HSP70 Ameliorates Septic Acute Kidney Injury via Binding with TRAF6 to Inhibit of Inflammation-Mediated Apoptosis.

Purpose: Acute kidney injury (AKI) is one of the most severe complications of sepsis, the pathological features of which are excessive inflammation and programmed cell death of resident renal cells. Heat shock protein 70 (HSP70) is a critical stress protein for repressing inflammation, however, its role in AKI is not fully understood. The current study aimed to determine the protective effect of HSP70 on septic AKI and its underlying mechanisms. Methods: Hsp70.1 knockout and wildtype mice were used for creating sepsis model by cecal ligation and puncture (CLP). Renal function, histological changes, pro-inflammatory cytokines, and apoptosis were analyzed with H&E, PAS, ELISA, western-blot, and immunofluorescence. Moreover, the effects of HSP70 on renal proximal tubular epithelial (HK-2) cells with LPS were assessed by measuring the levels of nuclear factor kappa B (NF-κB) signaling and downstream cytokines, viability, and apoptosis using western-blot, qRT-PCR, flow-cytometry, and immunofluorescence. Immunoprecipitate and immunoblotting were used for determining the interaction of HSP70 with tumor necrosis factor receptor-associated factor 6 (TRAF6). Exogenous HSP70 was applied to further identify its biological significance at the cellular and animal level. Results: Hsp70.1 deficiency significantly aggravated renal dysfunction with increasing serum levels of BUN, SCr, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL), and shortened survival in CLP mice. Furthermore, hsp70.1 knockout caused renal-tissue structural damage, especially proximal tubular, and inflammatory cascade and increased apoptotic cells, along with elevated Bax, caspase 3 and cleaved caspase 3, as well as decreased Bcl2 in vivo and vitro. Significantly, HSP70 directly interacted with TRAF6 in HK-2 cells, leading to suppression of inflammatory response and apoptosis. Moreover, exogenous HSP70 alleviated renal damage, decreased apoptosis and elevated survival rate in septic AKI in vivo and vitro. Conclusion: Our findings demonstrated that HSP70 played a critical role in sepsis-induced AKI via interaction with TRAF6 and inhibiting inflammation and apoptosis.

Comparison of Five Triage Tools for Identifying Mortality Risk and Injury Severity of Multiple Trauma Patients Admitted to the Emergency Department in the Daytime and Nighttime: A Retrospective Study.

Effective triage tools are indispensable for doctors to make a prompt decision for the treatment of multiple trauma patients in emergency departments (EDs). The Modified Early Warning Score (MEWS), National Early Warning Score (NEWS), standardized early warning score (SEWS), Modified Rapid Emergency Medicine Score (mREMS), and Revised Trauma Score (RTS) are five common triage tools proposed for trauma management. However, few studies have compared these tools in a multiple trauma cohort and investigated the influence of nighttime admission on the performance of these tools. This retrospective study was aimed at evaluating and comparing the performance of MEWS, NEWS, SEWS, mREMS, and RTS for identifying the mortality risk and trauma severity of patients with multiple trauma admitted to the ED during the daytime and nighttime. Retrospective data were collected from the medical records of patients with multiple trauma admitted in the daytime or nighttime to calculate scores for each triage tool. Logistic regression analysis was conducted on each triage tool for identifying in-hospital mortality and severe trauma (injury severity score > 15) in the daytime and nighttime. The performance of the tools was evaluated and compared by calculating area under the receiver operating characteristic curve (AUROC) of the retrospective logistic model of each tool. We collected data for 1,818 admissions, including 1,070 daytime and 748 nighttime admissions. A comparison of performance for identifying in-hospital mortality between daytime and nighttime yielded the following results (AUROC): MEWS (0.95 vs. 0.93, p = 0.384), NEWS (0.95 vs. 0.94, p = 0.708), SEWS (0.95 vs. 0.94, p = 0.683), mREMS (0.94 vs. 0.92, p = 0.286), and RTS (0.93 vs. 0.93, p = 0.87). Similarly, a comparison of performance for identifying trauma severity between daytime and nighttime yielded the following results (AUROC): MEWS (0.78 vs. 0.78, p = 0.95), NEWS (0.8 vs. 0.8, p = 0.885), SEWS (0.78 vs. 0.78, p = 0.818), mREMS (0.75 vs. 0.69, p = 0.019), and RTS (0.75 vs. 0.74, p = 0.619). All five scores are excellent triage tools (AUROC ≥ 0.9) for identifying in-hospital mortality for both daytime and nighttime admissions. However, they have only moderate effectiveness (AUROC < 0.9) at identifying severe trauma. The NEWS is the best triage tool for identifying severe trauma for both daytime and nighttime admissions. The MEWS, NEWS, SEWS, and RTS exhibited no significant differences in performance for identifying in-hospital mortality or severe trauma during the daytime or nighttime. However, the mREMS was better at identifying severe trauma during the daytime.

Identification and validation of candidate genes dysregulated in alveolar macrophages of acute respiratory distress syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a common cause of death in ICU patients and its underlying mechanism remains unclear, which leads to its high mortality rate. This study aimed to identify candidate genes potentially implicating in the pathogenesis of ARDS and provide novel therapeutic targets. METHODS: Using bioinformatics tools, we searched for differentially expressed genes (DEGs) in an ARDS microarray dataset downloaded from the Gene Expression Omnibus (GEO) database. Afterwards, functional enrichment analysis of GO, KEGG, GSEA and WGCNA were carried out to investigate the potential involvement of these DEGs. Moreover, the Protein-protein interaction (PPI) network was constructed and molecular complexes and hub genes were identified, followed by prognosis analysis of the hub genes. Further, we performed qRT-PCR, Western Blot and flow cytometry analysis to detect candidate genes of CCR2 and FPR3 in macrophage model of LPS-induced ARDS and primary alveolar macrophages(AMs). Macrophage chemotaxis was evaluated using Transwell assay. RESULTS: DEGs mainly involved in myeloid leukocyte activation, cell chemotaxis, adenylate cyclase-modulating G protein-coupled receptor signaling pathway and cytokine-cytokine receptor interaction. Basing on the constructed PPI network, we identified five molecular complexes and 10 hub genes potentially participating in the pathogenesis of ARDS. It was observed that candidate genes of CCR2 and FPR3 were significantly over-expressed in primary alveolar macrophages from ARDS patients and macrophgae model of LPS-induced ARDS. Moreover, in vitro transwell assay demonstrated that CCR2 and FPR3 down-regulation, respectively, inhibited LPS-triggered macrophage chemotaxis toward CCL2. Finally, a positive correlation between FPR3 and CCR2 expression was confirmed using pearson correlation analysis and Western Blot assay. CONCLUSIONS: Our study identified CCR2 and FPR3 as the candidate genes which can promote macrophage chemotaxis through a possible interaction between FPR3 and CCL2/CCR2 axis and provided novel insights into ARDS pathogenesis.

NLRP3 Inflammasome Activation by MicroRNA-495 Promoter Methylation May Contribute to the Progression of Acute Lung Injury.

Acute lung injury (ALI) is a pulmonary disorder that causes acute respiratory failure, thus leading to relative high mortality worldwide. However, the molecular mechanisms of ALI remain largely unknown. MicroRNA (miRNA)-dependent control of gene expression at a post-transcriptional level has been recently reported. Herein, we identify a candidate miRNA, miR-495, that affects the progression of ALI. Alveolar macrophages (NR8383) were treated with 1 µg/mL lipopolysaccharide (LPS) to establish a cell-injury model. Combined with the data from western blot, methylation-specific PCR, methylated DNA immunoprecipitation, and chromatin immunoprecipitation assays, NLRP3 inflammasome activation and methylation-dependent repression of miR-495 were found in LPS-exposed NR8383 cells. Dual-luciferase reporter gene assay and miR-495 gain-of-function experiments confirmed that NLRP3 was a target of miR-495. Next, the expression of miR-495 and NLRP3 was overexpressed or silenced to assess their effects on NLRP3 inflammasome activation, alveolar macrophage inflammation, and pyroptosis in vitro. As demonstrated, overexpressed miR-495 alleviated alveolar macrophage inflammation and pyroptosis and inhibited NLRP3 inflammasome activation by negatively regulating the NLRP3 gene. Consistently, elevated miR-495 alleviated lung injury and reduced the neutrophil infiltration and inflammation in rat models of LPS-induced ALI. Taken together, the data in our study demonstrated that methylation of the miR-495 promoter could downregulate miR-495, whose elevation could attenuate the activation of the NLRP3 inflammasome to protect against ALI, which provides novel therapeutic targets for ALI treatment.

The involvement of the laminin-integrin α7ß1 signaling pathway in mechanical ventilation-induced pulmonary fibrosis.

INTRODUCTION: The central objective of the study was to determine the possibility and potential mechanism by which the laminin-integrin α7ß1 signaling pathway acts on mechanical ventilation (MV)-induced pulmonary fibrosis in a rat model. METHODS: Fibrosis rat models were established via the mechanical injury method. Ninety rats were recruited and divided into the normal, low tidal volume (LVT), huge VT (HVT), Arg-Gly-Asp-Ser (RGDS), LVT + RGDS and HVT + RGDS groups. On day 0, 3, and 7 after model establishment, the pulmonary hydroxyproline content was measured using alkaline hydrolysis and the pulmonary index was also calculated. All rats in each group were executed on day 0, 3 and 7. The histopathological changes detected in the left pulmonary tissues were observed using hematoxylin-eosin (HE) and Masson staining methods. DISCUSSION: The mRNA and protein expressions of Wnt-5A, ß-catenin, E-cadherin and Collagen I in the Wnt/ß-catenin signaling pathway were detected using both reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting methods. Immunohistochemistry was employed to detect the fibronectin (FN) expression in the pulmonary tissues on the 7th day. All indexes in the RGDS and LVT + RGDS groups indicated no explicit differences compared with the normal group. In the LVT, HVT, HVT + RGDS groups, the respective weights of the rats and the expression of E-cadherin on the 7th day exhibited decreases, however the pulmonary index, hydroxyproline, pulmonary alveolar inflammation, pulmonary fibrosis, FN expression, and protein expressions of Wnt-5A, ß-catenin, and Collagen I all displayed increased levels (all P<0.05). The index changes detected in the HVT group were the most blatant results observed in the study. The rat pulmonary index on the 7th day, hydroxyproline (HYP), pulmonary alveolar inflammation, pulmonary fibrosis, FN expression, and protein expressions of Wnt-5A, ß-catenin, and type I-collagen were all down-regulated, in contrast the expression of E-cadherin was up-regulated in the LVT + RGDS and HVT + RGDS groups in comparison with the LVT and HVT groups, respectively (all P<0.05). CONCLUSIONS: The findings of the study suggested that RGDS could act to block the laminin-integrin α7ß1-signaling pathway, ultimately contributing to the inhibition of the progression of MV-induced pulmonary fibrosis.

Protective effect of hydrogen-saturated saline on acute lung injury induced by oleic acid in rats.

BACKGROUND: The purpose of the study is to investigate the role and mechanisms of hydrogen-saturated saline (HSS) in the acute lung injury (ALI) induced by oleic acid (OA) in rats. METHODS: Rats were treated with OA (0.1 mL/kg) to induce ALI and then administered with HSS (5 mL/kg) by intravenous (iv) and intraperitoneal (ip) injection, respectively. Three hours after the injection with OA, the arterial oxygen partial pressure (PaO2), arterial oxygen saturation (SaO2), carbon dioxide partial pressure (PaCO2), and bicarbonate (HCO3-) levels were analyzed using blood gas analyzer. In addition, the levels of malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), and interleukin 1ß (IL-1ß) and myeloperoxidase (MPO) activity were measured by commercial kits, and pathological changes of lung tissue were examined by HE staining. Finally, the correlations of MPO activity or MDA level with the levels of TNF-α or IL-1ß were analyzed by Pearson's correlation analysis. RESULTS: We found decreased PaO2 levels and the pathological changes of lung tissue of ALI after OA injection. In addition, OA increased the levels of MDA, TNF-α, and IL-1ß, as well as MPO activity in lung tissues (P < 0.05). However, after treatment with HSS, all of these changes were alleviated (P < 0.05), and these changes were mitigated when treated with HSS by ip then iv injection (P < 0.05). Furthermore, MDA level and MPO activity were positively correlated with TNF-α and IL-1ß levels in the lung tissue, respectively (P < 0.01). CONCLUSION: HSS attenuated ALI induced by OA in rats and might protect against ALI through selective resistance to oxidation and inhibiting inflammatory infiltration.

Effects on Proliferation and Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells Engineered to Express Neurotrophic Factors.

Mesenchymal stem cells (MSCs) are multipotential cells with capability to form colonies in vitro and differentiate into distinctive end-stage cell types. Although MSCs secrete many cytokines, the efficacy can be improved through combination with neurotrophic factors (NTFs). Moreover, MSCs are excellent opportunities for local delivery of NTFs into injured tissues. The aim of this present study is to evaluate the effects of overexpressing NTFs on proliferation and differentiation of human umbilical cord-derived mesenchymal stem cells (HUMSCs). Overexpressing NTFs had no effect on cell proliferation. Overexpressing NT-3, BDNF, and NGF also had no significant effect on the differentiation of HUMSCs. Overexpressing NTFs all promoted the neurite outgrowth of embryonic chick E9 dorsal root ganglion (DRG). The gene expression profiles of the control and NT-3- and BDNF-modified HUMSCs were compared using RNA sequencing and biological processes and activities were revealed. This study provides novel information about the effects of overexpressing NTFs on HUMSCs and insight into the choice of optimal NTFs for combined cell and gene therapy.