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.

Nobiletin protects against ferroptosis to alleviate sepsis-associated acute liver injury by modulating the gut microbiota.

Nobiletin (NOB), a plant-based polymethoxyflavone, is a promising protective agent against sepsis; yet the mechanisms were not fully elucidated. The gut microbiota is found to be strongly associated with sepsis-associated acute liver injury (SALI). Here, our study aimed to evaluate the protective effect of NOB on SALI and explore the underlying molecular mechanisms. Cecal ligation and puncture (CLP) was used to induce SALI in mice. NOB was administered by gavage for 7 days before CLP induction. The 16S rRNA gene sequencing and fecal microbiota transplantation (FMT) were performed to verify the function of the gut microbiota. The markers of ferroptosis, inflammation, gut microbiota composition, and liver injury were determined. NOB administration significantly alleviated hepatic ferroptosis and inflammation in septic mice. Meanwhile, NOB upregulated the expression levels of nuclear factor E2-related factor 2 (Nrf2) and its downstream protein heme oxygenase-1 (HO-1). The protective effect of NOB administration against ferroptosis in SALI mice was reversed by the Nrf2 inhibitor ML385. Additionally, increased abundances of Ligilactobacillus, Akkermansia, and Lactobacillus, and decreased abundances of Dubosiella and Bacteroides in the gut were observed under NOB administration, suggesting that NOB might modulate the gut microbiota composition of septic mice. Furthermore, gut microbiota ablation by antibiotic treatment partly reversed the protective effects of NOB on sepsis. FMT also confirmed that NOB inhibited ferroptosis and activated Nrf2 signalling in SALI mice by modulating the gut microbiota. These results revealed that, by modulating the gut microbiota, NOB attenuated ferroptosis in septic liver injury through upregulating Nrf2-Gpx4. Our findings provide novel insights into microbiome-based therapeutic approaches for sepsis.

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.

NF­κB/IκBα signaling pathways are essential for resistance to heat stress­induced ROS production in pulmonary microvascular endothelial cells.

The results of a previous study demonstrated that heat stress (HS) triggered oxidative stress, which in turn induced the apoptosis of epithelial cells. These results uncovered a novel mechanism underlying the activation of NF­κB in primary human umbilical vein endothelial cells. The present study aimed to further investigate the role of NF­κB/IκBα signaling pathways in the inhibition of HS­induced reactive oxygen species (ROS) generation and cytotoxicity in endothelial cells. The results of the present study demonstrated that HS triggered a significant amount of NF­κB and IκBα nuclear translocation without IκBα degradation in a time­dependent manner. Mutant constructs of IκBα phosphorylation sites (Ser32, Ser36) were employed in rat pulmonary microvascular endothelial cells (PMVECs). Cell Counting Kit­8 assays demonstrated that both the small interfering (si)RNA­mediated knockdown of p65 and IκBα mutant constructs significantly decreased cell viability and aggravated ROS accumulation in HS­induced rat PMVECs compared with the control. Additionally, western blot analysis revealed that p65 siRNA attenuated the protein expression of IκBα. However, IκBα mutant constructs failed to attenuate NF­κB activation and nuclear translocation, indicating that IκBα­independent pathways contributed to NF­κB activity and nucleus translocation in a time­dependent manner following HS. Collectively, the results of the present study suggested that the NF­κB/IκBα pathway was essential for resistance to HS­induced ROS production and cytotoxicity in rat PMVECs, and that it could be a potential therapeutic target to reduce the mortality and morbidity of heat stroke.

[Clinical features of severe or critical ill patients with COVID-19].

OBJECTIVE: To analyze the clinical features of severe or critical ill adult patients with coronavirus disease (COVID-19). METHODS: The clinical data of 75 patients with severe or critical COVID-19 in Honghu People's Hospital from January to March in 2020 were collected. RESULTS: Of the 75 patients with COVID-19 pneumonia, 41 were male (54.67%) and 34 were female (45.33%) with a mean age of 67.53 ±12.37 years; 43 patients had severe and 32 had critical COVID-19, and 49.3% of the patients had underlying diseases. The main clinical manifestations included fever (78.67%) and coughing (70.67%). Compared with the severe patients, the critically ill patients had higher proportions of patients over 60 years old with elevated white blood cell count, increased prothrombin time, and higher levels of hsCRP, PCT, D-dimer, ALT, LDH, cTnI and NT-proBNP. Univariate logistic regression analysis showed that an age over 60 years, leukocytosis, hs-CRP elevation, prolonged prothrombin time, and increased levels of D-dimer, NT-proBNP and cTnI were associated with severe COVID-19. Multivariate logistic regression showed that an age over 60 years (OR=8.165, 95% CI: 1.483-45.576, P=0.017), prolonged prothrombin time (OR=7.516, 95% CI: 2.568-21.998, P=0.006) and elevated NT-proBNP (OR=6.194, 95% CI: 1.305-29.404, P=0.022) were independent risk factors for critical type of COVID-19. CONCLUSIONS: An age over 60 years, a prolonged prothrombin time and elevated NT-proBNP level are important clinical features of critically ill patients with COVID-19, and can be deemed as early warning signals for critical conditions of the disease.

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.

Oxidative stress regulates mitogen­activated protein kinases and c­Jun activation involved in heat stress and lipopolysaccharide­induced intestinal epithelial cell apoptosis.

Heat stress and gut­derived endotoxinemia are common causes of multiple organ dysfunction syndrome in heat stroke patients. Evidence has demonstrated that cell apoptosis in the small intestine serves an important role in the pathogenesis of heatstroke, which leads to increased intestinal permeability to endotoxin or lipopolysaccharides (LPS) from the gut entering the circulation. However, little is known about the potential underlying mechanisms mediating heat stress combined with LPS­induced intestinal epithelial cell apoptosis. In the present study, LPS combined with heat stress induced production of reactive oxygen species (ROS), mitochondrial membrane potential disruption and cell apoptosis, which eventually led to increased intestinal permeability and reduced epithelial resistance in the IEC­6 cell line. Inductions in ROS, mitochondrial membrane potential disruption and cell apoptosis were detected by using an ROS assay kit, 5,5',6,6'­tetrachloro­1,1',3,3'tetraethylbenzimidazo carbocyanine iodide dye kit and annexin V­fluorescein isothiocyanate apoptosis kit, respectively. The effect of ROS on mitogen activated protein kinases (MAPKs) and c­Jun activation was investigated using the antioxidant drug, butylated hydroxyanisole (BHA) by western blotting. The results of the present study demonstrated that ROS is essential to activate p38, extracellular signal­regulated kinase (ERK) and c­Jun, but not c­Jun N­terminal kinase (JNK), in LPS combined with heat stress treated cells. Furthermore, ROS, and activation of p38, JNK and c­Jun, were revealed to serve pro­apoptosis roles which aggravated damage to epithelial barrier integrity, as assessed by flow cytometry using Annexin V­fluorescein isothiocyanate staining and pretreatment of cells with specific inhibitors of ROS, JNK, p38 and c­Jun (BHA, SP600125, SB203580 and c­Jun peptide, respectively). Transepithelial electrical resistance and horseradish peroxidase permeability were detected in cells treated with LPS combined with heat stress, which revealed that ERK serves an anti­apoptosis role, as determined by pretreatment of cells with PD98059, a specific inhibitor of ERK. In conclusion, these findings suggested a novel role of the ROS signaling pathway which involved activation of MAPKs and c­Jun, following LPS combined with heat stress­induced IEC­6 cell apoptosis and impairment of the epithelial barrier. These results may facilitate understanding of pathological conditions involving ROS, such as heat stroke.