and mitophagy machinery [38]. Then, we put focus on the involvement of sirtuins in the elaiophylin’s anti-mitophagy effect and found that SIRT1 was the significantly regulated protein among sirtuins by proteomic assay and Western blot analysis. SIRT1, one of the most extensively and thoroughly studied sirtuins family proteins, is a key player in maintaining homeostasis against DNA damage, aging, and apoptosis under oxidative stress [39]. Then, molecular docking further clarified that SIRT1 might be the direct target of elaiophylin. Further analysis revealed that elaiophylin impacted the expression and deacetylase activity of SIRT1. Mitophagy relies heavily on two factors: the PTEN-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase Parkin [41,42]. These proteins are responsible for sensing the function and health status of mitochondria and selecting damaged mitochondria for autophagy processing [43]. The link between SIRT1 and mitophagy was first demonstrated by Hwang’s group. They found that nicotinamide significantly extended the replication life of primary human fibroblasts by accelerating mitophagy degradation [44]. It was also shown that niacinamide-induced mitophagy is mediated by increased NAD+/NADH ratio and SIRT1 activation, and the SIRT1 activator SRT1720 can be used to mimic nicotinamide-induced mitochondrial phenotype [45]. Nowadays, more and more studies have evidenced that SIRT1 participates in the PINK1-Parkin labeled mitophagy process to regulate cellular oxidative stress, which may have therapeutic values [46,47,48,49]. To present, several studies reported that SIRT1 overexpression promotes the occurrence of LADC, which is closely related to its invasion and metastasis leading to poor prognosis [50,51,52]. Consistently, the current study indicated that elaiophylin increased the accumulation of autophagy surface marker protein LC3B and SQSTM1 (p62), decreased the expressions of PINK1 and Parkin, and inhibited the fusion of autophagosome in the mitochondria with lysosomes. However, SIRT1 overexpression only in A549 cells has no effect on the mitophagy. Meanwhile, SIRT1 overexpression plus SIRT1 activator SRT1720 treatment significantly attenuated the cellular effect of elaiophylin, indicating elaiophylin may exert its anti-mitophagy effect via regulating SIRT1 signaling. Future studies will investigate whether elaiophylin directly binds with SIRT1, probably using the SPR (surface plasmon resonance) method.As a NAD-dependent deacetylase, SIRT1 regulates downstream cellular pathways in relation to oxidative stress by deacetylating various transcription factors to maintain cell survival [53,54]. Previous studies have observed that the SIRT1/Nrf2 signaling pathway is associated with various cellular responses related to oxidative stress [55,56]. Recently, it has been reported that Nrf2 is an important downstream target of SIRT1. Nrf2 is known to be an important antioxidant sensor in cellular defense mechanisms, responding to oxidative damage [57,58]. In normal cells, activation of NRF2 helps prevent the initiation of cancer by chemical carcinogens; however, in many tumor types, NRF2 is permanently upregulated and its overexpressed target genes support the promotion and progression of cancer by suppressing oxidative stress [59]. Activation of Nrf2 has also been shown to protect cells from oxidative damage induced by electrophilic compounds [60]. Once Nrf2 is activated by electrophilic compounds, it translocates to the nucleus and binds to the electrophilic response element (ERE), which further regulates the proteins involved in electrophilic detoxification and elimination, thereby enhancing the cell’s antioxidant capacity [61]. It was noticed that SIRT1 significantly enhanced Nrf2 pathway activity and inhibited ROS overproduction by decreasing the expression of heme oxygenase 1(HO-1) to facilitate Nrf2 translocating to the cytoplasm [56,60]. A large number of studies have found that Nrf2 is highly expressed in A549 cells. Targeting Nrf2 activity can inhibit the growth of LADC and eliminate the resistance to chemotherapy [62,63,64]. In this study, we found that elaiophylin inhibited the deacetylation of Nrf2, leading to reduced stability of Nrf2 by promoting Nrf2 entering the cytoplasm. In addition, the reduced expression and transcriptional activity of Nrf2 were in a SIRT1-dependent manner. However, Nrf2 overexpression only in A549 cells has no effect on mitophagy. In addition, Nrf2 overexpression plus Nrf2 activator DMF treatment could significantly attenuate the anti-mitophagy effect of elaiophylin. Compared to the previous study in UM, we found that SIRT1 was the direct target of elaiophylin by molecular docking and the SIRT1-Nrf2 pathway was activated in regulating mitophagy upon elaiophylin, which was not consistent with the previous study. However, there are still some limitations to this study. First, surface plasmon resonance or other methods need to be conducted to confirm the direct interaction of elaiophylin and SIRT1, and then an orthotopic transplanted mouse model needs to be constructed in the further study which is closer to the environment of the tumor.Overall, our data suggested that elaiophylin has a potent cytotoxic effect on LADC by inhibiting mitophagy and increasing oxidative stress. Specifically, elaiophylin exerted its effect by suppressing the deacetylation of Nrf2 in a SIRT1-dependent manner, leading to the increase in non-functional Nrf2 in the cytoplasm (Figure 9). Therefore, as a new autophagy inhibitor, elaiophylin may be developed into a new therapeutic regimen for LADC. Of course, whether it can be used for clinical treatment still needs further research.