The deubiquitinase USP5 promotes cholangiocarcinoma progression by stabilizing YBX1.

AIMS: Ubiquitin specific peptidase 5 (USP5), a member of deubiquitinating enzymes, has garnered significant attention for its crucial role in cancer progression. This study aims to explore the role of USP5 and its potential molecular mechanisms in cholangiocarcinoma (CCA). MAIN METHODS: To explore the effect of USP5 on CCA, gain-of-function and loss-of-function assays were conducted in human CCA cell lines RBE and HCCC9810. The CCK8, colony-forming assay, EDU, flow cytometry, transwell assay and xenografts were used to assess cell proliferation, migration and tumorigenesis. Western blot and immunohistochemistry were performed to measure the expression of related proteins. Immunoprecipitation and immunofluorescence were applied to identify the interaction between USP5 and Y box-binding protein 1 (YBX1). Ubiquitination assays and cycloheximide chase assays were carried out to confirm the effect of USP5 on YBX1. KEY FINDINGS: We found USP5 is highly expressed in CCA tissues, and upregulated USP5 is required for the cancer progression. Knockdown of USP5 inhibited cell proliferation, migration and epithelial-mesenchymal transition (EMT) in vitro, along with suppressed xenograft tumor growth and metastasis in vivo. Mechanistically, USP5 could interact with YBX1 and stabilize YBX1 by deubiquitination in CCA cells. Additionally, silencing of USP5 hindered the phosphorylation of YBX1 at serine 102 and its subsequent translocation to the nucleus. Notably, the effect induced by USP5 overexpression in CCA cells was reversed by YBX1 silencing. SIGNIFICANCE: Our findings reveal that USP5 is required for cell proliferation, migration and EMT in CCA by stabilizing YBX1, suggesting USP5-YBX1 axis as a promising therapeutic target for CCA.

[Arterial partial pressure of oxygen and procalcitonin combined with ROX index predict the timing of tracheal intubation in patients with acute severe pancreatitis].

OBJECTIVE: To investigate the efficacy of arterial partial pressure of oxygen (PaO2), procalcitonin (PCT) combined with ROX index in predicting the timing of tracheal intubation in patients with acute severe pancreatitis (SAP). METHODS: A case-control study was conducted. A total of 148 patients with SAP admitted to Hunan Provincial People's Hospital from January 2019 to December 2022 were selected as the research objects. According to whether endotracheal intubation was used after admission during hospitalization, the patients were divided into the intubation group (102 cases) and non-intubation group (46 cases). Gender, age, white blood cell count (WBC), lymphocyte count (LYM), platelet count (PLT), C-reactive protein (CRP), hemoglobin (Hb), PCT, PaO2, arterial partial pressure of carbon dioxide (PaCO2), arterial bicarbonate ion (HCO3-) 1 day after admission, arterial lactic acid (Lac), lactate dehydrogenase (LDH), heart rate (HR), respiratory rate (RR), pulse oxygen saturation (SpO2), oxygenation index (PaO2/FiO2), blood pressure, worst ROX index (ROX index = SpO2/FiO2/RR) within 30 minutes of admission and 30 minutes before intubation of the two groups were measured. Multivariate Logistic regression was used to analyze the independent risk factors for the timing of endotracheal intubation in patients with SAP. The receiver operator characteristic curve (ROC curve) was used to determine the optimal predictive cut-off value for endotracheal intubation. RESULTS: There were no significant differences in age, gender, WBC, LYM, CRP, Hb, LDH, HR and blood pressure at admission between the two groups. The PLT, Lac, PCT and RR in the intubation group were significantly higher than those in the un-intubation group, and HCO3-, PaO2, SpO2, PaO2/FiO2, the worst ROX index within 30 minutes after admission and 30 minutes before intubation were significantly lower than those in the non-intubation group (all P < 0.05). Logistic regression analysis showed that the worst ROX index within 30 minutes before intubation was the largest negative influencing factor for the timing of tracheal intubation in SAP patients [odds ratio (OR) = 0.723, 95% confidence interval (95%CI) was 0.568-0.896, P = 0.000], followed by PaO2 (OR = 0.872, 95%CI was 0.677-1.105, P < 0.001). PCT was the positive influencing factor (OR = 1.605, 95%CI was 1.240-2.089, P < 0.001). ROC curve analysis showed that the area under the ROC curve (AUC) of PaO2, PCT, the worst ROX index within 30 minutes before intubation and the combination to evaluate the tracheal intubation time of patients with SAP were 0.715, 0.702, 0.722 and 0.808, the sensitivity was 78.1%, 75.0%, 81.5% and 89.3%, the specificity was 66.7%, 59.0%, 73.2% and 86.4%, and the best cut-off value was 60.23 mmHg (1 mmHg ≈ 0.133 kPa), 2.72 µg/L, 4.85, and 0.58, respectively. The AUC of the combination of PaO2, PCT and the worst ROX index within 30 minutes before intubation predicted the timing of tracheal intubation in patients with SAP was significantly greater than using each index alone (all P < 0.01). CONCLUSIONS: The worst ROX index within 30 minutes before intubation combined with PaO2 and PCT is helpful for clinicians to make a decision for tracheal intubation in patients with SAP.

Olmesartan alleviates SARS-CoV-2 envelope protein induced renal fibrosis by regulating HMGB1 release and autophagic degradation of TGF-ß1.

Background and aims: Renal damage in severe coronavirus disease 2019 (COVID-19) is highly associated with mortality. Finding relevant therapeutic candidates that can alleviate it is crucial. Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin-receptor blockers (ARBs) have been shown to be harmless to COVID-19 patients, but it remains elusive whether ACEIs/ARBs have protective benefits to them. We wished to determine if ACEIs/ARBs had a protective effect on the renal damage associated with COVID-19, and to investigate the mechanism. Methods: We used the envelope (E) protein of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) to induce COVID-19-like multiple organ damage and observed renal fibrosis. We induced the epithelial-mesenchymal transformation of HK-2 cells with E protein, and found that olmesartan could alleviate it significantly. The protective effects of olmesartan on E protein-induced renal fibrosis were evaluated by renal-function assessment, pathologic alterations, inflammation, and the TGF-ß1/Smad2/3 signaling pathway. The distribution of high-mobility group box (HMGB)1 was examined after stimulation with E protein and olmesartan administration. Results: E protein stimulated HMGB1 release, which triggered the immune response and promoted activation of TGF-ß1/Smad2/3 signaling: both could lead to renal fibrosis. Olmesartan regulated the distribution of HMGB1 under E protein stimulation. Olmesartan inhibited the release of HMGB1, and reduced the inflammatory response and activation of TGF-ß1/Smad2/3 signaling. Olmesartan increased the cytoplasmic level of HMGB1 to promote the autophagic degradation of TGF-ß1, thereby alleviating fibrosis further. Conclusion: Olmesartan alleviates E protein-induced renal fibrosis by regulating the release of HMGB1 and its mediated autophagic degradation of TGF-ß1.

Emerging role for branched-chain amino acids metabolism in fibrosis.

Fibrosis is a common pathological feature of organ diseases resulting from excessive production of extracellular matrix, which accounts for significant morbidity and mortality. However, there is currently no effective treatment targeting fibrogenesis. Recently, metabolic alterations are increasingly considered as essential factors underlying fibrogenesis, and especially research on metabolic regulation of amino acids is flourishing. Among them, branched-chain amino acids (BCAAs) are the most abundant essential amino acids, including leucine, isoleucine and valine, which play significant roles in the substance and energy metabolism and their regulation. Dysregulation of BCAAs metabolism has been proven to contribute to numerous diseases. In this review, we summarize the metabolic regulation of fibrosis and the changes in BCAAs metabolism secondary to fibrosis. We also review the effects and mechanisms of the BCAAs intervention, and its therapeutic targeting in hepatic, renal and cardiac fibrosis, with a focus on the fibrosis in liver and associated hepatocellular carcinoma.

Ceria nanoparticles ameliorate renal fibrosis by modulating the balance between oxidative phosphorylation and aerobic glycolysis.

BACKGROUND AND AIMS: Renal fibrosis is the common outcome in all progressive forms of chronic kidney disease. Unfortunately, the pathogenesis of renal fibrosis remains largely unexplored, among which metabolic reprogramming plays an extremely crucial role in the evolution of renal fibrosis. Ceria nanoparticles (CeNP-PEG) with strong ROS scavenging and anti-inflammatory activities have been applied for mitochondrial oxidative stress and inflammatory diseases. The present study aims to determine whether CeNP-PEG has therapeutic value for renal fibrosis. METHODS: The unilateral ureteral obstructive fibrosis model was used to assess the therapeutic effects in vivo. Transforming growth factor beta1-induced epithelial-to-mesenchymal transition in HK-2 cells was used as the in vitro cell model. The seahorse bioscience X96 extracellular flux analyzer was used to measure the oxygen consumption rate and extracellular acidification rate. RESULTS: In the present study, CeNP-PEG treatment significantly ameliorated renal fibrosis by increased E-cadherin protein expression, and decreased α-SMA, Vimentin and Fibronectin expression both in vitro and in vivo. Additionally, CeNP-PEG significantly reduced the ROS formation and improved the levels of mitochondrial ATP. The seahorse analyzer assay demonstrated that the extracellular acidification rate markedly decreased, whereas the oxygen consumption rate markedly increased, in the presence of CeNP-PEG. Furthermore, the mitochondrial membrane potential markedly enhanced, hexokinase 1 and hexokinase 2 expression significantly decreased after treatment with CeNP-PEG. CONCLUSIONS: CeNP-PEG can block the dysregulated metabolic status and exert protective function on renal fibrosis. This may provide another therapeutic option for renal fibrosis.

A novel role of BK potassium channel activity in preventing the development of kidney fibrosis.

Kidney fibrosis is a common characteristic of chronic kidney disease and while the large conductance voltage and calcium-activated potassium channel (BK) is widely expressed in kidneys, its role in kidney fibrosis is unknown. To evaluate this, we found that BK protein expression was decreased in the fibrotic kidneys. Accompanying this was increased fibrotic marker protein expression of fibronectin, vimentin and α-smooth muscle actin and increased mRNA expressions of fibronectin, α-smooth muscle actin, collagen III and collagen I. These changes occurred in the unilateral ureteral obstruction and folic acid models of fibrosis and were more pronounced in BK knockout than in wild-type mice. Activation of BK activity by chemical NS1619 or BMS191011 channel openers attenuated kidney fibrosis in these two models while protecting kidney function in wild-type mice. BK deficiency up-regulated transforming growth factor-ß (TGF-ß)/transcription factor Smad2/3 signaling in the fibrotic kidney, whereas activation of BK activity inhibited this signaling pathway both in vivo and in vitro. BK channel activation increased the degradation of TGF-ß receptors induced by TGF-ß1 in vivo and in vitro. Furthermore, in cell lines HK-2, NRK49, and NRK-52E, BK channel activation by NS1619 led to increased caveolae formation and facilitated localization of TGF-ß receptors in the microdomains of lipid rafts. Thus, our data demonstrated that BK activation has an anti-fibrotic effect on kidney fibrosis by inhibiting the TGF-ß signaling pathway through accelerating TGF-ß receptor degradation via the caveolae route. Hence, our study provides innovative insight into BK as a potential therapeutic target for the treatment of kidney fibrosis.

Activating BK channels ameliorates vascular smooth muscle calcification through Akt signaling.

Vascular calcification (VC) is characterized by pathological depositions of calcium and phosphate in the arteries and veins via an active cell-regulated process, in which vascular smooth muscle cells (VSMCs) transform into osteoblast/chondrocyte-like cells as in bone formation. VC is associated with significant morbidity and mortality in chronic kidney disease (CKD) and cardiovascular disease, but the underlying mechanisms remain unclear. In this study we investigated the role of large-conductance calcium-activated potassium (BK) channels in 3 experimental VC models. VC was induced in vascular smooth muscle cells (VSMCs) by ß-glycerophosphate (ß-GP), or in rats by subtotal nephrectomy, or in mice by high-dosage vitamin D3. We showed that the expression of BK channels in the artery of CKD rats with VC and in ß-GP-treated VSMCs was significantly decreased, which was functionally confirmed by patch-clamp recording. In ß-GP-treated VSMCs, BK channel opener NS1619 (20 µM) significantly alleviated VC by decreasing calcium content and alkaline phosphatase activity. Furthermore, NS1619 decreased mRNA expression of ostoegenic genes OCN and OPN, as well as Runx2 (a key transcription factor involved in preosteoblast to osteoblast differentiation), and increased the expression of α-SMA protein, whereas BK channel inhibitor paxilline (10 µM) caused the opposite effects. In primary cultured VSMCs from BK-/- mice, BK deficiency aggravated calcification as did BK channel inhibitor in normal VSMCs. Moreover, calcification was more severe in thoracic aorta rings of BK-/- mice than in those of wild-type littermates. Administration of BK channel activator BMS191011 (10 mg· kg-1 ·d-1) in high-dosage vitamin D3-treated mice significantly ameliorated calcification. Finally, co-treatment with Akt inhibitor MK2206 (1 µM) or FoxO1 inhibitor AS1842856 (3 µM) in calcified VSMCs abrogated the effects of BK channel opener NS1619. Taken together, activation of BK channels ameliorates VC via Akt/FoxO1 signaling pathways. Strategies to activate BK channels and/or enhance BK channel expression may offer therapeutic avenues to control VC.

The influx/efflux mechanisms of d-peptide ligand of nAChRs across the blood-brain barrier and its therapeutic value in treating glioma.

A d-peptide ligand of the nicotine acetylcholine receptors (nAChRs), termed DCDX, enables drug delivery to the brain when incorporated into liposomes and has shown promise as a nanocarrier for treating brain diseases. However, few reports have described the mechanisms whereby DCDX-modified liposomes traverse the blood-brain barrier (BBB). Here, we studied the molecular mechanisms enabling DCDX (and its associated liposomes) to cross an in vitro BBB using a simulated cerebral endothelium monolayer formed by brain capillary endothelial cells (bEnd.3 cells). We also examined the mechanisms whereby DCDX-modified liposomes cross the BBB in vivo using the brain efflux-index method. Transport of DCDX and its modified liposomes was dominantly mediated via the lipid raft/caveolae endocytic pathway. Both the endoplasmic reticulum (ER) and Golgi complex participated in delivering DCDX-modified liposomes to the plasma membrane (PM). DCDX-modified liposomes also participated in the endosome/lysosome pathway (with high-efficiency BBB crossing observed in vitro), while competing for the ER/Golgi/PM pathway. In addition, nAChR α7 did not promote the transportation of DCDX-modified liposomes in vivo or in vitro, as assessed with α7-knockout mice and by performing α-bungarotoxin (α-Bgt) binding-competition experiments. P-glycoprotein (P-gp) was identified as the main efflux transporter across the BBB, in vivo and in vitro. Using a xenograft nude mouse model of human glioblastoma multiforme, blocking the efflux function of P-gp with verapamil enhanced the therapeutic efficiency of DCDX-modified liposomes that were formulated with doxorubicin against glioblastoma. The findings of this study reveal novel mechanisms underlying crossing of the BBB by DCDX-modified liposomes, suggesting that DCDX-modified liposomes can potentially serve as a powerful therapeutic tool for treating glioma.

Structure and function of USP5: Insight into physiological and pathophysiological roles.

Deubiquitinase (DUB)-mediated cleavage of ubiquitin chains from substrate proteins plays a crucial role in various cellular processes, such as DNA repair and protein stabilization and localization. DUBs can be classified into five families based on their sequence and structural homology, and the majority belong to the ubiquitin-specific proteinase (USP) family. As one of the USPs, ubiquitin-specific proteinase 5 (USP5) is unique in that it can specifically recognize unanchored (not conjugated to target proteins) polyubiquitin and is essential for maintaining homeostasis of the monoubiquitin pool. USP5 has also been implicated in a wide variety of cellular events. In the present review, we focus on USP5 and provide a comprehensive overview of the current knowledge regarding its structure, physiological roles in multiple cellular events, and pathophysiological roles in relevant diseases, especially cancer. Signaling pathways and emerging pharmacological profiles of USP5 are also introduced, which fully embody the therapeutic potential of USP5 for human diseases ranging from cancer to neurological diseases.

Mechanism of Regulation of Big-Conductance Ca2+-Activated K+ Channels by mTOR Complex 2 in Podocytes.

Podocytes, dynamic polarized cells wrapped around glomerular capillaries, are an essential component of the glomerular filtration barrier. BK channels consist of one of the slit diaphragm (SD) proteins in podocytes, interact with the actin cytoskeleton, and play vital roles in glomerular filtration. Mechanistic target of rapamycin (mTOR) complexes regulate expression of SD proteins, as well as cytoskeleton structure, in podocytes. However, whether mTOR complexes regulate podocyte BK channels is still unclear. Here, we investigated the mechanism of mTOR complex regulation of BK channels via real-time PCR, western blot, immunofluorescence, and patch clamping. Inhibiting mTORC1 with rapamycin or downregulating Raptor had no significant effect on BK channel mRNA and protein levels and bioactivity. However, the dual inhibitor of mTORC1 and mTORC2 AZD8055 and short hairpin RNA targeting Rictor downregulated BK channel mRNA and protein levels and bioactivity. In addition, MK2206, GF109203X, and GSK650394, which are inhibitors of Akt, PKCα, and SGK1, respectively, were employed to test the downstream signaling pathway of mTORC2. MK2206 and GF109203X had no effect on BK channel protein levels. MK2206 caused an obvious decrease in the current density of the BK channels. Moreover, GSK650394 downregulated the BK channel protein and mRNA levels. These results indicate mTORC2 not only regulates the distribution of BK channels through Akt, but also modulates BK channel protein expression via SGK1 in podocytes.

[Value of the diaphragm movement index tested by ultrosonography for ventilation weaning].

OBJECTIVE: To evaluate the diaphragm movement index of mechanical ventilation weaning patients by ultrosonography, and to explore its value for weaning. METHODS: Forty patients undergoing invasive mechanical ventilation for at least 48 hours admitted to emergency intensive care unit (EICU) of Hunan Provincial People's Hospital from September 2017 to February 2018 were enrolled. Low level pressure support ventilation (PSV) was used for spontaneous breathing test (SBT), and bedside M-mode ultrasonography was used to assess the diaphragm movement index of the patient within 1 hour of SBT, including the excursion of the diaphragm, diaphragmatic-rapid shallow breathing index (D-RSBI). The rapid shallow breathing index (RSBI) was measured by ventilator. The patients who met the clinical weaning criteria were weaned. According to the success or failure of the weaning, the patients were divided into the successful weaning group and the failure weaning group. The receiver operating characteristic (ROC) curve was used to evaluate the predictive value of each indicator to the failure of the weaning. RESULTS: A total of 40 patients were enrolled in the final analysis, including 28 patients in the successful weaning group and 12 patients in the failure weaning group. The excursion of the diaphragm in the failure weaning group was significantly less than that in the successful weaning group (mm: 9.56±2.13 vs. 13.66±4.10, P < 0.01), and the D-RSBI and RSBI were significantly higher than those in the successful weaning group [D-RSBI (times×min-1×mm-1): 2.06±0.68 vs. 1.44±0.66, RSBI (times×min-1×L-1): 61.70±25.00 vs. 44.91±14.51, both P < 0.05]. The area under the ROC curve (AUC) of diaphragm displacement, D-RSBI, and RSBI was 0.830, 0.851 and 0.711, respectively, and the predicted value of diaphragm excursion and D-RSBI was higher. When the optimal critical value of diaphragmatic excursion was 11.15 mm, the sensitivity of predicting weaning failure was 83.3%, the specificity was 71.4%; when the optimal critical value of D-RSBI was 1.42 times×min-1×mm-1, the sensitivity of predicting the failure of weaning was 91.7%, and the specificity was 82.1%. CONCLUSIONS: Diaphragm excursion and D-RSBI of the diaphragmatic ultrosonography index could accurately predict the failure of the weaning, which was superior to the traditional RSBI in guiding weaning.

Non-invasive detection of the early phase of kidney injury by photoacoustic/computed tomography imaging.

The early diagnosis of kidney diseases, which can remarkably impair the quality of life and are costly, has encountered great difficulties. Therefore, the development of methods for early diagnosis has great clinical significance. In this study, we used an emerging technique of photoacoustic (PA) imaging, which has relatively high spatial resolution and good imaging depth. Two kinds of PA gold nanoparticle (GNP)-based bioprobes were developed based on their superior photo detectability, size controllability and biocompatibility. The kidney injury mouse model was developed by unilateral ureteral obstruction for 96 h and the release of obstruction model). Giving 3.5 and 5.5 nm bioprobes by tail vein injection, we found that the 5.5 nm probe could be detected in the bladder in the model group, but not in the control group. These results were confirmed by computed tomography imaging. Furthermore, the model group did not show changes in the blood biochemical indices (BUN and Scr) and histologic examination. The 5.5 nm GNPs were found to be the critical point for early diagnosis of kidney injury. This new method was faster and more sensitive and accurate for the detection of renal injury, compared with conventional methods, and can be used for the development of a PA GNP-based bioprobe for diagnosing renal injury.

[Clinical effect of pulmonary rehabilitation therapy including respiratory exercise and vibration expectoration on patients with pulmonary infection after abdominal surgery].

OBJECTIVE: To investigate the clinical effect of pulmonary rehabilitation therapy including respiratory exercise and vibration expectoration on patients with pulmonary infection after abdominal surgery. METHODS: A retrospective case control study was conducted. Seventy-six patients with pulmonary infection after abdominal surgery admitted to the First Affiliated Hospital of Hunan Normal University from September 2015 to September 2016 were enrolled. According to whether accept the pulmonary rehabilitation therapy or not, the patients were divided into two groups. In the control group (n = 35), the conventional expectoration method was adopted. The patients in pulmonary rehabilitation group (n = 41) received both methods of the control group and pulmonary rehabilitation treatment, including respiratory exercise (effective cough, lip reduction breathing), respiratory exercise device (respiratory exerciser tri-ball), and vibrated expectoration. The 24-hour sputum volume, degree of comfort, inflammatory and pulmonary function parameters, and recovery situation were recorded in the two groups. RESULTS: (1) There were no significant differences in the parameters of inflammation and pulmonary function before treatment between the two groups. After treatment, the white blood cell (WBC) and C-reactive protein (CRP) in both groups were significantly decreased, and the forced expiratory volume in 1 second (FEV1) and FEV1/forced vital capacity (FVC) were significantly increased. The above changes in pulmonary rehabilitation group were more significant than those of the control group [WBC (×109/L): 11.12±2.88 vs. 13.42±2.62 at 3 days, 8.22±1.48 vs. 9.27±1.92 at 5 days; CRP (mg/L): 13.47±4.77 vs. 16.03±4.94 at 3 days, 9.69±1.56 vs. 11.77±1.41 at 5 days; FEV1 (L): 2.48±0.14 vs. 2.29±0.16 at 3 days, FEV1/FVC: 0.78±0.04 vs. 0.75±0.04 at 3 days; all P < 0.05]. (2) The 24-hour sputum volume within 3 days of pulmonary rehabilitation group were significantly higher than that of the control group (mL: 30.51±4.15 vs. 18.30±3.64 at 1 day, 31.08±3.22 vs. 20.37±3.20 at 2 days, 29.03±2.55 vs. 19.03±2.51 at 3 days, all P < 0.01]. (3) In the pulmonary rehabilitation group, the recovery time of pulmonary infection symptoms (days: 5.44±1.45 vs. 6.20±1.55), the days of antibiotic use (days: 12.61±3.15 vs. 15.03±3.78), the time of getting out of the bed (days: 4.05±0.74 vs. 4.51±0.89), and the hospital days (days: 19.95±3.90 vs. 22.00±4.42) were significantly shorter than those of the control group (all P < 0.05), and the degree of comfort was significantly better than that of the control group (comfort score: 2.71±0.90 vs. 2.14±0.91, P < 0.01). CONCLUSIONS: The application of pulmonary rehabilitation including respiratory exercise and vibration expectoration in abdominal surgery patients with pulmonary infection can promote recovery, and it has a good clinical and practical application value.