Pyrogallol protects against influenza A virus-triggered lethal lung injury by activating the Nrf2-PPAR-γ-HO-1 signaling axis.

Pyrogallol, a natural polyphenol compound (1,2,3-trihydroxybenzene), has shown efficacy in the therapeutic treatment of disorders associated with inflammation. Nevertheless, the mechanisms underlying the protective properties of pyrogallol against influenza A virus infection are not yet established. We established in this study that pyrogallol effectively alleviated H1N1 influenza A virus-induced lung injury and reduced mortality. Treatment with pyrogallol was found to promote the expression and nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2) and peroxisome proliferator-activated receptor gamma (PPAR-γ). Notably, the activation of Nrf2 by pyrogallol was involved in elevating the expression of PPAR-γ, both of which act synergistically to enhance heme oxygenase-1 (HO-1) synthesis. Blocking HO-1 by zinc protoporphyrin (ZnPP) reduced the suppressive impact of pyrogallol on H1N1 virus-mediated aberrant retinoic acid-inducible gene-I-nuclear factor kappa B (RIG-I-NF-κB) signaling, which thus abolished the dampening effects of pyrogallol on excessive proinflammatory mediators and cell death (including apoptosis, necrosis, and ferroptosis). Furthermore, the HO-1-independent inactivation of janus kinase 1/signal transducers and activators of transcription (JAK1/STATs) and the HO-1-dependent RIG-I-augmented STAT1/2 activation were both abrogated by pyrogallol, resulting in suppression of the enhanced transcriptional activity of interferon-stimulated gene factor 3 (ISGF3) complexes, thus prominently inhibiting the amplification of the H1N1 virus-induced proinflammatory reaction and apoptosis in interferon-beta (IFN-ß)-sensitized cells. The study provides evidence that pyrogallol alleviates excessive proinflammatory responses and abnormal cell death via HO-1 induction, suggesting it could be a potential agent for treating influenza.

Insights into the mechanism of action of pterostilbene against influenza A virus-induced acute lung injury.

BACKGROUND: Severe respiratory system illness caused by influenza A virus infection is associated with excessive inflammation and abnormal apoptosis in alveolar epithelial cells (AEC). However, there are limited therapeutic options for influenza-associated lung inflammation and apoptosis. Pterostilbene (PTE, trans-3,5-dimethoxy-4-hydroxystilbene) is a dimethylated analog of resveratrol that has been reported to limit influenza A virus infection by promoting antiviral innate immunity, but has not been studied for its protective effects on virus-associated inflammation and injury in AEC. PURPOSE: Our study aimed to investigate the protective effects and underlying mechanisms of PTE in modulating inflammation and apoptosis in AEC, as well as its effects on macrophage polarization during influenza virus infection. STUDY DESIGN AND METHODS: A murine model of influenza A virus-mediated acute lung injury was established by intranasal inoculation with 5LD50 of mouse-adapted H1N1 viruses. Hematoxylin and eosin staining, immunofluorescence, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, western blotting, Luminex and flow cytometry were performed. RESULTS: PTE effectively mitigated lung histopathological changes and injury induced by H1N1 viruses in vivo. These beneficial effects of PTE were attributed to the suppression of inflammation and apoptosis in AEC, as well as the modulation of M1 macrophage polarization. Mechanistic investigations revealed that PTE activated the phosphorylated AMP-activated protein kinase alpha (P-AMPKα)/sirtui1 (Sirt1)/PPARγ coactivator 1-alpha (PGC1α) signal axis, leading to the inhibition of nuclear factor kappa-B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling induced by H1N1 viruses, thereby attenuating inflammation and apoptosis in AEC. PTE also forced activation of the P-AMPKα/Sirt1/PGC1α signal axis in RAW264.7 cells, counteracting the activation of phosphorylated signal transducer and activator of transcription 1 (P-STAT1) induced by H1N1 viruses and the augment of P-STAT1 activation in RAW264.7 cells with interferon-gamma (IFN-γ) pretreatment before viral infection, thereby reducing H1N1 virus-mediated M1 macrophage polarization as well as the enhancement of macrophages into M1 phenotypes elicited by IFN-γ pretreatment. Additionally, the promotion of the transition of macrophages towards the M2 phenotype by PTE was also related to activation of the P-AMPKα/Sirt1/PGC1α signal axis. Moreover, co-culturing non-infected AEC with H1N1 virus-infected RAW264.7 cells in the presence of PTE inhibited apoptosis and tight junction disruption, which was attributed to the suppression of pro-inflammatory mediators and pro-apoptotic factors in an AMPKα-dependent manner. CONCLUSION: In conclusion, our findings suggest that PTE may serve as a promising novel therapeutic option for treating influenza-associated lung injury. Its ability to suppress inflammation and apoptosis in AEC, modulate macrophage polarization, and preserve alveolar epithelial cell integrity highlights its potential as a therapeutic agent in influenza diseases.

Pyrogallol promotes growth arrest by activating the p53-mediated up-regulation of p21 and p62/SQSTM1-dependent degradation of ß-catenin in nonsmall cell lung cancer cells.

Pyrogallol (1,2,3-trihydroxybenzene), a polyphenolic natural compound, has attracted considerable attention with regard to its potential anticancer activity. However, further study is needed to elucidate the underlying mechanism related to the antiNSCLC activity of pyrogallol and provide a comprehensive theoretical basis for better clinical utilization of pyrogallol. Our current study aims to investigate the effects and potential underlying mechanisms of pyrogallol on the inhibition of NSCLC growth. Our results showed that pyrogallol treatment induced cell cycle arrest at the G2/M phase and apoptosis in two different NSCLC cell lines. Mechanistically, we found that the induction of cell cycle arrest in NSCLC cells at the G2/M phase by pyrogallol was due to the upregulation of p21 in a p53-dependent manner. And blockade of p53 and p21 effectively abolished the cell cycle arrest at the G2/M phase. Meanwhile, p53 inhibition has been found to abrogate the pyrogallol-induced apoptosis of the two NSCLC cells. Moreover, we revealed that the inhibitory effects of pyrogallol on ß-catenin signaling resulted from autophagy initiation depending on p53 activation, accompanied by an increase in p62/SQSTM1 expression, thus p62 subsequently interacting with ubiquitinated ß-catenin and facilitating autophagic destruction of ß-catenin. Furthermore, in vivo experiments demonstrated that pyrogallol exerted growth inhibition on NSCLC with low toxicity through the same molecular mechanism as observed in vitro. Our findings could contribute to the understanding of the mechanism by which pyrogallol negatively regulates NSCLC growth, which could be effective in treating NSCLC.

Rosmarinic acid treatment protects against lethal H1N1 virus-mediated inflammation and lung injury by promoting activation of the h-PGDS-PGD2-HO-1 signal axis.

BACKGROUND: Rosmarinic acid (RosA) is a natural phenolic compound that possesses a wide-range of pharmacological properties. However, the effects of RosA on influenza A virus-mediated acute lung injury remain unknown. In this study, we aimed to explore whether RosA could protect against H1N1 virus-mediated lung injury and elucidate the underlying mechanisms. METHODS: Mice were intragastrically administered with RosA for 2 days before intranasal inoculation of the H1N1 virus (5LD50) for the establishment of an acute lung injury model. At day 7 post-infection (p.i.), gross anatomic lung pathology, lung histopathologic, and lung index (lung weight/body weight) were examined. Luminex assay, multiple immunofluorescence and flow cytometry were performed to detect the levels of pro-inflammatory cytokines and apoptosis, respectively. Western blotting and plasmid transfection with hematopoietic-type PGD2 synthase (h-PGDS) overexpression were conducted to elucidate the mechanisms. RESULTS: RosA effectively attenuated H1N1 virus-triggered deterioration of gross anatomical morphology, worsened lung histopathology, and elevated lung index. Excessive pro-inflammatory reactions, aberrant alveolar epithelial cell apoptosis, and cytotoxic CD8+ T lung recruitment in the lung tissues induced by H1N1 virus infection were observed to be reduced by RosA treatment. In vitro experiments demonstrated that RosA treatment dose-dependently suppressed the increased levels of pro-inflammatory mediators and apoptosis through inhibition of nuclear factor kappa B (NF-κB) and P38 MAPK signaling pathways in H1N1 virus-infected A549 cells, which was accompanied by promoting activation of the h-PGDS-PGD2-HO-1 signal axis. Furthermore, we strikingly found that h-PGDS inhibition significantly abrogated the inhibitory effects of RosA on H1N1 virus-mediated activation of NF-κB and P38 MAPK signaling pathways, resulting in diminishing the suppressive effects on the increased levels of pro-inflammatory cytokines and chemokines as well as apoptosis. Finally, suppressing h-PGDS prominently abolished the protective effects of RosA on H1N1 virus-mediated severe pneumonia and lung injury. CONCLUSIONS: Taken together, our study demonstrates that RosA is a promising compound to alleviate H1N1 virus-induced severe lung injury through prompting the h-PGDS-PGD2-HO-1 signal axis.

Diosmetin alleviates benzo[a]pyrene-exacerbated H1N1 influenza virus-induced acute lung injury and dysregulation of inflammation through modulation of the PPAR-γ-NF-κB/P38 MAPK signaling axis.

The severity of a viral respiratory illness was greatly exacerbated after exposure to a contaminant containing benzo[a]pyrene (B[a]P). Flavonoid-rich fruit intake has gained intense interest due to their health-promoting benefits for viral respiratory diseases, including influenza viruses. In our study, diosmetin (3',5,7-trihydroxy-4'-methoxyflavone), a naturally occurring hydroxylated methoxyflavone that is abundant in Citrus fruits, was explored for its effects on B[a]P-exacerbated H1N1 influenza virus-mediated inflammation and lung injury. Initially, in vivo results demonstrated that diosmetin protected against H1N1 virus-elicited acute lung injury. Simultaneously, H1N1 virus or B[a]P-stimulated A549 cells treated with diosmetin inhibited NF-κB and P-P38 activation, resulting in suppression of pro-inflammatory cytokines and apoptosis. Interestingly, diosmetin obviously promoted the expression of PPAR-γ as well as nuclear translocation of PPAR-γ, whereas, PPAR-γ inhibition by GW9662 weakened the inhibitory effects of diosmetin on H1N1 virus or B[a]P-mediated activation of NF-κB and P-P38, elevated expression of pro-inflammatory mediators as well as apoptosis. Furthermore, it was surprising to discover that mice exposed to both B[a]P and H1N1 viruses contributed to exacerbated acute lung injury, which were significantly ameliorated by diosmetin administration. In vitro studies showed that A549 cells with the combination of B[a]P and H1N1 virus augmented NF-κB and P-P38 activation, accompanied by higher levels of pro-inflammatory mediators and apoptosis, all of which were also significantly reduced by diosmetin treatment. Repressing PPAR-γ abrogated the inhibitory effects of diosmetin on B[a]P-exacerbated H1N1 virus-mediated NF-κB and P-P38 activation, inflammation, and apoptosis in A549 cells. Our findings suggest that diosmetin protected against B[a]P-exacerbated H1N1 virus-mediated lung injury by suppressing the exacerbation of NF-κB and P38 kinase activation in a PPAR-γ-dependent manner, suggesting potential benefits for B[a]P-exacerbated influenza-related illness therapeutics.

5-Methoxyflavone alleviates LPS-mediated lung injury by promoting Nrf2-mediated the suppression of NOX4/TLR4 axis in bronchial epithelial cells and M1 polarization in macrophages.

BACKGROUND: Acute lung injury (ALI) arises from sepsis or bacterial infection, which are life-threatening respiratory disorders that cause the leading cause of death worldwide. 5-Methoxyflavone, a methylated flavonoid, is gaining increased attention for its various health benefits. In the current study, we investigated the potential effects of 5-methoxyflavone against LPS-mediated ALI and elucidated the corresponding possible mechanism. METHODS: A mouse model with ALI was established by intratracheal instillation of LPS, and lung pathological changes, signaling pathway related proteins and apoptosis in lung tissues were estimated by H&E staining, immunofluorescence and TUNEL assay, respectively. Cell viability was evaluated by MTT assay; protein levels of pro-inflammatory mediators were measured by ELISA assay; levels of ROS and M1 macrophage polarization were assayed by flow cytometry; the expression of Nrf2 signaling, NOX4/TLR4 axis and P-STAT1 were detected by western blotting. RESULTS: Our results showed that 5-methoxyflavone treatment inhibited LPS-induced expression of NOX4 and TLR4 as well as the activation of downstream signaling (NF-κB and P38 MAPK), which was accompanied by markedly decreased ROS levels and pro-inflammatory cytokines (IL-6, TNF-α, MCP-1, and IL-8) in BEAS-2B cells. Moreover, we revealed that these effects of 5-methoxyflavone were related to its Nrf2 activating property, and blockade of Nrf2 prevented its inhibitory effects on NOX4/TLR4/NF-κB/P38 MAPK signaling, thus abrogating the anti-inflammatory effects of 5-methoxyflavone. Besides, the Nrf2 activating property of 5-methoxyflavone in RAW264.7 cells led to inhibition of LPS/IFN-γ-mediated STAT1 signaling, resulting in suppression of LPS/IFN-γ-induced M1 macrophage polarization and the repolarization of M2 macrophages to M1. In a mouse model of LPS-induced ALI, 5-methoxyflavone administration ameliorated LPS-mediated lung pathological changes, the increased lung index (lung/body weight ratio), and epithelial cell apoptosis. Meanwhile, we found 5-methoxyflavone effectively suppressed the hyperactive signaling pathways and the production of excessive pro-inflammatory mediators. Moreover, 5-methoxyflavone reduced LPS-mediated M1 macrophage polarization associated with elevated P-STAT1 activation in the lung tissues. In addition, 5-methoxyflavone improved the survival of LPS-challenged mice. CONCLUSION: These results indicated that 5-methoxyflavone might be suitable for the development of a novel drug for ALI therapeutic.

5-Methoxyflavone-induced AMPKα activation inhibits NF-κB and P38 MAPK signaling to attenuate influenza A virus-mediated inflammation and lung injury in vitro and in vivo.

Influenza-related acute lung injury (ALI) is a life-threatening condition that results mostly from uncontrolled replication of influenza virus (IV) and severe proinflammatory responses. The methoxy flavonoid compound 5-methoxyflavone (5-MF) is believed to have superior biological activity in the treatment of cancer. However, the effects and underlying mechanism of 5-MF on IV-mediated ALI are still unclear. Here, we showed that 5-MF significantly improved the survival of mice with lethal IV infection and ameliorated IV-mediated lung edema, lung histological changes, and inflammatory cell lung recruitment. We found that 5-MF has antiviral activity against influenza A virus (IAV), which was probably associated with increased expression of radical S-adenosyl methionine domain containing 2 (RSAD2) and suppression of endosomal acidification. Moreover, IV-infected A549 cells with 5-MF treatment markedly reduced proinflammatory mediator expression (IL-6, CXCL8, TNF-α, CXCL10, CCL2, CCL3, CCL4, GM-CSF, COX-2, and PGE2) and prevented P-IKBα, P-P65, and P-P38 activation. Interestingly, we demonstrated that 5-MF treatment could trigger activation of AMP-activated protein kinase (AMPK)α in IV-infected A549 cells, as evidenced by activation of the AMPKα downstream molecule P53. Importantly, the addition of AMPKα blocker compound C dramatically abolished 5-MF-mediated increased levels of RSAD2, the inhibitory effects on H1N1 virus-elicited endosomal acidification, and the suppression expression of proinflammatory mediators (IL-6, TNF-α, CXCL10, COX-2 and PGE2), as well as the inactivation of P-IKBα, P-P65, and P-P38 MAPK signaling pathways. Furthermore, inhibition of AMPKα abrogated the protective effects of 5-MF on H1N1 virus-mediated lung injury and excessive inflammation in vivo. Taken together, these results indicate that 5-MF alleviated IV-mediated ALI and suppressed excessive inflammatory responses through activation of AMPKα signaling.

Pyrogallol enhances therapeutic effect of human umbilical cord mesenchymal stem cells against LPS-mediated inflammation and lung injury via activation of Nrf2/HO-1 signaling.

The main challenges in clinical applications of mesenchymal stem cells (MSCs) are attributed to their heterogeneity. It is believed that preconditioning of MSCs with active compounds may enhance the expression of potentially therapeutic molecules and thus achieve stable and effective therapeutic outcomes. In the present study, we investigated the mechanism by which pyrogallol increased the therapeutic efficacy of human umbilical cord mesenchymal stem cells (hUCMSCs) against LPS-induced acute lung injury (ALI). hUCMSCs with pyrogallol treatment increased expression of HO-1 at both mRNA and protein levels, accompanied by Kelch-Like ECH-Associated Protein 1 (Keap1) degradation, and upregulation of the Nrf2 protein levels as well as nuclear translocation of Nrf2. Moreover, the modulation of Keap1 and Nrf2 as well as HO-1 upregulation by pyrogallol was reversed by pretreatment with N-acetylcysteine (NAC) and a P38 kinase inhibitor (SB203580). Whereas, NAC pretreatment abrogated pyrogallol-mediated activation of P38 kinase, indicating that pyrogallol-derived ROS led to P38 kinase activation, thus promoting Nrf2/HO-1 signaling. Additionally, we found that the induction of p62 by the pyrogallol-mediated ROS/P38/Nrf2 axis interacted with Keap1 and resulted in autophagic degradation of Keap1, which created a positive feedback loop to further release of Nrf2. Furthermore, the increased expression of HO-1 in pyrogallol-pretreated hUCMSCs led to enhanced inhibitory effects on LPS-mediated TLR4/P-P65 signaling in BEAS-2B cells, resulting in increasing suppression of LPS-indued expression of a series of pro-inflammatory mediators. Compared to untreated hUCMSCs, Sprague-Dawley (SD) rats with pyrogallol-primed hUCMSCs transplantation showed enhanced improvements in LPS-mediated lung pathological alterations, the increased lung index (lung/body ratio), apoptosis of epithelial cells, the activation of TLR4/NF-κB signaling as well as the release of pro-inflammatory mediators. Together, these results suggested that hUCMSCs with pyrogallol pretreatment enhanced the therapeutic efficacy of hUCMSCs, which may provide a promising therapeutic strategy to maximize the therapeutic efficacy of hUCMSC-based therapy for treating LPS-associated ALI.

Arctiin suppresses H9N2 avian influenza virus-mediated inflammation via activation of Nrf2/HO-1 signaling.

BACKGROUND: H9N2 avian influenza viruses (AIVs) infect avian and mammalian hosts and provide internal genes for new emerging highly pathogenic avian viruses that cause severe pneumonia with high mortality, for which few medications are available. Arctiin, a bioactive lignan glycoside, has been reported to possess multiple pharmacological properties. However, the effect of arctiin on H9N2 virus infection is unclear. In the current study, we analyzed the effect of arctiin on H9N2 virus infection and the underlying molecular mechanism in vitro. METHODS: The antiviral effect against H9N2 virus was determined by plaque reduction assay (PRA) and progeny virus reduction assay. We employed MTT assay, qRT-PCR, ELISA, immunofluorescence and Western blotting to better understand the anti-inflammatory effect and corresponding mechanism of arctiin on H9N2 virus-infected cells. RESULTS: The results showed that arctiin had antiviral activity against H9N2 virus. Arctiin treatment reduced H9N2 virus-triggered proinflammatory cytokines, such as IL-6, and TNF-α. Moreover, arctiin significantly suppressed H9N2 virus-mediated expression of COX-2 and PGE2. Furthermore, we found that arctiin inhibited H9N2 virus-mediated activation of RIG-I/JNK MAPK signaling. Interestingly, arctiin treatment obviously reversed H9N2 virus-induced reduction of Nrf2, increased the nuclear translocation of Nrf2, and upregulated Nrf2 signaling target genes (HO-1 and SOD2). Zinc protoporphyrin (Znpp)-an HO-1 inhibitor-weakened the inhibitory effect of arctiin on H9N2 virus-induced RIG-I/JNK MAPK and proinflammatory mediators. CONCLUSION: Taken together, these results suggested that the anti-inflammatory effects of arctiin on H9N2 virus infection may be due to the activation of Nrf2/HO-1 and blocked RIG-I/JNK MAPK signaling; thus, arctiin may be a promising agent for prevention and treatment of H9N2 virus infections.