Single-cell immune profiling of mouse liver aging reveals Cxcl2+ macrophages recruit neutrophils to aggravate liver injury.

BACKGROUND AND AIMS: Immune cells play a crucial role in liver aging. However, the impact of dynamic changes in the local immune microenvironment on age-related liver injury remains poorly understood. We aimed to characterize intrahepatic immune cells at different ages to investigate key mechanisms associated with liver aging. APPROACH AND RESULTS: We carried out single-cell RNA sequencing on mouse liver tissues at 4 different ages, namely, the newborn, suckling, young, and aged stages. The transcriptomic landscape, cellular classification, and intercellular communication were analyzed. We confirmed the findings by multiplex immunofluorescence staining, flow cytometry, in vitro functional experiments, and chimeric animal models. Nine subsets of 89,542 immune cells with unique properties were identified, of which Cxcl2+ macrophages within the monocyte/macrophage subset were preferentially enriched in the aged liver. Cxcl2+ macrophages presented a senescence-associated secretory phenotype and recruited neutrophils to the aged liver through the CXCL2-CXCR2 axis. Through the secretion of IL-1ß and TNF-α, Cxcl2+ macrophages stimulated neutrophil extracellular traps formation. Targeting the CXCL2-CXCR2 axis limited the neutrophils migration toward the liver and attenuated age-related liver injury. Moreover, the relationship between Cxcl2+ macrophages and neutrophils in age-related liver injury was further validated by human liver transplantation samples. CONCLUSIONS: This in-depth study illustrates that the mechanism of Cxcl2+ macrophage-driven neutrophil activation involves the CXCL2-CXCR2 axis and provides a potential therapeutic strategy for age-related liver injury.

Machine learning-based identification of CYBB and FCAR as potential neutrophil extracellular trap-related treatment targets in sepsis.

Objective: Sepsis related injury has gradually become the main cause of death in non-cardiac patients in intensive care units, but the underlying pathological and physiological mechanisms remain unclear. The Third International Consensus Definitions for Sepsis and Septic Shock (SEPSIS-3) definition emphasized organ dysfunction caused by infection. Neutrophil extracellular traps (NETs) can cause inflammation and have key roles in sepsis organ failure; however, the role of NETs-related genes in sepsis is unknown. Here, we sought to identify key NETs-related genes associate with sepsis. Methods: Datasets GSE65682 and GSE145227, including data from 770 patients with sepsis and 54 healthy controls, were downloaded from the GEO database and split into training and validation sets. Differentially expressed genes (DEGs) were identified and weighted gene co-expression network analysis (WGCNA) performed. A machine learning approach was applied to identify key genes, which were used to construct functional networks. Key genes associated with diagnosis and survival of sepsis were screened out. Finally, mouse and human blood samples were collected for RT-qPCR verification and flow cytometry analysis. Multiple organs injury, apoptosis and NETs expression were measured to evaluated effects of sulforaphane (SFN). Results: Analysis of the obtained DEGs and WGCNA screened a total of 3396 genes in 3 modules, and intersection of the results of both analyses with 69 NETs-related genes, screened out seven genes (S100A12, SLC22A4, FCAR, CYBB, PADI4, DNASE1, MMP9) using machine learning algorithms. Of these, CYBB and FCAR were independent predictors of poor survival in patients with sepsis. Administration of SFN significantly alleviated murine lung NETs expression and injury, accompanied by whole blood CYBB mRNA level. Conclusion: CYBB and FCAR may be reliable biomarkers of survival in patients with sepsis, as well as potential targets for sepsis treatment. SFN significantly alleviated NETs-related organs injury, suggesting the therapeutic potential by targeting CYBB in the future.

Oxidative stress mediates hippocampal neuronal apoptosis through ROS/JNK/P53 pathway in rats with PTSD triggered by high-voltage electrical burn.

BACKGROUND: The pathogenesis of post-traumatic stress disorder (PTSD) triggered by high-voltage electrical burn (HVEB) remains unclear and the oxidative stress plays a role in this process. The purpose of this study is to investigate the underlying mechanism of oxidative stress mediates hippocampal neuronal apoptosis in rats with PTSD triggered by HVEB. MATERIALS AND METHODS: The PTSD rat model was developed by stimulating with high voltage electricity and screened using behavioral performance including Morris water maze (MWM), elevated plus-maze (EPM) and open-field test (OFT). The reactive oxygen species (ROS) generation was measured by DHE fluorescence staining or flow cytometry. Western blotting assay was used to detect the proteins of p-JNK, JNK, P53, PUMA, Bcl-2 and Bax in hippocampal tissue or HT22 cells treated with electrical stimulation. RESULTS: The serum MDA and 8-OHdG levels were increased (P < 0.001), while the activities of SOD and CAT were decreased (P < 0.001) significantly in patients with HVEB. Behavioral test results showed that high-voltage electric stimulation induced the PTSD-like symptoms and the ROS-JNK-P53 pathway was involved in the neuronal apoptosis in rats with PTSD induced by HVEB. In vitro experiments further confirmed the electrical stimulation induced neuronal apoptosis through ROS/JNK/P53 signaling pathway and the antioxidant NAC could rescued the ROS generation, activation of JNK/P53 proteins and improved the cell apoptosis rate in HT22 cells. Finally, the JNK inhibitor SP600125 could significantly inhibited the percentage of HT22 cell apoptosis induced by electrical stimulation (P < 0.001). CONCLUSIONS: These results indicated that oxidative stress mediates hippocampal neuronal apoptosis through ROS/JNK/P53 pathway in rats with PTSD triggered by HVEB.

MSC-derived extracellular vesicles as nanotherapeutics for promoting aged liver regeneration.

Aging is one of the critical factors to impair liver regeneration leading to a high incidence of severe complications after hepatic surgery in the elderly population without any effective treatment for clinical administration. As cell-free nanotherapeutics, mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been demonstrated the therapeutic potentials on liver diseases. However, the effects of MSC-EVs on the proliferation of aged hepatocytes are largely unclear. In this study, we found MSCs could reduce the expression of senescence-associated markers in the liver and stimulate its regeneration in aged mice after receiving a two-thirds partial hepatectomy (PHx) through their secreted MSC-EVs. Using RNA-Seq and AAV9 vector, we mechanistically found that these effects of UC-MSC-EVs partially attributed to inducing Atg4B-related mitophagy. This effect repairs the mitochondrial status and functions of aged hepatocytes to promote their proliferation. And protein mass spectrum analysis uncovered that DEAD-Box Helicase 5 (DDX5) enriches in UC-MSC-EVs, which interacts with E2F1 to facilitate its nuclear translocation for activating the expression of Atg4B. Collectively, our data show that MSC-EVs act nanotherapeutic potentials in anti-senescence and promoting regeneration of aged liver by transferring DDX5 to regulate E2F1-Atg4B signaling pathway that induce mitophagy, which highlights the clinical application valuation of MSC-EVs for preventing severe complications in aged population receiving liver surgery.

Classification of colorectal cancer into clinically relevant subtypes based on genes and mesenchymal cells.

BACKGROUND: Most studies on subtype identification of colorectal cancer (CRC) were based on expressions of either genes or immune cells. However, few studies have hitherto used the combination of genes with immune and stroma cells for subtype identification. METHODS: Dataset GSE17536 was obtained from the Gene Expression Omnibus (GEO) database. The xCell algorithm was used to estimate the composition and density of 64 cell types, including immune and stroma cell types. Clustering analysis was then conducted on the top 3000 most variable genes from a total of 20,174 genes for CRC subtype identification. We employed the ensemble method of Similarity network fusion and 112 Consensus Clustering (SNF-CC) for cancer subtype identification. Reactome pathway analysis was conducted to identify the impact of the representative genes on prognosis. The results were validated in independent gene expression data from dataset GSE17537. RESULTS: In this study, we identified 3 clinically relevant subtypes and their representative genes, immune and stroma cells. Moreover, we confirmed the correlation of these subtypes with their clinical characteristics. The representative genes of the subtype with poor prognosis correlated with extracellular matrix structural constituent, while the subtype with good prognosis correlated with Toll-like receptor signaling pathway or chemokine signaling pathway. However, different subtypes were associated with distinct cell subtypes; the subtype with poor prognosis had a high abundance of fibroblasts and endothelial cells; the subtype with median prognosis had a higher abundance of immune cells, such as CD4 + T-cell, Th2 cells and aDC; the subtype with good prognosis had a higher abundance of NKT. CONCLUSION: This study highlights the utility of immune and innate cells, especially during gene analysis, to provide the theoretical basis for personalized treatment in colorectal cancer patients.

Research of a Safe and Simplified Intertransverse Process Approach for the Lower Thoracic Interbody Surgery.

OBJECTIVE: To assess a safe surgical approach for intertransverse process lower thoracic intervertebral body fusion (ITIF) based on measurements from enhanced three-dimensional CT reconstruction, cadaver simulated operation, and patient operation. METHODS: Enhanced three-dimensional CT image reconstruction was performed for 20 healthy volunteers on thoracic segments T8-T12. The length of the transverse process (LTP), distance between the upper and lower transverse processes (DULTP), remote distance of the transverse process (RDTP), height of the extraforaminal intervertebral space (HEIS), and oblique diameter of the intervertebral space (ODIS) were measured and recorded. The blood vessels of the intertransverse lower thoracic region were observed, and their internal diameters were measured. The rib-intervertebral space relationship for T10/11 and T11/12 was measured in 104 patients of the thoracic skeleton. Then, based on the data from the CT measurements, simulated surgery was performed on six human cadavers at the T11/12 level. An ankylosing spondylitis (AS) patient with a fracture of the T10/11 level was eventually operated on with the ITIF technique. RESULTS: No significant difference was found between the lengths of the left and right thoracic transverse processes. The relationship of the values of the LTP and RDTP for the measured vertebrae were found to be as follows:T8 > T9 > T10 > T11 > T12. For HEIS and DULTP, T8-9 < T9-10 < T10-11 < T11-12. The results for the ODIS were as follows: T8-T9 < T9-T10 < T10-T11 < T11-T12. The blood vessel inner diameter of T11-12 was less than that of T10-11, while there was no significant difference between the diameters for T8-9 and T11-12. Almost half of the volunteer's T10/11 intervertebral spaces were covered posteriorly by the 11th rib (45.19% on left and 41.35% on right), while for most patients, the T11/12 intervertebral space was not covered by the 12th rib (98.08%). According to the cadaver experiments, intervertebral bone grafting and ipsilateral pedicle screw fixation were performed to simulate the operation. One patient with a combined AS and T10/11 fracture was then operated on with the ITIF technique and followed up for 3 years with satisfactory results. CONCLUSION: As verified by 3D CT reconstruction measurements, cadaver simulation surgery and patient operation with follow-up, the intertransverse process approach for some T10/T11 and almost all T11/T12 segments is a safe surgical pathway for operations such as ITIF, fracture bone grafting, clearance of focal lesions.

Determination of naloxone-3-glucuronide in human plasma and urine by HILIC-MS/MS.

A hydrophilic interaction chromatography-tandem mass spectrometric (HILIC-MS/MS) method was developed for the direct determination of naloxone-3-glucuronide (N3G) in human plasma and urine. After a straightforward sample preparation by protein precipitation, N3G was analyzed directly without the need for hydrolysis. Chromatographic separation was performed on a HILIC column. The mobile phase was composed of acetonitrile-10mmol/L ammonium formate (86:14, v/v), with a flow rate of 0.4mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via positive electrospray ionisation (ESI+) source. The linear calibration range was 0.5 to 200ng/mL in plasma and 10 to 5000ng/mL in urine (r(2)>0.99). The intra- and inter-day precision (relative standard deviation, RSD) values were below 15% and the accuracies (relative error, RE) were -7.1% to 2.8% in plasma and -1.3% to 10.3% in urine at three quality control levels. In human subjects receiving 100mg tilidine and 8mg naloxone, mean AUC0-24 of N3G was 160.93±52.77ng/mLh and mean Cmax was 75.33±25.27ng/mL. In 24-h urine samples, 8.0% of the dose was excreted in the form of N3G in urine. These results demonstrated a new method suitable for in vivo pharmacokinetic studies of N3G.