OBJECTIVE: To elucidate the role and underlying mechanism of ubiquitin-specific protease 35 (USP35) in ferroptosis of rheumatoid arthritis-fibroblast like synoviocytes (RA-FLS), thereby enhancing our comprehension of the pathogenesis of RA and identifying potential therapeutic targets for its treatment. METHODS: (1) RA-FLS were cultured in vitro and transduced with lentiviral vectors to establish stable cell lines: A USP35-knockdown line (short hairpin ribonucleic acid of USP35, shUSP35) and its control (negtive control of short hairpin ribonucleic acid, shNC), as well as a overexpression of USP35 line (USP35 OE) and its control (Vector). To investigate the role of USP35 in ferroptosis regulation, a ferroptosis model was induced in RA-FLS by treatment with 1 μmol/L Erastin. The cells were divided into six groups: shNC, shNC + Erastin, shUSP35 + Erastin, Vector, Vector + Erastin, and USP35 OE + Erastin. (2) Cell viability was detected using the cell counting kit-8 (CCK-8). (3) Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione/glutathione disulfide (GSH/GSSG) ratios, and Ferrous ion (Fe²⁺) levels were measured using specific assay kits to evaluate oxidative stress, lipid peroxidation, and glutathione redox status in the cells. (4) Protein expression levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were detected using Western blotting to investigate their potential involvement in USP35-mediated ferroptosis regulation. RESULTS: (1) Compared with the shNC +Erastin group, the cell viability of the shUSP35+Erastin group was significantly decreased (P < 0.001), while it was notably increased in the USP35 OE+Erastin group compared with the Vector+Erastin group (P < 0.001). These findings indicated that USP35 could alleviate the inhibitory effect of Erastin on RA-FLS cell viability. (2) In comparison to the shNC+Erastin group, the levels of ROS (P < 0.001), MDA (P < 0.05), and Fe²⁺ (P < 0.001) were significantly elevated, and the GSH/GSSG ratio was increased (P < 0.05) in the shUSP35+Erastin group. Conversely, the levels of ROS (P < 0.001), MDA (P < 0.05), and Fe²⁺ (P < 0.05) were significantly decreased, and the GSH/GSSG ratio was decreased (P < 0.05) in the USP35 OE+Erastin group compared with the Vector+Erastin group. These results suggested that USP35 could inhibit Erastin-induced oxidative stress and lipid peroxidation in RA-FLS. (3) In Erastin-induced RA-FLS, the expression of USP35 was positively correlated with the protein levels of SLC7A11 and GPX4, indicating a potential mechanism by which USP35 regulated ferroptosis in these cells. CONCLUSION USP35 inhibits ferroptosis in RA-FLS, potentially through the increased expression of SLC7A11 and GPX4.
Ferroptosis ; Humans ; Arthritis, Rheumatoid/metabolism* ; Synoviocytes/pathology* ; Reactive Oxygen Species/metabolism* ; Ubiquitin-Specific Proteases/metabolism* ; Fibroblasts/pathology* ; Cell Survival ; Piperazines/pharmacology* ; Endopeptidases/metabolism* ; Cells, Cultured ; Cell Line ; Amino Acid Transport System y+

Objective To investigate the correlation of serum levels of soluble programmed death ligand 1(sPD-L1),tumor necrosis factor-associated apoptosis-inducing ligand(TRAIL)and fibroblast growth factor 23(FGF-23)with risk stratification and death in patients with acute pulmonary embolism(APE).Methods A total of 113 pa-tients with APE admitted to the hospital from January 2022 to January 2024 were selected as APE group,and 50 healthy subjects were selected as control group.The 113 patients with APE were divided into high risk group(39 cases),medium risk group(45 cases)and low risk group(29 cases)by risk stratification.Accord-ing to the death of APE patients,they were divided into survival group(83 cases)and death group(30 cases).The levels of serum sPD-L1,TRAIL and FGF-23 were measured by enzyme-linked immunosorbent assay(ELISA).Multiple Logistic regression was used to analyze the risk factors affecting death of APE patients.Receiver operating characteristic(ROC)curve was drawn to analyze the value of serum sPD-L1,TRAIL and FGF-23 levels in evaluating mortality in APE patients.Pearson correlation analysis was used to investigate the correlation between serum sPD-L1,TRAIL and FGF-23 levels and cardiac function indicators in APE patients.Results The levels of serum sPD-L1,TRAILand FGF-23 in the APE group were significantly higher than those in the control group(P＜0.001).The serum sPD-L1,TRAIL and FGF-23 levels in the death group were significantly higher than those in the survival group(P＜0.001).The levels of serum sPD-L1,TRAIL and FGF-23 in the high-risk group were higher than those in the medium and low-risk groups,and the medium risk group was higher than the low-risk group,and the differences were statistically significant(P＜0.001).Multiple Logistic regression analysis showed that elevated levels of cardiac troponin Ⅰ(cTnⅠ),B type brain na-triuretic peptide(BNP),sPD-L1,TRAIL and FGF-23 were risk factors for mortality in APE patients(P＜0.05).ROC curve analysis showed that the combination of sPD-L1,TRAIL and FGF-23 had the largest area under curve for predicting death in APE patients,which was 0.924(95％CI:0.861-0.986),with a sensitivity of 96.8％and a specificity of 81.2％.Correlation analysis showed that the levels of serum sPD-L1,TRAIL and FGF-23 were positively correlated with cTnⅠ and BNP in APE patients(P＜0.001).Conclusion The lev-els of serum sPD-L1,TRAIL and FGF-23 are significantly elevated in APE patients,and their high expression is associated with high-risk stratification and mortality.The combination of these three indexes has good eval-uation value for APE patient mortality.

Objective:To evaluate whether the mechanism by which mesenchymal stem cell-derived exosomes (MSC-exo) mitigated endotoxin-induced lung injury was related to the heme oxygenase-1 (HO-1)/mitochondrial signaling pathway in alveolar macrophages of mice.Methods:In vivo experiment Eighteen C57BL/6 wild-type (WT) mice were divided into 3 groups ( n=6 each) using a random number table method: control group (C group), lipopolysaccharide (LPS) group (L group) and LPS + MSC-exo group (LM group). Six HO-1 conditional knockout mice (HO-1 -/-) were selected and served as HO-1 -/- + MSC-exo + LPS group (HML group). The model of endotoxin-induced lung injury was prepared by injection of LPS 15 mg/kg. MSC-exo (2×10 11 particles) was intravenously injected at 1 h before injection of LPS in LM group. MSC-exo (2×10 11 particles) was intravenously injected and 1 h later LPS was injected in HML group. The expression of HO-1 in macrophages was detected using immunofluorescence, lung injury was assessed following hematoxylin-eosin staining, the wet/dry weight ratio (W/D ratio) was determined, and the mitochondrial morphology was observed with a transmission electron microscope. Cell experiment Alveolar macrophages (MH-S) were divided into 4 groups ( n=20 each) using a random number table method: control group (C group), LPS+ phosphate buffer solution group (LP group), LPS+ MSC-exo group (LM group), and LPS+ MSC-exo+ HO-1 small-interfering RNA group (LMS group). Cells were incubated for 12 h with LPS 10 μg/ml in LP, LM and LMS groups. In addition, LM group was incubated with MSC-exo 100 μg/ml, LP group was incubated with the equal volume of phosphate buffer solution, and the alveolar macrophages were transfected with HO-1 small interfering RNA and incubated with MSC-exo 100 μg/ml in LMS group at the same time. The concentrations of interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in supernatant were measured by enzyme-linked immunosorbent assay, HO-1 expression was detected by Western blot, the mitochondrial membrane potential was measured using JC-1 staining, and the expression of reactive oxygen species (ROS) was detected by fluorescence. Results:In vivo experiment Compared to C group, the lung injury score and W/D ratio were significantly increased ( P<0.05), the fluorescence signal of HO-1 in macrophages was enhanced, and the damage to mitochondria was aggravated in L group. Compared to L group, the lung injury score and W/D ratio were significantly decreased ( P<0.05), the fluorescence signal of HO-1 in macrophages was enhanced, and the damage to mitochondria was reduced in LM group. Compared to LM group, the lung injury score and W/D ratio were significantly increased ( P<0.05), macrophages had no HO-1 fluorescence signal, and the damage to mitochondria was aggravated in HML group. Cell experiment Compared to C group, the concentrations of IL-1β and TNF-α in supernatant were significantly increased, the expression of HO-1 was up-regulated ( P<0.05), and the mitochondria predominantly exhibited green JC-1 fluorescence, accompanied by an enhanced ROS fluorescence signal in LP group. Compared to LP group, the concentrations of IL-1β and TNF-α in supernatant were significantly decreased, the expression of HO-1 was up-regulated ( P<0.05), and the mitochondria predominantly exhibited red JC-1 fluorescence, accompanied by a weakened ROS fluorescence signal in LM group. Compared to LM group, the concentrations of IL-1β and TNF-α in supernatant were significantly increased, the expression of HO-1 was down-regulated ( P<0.05), and the mitochondria predominantly exhibited green JC-1 fluorescence, accompanied by an enhanced ROS fluorescence signal in LMS group. Conclusions:The mechanism by which MSC-exo attenuates endotoxin-induced lung injury may be related to up-regulation of HO-1 expression in alveolar macrophages and reduction of mitochondrial damage in mice.

Objective:To explore the treatment methods and prognosis of pregnancy-related uterine arteriovenous malformation (UAVM).Methods:A retrospective analysis was conducted on clinical data from 81 patients with UAVM treated at Peking Union Medical College Hospital between March 2019 and March 2024. Clinical manifestations, diagnostic approaches, treatment strategies and prognosis were evaluated.Results:(1) General Information: the age of patients with UAVM was (32.7±4.6) years, with median gravidity and parity of 1 (quartile range: 1, 2) and 0 (0, 1), respectively. Pregnancy termination methods included surgical abortion or curettage in 46 cases (57%, 46/81), medical induction in 17 cases (21%, 17/81), spontaneous abortion in 16 cases (20%, 16/81), vaginal delivery in 1 case (1%, 1/81), and laparoscopic pregnancy tissue removal in 1 case (1%, 1/81). (2) Clinical manifestations: clinical presentations comprised vaginal bleeding in 59 cases [73%, 59/81; median blood loss: 740 ml (440, 1 360 ml)], massive hemorrhage in 9 cases (11%, 9/81, and bleeding combined with lower abdominal pain in 8 cases (10%, 8/81). Ultrasonography revealed intrauterine masses in 65 cases [80%, 65/81; median size: 2.5 cm (1.8, 4.2 cm)]. Elevated serum human chorionic gonadotrophin-β subunit (β-hCG) levels were observed in in 55 cases [85%, 55/65; median: 62.6 U/L (14.9, 300.1 U/L)]. The median time to UAVM diagnosis via ultrasound was 30.0 days (16.0, 52.0 days) after pregnancy termination, with median peak systolic velocity (PSV) and resistance index of 59.8 cm/s (45.0, 79.6 cm/s) and 0.39 (0.36, 0.43), respectively. (3) Treatment and prognosis: treatment modalities included expectant management in 49 cases (36%, 29/81), medication in 13 cases (16%, 13/81), lesion resection in 31 cases (38%, 31/81), and uterine artery angiography in 8 cases (10%, 8/81; 5 confirmed as arteriovenous fistula). The median time of PSV returning to normal after treatment was 53.8 days (36.0, 93.4 days). The average time for β-hCG returning to normal was (60.4±20.4) days. The median return time of menses was 59.0 days (43.0, 75.4 days).Conclusions:Pregnancy-related UAVM carries a high risk of life-threatening hemorrhage, necessitating management in centers equipped for emergency uterine artery embolization. Informed consent must emphasize disease progression risks and prognosis. Treatment stratification should integrate clinical parameters and imaging features.

ObjectiveTo explore the current situation of forensic ethics practice and education by designing a questionnaire on forensic ethics， with a view to exploring the path of forensic ethics education construction. MethodsA total of 667 valid questionnaires were collected using the online survey method， basically covering various regions across the country and all sub-specialties of forensic medicine. Descriptive analysis was used to analyze the relevant data. ResultsMost practitioners had relevant ethical reflections in the process of forensic practice. 69.12% of the respondents indicated that they had studied the relevant rules， but approximately half stated that there were no corresponding ethical norms or standard operating manuals. The specific behaviors violating ethics in different units were diverse. 23.04% of the respondents reported that they had encountered unethical behaviors， but only 4.9% of them reported such violations. In terms of forensic ethics education， 87.75% of the respondents believed that there were issues with the current model of forensic ethics education. Meanwhile， the respondents showed a high degree of recognition for receiving forensic ethics education， with 84.15% of respondents expressing willingness to participate in relevant courses. More than half of respondents were willing to participate in forensic ethics education during undergraduate studies， new employee training， and regular post-employment training. ConclusionCurrently， there is a problem of ethical neglect in forensic work in China. Combining ethics courses with professional courses at the practitioner training stage and providing regular training at the practice stage are effective measures to popularize forensic ethics knowledge， enhance ethical awareness， and improve the quality of practice.

Objective To analyze the risk factors affecting the progression-free survival(PFS)of patients with intrahepatic cholangiocarcinoma(ICC)after surgical resection,and a predictive nomogram model was constructed based on blood test indicators.Methods A total of 99 ICC patients who received their first treatment at Hunan Provincial People's Hospital from January 2020 to December 2022 were selected as research subjects,they were divided into training group(n=70)and validation group(n=29).The independent risk factors for postoperative recurrence were identified using least absolute shrinkage and selection operator(LASSO)regression analysis and multivariate Cox risk regression analysis.The accuracy and clinical utility of the method were further tested by consistency index,receiver operating characteristic(ROC)curve,calibration graph,decision curve analysis,and progression-free survival curve.Results LASSO and multivariate Cox risk regression analysis showed that patient's gender,tumor-lymph node metastasis,differentiation grade,tumor diameter,aspartate aminotransferase,alpha-fetoprotein,total bile acid after surgery,and monocyte changes before and after surgery were independent factors affecting the PFS of ICC patients.The predictive model based on the risk factor showed that the consistency index of the training group was 0.884 and that of the validation group was 0.838.The area under ROC curve for training group:PFS prediction at 6 months,1 year,and 2 years were 0.972,0.965 and 0.923,respectively;for validation group:PFS prediction at 6 months,1 year,and 2 years were 0.972,0.821 and 0.923,respectively.The risk score calculated according to the nomogram divided ICC patients into high-risk group and low-risk group with high postoperative recurrence risk,and the median PFS in high-risk group was significantly shorter than that in low-risk group(P＜0.05).Conclusion The nomogram constructed by this study has good predictive effect and can be used as a supplementary evaluation for predicting PFS after ICC patients.

Objective:To establish a gas chromatography-mass spectrometry method(GC-MS-MS)for determining the content of methyl p-toluenesulfonate(MTS),ethyl p-toluenesulfonate(ETS),and isopropyl p-toluenesulfonate(IPTS)in tosufloxacin tosylate hydrate.Methods:The HP-1MS(0.250 mm ×30 m,0.50 μm)column was used at progamming temperature,the injection temperature was 250 ℃.The MS conditions were as follow:the ionization mode was EI,the electron voltage was 70 V,the ion source temperature was 250 ℃,the scanning method was MRM,the quantitative ion pairs for MTS,ETS and IPTS were m/z 91 →65,m/z 155→91 and m/z 91→65.Results:The linearity ranges of MTS,ETS and IPTS were 48.779-975.59 ng·mL-1(r=0.998 5),54.586-1 091.7 ng·mL-1(r=0.998 4)and 46.241-924.82 ng·mL-1(r=0.999 8).The average recoveries were 99.47％,99.15％,98.83％(n=9).The MTS,ETS and IPTS were not detected in the 7 batches of tosufloxacin tosylate hydrate.Conclusion:The established method can be used for the determination of MTS,ETS,and IPTS content in tosufloxacin tosylate hydrate.

Objective To investigate the value of ultrasound for differentiating pancreatoblastoma(PB)and solid pseudopapillary tumor of pancreas(SPT)in children.Methods Data of 7 children with PB(PB group)and 22 with SPT(SPT group)were retrospectively analyzed.The clinical data and lesion's ultrasonic manifestations were compared between groups.Receiver operating characteristic(ROC)curves of clinical and ultrasound related parameters being significantly different between groups showed by univariate analysis were draw,and the area under the curve(AUC)were calculated to evaluate their efficacy for differentiating PB and SPT.Ultrasound parameters with P＜0.05 in univariate analysis were incorporated into binary logistic analysis,and a ultrasound regression model was constructed to distinguish PB and SPT,and its diagnostic efficacy was evaluated.Results Significant differences of children'age,gender,serum alpha fetoprotein level,and the shape,maximum diameter,texture,calcification and local invasion of lesions were found between groups(all P＜0.05).AUC of single serum alpha fetoprotein level,the shape,maximum diameter,texture,calcification and local invasion of lesion for differentiating PB and SPT was 1.000,0.766,0.854,0.776,0.789 and 0.714,respectively(all P＜0.05).The shape(OR=8.704,P=0.075)and maximum diameter of lesions(OR=1.695,P=0.042)showed with ultrasound were both important differentiating factors for PB and SPT,and AUC of the ultrasound regression model constructed based on them was 0.886.Conclusion Ultrasound could effectively differentiate PB and SPT in children.

Objective:To explore the effect of inhibiting miR-203a-3p gene expression on proliferation and apoptosis of rheu-matoid arthritis(RA)synovial fibroblasts.Methods:Divided fibroblast-like synovial cells MH7A into Control group(untransfected cells),anti-miR-NC group(transfected with anti-miR-NC),anti-miR-203a-3p group(transfected with anti-miR-203a-3p),anti-miR-203a-3p+LiCl group(transfected with anti-miR-203a-3p+signal pathway activator LiCl).qRT-PCR was used to detect expression level of miR-203a-3p;clone formation experiment,MTT experiment were used to detect cell proliferation;flow cytometry was used to de-tect cell apoptosis;Western blot was used to detect protein expressions of PCNA,Bcl-2,Bax,Wnt1,β-catenin.Results:Transfec-tion of miR-203a-3p reduced the number of clones and survival rate of MH7A cells,increased rate of apoptosis,reduced expression levels of PCNA,Bcl-2,Wnt1,β-catenin protein,increased expression level of Bax protein.The signaling pathway activator LiCl in-creases the number of clones and survival rate of MH7A cells transfected with miR-203a-3p,reduces the rate of apoptosis,increases the expression level of Wnt1,β-catenin,PCNA,Bcl-2 protein,and reduces expression level of Bax protein.Conclusion:miR-203a-3p may promote proliferation of RA synovial fibroblasts and inhibit cell apoptosis by activating Wnt/β-catenin.