Xiaoye Fan’s research while affiliated with Jilin University and other places

Objective: Acetaminophen (APAP) is one of the world's popular and safe painkillers, and overdose can cause severe liver damage and even acute liver failure. The effect and mechanism of the xanthohumol on acetaminophen-induced hepatotoxicity remains unclear. Methods: The hepatoprotective effects of xanthohumol were studied using APAP-induced HepG2 cells and acute liver injury of mouse, seperately. Results: In vitro, xanthohumol inhibited H2O2- and acetaminophen-induced cytotoxicity and oxidative stress. Xanthohumol up-regulated the expression of Nrf2. Further mechanistic studies showed that xanthohumol triggered Nrf2 activation via the AMPK/Akt/GSK3β pathway to exert a cytoprotective effect. In vivo, xanthohumol significantly ameliorated acetaminophen-induced mortality, the elevation of ALT and AST, GSH depletion, MDA formation and histopathological changes. Xanthohumol effectively suppressed the phosphorylation and mitochondrial translocation of JNK, mitochondrial translocation of Bax, the activation o cytochrome c, AIF secretion and Caspase-3. In vivo, xanthohumol increased Nrf2 nuclear transcription and AMPK, Akt and GSK3β phosphorylation in vivo. In addition, whether xanthohumol protected against acetaminophen-induced liver injury in Nrf2 knockout mice has not been illustated. Conclusion: Thus, xanthohumol exerted a hepatoprotective effect by inhibiting oxidative stress and mitochondrial dysfunction through the AMPK/Akt/GSK3β/Nrf2 antioxidant pathway.

Epidemiologic studies have shown that fine particulate matter 2.5 (PM2.5) exaggerates airway inflammation associated with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Daphnetin (Daph) is a natural compound with a variety of biological activities. At present, there are limited data on whether Daph can protect against cigarette smoke (CS)-induced chronic obstructive pulmonary disease (COPD) and PM2.5-CS-induced AECOPD. Therefore, this study systematically evaluated the effects of Daph on CS-induced COPD and PM2.5-CS-induced AECOPD and determined its mechanism of action. First, in vitro studies showed that PM2.5 exacerbated cytotoxicity and NLRP3 inflammasome-mediated pyroptosis induced by low-dose cigarette smoke extracts (CSE). However, the effect was reversed by si-NLRP3 and MCC950. Similar results were obtained in PM2.5-CS-induced AECOPD mice. The results of the mechanistic studies suggested that blocking NLRP3 abolished PM2.5 combined with cigarette induced cytotoxicity, lung damage, NLRP3 inflammasome activation and pyroptosis in vitro and in vivo. Second, Daph suppressed the expression of NLRP3 inflammasome and pyroptosis in BEAS-2B cells. Third, Daph significantly protected against CS-induced COPD and PM2.5-CS-induced AECOPD by inhibiting the NLRP3 inflammasome and pyroptosis in mice. Our findings identified the NLRP3 inflammasome as a critical contributor to PM2.5-CS-induced airway inflammation, and Daph as a negative regulator of NLRP3-mediated pyroptosis, which has implications for the pathophysiology of AECOPD.

Background and Purpose Our previous research showed that ferroptosis plays a crucial role in the pathophysiology of PM2.5‐induced lung injury. The present study aimed to investigate the protective role of the Nrf2 signalling pathway and its bioactive molecule tectoridin in PM2.5‐induced lung injury by regulating ferroptosis. Experimental Approach We examined the regulatory effect of Nrf2 on ferroptosis in PM2.5‐induced lung injury and Beas‐2b cells using Nrf2‐knockout (KO) mice and Nrf2 siRNA transfection. The effects and underlying mechanisms of tectoridin on PM2.5‐induced lung injury were evaluated in vitro and in vivo. Key Results Nrf2 deletion increased iron accumulation and ferroptosis‐related protein expression in vivo and vitro, further exacerbating lung injury and cell death in response to PM2.5 exposure. Tectoridin activated Nrf2 target genes and ameliorated cell death caused by PM2.5. In addition, tectoridin prevented lipid peroxidation, iron accumulation and ferroptosis in vitro, but in siNrf2‐treated cells, these effects almost disappeared. In addition, tectoridin effectively mitigated PM2.5‐induced respiratory system damage, as evaluated by HE, PAS, and inflammatory factors. Tectoridin also augmented the antioxidative Nrf2 signalling pathway and prevented changes in ferroptosis‐related morphological and biochemical indicators, including MDA levels, GSH depletion and GPX4 and xCT downregulation, in PM2.5‐induced lung injury. However, the effects of tectoridin on ferroptosis and respiratory injury were almost abolished in Nrf2‐KO mice. Conclusion and Implications Our data proposed the protective effect of Nrf2 activation on PM2.5‐induced lung injury by inhibiting ferroptosis‐mediated lipid peroxidation and highlight the potential of tectoridin as a PM2.5‐induced lung injury treatment.

The increasing abundance of fine particulate matter (PM2.5) in the environment has increased susceptibility to acute exacerbation of COPD (AECOPD). During PM2.5 exposure, excessive reactive oxygen species (ROS) production triggers a redox imbalance, which contributes to damage to organelles and disruption of homeostasis. At present, there are limited data on whether NOX4/Nrf2 redox imbalance increases susceptibility to acute exacerbation of COPD (AECOPD), and the underlying mechanism is unclear. Therefore, the current study was aimed to evaluate the role of NOX4/Nrf2 redox balance on AECOPD induced by PM2.5-CS-exposure. Here, we report that PM2.5 exacerbates cytotoxicity by enhancing NOX4/Nrf2 redox imbalance-mediated mitophagy. First, exposure to a low-dose of PM2.5 (200 μg/ml) significantly exacerbated oxidative stress and mitochondrial damage by increasing the ROS overproduction, enhancing the excessive NOX4/Nrf2 redox imbalance, decreasing the mitochondrial membrane potential (MMP), and enhancing the mitochondrial fragmentation that were caused by a low-dose of CSE (2.5%). Second, coexposure to PM2.5 and CSE (PM2.5-CSE) induced excessive mitophagy. Third, PM2.5 exacerbated CS-induced COPD, as shown by excessive inflammatory cell infiltration, inflammatory cytokine production and mucus hypersecretion, goblet cell hyperplasia, NOX4/Nrf2 redox imbalance, and mitophagy, these effects triggered excessive ROS production and mitochondrial damage in mice. Mechanistically, PM2.5-CS-induced excessive levels of mitophagy by triggering redox imbalance, leading to greater cytotoxicity and AECOPD; however, reestablishing the NOX4/Nrf2 redox balance via NOX4 blockade or mitochondria-specific ROS inhibitor treatment alleviated this cytotoxicity and ameliorated AECOPD. PM2.5 may exacerbate NOX4/Nrf2 redox imbalance and subsequently enhance mitophagy by increasing the ROS and mito-ROS levels, thereby increasing susceptibility to AECOPD.

Background and purpose: Increasing evidence suggests that ferroptosis plays a key role in the pathophysiology of acute kidney injury (AKI) induced by cisplatin. The Nrf2 signaling pathway regulates oxidative stress and lipid peroxidation and positively regulates cisplatin-induced AKI (CI-AKI). However, its effect as well as an alkaloid compound leonurine hydrochloride (LH) on ferroptosis after CI-AKI remain unclear. Experimental approach: The anti-ferroptotic effects of Nrf2 and LH were assessed using a mouse model of cisplatin-induced AKI. In vitro, the potential effects of LH on erastin- and RSL3-induced HK-2 human PTEC ferroptosis were examined. Key results: As expected, Nrf2 deletion induced ferroptosis-related protein expression and iron accumulation in vivo, further aggravating CI-AKI. LH activated Nrf2 and prevented iron accumulation, lipid peroxidation and ferroptosis in vitro, while these effects were abolished in siNrf2-treated cells. Moreover, LH potently ameliorated cisplatin-induced renal damage, as indicated by the assessment of SCr, BUN, KIM-1, and NGAL. Importantly, LH activated the Nrf2 antioxidative signaling pathway and prohibited changes in ferroptosis-related morphological and biochemical indicators, such as the MDA level, SOD and GSH depletion and GPX4 and xCT downregulation, in CI-AKI. Moreover, Nrf2 KO mice were more susceptible to ferroptosis after CI-AKI than control mice, and the protective effects of LH on AKI and ferroptosis were largely abolished in Nrf2 KO mice. Conclusion and implications: These data suggest that the renal protective effects of Nrf2 activation on CI-AKI are achieved at least partially by inhibiting lipid peroxide-mediated ferroptosis and highlight the potential of LH as a CI-AKI treatment.

Many natural flavonoids can activate nuclear factor erythroid 2-related factor 2 (Nrf2), which is pivotal for alleviating various diseases related to inflammation and oxidative stress, including pleurisy. Amentoflavone (AMF), a biflavonoid extracted from many plants, has some beneficial bioactivities, especially anti-inflammatory and antioxidative activities. We aimed to investigate whether AMF protects against pleurisy and lung injury induced by carrageenan (Car) by activating Nrf2. Pleurisy was induced in wild-type (WT) and Nrf2-deficient (Nrf2-/-) mice. Then, pleural exudate and lung tissue were collected for biochemical analysis, H&E staining, immunocytochemistry and western blotting. Our results indicated that AMF protected against Car-induced pleurisy and lung injury. The Wright-Giemsa and H&E staining results showed that AMF alleviated inflammatory effusion and pathological injury. In addition, AMF decreased SOD and GSH depletion and MDA and MPO generation in the lung tissue of mice. AMF activated Nrf2 through keap-1 dissociation and subsequently increased heme oxygenase-1 (HO-1), NAD(P)H-quinone oxidoreductase 1 (NQO1), and γ-glutamylcysteine ligase (GCL) levels. Furthermore, AMF suppressed IL-1β and TNF-α levels and increased IL-10 levels in pleural exudate by blocking the proinflammatory NF-κB, signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) pathways induced by Car. However, these antioxidative and anti-inflammatory effects were weakened in Nrf2-/- mice. Moreover, AMF failed to suppress the NF-κB and STAT3 pathways in Nrf2-/- mice. Our results demonstrated that AMF exerted anti-inflammatory and antioxidative effects in Car-induced lung injury and pleurisy in a Nrf2-dependent manner.

Background and purpose: Increasing evidence suggests that ferroptosis plays a key role in the pathophysiology of acute kidney injury induced by cisplatin. The Nrf2 signaling pathway regulates oxidative stress and lipid peroxidation and positively regulates cisplatin-induced AKI (CI-AKI). However, Nrf2 and its activator leonurine on ferroptosis after CI-AKI remain unclear. Experimental Approach: The anti-ferroptotic effects of Nrf2 and its activator leonurine were assessed using a mouse model of cisplatin-induced AKI. In vitro, the potential effects of leonurine on erastin- and RSL3-induced HK-2 human PTEC ferroptosis were examined. Key Results: As expected, Nrf2 deletion induced ferroptosis-related protein expression and iron accumulation in vivo, further aggravating CI-AKI. The Nrf2 activator leonurine prevented iron accumulation and lipid peroxidation and inhibited ferroptosis in vitro, while these effects were abolished in siNrf2-treated cells. Moreover, leonurine potently ameliorated cisplatin-induced renal damage, as indicated by the assessment of SCr, BUN, KIM-1, and NGAL. Importantly, leonurine activated the Nrf2 antioxidative signaling pathway and prohibited changes in ferroptosis-related morphological and biochemical indicators, such as the MDA level, SOD and GSH depletion and GPX4 and xCT downregulation, in CI-AKI. Moreover, Nrf2 KO mice were more susceptible to ferroptosis after CI-AKI than control mice, and the protective effects of leonurine on AKI and ferroptosis were largely abolished in Nrf2 KO mice. Conclusion and Implications: These data suggest that the renal protective effects of Nrf2 and its activator leonurine on CI-AKI are achieved at least partially by inhibiting lipid peroxide-mediated ferroptosis and highlight the potential of leonurine as a CI-AKI treatment.

Gentamicin (GM), an aminoglycoside antibiotic, is one of the most effective drugs used in the treatment of various types of bacterial infections, but the major adverse effect and drug-induced nephrotoxicity of GM limit its clinical applications. Daphnetin (Daph) is a natural coumarin derivative that is clinically used to treat rheumatoid arthritis and coagulopathy and exhibits antioxidant effects. However, the effect of Daph on GM-induced nephrotoxicity has not yet been elucidated. This study investigated Daph-mediated protection against GM-induced nephrotoxicity in mice and explored the underlying mechanisms of GM-induced renal dysfunction in mice. We found that Daph treatment significantly reduced GM-induced nephrotoxicity mainly by ameliorating renal injury in mice and attenuating cell damage in vitro. Mechanistically, we found that Daph upregulated the expression level of Nrf2 and its regulated antioxidant enzymes HO-1, NQO1, GCLC and GCLM in vivo and in vitro. GM upregulated the expression levels of NOX4, cleaved Caspase-3 and p53 and the BAX/BCL2 ratio in vivo to stimulate oxidative stress and apoptosis. However, Daph treatment significantly improved the oxidative stress and apoptosis caused by GM, thereby exerting antioxidative and antiapoptotic effects. Our study was the first to suggest that the natural product Daph protects against GM-induced nephrotoxicity through the activation of Nrf2 which regulates oxidative stress and apoptosis. The pharmacological activation of Nrf2 may be useful as a novel therapy to prevent renal injury.

Autophagy-mediated cell death plays a critical role in the pathogenesis of PMs-induced lung injury. Hyperoside (Hyp), a flavonoid glycosides, is known to exert protective effects on many diseases by inhibiting autophagic activity. The current study aimed to explore the protective effect and mechanism of Hyp against PMs-induced lung injury in PM2.5 challenged Beas-2b cells in vitro and BALB/C mice in vivo. In vitro, we found that the organic solvent-extractable fraction of SRM1649b (O-PMs) caused more severe cytotoxicity in Beas-2b cells than the water solvent-extractable fraction of SRM1649b (W-PMs). O-PMs treatment dose-dependently upregulated the expression of autophagy markers (beclin-1, p62, atg3 and LC3II) and apoptotic proteins. This cytotoxicity of O-PMs was attenuated by Hyp pretreatment in parallel with downregulation of the expression of autophagy markers, apoptotic proteins, and p-AMPK and upregulation of p-mTOR expression. Notably, the therapeutic effect of Hyp was attenuated by pretreated with AICAR (an AMPK inducer), but enhanced by CC and 3-MA treatment. In vivo, Hyp reduced pathological lung injury and decreased the levels of PMs-induced inflammatory cytokines (TNF-α and IL-6), and the number of total cells in the BALF by inhibiting AMPK/mTOR signaling. Furthermore, cotreatment with AICAR (500 mg/kg) reduced but did not abrogate the pulmonary protective effect of Hyp. These findings indicate that Hyp protects against PMs-induced lung injury by suppressing autophagy deregulation and apoptosis through regulation of the AMPK/mTOR pathway.

Background Ovarian cancer is one of the most common gynecological malignancies in the world. Daphnetin (Daph) was previously reported to possess antitumor potential, but its potential and molecular mechanisms in ovarian cancer remain poorly understood. Purpose In the current study, we aimed to explore the antitumor effect and detailed mechanisms of Daph in ovarian cancer cells. Methods The cytotoxic effect of Daph on ovarian cells was determined in vitro and in vivo. Cell growth, proliferation, apoptosis and ROS generation were measured by CCK8 assays, colony formation assays and flow cytometry. Western blotting was used to evaluate the related signal proteins. Immunofluorescence and transmission electron microscopy were used to evaluate markers of autophagy and autophagic flux. The antitumor effects were observed in the A2780 xenograft model. Moreover, Daph-induced autophagy was observed by enhanced LC3-II accumulation and endogenous LC3 puncta, and an autophagy inhibitor further enhanced the antitumor efficacy of Daph, which indicated that the cytoprotective role of autophagy in ovarian cancer. Results We found that Daph exhibited antitumor effects by inducing ROS-dependent apoptosis in ovarian cancer, which could be reversed by N-acetyl cysteine (NAC). The AMPK/Akt/mTOR pathway was involved in Daph-mediated cytoprotective autophagy, and when Daph-mediated the expression level of AMPK and autophagy were blocked, there was robust inhibition of cell proliferation and induction of apoptosis. In addition, in the A2780 xenograft model, combined treatment with Daph and an autophagy inhibitor showed obvious synergetic effects on the inhibition of cell viability and promotion of apoptosis, without any side effects. Conclusion Our results suggest that Daph triggers ROS-induced cell apoptosis and induces cytoprotective autophagy by modulating the AMPK/Akt/mTOR pathway. Moreover, the combination of Daph and autophagy inhibitor may be a potential therapeutic strategy for ovarian cancer.