OBJECTIVES: Metastasis is the primary cause of death in osteosarcoma, and current clinical treatments remain limited. BRD4, a key epigenetic regulator, has shown therapeutic promise in various cancers through its inhibition. However, the mechanistic role of BRD4 in osteosarcoma remains poorly understood. This study aims to elucidate the molecular mechanisms by which BRD4 regulate osteosarcoma progression and to explore novel therapeutic strategies. METHODS: Immunofluorescence was used to assess BRD4 expression levels in a tissue microarray containing 80 osteosarcoma samples from different patients. The Gene Expression Omnibus (GEO) dataset (GSE42352, containing survival data from 88 osteosarcoma patients) was downloaded to perform Kaplan-Meier survival analysis based on BRD4 gene expression levels. In vivo, an orthotopic intramedullary osteosarcoma model was established using HOS cells in C57 mice, followed by treatment with varying doses of the BRD4 inhibitor (+)-JQ1. Micro-CT, 3D reconstruction of bone tissue, and HE staining were employed to evaluate pathological changes in bone and intestinal lymph nodes. In vitro, cell viability was measured using the methyl thiazolyl tetrazolium (MTT) assay, while colony formation and Transwell assays assessed proliferative and invasive capacities. Chromatin-bound BRD4 was analyzed via co-immunoprecipitation combined with mass spectrometry (Co-IP/MS), and O-GlcNAc glycosylation sites and glycan chains of BRD4 were identified using Co-IP with Nano-LC MS/MS. Real-time PCR and Western blotting were used to analyze the relative mRNA and protein expression levels of target genes, respectively. RESULTS: BRD4 was positively expressed in 61.25% (49/80) of osteosarcoma tissues. Patients with high BRD4 expression exhibited significantly shorter survival times (P<0.05). In the orthotopic mouse model, intervention with (+)-JQ1, a potent and commonly used BETi, significantly inhibited tumor growth in vivo and reduced bone destruction (P<0.05). (+)-JQ1 treatment significantly suppressed the proliferation (P<0.001), invasion (P<0.001), and migration (P<0.05) of HOS cells. In osteosarcoma cells, BRD4 exhibited O-GlcNAc modifications at both N- and C- C-termini, particularly at Thr73, which is essential for protein stability. This modification also contributed to the activation of the EGFR tyrosine kinase inhibitor resistance pathway (KEGG Pathway: hsa01521). (+)-JQ1 treatment displaced BRD4 from enhancers and downregulated the transcription of pathway-related genes, such as EGFR and PDGFC, thereby suppressing the malignant behavior of osteosarcoma cells. CONCLUSIONS BRD4 promotes osteosarcoma progression via O-GlcNAc modification at Thr73 and plays a crucial role in tumor growth and metastasis.
Osteosarcoma/drug therapy* ; Humans ; Transcription Factors/metabolism* ; Animals ; Cell Cycle Proteins ; Mice ; Bone Neoplasms/drug therapy* ; Azepines/pharmacology* ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Triazoles/pharmacology* ; Mice, Inbred C57BL ; Nuclear Proteins/metabolism* ; Gene Expression Regulation, Neoplastic ; Male ; Bromodomain Containing Proteins

OBJECTIVE To optimize the water extraction process of Flos Lonicerae-Fructus Forsythuae and determine the range of water extraction process parameters.METHODS The active components were screened by network pharmacology,and the indica-tor ingredients were determined in combination with the quality markers under the relevant terms of Chinese Pharmacopoeia 2020 edition and the literature.Take extraction yield and the extraction rate of the indicative component as the critical quality attributes of the water extraction process to screen critical process parameters.The mathematical model was established by Box-Behnken experimental design to investigate the interaction between CQAs and CPPs and build the design space of the water extraction process of Flos Lonicerae-Fruc-tus Forsythuae.RESULTS The extraction percentages of phenolic acids,forsythoside A and forsythin were screened as the index components;specifications of medicinal slices,extraction time and water addition were the key process parameters.Based on the estab-lishment and optimization of the design space,the optimum water extraction process was obtained as follows:the medicinal slice of Lian-Qiao was broken into 0.8-1.2 cm,adding 12 times the amount of water in the first and extract for 30-50 min,10 times the a-mount of water in the second and extract for 25-30 min.CONCLUSION The verification results show that the measured value ob-tained by using the design space method to optimize the water extraction process is close to the predicted value,indicating that the method is stable and reliable,which can provide ideas for its further process development and quality control for the couple medicines of Flos Lonicerae-Fructus Forsythuae.

Objective:To investigate the incidence of postoperative complications in Chinese patients with gastric or colorectal cancer, and to evaluate the risk factors for postoperative complications.Methods:This was a national, multicenter, prospective, registry-based, cohort study of data obtained from the database of the Prevalence of Abdominal Complications After Gastro- enterological Surgery (PACAGE) study sponsored by the China Gastrointestinal Cancer Surgical Union. The PACAGE database prospectively collected general demographic characteristics, protocols for perioperative treatment, and variables associated with postoperative complications in patients treated for gastric or colorectal cancer in 20 medical centers from December 2018 to December 2020. The patients were grouped according to the presence or absence of postoperative complications. Postoperative complications were categorized and graded in accordance with the expert consensus on postoperative complications in gastrointestinal oncology surgery and Clavien-Dindo grading criteria. The incidence of postoperative complications of different grades are presented as bar charts. Independent risk factors for occurrence of postoperative complications were identified by multifactorial unconditional logistic regression.Results:The study cohort comprised 3926 patients with gastric or colorectal cancer, 657 (16.7%) of whom had a total of 876 postoperative complications. Serious complications (Grade III and above) occurred in 4.0% of patients (156/3926). The rate of Grade V complications was 0.2% (7/3926). The cohort included 2271 patients with gastric cancer with a postoperative complication rate of 18.1% (412/2271) and serious complication rate of 4.7% (106/2271); and 1655 with colorectal cancer, with a postoperative complication rate of 14.8% (245/1655) and serious complication rate of 3.0% (50/1655). The incidences of anastomotic leakage in patients with gastric and colorectal cancer were 3.3% (74/2271) and 3.4% (56/1655), respectively. Abdominal infection was the most frequently occurring complication, accounting for 28.7% (164/572) and 39.5% (120/304) of postoperative complications in patients with gastric and colorectal cancer, respectively. The most frequently occurring grade of postoperative complication was Grade II, accounting for 65.4% (374/572) and 56.6% (172/304) of complications in patients with gastric and colorectal cancers, respectively. Multifactorial analysis identified (1) the following independent risk factors for postoperative complications in patients in the gastric cancer group: preoperative comorbidities (OR=2.54, 95%CI: 1.51-4.28, P<0.001), neoadjuvant therapy (OR=1.42, 95%CI:1.06-1.89, P=0.020), high American Society of Anesthesiologists (ASA) scores (ASA score 2 points:OR=1.60, 95% CI: 1.23-2.07, P<0.001, ASA score ≥3 points:OR=0.43, 95% CI: 0.25-0.73, P=0.002), operative time >180 minutes (OR=1.81, 95% CI: 1.42-2.31, P<0.001), intraoperative bleeding >50 mL (OR=1.29,95%CI: 1.01-1.63, P=0.038), and distal gastrectomy compared with total gastrectomy (OR=0.65,95%CI: 0.51-0.83, P<0.001); and (2) the following independent risk factors for postoperative complications in patients in the colorectal cancer group: female (OR=0.60, 95%CI: 0.44-0.80, P<0.001), preoperative comorbidities (OR=2.73, 95%CI: 1.25-5.99, P=0.030), neoadjuvant therapy (OR=1.83, 95%CI:1.23-2.72, P=0.008), laparoscopic surgery (OR=0.47, 95%CI: 0.30-0.72, P=0.022), and abdominoperineal resection compared with low anterior resection (OR=2.74, 95%CI: 1.71-4.41, P<0.001). Conclusion:Postoperative complications associated with various types of infection were the most frequent complications in patients with gastric or colorectal cancer. Although the risk factors for postoperative complications differed between patients with gastric cancer and those with colorectal cancer, the presence of preoperative comorbidities, administration of neoadjuvant therapy, and extent of surgical resection, were the commonest factors associated with postoperative complications in patients of both categories.

The incidence of spinal infections,a relatively rare infectious disease,is on the rise due to the empirical use of antibiotics that increases the chances of infection with drug-resistant bacteria,as well as advances in testing technology that have led to an increase in detection rates.Identifying the type of pathogen to target antibi-otics is the key to treatment.However,conventionaldetection methods have low detection rates and are time-consum-ing,which are not conducive to the rapid and accurate diagnosis of spinal infection.Metagenomics next-generation sequencing(mNGS)is a detection technique that can sequence all nucleic acid fragments in samples,the emer-gence of which subverts traditional detection methods and plays an important role in the diagnosis and treatment of spinal infections.This article summarizes the application of mNGS in the diagnosis and treatment of spinal infection.

Objective To investigate the expression of miR-204 and silence information regulator 1(SIRT1)in colorectal cancer and their clinical value.Methods Cancer tissue specimens and paracancer tissue specimens of 60 patients with colorectal cancer treated in Department of Gastrointestinal Surgery of Affiliated Hospital of Jiaxing University from May 2018 to June 2020 were collected as study objects.Real time fluorogenic quantitative polymerase chain reaction was used to detect the gene expression of miR-204 and SIRT1,and the correlation between miR-204 and SIRT1 gene expression was compared by Pearson analysis.Immunohistochemical SABC method was used to detect SIRT1 protein expression,and relationship between different SIRT1 protein expression and clinicopathological features was compared.Kaplan-Meier method was used to analyze the survival difference of colorectal cancer patients with different SIRT1 protein expression.Results The mRNA expression of miR-204 gene in cancer tissues was significantly lower than that in paracancer tissues(P<0.05),and the mRNA expression of SIRT1 gene was significantly higher than that in paracancer tissues(P<0.05).Pearson correlation analysis showed that miR-204 was negatively correlated with SIRT1 mRNA expression in both paracancer tissues and cancer tissues(r=-0.647,-0.737,P<0.05).The expression of SIRT1 protein was correlated with the differentiation level,invasion level,lymph node metastasis and TNM stage of colorectal cancer(P<0.05),but not with patient age,gender,tumor size and tumor site(P>0.05).Kaplan-Meier analysis showed that the survival rate of patients with positive expression of SIRT1 in cancer tissues was significantly lower than that of patients with negative expression of SIRT1(χ2=5.001,P=0.025).Conclusion The expression of miR-204 is down-regulated and SIRT1 expression is up-regulated in colorectal cancer tissues,which may jointly promote the metastasis and invasion of colorectal cancer and affect the prognosis of patients through mutual influence.

Objective:To explore mechanism of Chaihu Longgu Muli Decoction and Ganmai Dazao Decoction in treatment of post-stroke depression(PSD)based on network pharmacology,molecular docking and animal experiment.Methods:TCMSP and other databases were used to predict active components and targets of Chaihu Longgu Muli Decoction and Ganmai Dazao Decoction.Targets of PSD were retrieved from PharmGKB and other databases,and"component-intersection target-disease"network was constructed by Cytoscape(v3.9.1)software.PPI network was constructed by String(v11.5)database,and GO enrichment and KEGG pathway analy-sis of intersection targets were performed by DAVID6.8 database.AutoDock vina(v1.1.2)software was used for molecular docking.Pymol(v 2.5)and other softwares were used to visualize optimal docking results.Animal experiments were setup in control group,model group,fluoxetine group,TCM group and TCM+fluoxetine group,neurobehavioral scores and expressions of neurotransmitters and inflammatory factors in brain tissues were detected.mRNA and protein expressions of key genes PPARG,MAPK3,AKT1,PIK3CA were detected by RT-qPCR and Western blot.Results:A total of 225 kinds of active ingredients of Chaihu Longgu Muli Decoction and Ganmai Dazao Decoction were obtained,which acted on 119 targets of PSD,among which key targets included MAPK3,AKT1,PIK3CA and PPARG,key pathways including MAPK signaling pathway,PI3K-Akt signaling pathway and etc.Compared with model group,MAPK3 mRNA and protein expressions were decreased,AKT1,PIK3CA,PPARG mRNA and protein expressions were increased in TCM group and TCM+fluoxetine group(P<0.05).Conclusion:Mechanism of Chaihu Longgu Muli Decoction and Ganmai Dazao Decoction in treatment of PSD may be related to inhibition of MAPK3 expression,promotion of AKT1,PIK3CA,PPARG expressions,alleviation of inflammatory response and oxidative stress in brain tissues.

Objective To explore the effect of fluid shear stress on the expression of glucose regulatory protein 78(GRP78)and C/EBP homologous protein(CHOP)in human umbilical vein endothelial cells(HUVECs).Methods HUVECs were used as experimental cells,and a fluid dynamics simulation experimental system was designed and constructed.Different fluid shear stresses were applied to the experimental cells by controlling the flow rate of the perfusion fluid in the experimental system.According to the fluid shear stress experienced by the experimental cells in the experimental system,they were divided into low shear stress group(group A;0.4Pa),medium shear stress group(group B;0.8 Pa)and high shear stress group(group C;1.2 Pa).Each group of HUVECs consisted of 3 cell slides,and each slide was repeatedly circulated through the perfusion solution of the experimental system for 12 h.Western blotting was used to detect the levels of GRP78 and CHOP proteins,and real-time quantitative reverse transcription polymerase chain reaction was used to determine the levels of GRP78 and CHOP and their messenger RNA(mRNA)in each group.GraphPad Prism 8.0 software was used to statistically analyze the data.Results(1)The relative expression levels of GRP78 protein in groups A,B and C were 1.33±0.46,0.93±0.34,0.64±0.30,respectively,the difference among groups was statistically significant(F=36.17,P＜0.05).The relative expression level of GRP78 protein in group A was higher than that in group B and group C(both P＜0.01),and the relative expression level of GRP78 protein in group B was higher than that in group C(P=0.0013).The relative expression levels of CHOP protein in the three groups were 1.29±0.38 in group A,0.90±0.34 in group B,and 0.59±0.29 in group C,the difference among groups was statistically significant(F=41.27,P＜0.05).The relative expression level of CHOP protein in group A was higher than that in group B and group C(both P＜0.01),and the relative expression level of CHOP protein in group B was higher than that in group C(P=0.0 004).(2)The relative expression levels of GRP78 mRNA in groups A,B,and C were 18.3±3.4,11.3±1.8,5.4±2.2,respectively,the difference among groups was statistically significant(F=189.20,P＜0.05).The relative expression level of GRP78 mRNA in group A was higher than that in group B and group C(both P＜0.01),and the relative expression level of GRP78 mRNA in group B was higher than that in group C(P＜0.01).The relative expression levels of CHOP mRNA in the three groups were 20.4±3.8 in group A,14.2±2.1 in group B,and 7.8±1.3 in group C,the difference among groups was statistically significant(F=171.80,P＜0.05).The relative expression level of CHOP mRNA in group A was higher than that in group B and group C(both P＜0.01),and the relative expression level of CHOP mRNA in group B was higher than that in group C(P＜0.01).Conclusion Low fluid shear stress may increase the protein and mRNA expression levels of GRP78 and CHOP in HUVECs.

Objective To establish interferon-induced transmembrane protein 3(Ifitm3)knockout mice and to explore the effects of Ifitm3 on the proliferation and differentiation of adult neural stem cells of mice(aNSCs).Methods IFITM3 knockout mice were established by the CRISPR/Cas9 method and identified by genotype identification and Western Blot.The differences between Ifitm3-knockout mice and wild-type mice were analyzed by hematoxylin-eosin(HE)staining and flow cytometry.The aNSCs of wild-type mice and Ifitm3-knockout mice were isolated and cultured,the number and size of neurospheres were detected,The ability of aNSCs to proliferate and differentiate were detected by quantitative reverse-transcription polymerase chain reaction,Western Blot,and immunofluorescence.Results Ifitm3-knockout mice were successfully established.The mice developed normally,and there were no obvious abnormalities either histopathologically or the immune system.In vitro experiments showed that Ifitm3 knockout inhibited the self-renewal potential of aNSCs,led to a decrease in the proliferation ability of aNSCs,and inhibited the differentiation of aNSCs into immature neurons and astrocytes.Conclusions This study finds that a lack of IFITM3 result in the ability of aNSCs to proliferate and differentiate decreased,IFITM3 may regulate the function of aNSCs.

Objective:To investigate the protective effect and possible mechanism of sulforaphane (SFN) on acute liver injury in mice induced by diquat (DQ) poisoning.Methods:Forty-eight male C57BL/6 mice were divided into Control group, DQ model group (DQ group), SFN intervention group (DQ+SFN group), and SFN control group (SFN group) using a random number table method, with 12 mice in each group. Acute liver injury mice model was established by one-time intraperitoneal injection of 1 mL of 40 mg/kg DQ solution at once. SFN group was injected with 1 mL of ddH 2O. After 4 hours of molding, 0.5 mL of 5 mg/kg SFN solution was injected into the peritoneal cavity of the DQ+SFN group and SFN group, once daily for 7 consecutive days. DQ group and Control group were injected with an equal amount of ddH 2O. Then, the mice were euthanized to collect liver tissue and blood samples, and the levels of plasma biomarkers alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as oxidative stress indicators such as superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) in liver tissue were measured. The changes of liver structure were observed under transmission electron microscopy. The apoptosis and reactive oxygen species (ROS) level in liver tissue were observed under fluorescence microscope. Western blotting was used to detect the protein expressions of nuclear factor E2-related factor 2 (Nrf2), hemeoxygenase-1 (HO-1), Kelch-like ECH-associated protein 1 (Keap1), and cleaved caspase-9 in liver tissue. Results:Compared with the Control group, the liver mitochondria in the DQ group showed severe swelling, partial dissolution of the matrix, and cristae rupture and loss; the levels of plasma AST and ALT significantly increased, the MDA content in the liver increased, the activities of SOD and GSH decreased, the level of ROS significantly increased, the number of apoptotic cells in the liver significantly increased, the protein expressions of Nrf2 and HO-1 significantly decreased, and the protein expressions of Keap1 and cleaved caspase-9 significantly increased. Compared with the DQ group, the mitochondrial damage in the DQ+SFN group was reduced, the levels of plasma AST and ALT were significantly reduced [ALT (U/L): 58.22±4.39 vs. 79.94±3.32, AST (U/L): 177.64±8.40 vs. 219.62±11.60, both P < 0.01], the liver MDA content decreased, and the activities of SOD and GSH increased [MDA (μmol/g: 5.63±0.18 vs. 5.96±0.29, SOD (kU/g): 102.05±4.01 vs. 84.34±5.34, GSH (mmol/g): 16.32±1.40 vs. 13.12±1.84, all P < 0.05], the production of ROS in liver tissue was significantly reduced [ROS (fluorescence intensity): 115.90±10.89 vs. 190.70±10.16, P < 0.05], and apoptotic cells were significantly reduced (cell apoptosis index: 4.39±1.00 vs. 10.71±0.56, P < 0.01), the protein expressions of Nrf2 and HO-1 were significantly increased, while the protein expressions of Keap1 and cleaved caspase-9 were significantly decreased (Nrf2/β-actin: 1.15±0.04 vs. 0.93±0.05, HO-1/β-actin: 1.75±0.12 vs. 0.78±0.04, Keap1/β-actin: 1.00±0.14 vs. 1.28±0.13, cleaved caspase-9/β-actin: 1.31±0.12 vs. 1.81±0.09, all P < 0.05). However, there was no statistically significant difference in various indicators between the SFN group and the Control group. Conclusion:SFN can activate the Keap1/Nrf2 signaling pathway to alleviate DQ induced acute liver injury in mice.

Objective To explore the patterns of the immunocyte infiltration in ovarian cancer,and their influence on clinical out-come and chemotherapy response.Methods The clinical information and gene expression profiles of 2206 ovarian cancer patients were downloaded from TCGA and GEO publicly databases.The CIBERSORT was used to evaluate the distribution of 22 immune cells and their effects on survival,and chemotherapy response.Unsupervised clustering was used to classify cases and analyze differences in clinical out-comes between subgroups.Immunofluorescence staining was performed to illustrate the macrophages infiltration in ovarian cancer samples collected from the First Affiliated Hospital,Sun Yat-sen University.Results Naive B cells,M2 macrophages,and resting memory CD4+T cells were significantly associated with poor prognosis,while follicular helper T cells were significantly associated with good prog-nosis.Plasma cells,M1 macrophages,and activated memory CD4+T cells were associated with chemotherapy sensitivity,and M2 macro-phages were associated with chemotherapy resistant.Furthermore,three subgroups were identified by unsupervised clustering which showed different distribution ratios of macrophages and T cells,and significant differences in overall survival among subgroups.The infil-tration of macrophages in metastasis tissue of ovarian cancer was more than that in primary tumor.Conclusion The immunocyte infiltra-tion in ovarian cancer is associated with clinical prognosis and chemotherapy resistance.Immune cell targeting therapy or combination chemotherapy may be the potential treatment choices for ovarian cancer.