Background/Aims: Metabolic dysfunction–associated steatotic liver disease (MASLD) is a chronic liver disease characterized by hepatic steatosis. Ubiquitin-specific protease 29 (USP29) plays pivotal roles in hepatic ischemiareperfusion injury and hepatocellular carcinoma, but its role in MASLD remains unexplored. Therefore, the aim of this study was to reveal the effects and underlying mechanisms of USP29 in MASLD progression. Methods: USP29 expression was assessed in liver samples from MASLD patients and mice. The role and molecular mechanism of USP29 in MASLD were assessed in high-fat diet-fed and high-fat/high-cholesterol diet-fed mice and palmitic acid and oleic acid treated hepatocytes. Results: USP29 protein levels were significantly reduced in mice and humans with MASLD. Hepatic steatosis, inflammation and fibrosis were significantly exacerbated by USP29 deletion and relieved by USP29 overexpression. Mechanistically, USP29 significantly activated the expression of genes related to fatty acid β-oxidation (FAO) under metabolic stimulation, directly interacted with long-chain acyl-CoA synthase 5 (ACSL5) and repressed ACSL5 degradation by increasing ACSL5 K48-linked deubiquitination. Moreover, the effect of USP29 on hepatocyte lipid accumulation and MASLD was dependent on ACSL5. Conclusions USP29 functions as a novel negative regulator of MASLD by stabilizing ACSL5 to promote FAO. The activation of the USP29-ACSL5 axis may represent a potential therapeutic strategy for MASLD.

Background/Aims: Metabolic dysfunction–associated steatotic liver disease (MASLD) is a chronic liver disease characterized by hepatic steatosis. Ubiquitin-specific protease 29 (USP29) plays pivotal roles in hepatic ischemiareperfusion injury and hepatocellular carcinoma, but its role in MASLD remains unexplored. Therefore, the aim of this study was to reveal the effects and underlying mechanisms of USP29 in MASLD progression. Methods: USP29 expression was assessed in liver samples from MASLD patients and mice. The role and molecular mechanism of USP29 in MASLD were assessed in high-fat diet-fed and high-fat/high-cholesterol diet-fed mice and palmitic acid and oleic acid treated hepatocytes. Results: USP29 protein levels were significantly reduced in mice and humans with MASLD. Hepatic steatosis, inflammation and fibrosis were significantly exacerbated by USP29 deletion and relieved by USP29 overexpression. Mechanistically, USP29 significantly activated the expression of genes related to fatty acid β-oxidation (FAO) under metabolic stimulation, directly interacted with long-chain acyl-CoA synthase 5 (ACSL5) and repressed ACSL5 degradation by increasing ACSL5 K48-linked deubiquitination. Moreover, the effect of USP29 on hepatocyte lipid accumulation and MASLD was dependent on ACSL5. Conclusions USP29 functions as a novel negative regulator of MASLD by stabilizing ACSL5 to promote FAO. The activation of the USP29-ACSL5 axis may represent a potential therapeutic strategy for MASLD.

Globally, over 300 million surgeries are performed each year, and more than 50% of surgeries involve patients aged 65 and older. Aging poses significant challenges to perioperative brain health, as the deterioration of brain structure and function increases susceptibility to postoperative neurological complications. Protecting perioperative brain health remains a worldwide clinical challenge. With senescence, the brain undergoes a progressive decline in homeostasis across various molecular, cellular, and regional functions. Anesthetics and surgical stimuli may accelerate the disruption of brain homeostasis and exacerbate age-related neurodegeneration. This review provides a framework for understanding how anesthesia and surgery can affect brain health in the aging population and contribute to postoperative neurological complications, with a particular focus on perioperative neurocognitive disorder.

Background/Aims: Metabolic dysfunction–associated steatotic liver disease (MASLD) is a chronic liver disease characterized by hepatic steatosis. Ubiquitin-specific protease 29 (USP29) plays pivotal roles in hepatic ischemiareperfusion injury and hepatocellular carcinoma, but its role in MASLD remains unexplored. Therefore, the aim of this study was to reveal the effects and underlying mechanisms of USP29 in MASLD progression. Methods: USP29 expression was assessed in liver samples from MASLD patients and mice. The role and molecular mechanism of USP29 in MASLD were assessed in high-fat diet-fed and high-fat/high-cholesterol diet-fed mice and palmitic acid and oleic acid treated hepatocytes. Results: USP29 protein levels were significantly reduced in mice and humans with MASLD. Hepatic steatosis, inflammation and fibrosis were significantly exacerbated by USP29 deletion and relieved by USP29 overexpression. Mechanistically, USP29 significantly activated the expression of genes related to fatty acid β-oxidation (FAO) under metabolic stimulation, directly interacted with long-chain acyl-CoA synthase 5 (ACSL5) and repressed ACSL5 degradation by increasing ACSL5 K48-linked deubiquitination. Moreover, the effect of USP29 on hepatocyte lipid accumulation and MASLD was dependent on ACSL5. Conclusions USP29 functions as a novel negative regulator of MASLD by stabilizing ACSL5 to promote FAO. The activation of the USP29-ACSL5 axis may represent a potential therapeutic strategy for MASLD.

Objective To obtain tree shrew Vasorin(VASN)recombinant protein through prokaryotic expression and purification,prepare monoclonal antibody against tree shrew VASN by immunizing mice with this protein,and preliminarily evaluate its application value.Methods Reverse transcription-polymerase chain reaction(RT-PCR)was used to amplify the full-length sequence of tree shrew VASN gene in vitro.The tree shrew VASN gene fragment was inserted into pET-30a vector to construct pET-30a-VASN recombinant plasmid.The recombinant plasmid was subjected to double digestion with BamH Ⅰ and Sal Ⅰfor identification,and its correctness was further verified by sequencing.The recombinant plasmid with correct sequencing was transformed into BL21(DE3)competent cells,and isopropyl β-D-thiogalactoside(IPTG)was used to induce expression of VASN recombinant protein.Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),and the VASN recombinant protein was purified by KCI.Purified recombinant protein was used to immunize BALB/c mice for four times,and serum antibody titer was detected by enzyme-linked immunosorbent assay(ELISA).Splenocytes from mice with serum antibody titer above 1:10 000 were used for cell fusion with myeloma cells.Hypoxanthine-aminopterin-thymidine(HAT)culture medium was first used to screen hybridoma cells.ELISA was used to screen positive hybridoma cell lines that could secrete specific antibodies,and monoclonal hybridoma cell lines were obtained by limiting dilution method.VASN monoclonal antibodies were prepared in large quantities by ascites induction method,purified using rProtein G,and the affinity and in vitro reaction specificity of the monoclonal antibodies were detected by ELISA and Western blotting.Results The full-length sequence of the tree shrew VASN gene was successfully amplified and the recombinant plasmid vector of tree shrew pET-30a-VASN was constructed.The sequence obtained by sequencing of the recombinant plasmid vector was identical to the tree shrew VASN target gene sequence.Recombinant protein VASN mainly existed in the form of inclusion bodies,and the purity after purification reached 90％,meeting the requirements of subsequent immunization experiments.After four immunizations with recombinant protein VASN,mouse serum antibody titer reached 1:729 000.Monoclonal positive hybridoma cell lines were obtained through ascites induction and purification,and the constant affinity value of monoclonal antibodies measured by ELISA reached 2.59x107 L/mol.Western blotting results showed that the tree shrew VASN monoclonal antibody could bind to tree shrew VASN recombinant protein,but it showed no binding reaction with porcine retinol-binding protein 4 recombinant protein,human VASN-leucine rich repeat recombinant protein,or bovine serum albumin.Anti-tree shrew VASN monoclonal antibody could specifically recognize VASN protein in tree shrew heart,liver,spleen,lung,kidney and muscle,with clear bands and clean background.Immunohistochemical detection results showed that this monoclonal antibody could recognize VASN protein in tree shrew spleen,lung,and tree shrew immortalized fibroblasts with high VASN mRNA expression levels,and the detection results were positive.Conclusion Monoclonal antibody against tree shrew VASN is successfully prepared.This antibody can be used for immunohistochemical detection of tree shrew immortalized fibroblasts,spleen tissue,and lung tissue,providing an important tool for further research on the function of VASN in tree shrew models.

Objective To obtain tree shrew Vasorin(VASN)recombinant protein through prokaryotic expression and purification,prepare monoclonal antibody against tree shrew VASN by immunizing mice with this protein,and preliminarily evaluate its application value.Methods Reverse transcription-polymerase chain reaction(RT-PCR)was used to amplify the full-length sequence of tree shrew VASN gene in vitro.The tree shrew VASN gene fragment was inserted into pET-30a vector to construct pET-30a-VASN recombinant plasmid.The recombinant plasmid was subjected to double digestion with BamH Ⅰ and Sal Ⅰfor identification,and its correctness was further verified by sequencing.The recombinant plasmid with correct sequencing was transformed into BL21(DE3)competent cells,and isopropyl β-D-thiogalactoside(IPTG)was used to induce expression of VASN recombinant protein.Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),and the VASN recombinant protein was purified by KCI.Purified recombinant protein was used to immunize BALB/c mice for four times,and serum antibody titer was detected by enzyme-linked immunosorbent assay(ELISA).Splenocytes from mice with serum antibody titer above 1:10 000 were used for cell fusion with myeloma cells.Hypoxanthine-aminopterin-thymidine(HAT)culture medium was first used to screen hybridoma cells.ELISA was used to screen positive hybridoma cell lines that could secrete specific antibodies,and monoclonal hybridoma cell lines were obtained by limiting dilution method.VASN monoclonal antibodies were prepared in large quantities by ascites induction method,purified using rProtein G,and the affinity and in vitro reaction specificity of the monoclonal antibodies were detected by ELISA and Western blotting.Results The full-length sequence of the tree shrew VASN gene was successfully amplified and the recombinant plasmid vector of tree shrew pET-30a-VASN was constructed.The sequence obtained by sequencing of the recombinant plasmid vector was identical to the tree shrew VASN target gene sequence.Recombinant protein VASN mainly existed in the form of inclusion bodies,and the purity after purification reached 90％,meeting the requirements of subsequent immunization experiments.After four immunizations with recombinant protein VASN,mouse serum antibody titer reached 1:729 000.Monoclonal positive hybridoma cell lines were obtained through ascites induction and purification,and the constant affinity value of monoclonal antibodies measured by ELISA reached 2.59x107 L/mol.Western blotting results showed that the tree shrew VASN monoclonal antibody could bind to tree shrew VASN recombinant protein,but it showed no binding reaction with porcine retinol-binding protein 4 recombinant protein,human VASN-leucine rich repeat recombinant protein,or bovine serum albumin.Anti-tree shrew VASN monoclonal antibody could specifically recognize VASN protein in tree shrew heart,liver,spleen,lung,kidney and muscle,with clear bands and clean background.Immunohistochemical detection results showed that this monoclonal antibody could recognize VASN protein in tree shrew spleen,lung,and tree shrew immortalized fibroblasts with high VASN mRNA expression levels,and the detection results were positive.Conclusion Monoclonal antibody against tree shrew VASN is successfully prepared.This antibody can be used for immunohistochemical detection of tree shrew immortalized fibroblasts,spleen tissue,and lung tissue,providing an important tool for further research on the function of VASN in tree shrew models.

OBJECTIVE: To investigate the regulatory effect of metformin on regulatory T cells (Treg) in acidic environment. METHODS: CD4 CD25 Treg cells were obtained by magnetic bead sorting. Treg and conventional T cells (Tcon) cells were cultured for 24-72 h in pH 7.4 or pH 6.7 medium, and the cell proliferation, apoptosis and Foxp3 expression were detected by flow cytometry. Real-time PCR was used to detect the expression levels of the genes related with glucose metabolism. Thirty-two C57BL/6 male mouse models bearing subcutaneous prostate cancer xenograft derived from RM-1 cells were randomized into 4 equal groups for treatment with PBS, metformin, tumor vaccine, or both metformin and the vaccine. The treatment started on the 4th day following tumor cell injection, and metformin (100 mg/kg) or PBS was administered by intraperitoneal injection on a daily basis; the vaccine was intramuscularly injected every 4 days. The tumor size was continuously monitored, and the mice were euthanized on day 25 after tumor implantation to obtain tumor and blood samples. Flow cytometry was used to detect the changes in CD4, CD8, CD4Foxp3 cell subsets in the tumor tissue and peripheral blood. RESULTS: Treg cells showed significantly enhanced proliferation ( < 0.05) while the proliferation of Tcon cells was suppressed in acidic medium ( < 0.001). Treg cells cultured in acidic medium showed significantly increased expressions of OXPHOS-related genes pgc1a ( < 0.001) and cox5b ( < 0.01), which did not vary significantly in Tcon cells in acidic medium. Treg cells exhibited significantly decreased apoptosis in acidic medium ( < 0.01) with increased Foxp3 cells ( < 0.001) and intracellular alkaline levels ( < 0.01). Metformin obviously reversed the acid tolerance of Treg cells without producing significant effect on Tcon cells. In the animal experiment, both metformin ( < 0.05) and vaccine ( < 0.01) alone reduced the tumor volume, but their combined treatment more potently reduced the tumor volume ( < 0.001). Metformin alone did not obviously affect CD4 cells or CD8 cells but significantly decreased the percentage of CD4Foxp3 ( < 0.05); the vaccine alone significantly increased CD4 cells and CD8 cells ( < 0.001) and also the percentage of CD4Foxp3 cells ( < 0.05). The combined treatment, while reducing the percentage of CD4Foxp3cells to a level lower than that in the vaccine group ( < 0.01), produced the strongest effect to increase CD4 cells and CD8 cells ( < 0.01). CONCLUSIONS Metformin can inhibit the proliferation and function of regulatory T cells in an acidic environment and enhance the effect of tumor vaccine by reducing the proportion of Treg cells to achieve the anti-tumor effect.
Animals ; Cell Proliferation ; Forkhead Transcription Factors ; Male ; Metformin ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory

Objective To evaluate the surgical approaches for gastric carcinoma accompanied by portal hypertension ( PHT).Methods The clinical data of 22 patients with PHT undergoing operation during 5 years were retrospectively analyzed.The liver function was Child's A in 12 cases, Child's B in 10 cases.Total gastrectomy + pericardial devascularization was performed in 11 cases, distal subtotal gastrectomy in 9 cases, distal subtotal gastrectomy + splenectomy in one, distal subtotal gastrectomy + pericardial devascularization in one.12 cases with Child's A underwent D2 lymph node (LN) dissection and 10 cases with Child's B were treated with D1 LN dissection.Liver biopsy was taken in all patients.Results Postoperative complications developed in 50% and mortality rate was 9%.The rate of liver function deterioration in patients of Child A ungergoing D2 lymph node dissection was 42% , and that of patients with Child B was 70%.The rate of postoperatiave complications in patients with Child A ungergoing D2 lymph node dissection was 25% , while that of patients with Child B was 80%.There was no significant difference in liver function deterioration rate between Child A and Child B (P ＞ 0.05) , but the rate of postoperative complications in Child A is much lower than those in Child B(P ＜ 0.05).The complication rate in patients receiving PHT targeting measures was 77% ,much higher than 11% in those without concurrent treatment of PHT ( P ＜ 0.05 ).Conclusions Individualized surgical approache is crucial for treatment of gastric carcinoma accompanied by PHT.Surgical treatment should be on the basis of liver function and the severity of PHT.

OBJECTIVE: To observe the effects of salvianolate on rats with postoperative intestinal adhesion and to explore the prevention mechanism. METHODS: Forty SD male rats with intestinal adhesion were randomly divided into four groups: untreated group, low-dose salvianolate-treated group (12 mg/kg), medium-dose salvianolate-treated group (24 mg/kg) and high-dose salvianolate-treated group (48 mg/kg), with another ten SD male rats as normal control. Intraperitoneal injection of glucose was administered to the rats in the normal control group and the untreated group, and intraperitoneal injection of salvianolate was administered to the rats in the low-, medium- and high-dose salvianolate-treated groups. They were all treated for 8 days and once a day. On the eighth day after surgery the blood samples of each group were collected. Grades of intestinal adhesion were ranked by macroscopic observation. The adhesive tissues between viscera and belly wall were taken for pathological observation. The levels of interleukin-1beta (IL-1beta), interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha) were determined by enzyme linked immunosorbent assay. RESULTS: Salvianolate can significantly reduce the extent of postoperative intestinal adhesion, obviously decrease the levels of IL-1beta, TNF-alpha and inhibit the hyperplasy of fibrous connective tissue. However, there was no significant impact on the level of IL-4. CONCLUSION: Salvianolate can reduce the extent of postoperative intestinal adhesion, decrease the expression of IL-1beta and TNF-alpha and inhibit the hyperplasy of fibrous connective tissue. This may be the mechanism of salvianolate in preventing intestinal adhesion.

Objective To discuss the predisposing factors,prognosis and treatment of pancreatic encephalopathy(PE) in acute pancreatitis(AP). Methods Nineteen cases of AP complicated with PE were retrospectively studied. Results The occurrence rate of PE was 12.0%. PE often occurred in association with such factors as hyperpyrexia, water-electrolyte disturbance, and hypoxemia. Among the 19 patients,11 patients received surgical operation and 8 were treated conservatively.The total fatality rate reached 52.6%(10/19), significantly higher than other concurrently treated cases of severe acute pancreatits(SAP) (20.7%,P