Objective:To discuss the effect of circadion rhythm gene TIMELESS(TIM)silencing on immune escape of the ovarian cancer cells,and to clarify its related mechanism.Methods:The CD8+T lymphocytes were isolated and identified by flow cytometry to detect the proportion of CD3+/CD8+cell subsets.The human ovarian cancer SK-OV-3 cells were cultured in vitro and divided into interference plasmid transfected with TIM small interfering(siRNA)(si-TIM),negative control plasmid(si-NC),programmed death ligand 1(PD-L1)over-expression plasmid(oe-PD-L1),and negative control plasmid(oe-NC)groups.The cells were further divided into blank control group(BC group,non-transfection),si-NC group(transfected with si-NC),si-TIM group(transfected with si-TIM),si-NC+oe-NC group(transfected with si-NC and oe-NC),and si-TIM+oe-PD-L1 group(transfected with si-TIM and oe-PD-L1).Real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the expression levels of TIM mRNA and protein in the SK-OV-3 cells to verify TIM gene silencing.The transfected SK-OV-3 cells were co-cultured with activated CD8+T lymphocytes and divided into BC group(SK-OV-3 cells cultured alone),BC/T group,si-NC/T group,si-TIM/T group,si-NC+oe-NC/T group,and si-TIM+oe-PD-L1/T group.CCK-8 method was used to detect the survival rates of the SK-OV-3 cells in various groups;flow cytometry was used to detect the apoptotic rates of the SK-OV-3 cells and positive expression rate of PD-L1 on surface of the cells in various groups;enzyme-linked immunosorbent assay(ELISA)method was used to detect the levels of interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α)in the co-culture supernatant;lactate dehydrogenase(LDH)release assay was used to detect the cytotoxicity of the CD8+T lymphocytes in various groups;RT-qPCR method was used to detect the expression levels of TIM and PD-L1 mRNA in the SK-OV-3 cells in various groups;Western blotting method was used to detect the expression levels of TIM and PD-L1 proteins in the SK-OV-3 cells in various groups.Results:After scparated with immune magnetic bead method,the proportion of CD8+T lymphocyte(CD3+/CD8+)subsets was(96.56%±0.59%),indicating high purity of the extracted CD8+T lymphocytes.Compared with BC group,the expression levels of TIM mRNA and protein in the cells in si-TIM group were significantly decreased(P<0.01),suggesting successful TIM gene silencing in the ovarian cancer SK-OV-3 cells.The CCK-8 results showed that compared with BC group,the survival rate of the SK-OV-3 cells in BC/T group was significantly decreased(P<0.01);compared with BC/T group,the survival rate of the SK-OV-3 cells in si-TIM/T group was significantly decreased(P<0.01).The flow cytometry results showed that compared with BC group,the apoptotic rate of the SK-OV-3 cells in BC/T group was significantly increased(P<0.01);compared with BC/T group,the apoptotic rate of the SK-OV-3 cells in si-TIM/T group was significantly increased(P<0.01);compared with si-TIM/T group,the apoptotic rate of the SK-OV-3 cells in si-TIM+oe-PD-L1/T group was significantly decreased(P<0.01).Compared with BC group,the positive expression rate of PD-L1 on surface of the SK-OV-3 cells in si-TIM group was significantly decreased(P<0.01).The ELISA results showed that compared with BC/T group,the levels of IFN-γ and TNF-α in the culture supernatant in si-TIM/T group were significantly increased(P<0.01);compared with si-TIM/T group,the levels of IFN-γ and TNF-α in the supernatant in si-TIM+oe-PD-L1/T group were significantly decreased(P<0.01).The LDH release assay results showed that compared with BC/T group,the cytotoxicity of the CD8+T lymphocytes in si-TIM/T group was significantly increased(P<0.01);compared with si-TIM/T group,the cytotoxicity of the CD8+T lymphocytes in si-TIM+oe-PD-L1/T group was significantly weakened(P<0.01).The RT-qPCR and Western blotting results showed that compared with BC group,the expression levels of PD-L1 mRNA and protein in the SK-OV-3 cells in si-TIM group were significantly decreased(P<0.01);compared with si-TIM group,the expression level of PD-L1 protein in the cells in si-TIM+oe-PD-L1 group was significantly increased(P<0.01).Conclusion:TIM gene silencing enhances the cytotoxic effect of CD8+T lymphocytes on ovarian cancer SK-OV-3 cells and inhibits immune escape,and its mechanism may be related to the regulation of PD-L1 protein expression.

Objective To develop a prognostic risk model for anoikis-related genes(ANRs)in bladder cancer,calculate risk scores,and analyze the relationship between bladder cancer patients with high and low risk scores and the tumor microenvironment.Methods Prognosis-related ANRs and clinically independent risk factors were screened by public database information and Cox regression analysis.Prognostic risk modeling was performed by least absolute shrinkage and selection operator(LASSO)analysis and column-line diagrams.Prognostic risk model accuracy was validated by kaplan-meier survival analysis and area under receiver operating characteristic curve(ROC)curve(AUC).The relationship between risk score and tumor microenvironment was explored by CIBERSORT(https://cibersortx.stanford.edu/)and single sample gene set enrichment analysis(ssGSEA).Results The prognostically relevant ANRs were B-lymphoblastoma-2-associated promoter(BAD),cell cycle protein-dependent kinase inhibitor 3(CDKN3),and proliferating cell nuclear antigen(PCNA),and the clinically independent risk factors were gender,age,clinical stage(T,N),and risk score.The prognostic risk model was expressed as risk score=(0.155 2 × BAD expression)+(0.2286 × CDKN3 expression)+(0.0114×PCNA expression)and column line graph.The lower the risk score the better the prognosis of bladder cancer patients,the AUC of the survival curves for 1,3 and 5 years were 0.732,0.620 and 0.541,respectively,and the column line graphs of the 1-,3-and 5-year calibration curves almost corresponded diagonally,reflecting the accuracy of the model.The high and low risk groups of the prognostic risk model showed great differences in immune cell infiltration in the tumor microenvironment of bladder cancer.Conclusion The established prognostic risk model for bladder cancer loss of apoptosis-related genes is highly accurate and can better assess the prognosis of bladder cancer patients,and bladder cancer patients with high and low risk scores are closely related to the tumor microenvironment.

Objective:To investigate the clinical characteristics of urea cycle disorder (UCD), the efficacy and safety of glyceryl phenylbutyrate (GPB) therapy in pediatric patients with UCD.Methods:This study was a retrospective, single-arm, multicenter clinical study. The clinical data of 20 pediatric patients with UCD who received GPB treatment at 9 hospitals nationwide between December 2021 and August 2024 were collected. The clinical manifestations, laboratory results, and molecular genetic characteristics were analyzed, ammonia levels and other laboratory results were evaluated pre-post GPB therapy by paired t-tests or Wilcoxon tests. Results:Among the 20 pediatric patients with UCD, there were 8 males and 12 females, and the onset age was 2.8 (1.4, 5.7) years. The ammonia levels were 174 (125, 342) μmol/L at first onset. The symptoms included vomiting in 6 cases, drowsiness in 5 cases, epilepsy in 5 cases, developmental delay in 5 cases, psychiatric and behavioral abnormalities in 3 cases, and lethargy in 1 case, and 18 cases exhibited abnormal liver function. Twenty cases included 6 UCD subtypes, with 11 cases being ornithine transcarbamylase deficiency. A total of 27 variants were identified, 11 (41%) of which were novel. The age of patients who began GPB therapy was 4.0 (1.5, 6.6) years. Ten cases stopped GPB after 4.2 (3.4, 5.3) months, with 4 patients undergoing liver transplantation and 6 discontinuing for financial reasons. The remaining ten patients continued GPB therapy for 11.6 (8.6, 14.0) months. The duration of GPB treatment was 6.0 (4.2, 12.3) months, at the final visit, the levels of ammonia, platelets and aspartate aminotransferase were lower compared to those of pre-treatment (all P<0.05). The serum albumin level was higher than that of pre-treatment ( P=0.016). Two patients suffered only one episode of acute hyperammonaemia, with ammonia levels of 232 and 141 μmol/L, respectively. Nine cases experienced adverse effects potentially related to GPB, decreased appetite in 6 cases, vomiting in 3 cases, abnormal skin oil odor in 2 cases, somnolence, fatigue and diarrhea each in 1 case, with symptoms improved within 6 (3, 10) days. Conclusions:UCD primarily manifests with neurological and gastrointestinal symptoms, and early diagnosis of UCD could be achieved through the analysis of ammonia. GPB may effectively reduce ammonia levels in UCD pediatric patients, with favorable safety and tolerability.

Hand degloving injury represents one of the most severe forms of hand trauma, characterised by challenging treatment and a complex prognostic outcome. It is crucial to effectively utilise the degloved tissues in emergency or primary repair of a hand degloving injury. This consensus provides a comprehensive review of the existing literature on definition, classification, emergency assessment, debridement, judgment of skin viability, in situ repair of the degloved skin, and adjunctive treatment for degloving injury of hand. Based on conclusion of both domestic and international experiences, this expert consensus on the classification of hand degloving injury and the emergency repair with the avulsed skin is established, aiming to provide a guidance to surgeons on standardised treatment strategy and improve the management of hand degloving injury.

With the continuous advancement of medical reform,the effectiveness of human resource management in pub-lic hospitals has become crucial for hospital development.This paper takes a tertiary first-class hospital in Northwest China as the research object,and uses the SWOT analysis method to comprehensively analyze the strengths,weaknesses,opportunities,and threats of its human resource development.Based on this,targeted countermeasures and suggestions are put forward,and the im-plementation results are summarized.The aim is to help the hospital's human resources achieve rapid development and enhance the hospital's competitiveness.

Objective The aim of this study was to clarify the role and mechanism of Pseudomonas aeruginosa vaccine subunit OprI in the fusion protein vaccine rePO(PcrV-OprI).Methods The in vitro stability of rePO,PcrV and OprI at 4 ℃,25 ℃,and 37 ℃ was examined.After immunizing mice with rePO,OprI and PcrV,respectively,the specific antibody potency in serum and the proportion of cells secreting IFN-γ and IL-4 in the spleen were examined;Additionally,detection of the levels of protein uptake by DC2.4 cells in vitro using laser confocal microscopy and flow cytometry,and their ability to promote the maturation of mouse bone marrow-derived dendritic cells(BMDC).Results The heat stability of fusion protein rePO was significantly better than that of PcrV.The induced anti-PcrV IgG and anti-OprI IgG potency of rePO was significantly higher than that of monomeric PcrV and OprI.Additionally,the number of cells secreting IFN-γ and IL-4 induced by immunization with rePO was significantly higher than that of PcrV and OprI.The uptake rate of fusion protein rePO by DC2.4 cells was significantly higher than that of PcrV and OprI.Furthermore,rePO promoted the maturation of mouse BMDC more effectively than PcrV and OprI.Conclusion OprI in the fusion protein rePO can significantly improve its thermal stability and immunogenicity,which lays the foundation for the successful development of Pseudomonas aeruginosa vaccine.

Objective: To investigate the mechanism of miR-224-5p on proliferation, apoptosis, invasion and migration of human hepatocellular carcinoma HepG2 cells. Methods : The RNA expression levels of miR-224-5p and early growth responsive gene 2(EGR2) in patients with hepatocellular carcinoma were obtained from the TCGA dataset. Normal human hepatocytes LO2 and hepatoma cells HepG2 were cultured in vitro, and the HepG2 cells were transfected with lentiviral vectors(knockdown of miR-224-5p), small interfering RNA fragments or overexpression vectors(interference and overexpression of EGR2). The expression levels of miR-224-5p and EGR2 in hepatocellular carcinoma cDNA chips and cells were detected by quantitative real-time PCR(qPCR). The expression level of EGR2 protein was detected by Western blot. Dual luciferase reporter gene assay was used to detect the binding of miR-224-5p to EGR2. HepG2 cells positive rate were detected by EdU assay, apoptosis rate was detected by flow cytometry, cell invasion number was detected by Transwell assay, and cell mobility was detected by scratch assay. Results : Compared with paracancerous tissues, the expression of miR-224-5p was increased and the expression of EGR2 mRNA decreased in HCC tissues. Compared with LO2 group, the expression of miR-224-5p in HepG2 cells increased, and the expression of EGR2 mRNA and protein decreased. Compared with the Lv-sh-NC group, the 24 h EdU positive cell rate, cell invasion number, and 48 h cell mobility of HepG2 cells in the Lv-sh-miR-224-5p group decreased, while the apoptosis rate increased. Compared with Oe-NC group, 24 h EdU positive cell rate, cell invasion number, and 48 h cell mobility of HepG2 cells in Oe-EGR2 group decreased, while apoptosis rate increased. Compared with Lv-sh-NC group, the expression of EGR2 protein in Lv-sh-miR-224-5p group increased. Compared with Lv-sh-miR-224-5p+si-NC group, 24 h EdU positive cell rate, cell invasion rate, and 48 h cell mobility of HepG2 cells in Lv-sh-miR-224-5p+si-EGR2 group increased, while apoptosis number decreased. Conclusion miR-224-5p can promote proliferation, invasion, and migration of HepG2 cells and inhibit apoptosis via binding with EGR2.

Objective To investigate the relationship between stationing years of personnel on an island and their traditional Chinese medicine(TCM)constitution,thus providing a reference for adjusting the health status of stationed personnel,preventing and treating diseases.Methods Based on The Scale of Constitution in Chinese Medicine Questionnaire,TCM constitution of 734 personnel stationed on an island was investigated.Pearson χ2 method was used for data analysis.Results Of the 734 personnel stationed on an island 345(47.0%)were of the balanced constitution type and 389(53.0%)were of the biased constitution types.Among the people with biased constitution types,composite constitution accounted for 80.5%(313 people)and the simple accounted for the rest(19.5%,76 people).The top three types of the biased were dampness heat(15.2%),qi deficiency(14.0%),and yin deficiency(10.7%).The distribution of TCM constitution types was significantly different in terms of stationing years on the island(P<0.05).There was a significant difference in the balanced type and yin deficiency type between people with stationing time≤2 years and>8 years(P<0.05).There was a significant difference in qi deficiency type and qi depression type between people with stationing time≤2 years and>5 years(P<0.05).Compared to people with stationing time≤2 years,significant difference was found in yang deficiency type in people with stationing time ranging from 2 to 5 years and those with>8 years(P<0.05).There were significant differences in the phlegm dampness type,blood stasis type,and specific diathesis type between people with stationing time≤2 years and people with stationing time ranging from>2～5 years and>8 years(P<0.05).There were significant differences in the dampness heat type between people with stationing time≤2 years and people with stationing time ranging from>5～8 years and>8 years,between pepole with stationing time ranging from>2～5 years and people with stationing time>8 years(P<0.05).Conclusion The dampness heat type,qi deficiency type and yin deficiency type are common biased TCM constitution in personnel stationed on islands.The longer the time spent on islands,the greater the possibility of forming biased constitution.

Objective To investigate the role of the BNIP3-PI3K/Akt signaling pathway in mediating the inhibitory effect of Buyang Huanwu Decoction(BYHWT)on mitochondrial autophagy in human synovial fibroblasts from rheumatoid arthritis patients(FLS-RA)cultured under a hypoxic condition.Methods Forty normal Wistar rats were randomized into two groups(n=20)for daily gavage of BYHWT or distilled water for 7 days to prepare BYHWT-medicated or control sera.FLS-RA were cultured in routine condition or exposed to hypoxia(10％O2)for 24 h wigh subsequent treatment with IL-1β,followed by treatment with diluted BYHWT-medicated serum(5％,10％and 20％)or control serum.AnnexinV-APC/7-AAD double staining and T-AOC kit were used for detecting apoptosis and total antioxidant capacity of the cells,and the changes in ROS,ATP level,mitochondrial membrane potential and Ca2+homeostasis were analyzed.The changes in mRNA and protein expressions of BNIP3,PI3K and AKT and mRNA expressions of LC3,Beclin-1 and P62 were detected using RT-qPCR and Western blotting.Results Treatment with BYHWT-medicated serum dose-dependently lowered apoptosis rate of IL-1β-induced FLS-RA with hypoxic exposure.The treatment significantly decreased T-AOC concentration,increased ROS production,autophagosome formation and ATPase levels,and lowered mitochondrial membrane potential and Ca2+level in the cells.In IL-1β-induced FLS-RA with hypoxic exposure,treatment with BYHWT-medicated serum significantly increased BNIP3 protein expression,decreased the protein expressions of PI3K and AKT,increased the mRNA expressions of BNIP3 and P62,and lowered the mRNA expressions of PI3K,AKT,LC3 and Beclin-1 without significantly affecting Beclin-1 protein expression.The cells treated with 5％and 10％BYHWT-medicated serum showed no significant changes in LC3 expression.Conclusion BYHWT inhibits mitochondrial autophagy in IL-1β-induced FLS-RA with hypoxic exposure possibly by inhibiting BNIP3-mediated PI3K/AKT signaling pathway.

OBJECTIVES: To investigate the role of the BNIP3-PI3K/Akt signaling pathway in mediating the inhibitory effect of Buyang Huanwu Decoction (BYHWT) on mitochondrial autophagy in human synovial fibroblasts from rheumatoid arthritis patients (FLS-RA) cultured under a hypoxic condition. METHODS: Forty normal Wistar rats were randomized into two groups (n=20) for daily gavage of BYHWT or distilled water for 7 days to prepare BYHWT-medicated or control sera. FLS-RA were cultured in routine condition or exposed to hypoxia (10% O₂) for 24 h wigh subsequent treatment with IL-1β, followed by treatment with diluted BYHWT-medicated serum (5%, 10% and 20%) or control serum. AnnexinV-APC/7-AAD double staining and T-AOC kit were used for detecting apoptosis and total antioxidant capacity of the cells, and the changes in ROS, ATP level, mitochondrial membrane potential and Ca²⁺ homeostasis were analyzed. The changes in mRNA and protein expressions of BNIP3, PI3K and AKT and mRNA expressions of LC3, Beclin-1 and P62 were detected using RT-qPCR and Western blotting. RESULTS: Treatment with BYHWT-medicated serum dose-dependently lowered apoptosis rate of IL-1β-induced FLS-RA with hypoxic exposure. The treatment significantly decreased T-AOC concentration, increased ROS production, autophagosome formation and ATPase levels, and lowered mitochondrial membrane potential and Ca²⁺ level in the cells. In IL-1β-induced FLS-RA with hypoxic exposure, treatment with BYHWT-medicated serum significantly increased BNIP3 protein expression, decreased the protein expressions of PI3K and AKT, increased the mRNA expressions of BNIP3 and P62, and lowered the mRNA expressions of PI3K, AKT, LC3 and Beclin-1 without significantly affecting Beclin-1 protein expression. The cells treated with 5% and 10% BYHWT-medicated serum showed no significant changes in LC3 expression. CONCLUSIONS BYHWT inhibits mitochondrial autophagy in IL-1β-induced FLS-RA with hypoxic exposure possibly by inhibiting BNIP3-mediated PI3K/AKT signaling pathway.
Drugs, Chinese Herbal/pharmacology* ; Arthritis, Rheumatoid/pathology* ; Animals ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Autophagy/drug effects* ; Humans ; Fibroblasts/cytology* ; Rats, Wistar ; Membrane Proteins/metabolism* ; Rats ; Phosphatidylinositol 3-Kinases/metabolism* ; Mitochondria/metabolism* ; Cells, Cultured ; Proto-Oncogene Proteins/metabolism* ; Apoptosis/drug effects* ; Cell Hypoxia ; Synovial Membrane/cytology* ; Male ; Mitochondrial Proteins