NOBILETIN AMELIORATES HEATSTROKE-INDUCED ACUTE LUNG INJURY BY INHIBITING FERROPTOSIS VIA P53/SLC7A11 PATHWAY.

ABSTRACT: The molecular mechanism for nobiletin's protective effect against heatstroke-induced acute lung injury (HS-ALI) remains largely unknown. Previous research has demonstrated that ferroptosis is an important pathogenic event in HS-ALI. Nobiletin is a natural polymethoxylated flavonoid. Herein, we investigated the potential contribution of nobiletin to HS-ALI by inhibiting ferroptosis. Heat stress was used to induce HS-ALI in mice, and mouse lung epithelial-12 (MLE-12) cells were stimulated by heat stress in vitro . Nobiletin was administrated by gavage for 2 h before HS induction. Biochemical kits, immunofluorescence staining, and western blotting were performed on the markers of ferroptosis. Our results showed that nobiletin administration significantly attenuated HS-induced lung injury and ferroptosis. Moreover, nobiletin pretreatment significantly reversed HS-induced p53 upregulation in vivo and in vitro . Pretreatment with a p53 agonist, tenovin-6, partly abolished the protective effect of nobiletin in mice with HS-ALI. Meanwhile, p53 knockdown significantly increased GPX4 and SLC7A11 expression levels compared with the HS group in HS-induced MLE-12 cells. Subsequently, nobiletin ameliorated HS-induced MLE-12 cells ferroptosis by activating the SLC7A11/GPX4 pathway, whereas p53 overexpression effectively abolished the protective effect of nobiletin. Taken together, our findings reveal that nobiletin attenuates HS-ALI by inhibiting ferroptosis through the p53/SLC7A11 pathway, indicating it to be a potential therapeutic agent for HS-ALI prevention and treatment.

SIRT1-mediated p53 deacetylation inhibits ferroptosis and alleviates heat stress-induced lung epithelial cells injury.

OBJECTIVE: Acute lung injury (ALI) is a common complication of heat stroke (HS) and a direct cause of death. However, the mechanism underlying ALI following HS remains unclear. METHOD: To investigate whether ferroptosis is involved in HS-ALI. We established a HS model of mice and mouse lung epithelial-2 cells (MLE-2). The severity of lung injury was measured by H&E staining, the wet-to-dry lung weight ratio, and Transmission electron microscopy. Potential markers of ferroptosis Fe2+, malondialdehyde (MDA), hydroxynonenal (4-HNE) and lipid peroxidation were detected. The percentages of cell death and viability induced by HS were assessed by LDH and CCK8 assays. SLC7A11, ACSL4, GPX4, SIRT1, p53, and p53 K382 acetylation levels were measured by Western blot. RESULTS: The administration of ferroptosis inhibitor ferrostatin-1(Fer-1) could significantly ameliorate lung injury, inhibiting levels of MDA and 4-HNE, and ameliorating HS-induced increased ACSL4, decreased SLC7A11 and GPX4, suggesting ferroptosis was involved in HS-induced ALI in vivo and in vitro. Moreover, SIRT1 expression decreased, and p53 K382 acetylation levels increased in MLE-2 cells. Activation of SIRT1 could improve lung epithelial ferroptosis caused by HS in vivo ang in vitro. Besides, the activation of SIRT1 could significantly reduce the p53 K382 acetylation levels, suggesting that activation of SIRT1 could prevent ferroptosis via inhibiting p53 acetylation. CONCLUSION: These findings substantiate the vital role of the SIRT1/p53 axis in mediating ferroptosis in HS-ALI, suggesting that targeting SIRT1 may represent a novel therapeutic strategy to ameliorate ALI during HS.

Cytosolic p53 Inhibits Parkin-Mediated Mitophagy and Promotes Acute Liver Injury Induced by Heat Stroke.

Heat stroke (HS) is a severe condition characterized by increased morbidity and high mortality. Acute liver injury (ALI) is a well-documented complication of HS. The tumor suppressor p53 plays an important role in regulation of mitochondrial integrity and mitophagy in several forms of ALI. However, the role of p53-regulated mitophagy in HS-ALI remains unclear. In our study, we discovered the dynamic changes of mitophagy in hepatocytes and demonstrated the protective effects of mitophagy activation on HS-ALI. Pretreatment with 3-MA or Mdivi-1 significantly exacerbated ALI by inhibiting mitophagy in HS-ALI mice. Consistent with the animal HS-ALI model results, silencing Parkin aggravated mitochondrial damage and apoptosis by inhibiting mitophagy in HS-treated normal human liver cell line (LO2 cells). Moreover, we described an increase in the translocation of p53 from the nucleus to the cytoplasm, and cytosolic p53 binds to Parkin in LO2 cells following HS. p53 overexpression using a specific adenovirus or Tenovin-6 exacerbated HS-ALI through Parkin-dependent mitophagy both in vivo and in vitro, whereas inhibition of p53 using siRNA or PFT-α effectively reversed this process. Our results demonstrate that cytosolic p53 binds to Parkin and inhibits mitophagy by preventing Parkin's translocation from the cytosol to the mitochondria, which decreases mitophagy activation and leads to hepatocyte apoptosis in HS-ALI. Overall, pharmacologic induction of mitophagy by inhibiting p53 may be a promising therapeutic approach for HS-ALI treatment.

Impact of UCP2 depletion on heat stroke-induced mitochondrial function in human umbilical vein endothelial cells.

OBJECTIVE: The incidence rate of heat stroke (HS) has increased, with high morbidity and mortality rates, in recent years. Previous studies have suggested that vascular endothelial cell injury is one of the main pathological features of HS. Uncoupling protein 2 (UCP2) exhibits antioxidant activity under various stress conditions. This study aims to investigate the role of UCP2 in HS-induced vascular endothelial injury. METHOD: To explore the mechanisms mediating vascular endothelial cell injury induced by HS, we established an HS model of HUVECs in vitro. The percentage of cell death and viability induced by HS were assessed using annexin V-FITC/PI staining and CCK8 assays. HS-induced mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining. HS-induced mitochondrial superoxide was measured by MitoSOX staining, and analyzed by flow cytometry. UCP2, Drp1, phosphorylated Drp1, OPA1, and Mfn2 expression levels were measured by western blotting. RESULTS: HS triggered mitochondrial fragmentation and UCP2 upregulation in a time-dependent manner in HUVECs. As a specific Drp1 inhibitor, Mdivi-1 pretreatment significantly promoted mitochondrial fission and apoptosis in HS-induced HUVECs. In addition, siRNA-mediated UCP2 knockdown further aggravated mitochondrial fragmentation and ΔΨm depolarization and increased mitochondrial ROS production and cell apoptosis in HS-induced HUVECs, which were abolished by Drp1 inhibition. CONCLUSION: Our results indicate that UCP2 protects against HS-induced vascular endothelial damage and that it enhances mitochondrial function. These findings reveal that UCP2 can be a potential contributor to mechanism-based therapeutic strategies for HS.

Erector spinae plane block for postoperative analgesia in breast and thoracic surgery: A systematic review and meta-analysis.

STUDY OBJECTIVE: The erector spinae plane block (ESPB) is a newly defined regional anesthesia technique first introduced in 2016. The aim of this study is to determine its analgesic efficacy compared with non-block care and thoracic paravertebral block (TPVB). DESIGN: We systematically searched PubMed, Web of Science citation index, Embase, the Cochrane Library, Google Scholar, and ClinicalTrials.gov register searched up to March 2020. We conducted a meta-analysis of randomized controlled trials (RCTs) that compared an ESPB to non-block care or TPVB for postoperative analgesia in breast and thoracic surgery patients. Primary outcome was 24-hour postoperative opioid consumption. Risk of bias was assessed using Cochrane methodology. RESULTS: 14 RCTs that comprised 1018 patients were included. Seven trials involved thoracic surgery patients and seven included breast surgery patients. Meta-analysis revealed that ESPB significantly reduced 24-hour opioid consumption compared with the non-block groups (-10.5 mg; 95% CI: -16.49 to -3.81; p = 0.002; I2 = 99%). Similarly, the finding was consistent in subgroup analysis between the breast surgery (-7.75 mg; 95%CI -13.98 to -1.51; p = 0.01; I2 = 97%) and thoracic surgery (-14.81 mg; 95%CI -21.18 to -8.44; p < 0.001; I2 = 96%) subgroups. The ESPB significantly reduced pain scores at rest or movement at various time points postoperatively compared with non-block group, and reduced the rate of postoperative nausea and vomiting (OR 0.48; 95%CI 0.27 to 0.86; p = 0.01; I2 = 0%). In contrast, there were no significative differences reported in any of the outcomes for ESPB versus TPVB strata. CONCLUSIONS: ESPB improved analgesic efficacy in breast and thoracic surgery patients compared with non-block care. Furthermore, current literature supported the ESPB offered comparable analgesic efficacy to a TPVB.

Heat stress induces RIP1/RIP3-dependent necroptosis through the MAPK, NF-κB, and c-Jun signaling pathways in pulmonary vascular endothelial cells.

Necroptosis represents a newly defined form of regulated necrosis and participates in various human inflammatory diseases. It remains unclear whether necroptosis is presented in heatstroke-induced lung injury. We show that heat stress(HS) triggered an significant upregulation of receptor-interacting protein 1 (RIP1) and mixed lineage kinase domain-like protein (MLKL) expression in a time-dependent manner, without a significant change of receptor-interacting protein 3 (RIP3). Furthermore, co-immunoprecipitation assays showed that RIP1 binds to RIP3 to form the necrosome in heat stress-induced PMVECs. In vitro, necrostatin-1 (Nec-1) pre-treatment reduced heat stress-induced PMVECs necroptosis, which also inhibited HMGB1 translocation from the nucleus into the cytoplasm. Similarly, inhibition for ERK (PD98059), NF-κB (BAY11-7082) and c-Jun (c-Jun peptide), respectively, also suppressed the HMGB1 cytoplasm translocation. Furthermore, siRNA-mediated RIP1/RIP3 knockdown negatively regulated the release of HMGB1 in HS-induced necroptosis through the ERK, NF-κB, and c-Jun signaling pathways. Our study reveals that HS induces RIP1/RIP3-dependent necroptosis through the MAPK, NF-κB, and c-Jun signaling pathways in PMVECs.