Tubule-mitophagic secretion of SerpinG1 reprograms macrophages to instruct anti-septic acute kidney injury efficacy of high-dose ascorbate mediated by NRF2 transactivation.

High-dose ascorbate confers tubular mitophagy responsible for septic acute kidney injury (AKI) amelioration, yet its biological roles in immune regulation remain poorly understood. Methods: The role of tubular mitophagy in macrophage polarization upon high-dose ascorbate treatment was assessed by fluorescence-activated cell sorter analysis (FACS) in vitro and by immunofluorescence in AKI models of LPS-induced endotoxemia (LIE) from Pax8-cre; Atg7 flox/flox mice. The underlying mechanisms were revealed by RNA-sequencing, gene set enrichment analysis (GSEA), luciferase reporter, chromatin immunoprecipitation (ChIP) and adeno-associated viral vector serotype 9 (AAV9) delivery assays. Results: High-dose ascorbate enables conversion of macrophages from a pro-inflammatory M1 subtype to an anti-inflammatory M2 subtype in murine AKI models of LIE, leading to decreased renal IL-1ß and IL-18 production, reduced mortality and alleviated tubulotoxicity. Blockade of tubular mitophagy abrogates anti-inflammatory macrophages polarization under the high-dose ascorbate-exposed coculture systems. Similar abrogations are verified in LIE mice with tubular epithelium-specific ablation of Atg7, where the high-dose ascorbate-inducible renal protection and survival improvement are substantially weaker than their control littermates. Mechanistically, high-dose ascorbate stimulates tubular secretion of serpin family G member 1 (SerpinG1) through maintenance of mitophagy, for which nuclear factor-erythroid 2 related factor 2 (NRF2) transactivation is required. SerpinG1 perpetuates anti-inflammatory macrophages to prevent septic AKI, while kidney-specific disruption of SerpinG1 by adeno-associated viral vector serotype 9 (AAV9)-short hairpin RNA (shRNA) delivery thwarts the anti-inflammatory macrophages polarization and anti-septic AKI efficacy of high-dose ascorbate. Conclusion: Our study identifies SerpinG1 as an intermediate of tubular mitophagy-orchestrated myeloid function during septic AKI and reveals a novel rationale for ascorbate-based therapy.

Interruption of neutrophil extracellular traps formation dictates host defense and tubular HOXA5 stability to augment efficacy of anti-Fn14 therapy against septic AKI.

The immunosuppressive, inflammatory microenvironment orchestrated by neutrophil extracellular traps (NETs) plays a principal role in pathogenesis of sepsis. Fibroblast growth factor-inducible molecule 14 (Fn14) has been established as a potential target for septic acute kidney injury (AKI), making further therapeutic benefits from combined NETs and Fn14 blockade possible. Methods: The concurrence of NETs and Fn14 in mice and patients with septic AKI were assessed by immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and in silico studies. Survival, histopathological and biochemical analyses of wild-type and PAD4-deficient CMV-Cre; PAD4 fl/fl mice with septic AKI were applied to evaluate the efficacy of either pharmacological or genetic NETs interruption in combination with Fn14 blockade. Molecular mechanisms underlying such effects were determined by CRISPR technology, fluorescence-activated cell sorter analysis (FACS), cycloheximide (CHX) pulse-chase, luciferase reporter and chromatin immunoprecipitation (ChIP) assay. Results: NETs formation is concurred with Fn14 upregulation in murine AKI models of abdominal, endotoxemic, multidrug-resistant sepsis as well as in serum samples of patients with septic AKI. Pharmacological or genetic interruption of NETs formation synergizes with ITEM-2, a monoclonal antibody (mAb) of Fn14, to prolong mice survival and provide renal protection against abdominal sepsis, the effects that could be abrogated by elimination of macrophages. Interrupting NETs formation predominantly perpetuates infiltration and survival of efferocytic growth arrest-specific protein 6+ (GAS6+) macrophages in combination with ITEM-2 therapy and enhances transcription of tubular cell-intrinsic Fn14 in a DNA methyltransferase 3a (DNMT3a)-independent manner through dismantling the proteasomes-mediated turnover of homeobox protein Hox-A5 (HOXA5) upon abdominal sepsis challenge or LPS stimuli. Pharmacological NETs interruption potentiates the anti-septic AKI efficacy of ITEM-2 in murine models of endotoxemic and multidrug-resistant sepsis. Conclusion: Our preclinical data propose that interrupting NETs formation in combination with Fn14 mAb might be a feasible therapeutic strategy for septic AKI.

Prognostic Immune-Related Analysis Based on Differentially Expressed Genes in Left- and Right-Sided Colon Adenocarcinoma.

BACKGROUND: Colon adenocarcinoma (COAD) can be divided into left-sided and right-sided COAD (LCCs and RCCs, respectively). They have unique characteristics in various biological aspects, particularly immune invasion and prognosis. The purpose of our study was to develop a prognostic risk scoring model (PRSM) based on differentially expressed immune-related genes (IRGs) between LCCs and RCCs, therefore the prognostic key IRGs could be identified. METHODS: The gene sets and clinical information of COAD patients were derived from TCGA and GEO databases. The comparison of differentially expressed genes (DEGs) of LCCs and RCCs were conducted with appliance of "Limma" analysis. The establishment about co-expression modules of DEGs related with immune score was conducted by weighted gene co-expression network analysis (WGCNA). Furthermore, we screened the module genes and completed construction of gene pairs. The analysis of the prognosis and the establishment of PRSM were performed with univariate- and lasso-Cox regression. We employed the PRSM in the model group and verification group for the purpose of risk group assignment and PRSM accuracy verification. Finally, the identification of the prognostic key IRGs was guaranteed by the adoption of functional enrichment, "DisNor" and protein-protein interaction (PPI). RESULTS: A total of 215 genes were screened out by differential expression analysis and WGCNA. A PRSM with 16 immune-related gene pairs (IRGPs) was established upon the genes pairing. Furthermore, we confirmed that the risk score was an independent factor for survival by univariate- and multivariate-Cox regression. The prognosis of high-risk group in model group (P < 0.001) and validation group (P = 0.014) was significantly worse than that in low-risk group. Treg cells (P < 0.001) and macrophage M0 (P = 0.015) were highly expressed in the high-risk group. The functional analysis indicated that there was significant up-regulation with regard of lymphocyte and cytokine related terms in low-risk group. Finally, we identified five prognostic key IRGs associated with better prognosis through PPI and prognostic analysis, including IL2RB, TRIM22, CIITA, CXCL13, and CXCR6. CONCLUSION: Through the analysis and screening of the DEGs between LCCs and RCCs, we constructed a PRSM which could predicate prognosis of LCCs and RCCs, and five prognostic key IRGs were identified as well. Therefore, the basis for identifying the benefits of immunotherapy and immunomodulatory was built.

LncRNA EGOT regulates the proliferation and apoptosis of colorectal cancer by miR-33b-5p/CROT axis.

Accumulating researches have proved that long noncoding RNAs (lncRNAs) regulate a variety of cellular processes during cancer progression. However, the detailed function of most lncRNAs in colorectal cancer (CRC) remains mostly unknown. This study was aimed at exploring the specific role of lncRNA EGOT in CRC. Data from this study revealed that EGOT expression was obviously upregulated in CRC tissues and cell lines, and high EGOT expression indicated poor overall survival of CRC patients. Besides, functional assays proved that EGOT knockdown inhibited cell proliferation and promoted cell apoptosis in CRC. Then, subsequent molecular mechanism assays uncovered that EGOT could bind with miR-33b-5p and negatively regulate miR-33b-5p expression. Additionally, CROT was a downstream target of miR-33b-5p. Further, rescued-function assays suggested that the suppressive influence of EGOT depletion on CRC progression was reversed by miR-33b-5p inhibition or CROT overexpression. In conclusion, lncRNA EGOT mediates the tumor-facilitating part in CRC via miR-33b-5p/CROT pathway.

Gene set enrichment analysis: A genome-wide expression profile-based strategy for discovering functional microRNA-disease relationships.

Objective To explore stable and functional microRNA (miRNA)-disease relationships using a genome-wide expression profile pattern matching strategy. Methods We applied the ranked microarray pattern matching strategy Gene Set Enrichment Analysis to identify miRNA permutations with similar expression patterns to diseases. We also used quantitative reverse transcription PCR to validate the predicted expression levels of miRNAs in three diseases: inflammatory bowel disease (IBD), oesophageal cancer, and colorectal cancer. Results We found that hsa-miR-200 c was upregulated more than 40-fold in oesophageal cancer. The expression of miR-16 and miR-124 was not consistently upregulated in IBD or colorectal cancer. Conclusions Our results suggest that this expression profile matching strategy can be used to identify functional miRNA-disease relationships.

MicroRNA-130b transcriptionally regulated by histone H3 deacetylation renders Akt ubiquitination and apoptosis resistance to 6-OHDA.

Apoptosis of DA neurons is a contributing cause of disability and death for Parkinson's disease (PD). Akt may become a potential therapeutic target for PD since Akt has been deactivated during DA neuron apoptosis. We previously demonstrated that Akt confers apoptosis resistance against 6-OHDA in DA neuron-like PC12 cells, yet the underlying mechanisms accounted for this are not fully understood. Here we report that microRNA-130b (miR-130b)-dependent and cylindromatosis (CYLD) repression-mediated Akt ubiquitination renders apoptosis resistance of PC12 cells to 6-OHDA, which elicits histone H3 deacetylation-induced transcriptional downregulation of miR-130b vice versa. CYLD deficiency ubiquitinates Akt at Lys63, thereby phosphorylating Akt and antagonizing 6-OHDA-initiated apoptosis. MiR-130b targetedly represses CYLD and increases apoptosis resistance to 6-OHDA. CYLD repression by miR-130b restores Akt ubiquitination and activation, GSK3ß and FoxO3a phosphorylation, FoxO3a removal from Bim promoter as well as Bim downregulation during 6-OHDA administration. CYLD deficiency-mediated Akt activation is instrumental for the apoptosis-resistant phenotypes of miR-130b. In addition, 6-OHDA transcriptionally downregulates miR-130b through recruitment of HDAC3 at the promoter. Furthermore, EPO potentiates the ability of miR-130b to activate Akt and augment apoptosis resistance. Our findings identify the apoptosis-resistant function of miR-130b and suggest that histone H3 deacetylation plays a pivotal role in regulating miR-130b transcription in response to 6-OHDA.

NF-κB-dependent transcriptional upregulation of cyclin D1 exerts cytoprotection against hypoxic injury upon EGFR activation.

Apoptosis of neural cells is one of the main pathological features in hypoxic/ischemic brain injury. Nuclear factor-κB (NF-κB) might be a potential therapeutic target for hypoxic/ischemic brain injury since NF-κB has been found to be inactivated after hypoxia exposure, yet the underlying molecular mechanisms of NF-κB inactivation are largely unknown. Here we report that epidermal growth factor receptor (EGFR) activation prevents neuron-like PC12 cells apoptosis in response to hypoxia via restoring NF-κB-dependent transcriptional upregulation of cyclin D1. Functionally, EGFR activation by EGF stimulation mitigates hypoxia-induced PC12 cells apoptosis in both dose- and time-dependent manner. Of note, EGFR activation elevates IKKß phosphorylation, increases IκBα ubiquitination, promotes P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as upregulates cyclin D1 expression. EGFR activation also abrogates the decrease of IKKß phosphorylation, reduction of IκBα ubiquitination, blockade of P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as downregulation of cyclin D1 expression induced by hypoxia. Furthermore, NF-κB-dependent upregulation of cyclin D1 is instrumental for the EGFR-mediated cytoprotection against hypoxic apoptosis. In addition, the dephosphorylation of EGFR induced by either EGF siRNA transfection or anti-HB-EGF neutralization antibody treatment enhances hypoxic cytotoxicity, which are attenuated by EGF administration. Our results highlight the essential role of NF-κB-dependent transcriptional upregulation of cyclin D1 in EGFR-mediated cytoprotective effects under hypoxic preconditioning and support further investigation of EGF in clinical trials of patients with hypoxic/ischemic brain injury.

Resolution acute respiratory distress syndrome through reversing the imbalance of Treg/Th17 by targeting the cAMP signaling pathway.

Acute respiratory distress syndrome (ARDS) is a severe cause of respiratory failure with a mortality rate as high as 40­46% and without any effective pharmacological treatment available. The present study provided a novel strategy for the treatment of ARDS by specifically interfering with cyclic adenosine monophosphate (cAMP) signaling. Pre-treatment with the phosphodiesterase antagonist pentoxifyllinum (PTX) obviously attenuated lung injury and reduced the mortality of mice with cecal ligature and puncture (CLP)­induced ARDS, while raising cAMP levels. In addition, pre­treatment with PTX attenuated CLP­induced increases in the number of T­regulatory cells (Tregs) and interleukin (IL)­17­producing T­helper lymphocytes (Th17) among spleen lymphocytes, while partially restoring the Treg/Th17 ratio. Correspondingly, CLP­induced increases in the secretion of IL­2, IL­6, IL­10 and IL­17 were attenuated. Furthermore, CLP­induced increases in forkhead box p3 and RAR­related orphan receptor Î³t (RORγt) expression as well as signal transducer and activator of transcription (STAT3) activation were attenuated by PTX. The results indicated that PTX­induced increases in cAMP may have partly restored the Treg/Th17 balance by modulating the transcription of Foxp3 and RORγt through the STAT3 pathway. In conclusion, the present study provided a novel treatment strategy for ARDS by modulating the balance of Treg/Th17 and the subsequent immune response via cAMP signaling, which requires pre-clinical and clinical validation.

VEGF-mediated NF-κB activation protects PC12 cells from damage induced by hypoxia.

Neuronal apoptosis is a contributing cause of disability and death in cerebral ischemia. Nuclear factor-κB (NF-κB) may become a potential therapeutic target for hypoxic/ischemic neuron damage because NF-κB is inactivated after hypoxia exposure. Vascular endothelial growth factor (VEGF) has been found to improve neurological function recovery in cerebral ischemic injury although the exact molecular mechanisms that underlie the neuroprotective function of VEGF remain largely unknown. Here we defined the mechanism by which VEGF antagonized neuron-like PC12 cells apoptosis induced by hypoxia mimetic agent cobalt chloride (CoCl2) is through restoration of NF-κB activity. Depletion of VEGF with small interfering RNA (siRNA) in PC12 cells conferred CoCl2-induced cytotoxicity which was mitigated by VEGF administration. Treatment of PC12 cells with VEGF attenuated the CoCl2-induced cytotoxicity in both dose- and time-dependent manner. Mechanistically, VEGF increased IκBα phosphorylation and ubiquitination, promoted P65 nuclear translocation as well as upregulated XIAP and CCND1 expression. Meanwhile, VEGF administration reversed the dysregulation of IκBα phosphorylation and ubiquitination, P65 nuclear translocation as well as XIAP and CCND1 expression induced by CoCl2. Notably, the VEGF-dependent cytoprotection was abolished by pretreatment with BAY 11-7085, a specific inhibitor of NF-κB. Our data suggest that VEGF/NF-κB signalling pathway represents an adaptive mechanism that protects neural cells against hypoxic damage.

Urinary netrin-1 and KIM-1 as early biomarkers for septic acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) during sepsis is associated with poor outcome. However, diagnosis of AKI with serum creatinine (SCr) level change is neither highly sensitive nor specific. Therefore, identification of novel biomarkers for early diagnosis of AKI is desirable. AIMS: To evaluate the capacity of combining urinary netrin-1 and human kidney injury molecule type 1 (KIM-1) in the early diagnosis of septic AKI. METHODS: We prospectively recruited 150 septic patients from Jun 2011 to Jun 2013 at Zhejiang Provincial People's Hospital, China. SCr, urinary netrin-1, and KIM-1 levels were recorded at 0, 1, 3, 6, 24, and 48 h of ICU admission and compared between AKI and non-AKI patients. In addition, we investigated the prognostic value of netrin-1 and KIM-1 between non-survivors and survivors in septic AKI patients. RESULTS: SCr levels started to show elevation after 24 h of ICU admission. However, netrin-1 levels increased significantly as early as 1 h, peaked at 3-6 h and remained elevated up to 48 h of ICU admission in septic AKI patients. KIM-1 increased significantly by 6 h, peaked at 24 h and remained significantly elevated until 48 h of ICU admission. Furthermore, we observed significant higher urinary KIM-1 levels at 24 h and 48 h in non-survivors compared to survivors in AKI patients. CONCLUSIONS: Our results suggest that both netrin-1 and KIM-1 are clinically useful as early biomarkers in the diagnosis of septic AKI. In addition, persistent elevation of urinary KIM-1 level may be associated with poor prognosis.

[Mechanism of liver injury in severe acute pancreatitis rats and role of melatonin].

OBJECTIVE: To investigate the activation level of nuclear factor-kappaB (NF-kappaB) in liver injury caused by severe acute pancreatitis (SAP) and the protective role of melatonin against liver injury. METHODS: Ninety-six Sprague-Dawley (SD) rats were randomly divided into 3 equal groups: SAP group undergoing injection of sodium taurocholate to establish SAP models, melatonin (Mel) treatment group undergoing intraperitoneal injection 50 mg/kg 30 minutes before the establishment of SAP models, and sham operation group (Sham group). 4, 12, 24, and 48 hours after the onset of operation, blood samples were collected from the inferior vena casa of 8 rats from each group to measure the serum level of amylase (AMY) and alanine transaminase (ALT) by iodine colorimetry, and to detect the serum level of tumor necrosis factor-alpha (TNF-alpha) by ELISA. The livers were taken out to undergo pathological examination. Immunohistochemistry was used to examine the percentage of nuclear factor (NF)-kappaB in the hepatocytes. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) was used to determine the extent of hepatic apoptosis. RESULTS: The AMY and ALT levels at different time points of the SAP and Mel subgroups were all significantly higher than those of the sham operation subgroups (all P<0.05), and the AMY and ALT levels at different time points of the Mel subgroup were all significantly lower than those of the SAP subgroups (all P<0.05). The liver NF-kappaB activation level and hepatocellular apoptosis index of the SAP group increased since the fourth hour after the operation, and peaked at the time point of 24 hour, all significantly higher than those of the sham operation group (all P<0.05), and then declined. The TNF-alpha level at the time points of 12, 24, and 48 h in the SAP group were all significantly higher than those of the other 2 groups (all P<0.05). The levels of TNF-alpha, AMY, and ALT, the activity of NF-kappaB, and the extent of hepatocellular apoptosis at any time points of the Mel group were all significantly lower than those of the SAP group, but significantly higher than those of the sham operation group. Microscopy showed that the liver pathological damages of the Mel group were milder than those of the SAP group. CONCLUSION: SAP with liver injury is associated with the hepatic NF-kappaB activation leading to the production of NF-kappaB dependent cytokines and chemokines such as TNF-alpha. Melatonin reduces the apoptosis and necrosis in liver by inhibiting the activity of NF-kappaB and decreasing the expression of TNF-alpha.