OBJECTIVETo explore the transcription regulation of 5-aza-2'-deoxycytidine(5-Aza-CdR) on SHP-1 gene and its effects on Daudi cell line growth.

METHODSMTT method and flow cytometry were used to detect the growth and apoptosis of Daudi cells after treated with different dosage of 5-Aza-CdIR. Bisulfite sequencing PCR ( BSP) , T-A cloning and sequence analysis were evaluated for methylation status. The SHP-I mRNA and protein were determined by reverse transcription polymerase chain reaction (RT-PCR) ,immunohistochemistry.

RESULTS(1)After 7 d treatment with 2. 00 micromol/L of 5-Aza-CdR, all cytosines (C) in Daudi cells genome DNA were converted to thymidine, and SHP-1 mRNA and protein expressed again in the cells while those Cs in CpG dinucleotides in untreated Daudi cells remained Cs; (2)5-Aza-CdR inhibited the cell growth,The effects within certain extent were dose and time dependent:after 72 h treatment with 5-Aza-CdR at 200. 00, 20. 00, 2. 00 and 0. 20 micromol/L, the inhibitive rates were 72. 0% , 65. 1%, 51. 5%, 28.8% ,23.4% respectively; (3) 5-Aza-CdR increased apoptosis rate of tumor cells with a dose and times dependent manner within certain extent, too:at the 1,3,5 d treatment with 5-Aza-CdR 2. 00 micromol/L,the apoptosis rates were 2. 3% ,10. 8 % and 17. 1% ; respectively. (4) 5-Aza-CdR also changed cell cycle of tumor cells: at 24 h treatment with 5-Aza-CdR 2.00 micromol/L,92. 7% tumor cells stopped at S phase and G, phase cells were increased gradually with time.

CONCLUSIONDNA promoter hypermethylation is associated with SHP-1 gene silence in Daudi lymphoma cell line. 5-Aza-CdR could effectively cause demethylation and inhibit the growth of tumor cell by reactivating the gene transcription.

Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA Methylation ; Dose-Response Relationship, Drug ; Humans ; Lymphoma ; genetics ; pathology ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction

AIM: To investigate the possible signaling pathway that promotes epithelial-mesenchymal transi-tion(EMT)of the lung cancer A549 cells stimulated with muscarinic receptor 3(M3R)agonist carbachol.METHODS:The lung cancer cells A549 were treated with 400 μmol/L carbachol.The morphological changes of the cells were observed under inverted phase contrast microscope.The migration and invasion abilites were measured by Wound healing and Tran-swell assays.qPCR was used to detect the mRNA level of vimentin and E-cadherin.The protein levels of p-AKT,vimentin and E-cadherin were determined by Western blot.RESULTS:After treatment with carbachol,the A549 cells showed loss of the close connection and the cell morphology was transformed from irregular polygon to spindle -like cells.The results of Wound healing and Transwell assays showed that the migration and invasion abilites of the A 549 cells were enhanced.Car-bachol increased the vimentin expression and decreased the E-cadherin expression at mRNA and protein level(P<0.05). The phosphorylation of AKT in the A549 cells was up-regulated(P<0.05).These changes was inhibited by M3R antago-nist 4-DAMP.CONCLUSION:Carbachol promotes EMT in the human lung cancer A 549 cells by activating PI3K/AKT signaling pathway.

Gallbladder adenoma,a benign tumor of mucosal epithelial origin,is considered to be a pre-cancer to gallbladder cancer(GBC),and its malignant transformation may occur in a relatively short time.The pathological process and molecular mechanism of gallbladder adenoma carcinogenesis are still controversial;ultrasound and CT are widely used to examine gallbladder diseases,and the application of contrast-enhanced ultrasound(CEUS)and high-frame-rate contrast-enhanced ultrasound(H-CEUS)has improved the diagnostic accuracy.In this paper,we summarize the literature on the clinicopathological features,imaging manifestations,gene expression,treatment,and clinical prognosis of gallbladder adenoma carcinogenesis.

Whole body vibration training (WBVT) is a new kind of therapeutic exercise, which can improve musculoskeletal function and motor performance by transferring vibration stimulation to the body to affect neuromuscular activity. In this paper, the clinical efficacy, mechanism and parameter setting of WBVT in the treatment of chronic ankle instability were introduced through a systematic review of relevant literatures, so as to provide theoretical basis for the clinical application of this technique.

Objective: To investigate clinicopathological features of multinodular and vacuolar neurodegenerative tumor (MVNT) of the cerebrum, and to investigate its immunophenotype, molecular characteristics and prognosis. Methods: Four cases were collected at the General Hospital of Southern Theater Command, Guangzhou, China and one case was collected at the First People's Hospital of Huizhou, China from 2013 to 2021. Clinical, histological, immunohistochemical and molecular characteristics of these five cases were analyzed. Follow-up was carried out to evaluate their prognoses. Results: There were four females and one male, with an average age of 42 years (range, 17 to 51 years). Four patients presented with seizures, while one presented with discomfort on the head. Pre-operative imaging demonstrated non-enhancing, T2-hyperintense multinodular lesions in the deep cortex and superficial white matter of the frontal (n=1) or temporal lobes (n=4). Microscopically, the tumor cells were mostly arranged in discrete and coalescent nodules primarily within the deep cortical ribbon and superficial subcortical white matter. The tumors were composed of large cells with ganglionic morphology, vesicular nuclei, prominent nucleoli and amphophilic or lightly basophilic cytoplasm. They exhibited varying degrees of matrix vacuolization. Vacuolated tumor cells did not show overt cellular atypia or any mitotic activities. Immunohistochemically, tumor cells exhibited widespread nuclear staining for the HuC/HuD neuronal antigens, SOX10 and Olig2. Expression of other neuronal markers, including synaptophysin, neurofilament and MAP2, was patchy to absent. The tumor cells were negative for NeuN, GFAP, p53, H3K27M, IDH1 R132H, ATRX, BRG1, INI1 and BRAF V600E. No aberrant molecular changes were identified in case 3 and case 5 using next-generation sequencing (including 131 genes related to diagnosis and prognosis of central nervous system tumors). All patients underwent complete or substantial tumor excision without adjuvant chemoradiotherapy. Post-operative follow-up information over intervals of 6 months to 8 years was available for five patients. All patients were free of recurrence. Conclusions: MVNT is an indolent tumor, mostly affecting adults, which supports classifying MVNT as WHO grade 1. There is no tumor recurrence even in the patients treated with subtotal surgical excision. MVNTs may be considered for observation or non-surgical treatments if they are asymptomatic.
Adult ; Female ; Humans ; Male ; Brain Neoplasms/pathology* ; Cerebrum/pathology* ; Neurons/metabolism* ; Seizures ; Temporal Lobe/pathology* ; Biomarkers, Tumor/metabolism*

The clinical comprehensive evaluation of drugs is an important basis for the return of clinical value, decision-making of medical and health authorities, and allocation of medical resources. In July 2021, the National Health Commission issued the Guidelines for the Management of Clinical Comprehensive Evaluation of Drugs(trial version 2021), which required the evaluation to be implemented from the six dimensions(safety, effectiveness, economy, innovation, suitability, and accessibility), and made detailed arrangements for the clinical comprehensive evaluation of drugs. As Chinese patent medicine differs from chemical medicines in terms of effective components and action modes, the clinical comprehensive evaluation of Chinese patent medicine should highlight the characteristics and advantages of traditional Chinese medicine(TCM) on the basis of general requirements of comprehensive clinical evaluation of drugs. At present, in the clinical comprehensive evaluation of Chinese patent medicine, unified report standards have not yet been generated, resulting in the uneven quality of existing reports. To standardize the clinical comprehensive evaluation report of Chinese patent medicine and improve its quality, the editorial team, based on the relevant policy documents of clinical comprehensive evaluation of drugs, formulated the clinical comprehensive evaluation report standards for Chinese patent medicine in combination with the previous practice and expert opinions. The report standards, containing seven sections with 15 items determined, focus on data source, evaluation content, evidence synthesis, quality control, and evaluation results supported with detailed interpretations to help researchers better understand and apply the report standards for clinical comprehensive evaluation of Chinese patent medicine, improve the report quality, and provide references for the decision-making by the national medical management authorities.
China ; Drugs, Chinese Herbal ; Information Storage and Retrieval ; Medicine, Chinese Traditional ; Nonprescription Drugs ; Quality Control

OBJECTIVE: To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD). METHODS: Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild-type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E-cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins. RESULTS: In the coculture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P < 0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS-treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa. CONCLUSION Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.
Humans ; Mice ; Animals ; Caco-2 Cells ; beta Catenin/metabolism* ; Culture Media, Conditioned/pharmacology* ; Tight Junctions/metabolism* ; Intestinal Mucosa ; Inflammatory Bowel Diseases ; Tight Junction Proteins/metabolism* ; Inflammation/metabolism* ; Fibroblasts/metabolism* ; Mice, Inbred C57BL ; Glycoproteins/metabolism* ; Wnt Proteins/pharmacology* ; Frizzled Receptors/metabolism*

BACKGROUND: Myocardial ischemia-reperfusion (I/R) is a serious and irreversible injury. Bone marrow-derived mesenchymal stem cells (MSCs) is considered to be a potential therapy for I/R injury due to the paracrine effects. High-mobility group box 1 (HMGB1) is a novel mediator in MSC and regulates the response of inflammation injury. Signal Transduction and Transcription Activator 3 (STAT3) is a critical transcription factor and important for release of paracrine factors. However, the relationship between HMGB1 and STAT3 in paracrine effect of MSC remains unknown. METHODS: In vitro, hypoxia/reoxygenation injury model was established by AnaeroPack System and examined by Annexin V flow cytometry, CCK8 assay and morphology observation. Detection of apoptotic proteins and protein expression of HMGB1 and STAT3 by Western blot. RESULTS: The conditioned medium of MSCs with or without LPS pretreatment was cocultured with H9C2 cells for 24 h before hypoxia treatment and MSC showed obvious cardiomyocytes protect role, as evidence by decreased apoptosis rate and improved cells viability, and LPS pretreated MSC exhibited better protect role than untreated MSC. However, such effect was abolished in HMGB1 deficiency group, silencing HMGB1 decreased the secretion of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin growth factor (IGF), cell viability, and the expression of STAT3. Furthermore, STAT3 silence attenuated the protective effect of LPS in MSC. CONCLUSIONS These findings suggested that LPS improved MSC-mediated cardiomyocytes protection by HMGB1/STAT3 signaling.

Objective: To explore the mechanism of intestinal tissue damage induced by macrophages activated by WNT2B high-expressed fibroblasts. Methods: This study involved biological information analysis, pathological tissue research and cell experimental research. The biological information of the colon tissue from the children with inflammatory bowel disease in previous study was analyzed again with single-cell sequencing. The pathological tissues were collected by colonoscopy from 10 children with Crohn's disease treated in the Department of Gastroenterology of Guangzhou Women and Children's Medical Center from July 2022 to September 2022. According to the findings of colonoscopy, tissues with obvious inflammation or ulceration were classified as the inflammatory group, while tissues with slight inflammation and no ulceration were classified as the non-inflammatory group. HE staining was performed to observe the pathological changes of the colon tissues. Macrophage infiltration and CXCL12 expression were detected by immunofluorescence. In terms of cell experiments, fibroblasts transfected with WNT2B plasmid or empty plasmid were co-cultured with salinomycin treated or non-treated macrophages, respectively; the expression of proteins through Wnt classical pathway were detected by western blotting. Macrophages treated with SKL2001 were used as the experimental group, and those with phosphate buffer as the control group. The expression and secretion of CXCL12 in macrophages were detected by quantitative Real-time PCR and enzyme-linked immunosorbent assay (ELISA). T-test or rank sum test were used for the comparison between groups. Results: Single-cell sequencing analysis suggested that macrophages were the main cells in inflammatory bowel disease colon tissue, and there was interaction between WNT2B high-expressed fibroblasts and macrophages. HE staining of the 10 patients ((9.3±3.8) years old, 7 males and 3 females) showed that the pathological score of colon tissue in the inflammatory group was higher than that in the non-inflammatory group (4 (3, 4) vs. 2 (1, 2) points, Z=3.05, P=0.002). Tissue immunofluorescence indicated that the number of infiltrating macrophages in the inflammatory group was significantly higher than that in the non-inflammatory group under high power field of view (72.8±10.4 vs.8.4±3.5, t=25.10, P<0.001), as well as the number of cells expressing CXCL12 (14.0±3.5 vs. 4.7±1.9, t=14.68, P<0.001). In cell experiments, western blotting suggested an elevated level of glycogen synthase kinase-3β phosphorylation in macrophages co-cultured with fibroblast transfected with WNT2B plasmid, and salinmycin could reverse this change. Real-time PCR suggested that the transcription level of CXCL12 in the experimental group was higher than that in the control group (6.42±0.04 vs. 1.00±0.03, t=183.00, P<0.001), as well as the expression and secretion of CXCL12 by ELISA ((465±34) vs. (77±9) ng/L, t=13.21, P=0.006). Conclusion: WNT2B high-expressed fibroblasts can secrete WNT2B protein and activate the Wnt classical signaling pathway thus enhancing the expression and secretion of CXCL12 in macrophages, inducing the development of intestinal inflammation of Crohn's disease.
Child ; Male ; Humans ; Female ; Child, Preschool ; Adolescent ; Crohn Disease ; Inflammatory Bowel Diseases ; Colon ; Inflammation ; Colonoscopy ; Glycoproteins ; Wnt Proteins