Fibroblasts overpressing WNT2b cause impairment of intestinal mucosal barrier / 南方医科大学学报

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.

Mechanism of intestinal injury induced by WNT2B high-expressed fibroblasts in Crohn's disease / 中华儿科杂志

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.

Genomewide DNA Methylation Responses in Patients with β-Thalassemia Treated with Yisui Shengxue Granules () / 中国结合医学杂志

OBJECTIVE@#To examine the clinical effects of Yisui Shengxue Granules () in the treatment of β-thalassemia and explore its mechanism on DNA methylation levels.@*METHODS@#A randomized placebo-controlled double-blinded trial was conducted. Forty patients with β-thalassemia were recruited and distributed randomly by envelope method into an experimental group and a control group, 20 patients in each group. The patients were given Yisui Shengxue Granules in the experimental group and placebo in the control group (12 g/bag three times a day) during a 3-month intervention. Before and after 1, 2, and 3 months of treatment, peripheral intravenous blood was sampled, and blood parameters such as hemoglobin (Hb), red blood cells (RBCs), reticulocytes (Ret), and fetal hemoglobin (HbF) were analyzed. Mononuclear cells from 5 patients, who showed an obvious treatment effect, were isolated by density gradient centrifugation. DNA methylation was analyzed using an Affymetrix USA GeneChip Human Promoter 1.0 Array and Input-promoter 1.0.@*RESULTS@#Compared with pre-treatment, there was an obvious increase in Hb and RBCs counts after 1, 2, and 3 months in the experiment group (P<0.01 or P<0.05). Meanwhile, HbF increased from the 2nd to the 3rd month (P<0.05). In the control group, Hb and RBCs showed no obvioas change. After 3-month treatment, DNA methylation results from 5 patients revealed that there were 24 hypomethylated genes and 3,685 hypermethylated genes compared with pre-treatment. Genes of insulin-like growth factor 1 receptor (IGF1R) and Janus kinase 3 (JAK3) revealed the most relations with other genes (degree: 21) and genes of 1-phosphatidylinositol-4, 5-bisphosphate phosphodiesterase gamma 2 (PLCG2) and mitogen-activated protein kinase 10 (MAPK10) showed a stronger intermediary role (betweenness centrality=0.04).@*CONCLUSIONS@#JAK3 and MAPK10 are two key genes in bone marrow and the lymphatic system, and JAK3 is likely to be related to hematopoietic cytokines in the process of early hematopoiesis. (Registration No. NCT01549080).

Response characteristics of neurons to tone in dorsal nucleus of the lateral lemniscus of the mouse / 生理学报

The dorsal nucleus of lateral lemniscus (DNLL) is a nucleus in the auditory ascending pathway, and casts inhibitory efferent projections to the inferior colliculus. Studies on the DNLL are less than studies on the auditory brain stem and inferior colliculus. To date, there is no information about response characteristics of neurons in DNLL of albino mouse. Under free field conditions, we used extracellular single unit recording to study the acoustic signal characteristics of DNLL neurons in Kunming mice (Mus musculus). Transient (36%) and ongoing (64%) firing patterns were found in 96 DNLL neurons. Neurons with different firing patterns have significant differences in characteristic frequency and minimal threshold. We recorded frequency tuning curves (FTCs) of 87 DNLL neurons. All of the FTCs exhibit an open "V" shape. There is no significant difference in FTCs between transient and ongoing neurons, but among the ongoing neurons, the FTCs of sustained neurons are sharper than those of onset plus sustained neurons and pauser neurons. Our results showed that the characteristic frequency of DNLL neurons of mice was not correlated with depth, supporting the view that the DNLL of mouse has no frequency topological organization through dorsal-ventral plane, which is different from cats and some other animals. Furthermore, by using rate-intensity function (RIF) analysis the mouse DNLL neurons can be classified as monotonic (60%), saturated (31%) and non-monotonic (8%) types. Each RIF type includes transient and ongoing firing patterns. Dynamic range of the transient firing pattern is smaller than that of ongoing firing ones (P < 0.01), suggesting that the inhibitory inputs may underlie the formation of transient firing pattern. Multiple firing patterns and intensity coding of DNLL neurons may derive from the projections from multiple auditory nuclei, and play different roles in auditory information processing.