AIM:To investigate the impact of recombinant human CC16 protein(rhCC16)on cigarette smoke extract(CSE)-induced senescence-associated secretory phenotype(SASP)in human bronchial epithelial cells(HBECs)and in the lung tissues of chronic obstructive pulmonary disease(COPD)mice,and to explore the underlying mechanism.METHODS:HBECs were induced into cellular senescence using 5%CSE.The senescent HBECs were treated with 250 ng/mL rhCC16,and the levels of reactive oxygen species(ROS)were assessed using the 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)method.The levels of trimethylated histone H3 at lysine 9(H3K9me3),a marker of senescence-associated heterochromatic foci(SAHF),were detected using a Western blot assay.RT-qPCR and ELISA were utilized to measure the mRNA expression and protein levels of SASP components including interleukin-1 beta(IL-1β),IL-6,IL-8,chemokine(C-X-C motif)ligand-1(CXCL-1),matrix metalloproteinase 1(MMP1)and MMP3.Passive smoking was con-ducted for six months to induce COPD in mice.RhCC16(2.5 μg/g body weight)or an equal volume of PBS(20 μL)was intranasally administered from the 16th week of smoking in the COPD+rhCC16 group or COPD+PBS group,respectively,with administration 2 hours before smoking.ROS levels in lung tissue cells were investigated using DCFH-DA staining.H3K9me3 levels in lung tissues were tested using Western blot assay.RT-qPCR and ELISA were performed to examine the mRNA expression and protein levels of IL-1β,IL-6,IL-8,CXCL-1,MMP1 and MMP3.RESULTS:DCFH-DA staining results showed that CSE stimulation increased ROS levels in HBECs,while rhCC16 treatment reduced them(P＜0.01).Western blot results indicated that CSE stimulation elevated H3K9me3 levels in HBECs,which were decreased with rhCC16 treatment(P＜0.01).RT-qPCR and ELISA assays demonstrated that CSE stimulation upregulated the mRNA and protein levels of IL-1β,IL-6,IL-8,CXCL-1,MMP1 and MMP3 in HBECs,which were reduced with rhCC16 admin-istration(P＜0.05).DCFH-DA staining results showed an increase in ROS levels in the lung tissues of COPD mice,which were decreased with rhCC16 administration(P＜0.01).Western blot data revealed an increase in H3K9me3 levels in the lung tissues of COPD mice,which were reduced with rhCC16 treatment(P＜0.01).RT-qPCR and ELISA assays demon-strated an upregulation of the mRNA and protein levels of IL-1β,IL-6,IL-8,CXCL-1,MMP1 and MMP3 in the lung tis-sues of COPD mice,which were reduced with rhCC16 treatment(P＜0.05).No statistically significant differences were ob-served in the above-mentioned indicators between the lung tissues of COPD and COPD+PBS mice(P＞0.05).CONCLU-SION:rhCC16 can effectively inhibit CSE-induced SASP in HBECs and in the lung tissues of COPD mice,with its under-lying mechanism potentially related to the inhibition of the ROS-H3K9me3 signaling pathway.

Objective:To investigate the role of mechanosensor Yes-associated protein 1 (YAP1) in urothelial cells in inducing bladder dysfunction in a partial bladder outlet obstruction (pBOO) model.Methods:Ten female C57BL/6 mice were included in this study and randomly divided into pBOO and sham groups based on body weight using a stratified pairing method, with 5 mice in each group. The pBOO group underwent proximal urethral ligation surgery, while the sham group underwent a sham operation. Two weeks after surgery, the urinary pattern was analyzed using the urine spot test. The significant increase in urine spot numbers indicated the successful establishment of the pBOO model. The mice were then sacrificed, and bladder tissues were weighed and stained with hematoxylin and eosin (HE) to observe morphological changes. The bladder urothelial layer was further isolated, and total cell proteins were extracted to detect the expression levels of YAP1 protein using Western blotting. Mouse immortalized bladder urothelial cells were divided into three experimental groups: the negative control (NC) group, which was treated with YAP1-NC lentivirus; the overexpression (OE) group, which was treated with YAP1-OE lentivirus to induce YAP1 protein overexpression; and the verteporfin treatment (VP) group, which was treated with verteporfin on the basis of the OE group. Real-time quantitative PCR and Western blotting were used to verify the transcription and expression levels of YAP1 protein, the co-transcriptional activator TEAD4 protein, and the phosphorylated protein DRP1-616 (at serine 616) of dynamin-related protein 1 (DRP1). An ATP detection kit was used to measure the ATP release concentration in the NC, OE, and VP groups. The interaction between YAP1 and TEAD4 was investigated using co-immunoprecipitation, and the expression of the mitochondrial marker translocase of the outer mitochondrial membrane 20 (Tom20) was observed using immunofluorescence staining.Results:The results of the urine spot test showed that the number of urine spots on the filter paper in the pBOO group was higher than that in the sham group within 6 hours [(283.0±9.1) spots vs. (3.7±0.3) spots, P<0.01], and the urine spots were scattered. The bladder wet weight in the pBOO group was significantly higher than that in the sham group [(105.70±6.84) mg vs. (22.33±1.20) mg, P<0.01]. Histological observations revealed reduced bladder mucosal folds and increased detrusor muscle thickness in the pBOO group. The expression of YAP1 protein in the bladder urothelial cells of the pBOO group was significantly upregulated compared to the sham group [(1.26±0.08) vs. (0.50±0.04), P<0.01]. In vitro experiments showed that compared to the NC group, the OE group had significantly increased expression of DRP1-616 [(0.94±0.05) vs. (0.33±0.01), P<0.01] and higher ATP release concentration [(24.45±0.16) μmol/mg vs. (19.67±0.42) μmol/mg, P<0.01]. In contrast, the VP group had significantly decreased expression of DRP1-616 [(0.29±0.04) vs. (0.94±0.05), P<0.01] and lower ATP release concentration [(10.55±0.01) μmol/mg vs. (24.45±0.16) μmol/mg, P<0.01] compared to the OE group. Co-immunoprecipitation experiments using YAP1 and TEAD4 antibodies showed that YAP1 and TEAD4 proteins could interact and form a transcriptional complex to regulate ATP release. Immunofluorescence staining revealed increased expression of Tom20 in the OE group compared to the NC group [(104.20±3.28) vs. (74.51±3.87), P<0.01]. Conclusions:In the pBOO-induced bladder dysfunction model, YAP1 is highly expressed in urothelial cells. YAP1 forms a transcriptional complex with TEAD4 to regulate ATP release by promoting mitochondrial fission via DRP1-616 expression, which is a key mechanism underlying pBOO-induced bladder dysfunction.

Objective:To analyze the interference of an exogenous glucose test on urinary creatinine-related renal injury biomarkers in patients with chronic kidney disease (CKD).Methods:This cross-sectional study enrolled CKD patients who visited Peking University First Hospital between October 2023 and March 2024. The patients (age: 50±18 years) included 90 males and 70 females. Fresh morning urine samples were collected, totaling 160 samples. Each urine sample was divided into 5 aliquots,each containing 225 μl. One aliquot received 75 μl of deionized water as the control. The other aliquots received 75 μl of glucose solutions at concentrations of 120, 480, 960, and 1200 mmol/L, resulting in final glucose concentrations of 30, 120, 240, and 300 mmol/L in the urine samples, respectively. Urinary creatinine in each sample was measured using both the enzymatic method and the picric acid (Jaffe) method. The following ratios were calculated: urinary albumin-to-creatinine ratio (uACR), urinary protein-to-creatinine ratio (uPCR), urinary transferrin-to-creatinine ratio (uTRF/uCr), urinary α1-microglobulin-to-creatinine ratio (uA1M/uCr), urinary immunoglobulin G-to-creatinine ratio (uIgG/uCr), and urinary N-acetyl-β-D-glucosaminidase-to-creatinine ratio (uNAG/uCr).Results:Under high glucose concentrations, significant differences ( P<0.05) were observed between the enzymatic method and the picric acid method in measuring urinary creatinine-related renal injury biomarkers. At glucose concentrations of 30, 120, 240, and 300 mmol/L, the mean percentage biases for creatinine measured by the enzymatic method were -0.19%, -0.27%, -0.20%, and -0.21%, respectively. The mean percentage biases for creatinine measured by the picric acid method were 0.78%, 1.26%, 1.35%, and 1.38%, respectively, showing an increasing deviation between the results before and after glucose addition as the glucose concentration rose. For uACR measurement, the mean absolute biases using the enzymatic method were -0.01, 1.27, 0.95, and 1.10 mg/g at the respective glucose concentrations. Using the picric acid method, the mean absolute biases for uACR were -11.69, -14.98, -16.91, and-18.51 mg/g. The biases of the picric acid method were significantly higher than the those of the enzymatic method, and the absolute value of the mean biases increased with rising glucose concentration. For uPCR, uTRF/uCr, uA1M/uCr, uNAG/uCr, and uIgG/uCr, the deviations measured by the enzymatic method were consistently smaller than those measured by the picric acid method. Conclusions:The measurement of creatinine and related renal injury biomarkers by the enzymatic method is less affected by glucose concentration. In contrast, the measurement results obtained using the picric acid method are significantly affected by glucose concentration.

AIM:To investigate the impact of recombinant human CC16 protein(rhCC16)on cigarette smoke extract(CSE)-induced senescence-associated secretory phenotype(SASP)in human bronchial epithelial cells(HBECs)and in the lung tissues of chronic obstructive pulmonary disease(COPD)mice,and to explore the underlying mechanism.METHODS:HBECs were induced into cellular senescence using 5%CSE.The senescent HBECs were treated with 250 ng/mL rhCC16,and the levels of reactive oxygen species(ROS)were assessed using the 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)method.The levels of trimethylated histone H3 at lysine 9(H3K9me3),a marker of senescence-associated heterochromatic foci(SAHF),were detected using a Western blot assay.RT-qPCR and ELISA were utilized to measure the mRNA expression and protein levels of SASP components including interleukin-1 beta(IL-1β),IL-6,IL-8,chemokine(C-X-C motif)ligand-1(CXCL-1),matrix metalloproteinase 1(MMP1)and MMP3.Passive smoking was con-ducted for six months to induce COPD in mice.RhCC16(2.5 μg/g body weight)or an equal volume of PBS(20 μL)was intranasally administered from the 16th week of smoking in the COPD+rhCC16 group or COPD+PBS group,respectively,with administration 2 hours before smoking.ROS levels in lung tissue cells were investigated using DCFH-DA staining.H3K9me3 levels in lung tissues were tested using Western blot assay.RT-qPCR and ELISA were performed to examine the mRNA expression and protein levels of IL-1β,IL-6,IL-8,CXCL-1,MMP1 and MMP3.RESULTS:DCFH-DA staining results showed that CSE stimulation increased ROS levels in HBECs,while rhCC16 treatment reduced them(P＜0.01).Western blot results indicated that CSE stimulation elevated H3K9me3 levels in HBECs,which were decreased with rhCC16 treatment(P＜0.01).RT-qPCR and ELISA assays demonstrated that CSE stimulation upregulated the mRNA and protein levels of IL-1β,IL-6,IL-8,CXCL-1,MMP1 and MMP3 in HBECs,which were reduced with rhCC16 admin-istration(P＜0.05).DCFH-DA staining results showed an increase in ROS levels in the lung tissues of COPD mice,which were decreased with rhCC16 administration(P＜0.01).Western blot data revealed an increase in H3K9me3 levels in the lung tissues of COPD mice,which were reduced with rhCC16 treatment(P＜0.01).RT-qPCR and ELISA assays demon-strated an upregulation of the mRNA and protein levels of IL-1β,IL-6,IL-8,CXCL-1,MMP1 and MMP3 in the lung tis-sues of COPD mice,which were reduced with rhCC16 treatment(P＜0.05).No statistically significant differences were ob-served in the above-mentioned indicators between the lung tissues of COPD and COPD+PBS mice(P＞0.05).CONCLU-SION:rhCC16 can effectively inhibit CSE-induced SASP in HBECs and in the lung tissues of COPD mice,with its under-lying mechanism potentially related to the inhibition of the ROS-H3K9me3 signaling pathway.

Objective:To investigate the relationship of several adiposity-related anthropometric parameters, including BMI, waist circumference (WC), waist-to-hip ratio (WHR), body fat percentage (BFP) and indoles in plasma with the incidence of atherosclerotic cardiovascular disease (ASCVD) in adults in China.Methods:In China Kadoorie Biobank (CKB) study, blood samples were collected from 2 183 participants in the first resurvey in 2008 to detect indoles. Participants' body weight, body height, WC, hip circumference, and BFP were measured at baseline survey in 2004 and resurvey in 2008, the BMI and WHR were calculated with standardized methods. The long-term follow-up of all participants started from the completion of the resurvey in 2008 until the occurrence of incident ASCVD, death, loss to follow-up or until December 31, 2018. CKB ascertained outcome status (incident ASCVD) through death and disease registries and national health insurance databases, supplemented by active follow-up. Multivariate linear regression model was used to estimate the associations of anthropometric measurements at baseline survey and the first resurvey, and changes in these measurements with 3 indoles [indole, indole-3-acetic acid (IAA), and indole-3-propionic acid (IPA)]. Cox proportional hazard regression model was used to estimate the associations between indoles and the risk for ASCVD.Results:Anthropometric measurements at baseline survey or the first resurvey were negatively associated with plasma IPA level. The regression coefficient ( β) of baseline BMI (per 1.0 kg/m 2) with 0.1 standard deviation ( SD) IPA was -0.23 (95% CI: -0.36 - -0.10) (false discovery rate=0.004). After adjusting for baseline BMI, the β of baseline WC, WHR and BFP with 0.1 SD IPA were -0.09 (95% CI: -0.18 - -0.01), -0.12 (95% CI: -0.19 - -0.05), and -0.20 (95% CI: -0.32 - -0.08), respectively. The annual change in BMI (difference between BMI in 2008 and 2004 divided by the time interval) was associated with indole and IAA, with β of 1.40 (95% CI: 0.58 - 2.21) and -1.07 (95% CI: -1.91 - -0.23), respectively, at each 0.1 increase of SD. Over a median ( Q1, Q3) follow-up of 10.46 (10.36, 10.53) years after 2008 resurvey, 236 cases of ASCVD were recorded. IAA and IPA levels were negatively associated with the risk for ASCVD, with hazard ratios for one SD increase of IAA and IPA of 0.87 (95% CI: 0.76 - 0.99) and 0.84 (95% CI: 0.73 - 0.96), respectively. Conclusions:Our results suggested that anthropometric measurements and their changing trends affect the levels of plasma imicrobial tryptophan metabolite levels, decreased levels of IAA and IPA levels are associated with increased risk of ASCVD and indoles in plasma including IPA and IAA might be the mediating factors for adiposity-induced ASCVD.

Objective:To analyze the prospective associations between liver biomarkers and mortality among Chinese middle-aged and elderly populations and to evaluate the mortality risk predictive value.Methods:A total of 22 758 participants from the 3 rd resurvey of the China Kadoorie Biobank were included. Cox proportional hazard models were used to analyze the prospective associations of 5 liver biomarkers with mortality. These liver biomarkers included two liver imaging biomarkers (liver fat attenuation parameter, liver stiffness measurement) and three serum liver enzyme biomarkers [gamma-glutamyl transferase (GGT), ALT, and AST]. Restricted cubic spline was used to assess the nonlinear associations between biomarkers and mortality. The area used the receiver operating characteristic curve (AUC) to evaluate the predictive ability of the models after incorporating liver biomarkers into traditional prediction models for mortality. Results:The mean age of the participants was (65.2±9.1) years, with a median follow-up of 1.5 years, during which 307 deaths occurred. Compared to individuals without hepatic steatosis, those with severe hepatic steatosis had a 79% higher risk of mortality, with a HR of 1.79 (95% CI: 1.06-3.03). Compared to individuals without hepatic fibrosis, those with advanced fibrosis and cirrhosis had higher mortality risks of 48% and 91%, respectively (both P<0.05). For each standard deviation increase in GGT, the mortality risk increased by 10% ( HR=1.10, 95% CI: 1.05-1.15), with the positive association plateauing at higher GGT levels. AST exhibited a U-shaped association with mortality risk. The AUC of the prediction model adding liver biomarkers into traditional prediction factors was 0.718 (95% CI: 0.679-0.757), with an increase of 0.030 ( P<0.001) compared with the traditional model. Conclusions:Severe hepatic steatosis, higher levels of hepatic fibrosis, and elevated GGT levels are significantly associated with higher mortality risk. AST shows a U-shaped nonlinear association with mortality risk. Incorporating liver biomarkers into traditional risk prediction models enhance the ability to predict mortality.

Objective:To investigate the prospective associations between plasma metabolites and the risks of all-cause and cause-specific mortality among Chinese adults.Methods:This study analyzed plasma metabolomics data from 2 183 healthy adults in the China Kadoorie Biobank (CKB), measured using targeted mass spectrometry. Cox proportional hazards regression models were used to examine the associations between 630 metabolites and the risk of all-cause mortality. Cause-specific hazard regression models evaluated the associations between metabolites and cardiovascular disease (CVD) risks, cancer, and other-cause mortality. Stepwise regression was used to identify key metabolites independently associated with all-cause mortality, and the area under the receiver operating characteristic curve (AUC) was calculated to assess the improvement in predictive performance when these metabolites were added to traditional risk prediction models.Results:The mean age of the participants was (53.2±9.8) years, 65.1% of whom were female. During a median follow-up of 14.5 years, 231 deaths occurred. A total of 44 metabolites were significantly associated with the risk of all-cause mortality [false discovery rate (FDR)-adjusted P<0.05], primarily including triglycerides, ceramides, and amino acids. Additionally, 29 and 15 metabolites were found to be associated with cancer and other-cause mortality, respectively, but no metabolites were significantly associated with CVD mortality after FDR corrections. Adding 14 metabolites independently associated with all-cause mortality into the traditional prediction model significantly improved its predictive performance. Specifically, incorporating metabolites into the traditional model, which already included laboratory biomarkers, increased the AUC to 0.798 (95% CI: 0.755-0.843), an improvement of 0.088 compared to the traditional model ( P<0.001). Conclusions:Multiple metabolites are significantly associated with mortality risk and can substantially improve the accuracy of mortality risk prediction models. These findings provide new insights into the physiological mechanisms of aging and offer valuable clues for personalized health risk assessment.

Objective:To investigate the role of mechanosensor Yes-associated protein 1 (YAP1) in urothelial cells in inducing bladder dysfunction in a partial bladder outlet obstruction (pBOO) model.Methods:Ten female C57BL/6 mice were included in this study and randomly divided into pBOO and sham groups based on body weight using a stratified pairing method, with 5 mice in each group. The pBOO group underwent proximal urethral ligation surgery, while the sham group underwent a sham operation. Two weeks after surgery, the urinary pattern was analyzed using the urine spot test. The significant increase in urine spot numbers indicated the successful establishment of the pBOO model. The mice were then sacrificed, and bladder tissues were weighed and stained with hematoxylin and eosin (HE) to observe morphological changes. The bladder urothelial layer was further isolated, and total cell proteins were extracted to detect the expression levels of YAP1 protein using Western blotting. Mouse immortalized bladder urothelial cells were divided into three experimental groups: the negative control (NC) group, which was treated with YAP1-NC lentivirus; the overexpression (OE) group, which was treated with YAP1-OE lentivirus to induce YAP1 protein overexpression; and the verteporfin treatment (VP) group, which was treated with verteporfin on the basis of the OE group. Real-time quantitative PCR and Western blotting were used to verify the transcription and expression levels of YAP1 protein, the co-transcriptional activator TEAD4 protein, and the phosphorylated protein DRP1-616 (at serine 616) of dynamin-related protein 1 (DRP1). An ATP detection kit was used to measure the ATP release concentration in the NC, OE, and VP groups. The interaction between YAP1 and TEAD4 was investigated using co-immunoprecipitation, and the expression of the mitochondrial marker translocase of the outer mitochondrial membrane 20 (Tom20) was observed using immunofluorescence staining.Results:The results of the urine spot test showed that the number of urine spots on the filter paper in the pBOO group was higher than that in the sham group within 6 hours [(283.0±9.1) spots vs. (3.7±0.3) spots, P<0.01], and the urine spots were scattered. The bladder wet weight in the pBOO group was significantly higher than that in the sham group [(105.70±6.84) mg vs. (22.33±1.20) mg, P<0.01]. Histological observations revealed reduced bladder mucosal folds and increased detrusor muscle thickness in the pBOO group. The expression of YAP1 protein in the bladder urothelial cells of the pBOO group was significantly upregulated compared to the sham group [(1.26±0.08) vs. (0.50±0.04), P<0.01]. In vitro experiments showed that compared to the NC group, the OE group had significantly increased expression of DRP1-616 [(0.94±0.05) vs. (0.33±0.01), P<0.01] and higher ATP release concentration [(24.45±0.16) μmol/mg vs. (19.67±0.42) μmol/mg, P<0.01]. In contrast, the VP group had significantly decreased expression of DRP1-616 [(0.29±0.04) vs. (0.94±0.05), P<0.01] and lower ATP release concentration [(10.55±0.01) μmol/mg vs. (24.45±0.16) μmol/mg, P<0.01] compared to the OE group. Co-immunoprecipitation experiments using YAP1 and TEAD4 antibodies showed that YAP1 and TEAD4 proteins could interact and form a transcriptional complex to regulate ATP release. Immunofluorescence staining revealed increased expression of Tom20 in the OE group compared to the NC group [(104.20±3.28) vs. (74.51±3.87), P<0.01]. Conclusions:In the pBOO-induced bladder dysfunction model, YAP1 is highly expressed in urothelial cells. YAP1 forms a transcriptional complex with TEAD4 to regulate ATP release by promoting mitochondrial fission via DRP1-616 expression, which is a key mechanism underlying pBOO-induced bladder dysfunction.

Objective To explore the detection rate and distribution characteristics of gene mutations in pa-tients with non-small cell lung cancer,and to analyze their relationship with clinicopathological characteristics.Methods The genetic testing results and clinicopathological data of 213 patients with non-small cell lung cancer who underwent high-throughput genetic testing at the Gene Detection Center,Changchun Cancer Hos-pital from April 2020 to December 2023 were collected.High-throughput sequencing technology was used to detect mutations in 26 genes,and the relationship between the mutation frequency and its distribution and the clinicopathological characteristics of patients was analyzed.Results Among 213 patients with non-small cell lung cancer,192 cases(90.14％)had at least one gene mutation detected.Among them,the genes with rela-tively high mutation frequencies were TP53(60.56％),EGFR(46.48％),KRAS(14.55％),ALK fusion(11.74％),and PIK3CA(8.92％).There were 132 cases(30.28％)of evidence level for Class 1 drugs and 11 cases(2.52％)of evidence level for Class 2 drugs.The incidence of EGFR gene mutations was higher in women,non-smokers and patients with lung adenocarcinoma(P＜0.05).TP53 mutations usually occured in women,smokers and patients with stage Ⅳ.ALK mutations were more common in young patients,while KRAS mutations were more frequently seen in male smokers.Conclusion Analyzing the distribution charac-teristics of gene mutations in non-small cell lung cancer and their relationship with clinicopathological charac-teristics can provide a scientific basis for further optimizing genetic testing for patients with non-small cell lung cancer and offer guidance for clinical treatment.

Objective To establish a model that integrates ultrasound features and immunological characteristics for predicting the efficacy of neoadjuvant chemotherapy(NAC)in breast cancer patients.Methods A total of 203 breast cancer patients undergoing preoperative NAC at the Fifth Medical Center of the PLA General Hospital between July 2021 and July 2024 were screened.In line with the inclusion/exclusion criteria,177 patients were included.Data on ultrasound and immunohistochemistry was collected.These patients were divided into pathological complete response(pCR)and non-pathological complete response(non-pCR)groups based on postoperative pathology.Factors with P＜0.0 1 in univariate analysis were evaluated using multivariate Logistic regression.Independent predictive factors were used to construct and validate the ultrasound-immunohistochemical model via Bootstrap.Results The reduction rateof the maximum diameter of lesions,posterior echo attenuation,PR status and HER-2 status were identified as independent predictors of pCR(all P＜0.05).The model proved to be highly accurate and stable.Conclusion The model that combines ultrasound and immunohistochemical features can effectively evaluatep CR after NAC in breast cancer patients.