Objective:To elucidate the molecular mechanisms through which high-dose dexamethasone exerts long-term effects on bone marrow mesenchymal stem cells (BMSCs), specifically its role in suppressing osteogenic differentiation, accelerating cellular senescence, triggering the senescence-associated secretory phenotype (SASP), and inducing apoptosis.Methods:Primary rat BMSCs were isolated and treated with high-dose dexamethasone (1×10 -4 mol/L) to establish the experimental group, while untreated cells served as the control. The gene and protein expression levels of osteogenic markers, bone alkaline phosphatase (bALP) and Runt-related transcription factor 2 (Runx2), were analyzed in both groups. Cellular senescence was evaluated using senescence-associated β-galactosidase (SA-β-gal) staining. The expression of senescence-related markers (P16 and P21), components of the senescence-associated secretory phenotype (SASP), including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interferon (IFN)-γ, as well as apoptosis-related proteins (Bcl-2, Bax, and Cleaved-Caspase-3), and key factors of the Nrf2/HO-1 signaling pathway were assessed at both transcriptional and protein levels using qRT-PCR, immunofluorescence, and Western-blot analyses. These comprehensive evaluations aimed to determine the senescent state, apoptotic features, and alterations in osteogenic differentiation of BMSCs. Results:Following treatment with dexamethasone and subsequent withdrawal, both qRT-PCR and Western blot analyses indicated a significant reduction in the expression of the osteogenic markers bALP and Runx2 at both mRNA and protein levels. The proportion of SA-β-gal positive cells was markedly higher in the dexamethasone group (74.33%±6.89%) than in the control group (20.30%±1.57%, t=17.300, P<0.001). qRT-PCR analysis revealed upregulated mRNA expression of the senescence-related genes P16 and P21 after dexamethasone treatment, which was further supported at the protein level by immunofluorescence showing increased P21 expression. Western-blot results confirmed that protein expression levels of P16 and P21 were significantly elevated in the dexamethasone group (7.025±0.255 and 6.362±0.456, respectively) compared with the control group (1.016±0.115 and 0.816±0.172; both P<0.05). Furthermore, gene expression levels of the senescence-associated secretory phenotype (SASP) factors TNF-α and IL-1β were significantly increased (TNF-α: 3.539±0.599 vs. 0.742±0.095; IL-1β: 4.469±0.331 vs. 0.799±0.175; both P<0.05), and their protein expression was consistently upregulated as validated by Western-blot. Additionally, protein expression levels of TNF-α, IL-1β, and IFN-γ were significantly higher in the dexamethasone-treated group (3.476±0.932 vs. 0.945±0.095; 4.111±0.220 vs. 0.762±0.105; 2.155±0.240 vs. 0.656±0.104; all P<0.05).Western-blot analysis also demonstrated that protein expression of Nrf2 and HO-1 was significantly suppressed in the dexamethasone group (0.21±0.07 and 0.19±0.06, respectively) compared with the control group (1.13±0.15 and 0.92±0.21; P<0.05). Moreover, Western-blot analysis revealed that the expression levels of the pro-apoptotic proteins Bax and Cleaved-Caspase-3 were significantly up, regulated in the dexamethasone, treated BMSCs (Bax: 3.673±0.397 vs. 0.453±0.111; Cleaved-Caspase-3: 3.863±0.399 vs. 0.465±0.057), while the expression of the anti-apoptotic protein Bcl-2 was markedly down, regulated (0.959±0.073 vs. 2.126±0.195), with all differences being statistically significant ( P<0.05). Conclusions:High-dose dexamethasone treatment of BMSCs, followed by withdrawal of dexamethasone, induces cellular senescence and enhances the expression of the senescence-associated secretory phenotype (SASP) through suppression of the Nrf2/HO-1 signaling pathway. Concurrently, it promotes apoptosis by activating the mitochondrial apoptotic pathway, collectively leading to impaired osteogenic differentiation of BMSCs.

Objective:To elucidate the molecular mechanisms through which high-dose dexamethasone exerts long-term effects on bone marrow mesenchymal stem cells (BMSCs), specifically its role in suppressing osteogenic differentiation, accelerating cellular senescence, triggering the senescence-associated secretory phenotype (SASP), and inducing apoptosis.Methods:Primary rat BMSCs were isolated and treated with high-dose dexamethasone (1×10 -4 mol/L) to establish the experimental group, while untreated cells served as the control. The gene and protein expression levels of osteogenic markers, bone alkaline phosphatase (bALP) and Runt-related transcription factor 2 (Runx2), were analyzed in both groups. Cellular senescence was evaluated using senescence-associated β-galactosidase (SA-β-gal) staining. The expression of senescence-related markers (P16 and P21), components of the senescence-associated secretory phenotype (SASP), including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interferon (IFN)-γ, as well as apoptosis-related proteins (Bcl-2, Bax, and Cleaved-Caspase-3), and key factors of the Nrf2/HO-1 signaling pathway were assessed at both transcriptional and protein levels using qRT-PCR, immunofluorescence, and Western-blot analyses. These comprehensive evaluations aimed to determine the senescent state, apoptotic features, and alterations in osteogenic differentiation of BMSCs. Results:Following treatment with dexamethasone and subsequent withdrawal, both qRT-PCR and Western blot analyses indicated a significant reduction in the expression of the osteogenic markers bALP and Runx2 at both mRNA and protein levels. The proportion of SA-β-gal positive cells was markedly higher in the dexamethasone group (74.33%±6.89%) than in the control group (20.30%±1.57%, t=17.300, P<0.001). qRT-PCR analysis revealed upregulated mRNA expression of the senescence-related genes P16 and P21 after dexamethasone treatment, which was further supported at the protein level by immunofluorescence showing increased P21 expression. Western-blot results confirmed that protein expression levels of P16 and P21 were significantly elevated in the dexamethasone group (7.025±0.255 and 6.362±0.456, respectively) compared with the control group (1.016±0.115 and 0.816±0.172; both P<0.05). Furthermore, gene expression levels of the senescence-associated secretory phenotype (SASP) factors TNF-α and IL-1β were significantly increased (TNF-α: 3.539±0.599 vs. 0.742±0.095; IL-1β: 4.469±0.331 vs. 0.799±0.175; both P<0.05), and their protein expression was consistently upregulated as validated by Western-blot. Additionally, protein expression levels of TNF-α, IL-1β, and IFN-γ were significantly higher in the dexamethasone-treated group (3.476±0.932 vs. 0.945±0.095; 4.111±0.220 vs. 0.762±0.105; 2.155±0.240 vs. 0.656±0.104; all P<0.05).Western-blot analysis also demonstrated that protein expression of Nrf2 and HO-1 was significantly suppressed in the dexamethasone group (0.21±0.07 and 0.19±0.06, respectively) compared with the control group (1.13±0.15 and 0.92±0.21; P<0.05). Moreover, Western-blot analysis revealed that the expression levels of the pro-apoptotic proteins Bax and Cleaved-Caspase-3 were significantly up, regulated in the dexamethasone, treated BMSCs (Bax: 3.673±0.397 vs. 0.453±0.111; Cleaved-Caspase-3: 3.863±0.399 vs. 0.465±0.057), while the expression of the anti-apoptotic protein Bcl-2 was markedly down, regulated (0.959±0.073 vs. 2.126±0.195), with all differences being statistically significant ( P<0.05). Conclusions:High-dose dexamethasone treatment of BMSCs, followed by withdrawal of dexamethasone, induces cellular senescence and enhances the expression of the senescence-associated secretory phenotype (SASP) through suppression of the Nrf2/HO-1 signaling pathway. Concurrently, it promotes apoptosis by activating the mitochondrial apoptotic pathway, collectively leading to impaired osteogenic differentiation of BMSCs.

Objective:To investigate the surgical efficacy and prognosis influencing factors of hilar cholangiocarcinoma based on multidisciplinary diagnosis and treatment.Methods:The retrospective cohort study was conducted. The clinicopathological data of 91 patients with hilar cholangiocarcinoma who underwent surgery in Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School from April 2004 to April 2021 were collected. There were 59 males and 32 females, aged (61±10)years. Patients who were admitted from April 2004 to March 2014 underwent traditional surgical diagnosis and treatment, and patients who were admitted from April 2014 to April 2021 underwent multidisciplinary diagnosis and treatment. Observation indica-tors: (1) surgical situations; (2) postoperative situations; (3) postoperative pathological examina-tions; (4) postoperative prognosis analysis; (5) influencing factors of postoperative prognosis. Follow-up was conducted using telephone interview and outpatient examination. Patients were followed up once every 6 months after surgery to detect survival. The follow-up was up to April 2023. Measure-ment data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the independent sample t test. Measurement data with skewed distribution were represented as M(range), and comparison between groups was conducted using the Mann-Whitney U test. Comparison of ordinal data was conducted using the rank sum test. Count data were described as absolute numbers or percentages, and comparison between groups was conducted using the chi-square test or Fisher exact probability. The Kaplan-Meier method was used to draw survival curve and calculate survival rate. The Log-Rank test was used for survival analysis. Univariate and multivariate analyses were conducted using the COX proportional hazard model. Results:(1) Surgical situations. Of the 91 patients, there were 65 cases receiving hemi- or expanded hemi-hepatectomy, 13 cases receiving tri-hepatectomy, 9 cases receiving partial hepatectomy, 4 cases receiving extrahepatic bile duct resection. There were 24 cases receiving combined vein resection and reconstruction, 8 cases receiving combined pancreaticoduodenectomy, 6 cases receiving com-bined hepatic artery resection and reconstruction, including 24 cases receiving extended radical surgery (tri-hepatectomy, hepatic artery resection and reconstruction, hepatopancreaticoduodenec-tomy). The operation time, volume of intraoperative blood loss and intraoperative blood transfusion rate of 91 patients was (590±124)minutes, 800(range, 500?1 200)mL and 75.8%(69/91), respectively. Of the 91 patients, cases receiving extended radical surgery, the volume of intraoperative blood loss were 4, 650(range, 300?1 000)mL in the 31 patients who were admitted from April 2004 to March 2014, versus 20, 875 (range, 500?1 375)mL in the 60 patients who were admitted from April 2014 to April 2021, showing significant differences between them ( χ2=4.39, Z=0.31, P<0.05). (2) Post-operative situations. The postoperative duration of hospital stay and cases with postoperative infectious complications were (27±17)days and 50 in the 91 patients. Cases with abdominal infection, cases with infection of incision, cases with bacteremia and cases with pulmonary infection were 43, 7, 5, 8 in the 91 patients. One patient might have multiple infectious complications. Cases with bile leakage, cases with delayed gastric emptying, cases with chylous leakage, cases with liver failure, cases with pancreatic fistula, cases with intraperitoneal hemorrhage, cases with reoperation, cases dead during the postoperative 90 days were 30, 9, 9, 6, 5, 3, 6, 3 in the 91 patients. Cases with abdominal infection was 10 in the 31 patients who were admitted from April 2004 to March 2014, versus 33 in the 60 patients who were admitted from April 2014 to April 2021, showing a significant difference between them ( χ2=4.24, P<0.05). Cases dead during the postoperative 90 days was 3 in the 31 patients who were admitted from April 2004 to March 2014, versus 0 in the 60 patients who were admitted from April 2014 to April 2021, showing a significant difference between them ( P<0.05). (3) Post-operative pathological examinations. Of the 91 patients, cases with Bismuth type as type Ⅰ?Ⅱ, type Ⅲ, type Ⅳ, cases with T staging as Tis stage, T1 stage, T2a?2b stage, T3 stage, T4 stage, cases with N staging as N0 stage, N1 stage, N2 stage, cases with M staging as M0 stage, M1 stage, cases with TNM staging as 0 stage, Ⅰ stage, Ⅱ stage, Ⅲ stage, ⅣA stage, ⅣB stage, cases with R 0 radical resection, cases with R 1 or R 2 resection were 15, 46, 30, 1, 9, 25, 30, 26, 49, 36, 6, 85, 6, 1, 7, 13, 58, 6, 6, 63, 28. Cases with R 0 radical resection, cases with R 1 or R 2 resection were 15, 16 in the 31 patients who were admitted from April 2004 to March 2014, versus 48, 12 in the 60 patients who were admitted from April 2014 to April 2021, showing a significant difference between them ( χ2=9.59, P<0.05). (4) Postoperative prognosis analysis. Of the 91 patients, 3 cases who died within 90 days after surgery were excluded, and the 5-year overall survival rate and median overall survival time of the rest of 88 cases were 44.7% and 55 months. The 5-year overall survival rate was 33.5% in the 28 patients who were admitted from April 2004 to March 2014, versus 50.4% in the 60 patients who were admitted from April 2014 to April 2021, showing a significant difference between them ( χ2=5.31, P<0.05). Results of further analysis showed that the corresponding 5-year overall survival rate of cases without lymph node metastasis was 43.8% in the 16 patients who were admitted from April 2004 to March 2014, versus 61.6% in the 31 patients who were admitted from April 2014 to April 2021. There was a significant difference in the 5-year overall survival rate between these patients without lymph node metastasis ( χ2=3.98, P<0.05). The corresponding 5-year overall survival rate of cases with lymph node metastasis was 18.5% in the 12 patients who were admitted from April 2004 to March 2014, versus 37.7% in the 29 patients who were admitted from April 2014 to April 2021. There was no significant difference in the 5-year overall survival rate between these patients with lymph node metastasis ( χ2=2.25, P>0.05). (5) Influencing factors of postoperative prognosis. Results of multivariate analysis showed that poorly differentiated tumor and R 1 or R 2 resection were inde-pendent risk factors influencing prognosis after surgical treatment of hilar cholangiocarcinoma ( hazard ratio=2.62, 2.71, 95% confidence interval as 1.30?5.29, 1.30?5.69, P<0.05). Conclusions:Compared with traditional surgical diagnosis and treatment, treatment of hilar cholangiocarcinoma based on multidisciplinary diagnosis and treatment can expand surgical indications, reduce proportion of dead patients within 90 days after surgery, improve proportation of radical resection and long-term survival rate. Poorly differentiated tumor and R 1 or R 2 resection are independent risk factors influencing prognosis after surgical treatment of hilar cholangiocarcinoma.

Non-small cell lung cancer (NSCLC) is the most common pathological type of lung cancer. The systemic antitumor therapy of advanced NSCLC has undergone renovations of chemotherapy, targeted therapy and immunotherapy, which results in greatly improved survival for patients with advanced NSCLC. Immune checkpoint inhibitors (ICIs), especially targeting programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1), has changed the treatment paradigm of NSCLC. ICIs have become the standard treatment for advanced NSCLC without epidermal growth factor receptor(EGFR) mutation or anaplastic lymphomakinase(ALK) translocation in the first- or second-line setting, and for locally advanced NSCLC following concurrent radiotherapy and chemotherapy. ICIs are also promising in adjuvant/neoadjuvant therapy. More and more ICIs have been approved domestically for the treatment of NSCLC. Led by the NSCLC expert committee of Chinese Society of Clinical Oncology (CSCO), this consensus was developed and updated based on thoroughly reviewing domestic and foreign literatures, clinical trial data, systematic reviews, experts' discussion and the consensus(2019 version). This consensus will aid domestic clinicians in the treatment of NSCLC with ICIs.
.

Non-small cell lung cancer (NSCLC) is the most common pathological type of lung cancer, most NSCLC patients are at advanced stage at the time of diagnosis. For patients without sensitive driven-oncogene mutations, chemotherapy is still the main treatment at present, the overall prognosis is poor. Improving outcomes and obtaining long-term survival are the most urgent needs of patients with advanced NSCLC. In recent years, immunotherapy has developed rapidly. Immune checkpoint inhibitors (ICIs), especially targeting programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1), have made a breakthrough in the treatment of NSCLC, beneficial to patients' survival and changed the treatment pattern for NSCLC. It shows more and more important role in the treatment of NSCLC. Led by NSCLC expert committee of Chinese society of clinical oncology (CSCO), relevant experts in this field were organized. On the basis of referring to domestic and foreign literature, systematically evaluating the results of Chinese and foreign clinical trials, and combining the experiences of the experts, the experts group reached an agreement to develop this consensus. It will guide domestic counterparts for better application of ICIs to treat NSCLC.

Objective To evaluate the prevalence and risk factors of progressive liver fibrosis in patients with non-alcoholic fatty liver disease (NAFLD).Methods A total of 2 054 subjects who underwent health check up and were diagnosed as NAFLD in 9 institutions were included in the study.Blood routine and biochemical findings were collected to calculate aspartate aminotransferase-to-platelet ratio index (APRI).Subjects were divided into three groups according to diagnostic threshold of liver fibrosis:APRI ＜0.43 group,APRI 0.43-0.53 group and APRI ≥ 0.54 group.The correlation between APRI and biochemical variables was analyzed,and the risk factors of progressive fibrosis were also analyzed.Results Among 2 054 subjects (male/female 1 598/456) there were 61 cases with APRI ≥ 0.98 (2.97％,progressive fibrosis),318 with APRI ≥0.54 (15.48％),1 475 with APRI ＜ 0.43 (71.81％),261 with APRI 0.43-0.53 (12.71％).Logistic stepwise regression analysis showed that TG (P =0.002,OR =1.095,95％CI:1.033-1.161),2 hPG(P =0.000,OR =1.103,95％ CI:1.058-1.151,BUN(P =0.034,OR =1.215,95 ％ CI:1.014-1.454) were risk factors,and H DL-C (P =0.034,OR =0.353,95 ％ CI:O.135-0.924) was a protective factor for the progression of fibrosis.Conclusion The progressive fibrosis in patients with NAFLD is closely associated to blood glucose and lipid metabolism disorder.

Objective To investigate the expression profile variation of long non-coding RNAs (lncRNAs) in ankylosing sporidylitis (AS) and explore the role of lncRNAs in the pathogenesis of AS.Methods The peripheral blood mononuclear cells of AS patients and health controls (HC) were used to detect for differently expressed lncRNAs by microarray.The roles of lncRNAs were predicted with GO and pathway analysis.The results were verified by real time-polymerase chain reaction (PCR).Results A total of 148 lncRNAs and 134 mRNAs were detected,which had more than 2-fold differentially expressed in AS patients.Bioinformatics analysis found that GO term enrichment included protein binding,regulation of transcription,metabolism,signal transduction,et al.and might involve in toll-like receptor pathway,protein kinase,complement pathway,notch signaling pathway and so on.The expressions of three lncRNAs were estimated by real time-PCR which found that consistent with that of microarrays.Among these,D90064 was the most aberrantly expressed lncRNAs.Conclusions Several lncRNAs expression was changed significantly in AS patients in comparison with HC,which implies that those different lncRNAs may have an important role in the development and progression of AS.

Objective To investigate the single nucleotide polymorphisms(SNPs) rs3733591(C>T) of SLC2A9 gene in Chinese Han population, and to explore the association of this gene polymorphisms with gout susceptibility, tophi, serum uric acid levels, other clinical and laboratory data and the levels of SLC2A9 mRNA of peripheral blood mononuclear cells(PBMCs). Methods ① A total of 297 primary gout arthritis patients(GA) and 211 normal controls(NC) were enrolled into this study. The clinical and laboratory data of patients were collected. The genotypes and alleles frequencies were measured by using TaqMan ?SNP Geno-typing Assays and the possible association between gene polymorphism of SLC2A9 and gout was investigated by Chi-square test. The odds ratios(OR) and 95% confidence intervals(95%CI) were calculated. ② The lev-els of SLC2A9 mRNA on PBMCs of 86 gout patients(46 patients in remission) and controls were measured by real-time quantitative polymerase chain reaction (RT-qPCR). The nonparametric test was used to analyze the expression in different groups. Results The frequencies of genotypes and alleles of rs3733591(C>T) in gout patients were different from controls(P<0.05). The frequency of TT genotype was significantly lower than that in controls (P<0.05) and the relative risk of this genotype to develop gout was 0.647 (95%CI: 0.452-0.925). Moreover, the frequency of T allele in cases was much lower than in controls (60.9% vs 69.2%, χ2=7.324, P=0.007, OR=0.695), but the frequency of C allele was much higher(39.1% vs 30.8%, χ2=1.440, P=0.007, OR=1.440). Interestingly, the levels of SLC2A9 mRNA on PBMCs in gout patients who carried TC genotype of rs3733591 was higher than those who carried TT genotype(P<0.05). There was no difference in the expression of SLC2A9 mRNA on PBMCs among different genotype carriers of rs3733591 in controls (P>0.05). However, there was no significant difference in the distribution of genotypes and alleles between 30 tophaceous gout patients and 190 non-tophaceous gout patients(P>0.05). Conclusion Results of present study suggest the rs3733591(C>T) polymorphism of the SLC2A9 gene might be associated with gout development, but not with tophaceous gout. The C allele predisposes to gout, and TT genotype and T allele might protect Chinese Han population from developing gout. The rs3733591(C>T) polymorphism probably affects the susceptibility to gout by influencing the f expression of SLC2A9 mRNA susceptibility.

Objective To measure the level of androgen receptor (AR) mRNA in peripheral blood monocytes (PBMCs) and serum testosterone level of patients with gouty arthritis (GA) and healthy controls (HC),and to explore the role of testosterone and AR in the pathogenesis of GA.Methods Chemilluminescence was used to detect the level of serum testosterone in GA [including 119 acute GA (AGA) and 60 nonacute GA (NAGA) patients] and 47 HC group.Real-time quantitative polymerase chain reaction (RT-qPCR)was used to measure AR mRNA in PBMCs from 41 GA and 35 HC.Western blotting was used to measure PBMCs AR in GA and HC for each 6 cases.One-way ANOVA,t test and Spearman's correlation were adopted for statistical analysis.Results Serum testosterone was significantly reduced in AGA and NAGA group compared to that in HC group [(6.1±1.5) ng/ml,P＜0.01,respectively],and the expression was lower in the AGA [(3.7±1.4) ng/ml] group [(4.9±2.0) ng/ml] than that in the NAGA group (P＜0.01).The level of AR mRNA and protein was much lower in the GA group than that in the HC group (P＜0.01,respectively).Negative correlations was detected between AR mRNA and uric acid in GA patients.There was negative correlation between serum testosterone and VLDL,GLU; meanwhile,positive correlation was found between serum testosterone and HDL (P＜0.05,respectively) in NAGA patients.There were no correlations between testosterone and other laboratory data.There was no correlation between AR and other laboratory data in GA patients and healthy controls (P＞0.05,respectively).Conclusion Altered expression of testosterone and its receptor may be involved in the pathogenesis of gouty inflammation.Further study will be needed to shed light on the exact role of androgen and AR in gout.

Objective To investigate the role of adiponectin (ADP) and its receptors (ADR) in pati ents with primary gouty arthritis (GA).Methods Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of plasma ADP in 88 GA and 80 healthy controls (NC).Real time quantitative polymerase chain reaction (RT-qPCR) was employed to study the expression of ADR1 and ADR2 mRNA in peripheral blood mononuclear cells (PBMCs).The biochemical indicator of TG,HDL,LDL,VLDL,apoA1,apoB100 and uric acid (UA) were detected at the same time.T test,Spearman's correlations and regression analysis were used for statistical analysis.Results The concentration of plasma ADP was significantly lower in GA than that in NC [(6±7) μg/ml,(8±6) μg/ml,t=-3.71,P＜0.01 ],the expression of ADR1 and ADR2 mRNA in GA (ADR1:0.09±0.08,ADR2:0.0122±0.0164) was significantly increased when compared to the NC (ADR1:0.05±0.03,ADR2:0.0054±0.0024) (t=2.71,2.35,P＜0.05).In the GA patients,the level of ADP was negatively correlated with the lymphocytes count (LY) and UA (r=-0.32,-0.36,P＜0.05),and it was positively correlated with ESR and LDL level (r=0.31,0.39,P＜0.05).The expression of ADR1 mRNAwas negatively correlated with TG (r=-0.43,P＜0.05 ),but positivdy correlated with ESR and CRP level (r=0.45,0.57,P＜0.05).The expression of ADR2 mRNA was negatively correlated with glucose and UA(r=-0.50,-0.59,P＜0.05).Conclusion Altered expression of ADP and its receptors may be involved in thepathogenesis of gouty inflammation.