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.

Effects of Foxp3 gene silencing on the expression of inflammatory cytokines and the proliferation and migration of human periodontal ligament fibroblasts in an inflammatory environment / 华西口腔医学杂志

OBJECTIVES@#This study aimed to clarify the effects of Foxp3 silencing on the expression of inflammatory cytokines in human periodontal ligament cells (hPDLFs) in an inflammatory environment and on cell proliferation and invasiveness, as well as to explore the role of Foxp3 gene in the development of periodontitis.@*METHODS@#An small interfering RNA (siRNA) construct specific for Foxp3 was transfected into hPDLFs. Foxp3 silencing efficiency was verified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, and the siRNA with the optimum silencing effect of Foxp3 gene was screened. Using lipopolysaccharide to simulate an inflammatory environment in vitro, CCK-8 detected the effect of silencing Foxp3 on hPDLFs proliferation under inflammatory conditions. Wound-healing experiments and transwell assays were conducted to detect the effect of silencing Foxp3 on hPDLF migration under inflammatory conditions. The expression of the inflammatory cytokines interleukin (IL)-6 and IL-8 was detected by RT-PCR and Western blotting under inflammatory conditions.@*RESULTS@#After siRNA transfection, RT-PCR and Western blotting analyses showed that the expression of Foxp3 mRNA in the Foxp3-si3 group decreased significantly (t=21.03, P<0.000 1), and the protein expression of Foxp3 also decreased significantly (t=12.8, P<0.001). In the inflammatory environment, Foxp3 gene silencing had no significant effect on hPDLFs proliferation (P>0.05), and Foxp3 gene silencing promoted hPDLFs migration (P<0.05). Moreover, the expression of IL-6 and IL-8 increased (P<0.05).@*CONCLUSIONS@#In an inflammatory environment, Foxp3 gene silencing promoted hPDLFs migration but had no significant effect on hPDLFs proliferation. The expression of inflammatory factors expressed in hPDLFs increased after Foxp3 gene silencing, indicating that Foxp3 gene inhibited inflammation in periodontitis.

Effect of tumor-stromal fibroblasts on the biological behavior of salivary gland pleomorphic adenoma cells in vitro / 华西口腔医学杂志

OBJECTIVES@#This study aims to investigate the effects of tumor-stromal fibroblasts (TSFs) on the proliferation, invasion, and migration of salivary gland pleomorphic adenoma (SPA) cells in vitro.@*METHODS@#Salivary gland pleomorphic adenoma cells (SPACs), TSFs, and peri-tumorous normal fibroblasts (NFs) were obtained by tissue primary culture and identified by immunocytochemical staining. The conditioned medium was obtained from TSF and NF in logarithmic phase. SPACs were cultured by conditioned medium and treated by TSF (group TSF-SPAC) and NF (group NF-SPAC). SPACs were used as the control group. The proliferation, invasion, and migration of the three groups of cells were detected by MTT, transwell, and scratch assays, respectively. The expression of vascular endothelial growth factor (VEGF) in the three groups was tested by enzyme linked immunosorbent assay (ELISA).@*RESULTS@#Immunocytochemical staining showed positive vimentin expression in NF and TSF. Results also indicated the weak positive expression of α-smooth muscle actin (SMA) and fibroblast activation protein (FAP) in TSFs and the negative expression of α-SMA and FAP in NFs. MTT assay showed that cell proliferation in the TSF-SPAC group was significantly different from that in the NF-SPAC and SPAC groups (P<0.05). Cell proliferation was not different between the NF-SPAC and SPAC groups (P>0.05). Transwell and scratch assays showed no difference in cell invasion and migration among the groups (P>0.05). ELISA showed that no significant difference in VEGF expression among the three groups (P>0.05).@*CONCLUSIONS@#TSFs may be involved in SPA biological behavior by promoting the proliferation of SPACs but has no effect on the invasion and migration of SPACs in vitro. Hence, TSF may be a new therapeutic target in SPA treatment.

Desmoid-type fibromatosis of the mesentery: a clinicopatho-logical and genetic analysis of 9 cases / 浙江大学学报·医学版

Nine cases of mesenteric desmoid-type fibromatosis were diagnosed and treated in Taizhou Hospital, Wenzhou Medical University between January 2010 and May 2022, including 2 females and 7 males, aged 16 to 59 years. The lesions were in the mesentery of small intestine with 7 cases, ileocecal junction with 1 cases and transverse colon with 1 case. The tumors had an unclear boundary and no envelope, the section was solid, gray and tough. The mean maximum diameter was (10.7±8.5) cm (range 3.5-33.0 cm). Microscopically, fusiform fibroblasts and myofibroblasts were parallel, bunched or staggered, buried in a large amount of extracellular collagen. The cell morphology was relatively consistent, without obvious atypia, and mitosis was rare. Immunohistochemistry showed that the tumor cells were positive for vimentin (9/9), β-catenin (9/9), while smooth muscle actin (5/9) stains were focally positive. Ki-67 proliferation index was 1%-10%. Cytokeratin Pan, S-100, STAT6, CD117, DOG1, CD34, desmin and anaplastic lymphoma kinase stains were negative. Genetic analysis showed that there were 7 cases of c.121G>A(p.Thr41Ala) mutation of CTNNB1 gene, 1 case of c.121G>A(p.Thr41Ala) and 1 case of c.134C>T(p.Ser45Phe) double mutation, and 1 case of wild type. Tumors were surgically resected in all 9 cases. Eight cases had no recurrence or metastasis, 1 case had recurrence 6 months later, and no recurrence or metastasis after additional surgical resection.

Low-dose paclitaxel modulates the cross talk between the JNK and Smad signaling in primary biliary fibroblasts

SUMMARY OBJECTIVE: The objective of this study was to explore the molecular mechanism underlying the occurrence of benign bile duct stricture and the target of low-dose paclitaxel in the prevention of benign bile duct stricture. METHODS: Under the stimulation of transforming growth factor beta 1, the expression of collagen type I and connective tissue growth factor were detected on isolated primary fibroblasts. The phosphorylation levels of JNK and Smad2L were detected using Western blot. The effect of low-dose paclitaxel on the transforming growth factor beta 1-induced inhibition of type I collagen and connective tissue growth factor expression and JNK and Smad2L phosphorylation was also observed. RESULTS: Transforming growth factor beta 1 induced the secretion of type I collagen and connective tissue growth factor as well as JNK phosphorylation in biliary fibroblasts. The JNK inhibitor or siRNA-Smad2 inhibited the transforming growth factor beta 1-induced secretion of type I collagen and connective tissue growth factor. Low-dose paclitaxel inhibited the expression of type I collagen induced by transforming growth factor beta 1 and may inhibit the secretion of collagen in biliary fibroblasts. CONCLUSION: The activation of JNK/Smad2L induced by transforming growth factor beta 1 is involved in the occurrence of benign bile duct stricture that is mediated by the overexpression of type I collagen and connective tissue growth factor, and low-dose paclitaxel may inhibit the phosphorylation of JNK/Smad2L.

Transition of autophagy and apoptosis in fibroblasts depends on dominant expression of HIF-1α or p53 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

It has been revealed that hypoxia is dynamic in hypertrophic scars; therefore, we considered that it may have different effects on hypoxia-inducible factor-1α (HIF-1α) and p53 expression. Herein, we aimed to confirm the presence of a teeterboard-like conversion between HIF-1α and p53, which is correlated with scar formation and regression. Thus, we obtained samples of normal skin and hypertrophic scars to identify the differences in HIF-1α and autophagy using immunohistochemistry and transmission electron microscopy. In addition, we used moderate hypoxia in vitro to simulate the proliferative scar, and silenced HIF-1α or p53 gene expression or triggered overexpression to investigate the changes of HIF-1α and p53 expression, autophagy, apoptosis, and cell proliferation under this condition. HIF-1α, p53, and autophagy-related proteins were assayed using western blotting and immunofluorescence, whereas apoptosis was detected using flow cytometry analysis, and cell proliferation was detected using cell counting kit-8 (CCK-8) and 5-bromo-2'-deoxyuridine (BrdU) staining. Furthermore, immunoprecipitation was performed to verify the binding of HIF-1α and p53 to transcription cofactor p300. Our results demonstrated that, in scar tissue, HIF-1α expression increased in parallel with autophagosome formation. Under hypoxia, HIF-1α expression and autophagy were upregulated, whereas p53 expression and apoptosis were downregulated in vitro. HIF-1α knockdown downregulated autophagy, proliferation, and p300-bound HIF-1α, and upregulated p53 expression, apoptosis, and p300-bound p53. Meanwhile, p53 knockdown induced the opposite effects and enhanced HIF-1α, whereas p53 overexpression resulted in the same effects and reduced HIF-1α. Our results suggest a teeterboard-like conversion between HIF-1α and p53, which is linked with scar hyperplasia and regression.

Regulatory effects and signaling mechanism of sodium ferulate on the proliferation and apoptosis of human skin hypertrophic scar fibroblasts / 中华烧伤杂志

Objective: To investigate the regulatory effects and signaling mechanism of sodium ferulate on the proliferation and apoptosis of human skin hypertrophic scar fibroblasts (HSFbs). Methods: The experimental research methods were used. The 4th-6th passage of HSFbs from human skin were used for the following experiments. HSFbs were co-cultured with sodium ferulate at final mass concentrations of 1, 1×10-1, 1×10-2, 1×10-3, 1×10-4, 1×10-5, and 1×10-6 mg/mL for 48 hours, and methyl thiazolyl tetrazolium method was used to determine the cell absorbance values and linear regression was used to analyze the half lethal concentration (LC50) of sodium ferulate (n=6). HSFbs were co-cultured with sodium ferulate at final mass concentrations of 0.1, 0.2, 0.3, and 0.4 mg/mL for 24, 48, 72, and 96 hours, and methyl thiazolyl tetrazolium method was used to determine the cell absorbance values and the cell proliferation inhibition rate was calculated (n=3). According to the random number table, the cells were divided into 0.300 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, 0.003 mg/mL sodium ferulate group treated with sodium ferulate at corresponding final mass concentrations, and negative control group without any treatment. After 72 hours of culture, the cell absorbance values were determined by methyl thiazolyl tetrazolium method (n=5), the microscopic morphology of cells was observed by transmission electron microscope (n=3), the cell apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay and the apoptosis index was calculated (n=4), the protein expressions of B lymphocystoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and cysteine aspartic acid specific protease-3 (caspase-3) were determined by immunohistochemistry (n=4), and the protein expressions of transformed growth factor β1 (TGF-β1), phosphorylated Smad2/3, phosphorylated Smad4, and phosphorylated Smad7 were detected by Western blotting (n=4). Data were statistically analyzed with one-way analysis of variance and Dunnett test. Results: The LC50 of sodium ferulate was 0.307 5 mg/mL. After being cultured for 24-96 hours, the cell proliferation inhibition rates of cells treated with sodium ferulate at four different mass concentrations tended to increase at first but decrease later, which reached the highest after 72 hours of culture, so 72 hours was chosen as the processing time for the subsequent experiments. After 72 hours of culture, the cell absorbance values in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group were 0.57±0.06, 0.53±0.04, 0.45±0.05, respectively, which were significantly lower than 0.69±0.06 in negative control group (P<0.01). After 72 hours of culture, compared with those in negative control group, the cells in the three groups treated with sodium ferulate showed varying degrees of nuclear pyknosis, fracture, or lysis, and chromatin loss. In the cytoplasm, mitochondria were swollen, the rough endoplasmic reticulum was expanded, and local vacuolation gradually appeared. After 72 hours of culture, compared with that in negative control group, the apoptosis indexes of cells were increased significantly in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group (P<0.05 or P<0.01). After 72 hours of culture, compared with those in negative control group, the protein expressions of Bcl-2 of cells in 0.300 mg/mL sodium ferulate group was significantly decreased (P<0.01), the protein expressions of Bax of cells in 0.030 mg/mL sodium ferulate group and 0.300 mg/mL sodium ferulate group were significantly increased (P<0.05), and the protein expression of caspase-3 of cells in 0.300 mg/mL sodium ferulate group was significantly increased (P<0.01). After 72 hours of culture, compared with those in negative control group, the protein expression levels of TGF-β1, phosphorylated Smad2/3, and phosphorylated Smad4 of cells in 0.030 mg/mL sodium ferulate group and 0.300 mg/mL sodium ferulate group were significantly decreased (P<0.05 or P<0.01), and the protein expression levels of phosphorylated Smad7 of cells in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group were significantly increased (P<0.01). Conclusions: Sodium ferulate can inhibit the proliferation of HSFbs of human skin and promote the apoptosis of HSFbs of human skin by blocking the expression of key proteins on the TGF-β/Smad signaling pathway and synergistically activating the mitochon- drial apoptosis pathway.

Research advances on the characteristics of fibroblast in keloid / 中华烧伤杂志

In re-cent 20 years, the development of cell biology technology has promoted the research of keloid. Keloid fibroblasts (KFbs) are the main effector cells in keloid, which are closely related to the occurrence and development of keloid. It is significantly different in terms of biological characteristics and gene expression between KFbs and normal fibroblasts. This articles reviews the characteristics of KFbs from multiple perspectives, describing its biological character- istics in details including microstructures, metabolic character- istics, and proliferation properties, and introducing the main characteristics of heterogeneity and genomics of KFbs. The further research on KFbs will help to elucidate the pathogenesis of keloids and provide valuable strategies for the prevention and treatment of keloids.

Transforming growth factor-β1 induces transformation of rat meningeal fibroblasts into myofibroblasts by upregulating Shh signaling / 南方医科大学学报

OBJECTIVE@#To investigate the effect of TGF-β1 on Shh signaling pathway during the transformation of meningeal fibroblasts into myofibroblasts.@*METHODS@#Primary meningeal fibroblasts were isolated from neonatal (24 h) SD rats and purified using type Ⅳ collagenase. The isolated cells were treated with 10 ng/mL TGF-β1 alone or in combination with 20 μmol/L SB-431542 (a TGF-β1 receptor inhibitor) for 72 h, and the changes in proliferation and migration abilities of the fibroblasts were assessed with CCK-8 assay and cell scratch test. The expression of fibronectin (Fn) was detected with immunofluorescence assay, and Western blotting was performed to examine the expressions of Fn, α-SMA and Shh protein in the cells; the expression of Shh mRNA was detected with real-time fluorescence quantitative PCR.@*RESULTS@#TGF-β1 treatment obviously enhanced the proliferation and migration of primary meningeal fibroblasts (P < 0.05), and promoted the transformation of meningeal fibroblasts into myofibroblasts and the secretion of Fn (P < 0.05). TGF-β1 treatment also upregulated the expression of Shh at both protein and mRNA levels (P < 0.05). Treatment with SB-431542 partially blocked the effect of TGF-β1 on the transformation of meningeal fibroblasts (P < 0.05).@*CONCLUSION@#TGF-β1 can induce the transformation of meningeal fibroblasts into myofibroblasts by up-regulating Shh expression in Sonic Hedgehog signaling pathway.

C/EBPb converts bovine fibroblasts to adipocytes without hormone cocktail induction

BACKGROUND: Adipogenesis and fibrogenesis can be considered as a competitive process in muscle, which may affect the intramuscular fat deposition. The CCAAT/enhancer-binding protein beta (C/EBPb) plays an important role in adipogenesis, which is well-characterized in mice, but little known in bovine so far. RESULTS: In this study, real-time qPCR revealed that the level of C/EBPb was increased during the developmental stages of bovine and adipogenesis process of preadipocytes. Overexpression of C/EBPb promoted bovine fibroblast proliferation through mitotic clonal expansion (MCE), a necessary process for initiating adipogenesis, by significantly downregulating levels of p21 and p27 (p < 0.01). Also, the PPARc expression was inhibited during the MCE stage (p < 0.01). 31.28% of transfected fibroblasts adopted lipid-laden adipocyte morphology after 8 d. Real-time qPCR showed that C/EBPb activated the transcription of early stage adipogenesis markers C/EBPa and PPARc. Expression of ACCa, FASN, FABP4 and LPL was also significantly upregulated, while the expression of LEPR was weakened. CONCLUSIONS: It was concluded C/EBPb can convert bovine fibroblasts into adipocytes without hormone induction by initiating the MCE process and promoting adipogenic genes expression, which may provide new insights into the potential functions of C/EBPb in regulating intramuscular fat deposition in beef cattle.

The action of subconjunctival cyclophosphamide in rabbits / Ação da ciclofosfamida subconjuntival em coelhos

ABSTRACT Objective: To observe clinically, in rabbits, the side effects of topical injection of subconjunctival cyclophosphamide, studying its role as an antifibrotic drug. Methods: Prospective study in 20 albino rabbits of New Zealand race. All rabbits were treated with cyclophosphamide, 10mg/ml in a volume of 0.3 ml, in the left eye through subconjunctival injection. They were evaluated for 1, 7, 30, and 60 days after the procedure. All the animals were examined for the detection of ocular reactions such as necrosis, hyperemia, chemosis, secretion, opacity, and iritis. Other side effects as changes in the behavior, in the feed, and the water consumption were also evaluated. Results: It was observed that from the 20 rabbits studied, three rabbits (15%) showed side effects only at the 24 hours analysis. One rabbit (5%) presented hyperemia, one rabbit (5%) had hyperemia associated with iritis, and one rabbit (5%) presented hyperemia associated with secretion. These reactions were not observed at 1, 7, 30, and 60 days. Conclusion: Cyclophosphamide subconjunctival injection induces minor side effects on the conjunctiva of rabbits such as hyperemia, associated with iritis and secretion.

RESUMO Objetivo: Observar clinicamente os efeitos colaterais de injeção subconjuntival de ciclofosfamida, pensando em sua ação como um agente antifibrótico. Métodos: Estudo prospectivo realizado com 20 coelhos albinos da raça Nova Zelândia. Todos os coelhos foram submetidos a 0,3 ml de injeção subconjuntival de ciclofosfamida 10mg/ml no olho esquerdo e foram avaliados de acordo com os efeitos locais no primeiro dia após a injeção, 7, 30 e 60 dias. Foram examinados para detecção de reações oculares como necrose, hiperemia, quemose, secreção, opacidade corneana, irite além de alterações comportamentais e variação no consumo de água e alimentação. Resultados: Dos 20 coelhos estudados, apenas 3 apresentaram reações oculares e somente na leitura de 24 horas. Um coelho (5%) apresentou hiperemia, 1 coelho (5%) apresentou hiperemia associada a presença de irite e 1 coelho (5%) apresentou hiperemia associada a presença de secreção. As reações não foram mais observadas durante os exames de 7, 30 e 60 dias. Conclusão: A ciclofosfamida subconjuntival causou poucos efeitos colaterais na conjuntiva dos coelhos. Os únicos efeitos encontrados foram hiperemia, irite e secreção.

Human esophageal fibroblast-derived exosomal miR-21 reduced the cisplatin sensitivity to esophageal carcinoma EC9706 cells

The objective of this study was to investigate the effect of human esophageal fibroblast-derived exosomal miR-21 on cisplatin sensitivity against esophageal squamous EC9706 cells. EC9706 cells were co-cultured indirectly with human esophageal fibroblasts (HEF) or miR-21 mimics transfected-HEF in the transwell system. The exosomes in HEF-culture conditioned medium were extracted by differential ultracentrifugation. EC9706 cells were co-cultured with HEF-derived exosomes directly. The cisplatin sensitivity against EC9706 cells was revealed via half maximal inhibitory concentration (IC50) values using MTT assay. The expressions of miR-21, programmed cell death 4 (PDCD4) mRNA, and gene of phosphate and tension homology deleted on chromosome ten (PTEN) mRNA were determined by qRT-PCR. The changes of the protein level were detected using western blot assay. IC50 values of cisplatin against EC9706 cells were increased after EC9706 cells were co-cultured with either HEF or exosomes derived from miR-21 mimics-transfected HEF. Following the increased level of miR-21, the mRNA expression and protein levels of PTEN and PDCD4 were decreased in EC9706 cells. The cisplatin sensitivity to EC9706 cells was reduced by HEF-derived exosomal miR-21 through targeting PTEN and PDCD4. This study suggested that non-tumor cells in the tumor micro-environment increased the tumor anti-chemotherapy effects through their exosomes.

IL-17A activates mouse lung fibroblasts through promoting chemokine CXCL12 secretion / 浙江大学学报·医学版

OBJECTIVE@#To investigate the role of IL-17A in promoting the activation of lung fibroblasts and the secretion of chemokine CXCL12, and to explore the possible mechanism.@*METHODS@#Lung tissues of BALB/c mice were collected after intraperitoneal injection of recombinant mouse IL-17A (rmIL-17A). Real-time RT-PCR and Western blotting were used to detect the expression levels of α-smooth muscle actin (α-SMA) and collagen I in lung tissues, and immunohistochemical staining and real-time RT-PCR were used to determine the expression of CXCL12. Normal mouse primary lung fibroblasts were isolated and cultured, and identified by immunofluorescence staining with optical microscopy. Cells and supernatant of culture medium were collected after stimulation with rmIL-17A at different concentrations. mRNA levels of α-SMA, collagen I, and CXCL12 in the cells were determined by real-time RT-PCR, and the levels of collagen I and CXCL12 in the supernatant of culture medium were determined by ELISA.@*RESULTS@#The mRNA and protein levels of α-SMA and collagen I in the lung tissue of mice injected with rmIL-17A were significantly increased compared with the control group (all @*CONCLUSIONS@#s: IL-17A can promote the activation of lung fibroblasts and translation into myofibroblast. The secretion of collagen is increased, which promote the deposition of extracullular matrix, and leads to the occurrence and development of lung fibrosis. CXCL12, a chemokine secreted by activated fibroblasts, may be involved in this process.

Effect of ScLL and 15d-PGJ2 on viability and cytokine release in LPS-stimulated fibroblasts: an in vitro study

Abstract This study evaluated the effect of a cyclopentenone-type PG, 15-Deoxy-Δ12,14-PG J2 (15d-PGJ2), and lectin (ScLL) on the viability of human gingival fibroblasts (HGFs), and on IL-6 and TGFβ-1 release by these fibroblasts, stimulated with lipopolysaccharide (LPS). HGFs were stimulated with LPS 10 μg/ml and treated with 15d-PGJ2 1 and 2 μg/ml, and ScLL 2 and 5 μg/ml, for 1 and 3h, and then evaluated for viability by MTT assay. Supernatant was collected to detect IL-6 and TGFβ-1 release, by ELISA. Positive control was cells kept in Dulbecco's Modified Eagle's Medium, and negative control was those kept in LPS. Data were analyzed by ANOVA and Dunnett's test (α = 0.05). No significant difference was found in viability among experimental groups at 1h (p > 0.05). Percentage of ScLL 5 µg/ml viable cells was similar to that of positive control at evaluated periods (p > 0.05), whereas the other groups had lower levels than the positive control (p < 0.05). IL-6 release was statistically higher for ScLL 5 μg/ml and 15d-PGJ2 2 µg/ml at 1h, compared with the other treated groups and positive control (p < 0.05). No significant differences were found among the groups at 3h (p > 0.05), except for ScLL 2 µg/ml and 15d-PGJ2 1 µg/ml, which showed lower IL-6 release compared with that of negative control (p < 0.05). No significant difference was found among the groups for TGFβ-1 release (p > 0.05). Results indicated that ScLL 5 μg/ml did not interfere in viability, and ScLL 2 µg/ml and 15d-PGJ2 1 µg/ml demonstrated reduced IL-6 release. Tested substances had no effect on TGFβ-1 release.

Effect of cytoglobin overexpression on extracellular matrix component synthesis in human tenon fibroblasts

BACKGROUND: Conjunctival filtering bleb scar formation is the main reason for the failure of glaucoma filtration surgery. Cytoglobin (Cygb) has been reported to play an important role in extracellular matrix (ECM) remodeling, fibrosis and tissue damage repairing. This study aimed to investigate the role of Cygb in anti-scarring during excessive conjunctival wound healing after glaucoma filtration surgery. METHODS: Cygb was overexpressed in human tenon fibroblasts (hTFs) by transfecting hTFs with lentiviral particles encoding pLenti6.2-FLAG-Cygb. Changes in the mRNA and protein levels of fibronectin, collagen I, collagen III, TGF-ß1, and HIF1α were determined by RT-PCR and western blotting respectively. RESULTS: After Cygb overexpression, hTFs displayed no significant changes in visual appearance and cell counts compared to controls. Whereas, Cygb overexpression significantly decreased the mRNA and protein expression levels of collagen I, collagen III and fibronectin compared with control (p < 0.01). There was also a statistically significant decrease in the mRNA and protein levels of TGF-ß1 and HIF-1α in hTFs with overexpressed Cygb compared with control group (p < 0.05). CONCLUSION: Our study provided evidence that overexpression of Cygb decreased the expression levels of fibronectin, collagen I, collagen III, TGF-ß1 and HIF-1α in hTFs. Therefore, therapies targeting Cygb expression in hTFs may pave a new way for clinicians to solve the problem of post-glaucoma surgery scarring.

The E3 ligase MuRF2 plays a key role in the functional capacity of skeletal muscle fibroblasts

Fibroblasts are a highly heterogeneous population of cells, being found in a large number of different tissues. These cells produce the extracellular matrix, which is essential to preserve structural integrity of connective tissues. Fibroblasts are frequently engaged in migration and remodeling, exerting traction forces in the extracellular matrix, which is crucial for matrix deposition and wound healing. In addition, previous studies performed on primary myoblasts suggest that the E3 ligase MuRF2 might function as a cytoskeleton adaptor. Here, we hypothesized that MuRF2 also plays a functional role in skeletal muscle fibroblasts. We found that skeletal muscle fibroblasts express MuRF2 and its siRNA knock-down promoted decreased fibroblast migration, cell border accumulation of polymerized actin, and down-regulation of the phospho-Akt expression. Our results indicated that MuRF2 was necessary to maintain the actin cytoskeleton functionality in skeletal muscle fibroblasts via Akt activity and exerted an important role in extracellular matrix remodeling in the skeletal muscle tissue.

Effects of the application of the amniotic membrane in the healing process of skin wounds in rats

Abstract Purpose: To evaluate the efficacy of the application of the human amniotic membrane (HAM) on the inflammatory process, fibroblast proliferation, formation of collagenand reduction of skin wound areas in rats. Methods: Thirty six rats were submitted to a surgical injury induction and divided into two groups (n = 18): group C (control) and T (treated with the HAM). The macroscopic evolution in the wound area and the histological characteristics of the skin samples were evaluated. Results: The regression of the wound area was greater in group T. The histological analysis revealed a significant reduction (p < 0.05) in the inflammatory infiltrate in group T at all experimental periods compared with that in the control group. Furthermore, the group T presented a significant increase in the proliferation of fibroblasts at 14 and 21 days compared with group C (p < 0.05). Regarding the deposition of mature collagen fibers, there was an increase in the replacement of type III collagen by type I collagen in group T (p < 0.05). Conclusion: Treatment with the HAM reduced the healing time as well as the inflammatory responses, increased the proliferation of fibroblasts, and induced a higher concentration of mature collagen fibers.

Influence of vitamin D receptor FokI polymorphism on expression of CYP24A1 in periodontal cells / 北京大学学报(医学版)

OBJECTIVE@#There is asingle nucleotide polymorphism (SNP) in the exon 2 of the vitamin D receptor (VDR) gene that can be distinguished using the restriction endonuclease FokI, and accordingly divided into three genotypes: FF, Ff and ff. VDR-FokI polymorphism was the only known SNP that could alter the protein structure of VDR. CYP24A1 is the gene encoding vitamin D 24 hydroxylase and is a vitamin D responsive gene. The influence of rs2228570 on transcriptional activation by VDR in human gingival fibroblasts (hGF) and periodontal ligament cells (hPDLC) was investigated in this study.@*METHODS@#hGF and hPDLC of 12 donors' were primarily cultured and genomic DNA was extracted. A part of genomic DNA with the length of 267 bp was obtained using PCR, which contained the SNP. VDR-Fok I genotypes were determined according to the results of restriction fragment length polymorphism. hGF and hPDLC were stimulated with 10 nmol/L 1α,25 dihydroxy vitamin D3 (1,25OH2D3) or 1 000 nmol/L 25 hydroxy vitamin D3 (25OHD3) for 48 h before RNA was extracted. Then VDR antagonist ZK159222 was used or not used during 1,25OH2D3 or 25OHD3 stimulation with hGF and hPDLC. After 1,25OH2D3 stimulation for 48 h, the proteins in hGF and hPDLC were also collected. The protein expressions of CYP24A1 and VDR were detected using Western blot.@*RESULTS@#Among the 12 donors' cell cultures, the number of FF, ff and Ff genotypes was 4, 3 and 5, respectively.After stimulation with 1,25OH2D3 or 25OHD3 for 48 h,CYP24A1 mRNA levels in FF-hGF were significantly higher than those in other hGF genotypes(1,25OH2D3: F=31.147, P<0.01; 25OHD3: F= 32.061,P <0.01), as was in FF-hPDLC (1,25OH2D3: F=23.347, P<0.01; 25OHD3: F=32.569,P<0.01). When ZK159222 was used before 1,25OH2D3 stimulation, this statistically significant difference disappeared (hGF: F=0.246, P=0.787; hPDLC: F=0.574, P=0.583). When ZK159222 was used before 25OHD3 stimulation, the trend was similar (hGF: F=1.636, P=0.248; hPDLC: F=0.582, P=0.578).After stimulation with 1,25OH2D3 for 48 h, CYP24A1 protein levels in FF-hGF were significantly higher than those in the other hGF genotypes (F=12.368, P <0.01), as was in FF-hPDLC (F=15.749, P <0.01). In hGF and hPDLC, the mRNA or protein expression of VDR of different genotypes was not significantly different under different stimulation conditions.The paired comparison showed that there was no statistically significant difference between the expression of CYP24A1 in hGF and that in hPDLC under all the stimulation conditions, as was the expression of VDR.@*CONCLUSION@#In hGF and hPDLC, the FF-VDR genotype is associated with the more remarkable up-regulation of CYP24A1than the other genotypes, indicating that transcriptional activation of FF-VDR might be higher than those of other vitamin D receptors.

Adipogenic and Lipolytic Effects of Ascorbic Acid in Ovariectomized Rats

PURPOSE: Ascorbic acid has been reported to have an adipogenic effect on 3T3-L1 preadipocytes, while evidence also suggests that ascorbic acid reduces body weight in humans. In this study, we tested the effects of ascorbic acid on adipogenesis and the balance of lipid accumulation in ovariectomized rats, in addition to long-term culture of differentiated 3T3-L1 adipocytes. MATERIALS AND METHODS: Murine 3T3-L1 fibroblasts and ovariectomized rats were treated with ascorbic acid at various time points. In vitro adipogenesis was analyzed by Oil Red O staining, and in vivo body fat was measured by a body composition analyzer using nuclear magnetic resonance. RESULTS: When ascorbic acid was applied during an early time point in 3T3-L1 preadipocyte differentiation and after bilateral ovariectomy (OVX) in rats, adipogenesis and fat mass gain significantly increased, respectively. However, lipid accumulation in well-differentiated 3T3-L1 adipocytes showed a significant reduction when ascorbic acid was applied after differentiation (10 days after induction). Also, oral ascorbic acid administration 4 weeks after OVX in rats significantly reduced both body weight and subcutaneous fat layer. In comparison to the results of ascorbic acid, which is a well-known cofactor for an enzyme of collagen synthesis, and the antioxidant ramalin, a potent antioxidant but not a cofactor, showed only a lipolytic effect in well-differentiated 3T3-L1 adipocytes, not an adipogenic effect. CONCLUSION: Taking these results into account, we concluded that ascorbic acid has both an adipogenic effect as a cofactor of an enzymatic process and a lipolytic effect as an antioxidant.

Expression dynamics of caveolin-1 in fibroblasts of newborn rats with chronic lung disease and its impact on lung fibroblast proliferation

Abstract Purpose: To evaluate the changes of caveolin-1 in lung fibroblasts in newborn Wistar rats when exposed to hyperoxic conditions, as well as lung fibroblasts cell cycle. Methods: One hundred newborn Wistar rats were randomly divided (50 rats/group) into experimental and control groups, exposed to hyperoxic conditions or normal air, respectively. The fraction of inspired oxygen (FiO2) in the experimental group was 90%, whereas this value was 21% in the control group. Lung fibroblasts were collected on days 3, 7, and 14 of the experiment. Caveolin-1 expression dynamics in lung fibroblasts was assayed in each group by immunofluorescence and Western blot analyses. Flow cytometry (FCM) was used to assess the proportions of lung fibroblasts at different stages of the cell cycle. Results: On day 3, no significant difference in caveolin-1 expression was observed between the hyperoxic and control groups; however, on days 7 and 14, caveolin-1 expression was significantly lower in the hyperoxic group than in the control (P<0.05). No apparent differences were observed in caveolin-1 expression in the control group at the different time points. Using FCM analysis, we showed that the proportion of lung fibroblasts in G0/G1 phase in the hyperoxic group decreased compared to that of the control group on day 7, while the proportion of S-phase cells increased (P<0.05). These differences were more significant when the groups were compared on day 14 (P<0.01). Conclusion: After seven days the exposure to hyperoxic conditions, lung fibroblasts proliferated and caveolin-1 expression decreased.