TGF-β1 In Group Research Articles

Conbercept reverses TGF-β2-induced epithelial-mesenchymal transition in human lens epithelial cells by regulating the TGF-β/Smad signaling pathway

To investigate the mechanism by which conbercept reverses transforming growth factor-β2 (TGF-β2)-induced epithelial-mesenchymal transition (EMT) in human lens epithelial cells (HLECs). Cultured HLEC SRA01/04 cells were treated with TGF-β2, conbercept, or both, and the changes in cell proliferation, apoptosis, and migration were observed using MTT assay, flow cytometry, scratch assay, and Transwell assay. Western blotting and qRT-PCR were used to detect the changes in the expression of EMT-related epithelial cell markers (E-Cadherin, α-SMA, and Snail), extracellular matrix components, and genes related to the TGF-β/Smad signaling pathway. Conbercept significantly reduced TGF-β2-induced EMT of SRA01/04 cells, decreased the expression levels of mesenchymal and extracellular matrix markers α-SMA, Snail, collagen I, collagen IV, and FN1, and upregulated the protein and mRNA expressions of E-cadherin (P <0.05). Transwell assay showed significantly lower cell migration ability in TGF-β2+conbercept group than in TGF-β2 group (P <0.05). Conbercept also inhibited the increase in Smad2/3 phosphorylation levels in HLEC-SRA01/04 cells with TGF-β2-induced EMT (P <0.01). Conbercept inhibits TGF-β2 induced EMT by downregulating the expression of pSmad2/3 in TGF-β/Smad signaling pathway, indicating a potential therapeutic strategy against visual loss induced by posterior capsule opacification.

Bone morphogenetic protein-6 suppresses TGF-β2-induced epithelial-mesenchymal transition in retinal pigment epithelium.

To evaluate the effect of bone morphogenetic protein-6 (BMP-6) on transforming growth factor (TGF)-β2-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE). Adult retinal pigment epithelial cell line (ARPE-19) were randomly divided into control, TGF-β2 (5 µg/L), and BMP-6 small interfering RNA (siRNA) group. The cell morphology was observed by microscopy, and the cell migration ability were detected by Transwell chamber. The EMT-related indexes and BMP-6 protein levels were detected by Western blotting. Furthermore, a BMP-6 overexpression plasmid was constructed and RPE cells were divided into the control group, TGF-β2+empty plasmid group, BMP-6 overexpression group, and TGF-β2+BMP-6 overexpression group. The EMT-related indexes and extracellular regulated protein kinases (ERK) protein levels were detected. Compared with the control group, the migration of RPE cells in the TGF-β2 group was significantly enhanced. TGF-β2 increased the protein expression levels of α-smooth muscle actin (α-SMA), fibronectin and vimentin but significantly decreased the protein levels of E-cadherin and BMP-6 (P<0.05) in RPE. Similarly, the migration of RPE cells in the BMP-6 siRNA group was also significantly enhanced. BMP-6 siRNA increased the protein expression levels of α-SMA, fibronectin and vimentin but significantly decreased the protein expression levels of E-cadherin (P<0.05). Overexpression of BMP-6 inhibited the migration of RPE cells induced by TGF-β2 and prevented TGF-β2 from affecting EMT-related biomarkers (P<0.05). BMP-6 prevents the EMT in RPE cells induced by TGF-β2, which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy.

Secreted frizzled-related protein 5 protects against renal fibrosis by inhibiting Wnt/β-catenin pathway.

Renal fibrosis (RF) is an important pathogenesis for renal function deterioration in chronic kidney disease. Secreted frizzled-related protein 5 (SFRP5) is an anti-fibrotic adipokine but its direct role on RF remains unknown. It was aimed to study the protective effect of SFRP5 against RF and interference with Wnt/β-catenin signaling pathway for the first time. First, the therapeutic efficacy of SFRP5 was evaluated by adenovirus overexpression in rats with unilateral ureteral obstruction (UUO) in vivo. Thirty-six rats were randomly divided into the sham, UUO, and SFRP5 (UUO + Ad-SFRP5) groups. Half rats in each group were selected at random for euthanasia at 7 days and the others until 14 days. Then, the transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) was established in HK-2 cells in vitro. The cells were divided into four groups: the control group, the TGF-β1 group, the TGF-β1 + SFRP5 group, and the TGF-β1 + SFRP5 + anti-SFRP5 group. The makers of EMT and Wnt/β-catenin pathway proteins were investigated. In the UUO model, expression of SFRP5 showed compensatory upregulation, and adenoviral-mediated SFRP5 over-expression remarkably attenuated RF, as demonstrated by maintenance of E-cadherin and suppression of α-smooth muscle actin (SMA). In vitro, SFRP5 was shown to inhibit TGF-β1-mediated positive regulation of α-SMA, fibronectin, collagen I but negative regulation of E-cadherin. Furthermore, SFRP5 abrogated activation of Wnt/β-catenin, which was the essential pathway in EMT and RF pathogenesis. The changes after a neutralizing antibody to SFRP5 confirmed the specificity of SFRP5 for inhibition. These findings suggest that SFRP5 can directly ameliorate EMT and protect against RF by inhibiting Wnt/β-catenin pathway.

TGF-β3 increases the severity of radiation-induced oral mucositis and salivary gland fibrosis in a mouse model

Purpose Toxicities from head and neck (H&N) radiotherapy (RT) may affect patient quality of life and can be dose-limiting. Proteins from the transforming growth factor beta (TGF-β) family are key players in the fibrotic response. While TGF-β1 is known to be pro-fibrotic, TGF-β3 has mainly been considered anti-fibrotic. Moreover, TGF-β3 has been shown to act protective against acute toxicities after radio- and chemotherapy. In the present study, we investigated the effect of TGF-β3 treatment during fractionated H&N RT in a mouse model. Materials and methods 30 C57BL/6J mice were assigned to three treatment groups. The RT + TGF-β3 group received local fractionated H&N RT with 66 Gy over five days, combined with TGF-β3-injections at 24-hour intervals. Animals in the RT reference group received identical RT without TGF-β3 treatment. The non-irradiated control group was sham-irradiated according to the same RT schedule. In the follow-up period, body weight and symptoms of oral mucositis and lip dermatitis were monitored. Saliva was sampled at five time points. The experiment was terminated 105 d after the first RT fraction. Submandibular and sublingual glands were preserved, sectioned, and stained with Masson’s trichrome to visualize collagen. Results A subset of mice in the RT + TGF-β3 group displayed increased severity of oral mucositis and increased weight loss, resulting in a significant increase in mortality. Collagen content was significantly increased in the submandibular and sublingual glands for the surviving RT + TGF-β3 mice, compared with non-irradiated controls. In the RT reference group, collagen content was significantly increased in the submandibular gland only. Both RT groups displayed lower saliva production after treatment compared to controls. TGF-β3 treatment did not impact saliva production. Conclusions When repeatedly administered during fractionated RT at the current dose, TGF-β3 treatment increased acute H&N radiation toxicities and increased mortality. Furthermore, TGF-β3 treatment may increase the severity of radiation-induced salivary gland fibrosis.

Effect of curcumin nanoparticles on proliferation and migration of mouse airway smooth muscle cells and airway inflammatory infiltration.

Curcumin (CUR) possesses the capability to inhibit various inflammatory factors, exert anti-inflammatory effects, and alleviate asthma attacks; however, its hydrophobicity and instability significantly impede its clinical application. In this study, we synthesized CUR-loaded nanoparticles (CUR-NPs) and evaluated their impact on the proliferation, migration, and inflammatory infiltration of mouse airway smooth muscle cells (ASMCs), while investigating their underlying mechanisms. To achieve this objective, ASMCs were isolated from BALB/c mice and subjected to TGF-β1-induced cell proliferation and migration. Our findings demonstrate that CUR-NPs effectively regulate the release of CUR within cells with superior intracellular uptake compared to free CUR. The CCK-8 assay results indicate that the blank carrier does not exhibit any cytotoxic effects on cells, thus rendering the impact of the carrier itself negligible. The TGF-β1 group exhibited a significant increase in cell proliferation, whereas treatment with CUR-NPs significantly suppressed TGF-β1-induced cell proliferation. The findings from both the cell scratch assay and transwell assay demonstrated that TGF-β1 substantially enhanced cell migration, while CUR-NPs treatment effectively attenuated TGF-β1-induced cell migration. The Western blot analysis demonstrated a substantial increase in the expression levels of TGF-β1, p-STAT3, and CTGF in ASMCs following treatment with TGF-β1 when compared to the control group. Nevertheless, this effect was effectively counteracted upon administration of CUR-NPs. Furthermore, an asthma mouse model was successfully established and CUR-NPs were administered through tail vein injection. The serum levels of TGF-β1 and the expression levels of TGF-β1, p-STAT3, and CTGF proteins in the lung tissue of mice in the model group exhibited significant increases compared to those in the control group. However, CUR-NPs treatment effectively attenuated this change. Our research findings suggest that CUR-NPs possess inhibitory effects on ASMC proliferation, migration, and inflammatory infiltration by suppressing activation of the TGF-β1/p-STAT3/CTGF signaling pathway, thereby facilitating inhibition of airway remodeling.

The Expression of Circ_0000615 in Tenon’s Capsule Fibroblasts and Its Effect on Cell Proliferation and Migration

To explore the expression of circ_0000615 in HTFs and its effect on cell proliferation and migration. With in vitro culture of HTFs, qRT-PCR was performed to detect the expression of circ_0000615 in HTFs. Cells in logarithmic phase were taken for subsequent experiments, and the following groups were constructed, including HTF blank control group (C group); HTFs+10 ng/mL TGF-β1 group (TGF-β1 group); HTFs+si-NC group (si-NC group); and HTFs+si-circ_0000615 group (si-circ_0000615 group). CCK-8 assay was performed to detect cell proliferation, Cell Monoclonal Assay was used to detect Cell Monoclonal Formationand, Transwell assay was conducted simultaneously to detect cell migration. According to the results of qRT-PCR, compared with C group, after induction of HTFs with TGF-β1 for 24 h and 48 h, TGF-β1 group showed significantly increased expressions of circ_0000615, with statistically significant differences (P &lt; 0.05). After induction of HTFs with TGF-β1, compared with C group, TGF-β1 group had enhanced cell proliferation, monoclonal formation and migration, showing statistically significant differences (P &lt; 0.05). Furthermore, after cell transfections for HTFs, compared with si-NC group, si-circ_0000615 group showed obviously downregulated expression of circ_0000615 in HTFs, accompanied by evidently weakened cell proliferation, monoclonal formation and migration, statistically significant differences (P &lt; 0.05). Circ_0000615 is highly expressed in HTFs. A silenced expression of circ_0000615 may inhibit the proliferation and migration of HTFs.

Abstract 13116: Profiling the Role of Gut Microbiota-Derived Trimethylamine- N -Oxide in Cardiac Fibrosis — Evidence From a Primary Human in vitro Model

Introduction: The gut derived trimethylamine- N -oxide (TMAO) is correlated with increased atrial inflammation and fibrosis and associated with atrial fibrillation, heart failure, and stroke. However, the mechanisms mediating these links remain unresolved. Hypothesis: TMAO and its precursor metabolite L-carnitine (LCART) induce the transformation of atrial fibroblasts into pro-fibrotic phenotypes. Aim: To characterise the association between TMAO and cardiac fibrosis. Methods: Primary human cardiac (atrial) fibroblasts (hCFs) were sourced from healthy donors. hCFs were starved for 24 h and treated with PBS (control), 20 ng/mL transforming growth factor beta 1 (TGFβ1), 10 mM TMAO, or 1.0 mM LCART for 72 h (n=6 in each), then analysed by flow cytometry for α-smooth muscle actin (αSMA) expression. Unbiased proteomics was performed using LC-MS/MS to determine protein expression profile. Results: Analysis of αSMA expression revealed 3 hCF states: quiescent (Fbs), quiescent-to-myofibroblast (intermediate [Fbs-myoFbs]), and fully activated myofibroblast (myoFbs), (Fig-A). Compared to controls, there was no difference in the intermediate state after TGFβ1, TMAO, or LCART treatment (p=0.30). TMAO and LCART resulted in a significant induction of myofibroblast states compared to controls and TGFβ1 group. Unbiased proteomics identified 92, 65, and 43 proteins to be overexpressed and 84, 46, and 78 proteins downregulated following TGFβ1, TMAO, and LCART treatments (Fig-B). Both TGFβ1 and TMAO demonstrated shared enrichment for oxidative stress-regulated ferroptosis pathway (fold enrichment [FE] 31.8 and 22.2, p &lt;.01 and 0.03); LCART showed enrichment for cell motility (FE 3.8, p=0.03). Conclusions: TMAO and its precursor L-carnitine are associated with activation of pro-fibrotic states, which may be due to oxidative stress and abnormal cell migration related mechanisms.

Effect of entecavir on IL-6/STAT3/SOCS3 pathway in patients with chronic hepatitis B-induced liver fibrosis

Purpose: To evaluate the impact of entecavir (ENT) on patients with liver fibrosis resulting from chronic hepatitis B (CHB) and its association with the interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3)/suppressor of cytokine signaling 3 (SOCS3) pathway.Methods: Thirty-one patients with liver fibrosis received ENT at a dose of 0.5 mg/day for 48 weeks. Relevant protein levels in patient’s serum before and after treatment were assayed using enzyme-linked immunosorbent assay (ELISA). Furthermore, human hepatic stellate cells (HSCs) were cultured in vitro and divided into three groups: control, transforming growth factor beta 1 (TGF-β1) induction (TGF-β1 group), and ENT treatment (TGF-β1 + ENT group). Protein levels in the supernatant were assayed using ELISA, while the expression levels of related genes were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Expression of α-SMA was visualized using immunofluorescence assay and the relevant protein levels were determined by Western blotting.Results: Treatment with ENT significantly decreased (p &lt; 0.01) IL-6 and STAT3 expression, increased SOCS3 expression and significantly reduced (p &lt; 0.01) the concentrations of hyaluronic acid (HA), type IV collagen (IVC), laminin (LN) and pro-collagen type III (PCIII) in patients with liver fibrosis. TGF-β1 significantly (p &lt; 0.01) elevated IL-6, STAT3 and Col-I expressions and a tissue inhibitor of metalloproteinases-1 (TIMP-1) suppressed the expression of SOCS3 in human HSCs and induced fibrosis. Entecavir mitigated TGF-β1-induced fibrogenesis in HSCs (p &lt; 0.01).Conclusion: Entecavir has a positive effect on liver fibrosis resulting from CHB by regulating IL- 6/STAT3/SOCS3 pathway. Future research will focus on conducting larger clinical trials to further validate these findings and explore the long-term effects of ENT on liver fibrosis progression and patient outcomes.

Effect of alpha lipoic acid on epithelial mesenchymal transition in SKOV-3 cells

BackgroundOvarian cancer is the fifth leading cause of cancer-related death in women. Patients are usually diagnosed with advanced tumor metastass. Epithelial over cancer cells spread from primary tumor by undergoing epithelial mesenchymal transition (EMT). It has been suggested that alpha lipoic acid (ALA), a natural antioxidant lipophilic compound, reduces the oxidative stress by causing apoptosis and inhibition of proliferation of cell in cancer cells. The aim of our study was to establish a transforming growth factor β1 (TGF β1) dependent epithelial mesenchymal transition model in the SKOV-3 ovarian adenocarcinoma cell line which is an epithelial subtype of ovarian cancer and to investigate the effects of alpha lipoic acid on EMT and ovarian cancer migration. MethodsFor establish an EMT model, SKOV-3 cells were treated with different dose of TGF β1 and XTT cell viability kit was used to find IC 50 dose of ALA. Four different groups that are control, TGF β1, ALA and ALA + TGF β1 were created. Changes in the expression of genes related to EMT markers that are E-cadherin, vimentin, Snail, Slug, Twist and Zeb were analyzed with quantitative real-time PCR. These proteins were determined with the immunocytochemistry method. The migration capacity was analyzed with wound healing assay. Matrigel invasion capacity test was used to show invasion and colonization test to show colonization. ResultsThe dose of TGF β1 was determined 100 ng/ml at 72 h, the IC50 dose of ALA 219.033 µM at 48 h was determined. EMT markers in the TGF β1 group were compatible with EMT and it was shown to inhibit EMT in the groups given ALA. According to wound healing, colonization and invasion experiments, proliferation and invasion increased in TGF β1 group, but decreased in ALA and combined groups (p < 0.05). ConclusionThese results indicate that ALA suppresses the metastasis of ovarian cancer cells by regulating EMT, implying that ALA might be a potential therapeutic agent for the treatment of ovarian cancer.

Effect of Tuina along "bladder meridian" alleviating intervertebral disc degeneration by regulating the transforming growth factor-β1/Smad signaling pathway in a rabbit model.

The aim of this study was to investigate the protective effects of Tuina (a traditional Chinese massage therapy) on intervertebral disc (IVD) degeneration and the regulatory mechanisms of the transforming growth factor-β1 (TGF-β1)/small mothers against decapentaplegic (Smad) signaling pathway. Thirty New Zealand white rabbits were randomized into five groups: the control group, model group, model + Tuina group (Tuina group), model + TGF-β1 group (TGF-β1 group), and model + TGF-β1 inhibitor SB431542 group (SB431542 group). The model was established by posterolateral annulus fibrosus puncturing (AFP). Recombinant TGF-β1 and inhibitor SB431542 was injected into the TGF-β1 group and SB431542 group with a microsyringe, respectively. The rabbits in the Tuina group received Tuina treatment along the bladder meridian for 4 weeks. Magnetic resonance imaging (MRI) was performed on rabbits before AFP and after 4 weeks of intervention. Lumbar IVDs (L2-L3 to L4-L5) were harvested after intervention. Histopathological changes in the IVDs were measured by hematoxylin and eosin (HE) staining. Type I collagen was analyzed by immunohistochemistry detection. The expression level of matrix metalloproteinase-3 (MMP3) was determined by enzyme-linked immunosorbent assay. Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated nick end labeling and Western blotting. Real-time polymerase chain reaction and Western blotting were used to analyze the expression of TGF-β1 and Smad2/3/4 and a disintegrin and metalloproteinase with thrombospondin motifs 5. Posterolateral AFP induced IVD degeneration in rabbits with histopathological damage and noticeable changes in MRI images. Tuina alleviated histo-pathological changes and reversed the expression of extracellular matrix degeneration-related molecules and apoptosis-related proteins. Furthermore, AFP induced the activation of TGF-β1 and Smad2/3/4, whereas Tuina therapy markedly reduced the protein expression of Smad2/3 and the gene expression of TGF-β1 and Smad2/3/4. Additionally, the TGF-β1/Smad signaling pathway was activated in the TGF-β1 group, while the TGF-β1/Smad signaling pathway was inhibited in the SB431542 group. Posterolateral AFP induced disc degeneration as determined by MRI assessment and histological analysis. Tuina alleviated disc degeneration, possibly by inhibiting the fibrotic response mediated by the TGF-β1/Smad pathway, thus alleviating extracellular matrix degeneration and reducing cell apoptosis.

Ruyong formula improves thymus function of CUMS-stimulated breast cancer mice

Ethnopharmacological relevanceRuyong Formula (RYF) is a famous Chinese herbal formula composed of 10 traditional Chinese herbs. It has been used as a therapeutic agent for breast cancer patients with depressive symptoms in China. However, its underlying pharmacological mechanism remains unclear. Aim of the studyThis study aimed to explore the mechanism of RYF on the changes of thymus immune function in breast cancer body under mood disorders such as depression/anxiety. Materials and methodsThe chronic unpredictable mild stress (CUMS) was used to stimulate 4T1 breast cancer mice. The behavioral changes, 5-hydroxytryptamine (5-HT) level in brain, cytokeratin 5 (CK5) and 8 (CK8) expression in thymus, the proportion of T cell subsets, the thymic output, phenotypic changes of thymus epithelial cells (TECs), the expression levels of immune-related factors and downstream proteins of TSLP were analyzed after RYF treatment. ResultsIn CUMS stimulated group, the level of 5-HT in brain was significantly increased after RYF treatment. The output function of the thymus was improved, and the number of TECs in the medulla (CK5+), the proportion of CD3+CD4−CD8− (Double negative) and CD3+CD4+CD8+ (Double positive) T cells were all increased. The mRNA level of TSLP in mouse thymus was significantly decreased, but increased for IL-7. The protein levels of TSLP and Vimentin were decreased, but increased for p-STAT3, p-JAK2, E-cadherin, and p-PI3K p55 in vivo. In vitro study was showed the levels of Snail 1, Zeb 1 and Smad increased significantly in TGF-β1 group, and RYF could reverse their expression. ConclusionsRYF could restore the structure and function of the thymus in depressed breast cancer mice by reversing the phenotypic changes of TECs and activating the JAK2/STAT3/PI3K pathway.

Adiponectin inhibits TGF-β1-induced skin fibroblast proliferation and phenotype transformation via the p38 MAPK signaling pathway.

The aim of this study was to investigate the effects of adiponectin (APN) on the proliferation and phenotypic transformation of human skin fibroblasts (HSFs) induced by TGF-β1. Primary fibroblast cultures were collected from prepuce surgery, and the cell viability and proliferative activity of HSFs were detected by Cell Counting Kit-8 and EdU assays. In addition, cell migration was detected by Transwell assay. The protein levels of related genes in HSF were detected by Western blotting. The results showed that the proliferation and migration abilities of HSF in the TGF-β1 group were significantly improved, and the relative protein expression levels of PCNA, α-SMA, and Collagen I in the TGF-β1 group were greatly increased. Furthermore, TGF-β1 stimulated the phosphorylation of p38 in HSF, while APN pretreatment significantly inhibited the TGF-β1-induced phosphorylation of p38. Additionally, blocking the p38 MAPK signaling pathway relieved the injury in the HSF induced by TGF-β1 and enhanced the therapeutic effect of APN in the TGF-β1-treated HSF. In conclusion, APN inhibits TGF-β1-induced HSF proliferation and myofibroblast phenotypic transformation by activating the p38 MAPK signaling pathway. APN is expected to become a potential target for preventing and treating skin fibrosis and pathological scars.

Effects of atorvastatin on the function of Tenon's capsule fibroblasts in human eyes.

This study aimed to explore the role of atorvastatin (ATO) in the prevention and treatment of the scarring of filtration channels after glaucoma surgery. Human Tenon's capsule fibroblasts (HTFs) were co-cultured with various concentrations of ATO. First, Cell Counting Kit-8 assay was performed to evaluate the effects of various concentrations of ATO on the viability of HTFs. Then, after the ATO stimulated the HTFs for 24h, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to evaluate the apoptosis of HTFs. Transwell assay was also performed to evaluate the migration of HTFs. Moreover, enzyme-linked immunosorbent assay (ELISA) was performed to detect the protein expression levels of transforming growth factor-β1 (TGF-β1) and TGF-β2 in the cell culture supernatant of HTFs. Western blot was carried out to detect the protein expression levels of smooth muscle actin (SMA), p38, Smad3, fibronectin, collagen I and collagen III in different groups. The results revealed that ATO could inhibit the proliferation and migration of HTFs. Based on the TUNEL assay, 100μM and 150μM ATO could induce cell apoptosis. The ELISA results indicated that ATO could down-regulate the expression level of TGF-β2, and western blot analysis revealed that the protein expression levels of SMA, p38, Smad3, fibronectin, collagen I and collagen III in the TGF-β2 group were all up-regulated compared with the control group, whereas the addition of ATO could reverse this up-regulation. ATO could inhibit the proliferation and migration of HTFs and induce their apoptosis. It was preliminary proven that ATO could inhibit the signaling pathway induced by TGF-β. It is suggested that ATO could be a basis for the treatment of the scarring of filtration channels after glaucoma surgery.

Poster 136: Augmented Meniscus Radial Tear Healing via Local Transforming Growth Factor-beta 1 (TGF- β1) Gene Therapy in a Rat Model

Objectives: Meniscus tears cause significant musculoskeletal impairment and are among the most common knee injuries. Tears in the inner third of the meniscus have intrinsically poor healing capacity due to insufficient blood supply. In an effort to improve healing, use of biologic augments is a promising area of investigation. Tissue engineering strategies, such as growth factor gene therapy, have emerged as encouraging approaches to improving meniscal healing. Transforming Growth Factor-Beta 1 (TGF-β1) gene therapy has shown promise in vitro and ex vivo as a therapeutic strategy, but its effects on meniscus healing in vivo remain unknown. This study aimed to determine whether local delivery of viral vectors carrying the TGF-β1 gene could improve the healing of a meniscus tear involving the avascular zone in vivo in a rat model. Methods: Recombinant adenoviruses were generated using the AdEasy system. Briefly, the coding regions of mouse TGFβ1 were PCR amplified and cloned into an adenoviral shuttle vector and subsequently used to generate recombinant adenoviruses in HEK293 cells. The resulting adenoviruses were designated as Adv-TGF-β1. An analogous adenovirus expressing only GFP (Adv-GFP) was used as mock virus controls. To achieve local delivery of viral vectors to the injury site, we utilized a thermoresponsive biodegradable polydiolcitrate-gelatin delivery scaffold (PPCNg). A full-thickness radial tear was made in the medial meniscus of the left hind leg of twelve Wistar rats. Rats were divided into two groups: one receiving a mixture of PPCNg+TGF-β1 (n = 3) and the other receiving PPCNg+GFP (n = 3). A 4ºC pre-chilled solution of PPCNg seeded with Adv-TGF-β1 or Adv-GFP at 1011 infectious viral particles/injection was administered at the site of the induced meniscus tear. After 3 weeks, rats were euthanized, and the menisci were histologically evaluated by hematoxylin and eosin (H&amp;E) staining. The histologic assessment and scoring of meniscus tissue wound healing were done by one blinded and one unblinded observer, each using selected wound healing parameters as described in Table 1, and the results were averaged. The intra-class correlation coefficient (ICC) was computed based on a mean rating (k=2), absolute agreement, and two-way random effects model to assess the agreement between the wound healing score rating of the two observers. Statistical significance between groups was determined using the Mann-Whitney Wilcoxon rank sum test. A value of p &lt; 0.05 was considered statistically significant. All animal procedures were performed according to an Institutional Animal Care and Use Committee protocol at the home institution. Results: Immediately after and 3 weeks post-surgery, there was no evidence of infection, dehiscence, or functional deficit. Histologic evaluation of the sections (n = 8/group) of harvested rat menisci (n = 2/group) revealed improved healing of tears in the PPCNg+TGF-β1 group compared to the control group (5.7 ± 0.48 vs. 5.0 ± 0.89, p = 0.011) (Figure 1B). The lesion area demonstrated significantly greater neovascularization (2.75 ± 0.45 vs. 2.06 ± 0.68, p = 0.004), better collagen organization (1.94 ± 0.25 vs. 1.62 ± 0.50, p = 0.038), and increased hypercellularity (1.0 ± 0.00 vs. 1.3 ± 0.48, p = 0.018) in the PPCNg+TGF-β1 group compared to the controls (Figure 1C). Moderate absolute agreement between the two raters was observed in their wound healing score ratings (ICC = 0.615). Conclusions: Our results indicate that PPCNg-mediated delivery of Adv-TGF-β1 enhanced the early (3 weeks) healing of the meniscus lesion in rats compared with control (Adv-GFP) gene therapy. Further, localized administration of Adv-TGF-β1 induced greater neovascularization, better organization of collagen fibers, and increased hypercellularity at the injury site in the Adv-TGFβ-1 rat group compared with controls at 3 weeks post-injury. These results together suggest that TGF-β1 gene therapy may enhance meniscus healing by inducing more cellular proliferation and infiltration and greater neovascularization at the injury site to serve as nourishment for the establishment of new wound matrix, leading to more significant deposition and remodeling of mature collagen matrix. Further study that includes larger sample sizes, confirmation of successful gene transfer to meniscal cells around the tear site, and investigations of both later time points of wound healing and the mechanisms by which local TGF-β1 gene therapy may modulate augmented meniscus healing in this rat model of meniscal defect is necessary to better determine the potential future clinical utility of local TGF-β1 gene therapy to treat of meniscus tears with poor healing capacity. [Table: see text]

NT-3 Combined with TGF-β Signaling Pathway Enhance the Repair of Spinal Cord Injury by Inhibiting Glial Scar Formation and Promoting Axonal Regeneration.

This study investigated the mechanism of neurotrophin-3 (NT-3) in promoting spinal cord injury repair through the transforming growth factor-beta (TGF-β) signaling pathway. A mouse model of spinal cord injury was established. Forty C57BL/6J mice were randomized into model, NT-3, NT-3 + TGF-β1 and NT-3 + LY364947 groups. The Basso-Beattie-Bresnahan (BBB) scores of the NT-3 and NT-3 + LY364947 groups were significantly higher than the model group. The BBB score of the NT-3 + TGF-β1 group was significantly lower than NT-3 group. Hematoxylin-eosin staining and transmission electron microscopy showed reduction in myelin sheath injury, more myelinated nerve fibers in the middle section of the catheter, and relatively higher density and more neatly arranged regenerated axons in the NT-3 and NT-3 + LY364947 groups compared with the model and NT-3 + TGF-β1 groups. Immunofluorescence, TUNEL and Western blot analysis showed that compared with model group, the NEUN expression increased, and the apoptosis and Col IV, LN, CSPG, tenascin-C, Sema 3A, EphB2 and Smad2/3 protein expression decreased significantly in the NT-3 and NT-3 + LY364947 groups; the condition was reversed in the NT-3 + TGF-β1 group compared with the NT-3 group. NT-3 combined with TGF-β signaling pathway promotes astrocyte differentiation, reduces axon regeneration inhibitory molecules, apoptosis and glial scar formation, promotes axon regeneration, and improves spinal cord injury.

Eriocalyxin B mediates the migration and inflammation of TGF β2 induced human lens epithelial cells by inhibiting JAK/STAT3 pathway

Purpose: To study the role of Eriocalyxin B (EriB) in the migration and inflammation of TGF-β2-induced human lens epithelial cells (hLECs), and to elucidate the molecular mechanisms involved. Methods: The hLECs cultured in vitro were divided into 5 groups, viz, control, TGFβ2, TGFβ2+2 μM EriB, TGFβ2+4 μM EriB, and TGFβ2+8 μM EriB groups. CCK-8, clone formation and Edu labeling assays were performed to assess the effect of EriB on the proliferation of hLECs cells. To determine the role of EriB in cell migration, Transwell and wound healing assays were used. The levels of vimentin, α-SMA, snail, TNF-α，IL-1β，IL-6, P65, p-P65, p-JAK2, JAK2, p-STAT3, STAT3, and β-catenin in hLECs cells were evaluated by enzyme-linked immunosorbent assay (ELISA) and western blot analysis in order ascertain the signaling pathways involved. Results: The rate of cell proliferation significantly decreased in TGFβ2+2μM EriB, TGFβ2+4μM and TGFβ2+8μM groups compared with TGFβ2 group (p &lt; 0.001). In addition, the migration of hLECs cells and epithelial mesenchymal transition were inhibited by EriB in a dose-dependent way (p &lt; 0.001). ELISA results showed that compared to TGFβ2 group, TNF-α, IL-1β and IL-6 levels in EriB group significantly decreased (p &lt; 0.001). The levels of TNF-α, IL-1β, IL-6, p-P65/P65, p-JAK2/JAK2, p-STAT3/STAT3 and metastasis-associated proteins (α-SMA and snail) in hLECs cells were downregulated by EriB (p &lt; 0.001). Furthermore, vimentin level was increased by EriB (p &lt; 0.001). Conclusion: The results show that EriB inhibits the growth, metastasis and inflammation of hLECs cells by inhibiting JAK/STAT3 pathway, thus indicating that this pathway is a potential therapeutic target for treating cataract

PD02-02 THE ROLE OF TRANSFORMING GROWTH FACTOR Β IN A PROSTATIC FIBROSIS UNDER SUPPRESSION OF DIHYDROTESTOSTERONE IN HIGH FAT DIET INDUCED OBESITY RAT MODEL

You have accessJournal of UrologyCME1 Apr 2023PD02-02 THE ROLE OF TRANSFORMING GROWTH FACTOR Β IN A PROSTATIC FIBROSIS UNDER SUPPRESSION OF DIHYDROTESTOSTERONE IN HIGH FAT DIET INDUCED OBESITY RAT MODEL Shinsuke Mizoguchi, Kenichi Mori, Hiromitsu Mimata, and Toshitaka Shin Shinsuke MizoguchiShinsuke Mizoguchi More articles by this author , Kenichi MoriKenichi Mori More articles by this author , Hiromitsu MimataHiromitsu Mimata More articles by this author , and Toshitaka ShinToshitaka Shin More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003219.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Although 5 alpha reductase inhibitors (5ARIs) can reduce prostatic volume by blocking production of dihydrotestosterone (DHT) and is widely used to treat symptomatic benign prostatic hyperplasia (BPH), the histological response to the treatment by 5ARIs is often heterogeneous and lower urinary tract symptoms can persist. Thus, a 5ARIs induced pathway related to treatment refractory is supposed to exist. To elucidate the pathways implicated to unsuccessful treatment for BPH patients, this study investigated changes in morphological and gene expression profiles in the prostate under DHT suppression using high fat diet induced obesity rat model. METHODS: Male Wistar rats at 8 weeks old were divided into four groups; rats with normal diet group (ND, n=5), rats with high fat diet induced obesity group (HFD, n=5), HFD treated by dutasteride (D, n=4), and HFD treated by dutasteride and tranilast (D+T, n=4), which is known to inhibit TGFβ. The high fat diet contains 32% fat. These rats were maintained for twelve weeks followed by two weeks treatment by oral administration of placebo or dutasteride (0.5 mg/kg) or tranilast (150 mg/kg) daily. Lateral lobes of prostate were harvested to evaluate prostatic weight, histological change and mRNA expression of IL1β and TGFβ in the prostate by qPCR. RESULTS: HFD showed significantly increased body weight, visceral fat and prostatic weight as well as significant upregulation of gene expressions in IL1β and TGFβ compared to ND. In contrast, HFD treated by dutasteride demonstrated significantly decreased prostatic weight in association with downregulation of IL1β and upregulation of TGFβ with compared to HFD with placebo. Furthermore, dutasteride administration induced prostatic fibrosis as evidenced by masson trichrome staining whereas the addition of tranilast to dutasteride suppressed prostatic tissue fibrosis in association with reduced weight of prostatic tissue and downregulation of TGFβ in group D+T compared to group D. CONCLUSIONS: This study demonstrated prostatic fibrosis in association with upregulation of TGFβ as well as decreased prostatic weight and improvement of prostatic fibrosis by TGFβ inhibition. These results suggest that TGFβ signaling has an important role in development of prostatic fibrosis under 5ARIs treatment possibly leading to treatment refractory. Therefore, TGFβ signaling pathway could be an therapeutic target for BPH. Source of Funding: JSPS KAKENHI Grant Number 20K18094 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e69 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shinsuke Mizoguchi More articles by this author Kenichi Mori More articles by this author Hiromitsu Mimata More articles by this author Toshitaka Shin More articles by this author Expand All Advertisement PDF downloadLoading ...

Role of TGF-β3 and bone marrow mesenchymal stem cells on regeneration of myometrial injury in rats

ObjectiveThis study aimed to explore therapeutic effect of bone marrow mesenchymal stem cells (BMSCs) and TGF-β3 in regenerative injuries in the myometrium of the uterus in rats. MethodsTwenty-four pregnant rats were randomly assigned into four groups and their pregnancy was terminated on day 19 of pregnancy: 1. Blank scar uterus control group injected with the physiological saline, 2. TGF-β3 group injected with TGF-β3, 3. BMSCs group injected with BMSCs, 4. BMSCS + TGF-β3 injected with TGF-β3 and BMSCs. ResultsMSCs expressing specific surface antigens had multidirectional differentiation potential and showed a tendency to home to the site of injury, and the macroscopic appearance of the cell-treated uterus was smoother and tougher in the three groups than in the control group. The uterus improved by the TGF-β3+MSCs group had the best recovery showing significant glandular and vascular nature, with better repair effect and promoted endometrial cell proliferation. Proteins related to endometrial cell proliferation showed that the expression of Ki-67 was significantly increased in three intervention groups, and a positive signal of waveform protein was observed in the uterine stromal cells. The levels of waveform protein and CK-19 protein were significantly lower in the control group compared to their levels in the three interventional groups. Injection of BMSCs and TGF-β3 resulted in the repair of whole damaged myometrium and significant improvement in function compared to other groups, whereas TGF-β3 significantly reduced collagen fiber production. ConclusionsBMSCs can differentiate into uterine smooth muscle cells, repair uterine scar tissue and promote uterine scar healing.

AICAR attenuates postoperative abdominal adhesion formation by inhibiting oxidative stress and promoting mesothelial cell repair.

Postoperative abdominal adhesion is one of most common complications after abdominal operations. 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) is an adenosine 5'-monophosphate activated protein kinase (AMPK) pathway agonist that inhibits inflammation, reduces cell fibrosis and cellular reactive oxygen species (ROS) injury, promotes autophagy and mitochondrial function. This study aimed to explore the mechanism of AICAR in inhibiting adhesion formation. Forty rats were randomly divided into five groups. All of the rats except the sham group received cecal abrasion to establish an adhesion model. The rats in the sodium hyaluronate group were treated with 2 mL sodium hyaluronate before closing the peritoneal cavity. The AICAR 1 and 2 groups were treated with 100 mg/kg and 200 mg/kg AICAR, respectively. Seven days after the operation, all of the rats were euthanized, and the adhesion condition was evaluated by Nair's system. Inflammation was assessed by Eosin-hematoxylin (HE) staining and transforming growth factor-β (TGF-β1) detection. Oxidative stress effect was determined by ROS, nitric oxide (NO) level, superoxide dismutase (SOD), catalase, glutathione peroxidase (Gpx) and malondialdehyde (MDA) levels in adhesion tissue. Then, Sirius red picric acid staining was used to detect the fiber thickness. Immunohistochemical staining of cytokeratin-19 (CK-19), alpha-smooth muscle actin (α-SMA) and nuclear factor erythroid 2-related factor 2 (Nrf2) was also performed. Finally, HMrSV5 cells were treated with TGF-β1 and AICAR, the mRNA expression of E-cadherin, α-SMA and vimentin was assessed by q-PCR and cellular immunofluorescent staining. The rats in the AICAR-treated group had fewer adhesion formation incidences and a reduced Nair's score. The inflammation was determined by HE staining and TGF-β1 concentration. The ROS, SOD, Catalase, Gpx, MDA levels and fiber thickness were decreased by AICAR treatments compared to the control. However, the NO production, Nrf2 levels and peritoneal mesothelial cell integrity were promoted after AICAR treatments. In vitro work, AICAR treatments reduced E-cadherin, α-SMA and vimentin mRNA level compared to that in the TGF-β1 group. AICAR can inhibit postoperative adhesion formation by reducing inflammation, decreasing oxidative stress response and promoting peritoneal mesothelial cell repair.

Metformin Mitigated Obesity-Driven Cancer Aggressiveness in Tumor-Bearing Mice.

Metformin may offer benefits to certain cancer populations experiencing metabolic abnormalities. To extend the anticancer studies of metformin, a tumor model was established through the implantation of murine Lewis Lung Carcinoma (LLC) cells to Normal Diet (ND)-fed and High-Fat Diet (HFD)-fed C57BL/6 mice. The HFD-fed mice displayed metabolic and pro-inflammatory alterations together with accompanying aggressive tumor growth. Metformin mitigated tumor growth in HFD-fed mice, paralleled by reductions in circulating glucose, insulin, soluble P-selectin, TGF-β1 and High Mobility Group Box-1 (HMGB1), as well as tumor expression of cell proliferation, aerobic glycolysis, glutaminolysis, platelets and neutrophils molecules. The suppressive effects of metformin on cell proliferation, migration and oncogenic signaling molecules were confirmed in cell study. Moreover, tumor-bearing HFD-fed mice had higher contents of circulating and tumor immunopositivity of Neutrophil Extracellular Traps (NETs)-associated molecules, with a suppressive effect from metformin. Data taken from neutrophil studies confirmed the inhibitory effect that metformin has on NET formation induced by HMGB1. Furthermore, HMGB1 was identified as a promoting molecule to boost the transition process towards NETs. The current study shows that metabolic, pro-inflammatory and NET alterations appear to play roles in the obesity-driven aggressiveness of cancer, while also representing candidate targets for anticancer potential of metformin.