mRNA Expression Levels Of TGF-β1 Research Articles

Investigation of TGF-β1 gene variant and expression in a group of Iranian women with endometriosis.

Endometriosis is defined as a common gynecologic and inflammatory disease. Transforming growth factor-beta 1 (TGF-β1) gene and its protein level might play a role in the pathogenesis of endometriosis. The present study aimed for the first time to assess the associations between endometriosis risk and -509 C/T (rs1800469) variant of the TGF-β1 gene as well as TGF-β1 mRNA expression in eutopic endometrium tissue of patients with and without endometriosis among a group of Iranian women. Genotyping was carried out in 100 endometriosis patients (cases) with confirmed histological diagnosis of endometriosis and 197 non-endometriosis subjects (controls). The expression level of TGF-β1 mRNA was determined using Real-Time PCR assay in 15 eutopic endometrium tissue of women with endometriosis and 15 healthy controls. There was a significant association for allele and genotype frequencies of rs1800469 variant and endometriosis. No significant difference for TGF-β1 expression was observed between eutopic endometrium of patients and healthy group. Also, evaluation of TGF-β1expression across the menstrual cycle showed the same level of TGF-β1 among case and control subjects. Our investigations indicated enough evidence for the effect of TGF-β1 genetic variant on endometriosis risk in an Iranian population. Furthermore, we could not find any relations between TGF-β1 mRNA expression and susceptibility to endometriosis.

MiR-124-3p targeting of TGF-β1 inhibits the proliferation of hypertrophic scar fibroblasts.

microRNAs are involved in a variety of physiological and pathophysiological processes, but their role in the pathogenesis of hypertrophic scars (HS) is not fully understood. Transforming growth factor β1 (TGF-β1) plays an important role in the genesis and development of HS. In this study, we hypothesized that a post-translational miRNA mechanism regulates the expression of TGF-β1 in HS fibroblasts (HSFBs) and participates in the development of HS. Predictions from EBCORI, PicTar and miRBase databases showed that miR-124-3p can target and regulate the expression of TGF-β1. We collected HS tissue and corresponding normal tissue from 25 patients with HS who had been operated on for the first time. The expression level of miR-124-3p in HS tissue was significantly lower than in normal tissue, while the expression level of TGF-β1 mRNA was significantly higher than in normal tissue (p < 0.05), showing a negative correlation between them. Results from a luciferase reporter assay showed that miR-124-3p targets the 3'-UTR of TGF-β1 and inhibits its expression. After miR-124-3p mimics were transfected into HSFBs, the expression of TGF-β1, α-smooth muscle actin (α-SMA), collagen I, survivin, and Bcl-2 were reduced and the expression of Bax was increased, with significant decreases in DNA synthesis, proliferation and survival. However, after a miR-124-3p inhibitor was transfected into HSFBs, these effects were reversed as the expression of TGF-β1, α-SMA, collagen I, survivin, and Bcl-2 increased, expression of Bax decreased, and DNA synthesis, proliferation and survival cells increased significantly. miR-124-3p can inhibit the proliferation of HSFBs by targeting TGF-β1, and miR-124-3p may thus be a potential therapeutic target in HS.

Changes in Smad1/5/9 expression and phosphorylation in astrocytes of the rat hippocampus after transient global cerebral ischemia

Smad proteins are known to transduce the actions of the transforming growth factor-β (TGF-β) family including TGF-βs, activins, and bone morphogenetic proteins (BMPs). We previously reported that Smad1/5/9 immunoreactivity was observed in astrocytes of various rat brain regions including the hippocampus, suggesting that Smad1/5/9 may be associated with the physiology of astrocytes. However, the Smad1/5/9 expression and activation in the hippocampal astrocytes after global cerebral ischemia has not been yet elucidated. In this study, we examined temporal changes in the expression and phosphorylation of Smad1/5/9 in the hippocampus using a rat model of global cerebral ischemia. Furthermore, we examined the candidate ligand involved in the phosphorylation of Smad1/5/9 in the hippocampus after ischemia. Pyramidal neuronal cell death in the CA1 regions was visible at 3 days, and maximum death occurred within 7 days after ischemia. At 7 days after ischemia, astrocytes that showed strong immunoreactivity for Smad1/5/9 were frequently observed in the CA1 region. Additionally, there was an increase in phosphorylated Smad1/5/9 (phospho-Smad1/5/9) -immunopositive astrocytes in the CA1 region 7 days after ischemia. Real-time PCR analysis showed an increase in the expression level of TGF-β1 mRNA in the hippocampus after ischemia. Intracerebroventricular injection of SB525334, an inhibitor of TGF-β/Smad signaling, reduced immunoreactivity for phospho-Smad1/5/9 in astrocytes. These results suggest that TGF-β1 may be a key molecule for ischemia-induced Smad1/5/9 phosphorylation in astrocytes, and TGF-β1-Smad1/5/9 signaling may play a role in post-ischemic events, including brain inflammation or tissue repair rather than neuroprotection of the hippocampus.

Promising Effects of Zerumbone on the Regulation of Tumor-promoting Cytokines Induced by TNF-α-activated Fibroblasts.

Inflammation plays an important role in the development of several cancers. Inflammatory cytokines, including tumor necrosis factor-α (TNF-α), are associated with the induction of inflammation. Chronic inflammation contributes to the progression of cancer through several mechanisms, including increased cytokine production and activation of transcription factors, such as nuclear factor-κB (NF-κB). Zerumbone (ZER), a component of subtropical ginger (Zingiber zerumbet Smith), seems to have anti-inflammatory, anti-cancer, and antioxidant activities. In this study, we aimed to explore the protective function and mechanisms of ZER against TNF-α-induced cancer-promoting cytokines. We found that the viability of stimulated human fibroblast cell lines was reduced after treatment with ZER (IC50=18 µmol/L), compared to un-stimulated fibroblasts (IC50=40 µmol/L). Besides, ZER inhibited mRNA expression and protein secretion of transforming growth factor-β (TGF-β), interleukin-33 (IL-33), monocyte chemoattractant protein-1 (MCP-1), and stromal cell-derived factor 1 (SDF-1), which were produced by TNF-α-induced fibroblasts, as measured by quantitative real time-PCR (qRT-PCR) and ELISA assays. The mRNA expression levels of TGF-β, IL-33, SDF-1, and MCP-1 showed 8, 5, 2.5, and 4-fold reductions, respectively. Moreover, secretion of TGF-β, IL-33, SDF-1, and MCP-1 was reduced to 3.65±0.34 ng/mL, 6.3±0.26, 1703.6±295.2, and 5.02±0.18 pg/mL, respectively, compared to the untreated group. In addition, the conditioned media (CM) of TNF-α-stimulated fibroblasts increased the NF-κB expression in colorectal cancer cell lines (HCT-116 and Sw48), while in the vicinity of ZER, the expression of NF-κB was reversed. Considering the significant effects of ZER, this component can be used as an appropriate alternative herbal treatment for cancer-related chronic inflammation.

Research on feasibility of in vitro inflammatory wound microenvironment simulated by using inflammatory wound tissue homogenate of mice

Research on feasibility of in vitro inflammatory wound microenvironment simulated by using inflammatory wound tissue homogenate of mice

Distinct prognostic values and antitumor effects of tumor growth factor β1 and its receptors in gastric cancer.

Gastric cancer (GC) is one of the most common malignancies and is the second leading cause of cancer-associated mortality world-wide. In the present study, the prognostic value and antitumor effects of transforming growth factor β1 (TGFβ1) and its receptors in GC were explored. The online Kaplan-Meier plotter database was used to investigate the prognostic values of TGFβ1 and its receptors. The present study demonstrated that low mRNA expression levels of TGFβ1 and its 3 receptors, transforming growth factor β1 (TGFβR1), TGFβR2 and TGFβR3, was associated with improved overall survival time in patients with GC. Cell Counting Kit-8 and Transwell assays were used to confirm the effects of TGFβ1, TGFβR1, TGFβR2 and TGFβR3 on the proliferation, migration and invasiveness of the AGS and MKN45 GC cell lines. It was found that the knockdown of these genes blocked cell proliferation, migration and invasion in GC cells. To the best of our knowledge, the present study is the first to determine the role of TGFβR1 and TGFβR3 in GC cells. The results indicate that in addition to TGFβ1 and TGFβR2, TGFβR1 also plays a specific role in the occurrence and development of tumors. Thus, these markers may be considered as potential prognostic indicators in human GC. The findings of the present study indicate that not only TGFβ1 and TGFβR2, but also TGFβR1 is involved in the progression of GC. The findings of the present study provide new ideas and approaches for the treatment of patients with GC.

Curcumin protects bone biomechanical properties and microarchitecture in type 2 diabetic rats with osteoporosis via the TGFβ/Smad2/3 pathway.

Type 2 diabetic osteoporosis (T2DOP) has become a common secondary cause of osteoporosis that accelerates bone loss and leads to bone fractures. The aim of the current study was to investigate the association between the anti-osteoporotic effect of curcumin (Cur) and the transforming growth factor (TGF)β/Smads signaling pathway. Male Sprague-Dawley rats were used in the experiments. The type 2 diabetes mellitus (T2DM) animals were treated with Cur for 8 weeks and blood lipid markers, bone microstructure and bone biomechanics were then evaluated. The mRNA expression levels of TGFβ1, type I TGFβ receptor (TβRI), TβRII and Smad2/3 were determined using reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry. The body weight of rats with type 2 diabetes-induced osteoporosis increased (P<0.05), while the lipid (total cholesterol, triglyceride and low-density lipoprotein) and fasting blood glucose levels were decreased by Cur (P<0.05). In addition, Cur significantly improved bone biomechanical properties (maximum load, breaking load, elastic load and the bone rigidity coefficient) and preserved bone microarchitecture (P<0.05). The RT-qPCR and IHC results revealed that Cur increased TGFβ1, TβRI, TβRII and Smad2/3 expression levels and promoted Smad2/3 phosphorylation in bones. The present results also indicated that Cur regulated lipid and glucose levels, improved bone biomechanical properties and preserved bone microarchitecture, and that these effects may be mediated via TGFβ/Smad2/3 pathway activation.

Transient Receptor Potential Channel Canonical Type 3 Deficiency Antagonizes Myofibroblast Transdifferentiation In Vivo.

Objective Myofibroblast transformation has been shown to be associated with the reactive oxygen species- (ROS-) producing enzyme NADPH oxidase (Nox4). Inhibition of transient receptor potential channel canonical type 3 (TRPC3) attenuates mitochondrial calcium handling and ROS production in the vasculature of hypertensive rats. However, it remains elusive whether TRPC3 regulates mitochondrial calcium and ROS production and participates in myofibroblast transdifferentiation during wound healing. Methods and Results In this study, we demonstrated that activation of TRPC3 by transforming growth factor β (TGFβ (TGFαSMA). Inhibition of TRPC3 with its specific inhibitor, Pyr3, significantly decreased TGFβ (TGFαSMA). Inhibition of TRPC3 with its specific inhibitor, Pyr3, significantly decreased TGFβ (TGFβ (TGFTrpc3−/− mice exhibited significantly attenuated myofibroblast transdifferentiation, as demonstrated by decreased αSMA). Inhibition of TRPC3 with its specific inhibitor, Pyr3, significantly decreased TGFβ (TGFβ (TGFTrpc3−/− mice exhibited significantly attenuated myofibroblast transdifferentiation, as demonstrated by decreased Trpc3+/+ mice. In addition, Trpc3−/− mice exhibited significantly attenuated myofibroblast transdifferentiation, as demonstrated by decreased Conclusions Our data indicate that TGFβ1-mediated activation of TRPC3 enhances mitochondrial calcium and ROS production, which promotes myofibroblast transdifferentiation and HTS formation. Inhibition of the TRPC3-mediated Nox4/pSmad2/3 pathway may be a useful strategy to limit HTS formation after injury.β (TGF

Acute PFOA exposure promotes epigenomic alterations in mouse kidney tissues

Perfluorooctanoic acid (PFOA), a manufactured perfluorochemical is a common surfactant and environmental pollutant found in various consumer products and water sources. Epidemiological studies have demonstrated its association with kidney dysfunction. However, the mechanisms that trigger kidney dysfunction following PFOA exposure is a gap in the field. The work presented explores the potential epigenetic indicators of kidney disease due to exposure to PFOA. In this study, 30 days old CD-1 mice were exposed to 1, 5, 10, or 20 mg/kg/day of PFOA for 10 days. Following acute oral exposure, epigenetic alterations and expression levels of various markers of fibroblast activation were evaluated in kidney tissues. We noted that PFOA-exposed mice exhibited differential methylation yielding 879 differentially methylated regions compared to vehicle. The mRNA expression revealed significant increase in Dnmt1 with decreased Rasal1 expression at higher levels of PFOA exposure suggestive of Rasal1 hypermethylation (an early indicator of fibroblast activation in kidney). Like Dnmt1, we also observed significant increase in Hdac1, 3 and 4. These are class I & II HDACs which are known to be critically altered in some renal diseases. Further, the mRNA expression levels of TGF-β and α-SMA significantly increased compared to vehicle. The KEGG and Go enrichment pathway analysis of reduced representation bisulfite data also revealed pathways implicated in renal fibrosis. Our study shows clear evidence of epigenetic alterations (DNA methylation and HDAC expression changes) in tissues from mouse kidney following PFOA exposure. Our results also suggest that epigenetic alterations in kidney promote the expression of early markers of fibroblast activation.

Regulatory mechanism of TGF-β1/SGK1 pathway in tubulointerstitial fibrosis of diabetic nephropathy.

The aim of this study was to clarify the potential function of transforming growth factor-β1/serum/glucocorticoid-regulated kinase 1 (TGF-β1/SGK1) pathway in diabetic nephropathy-induced tubulointerstitial fibrosis. Type 2 diabetes mellitus (T2DM) model was successfully established in rats by high-sucrose-high-fat diet combined with streptozotocin (STZ) induction. Subsequently, blood glucose level, renal function and pathological changes in kidneys of T2DM and control rats were evaluated. Western blot and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) were conducted to determine the protein and mRNA expression levels of TGF-β1, SGK1, fibronectin (FN) and α-smooth muscle actin (α-SMA) in rat kidney tissues, respectively. Blood glucose (BG), glycosylated hemoglobin (GHb), serum creatinine (Scr) and blood urea nitrogen (BUN) in T2DM rats were significantly higher than those of control rats (p<0.05). The morphology of glomeruli and renal tubules in rats of control group were normal. In contrast, T2DM rats showed significant lesions in glomeruli, renal tubules, and renal interstitium. Furthermore, the relative expression levels of TGF-β1, SGK1, FN, and α-SMA in kidney tissues of T2DM rats were remarkably higher than those of controls (p<0.05). The TGF-β1/SGK1 pathway is closely related to tubulointerstitial fibrosis in T2DM rats.

Hepatic Artery Embolization Induces the Local Overexpression of Transforming Growth Factor β1 in a Rat Hepatoma Model

Introduction: The underlying mechanism involved in the recurrence of hepatoma after hepatic arterial embolization (HAE) is not adequately examined. An immunosuppressive cytokine, transforming growth factor β1 (TGF-β1), can lead to tumor progression and is affected by hypoxia in various cancers. The study aimed to assess the effect of HAE on the expression of TGF-β1 in a rat hepatoma model. Methods: Sprague-Dawley rats bearing N1S1 hepatoma cells underwent HAE (HAE group, n = 5) or sham treatment (sham group, n = 4). The animals were euthanized at 48 h, and liver tissues were harvested. Immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR) were performed to compare the expression of TGF-β1 and hypoxia-inducible factor 1α (HIF-1α) between the HAE and sham groups. In vitro experiments with the N1S1 cell line were also performed under normoxic (21% O₂) or hypoxic (1% O₂) conditions for 48 h, and the expression of TGF-β1 and HIF-1α was assessed with western blotting and enzyme-linked immunosorbent assay. Statistical data comparisons were performed by Student t test. Results: IHC showed that both the TGF-β1-positive and HIF-1α-positive tumor peripheral areas were larger in the HAE group (6.59 ± 2.49 and 10.26 ± 4.14%; p < 0.001, respectively) than in the sham group (0.34 ± 0.41 and 0.40 ± 0.84% respectively). Similarly, qPCR showed that the mRNA expression levels of TGF-β1 and HIF-1α were higher (1.95 ± 0.38-fold and 1.62 ± 0.37-fold; p < 0.001 and p = 0.002, respectively) in the HAE group than those in the sham group. TGF-β1 expression was suppressed when HIF-1α inhibitors were added (p = 0.001), and HIF-1α expression was upregulated when exogenous TGF-β1 was added (p = 0.033) in N1S1 cells. Conclusion: HAE enhanced local TGF-β1 expression in a rat hepatoma model. In vitro experiments suggest that HAE-induced hypoxic stress may trigger the interdependent expression of TGF-β1 and HIF-1α.

High Expression of TGF-β1 Predicting Tumor Progression in Skull Base Chordomas

To investigate the expression characteristics and prognostic value of transforming growth factor β1 (TGF-β1) in primary skull base chordomas (SBCs). The mRNA expression levels of TGF-β1 were measured in 57 frozen samples from patients with primary SBCs. Clinical data collection, follow-up, correlations, and survival analyses were performed. In the series of 57 patients (29 men and 28 women) with primary SBCs, the mean value of TGF-β1 mRNA was 1.713 with a median of 0.904. Twenty-four SBCs were soft type and 33 were hard type. The Mann-Whitney U test revealed that the expression level of TGF-β1 mRNA in hard type SBCs was significantly higher than the expression level found in the soft type (P= 0.03). The independent-samples median test suggested that the expression level of TGF-β1 mRNA in female patients' SBCs was significantly higher than that in male patients' SBCs (P= 0.01). Expression differences of TGF-β1 were not seen among different pathological subtypes, tumor blood supply, or degree of resection. The Spearman rank correlation coefficient clarified that TGF-β1 mRNA levels were not correlated with tumor diameter, preoperative Karnofsky Performance Status (KPS), postoperative KPS, follow-up KPS, age, or intraoperative blood loss. The multivariate Cox analysis revealed that pathological subtype (P= 0.008), expression level of TGF-β1 mRNA (P= 0.01), and tumor texture (P= 0.03) were all independent prognostic factors for tumor progression. SBCs in female patients and SBCs with hard texture were prone to have high TGF-β1 mRNA expression. High expression of TGF-β1, hard tumor texture, and conventional subtype were all independent risk factors for tumor progression.

Hyaluronic acid hydrogel loaded by adipose stem cells enhances wound healing by modulating IL-1β, TGF-β1, and bFGF in burn wound model in rat.

Application of hydrogels can be an effective technique in transferring the adipose-derived stem cells (ASCs) to injured tissue and their protection from further complications. Besides, acellular dermal matrix (ADM) has successfully been used in treatment of wounds. In this study, a combination of hylauronic acid (HA) and ASCs (HA/ASCs) was applied on burn wounds and the injured area was then covered by an ADM dressing in a rat model (ADM-HA/ASCs). Wound healing was evaluated by histopathological, histomorphometrical, molecular, biochemical, and scanning electron microscopy assessments on days 7, 14, and 28 post-wounding. ADM-HA/ASCs stimulated healing significantly more than the ADM-HA and ADM treated wounds, as it led to reduced inflammation, and improved angiogenesis and enhanced granulation tissue formation. Expression of interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) was lower in the ADM-HA/ASCs treated wounds than the ADM-HA and ADM groups, at the seventh post-wounding day. ADM-HA/ASCs also enhanced the expression level of TGF-β1 mRNA at 14 day post-wounding that was parallel to the experimental data from histological and biochemical assessments and confirmed the positive role of ASCs in repair of burn wounds. Additionally, increase in basic fibroblast growth factor (bFGF) expression and decreased TGF-β1 level on the 28th post-wounding day indicated the anti-scarring activity of ASCs. HA loaded by adipose stem cells can represent a promising strategy in accelerating burn wound healing.

THE EFFECT OF ALPHA-MANGOSTIN ON TRANSFORMING GROWTH FACTOR BETA 1 (TGF-β1) AND MATRIX METALLOPROTEINASE-3 EXPRESSION IN TGF-β-INDUCED HEPATIC STELLATE CELLS

Objective: Alpha-mangostin (α-MG) has been shown to possess antifibrotic effects. However, the specific mechanism of action of this compoundremains poorly understood. Therefore, the aim of this study was to investigate the effect of α-MG on the expression levels of transforming growthfactor (TGF)-β1 and matrix metalloproteinase-3 (MMP3) in hepatic stellate cells (HSCs) induced by TGF-β.Methods: Immortalized HSCs and LX-2 cells were incubated with TGF-β with or without α-MG (5 and 10 μM). The viability of LX-2 cells was assessedusing the Trypan Blue Exclusion Method. The effect of α-MG on cell morphology and the mRNA expression levels of TGF-β1, TGF-β receptor, and MMP3was then evaluated.Results: TGF-β enhanced the proliferation of HSCs and caused significant increases in the expression levels of TGF-β1, TGF-β receptor, and MMP3.α-MG treatment reduced the proliferation of HSCs and decreased the expression levels of TGF-β1, TGF-β1 receptor, and MMP3.Conclusion: α-MG is a potential antifibrotic agent due to its antiproliferative and antifibrogenic effects, mainly by suppressing the expression of TGF-βand MMP3 on the surfaces of activated HSCs.

Antifibrotic effect of pirfenidone on orbital fibroblasts in patients with thyroid-associated ophthalmopathy and its mechanisms

Objective: To investigate the effects of pirfenidone on orbital fibroblasts (OFs) from patients with thyroid-associated ophthalmopathy (TAO) and its underlying mechanisms. Methods: OFs from patients with TAO were isolated and cultured in DMEM. Cells were divided into four groups and treated with 0, 250, 500 and 1 000 μg/ml pirfenidone for 24, 48 or 72 hours, respectively. Cell proliferation was detected by tetramethyl azo salt (MTT) assay, and cell viability was determined by trypan blue. Transforming growth factor (TGF) β1 mRNA level was determined by real-time fluorescence quantitative PCR (RT-qPCR). Type Ⅰ and type Ⅲ collagen secreted from cultured cells were measured by enzyme-linked immuno sorbent assay (ELISA). Results: (1) The primary cultured OFs had typical fibroblast spindle-like morphology. (2) MTT assay showed that pirfenidone treatment significantly inhibited the proliferation of OFs in a dose-dependent manner (P<0.05) with the proliferation rates of pirfenidone treated groups of -15.31%, -24.92%, -48.53% from 250, 500, 1 000 μg/ml after 72 h, respectively, in which the inhibition effect of 1 000 μg/ml pirfenidone was significantly different from the other two treated groups (P<0.05). There were no significant differences in the inhibitory effect of the same concentration group among different time points at 24 h, 48 h and 72 h (P>0.05). Trypan blue showed that the survival rate of OFs in different concentrations of pirfenidone from 0,250, 500, 1 000 μg/ml at 72 h were 78.37%, 79.21%, 78.24% and 76.28%, respectively. There were no significant differences between each drug treated and the control group (P>0.05). (3) RT-qPCR results showed that the mRNA expression levels of TGFβ1 at 250, 500, 1 000 μg/ml pirfenidone treated groups at 72 h were 0.760±0.010, 0.440±0.006, and 0.290±0.002, respectively. Compared with the control group (0.950±0.014), the differences were statistically significant (all P<0.05). Moreover, TGFβ1 mRNA expression level in 1 000 μg/ml pirfenidone treated group was significantly lower than those in the other two treated groups (all P<0.05). The secretion of type Ⅰ collagen (0.633±0.006, 0.527±0.003 and 0.402±0.008) and type Ⅲ collagen (0.511±0.003, 0.439±0.007 and 0.223±0.006) in 250, 500 and 1 000 μg/ml pirfenidone treated groups at 72 h were significantly lower than those in the control group (0.794±0.005, 0.527±0.007, all P<0.05). Type Ⅰ and type Ⅲ collagen secretion in 1 000 μg/ml pirfenidone treated group were significantly lower than those in the other two groups (P<0.05). Conclusions: Pirfenidone inhibits the cell proliferation, TGFβ1 expression and collagen secretion of OFs, which may contribute to the anti-fibrotic effect of pirfenidone.

Analysis of differential gene expressions of inflammatory and repair-related factors in chronic refractory wounds in clinic

Objective To compare the tissue morphology and gene expressions of inflammatory and repair-related factors in chronic refractory wound tissue including pressure ulcers and diabetic feet. Methods During August 2016 to September 2017, 10 samples of prepuce were collected after circumcision of 10 urological patients [all male, aged (38±4) years old] admitted in the First Affiliated Hospital of Nanchang University and included in normal skin group, samples of tissue around the edge of wounds with blood supply were collected from 9 heat or electric burn patients [6 male patients, 3 female patients, aged (51±8) years old], 13 pressure ulcer patients [9 male patients, 4 female patients, aged (51±14) years old] and 10 diabetic foot patients [8 male patients, 2 female patients, aged (61±10) years old] during the operations. The samples were divided into burn wound group (9 samples), pressure ulcer group (13 samples), and diabetic foot group (10 samples). Ten slices were taken from pressure ulcer group and diabetic foot group respectively, and 5 slices in each group were used to observe the tissue morphology and expressions of Ki67 and CD31 of wounds respectively with immunofluorescence method. Ten samples from normal skin group, 9 samples from burn wound group, 13 samples from pressure ulcer group, and 10 samples from diabetic foot group were collected for analysis of mRNA expressions of vascular endothelial growth factor 192 (VEGF192), transforming growth factor β (TGF-β), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) , interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α) by real time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with Mann-Whitney U test and Kruskal-Wallis rank-sum test. Results (1) The expression level of Ki67 in diabetic foot group (390±100) was higher than that of pressure ulcer group (182±14, Z=-2.611, P 0.05), while the mRNA expression levels of TGF-β in wounds of pressure ulcer group and diabetic foot group were significantly decreased (H=18.04, 14.50, P 0.05). (4) Compared with those of normal skin group, the mRNA expression levels of VCAM-1 in wounds of burn wound group and pressure ulcer group were significantly increased (H=-22.50, -11.50, P 0.05); the mRNA expression level of ICAM-1 in wounds of burn wound group showed no significant difference (H=-9.50, P>0.05), and the levels of ICAM-1 in wounds of pressure ulcer group and diabetic foot group were significantly decreased (H=16.50, 16.50, P 0.05). (5) Compared with those of normal skin group, except for the mRNA expression level of IL-1β in wounds of diabetic foot group showed no significant difference (H=-10.00, P>0.05), the mRNA expression levels of IL-1β in wounds of burn wound group and pressure ulcer group were significantly increased (H=-32.50, -21.50, P 0.05), the mRNA expression levels of TNF-α in wounds of pressure ulcer group and diabetic foot group were significantly decreased (H=18.04, 14.50, P 0.05). Conclusions The phenotypes of diabetic foot and pressure ulcer vary from the expressions levels of proliferating cell nuclear antigen and blood vessels forming ability to the expression levels of growth factors, cell adhesion factors, and inflammatory cytokines. Key words: Pressure ulcer; Diabetic foot; Cytokines; Gene expression; Chronic refractory wounds

TGF‑β1 promotes the invasion and migration of papillary thyroid carcinoma cells by inhibiting the expression of lncRNA‑NEF

Increased expression levels of transforming growth factor-β1 (TGF-β1) are associated with metastasis in papillary thyroid carcinoma (PTC), although the mechanisms involved remain unknown. The present study aimed to investigate these mechanisms. The results demonstrated that the expression levels of TGF-β1 mRNA were higher and those of lncRNA-NEF were lower in tumor tissues compared with in healthy tissues. High levels of TGF-β1 mRNA and low levels of lncRNA-NEF were also detected in the serum of patients with PTC compared with in healthy controls. Serum levels of TGF-β1 and lncRNA-NEF were significantly associated with lymph node metastasis. Furthermore, TGF-β1 promoted cell migration and invasion of PTC cell lines, but not the normal cell line, whereas lncRNA-NEF overexpression inhibited these phenomena. TGF-β1 also inhibited lncRNA-NEF expression in PTC cell lines, but not in the normal cell line. Conversely, lncRNA-NEF overexpression exhibited no effects on TGF-β1. In conclusion, the present study suggested that TGF-β1 may promote the invasion and migration of PTC cells by inhibiting the expression of lncRNA-NEF.

Suppression of neovascularization in corneal stroma in a TRPA1-null mouse

Corneal neovascularization and inflammatory fibrosis induced by severe injury or infection leads to tissue opacification and even blindness. Transient receptor potential (TRP) channel subtypes contribute to mediating these maladaptive responses through their interactions with other receptors. TRPV1 is one of the contributing channel isoforms inducing neovascularization in an alkali burn mouse wound healing model. VEGF-A upregulation contributes to neovascularization through interaction with its cognate receptors (VEGFR). Since the TRP isoform in this tissue, TRPA1, is also involved, we determined here if one of the pathways mediating neovascularization and immune cell infiltration involve an interaction between VEGFR and TRPA1 in a cauterization corneal mouse wound healing model. Localization of TRPA1 and endothelial cell (EC) CD31 immunostaining pattern intensity determined if TRPA1 expression was EC delimited during cauterization induced angiogenesis. Quantitative RT-PCR evaluated the effects of the absence of TRPA1 function on VEGF-A and TGF-β1 mRNA expression during this process. Macrophage infiltration increased based on rises in F4/80 antigen immunoreactivity. TRPA1 immunostaining was absent on CD31-immunostained EC cells undergoing neovascularization, but it was present on other cell type(s) adhering to EC in vivo. Absence of TRPA1 expression suppressed both stromal neovascularization and inhibited macrophage infiltration. Similarly, the increases occurring in both VEGF-A and TGF-β1 mRNA expression levels in WT tissue were blunted in the TRPA1−/− counterpart. On the other hand, in the macrophages their levels were invariant and their infiltration was inhibited. To determine if promotion by TRPA1 of angiogenesis was dependent on its expression on other unidentified cell types, the effects were compared of pharmacological manipulation of TRPA1 activity on EC proliferation tube formation and migration. In the presence and absence of a fibroblast containing feeder layer. Neither VEGF-induced increases in human vascular endothelial cell (HUVEC) proliferation nor migration were changed by a TRPA1 antagonist HC-030031 in the absence of a feeder layer. However, on a fibroblast feeder layer this antagonist suppressed HUVEC tube formation. In conclusion, during corneal wound healing transactivation by VEGFR of TRPA1 contributes to mediating neovascularization and macrophage infiltration. Such crosstalk is possible because of close proximity between VEGFR delimited expression on EC and TRPA1 expression restricted to cell types adhering to EC.

CD5+ B lymphocytes secrete IL-10 rather than TGF-β1 which control the immune response in autoimmune haemolytic anaemia/Evans syndrome

Objective: To investigate the quantity and secretion function of cytokines-secreted CD5+ B lymphocytes in Autoimmune Haemolytic Anaemia (AIHA)/Evans syndrome (ES) patients.Methods: Twenty-five untreated AIHA/ES patients, 28 remission AIHA/ES patients and 25 healthy controls (HCs) were enrolled in this study. The quantity of CD5+B lymphocytes which produce interleukin-10 (IL-10) (CD5+IL-10+) and transforming growth factor (TGF-β1) (CD5+TGF-β1+) were detected by flow cytometry (FCM). CD5+ B lymphocytes were sorted from peripheral blood (PB) by FCM and the expression of IL-10 and TGF-β1 mRNA in CD5+ B cells were measured by real-time PCR (RT-PCR).Results: The percentage of CD5+IL-10+B cells in CD5+ B lymphocytes in newly diagnosed patients was 82.18 ± 14.78%, which being significantly higher than that of remission AIHA/ES patients (56.68 ± 24.39%) and HCs (51.90 ± 22.95%) (p < .05). The percentage of CD5+IL-10+ B cells in CD5+ B lymphocytes in newly diagnosed patients was negatively correlated with haemoglobin (Hb), complement 3 (C3) (p < .05) and positively correlated with lactate dehydrogenase (LDH), total bilirubin (TBIL) and indirect unconjugated bilirubin (IBIL) (p < .05). The expression level of IL-10 mRNA in CD5+ B lymphocytes of newly diagnosed patients (49.34 ± 22.84) was higher than that of remission patients (3.97 ± 3.83) and HCs (1.78 ± 1.66) (p < .05). There was no significant difference among three groups with the proportion of CD5+TGF-β1+ B lymphocytes and the expression level of TGF-β1 mRNA in CD5+B lymphocytes (p > .05).Conclusions: CD5+ B lymphocytes could secrete IL-10 rather than TGF- β1 which control the immune response in AIHA/ES.

Vitamin D supplementation decreases the expression of TGF-β1 in rats with asthma and thereby suppressing airway remodeling

Objective To investigate the preventive and therapeutic effects of 1, 25-(OH)2D3 on airway remodeling in asthmatic rats, and to explore the effect of 1, 25-(OH)2D3 on the expression of transforming growth factor-β1 (TGF-β1) and the related mechanism between them. Methods Seventy male Sprague Dawley (SD) rats were randomly divided into seven groups: normal control group (group A, n=10), asthmatic group (group B, n=10), dexamethasone group (group C, n=10), 1, 25-(OH)2D3 2 μg/(kg·d) group (group D, n=10), 1, 25-(OH)2D3 4 μg/(kg·d) group (group E, n=10), 1, 25-(OH)2D3 8 μg/(kg·d) group (group F, n=10), 1, 25(OH)2D3 16 μg/(kg·d) group (group G, n=10). Rats were sensitized and challenged with ovalbumin (OVA) to establish the asthmatic model in B, C, D, E, F, G group respectively. Rats in group B and group C were fed with normal saline 10 ml/(kg·d) and dexamethasone 2 mg/(kg·d) before atomization. While group D-G was 1, 25- (OH)2D3 intervention group, rats were fed with vitamin D 2, 4, 8, 16 μg/(kg·d) before atomization. The number of total and different cells of the bronchoalveolar lavage fluid (BALF) from the 7 groups was counted for analysis; The change of the thickness, the area of airway wall and the number of ASMC nucleus and pulmonary tissue changes were observed by microscope. The protein and mRNA expression of TGF-β1 in the lung tissue was measured by immunochemistry and real-time polymerase chain reaction (RT-PCR). Results ⑴ The airway remodeling and pulmonary inflammatory exudation in the B group were significantly heavier than those in the A, C and G groups (P<0.01); and the difference between groups of different doses of vitamin D [D group (minimum dose) and G group(the maximum dose) ] was statistically significant (P<0.01). ⑵ The results of TGF-β1 mRNA and protein levels in rats of each group showed that the expression level of TGF-β1 mRNA and integrated option density (IOD) value in A group were significantly lower than those in other groups (P<0.01); the expression level of TGF-β1 mRNA and IOD value in B group was higher than the other groups (P<0.01); Compared with the B group, the expression level of TGF-β1 mRNA and IOD value in G group (large dose vitamin D group) was decreased, with statistically difference. Conclusions In the rat model of asthma, 1, 25-(OH)2D3 may alleviate airway remodeling by regulating the expression of TGF- beta 1 and play its opposite biological effect in the prevention and treatment of asthma. Key words: Asthma; Rats; Transforming growth factor beta1; Vitamin D; Airway remodeling