Objective:To investigate the role of cyclooxygenase-2 (COX-2) in glycochenodeoxycholic acid (GCDC)-induced apoptosis of mouse extrahepatic biliary epithelial cells (EBECs) and clarify its possible mechanism.Methods:EBECs were cultured in vitro and infected with RNAi-COX-2 lentivirus (GCDC+ shCOX-2 group). EBECs were then treated with different concentrations (0, 25, 50, 100, 200, 400, 800 μmol/L) of GCDC (GCDC group). Cell proliferation activity was detected by cell counting kit-8 (CCK-8) assay; lactate dehydrogenase (LDH) release rate was measured by colorimetry; apoptosis was analyzed by flow cytometry; caspase-3 activity was detected by fluorescent probe method; COX-2 mRNA expression was determined by real-time quantitative polymerase chain reaction (qRT-PCR); protein levels of COX-2, autophagy-related proteins LC3, p62, Beclin-1, and apoptosis-related proteins Bax, Bcl-2, cleaved caspase-3 were evaluated by Western blot.Results:Compared with the 0 μmol/L GCDC group, the apoptosis level, LDH release rate, and caspase-3 activity in the 50, 100, and 200 μmol/L GCDC groups gradually increased (all P<0.05) in a concentration-dependent manner. After RNAi-COX-2 lentivirus infection, COX-2 mRNA and protein expression levels in EBECs significantly decreased (all P<0.05). Compared with the GCDC group, the apoptosis rate of EBECs in the GCDC+ shCOX-2 group significantly decreased ( P<0.05). The GCDC+ shCOX-2 group showed increased cell proliferation activity, downregulated protein expressions of Bax, cleaved caspase-3, and p62, and upregulated protein expressions of Bcl-2, LC3 II/I, and Beclin-1 (all P<0.05). Conclusions:Inhibition of COX-2 improves GCDC-induced apoptosis of EBECs, and the mechanism may be related to the activation of the autophagy pathway.

Objective:To investigate the role of cyclooxygenase-2 (COX-2) in glycochenodeoxycholic acid (GCDC)-induced apoptosis of mouse extrahepatic biliary epithelial cells (EBECs) and clarify its possible mechanism.Methods:EBECs were cultured in vitro and infected with RNAi-COX-2 lentivirus (GCDC+ shCOX-2 group). EBECs were then treated with different concentrations (0, 25, 50, 100, 200, 400, 800 μmol/L) of GCDC (GCDC group). Cell proliferation activity was detected by cell counting kit-8 (CCK-8) assay; lactate dehydrogenase (LDH) release rate was measured by colorimetry; apoptosis was analyzed by flow cytometry; caspase-3 activity was detected by fluorescent probe method; COX-2 mRNA expression was determined by real-time quantitative polymerase chain reaction (qRT-PCR); protein levels of COX-2, autophagy-related proteins LC3, p62, Beclin-1, and apoptosis-related proteins Bax, Bcl-2, cleaved caspase-3 were evaluated by Western blot.Results:Compared with the 0 μmol/L GCDC group, the apoptosis level, LDH release rate, and caspase-3 activity in the 50, 100, and 200 μmol/L GCDC groups gradually increased (all P<0.05) in a concentration-dependent manner. After RNAi-COX-2 lentivirus infection, COX-2 mRNA and protein expression levels in EBECs significantly decreased (all P<0.05). Compared with the GCDC group, the apoptosis rate of EBECs in the GCDC+ shCOX-2 group significantly decreased ( P<0.05). The GCDC+ shCOX-2 group showed increased cell proliferation activity, downregulated protein expressions of Bax, cleaved caspase-3, and p62, and upregulated protein expressions of Bcl-2, LC3 II/I, and Beclin-1 (all P<0.05). Conclusions:Inhibition of COX-2 improves GCDC-induced apoptosis of EBECs, and the mechanism may be related to the activation of the autophagy pathway.

Objective:To compare and analyze the differences in clinical and pathological features of uterine smooth muscle tumor of uncertain malignant potential (STUMP), common uterine leiomyoma (UL), and cellular uterine leiomyoma (CUL), and to identify biomarkers for predicting STUMP recurrence.Methods:A total of 72 cases of STUMP patients (STUMP group) treated at the First Affiliated Hospital of Nanjing Medical University and the First Affiliated Hospital of Soochow University were collected from June 2015 to March 2024. Additionally, 72 cases of UL and 72 cases of CUL (UL group and CUL group) in the same period were collected as controls. The clinical and pathological features of the three groups were compared, and the recurrence rates and related factors affecting STUMP recurrence were analyzed.Results:(1) Comparison of clinical and pathological features: there were statistically significant differences in age, history of myomectomy, and preoperative serum lactate dehydrogenase (LDH) levels among STUMP, UL, and CUL groups (all P<0.05). STUMP group were significantly older than UL group ( P<0.05). The proportions of STUMP group with a history of myomectomy and elevated preoperative serum LDH levels were significantly higher than those in UL and CUL groups (all P<0.05). On ultrasound, 16 cases of STUMP patients (22%, 16/72), 2 cases of UL patients (3%, 2/72), and 8 cases of CUL patients (11%, 18/72) had unclear fibroid borders, with significant differences between the three groups ( χ2=12.94, P=0.002), with STUMP group significantly higher than UL group ( P<0.05). Regarding immunohistochemistry, the proportion of p16 positivity, p53 mutations, and nuclear antigen associated with cell proliferation (Ki-67) >10% were significantly higher in STUMP group compared to UL group ( P<0.05). In terms of surgical approach, 52 cases of STUMP patients (72%, 52/72) underwent hysterectomy, compared to 27 cases of UL patients (38%, 27/72) and 38 cases of CUL patients (53%, 38/72), with a significant difference between the three groups ( χ2=17.89, P=0.001). The proportion of patients who underwent myomectomy was significantly lower in STUMP group compared to UL and CUL groups (both P<0.05). Among the 20 cases of STUMP patients who underwent myomectomy, 6 patients had a subsequent total hysterectomy after being diagnosed with STUMP. (2) Comparison of recurrence: the median follow-up time for the STUMP, UL, and CUL groups was 38, 12, and 29 months, respectively. During the follow-up period, 3 cases (6%, 3/53) in STUMP group, 4 cases (7%, 4/55) in UL group, and 8 cases (13%, 8/62) in CUL group had recurrence, with no significant differences between the three groups ( χ2=1.91, P=0.411). Among the 3 cases of STUMP patients with recurrence (in the pelvic cavity, liver, and abdominal wall), 2 cases had STUMP pathology on recurrence, and 1 case progressed to well-differentiated uterine leiomyosarcoma. (3) Related factors affecting STUMP recurrence: when comparing preoperative body mass index, serum LDH levels, and Ki-67 positivity ≥30% between recurrent and non-recurrent patients, significant differences were observed (all P<0.05). Univariate logistic regression analysis showed that Ki-67 positivity ≥30% was a significant risk factor for STUMP recurrence ( OR=24.67, 95% CI: 1.70-357.36, P=0.019). Conclusions:Factors such as age, history of myomectomy, preoperative serum LDH levels, and ultrasound findings of unclear fibroid borders are helpful for distinguishing STUMP from UL and CUL. Elevated preoperative serum LDH levels and Ki-67 positivity ≥30% have predictive value for STUMP recurrence. Active postoperative follow-up is essential, whether STUMP patients undergo myomectomy or hysterectomy.

Objective:To compare and analyze the differences in clinical and pathological features of uterine smooth muscle tumor of uncertain malignant potential (STUMP), common uterine leiomyoma (UL), and cellular uterine leiomyoma (CUL), and to identify biomarkers for predicting STUMP recurrence.Methods:A total of 72 cases of STUMP patients (STUMP group) treated at the First Affiliated Hospital of Nanjing Medical University and the First Affiliated Hospital of Soochow University were collected from June 2015 to March 2024. Additionally, 72 cases of UL and 72 cases of CUL (UL group and CUL group) in the same period were collected as controls. The clinical and pathological features of the three groups were compared, and the recurrence rates and related factors affecting STUMP recurrence were analyzed.Results:(1) Comparison of clinical and pathological features: there were statistically significant differences in age, history of myomectomy, and preoperative serum lactate dehydrogenase (LDH) levels among STUMP, UL, and CUL groups (all P<0.05). STUMP group were significantly older than UL group ( P<0.05). The proportions of STUMP group with a history of myomectomy and elevated preoperative serum LDH levels were significantly higher than those in UL and CUL groups (all P<0.05). On ultrasound, 16 cases of STUMP patients (22%, 16/72), 2 cases of UL patients (3%, 2/72), and 8 cases of CUL patients (11%, 18/72) had unclear fibroid borders, with significant differences between the three groups ( χ2=12.94, P=0.002), with STUMP group significantly higher than UL group ( P<0.05). Regarding immunohistochemistry, the proportion of p16 positivity, p53 mutations, and nuclear antigen associated with cell proliferation (Ki-67) >10% were significantly higher in STUMP group compared to UL group ( P<0.05). In terms of surgical approach, 52 cases of STUMP patients (72%, 52/72) underwent hysterectomy, compared to 27 cases of UL patients (38%, 27/72) and 38 cases of CUL patients (53%, 38/72), with a significant difference between the three groups ( χ2=17.89, P=0.001). The proportion of patients who underwent myomectomy was significantly lower in STUMP group compared to UL and CUL groups (both P<0.05). Among the 20 cases of STUMP patients who underwent myomectomy, 6 patients had a subsequent total hysterectomy after being diagnosed with STUMP. (2) Comparison of recurrence: the median follow-up time for the STUMP, UL, and CUL groups was 38, 12, and 29 months, respectively. During the follow-up period, 3 cases (6%, 3/53) in STUMP group, 4 cases (7%, 4/55) in UL group, and 8 cases (13%, 8/62) in CUL group had recurrence, with no significant differences between the three groups ( χ2=1.91, P=0.411). Among the 3 cases of STUMP patients with recurrence (in the pelvic cavity, liver, and abdominal wall), 2 cases had STUMP pathology on recurrence, and 1 case progressed to well-differentiated uterine leiomyosarcoma. (3) Related factors affecting STUMP recurrence: when comparing preoperative body mass index, serum LDH levels, and Ki-67 positivity ≥30% between recurrent and non-recurrent patients, significant differences were observed (all P<0.05). Univariate logistic regression analysis showed that Ki-67 positivity ≥30% was a significant risk factor for STUMP recurrence ( OR=24.67, 95% CI: 1.70-357.36, P=0.019). Conclusions:Factors such as age, history of myomectomy, preoperative serum LDH levels, and ultrasound findings of unclear fibroid borders are helpful for distinguishing STUMP from UL and CUL. Elevated preoperative serum LDH levels and Ki-67 positivity ≥30% have predictive value for STUMP recurrence. Active postoperative follow-up is essential, whether STUMP patients undergo myomectomy or hysterectomy.

Objective:To investigate the effect of Edaravone and dexborneol(Eda.B)on oxidative stress pathway in peripheral blood of elderly patients with acute ischemic stroke.Methods:A total of 87 elderly patients with acute ischemic stroke in the Department of Neurology, Qinghai University Affiliated Hospital from July 2021 to January 2022 were selected as the study subjects.According to the random number table, they were divided into control group(44 cases)and edaravone dexborneol group(43 cases). Each group was divided into <12 h group, 12-24 h group and 24-48 h group according to the time of onset.Peripheral blood was collected in each group at admission and discharge, respectively.The serum levels of reactive oxygen species(ROS), Kelch-like epichlorohydrin-associated protein 1(Keap1), nuclear factor-E2-associated factor 2(Nrf2), heme oxygenase-1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1), tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6), as well as superoxide dismutase(SOD)activity and malondialdehyde(MDA)content were detected.Results:Elderly patients with acute ischemic stroke receving Eda.B treatment after admission could reduce the serum concentration of ROS, TNF-α and IL-6, as well as MDA content, and increase the concentration of Keap1, Nrf2, HO-1 and NQO1 and SOD activity.Except for ROS concentration in <12 h group and SOD activity in <12 h and 12 h-24 h groups, the differences between the other groups were statistically significant( P<0.05 for all). Compared with the control group, the serum concentration of TNF-α and IL-6 of patients in the Eda.B group at discharge decreased, while the concentration of Nrf2(24-48 h group)and HO-1(24-48 h group), and SOD activity increased, the differences were statistically significant( P<0.05 for all). In the control group at discharge, the concentrations of ROS(24-48 h group), TNF-α(<12 h group, 24-48 h group)and IL-6, as well as MDA content decreased, while the concentrations of Keap1, Nrf2(<12 h group, 12-24 h group)and HO-1(<12 h group, 12-24 h group)increased, the differences were also statistically significant( P<0.05 for all). Compared with admission, the concentration of Keap1(24-48 h group)and HO-1(24-48 h group), the activity of SOD(<12 h group, 12-24 h group)increased and the content of MDA(12-24 h group)in the Eda.B group decreased at discharge( P<0.05 for all). Conclusions:Eda.B can reduce oxidative stress and inflammatory response in peripheral blood of elderly patients with acute ischemic stroke by acting on the Keap1/Nrf2 pathway.

Objective To explore the antitumor small molecules targeting the ubiquitin–proteasome system (UPS) on the basis of active molecules from traditional Chinese medicine. Methods Ub^G76V-GFP stably expressing cell line was constructed to screen novel small molecule inhibitors targeting UPS. The fluorogenic substrates of Suc-LLVY-AMC, Z-LLE-AMC, and Boc-LRR-AMC were used to assess the effect of dioscin on the 20S proteasome hydrolase activity. The Ub-AMC substrate was used to evaluate the effect of dioscin on the intracellular deubiquitinating enzyme activity. Western blot was used to detect the effect of dioscin on intracellular ubiquitination levels. CCK-8 and colony formation assays were used to detect the inhibitory effect of dioscin on the tumor cell proliferation. Results Dioscin is a UPS inhibitor discovered through the Ub^G76V-GFP reporter system. It enhances intracellular ubiquitination and inhibits tumor cell proliferation and colony formation by targeting deubiquitinating enzymes. Conclusion Dioscin could significantly inhibit tumor cell proliferation by targeting ubiquitin–proteasome.

Objective:To explore the effects of diary research-based communication pattern on migration stress of first-degree relatives of elderly patients transferred out of ICU.Methods:Totally 86 elderly patients transferred out of ICU postoperatively at the 2nd Affiliated Hospital of Wenzhou Medical University from October 2017 to April 2018 were selected by convenient sampling and divided into the control group ( n=43, admitted between October and December 2017) and the observation group ( n=43, admitted between January and April 2018) according to admission time. Patients in the control group underwent routine communication, while patients in the observation group conducted communication based on diary research. The migration stress levels of the first-degree relatives were compared between the two groups. Results:There was no significant difference in migration stress levels between the first-degree relatives of the two groups before the intervention ( P>0.05) . After the intervention, the level of migration stress of the first-degree relatives in the observation group was lower than that in the control group, and the difference was statistically significant ( t=3.598, P<0.01) . Conclusions:The communication model based on diary research can effectively reduce the level of migration stress among the first-degree relatives of elderly patients who are transferred out of ICU postoperatively.

Objective To investigate the change of circulating follicular helper T cells (cTfh) in patients with anti-neutrophil cytoplasmic myeloperoxidase antibody-associated vasculitis (MPO-AAV), and to analyze the relationship between cTfh and disease activity. Methods Thirty-eight untreated MPO-AAV patients (patient group) and thirty-eight healthy volunteers (control group) were enrolled in this study. cTfh and membrane expression of inducible co-stimulator(ICOS)and programmed cell death protein 1(PD-1) were detected by flow cytometry (FCM). Serum anti-neutrophil cytoplasmic myeloperoxidase antibody (MPO-ANCA) was measured by ELISA. Disease activity was evaluated by Birmingham vasculitis activity score (BVAS). Results Compared with those in control group, the proportions of cTfh, ICOS+Tfh and PD-1+Tfh cells in patient group were significantly higher [(25.9±3.8)％vs. (21.0±5.3)％, P<0.001;(1.8±0.8)％vs. (0.8±0.5)％, P<0.001 and (10.2±2.8)％vs. (8.2±2.2)％, P=0.001, respectively]. Meanwhile, the expression of ICOS and PD-1 on cTfh in patient group was markedly more intensive (59.6±10.0 vs.49.2±6.9, P<0.001 and 532.6±104.2 vs. 485.1±73.4, P=0.025, respectively). In patient group, the proportion of cTfh was positively correlated with the ratio of ICOS+Tfh, the expression of ICOS, the level of MPO-ANCA and BVAS (r=0.407, P=0.011; r=0.705, P<0.001; r=0.737, P<0.001 and r=0.663, P<0.001, respectively). The expression intensity of ICOS on cTfh was positively associated with ICOS+Tfh ratio, serum MPO-ANCA and BVAS (r=0.388, P=0.016; r=0.645, P<0.001 and r=0.653, P<0.001, respectively). Nevertheless, the expression of PD-1 on cTfh was only positively correlated with the ratio of PD-1+Tfh (r=0.473, P=0.003). Conclusions Enhanced cTfh in patients with MPO-AAV might produce MPO-ANCA, which is related to the aggravation of MPO-AAV. Thus, cTfh and its ICOS could be potentially targeted for the treatment of MPO-AAV.

Objective To explore preliminarily the role of the E2 subunit of pymvate dehydrogenase (PDC-E2) modified by xenobiotics (e.g.2-octynic acid,2-OA) in the pathogenesis of primary biliary cirrhosis (PBC).Methods Patients of PBC (102 cases),primary sclerosing cholangitis (PSC,34 cases) and healthy controls (HC,50 cases) were selected.The anti-PDC-E2,anti-2-OA and anti-lipoic acid (LA) antibody in the peripheral blood of the 3 groups were tested by enzyme linked immunosorbent assay (ELISA).By inhibitive ELISA (iELISA),30 of the 102 PBC patients with anti-PDC-E2 antibody but without anti-2-OA antibody were selected to detect whether there was any new epitope on the PEC-E2 conjugated with 2-OA.The chi-square test and Fisher exact test were taken to analyze the enumeration data.The two-tailed unpaired t test with Welch's correction was used to compare the measurement data.Spearman rank correlation analysis was also employed for proper test.Results The positive rate of anti-PDC-E2,anti-LA and anti-2-OA antibody in PBC patients was 94.1％(96/102),73.5％(73/102) and 53.9％(55/102) respectively,all of which were statistically significantly higher than those in healthy controls group but were of no significant difference between PSC and healthy controls group.There was no significant relevance between the levels of Anti-LA and anti-2-OA antibody in the PBC group (r=-0.065,P=0.520).The iELISA results showed that the antibody,which only identified the epitopes on 2-OA-PDC-E2 induced by the 2-OA conjugation with PDC-E2,existed in 40％(12/30) of the PBC patients,and more interestingly,this antibody was predominantly appeared in PBC patients at their early clinical stage.Conclusion There are anti-LA antibody and anti-2-OA antibody in PBC patients,which have shown no significant association with each other.It is very likely that new antigenic conformational epitopes on PDC-E2 modified by 2-OA would emerge,which might led to the immune response in the individuals who are susceptible to PBC,and thus contribute for the breaking of PDC-E2 immune tolerance,and PBC occurrence finally.

Objective To investigate the expression of CD55 and myeloperoxidase (MPO) on neutrophils in patients with MPO-specific anti-neutrophil cytoplasmic antibody associated vasculitis(MPO-AAV), and analyze the relationship between the expression and clinical manifestation.Methods Forty untreated patients with active MPO-AAV (patient group) and 30 healthy volunteers (control group) were enrolled in this study.The CD55 on neutrophils and both membrane and cytoplasmic MPO were detected by flow cytometry.Serum fragment-from the activated complement factor B(Ba) and MPO were measured by ELISA.The clinical activity of vasculitis was valued by Birmingham vasculitis activity score-version 3(BVAS-V3).The significance of laboratory data was evaluated by Spearman correlation test and multivariate linear regression analysis.Results (1)The mean fluorescence intensity(MFI) of CD55 expressed on neutrophils was significantly higher than that in control group[4 068.6±2 306.0 vs 2 999.5±1 504.9,P=0.033].Similar results of serum MPO and Ba in patient group were found compared to controls [500.0(381.0, 612.7) IU/L vs 286.9(225.5, 329.1) IU/L,P<0.001;35.2(25.2, 79.5) ng/L vs 18.0(15.0, 28.0) ng/L,P<0.001], respectively.However, MIF of cytoplasmic MPO in patients was significantly lower than that of control group(1 577.1±1 175.9 vs 3 105.3±2 323.0,P=0.003).(2) In patient group, cytoplasmic intensity of MPO was negatively associated with the serum levels of MPO(r=-0.710,P<0.001) and Ba (r=-0.589,P=0.001).Moreover, serum MPO was positively associated with serum Ba(r=0.691,P<0.001).Membrane intensity of CD55 on neutrophils was positively correlated with patient age (r=0.514, P=0.001), C reactive protein (r=0.376, P=0.018), peripheral neutrophils count (r=0.485, P=0.001) and BVAS-V3 (r=0.484, P=0.002), whereas negative correlation between membrane CD55 and disease duration was seen (r=-0.403,P=0.01).(3) The result of multiple linear regression analysis showed there was statistically significant positive correlation between MFI of CD55 expressed on neutrophils and BVAS-V3 (β=0.001,P=0.027).Conclusions In MPO-AAV,CD55 expression on neutrophils is markedly enhanced, which is one of the independent risk factors related to disease activity.It might protect neutrophils from attacking AAV, CD55 expression on neutrophils is markedly enhanced, which is one of the independent risk factors related to disease activity.It might protect neutrophils from attacking by complement alternative pathway.Activated neutrophils release more MPO and lysosome to intensify the inflammation reaction and aggravate the disease.Thus CD55 might become a new potential target for the treatment of this disease in the future.