LPS-induced monocarboxylate transporter-1 inhibition facilitates lactate accumulation triggering epithelial-mesenchymal transformation and pulmonary fibrosis.

The epithelial-mesenchymal transformation (EMT) process of alveolar epithelial cells is recognized as involved in the development of pulmonary fibrosis. Recent evidence has shown that lipopolysaccharide (LPS)-induced aerobic glycolysis of lung tissue and elevated lactate concentration are associated with the pathogenesis of sepsis-associated pulmonary fibrosis. However, it is uncertain whether LPS promotes the development of sepsis-associated pulmonary fibrosis by promoting lactate accumulation in lung tissue, thereby initiating EMT process. We hypothesized that monocarboxylate transporter-1 (MCT1), as the main protein for lactate transport, may be crucial in the pathogenic process of sepsis-associated pulmonary fibrosis. We found that high concentrations of lactate induced EMT while moderate concentrations did not. Besides, we demonstrated that MCT1 inhibition enhanced EMT process in MLE-12 cells, while MCT1 upregulation could reverse lactate-induced EMT. LPS could promote EMT in MLE-12 cells through MCT1 inhibition and lactate accumulation, while this could be alleviated by upregulating the expression of MCT1. In addition, the overexpression of MCT1 prevented LPS-induced EMT and pulmonary fibrosis in vivo. Altogether, this study revealed that LPS could inhibit the expression of MCT1 in mouse alveolar epithelial cells and cause lactate transport disorder, which leads to lactate accumulation, and ultimately promotes the process of EMT and lung fibrosis.

Retinol and retinol binding protein 4 levels and COVID-19: a Mendelian randomization study.

BACKGROUND: The Corona Virus Disease 2019 (COVID-19) pandemic has struck globally. Whether the related proteins of retinoic acid (RA) signaling pathway are causally associated with the risk of COVID-19 remains unestablished. We conducted a two-sample Mendelian randomization (MR) study to assess the associations of retinol, retinol binding protein 4 (RBP4), retinol dehydrogenase 16 (RDH16) and cellular retinoic acid binding protein 1 (CRABP1) with COVID-19 in European population. METHODS: The outcome utilized the summary statistics of COVID-19 from the COVID-19 Host Genetics Initiative. The exposure data were obtained from public genome wide association study (GWAS) database. We extracted SNPs from exposure data and outcome data. The inverse variance weighted (IVW), MR-Egger and Wald ratio methods were employed to assess the causal relationship between exposure and outcome. Sensitivity analyses were performed to ensure the validity of the results. RESULTS: The MR estimates showed that retinol was associated with lower COVID-19 susceptibility using IVW (OR: 0.69, 95% CI: 0.53-0.90, P: 0.0065), whereas the associations between retinol and COVID-19 hospitalization or severity were not significant. RBP4 was associated with lower COVID-19 susceptibility using the Wald ratio (OR: 0.83, 95% CI: 0.72-0.95, P: 0.0072). IVW analysis showed RDH16 was associated with increased COVID-19 hospitalization (OR: 1.10, 95% CI: 1.01-1.18, P: 0.0199). CRABP1 was association with lower COVID-19 susceptibility (OR: 0.95, 95% CI: 0.91-0.99, P: 0.0290) using the IVW. CONCLUSIONS: We found evidence of possible causal association of retinol, RBP4, RDH16 and CRABP1 with the susceptibility, hospitalization and severity of COVID-19. Our study defines that retinol is significantly associated with lower COVID-19 susceptibility, which provides a reference for the prevention of COVID-19 with vitamin A supplementation.

Integrin ß3 Mediates Sepsis and Mechanical Ventilation-Associated Pulmonary Fibrosis Through Glycometabolic Reprogramming.

Mechanical ventilation (MV) has become a clinical first-line treatment option for patients with respiratory failure. However, it was unclear whether MV further aggravates the process of sepsis-associated pulmonary fibrosis and eventually leads to sepsis and mechanical ventilation-associated pulmonary fibrosis (S-MVPF). This study aimed to explore the mechanism of S-MVPF concerning integrin ß3 activation in glycometabolic reprogramming of lung fibroblasts. We found that MV exacerbated sepsis-associated pulmonary fibrosis induced by lipopolysaccharide, which was accompanied by proliferation of lung fibroblasts, increased deposition of collagen in lung tissue, and increased procollagen type I carboxy-terminal propeptide in the bronchoalveolar lavage fluid. A large number of integrin ß3- and pyruvate kinase M2-positive fibroblasts were detected in lung tissue after stimulation with lipopolysaccharide and MV, with an increase in lactate dehydrogenase A expression and lactate levels. S-MVPF was primarily attenuated in integrin ß3-knockout mice, which also resulted in a decrease in the levels of pyruvate kinase M2, lactate dehydrogenase A, and lactate. In conclusion, MV aggravated sepsis-associated pulmonary fibrosis, with glycometabolic reprogramming mediated by integrin ß3 activation. Thus, integrin ß3-mediated glycometabolic reprogramming might be a potential therapeutic target for S-MVPF.

Development and validation of a nomogram to predict the risk of renal replacement therapy among acute kidney injury patients in intensive care unit.

BACKGROUND: There are no universally accepted indications to initiate renal replacement therapy (RRT) among patients with acute kidney injury (AKI). This study aimed to develop a nomogram to predict the risk of RRT among AKI patients in intensive care unit (ICU). METHODS: In this retrospective cohort study, we extracted AKI patients from Medical Information Mart for Intensive Care III (MIMIC-III) database. Patients were randomly divided into a training cohort (70%) and a validation cohort (30%). Multivariable logistic regression based on Akaike information criterion was used to establish the nomogram. The discrimination and calibration of the nomogram were evaluated by Harrell's concordance index (C-index) and Hosmer-Lemeshow (HL) test. Decision curve analysis (DCA) was performed to evaluate clinical application. RESULTS: A total of 7413 critically ill patients with AKI were finally enrolled. 514 (6.9%) patients received RRT after ICU admission. 5194 (70%) patients were in the training cohort and 2219 (30%) patients were in the validation cohort. Nine variables, namely, age, hemoglobin, creatinine, blood urea nitrogen and lactate at AKI detection, comorbidity of congestive heart failure, AKI stage, and vasopressor use were included in the nomogram. The predictive model demonstrated satisfying discrimination and calibration with C-index of 0.938 (95% CI, 0.927-0.949; HL test, P = 0.430) in training set and 0.935 (95% CI, 0.919-0.951; HL test, P = 0.392) in validation set. DCA showed a positive net benefit of our nomogram. CONCLUSION: The nomogram developed in this study was highly accurate for RRT prediction with potential application value.

The role of macrophage-fibroblast interaction in lipopolysaccharide-induced pulmonary fibrosis: an acceleration in lung fibroblast aerobic glycolysis.

Recent evidence has shown that lipopolysaccharide (LPS)-induced aerobic glycolysis of lung fibroblasts is closely associated with the pathogenesis of septic pulmonary fibrosis. Nevertheless, the underlying mechanism remains poorly defined. In this study, we demonstrate that LPS promotes c-Jun N-terminal kinase (JNK) signaling pathway activation and endogenous tumor necrosis factor-α (TNF-α) secretion in pulmonary macrophages. This, in turn, could significantly promote aerobic glycolysis and increase lactate production in lung fibroblasts through 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3) activation. Culturing human lung fibroblast MRC-5 cell line with TNF-α or endogenous TNF-α (cell supernatants of macrophages after LPS stimulation) both enhanced the aerobic glycolysis and increased lactate production. These effects could be prevented by treating macrophages with JNK pathway inhibitor, by administering TNF-α receptor 1 (TNFR1) siRNA, PFKFB3 inhibitor, or by silencing PFKFB3 with fibroblasts-specific shRNA. In addition, the inhibition of TNF-α secretion and PFKFB3 expression prevented LPS-induced pulmonary fibrosis in vivo. In conclusion, this study revealed that LPS-induced macrophage secretion of TNF-α could initiate fibroblast aerobic glycolysis and lactate production, implying that inflammation-metabolism interactions between lung macrophages and fibroblasts might play an essential role in LPS-induced pulmonary fibrosis.

Prediction of high-flow nasal cannula outcomes at the early phase using the modified respiratory rate oxygenation index.

BACKGROUND: This study was designed to explore the early predictive value of the respiratory rate oxygenation (ROX) index modified by PaO2 (mROX) in high-flow nasal cannula (HFNC) therapy in patients with acute hypoxemia respiratory failure (AHRF). METHOD: Seventy-five patients with AHRF treated with HFNC were retrospectively reviewed. Respiratory parameters at baseline and 2 h after HFNC initiation were analyzed. The predictive value of the ROX (ratio of pulse oximetry/FIO2 to respiratory rate) and mROX (ratio of arterial oxygen /FIO2 to respiratory rate) indices with two variations by adding heart rate to each index (ROX-HR and mROX-HR) was evaluated. RESULTS: HFNC therapy failed in 24 patients, who had significantly higher intensive care unit (ICU) mortality and longer ICU stay. Both the ROX and mROX indices at 2 h after HFNC initiation can predict the risk of intubation after HFNC. Two hours after HFNC initiation, the mROX index had a higher area under the receiver operating characteristic curve (AUROC) for predicting HFNC success than the ROX index. Besides, baseline mROX index of greater than 7.1 showed a specificity of 100% for HFNC success. CONCLUSION: The mROX index may be a suitable predictor of HFNC therapy outcomes at the early phase in patients with AHRF.

PI3K-Akt-mTOR/PFKFB3 pathway mediated lung fibroblast aerobic glycolysis and collagen synthesis in lipopolysaccharide-induced pulmonary fibrosis.

Metabolic reprogramming plays a critical role in many diseases. A recent study revealed that aerobic glycolysis in lung tissue is closely related to pulmonary fibrosis, and also occurs during lipopolysaccharide (LPS)-induced sepsis. However, whether LPS induces aerobic glycolysis in lung fibroblasts remains unknown. The present study demonstrated that LPS promotes collagen synthesis in the lung fibroblasts through aerobic glycolysis via the activation of the PI3K-Akt-mTOR/PFKFB3 pathway. Challenging the human lung fibroblast MRC-5 cell line with LPS activated the PI3K-Akt-mTOR pathway, significantly upregulated the expression of 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), enhanced the aerobic glycolysis, and promoted collagen synthesis. These phenomena could be reversed by the PI3K-Akt inhibitor LY294002, mTOR inhibitor rapamycin, PFKFB3 inhibitor 3PO, or PFKFB3 silencing by specific shRNA, or aerobic glycolysis inhibitor 2-DG. In addition, PFKFB3 expression and aerobic glycolysis were also detected in the mouse model of LPS-induced pulmonary fibrosis, which could be reversed by the intraperitoneal injection of PFKFB3 inhibitor 3PO. Taken together, this study revealed that in LPS-induced pulmonary fibrosis, LPS might mediate lung fibroblast aerobic glycolysis through the activation of the PI3K-Akt-mTOR/PFKFB3 pathway.

Lipopolysaccharide promotes lung fibroblast proliferation through autophagy inhibition via activation of the PI3K-Akt-mTOR pathway.

Pulmonary fibrosis is a major cause of death in patients with acute respiratory distress syndrome (ARDS). Our previous study revealed that lipopolysaccharide (LPS) challenge could lead to mouse lung fibroblast proliferation. Additionally, inhibition of autophagy in lung fibroblasts was also reported to be crucial during the process of pulmonary fibrosis. However, the correlation between proliferation and inhibition of autophagy of lung fibroblasts and the underlying mechanism remain unknown. In this study, we report that autophagy was inhibited in mouse lung fibroblasts after LPS challenge, and was accompanied by activation of the PI3K-Akt-mTOR signaling pathway. Treating mouse lung fibroblasts with LPS resulted in mTOR and Akt phosphorylation, p62 up-regulation, and significant down-regulation of autophagosomes, which could be reversed by PI3K-Akt inhibitors (Ly294002) or mTOR inhibitors (rapamycin, RAPA). Furthermore, either LPS or hydroxychloroquine (HCQ), an autophagy inhibitor, could promote mouse lung fibroblast proliferation, which could be reversed by RAPA application. The present research therefore reveals that LPS promotes lung fibroblast proliferation through autophagy inhibition via activation of the PI3K-Akt-mTOR pathway.

Thy-1 depletion and integrin ß3 upregulation-mediated PI3K-Akt-mTOR pathway activation inhibits lung fibroblast autophagy in lipopolysaccharide-induced pulmonary fibrosis.

Lipopolysaccharide (LPS)-induced autophagy inhibition in lung fibroblasts is closely associated with the activation of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K-Akt-mTOR) pathway. However, the underlying mechanism remains unknown. In this study, we demonstrated that LPS activated the PI3K-Akt-mTOR pathway and inhibited lung fibroblast autophagy by depleting thymocyte differentiation antigen-1 (Thy-1) and upregulating integrin ß3 (Itgb3). Challenge of the human lung fibroblast MRC-5 cell line with LPS resulted in significant upregulation of integrin ß3, activation of the PI3K-Akt-mTOR pathway and inhibition of autophagy, which could be abolished by integrin ß3 silencing by specific shRNA or treatment with the integrin ß3 inhibitor cilengitide. Meanwhile, LPS could inhibit Thy-1 expression accompanied with PI3K-Akt-mTOR pathway activation and lung fibroblast autophagy inhibition; these effects could be prevented by Thy-1 overexpression. Meanwhile, Thy-1 downregulation with Thy-1 shRNA could mimic the effects of LPS, inducing the activation of PI3K-Akt-mTOR pathway and inhibiting lung fibroblast autophagy. Furthermore, protein immunoprecipitation analysis demonstrated that LPS reduced the binding of Thy-1 to integrin ß3. Thy-1 downregulation, integrin ß3 upregulation and autophagy inhibition were also detected in a mouse model of LPS-induced pulmonary fibrosis, which could be prohibited by intratracheal injection of Thy-1 overexpressing adeno-associated virus (AAV) or intraperitoneal injection of the integrin ß3 inhibitor cilengitide. In conclusion, this study demonstrated that Thy-1 depletion and integrin ß3 upregulation are involved in LPS-induced pulmonary fibrosis, and may serve as potential therapeutic targets for pulmonary fibrosis.

Lipopolysaccharide induced the proliferation of mouse lung fibroblasts by suppressing FoxO3a/p27 pathway.

Aberrant aggregation and activation of lung fibroblasts is a key process in pulmonary fibrosis, but the underlying mechanism remains enigmatic. Forkhead Box O3a (FoxO3a) is considered to be an important transcription factor that could regulate both cell cycle and cell viability. To investigate the role of FoxO3a on LPS-induced lung fibroblast proliferation, we transfected FoxO3a-SiRNA or FoxO3a-OE lentivirus into cultured mouse lung fibroblasts to knockdown or overexpress FoxO3a and pretreated mouse lung fibroblasts with gefitinib to enhance FoxO3a activity. The proliferation of lung fibroblasts was evaluated by CCK8 assay, the expression of FoxO3a, phosphorylated FoxO3a (p-FoxO3a) and p27 were measured by Western blot. We found that the proliferation of mouse lung fibroblasts mediated by LPS is accompanied by the inactivation of FoxO3a. The knockdown of FoxO3a could further decreased the expression of p27 mediated by LPS, while the overexpression of FoxO3a significantly increased the expression of p27 and suppressed LPS-induced lung fibroblast proliferation. Upon treating fibroblasts with gefitinib, the phosphorylation of FoxO3a was reduced and FoxO3a translocated into the nucleus, the expression of p27 was significantly increased and the proliferation of lung fibroblasts mediated by LPS could also be inhibited effectively. The results indicate that overexpression and reduced phosphatase activity of FoxO3a inhibit LPS-induced lung fibroblast proliferation through the activation of FoxO3a/p27 signaling pathways. Thus, to enhance FoxO3a activity could be a potential therapeutic target for LPS-induced pulmonary fibrosis.

Liver-specific deletion of LASS2 delayed regeneration of mouse liver after partial hepatectomy.

The capacity of liver regeneration is critical for patients with liver diseases. However, cellular and molecular mechanisms of liver regeneration are still incompletely defined. Here, we assessed roles of LASS2 in liver regeneration following partial hepatectomy (PHx) in mice. Our results showed that protein level of LASS2 remarkably increased during liver regeneration after PHx in wildtype (WT) mice. Comparing to WT mice, liver regeneration index after PHx was significantly decreased from day 1 to day 5 in liver-specific LASS2 knockout (LASS2-LKO) mice. Interestingly, liver mass of LASS2-LKO mice could sufficiently recover at day 14 after PHx. Immunohistochemistry (IHC) and western blot analyses revealed that proliferation markers, such as PCNA and Ki67, were potently reduced during liver regeneration in LASS2-LKO mice. In addition, several cell cycle related molecules, such as cyclin A, CDK2 and p-Rb, were decreased in LASS2-LKO mice after PHx. Co-immunoprecipitation assay further revealed a decreased formation of CDK4/cyclin D1 complex after PHx in LASS2-LKO mice. However, phosphorylation of Akt was significantly activated from day 2 after PHx in LASS2-LKO mice when compared with that in WT mice, which may explain the recovery of liver mass at the late stage of liver regeneration in LASS2-LKO mice. Taken together, we conclude that LASS2 plays an important role in efficient liver regeneration in response to PHx.

High-mobility group box 1 accelerates lipopolysaccharide-induced lung fibroblast proliferation in vitro: involvement of the NF-κB signaling pathway.

The mechanism underlying lipopolysaccharide (LPS)-induced aberrant proliferation of lung fibroblasts in Gram-negative bacilli-associated pulmonary fibrosis is unknown. High-mobility group box 1 (HMGB1) is a ubiquitous nuclear protein that is released from the nuclei of lung fibroblasts after LPS stimulation. It can exasperate LPS-induced inflammation and hasten cell proliferation. Thus, this study investigated the effects of LPS- and/or HMGB1-stimulating murine lung fibroblasts on gene expression using various assays in vitro. Thiazolyl-diphenyl-tetrazolium bromide (MTT) assay data showed that either LPS or HMGB1 could induce lung fibroblast proliferation. Endogenous HMGB1 secreted from lung fibroblasts was detected by enzyme-linked immunosorbent assay (ELISA) 48 h after LPS stimulation. Pretreatment with an anti-HMGB1 antibody inhibited the proliferative effects of LPS on lung fibroblasts. DNA microarray data showed that the NF-κB signaling genes were upregulated in cells after stimulated with LPS, HMGB1, or both. Secretion of matrix metalloproteinase (MMP)-2 and MMP-9, and tissue inhibitor of metalloproteinase 2 (TIMP-2) was significantly upregulated after treatment with LPS, HMGB1, or their combination. However, an NF-κB inhibitor was able to downregulate levels of these proteins. In addition, levels of Toll-like receptor 4 (TLR4), Toll-like receptor 2 (TLR2), and receptors for advanced glycation end products (RAGE) mRNA and proteins were also upregulated in these cells after LPS treatment and further upregulated by LPS plus HMGB1. In conclusion, the data from the current study demonstrate that LPS-induced lung fibroblast secretion of endogenous HMGB1 can augment the proproliferative effects of LPS and, therefore, may play a key role in exacerbation of pulmonary fibrosis. The underlying molecular mechanisms are related to the activation of the TLR4/NF-κB signaling pathway and its downstream targets.

HDAC is essential for epigenetic regulation of Thy-1 gene expression during LPS/TLR4-mediated proliferation of lung fibroblasts.

Lipopolysaccharide (LPS)-induced proliferation of lung fibroblasts is closely correlated with loss of gene expression of thymocyte differentiation antigen-1 (Thy-1), accompanied with deacetylation of histones H3 and H4 at the Thy-1 gene promoter region; however, the mechanism remains enigmatic. We report here that LPS downregulates Thy-1 gene expression by activating histone deacetylases (HDACs) via Toll-like receptor 4 (TLR4) signaling. Treatment of primary cultured mouse lung fibroblasts with LPS resulted in significant upregulation of TLR4 and enhanced cell proliferation that was abolished by silencing TLR4 with lentivirus-delivered TLR4 shRNA. Interestingly, LPS increased the mRNA and protein levels of HDAC-4, -5, and -7, an effect that was abrogated by HDAC inhibitor trichostatin A (TSA) or TLR4-shRNA-lentivirus. Consistent with these findings, Ace-H3 and Ace-H4 were decreased by LPS that was prevented by TSA. Most importantly, chromosome immunoprecipitation (ChIP) analysis demonstrated that LPS decreased the association of Ace-H4 at the Thy-1 promoter region that was efficiently restored by pretreatment with TSA. Accordingly, LPS decreased the mRNA and protein levels of Thy-1 that was inhibited by TSA. Furthermore, silencing the Thy-1 gene by lentivirus-delivered Thy-1 shRNA could promote lung fibroblast proliferation, even in the absence of LPS. Conversely, overexpressing Thy-1 gene could inhibit lung fibroblast proliferation and reduce LPS-induced lung fibroblast proliferation. Our data suggest that LPS upregulates and activates HDACs through TLR4, resulting in deacetylation of histones H3 and H4 at the Thy-1 gene promoter that may contribute to Thy-1 gene silencing and lung fibroblast proliferation.

METFORMIN MITIGATES SEPSIS-ASSOCIATED PULMONARY FIBROSIS BY PROMOTING AMPK ACTIVATION AND INHIBITING HIF-1α-INDUCED AEROBIC GLYCOLYSIS.

ABSTRACT: Recent research has revealed that aerobic glycolysis has a strong correlation with sepsis-associated pulmonary fibrosis (PF). However, at present, the mechanism and pathogenesis remain unclear. We aimed to test the hypothesis that the adenosine monophosphate-activated protein kinase (AMPK) activation and suppression of hypoxia-inducible factor 1α (HIF-1α)-induced aerobic glycolysis play a central role in septic pulmonary fibrogenesis. Cellular experiments demonstrated that lipopolysaccharide increased fibroblast activation through AMPK inactivation, HIF-1α induction, alongside an augmentation of aerobic glycolysis. By contrast, the effects were reversed by AMPK activation or HIF-1α inhibition. In addition, pretreatment with metformin, which is an AMPK activator, suppresses HIF-1α expression and alleviates PF associated with sepsis, which is caused by aerobic glycolysis, in mice. Hypoxia-inducible factor 1α knockdown demonstrated similar protective effects in vivo . Our research implies that targeting AMPK activation and HIF-1α-induced aerobic glycolysis with metformin might be a practical and useful therapeutic alternative for sepsis-associated PF.

Pharmacological inhibition of the ACE/Ang-2/AT1 axis alleviates mechanical ventilation-induced pulmonary fibrosis.

Mechanical ventilation (MV) is an essential therapy for acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. However, it can also induce mechanical ventilation-induced pulmonary fibrosis (MVPF) and the underlying mechanism remains unknown. Based on a mouse model of MVPF, the present study aimed to explore the role of the angiotensin-converting enzyme/angiotensin II/angiotensin type 1 receptor (ACE/Ang-2/AT1R) axis in the process of MVPF. In addition, recombinant angiotensin-converting enzyme 2(rACE2), AT1R inhibitor valsartan, AGTR1-directed shRNA and ACE inhibitor perindopril were applied to verify the effect of inhibiting ACE/Ang-2/AT1R axis in the treatment of MVPF. Our study found MV induced an inflammatory reaction and collagen deposition in mouse lung tissue accompanied by the activation of ACE in lung tissue, increased concentration of Ang-2 in bronchoalveolar lavage fluid (BALF), and upregulation of AT1R in alveolar epithelial cells. The process of pulmonary fibrosis could be alleviated by the application of the ACE inhibitor perindopril, ATIR inhibitor valsartan and AGTR1-directed shRNA. Meanwhile, rACE2 could also alleviate MVPF through the degradation of Ang-2. Our finding indicated the ACE/Ang-2/AT1R axis played an essential role in the pathogenesis of MVPF. Pharmacological inhibition of the ACE/Ang-2/AT1R axis might be a promising strategy for the treatment of MVPF.

Development and validation of a deep learning-based framework for automated lung CT segmentation and acute respiratory distress syndrome prediction: a multicenter cohort study.

Background: Acute respiratory distress syndrome (ARDS) is a life-threatening condition with a high incidence and mortality rate in intensive care unit (ICU) admissions. Early identification of patients at high risk for developing ARDS is crucial for timely intervention and improved clinical outcomes. However, the complex pathophysiology of ARDS makes early prediction challenging. This study aimed to develop an artificial intelligence (AI) model for automated lung lesion segmentation and early prediction of ARDS to facilitate timely intervention in the intensive care unit. Methods: A total of 928 ICU patients with chest computed tomography (CT) scans were included from November 2018 to November 2021 at three centers in China. Patients were divided into a retrospective cohort for model development and internal validation, and three independent cohorts for external validation. A deep learning-based framework using the UNet Transformer (UNETR) model was developed to perform the segmentation of lung lesions and early prediction of ARDS. We employed various data augmentation techniques using the Medical Open Network for AI (MONAI) framework, enhancing the training sample diversity and improving the model's generalization capabilities. The performance of the deep learning-based framework was compared with a Densenet-based image classification network and evaluated in external and prospective validation cohorts. The segmentation performance was assessed using the Dice coefficient (DC), and the prediction performance was assessed using area under the receiver operating characteristic curve (AUC), sensitivity, and specificity. The contributions of different features to ARDS prediction were visualized using Shapley Explanation Plots. This study was registered with the China Clinical Trial Registration Centre (ChiCTR2200058700). Findings: The segmentation task using the deep learning framework achieved a DC of 0.734 ± 0.137 in the validation set. For the prediction task, the deep learning-based framework achieved AUCs of 0.916 [0.858-0.961], 0.865 [0.774-0.945], 0.901 [0.835-0.955], and 0.876 [0.804-0.936] in the internal validation cohort, external validation cohort I, external validation cohort II, and prospective validation cohort, respectively. It outperformed the Densenet-based image classification network in terms of prediction accuracy. Moreover, the ARDS prediction model identified lung lesion features and clinical parameters such as C-reactive protein, albumin, bilirubin, platelet count, and age as significant contributors to ARDS prediction. Interpretation: The deep learning-based framework using the UNETR model demonstrated high accuracy and robustness in lung lesion segmentation and early ARDS prediction, and had good generalization ability and clinical applicability. Funding: This study was supported by grants from the Shanghai Renji Hospital Clinical Research Innovation and Cultivation Fund (RJPY-DZX-008) and Shanghai Science and Technology Development Funds (22YF1423300).

Epigenetic regulation of Thy-1 gene expression by histone modification is involved in lipopolysaccharide-induced lung fibroblast proliferation.

Lipopolysaccharide (LPS)-induced pulmonary fibrosis is characterized by aberrant proliferation and activation of lung fibroblasts. Epigenetic regulation of thymocyte differentiation antigen 1 (Thy-1) is associated with lung fibroblast phenotype transformation that results in aberrant cell proliferation. However, it is not clear whether the epigenetic regulation of Thy-1 expression is required for LPS-induced lung fibroblast proliferation. To address this issue and better understand the relative underlying mechanisms, we used mouse lung fibroblasts as model to observe the changes of Thy-1 expression and histone deacetylation after LPS challenge. The results showed that cellular DNA synthesis, measured by BrdU incorporation, was impacted less in the early stage (24 hrs) after the challenge of LPS, but significantly increased at 48 or 72 hrs after the challenge of LPS. Meanwhile, Thy-1 expression, which was detected by real-time PCR and Western blot, in lung fibroblasts decreased with increased time after LPS challenge and diminished at 72 hrs. We also found that the acetylation of either histone H3 or H4 decreased in the LPS-challenged lung fibroblasts. ChIP assay revealed that the acetylation of histone H4 (Ace-H4) decreased in the Thy-1 promoter region in response to LPS. In addition, all the above changes could be attenuated by depletion of TLR4 gene. Our studies indicate that epigenetic regulation of Thy-1 gene expression by histone modification is involved in LPS-induced lung fibroblast proliferation.

Association of geriatric nutritional risk index with all-cause hospital mortality among elderly patients in intensive care unit.

Background: Malnutrition is associated with poor outcomes for geriatric patients in intensive care unit (ICU). It is important to identify patients at risk of malnutrition and provide individual nutrition support. The assessment of malnutrition risk is not easy for these patients due to their cognitive impairment. Geriatric nutrition risk index (GNRI) is a simple and objective scoring tool to evaluate the risk of malnutrition in elderly patients. In this study, we aimed to see whether GNRI score was appropriate to predict clinical outcomes among geriatric patients in the setting of ICU. Materials and methods: Elderly patients with age ≥ 65 years were extracted from Medical Information Mart for Intensive Care IV (MIMIC-IV) database. Categories based on GNRI were classified as major risk (GNRI <82), moderate risk (GNRI 82 to <92), low risk (GNRI 92 to ≤98), and no risk (GNRI >98). The primary outcome was all-cause hospital mortality. Multivariable Cox proportional hazards regression models and restricted cubic spline were used to investigate associations of GNRI with hospital mortality, respectively. A two-piecewise linear regression model was applied to examine the inflection point of GNRI on hospital mortality. To reduce selection bias, propensity score matching (PSM) was used in a 1:1 ratio. Results: A total of 3,696 geriatric patients were finally included with median age 75 (69, 81) years. The prevalence of major risk was 28.6%. In the fully adjusted model, GNRI categories featured a negative trend with hospital mortality (p for trend = 0.037). Restricted cubic spline analysis demonstrated an L-shaped relationship between GNRI and hospital mortality before and after matching. The inflection point was 78.7. At the left side of inflection point, GNRI levels were significantly negatively associated with hospital mortality (HR = 0.96, 95% CI: 0.94-0.98; p < 0.001) and featured no significant relations at the right side. Multiple linear regression also showed that GNRI was negatively associated with length of stay in hospital. Conclusion: The major risk of malnutrition defined by GNRI was able to predict poor prognosis for geriatric patients admitted to ICU.

[Research on the mechanism of mechanical ventilation induced endoplasmic reticulum stress promoting mechanical ventilation-induced pulmonary fibrosis].

OBJECTIVE: To demonstrate the mechanism of mechanical ventilation (MV) induced endoplasmic reticulum stress (ERS) promoting mechanical ventilation-induced pulmonary fibrosis (MVPF), and to clarify the role of angiotensin receptor 1 (AT1R) during the process. METHODS: The C57BL/6 mice were randomly divided into four groups: Sham group, MV group, AT1R-shRNA group and MV+AT1R-shRNA group, with 6 mice in each group. The MV group and MV+AT1R-shRNA group mechanically ventilated for 2 hours after endotracheal intubation to establish MVPF animal model (parameter settings: respiratory rate 70 times/minutes, tidal volume 20 mL/kg, inhated oxygen concentration 0.21). The Sham group and AT1R-shRNA group only underwent intubation after anesthesia and maintained spontaneous breathing. AT1R-shRNA group and MV+AT1R-shRNA group were airway injected with the adeno-associated virus one month before modeling to inhibit AT1R gene expression in lung tissue. The expressions of AT1R, ERS signature proteins [immunoglobulin heavy chain-binding protein (BIP), protein disulfide isomerase (PDI)], fibrosis signature proteins [collagen I (COL1A1), α-smooth muscle actin (α-SMA)] in lung tissues were detected by immunofluorescence and Western blotting. Hematoxylin-eosin (HE) staining was used to evaluate lung injury and Masson staining was used to evaluate pulmonary fibrosis. RESULTS: Compared with the Sham group, the degree of pulmonary fibrosis and lung injury were more significant in the MV group. In the MV group, the protein expressions of AT1R, BIP, PDI, COL1A1 and α-SMA were increased (AT1R/ß-actin: 1.40±0.02 vs. 1, BIP/ß-actin: 2.79±0.07 vs. 1, PDI/ß-actin: 2.07±0.02 vs. 1, COL1A1/α-Tubulin: 2.60±0.15 vs. 1, α-SMA/α-Tubulin: 2.80±0.25 vs. 1, all P < 0.01). The number of E-cad+/AT1R+ and E-cad+/BIP+ cells in lung tissue increased, and the fluorescence intensity of COL1A1 and α-SMA increased. Compared with the MV group, the degree of pulmonary fibrosis and lung injury were significantly relieved in the MV+AT1R-shRNA group. In the MV+AT1R-shRNA group, the protein expressions of AT1R, BIP, PDI, COL1A1 and α-SMA were decreased (AT1R/ß-actin: 0.53±0.03 vs. 1.40±0.02, BIP/ß-actin: 1.73±0.15 vs. 2.79±0.07, PDI/ß-actin: 1.04±0.07 vs. 2.07±0.02, COL1A1/α-Tubulin: 1.29±0.11 vs. 2.60±0.15, α-SMA/α-Tubulin: 1.27±0.10 vs. 2.80±0.25, all P < 0.01). The number of E-cad+/AT1R+ and E-cad+/BIP+ cells in lung tissue decreased, and the fluorescence intensity of COL1A1 and α-SMA decreased. There was no statistically significant difference in the indicators between AT1R-shRNA group and Sham group. CONCLUSIONS: MV up-regulate the expression of AT1R in alveolar epithelial cells, activate the AT1R pathway, induce ERS and promote the progression of MVPF.

Fibrotic extracellular vesicles contribute to mechanical ventilation-induced pulmonary fibrosis development by activating lung fibroblasts via JNK signalling pathway: an experimental study.

Recent research has revealed that mechanical ventilation (MV) could initiate ventilator-induced lung injury along with the initiation of the process of pulmonary fibrosis (PF), leading to MV-induced PF (MVPF). However, the underlying mechanism remains unclear. This study aimed to explore the role of MV-induced extracellular vesicles (MV-EVs) and the c-Jun N-terminal kinase (JNK) signalling pathway in the pathogenesis of MVPF in vivo and in vitro. The process of MV is accompanied by the secretion of MV-EVs, which could induce lung fibroblast activation. Furthermore, single-cell RNA-sequencing analysis revealed that the JNK pathway in lung fibroblasts was activated after MV initiation. Inhibiting the JNK pathway could both restrain MV-EV-induced lung fibroblast activation in vitro or reduce the severity of MVPF in vivo. In conclusion, this study demonstrated that MV-EVs contribute to MVPF progression by activating lung fibroblasts via the JNK signalling pathway and that inhibiting the secretion of EV and the activation of the JNK signalling pathway is a promising strategy for treating MVPF.