Comparative analysis of monocyte-derived dendritic cell phenotype and T cell stimulatory function in patients with acute-on-chronic liver failure with different clinical parameters.

Background: Acute-on-Chronic Liver Failure (ACLF) patients experience systemic inflammation as well as immune dysfunction and exhaustion. The phenotype and functionality of monocyte-derived dendritic cells in ACLF patients with different clinical parameters have not been elucidated. Methods: This study included 37 cases of ACLF, 20 cases of Chronic Hepatitis B (CHB) patients, and 12 healthy controls. Demographic and laboratory parameters were collected from the enrolled patients. Peripheral blood samples were obtained from the participants. Monocyte-derived dendritic cells were induced and cultured, followed by co-culturing with T cells from the patients. Cell surface markers and intracellular markers were analyzed using flow cytometry. The relationship between these markers and clinical parameters was compared. Results: Our study found that ACLF patients had lower expression levels of HLA-DR, CD86, and CD54 on monocyte-derived dendritic cells compared to both CHB patients and healthy controls. IL-4, GM-CSF, and alcohol were found to promote the expression of HLA-DR, CD86, and CD54 on monocyte-derived dendritic cells. In ACLF patients, higher levels of procalcitonin (PCT), lower levels of albumin, decreased prothrombin activity and deceased patients were associated with lower expression of HLA-DR, CD86, and CD54 on monocyte-derived dendritic cells. Peripheral blood mononuclear cells (PBMCs), after removing adherent cells, were co-cultured with monocyte-derived DC. Our study revealed that patients with infection and low albumin levels exhibited a decreased proportion of T cell subsets within PBMCs. Additionally, these patients' T cells showed lower levels of Ki-67 and interferon-gamma (IFN-γ) production. Conclusion: ACLF patients exhibit varying clinical states, with differences in the phenotype and the ability of monocyte-derived dendritic cells to stimulate T cells. Alcohol can stimulate the maturation of monocyte-derived dendritic cells.

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.

Retraction Note: Construction of Gpm6a/ReelinGFPCreERT2 by BAC recombination using a specific gene in hepatic mesothelial or stellate cells.

[This retracts the article on p. 224 in vol. 23, PMID: 28127196.].

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.

Construction of Gpm6a/ReelinGFPCreERT2 by BAC recombination using a specific gene in hepatic mesothelial or stellate cells.

AIM: To prepare a Gpm6a/ReelinGFPCreERT2 construct with a rapid and reliable strategy using a bacterial artificial chromosome (BAC). METHODS: Gpm6a and Reelin BACs were purified and transformed into SW102 E. coli by electroporation. The GFPCreERT2 fragment was prepared from a shuttle vector and transformed into SW102 E. coli carrying a BAC. Homologous recombination was induced in SW102 E. coli. Recombinant clones were screened and confirmed by PCR and restriction enzyme digestion. Recombinant clones were transformed into SW102 E. coli to remove the kanamycin unit. RESULTS: A complete BAC was successfully transformed into SW102 E. coli by electroporation because BAC purified from SW102 E. coli showed the same pattern as the original BAC with BamHI digestion. The GFPCreERT2 fragment was deemed to have been prepared successfully because we obtained the same size fragment as expected. Homologous recombination was induced, and GFPCreERT2 was deemed to have been inserted into the correct site of the BAC because we found the band change was the same as the expected pattern after restriction enzyme digestion. The kanamycin unit was deemed to have been removed successfully because we obtained different sizes of bands that were consistent with the results expected by PCR with different primers. CONCLUSION: The construct of Gpm6aGFPCreERT2 or ReelinGFPCreERT2 was prepared successfully, which will establish a foundation for tracing the hepatic stellate cell lineage and studying its function.

Augmenter of liver regeneration protects against carbon tetrachloride-induced liver injury by promoting autophagy in mice.

BACKGROUND: Augmenter of liver regeneration (ALR) exerts strong hepatoprotective properties in various animal models of liver injury, but its protective mechanisms have not yet been explored. Autophagy is a recently recognized rudimentary cellular response to inflammation and injury. The aim of this study was to test the hypothesis that ALR may protect against acute liver injury through the autophagic pathway. METHODS: The level and role of ALR in liver injury were studied in a mouse model of acute liver injury induced by carbon tetrachloride (CCl4). The effect of ALR on autophagy was analyzed in vitro and in vivo. After autophagy was inhibited by 3-methyladenine (3-MA), apoptosis and proliferation were detected in the mouse model with acute liver injury. The ALR and autophagic levels were measured in patients with liver cirrhosis (LC) and acute liver failure (ALF), respectively. RESULTS: During the progression of acute liver injury, the ALR levels increased slightly in early stage and significantly decreased in late stage in mice. Treatment with an ALR plasmid via tail vein injection protected mice against acute liver injury. The protective effect of ALR relied on the induction of autophagy, which was supported by the following evidence: (1) ALR overexpression directly induced autophagy flux in vitro and in vivo; and (2) ALR treatment suppressed apoptosis and promoted proliferation in mice exposed to CCl4, but the inhibition of autophagy reversed these effects. More importantly, the ALR levels decreased in patients with LC and ALF compared with normal controls. CONCLUSION: We demonstrated that ALR ameliorated liver injury via an autophagic mechanism, which indicates a potential therapeutic application for liver injury.

Naringin in Ganshuang Granule suppresses activation of hepatic stellate cells for anti-fibrosis effect by inhibition of mammalian target of rapamycin.

A previous study has demonstrated that Ganshuang granule (GSG) plays an anti-fibrotic role partially by deactivation of hepatic stellate cells (HSCs). In HSCs activation, mammalian target of rapamycin (mTOR)-autophagy plays an important role. We attempted to investigate the role of mTOR-autophagy in anti-fibrotic effect of GSG. The cirrhotic mouse model was prepared to demonstrate the anti-fibrosis effect of GSG. High performance liquid chromatography (HPLC) analyses were used to identify the active component of GSG. The primary mouse HSCs were isolated and naringin was added into activated HSCs to observe its anti-fibrotic effect. 3-methyladenine (3-MA) and Insulin-like growth factor-1 (IGF-1) was added, respectively, into fully activated HSCs to explore the role of autophagy and mTOR. GSG played an anti-fibrotic role through deactivation of HSCs in cirrhotic mouse model. The concentration of naringin was highest in GSG by HPLC analyses and naringin markedly suppressed HSCs activation in vitro, which suggested that naringin was the main active component of GSG. The deactivation of HSCs caused by naringin was not because of the autophagic activation but mTOR inhibition, which was supported by the following evidence: first, naringin induced autophagic activation, but when autophagy was blocked by 3-MA, deactivation of HSCs was not attenuated or reversed. Second, naringin inhibited mTOR pathway, meanwhile when mTOR was activated by IGF-1, deactivation of HSCs was reversed. In conclusion, we have demonstrated naringin in GSG suppressed activation of HSCs for anti-fibrosis effect by inhibition of mTOR, indicating a potential therapeutic application for liver cirrhosis.

[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.

[Compound nutrients promote liver rehabilitation and regeneration in rats with CCl4;-induced liver cirrhosis].

OBJECTIVE: To explore the effects of compound nutrients on liver rehabilitation and regeneration in rats with CCl4;-induced liver cirrhosis. METHODS: The rat cirrhotic models were prepared by injecting intraperitoneally the mixture of CCl4; (40%) and oil (60%) by 2 mL/kg body weight twice a week for 12 weeks. The nutrition treatment group was treated with compound nutrients and the spontaneous recovery group was not treated with nutrients after stopping CCl4; injection. Then liver tissues and blood samples were harvested to detect the expressions of augmenter of liver regeneration (ALR) and proliferating cell nuclear antigen (PCNA) by immunohistochemistry, the level of hepatocyte growth factor (HGF) by ELISA, the levels of albumin (ALB), alanine aminotransferase (ALT), total bilirubin (TBIL), glucose, triglyceride (TG) and cholesterol (CHOL) by automatic biochemical analyzer, and the levels of amino acids in blood plasma by mass spectrometry. RESULTS: The expressions of ALR and PCNA were higher in nutrition treatment group than in spontaneous recovery group. The level of HGF in serum was higher in nutrition treatment group than in spontaneous recovery group (98.92 ± 2.42 vs 60.99 ± 27.63, t = 3.349, P = 0.020). The levels of ALT (60.18 ± 6.39 vs 84.6 ± 17.91, t = 3.146, P = 0.019) and TBIL (2.08 ± 0.46 vs 6.97 ± 2.58, t = 4.56, P = 0.001) were lower in nutrition treatment group than in spontaneous recovery group. The level of ALB was higher in nutrition treatment group than in spontaneous recovery group (33.15 ± 1.36 vs 24.62 ± 2.48, t = 7.39, P=0.000). The level of branched chain amino acids (BCAA) in blood plasma was higher in nutrition treatment group than in spontaneous recovery group (381.53 ± 35.86 vs 283.05 ± 79.14, t = 2.78, P = 0.02). CONCLUSION: Compound nutrients can be good for liver rehabilitation and regeneration in CCl4;-induced cirrhotic rat model, and the liver regeneration may relate to high BCAA level in blood plasma.