Exosome GLUT1 derived from hepatocyte identifies the risk of non-alcoholic steatohepatitis and fibrosis.

BACKGROUND AND AIMS: It is particularly important to identify the progression of non-alcoholic fatty liver disease (NAFLD) for prognosis evaluation and treatment guidance. The aim of this study was to explore the clinic use of exosomal protein-based detection as a valuable non-invasive diagnostic method for NAFLD. METHODS: Exosomes were extracted from plasma of patients with NAFLD using Optima XPN-100 ultrafast centrifuge. The patients were recruited from outpatients and inpatients of Beijing Youan Hospital Affiliated to Capital Medical University. The exosomes were stained with fluorescent-labeled antibody and determined by ImageStream® X MKII imaging flow cytometry. Generalized linear logistic regression model was used to evaluate the diagnostic value of hepatogenic exosomes in NAFLD and liver fibrosis. RESULTS: The percentage of hepatogenic exosomes glucose transporter 1 (GLUT1) in patients with non-alcoholic steatohepatitis (NASH) was significantly higher than that in patients with non-alcoholic fatty liver (NAFL). According to liver biopsy, we found that the percentage of hepatogenic exosomes GLUT1 in patients with advanced NASH (F2-4) was significantly higher than that in patients with early NASH (F0-1), and the same trend was observed in exosomes with CD63 and ALB. Compared with other clinical fibrosis scoring criteria (FIB-4, NFS, etc.), the diagnostic performance of hepatogenic exosomes GLUT1 was the highest and the area under the receiver-operating curves (AUROC) was 0.85 (95% CI 0.77-0.93). Furthermore, the AUROC of hepatogenic exosomes GLUT1 combined with fibrosis scoring was as high as 0.86-0.91. CONCLUSION: Hepatogenic exosome GLUT1 can be a molecular biomarker for early warning of NAFLD to distinguish the NAFL and NASH, and it also can be used as a novel non-invasive diagnostic biomarker for the staging liver fibrosis in NAFLD.

ASPP2 Coordinates ERS-Mediated Autophagy and Apoptosis Through mTORC1 Pathway in Hepatocyte Injury Induced by TNF-α.

Background: Though ASPP2 plays an important role in regulating cell apoptosis and autophagy in case of liver injury, there remains a lack of clarity on the molecular mechanism of ASPP2 regulating autophagy and apoptosis. Methods: A hepatocyte injury model was constructed using HL7702 cell line and TNF-α. The cells were treated by ASPP2 overexpression adenovirus or short hairpin RNA lentivirus and endoplasmic reticulum stress (ERS) or the mammalian target of rapamycin (mTOR) inhibitor or agonist, respectively. The autophagy was detected by means of western blot and Green fluorescent protein-labeled- Microtubule-associated protein light chain 3 (GFP-LC3) plasmid transfection, while the apoptosis was detected through western blot, flow cytometry and TUNEL assay. Besides, the proteins related to ERS and mTOR were detected by western blot. Results: The low level of ASPP2 expression was accompanied by high-level autophagy and low-level apoptosis and vice versa in case of hepatocyte injury induce by TNF-α. By upregulating the proteins related to mTORC1 and ERS, ASPP2 induced apoptosis but inhibited autophagy. However, the effect of ASPP2 on autophagy and apoptosis can be reversed by the use of mTORC1 and ERS interfering agent, which indicates that ASPP2 regulated autophagy and apoptosis through mTORC1and ERS pathway. ERS treatment made no difference to the expression of ASPP2 and mTOR-related proteins, which suggests the possibility that the regulation of ERS on apoptosis and autophagy could occur in the downstream of ASPP2 and mTOR. Conclusion: ASPP2 could inhibit autophagy and induce apoptosis through mTORC1-ERS pathway in case of the hepatocyte injury induce by TNF-α. The role of ASPP2-mTORC1-ERS axis was verified in hepatocyte injury, which suggests the possibility that ASPP2 is an important regulatory molecule for the survival and death of hepatocyte.

Hepatocyte-derived exosome may be as a biomarker of liver regeneration and prognostic valuation in patients with acute-on-chronic liver failure.

BACKGROUND: The assessment of liver regeneration is particularly critical for patients with acute-on-chronic liver failure (ACLF). Exosome has both the advantages of specificity of liver biopsy and noninvasion of peripheral blood, which may be the potential biomarker of liver disease. METHODS: The patients with chronic hepatitis B (CHB) and ACLF were enrolled from outpatients and inpatients in Beijing Youan Hospital, Capital Medical University. The exosomes in plasma were extracted by ultracentrifuge using Optima XPN-100 Ultracentrifuge. Exosomes were dyed with fluorescent direct-labeled antibody and the expression profile was assayed using ImageStream® X MKII Imaging Flow Cytometer. RESULTS: The percentage of exosomes with ALB and CD63 was significant higher in ACLF than that in CHB. The percentage of exosomes with ALB and CD63 and VEGF increased in CHB, but decreased in ACLF. The exosomes with ALB, CD63, and VEGF were significant more in survival group than that in dead group in patients with ACLF. The sensitivity and specificity of exosomes with CD63, ALB, and VEGF were significantly higher than the other markers of liver regeneration and prognostic valuation in patients with ACLF including AFP. The hepatocyte-derived exosomes expression profile had no difference in different stages and different AFP levels of patients with ACLF. CONCLUSION: The exosomes profile with ALB and VEGF may be a more accurate and specific biomarker of liver regeneration and prognostic valuation than AFP in patients with ACLF. In addition, the exosomes profile with CD63 and ALB may be an early-warning marker in patients with ACLF.

Advances on liver cell-derived exosomes in liver diseases.

Exosomes are extracellular vesicles with diameters ranging from 30 to 150 nm, which contain several donor cell-associated proteins as well as mRNA, miRNA, and lipids and coordinate multiple physiological and pathological functions through horizontal communication between cells. Almost all types of liver cells, such as hepatocytes and Kupffer cells, are exosome-releasing and/or exosome-targeted cells. Exosomes secreted by liver cells play an important role in regulating general physiological functions and also participate in the onset and development of liver diseases, including liver cancer, liver injury, liver fibrosis and viral hepatitis. Liver cell-derived exosomes carry liver cell-specific proteins and miRNAs, which can be used as diagnostic biomarkers and treatment targets of liver disease. This review discusses the functions of exosomes derived from different liver cells and provides novel insights based on the latest developments regarding the roles of exosomes in the diagnosis and treatment of liver diseases.

The inhibition of IL-2/IL-2R gives rise to CD8+ T cell and lymphocyte decrease through JAK1-STAT5 in critical patients with COVID-19 pneumonia.

Although most patients with COVID-19 pneumonia have a good prognosis, some patients develop to severe or critical illness, and the mortality of critical cases is up to 61.5%. However, specific molecular information about immune response in critical patients with COVID-19 is poorly understood. A total of 54 patients were enrolled and divided into three groups, among which 34 were common, 14 were severe, and 6 were critical. The constitution of peripheral blood mononuclear cells (PBMC) in patients was analyzed by CyTOF. The profile of cytokines was examined in plasma of patients using luminex. The IL-2 signaling pathway was investigated in the PBMC of patients by qRT-PCR. The count and percentage of lymphocytes were significantly decreased in critical patients compared to common and severe patients with COVID-19 pneumonia. The count of T cells, B cells, and NK cells was remarkably decreased in critical patients compared to normal controls. The percentage of CD8+ T cells was significantly lower in critical patients than that in common and severe patients with COVID-19 pneumonia. The expression of IL-2R, JAK1, and STAT5 decreased in PBMC of common, severe, and critical patients, but IL-2 level was elevated in severe patients and decreased in critical patients with COVID-19 pneumonia. The decrease of CD8+ T cells in critical patients with COVID-19 pneumonia may be related to the IL-2 signaling pathway. The inhibition of IL-2/IL-2R gives rise to CD8+ T cell and lymphocyte decrease through JAK1-STAT5 in critical patients with COVID-19 pneumonia.

[TP53BP2/ASPP2 inhibits autophagy of HepG2 cells by activating mTOR pathway in a p53-independent manner].

Objective To study the regulative effect of tumor protein p53 binding protein 2/p53 apoptosis-stimulating protein 2 (TP53BP2/ASPP2) on the autophagy of HepG2 human hepatoma cells and its mechanism. Methods The expression of ASPP2 was up-regulated or down-regulated by HepG2 cells infected with adenovirus and lentivirus. HepG2 cells were cultured in medium without fetal bovine serum for 24 hours. Western blotting was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3), beclin1, P62, autophagy-related gene 5 (ATG5), ATG7, and mammalian target of rapamycin (mTOR) pathway-associated protein mTOR, phospho-mTOR (p-mTOR), eukaryotic transcription initiation factor 4E binding protein 1 (4EBP1), phospho-4EBP1 (p-4EBP1), ribosomal protein S6, phospho-S6 (p-S6), ribosomal S6 kinase B1 (RPS6KB1/p70S6K), phospho-p70S6K (p-p70S6K). Fluorescence microscopy was used to detect the green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) fusion protein expressed by the cells. At the same time, the Hep3B cells without p53 expression were infected with lentivirus, and the levels of ASPP2 in the cells were knocked down. Western blotting was performed to detect the expression of ASPP2 and mTOR pathway-associated protein mTOR, p-mTOR, 4EBP1, p-4EBP1, S6, p-S6, RPS6KB1/p70S6K, p-p70S6K. Results When the expression of ASPP2 was up-regulated, the mTOR complex 1 (mTORC1) pathway was activated, and the expression of autophagy-related proteins and the number of autophagosomes were reduced. After the expression of ASPP2 was down-regulated, the mTORC1 pathway was inhibited, and the expression level of autophagy-related proteins and the number of autophagosomes increased. In Hep3B cells with p53 expression silence, the mTORC1 pathway was still inhibited when ASPP2 wasdown-regulated. Conclusion ASPP2 inhibits the autophagy of HepG2 cells by activating mTOR pathway in a p53-independent manner.

Deficiency of apoptosis-stimulating protein 2 of p53 protects mice from acute hepatic injury induced by CCl4 via autophagy.

BACKGROUND: Apoptosis-stimulating protein 2 of p53 (ASPP2) has a variety of biological functions, and is involved in cellular apoptosis, autophagy and inflammatory reaction. However, the role of ASPP2 in acute hepatic injury remains unclear. METHODS: We established an animal model of acute hepatic injury by intraperitoneal injection of CCl4. The expression profile of ASPP2 was measured in wild type (ASPP2+/+) mice with acute hepatic injury induced by CCl4. Hepatic pathological changes and liver function, apoptosis, inflammation and autophagic levels were measured in ASPP2+/+and ASPP2 haploid deletion (ASPP2+/-) mice with acute hepatic injury, respectively. After 3-methyladenine (3-MA) treatment, indicators of hepatic injury were observed in ASPP2+/+and ASPP2+/- mice with CCl4 injection. RESULTS: During the development of acute hepatic injury, ASPP2 expression significantly upregulated at 24 h and 48 h after CCl4 injection. ASPP2 haplotype deletion protected against acute hepatic injury, and this was mainly reflected in decreased ALT and AST levels, less hepatic tissue hemorrhage and necrosis, and reduced cellular inflammation and apoptosis in ASPP2+/- mice compared with ASPP2+/+ mice with acute hepatic injury. ASPP2 haploid deletion activates autophagy in mice with acute hepatic injury, and protects mice from acute hepatic injury via the autophagic signal pathway. CONCLUSION: ASPP2 haplotype deletion protected mice against acute hepatic injury through autophagy activation, which inhibited inflammation and apoptosis in acute hepatic injury.

Ultrasound-assisted microsurgical left spermatic-inferior epigastric vein anastomosis for treating nutcracker syndrome-associated varicocele.

OBJECTIVES: The study summarizes the effectiveness of ultrasound-assisted microsurgical left spermatic-inferior epigastric vein anastomosis for treating nutcracker syndrome (NCS)-associated varicocele. METHODS: Cases of NCS-associated varicocele were recruited between December 2012 and December 2018. Prior to the operation, all patients were tested for the internal diameter and blood flow velocity of left renal vein, testicular volume, maximum venous diameter and venous retrograde flow in the pampiniform plexus of veins during the Valsalva maneuver by Color Doppler ultrasound. Moreover, the direction of left spermatic and inferior epigastric vein was marked. RESULTS: All patients underwent ligation of the internal spermatic veins and left spermatic-inferior epigastric vein anastomosis under microscopy. Color Doppler ultrasound, urinary and semen analysis (above age 18 years old) were reviewed during the follow-up period. 53 patients (94.6%) underwent spermatic-inferior epigastric vein anastomosis with the mean operation time of 78.4 ± 14.2 min. The hospital stay was 4-7 days. Scrotal hydrocele, wound infection and testicular atrophy did not occur after operation. However, there were 5 cases of left varicocele recurrence and 2 cases of vascular anastomotic thrombosis. 51 cases had decrease in blood peak flow rate of left renal vein and improvement in nutcracker syndrome while scrotal bulge symptoms resolved in 26 cases. 10 cases had microscopic hematuria disappearance with symptom improvement in 2 cases. 19 cases of left testicular hypotrophy experience no further deterioration after surgery, of which 16 cases had catch-up testicular growth. CONCLUSION: Ultrasound-assisted microsurgical left spermatic-inferior epigastric vein anastomosis assisted is safe, easy and effective for treating nutcracker syndrome-associated varicocele.

Deficiency of apoptosis-stimulating protein two of p53 ameliorates acute kidney injury induced by ischemia reperfusion in mice through upregulation of autophagy.

Acute kidney injury (AKI) has become a common disorder with a high risk of morbidity and mortality, which remains major medical problem without reliable and effective therapeutic intervention. Apoptosis-stimulating protein two of p53 (ASPP2) is a proapoptotic member that belongs to p53 binding protein family, which plays a key role in regulating apoptosis and cell growth. However, the role of ASPP2 in AKI has not been reported. To explore the role of ASPP2 in the progression of AKI, we prepared an AKI mouse model induced by ischaemia reperfusion (I/R) in wild-type (ASPP2+/+ ) mice and ASPP2 haploinsufficient (ASPP2+/- ) mice. The expression profile of ASPP2 were examined in wild-type mice. The renal injury, inflammation response, cellular apoptosis and autophagic pathway was assessed in ASPP2+/+ and ASPP2+/- mice. The renal injury, inflammation response and cellular apoptosis was analysed in ASPP2+/+ and ASPP2+/- mice treated with 3-methyladenine or vehicle. The expression profile of ASPP2 showed an increase at the early stage while a decrease at the late stage during renal injury. Compared with ASPP2+/+ mice, ASPP2 deficiency protected mice against renal injury induced by I/R, which mainly exhibited in slighter histologic changes, lower levels of blood urea nitrogen and serum creatinine, and less apoptosis as well as inflammatory response. Furthermore, ASPP2 deficiency enhanced autophagic activity reflecting in the light chain 3-II conversion and p62 degradation, while the inhibition of autophagy reversed the protective effect of ASPP2 deficiency on AKI. These data suggest that downregulation of ASPP2 can ameliorate AKI induced by I/R through activating autophagy, which may provide a novel therapeutic strage for AKI.

Protective Effect of Ganshuang Granules () on Liver Cirrhosis by Suppressing Regulatory T Cells in Mouse Model.

OBJECTIVE: To investigate the potential antifibrotic mechanisms of Chinese medicine Ganshuang Granules (, GSG) and to provide clinical therapeutic evidence of its effects. METHODS: A cirrhotic mouse model was established by intraperitoneally injecting a mixture of CCl4 (40%) and oil (60%) at 0.2 mL per 100 g of body weight twice a week for 12 weeks. After 12-week modeling, GSG was intragastric administrated to the mice for 2 weeks, and the mice were divided into low-, medium- and high-dose groups at doses of 1, 2 and 4 g/(kg·day), respectively. Liver morphology changes were observed using Masson's trichrome staining and B-ultrasound. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hyaluronic acid (HA) in serum were detected using an automatic biochemistry analyzer. The expressions of desmin, smooth muscle actin (SMA) and Foxp3 in liver were detected by immunoflfluorescence. The regulatory T cell (Treg) frequency was determined through flflow cytometry analysis. Collagen-I, SMA, IL-6, tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and transforming growth factor ß1 (TGF-ß1) expression levels were measured using quantitative polymerase chain reaction (qPCR). RESULTS: Masson's staining result showed fewer pseudolobule structures and fibrous connective tissue in the GSG-treatment groups than in the spontaneous recovery group. Ultrasonography showed that GSG treatment reduced the number of punctate hyperechoic lesions in mice cirrhotic livers. The serum ALT, AST, HA levels were significantly ameliorated by GSG treatment (ALT: F=8.104, P=0.000; AST: F=7.078, P=0.002; and HA: F=7.621, P=0.001). The expression levels of collagen-I and SMA in the cirrhotic livers were also attenuated by GSG treatment (collagen-I: F=3.938, P=0.011; SMA: F=4.115, P=0.009). Tregs, which were elevated in the fibrotic livers, were suppressed by GSG treatment (F=8.268, P=0.001). The expressions of IL-6, TNF-α and IL-1ß increased, and TGF-ß levels decreased in the cirrhotic livers after GSG treatment (IL-6: F=5.457, P=0.004; TNF-α: F=6.023, P=0.002; IL-1ß: F=6.658, P=0.001; and TGF-ß1: F=11.239, P=0.000). CONCLUSIONS: GSG promoted the resolution/regression of cirrhosis and restored liver functions in part by suppressing Treg cell differentiation, which may be mediated by hepatic stellate cells.

Deficiency of apoptosis-stimulating protein two of p53 promotes liver regeneration in mice by activating mammalian target of rapamycin.

Although liver regeneration has been intensively studied in various ways, the mechanisms underlying liver regeneration remain elusive. Apoptosis-stimulating protein two of p53 (ASPP2) was discovered as a binding partner of p53 and plays an important role in regulating cell apoptosis and growth. However, the role of ASPP2 in hepatocyte proliferation and liver regeneration has not been reported. The expression profile of ASPP2 was measured in a mouse model with 70% partial hepatectomy (PHX). Liver regeneration and hepatocyte proliferation were detected in wild-type (ASPP2+/+) and ASPP2 haploinsufficient (ASPP2+/-) mice with PHX. The mammalian target of rapamycin (mTOR) and autophagy pathways were analyzed in the ASPP2+/+ and ASPP2+/- mice with PHX. After rapamycin or 3-methyladenine (3-MA) treatment, hepatocyte proliferation and liver regeneration were analyzed in the ASPP2+/+ and ASPP2+/- mice with PHX. ASPP2 expression was shown to be upregulated at the early stage and downregulated at the late stage. Compared to the ASPP2+/+ mice, liver regeneration was enhanced in ASPP2+/- mice with 70% PHX. In addition, compared to the ASPP2+/+ mice, the mTORC1 pathway was significantly upregulated and the autophagic pathway was downregulated in ASPP2+/-mice with 70% PHX. Inhibition of the mTORC1 pathway significantly suppressed liver regeneration in ASPP2+/- mice with 70% PHX. In contrast, disruption of the autophagic pathway further enhanced liver regeneration in ASPP2+/- mice with 70% PHX. ASPP2 deficiency can promote liver regeneration through activating the mTORC1 pathway, which further regulates downstream molecules, such as those related to autophagy and p70S6K expression in mouse model post-PHX.

[8.5/11.5F transurethral seminal vesiculoscopy in the diagnosis and treatment of refractory hematospermia].

OBJECTIVE: To investigate the clinical application value of 8.5/11.5 F transurethral seminal vesiculoscopy in the diagnosis and treatment of refractory hematospermia. METHODS: We retrospectively analyzed 78 cases of refractory hematospermia diagnosed and treated by 8.5/11.5 F transurethral seminal vesiculoscopy from June 2012 to June 2014. The patients underwent serum PSA examination, transrectal ultrasonography, seminal vesicle ultrasonography, and pelvis CT or MRI before surgery, and all received transurethral seminal vesiculoscopy under the 8.5/11.5 F rigid ureteroscope. RESULTS: Operations were all successfully accomplished, which revealed abnormal opening of the ejaculatory duct in 5 cases, mucosal inflammatory hyperemia in the prostatic utricle and seminal vesicle in 78, dark red mucilage substance in the seminal vesicle in 34, seminal vesicle stones in 19, small polyp in the seminal vesicle in 2, and ejaculatory duct or seminal vesicle cyst in 4. All the patients received symptomatic treatment during the surgery. After surgery, hematouria was found in 13 cases, which disappeared within 2 weeks, pelvic hematoma in 1 case, which was cured by conservative treatment within 3 months, and epididymitis in 2 cases, which was controlled by anti-infection treatment. Hematospermia recurred in 3 cases during the 1-year postoperative follow-up. CONCLUSION: 8.5/11.5 F transurethral seminal vesiculoscopy, with its advantages of easy operation, wide field of vision, large channel for operation, and few complications, deserves general clinical application in the diagnosis and treatment of refractory hematospermia.

Autophagy in anti-apoptotic effect of augmenter of liver regeneration in HepG2 cells.

AIM: To investigate the role of autophagy in the anti-apoptotic effect of augmenter of liver regeneration (ALR). METHODS: Autophagy was induced through serum deprivation. An ALR-expressing plasmid was transfected into HepG2 cells, and autophagic flux was determined using fluorescence microscopy, electron microscopy, Western blot and quantitative polymerase chain reaction (qPCR) assays. After ALR-expressing plasmid transfection, an autophagy inhibitor [3-methyladenine (3-MA)] was added to HepG2 cells, and apoptosis was observed using fluorescence microscopy and flow cytometry. RESULTS: Autophagy was activated in HepG2 cells, peaking at 24 h after serum deprivation. Microtubule-associated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells, fluorescence microscopy, electron microscopy and qPCR studies showed the similar trend, and p62 levels showed the opposite trend, which indicated that ALR may play an important role in increasing autophagy flux. The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone. Therefore, the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited, indicating that the anti-apoptotic effect of ALR may be related to autophagy. CONCLUSION: ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease.

Study on the determination of polyphenols in tobacco by HPLC coupled with ESI-MS after solid-phase extraction.

A high-performance liquid chromatography method coupled with electrospray ionization-mass spectrometry for the determination of polyphenols in tobacco is studied. The polyphenols are extracted from a tobacco sample by being refluxed in a boiling water bath with 80% methanol and purified by solid-phase extraction with a C18 cartridge. The chlorogenic acid, rutin, scopoletin, caffeic acid, scopolin, and other polyphenols are satisfactorily separated on a Nova-Pak C18 chromatographic column (3.9 x 150 mm) with methanol and 0.05 mol/L potassium dihydrogen phosphate buffer solution gradient elution as mobile phase at a flow rate of 0.5 mL/min. Each of the polyphenols is monitored by photodiode array detector at its maximum wavelength: chlorogenic acid, 326.1 nm; rutin, 354.8 nm; scopoletin, 344.0 nm; caffeic acid, 323.7 nm; and scopolin, 365.2 nm. The limits of detection are: 100 ng/mL for chlorogenic acid, 125 ng/mL for rutin, 60 ng/mL for scopoletin, 50 ng/mL for caffeic acid, and 100 ng/mL for scopolin. The key polyphenols in tobacco are identified by comparing the retention time, the UV-spectrum, and the mass spectra with those of the standards. The recovery of tobacco polyphenols is 94-105%, and the relative standard deviations are 1.28-1.49%. This method is successfully applied to qualitatively and quantitatively analyze the polyphenols in tobacco with good results.