Legumain is a paracrine regulator of osteoblast differentiation and mediates the inhibitory effect of TGF-ß1 on osteoblast maturation.

Transforming growth factor-beta 1 (TGF-ß1) is a critical regulator of skeletal homeostasis and has diverse effects on osteoblastogenesis. To date, the mechanisms behind the intriguing inhibitory effect of TGF-ß1 on osteoblast maturation are not fully understood. Here, we demonstrate a novel mechanism by which TGF-ß1 modulates osteoblast maturation through the lysosomal protease legumain. We observed that addition of TGF-ß1 to osteogenic cultures of human bone marrow derived mesenchymal stromal (stem) cells enhanced legumain activity and secretion, in-spite of decreased legumain mRNA expression, suggesting post-transcriptional regulation. We further showed that osteogenic cells internalize and activate prolegumain, associated with inhibited osteoblast maturation, revealing legumain as a paracrine regulator of osteoblast maturation. Interestingly, TGF-ß1 treatment exacerbated legumain internalization and activity, and showed an additive effect on legumain-induced inhibition of osteoblast maturation. Importantly, pharmacological inhibition of legumain abolished the inhibitory effect of TGF-ß1 on osteoblast maturation. Our findings reveal that TGF-ß1 inhibits osteoblast maturation by stimulating secretion and activity of endogenous legumain, as well as enhancing internalization and activation of extracellular prolegumain. Therefore, our study provides a deeper understanding of the complex regulation of osteoblastogenesis and unveils a novel TGF-ß1-legumain axis in regulation of osteoblast maturation, offering novel insights for possible therapeutic interventions related to bone diseases associated with aberrant TGF-ß1 signaling.

Blocking TSP1 Ameliorates Diabetes Mellitus-Induced Erectile Dysfunction by Inhibiting the TGF-ß/SMAD Pathway.

PURPOSE: To examine the role and mechanism of thrombospondin-1 (TSP1) in the development of fibrosis in diabetes mellitus-induced erectile dysfunction (DMED). MATERIALS AND METHODS: DMED was induced by intraperitoneal streptozotocin injection. All rats were categorized into three groups: control group (n=8), DMED group (n=8) and DMED+Leu-Ser-Lys-Leu (LSKL) group (n=8). After eight weeks following the induction of diabetes mellitus, the DMED+LSKL group was subjected to intraperitoneal injections of LSKL twice weekly for four weeks. To measure intracavernous pressure (ICP), a 25-gauge needle connected to a PE tube containing heparin was inserted into the corpus cavernosum (CC). Additionally, a needle was inserted into the carotid artery to measure mean arterial pressure (MAP). Sirius red staining and Masson trichrome staining were utilized to assess CC fibrosis. Moreover, high glucose (HG)-induced CC smooth muscle cells (CCSMCs) and CC fibroblasts (CCFs) were treated with or without LSKL. Western blotting and immunofluorescence were utilized to assess the phosphorylation and expression of related proteins. RESULTS: Compared with those in the control group, the ratio of the maximum ICP to the MAP markedly decreased in the DMED group, as did the ratio of smooth muscle to collagen and the ratio of collagen I to collagen III. These ratios were greater in the DMED+LSKL group than in the DMED group. TSP1 was highly expressed in the CC of DMED rats. In vitro experiments indicated that TSP1 expression significantly increased in the medium of CCSMCs and CCFs cultured in HG media and that the TGF-ß pathway was activated in CCSMCs. Collagen IV was overexpressed in CCSMCs, indicating severe fibrosis was severe. Adding LSKL or knocking TSP1 down can prevent the activation of TGF-ß signaling, as well as the overexpression of collagen IV in CCSMCs promoted by TSP1 secreted from CCSMCs itself or CCFs. CONCLUSIONS: TSP1 expression is increased in the CC of DMED rats. HG-induced TSP1 secretion via autocrine signaling from CCSMCs and/or paracrine signaling from CCFs to accelerate penile fibrosis. LSKL, an antagonist of TSP1, could improve erectile dysfunction by inhibiting the TGF-ß/SMAD pathway.

Picroside â ¡ alleviates renal fibrosis through YY1-dependent transcriptional inhibition of TGFß1.

Diabetic Nephropathy (DN) has become the leading cause of end-stage renal disease worldwide. Studies have indicated that Transforming Growth Factor beta1 (TGFß1) is the most potent factor contributing to renal fibrosis, and understanding the exact pathogenic mechanism of renal fibrosis is crucial for alleviating the condition. Previous research has identified Yin Yang 1 (YY1) as an effective inhibitor of TGF-ß1. Our study, through dual-luciferase reporter gene assays and Western blot experiments, screened and obtained the small molecule compound PdⅡ. Subsequently, validation in a high-glucose-induced renal mesangial cell injury model showed that PdⅡ treatment significantly increased the expression of YY1 protein and mRNA, while correspondingly reducing the expression of TGFß1 protein and mRNA. Dual-luciferase reporter gene assay results revealed that, compared to the control group, the luciferase transcription activity of YY1 molecules increased in the PdⅡ treatment group, and the luciferase transcription activity of TGFß1 decreased. By further designing mutations in the binding sites between TGFß1 and YY1 on the promoter, transfecting fluorescent enzyme reporter gene plasmids with TGFß1 mutant promoter into mesangial cells damaged by high glucose, and then treating the cells with PdⅡ, it was observed that the luciferase transcription activity of TGFß1 did not decrease. Therefore, these results suggest that PdⅡ may inhibit TGFß1 transcriptional activity by activating YY1, thereby slowing down the progression of diabetic nephropathy.

Berberine suppressed the epithelial-mesenchymal transition (EMT) of colon epithelial cells through the TGF-ß1/Smad and NF-κB pathways associated with miRNA-1269a.

Objective: To explore the mechanisms of the TGF-ß1/Smad and NF-κB pathways in the effect of berberine (BBR) on colon cancer epithelial-mesenchymal transition (EMT) and their regulatory relationships with microRNAs (miRNAs). Methods: TGF-ß1 was used to induce EMT in normal colon epithelial HCoEpiC cells and colon cancer HT29 cells in vitro. After BBR intervention, the expression of EMT-related markers and the major molecules involved in the TGF-ß1/Smad and NF-κB pathways were detected via western blotting. Cell migration was detected via wound healing assays. SMAD2 and NF-κB p65 were overexpressed and transfected into cells, and the inhibitors SB431542 and BAY 11-7082 were added to block the TGF-ß1/Smad and NF-κB pathways, respectively. The mRNA expression levels of related microRNA genes were detected by using RT‒PCR. Results: Treatment with 10 ng/ml TGF-ß1 for 72 h significantly induced EMT in HCoEpiC and HT29 cells, which was repressed by BBR. BBR significantly inhibited the TGF-ß1-induced migration of HCoEpiC and HT29 cells and the TGF-ß1-promoted expression of p-Smad2/3, NF-κB p65, and p-IκBα. Compared to those in the group treated with TGF-ß1, the expression of NF-κB p65 and p-Smad2 in the group treated with NF-κB pathway inhibitor BAY 11-7082 was decreased (P < 0.05), and TGF-ß1 signalling inhibitor SB431542 significantly reduced the expression of NF-κB p65 (P < 0.05). Overexpression of NF-κB p65 and SMAD2 in HT29 cells decreased the expression of E-cadherin and caused a relative increase in N-cadherin. BBR mediated the expression profile of microRNAs in TGF-ß1-induced HCoEpiC cells, but this pattern differed from that in HT29 cells. SB431542 and BAY 11-7082 significantly reduced the mRNA level of miR-1269a in HCoEpiC and HT29 cells (P < 0.05). Overexpressed NF-κB p65 and SMAD2 increased the mRNA level of miR-1269a in both cell lines; however, this increase was significantly lower than that in the TGF-ß1 treatment group (P < 0.05). Conclusion: BBR can significantly inhibit TGF-ß1-induced EMT in normal and cancerous colon epithelial cells through the inhibition of the TGF-ß1/Smad and NF-κB p65 pathways. TGF-ß1/Smads can promote the NF-κB p65 pathway, which is a common target of miR-1269a, and can partially regulate the expression of miR-1269a.

Reduced Cell Migration in Human Chondrocyte Sheets Increases Tissue Stiffness and Cartilage Protein Production.

BACKGROUND: Chondrogenic differentiation medium (CDM) is usually used to maintain chondrogenic activity during chondrocyte sheet production. However, tissue qualities remain to be determined as to what factors improve cell functions. Moreover, the relationship between CDM and cell migration proteins has not been reported. METHOD: In this study, the effect of CDM on the behavior of chondrocyte sheets was investigated. Structural analysis, mechanical testing and proteomics were performed to observe tissue qualities. The relationship between CDM and cell migration proteins were investigated using time-lapse observations and bioinformatic analysis. RESULTS: During 48 h, CDM affected the chondrocyte behaviors by reducing cell migration. Compared to the basal medium, CDM impacted the contraction of monolayered chondrocyte sheets. At day 7, the contracted sheets increased tissue thickness and improved tissue stiffness. Cartilage specific proteins were also upregulated. Remarkedly, the chondrocyte sheets in CDM displayed downregulated proteins related to cell migration. Bioinformatic analysis revealed that TGFß1 was shown to be associated with cartilage functions and cell migration. Pathway analysis of chondrocyte sheets in CDM also revealed the presence of a TGFß pathway without activating actin production, which might be involved in synthesizing cartilage-specific proteins. Cell migration pathway showed MAPK signaling in both cultures of the chondrocyte sheets. CONCLUSION: Reduced cell migration in the chondrocyte sheet affected the tissue quality. Using CDM, TGFß1 might trigger cartilage protein production through the TGFß pathway and be involved in cell migration via the MAPK signaling pathway. Understanding cell behaviors and their protein expression would be beneficial for developing high-quality tissue-engineered cartilage.

Alkali-extracted proteins from the tooth dentin matrix as a mixture of bioactive molecules for cartilage repair and regeneration.

Introduction: Dentin matrix extracted protein (DMEP) is a mixture of proteins extracted from the organic matrix of a natural demineralized dentin matrix that is rich in a variety of growth factors. However, the effect of DMEP on cartilage regeneration is unclear. The aim of this study was to investigate the efficacy of DMEP extracted via a novel alkali conditioning method in promoting cartilage regeneration. Methods: Alkali-extracted DMEP (a-DMEP) was obtained from human dentin fragments using pH 10 bicarbonate buffer. The concentration of chondrogenesis-related growth factors in a-DMEP was measured via enzyme-linked immunosorbent assay (ELISA). Human bone marrow mesenchymal stem cells (hBMMSCs) in pellet form were induced with a-DMEP. Alcian blue and Safranin O staining were performed to detect cartilage matrix formation, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess chondrogenic-related gene expression in the pellets. Rabbit articular osteochondral defects were implanted with collagen and a-DMEP. Cartilage regeneration was assessed with histological staining 4 weeks after surgery. Results: Compared with traditional neutral-extracted DMEP, a-DMEP significantly increased the levels of transforming growth factor beta 1(TGF-ß1), insulin-like growth factor-1(IGF-1) and basic fibroblast growth factor (bFGF). After coculture with hBMMSC pellets, a-DMEP significantly promoted the expression of the collagen type II alpha 1(COL2A1) and aggrecan (ACAN) genes and the formation of cartilage extracellular matrix in cell pellets. Moreover, compared with equivalent amounts of exogenous human recombinant TGF-ß1, a-DMEP had a stronger chondrogenic ability. In vivo, a-DMEP induced osteochondral regeneration with hyaline cartilage-like structures. Conclusions: Our results showed that a-DMEP, a compound of various proteins derived from natural tissues, is a promising material for cartilage repair and regeneration.

Saponin â from Shuitianqi () inhibits metastasis by negatively regulating the transforming growth factor-ß1/Smad7 network and epithelial-mesenchymal transition in the intrahepatic metastasis Bagg's Albino/c mouse model.

OBJECTIVE: To examine the influence of Saponin I from Shuitianqi (Rhizoma Schizocapasae Plantagineae) (SSPH I) on hepatocellular carcinoma (HCC) metastasis, and to elucidate the underlying mechanism. METHODS: The intrahepatic metastasis Bagg's Albino/c (BALB/c) mouse model was established with human hepatocellular carcinomas (HepG2) cells, then treated with normal saline (once per day), cisplatin (2 mg/kg, once every 2 d), and SSPH Ⅰ (25, 50, and 75 mg/kg, once per day). Then, we assessed alterations in the hepatic pathology and target protein expressions in the intrahepatic metastasis BALB/c mouse model using a series of molecular biology techniques. RESULTS: Based on our analysis, SSPH Ⅰ significantly alleviated hepatocyte necrosis and tumor cells infiltration. Moreover, SSPH Ⅰ suppressed extracellular matrix (ECM) degradation and angiogenesis viaa decrease in matrix etalloproteinase-2 (MMP-2), MMP-9, CD31, CD34, and vascular endothelial growth factor (VEGF) levels. Furthermore, SSPH Ⅰ repressed invasion and meta-stasis by suppressing the transforming growth factor-ß1 (TGF-ß1)/Smad7 axis and epithelial-mesenchymal transition (EMT), as evidenced by the scarce TGF-ß1, N-cadherin, and Vimentin expressions, and elevated Smad7 and E-cadherin expressions. CONCLUSION: The SSPH Ⅰ-mediated negative regulation of the TGF-ß1/Smad7 axis and EMT are critical for the inhibition of HCC invasion and metastasis.

Hsa-miR-134-5p predicts cardiovascular risk in circulating mononuclear cells and improves angiogenic action of senescent endothelial progenitor cells.

This research explores the role of microRNA in senescence of human endothelial progenitor cells (EPCs) induced by replication. Hsa-miR-134-5p was found up-regulated in senescent EPCs where overexpression improved angiogenic activity. Hsa-miR-134-5p, which targeted transforming growth factor ß-activated kinase 1-binding protein 1 (TAB1) gene, down-regulated TAB1 protein, and inhibited phosphorylation of p38 mitogen-activated protein kinase (p38) in hsa-miR-134-5p-overexpressed senescent EPCs. Treatment with siRNA specific to TAB1 (TAB1si) down-regulated TAB1 protein and subsequently inhibited p38 activation in senescent EPCs. Treatment with TAB1si and p38 inhibitor, respectively, showed angiogenic improvement. In parallel, transforming growth factor Beta 1 (TGF-ß1) was down-regulated in hsa-miR-134-5p-overexpressed senescent EPCs and addition of TGF-ß1 suppressed the angiogenic improvement. Analysis of peripheral blood mononuclear cells (PBMCs) disclosed expression levels of hsa-miR-134-5p altered in adult life, reaching a peak before 65 years, and then falling in advanced age. Calculation of the Framingham risk score showed the score inversely correlates with the hsa-miR-134-5p expression level. In summary, hsa-miR-134-5p is involved in the regulation of senescence-related change of angiogenic activity via TAB1-p38 signalling and via TGF-ß1 reduction. Hsa-miR-134-5p has a potential cellular rejuvenation effect in human senescent EPCs. Detection of human PBMC-derived hsa-miR-134-5p predicts cardiovascular risk.

Pregnancy risk in beef and dairy cows after supplementing semen with transforming growth factor beta-1 at the time of artificial insemination.

Our objective was to determine if the addition of a concentrated human recombinant transforming growth factor beta-1 (TGF) to bovine semen at the time of AI would result in increased risk of pregnancy in beef and dairy cows. Suckled beef cows (n = 1,132) in 11 herds across 2 states and lactating dairy cows (n = 2,208) in one organic-certified herd were enrolled. Beef cows received fixed-time AI (FTAI) following a 7 d CO-Synch + controlled internal drug release estrous synchronization protocol. Dairy cows were inseminated following observation of natural estrus expression. Cows received either no treatment as a control (CON) or 10 ng of TGF in 10 µL added through the cut-end of a thawed straw of semen immediately prior to AI. At the time of FTAI of beef cows, the mean ±â€…SD age was 5.0 ±â€…2.4 yr, BCS was 5.3 ±â€…0.7, and days postpartum was 78.2 ±â€…15.5 d. The overall pregnancy risk (PR) in beef cows was 55.2% to AI and 90.5% season-long. PR in beef cows was not affected (P = 0.27) by the addition of TGF (53.1% vs. 58.1%). Furthermore, there was no difference (P = 0.88) for season-long PR in beef cows that received TGF (91.2% vs. 91.5%). At the time of insemination of dairy cows, the mean ±â€…SD lactation was 3.0 ±â€…1.3 lactations, BCS was 2.9 ±â€…0.3, days in milk was 115.6 ±â€…56.6 d, and cows had received 2.4 ±â€…1.5 inseminations/cow. The overall pregnancy risk to AI in dairy cows was 23.1%. PR to AI for dairy cows was not affected (P = 0.32) by addition of TGF (22.0% vs. 23.8%). In conclusion, PR to AI was not affected by addition of TGF to thawed semen immediately prior to AI in beef or dairy cows.

Seminal plasma is the fluid portion of the ejaculate that is routinely removed or significantly diluted when preparing semen for artificial insemination. Seminal plasma has been shown to elicit changes to the tissues of the uterus at the time of insemination that improves pregnancy outcomes in rodents and swine. Here, we supplemented the molecule of seminal plasma, transforming growth factor beta-1, to semen at the time of artificial insemination in an attempt to improve pregnancy rates in beef and dairy cattle. In total, 3,340 cows were inseminated; half received no treatment, and the other half received a supplementation of transforming growth factor beta-1. We found that supplementing transforming growth factor beta-1 did not improve the pregnancy rate in beef or dairy cattle. We conclude that the pregnancy rate was not affected by the supplementation of transforming growth factor beta-1 to semen at the time of insemination. Future studies should consider the effects of transforming growth factor beta-1 on other pregnancy outcomes, such as calving rate, birth weight, and postnatal growth.

MicroRNA-29b attenuates fibrosis in a rat model of Peyronie's disease.

BACKGROUND: Peyronie's disease is characterized by the formation of fibrotic plaques in the penile tunica albuginea. Effective treatments are limited, warranting the investigation of new promising therapies, such as the application of microRNAs that regulate fibrosis-related genes. OBJECTIVE: We aimed to investigate the therapeutic potential of mimicking microRNA-29b in a fibrin-induced rat model of Peyronie's disease. MATERIAL/METHODS: The study was designed in two phases. To establish an optimal Peyronie's disease model, rats received either human fibrin and thrombin or saline solutions into the tunica albuginea on days 0 and 5. The animal model validation was done through expression and histopathological analyses, the latest by an experienced uropathologist. After validation, we performed microRNA-29b treatment on days 14, 21, and 28 of the study. This phase had control (normal saline) and scramble (microRNA scramble) groups. The mid-penile shaft was removed on day 30 for histological examination and molecular analyses in both study stages. RESULTS: The control group displayed typical tunica albuginea histologic architecture in the animal model validation. In Peyronie's disease group, the Hematoxylin and eosin and Masson Trichrome staining methods demonstrated an interstitial inflammatory process with concomitant dense fibrotic plaques as well as disarrangement of collagen fibers. Additionally, we found out that reduced microRNA-29b (p = 0.05) was associated with significantly increased COL1A1 and transforming growth factor ß1 genes and proteins (p > 0.05) in the Peyronie's disease group. After treatment with mimic microRNA-29b stimulation, the Hematoxylin & eosin and Masson Trichrome staining revealed a discrete and less dense fibrotic plaque. This result was associated with significantly decreasing expression of COL1A1, COL3A1, and transforming growth factor ß1 genes and proteins (p < 0.05). DISCUSSION: The fibrin-induced animal model showed significant histopathological and molecular changes compared to the Control group, suggesting that our model was appropriate. Previous findings have shown that increased expression of microRNA-29b was associated with decreased pathological fibrosis. In the present study, treatment with microRNA-29b decreased the gene and protein expression of collagens and transforming growth factor ß1. This study reveals the therapeutic potential for Peyronie's disease involving molecular targets. CONCLUSION: MicroRNA-29b application on the rat's tunica albuginea attenuated fibrosis, arising as a novel potential strategy for Peyronie's disease management.

Ziziphus spina-christi L. extract attenuates bleomycin-induced lung fibrosis in mice via regulating TGF-ß1/SMAD pathway: LC-MS/MS Metabolic profiling, chemical composition, and histology studies.

Ancient Egyptians (including Bedouins and Nubians) have long utilized Ziziphus spina-christi (L.), a traditional Arabian medicinal herb, to alleviate swellings and inflammatory disorders. It is also mentioned in Christian and Muslim traditions. Ziziphus spina-christi L. (Family: Rhamnaceae) is a plentiful source of polyphenols, revealing free radical scavenging, antioxidant, metal chelating, cytotoxic, and anti-inflammatory activities. Herein, different classes of the existing bioactive metabolites in Z. spina-christi L. were detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the first time. The study also aimed to assess the anti-inflammatory and antifibrotic properties of Z. spina-christi L. extract against bleomycin-induced lung fibrosis in an experimental mouse model. 32 male Swiss Albino mice were assigned into 4 groups; the first and second were the normal control group and the bleomycin positive control (single 2.5 U/kg bleomycin intratracheal dose). The third and fourth groups received 100 and 200 mg/kg/day Z. spina-christi L. extract orally for 3 weeks, 2 weeks before bleomycin, and 1 week after. The bioactive metabolites in Z. spina-christi L. extract were identified as phenolic acids, catechins, flavonoids, chalcones, stilbenes, triterpenoid acids, saponins, and sterols. The contents of total phenolic compounds and flavonoids were found to be 196.62 mg GAE/gm and 33.29 mg QE/gm, respectively. In the experimental study, histopathological examination revealed that lung fibrosis was attenuated in both Z. spina-christi L.- treated groups. Z. spina-christi L. extract downregulated the expression of nuclear factor kappa B (NF-κB) p65 and decreased levels of the inflammatory markers tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and c-Jun N-terminal kinase (JNK) in lung tissue. Z. spina-christi L. also downregulated the expression of the fibrotic parameters collagen-1, alpha-smooth muscle actin (α-SMA), transforming growth factor-beta 1 (TGF-ß1), matrix metalloproteinase-9 (MMP-9) and SMAD3, with upregulation of the antifibrotic SMAD7 in lung tissue. Overall, the present study suggests a potential protective effect of Z. spina-christi L. extract against bleomycin-induced lung fibrosis through regulation of the TGF-ß1/SMAD pathway.

The mitigating effect of para-hydroxycinnamic acid in bleomycin-induced pulmonary fibrosis in mice through targeting oxidative, inflammatory and fibrotic pathways.

This study investigated the therapeutic benefits of para-hydroxycinnamic acid in mice with bleomycin-induced lung fibrosis. Forty male BALB/c mice were randomly assigned to four groups: normal, which received 0.9% normal saline; induced, which received a single dose of bleomycin (5 mg/kg) by oropharyngeal challenge; pirfenidone-treated; and para-hydroxycinnamic acid-treated, which challenged with bleomycin and received a daily oral dose of 300 and 50 mg/kg, respectively, from day 7 to day 21. Tissue pro-fibrotic and inflammatory cytokines, oxidative indicators, pulmonary histopathology, immunohistochemistry of fibrotic proteins and the assessment of gene expression by RT-qPCR were evaluated on day 22 after euthanizing animals. Pirfenidone and para-hydroxycinnamic acid managed to alleviate the fibrotic endpoints by statistically improving the weight index, histopathological score and reduced expression of fibrotic-related proteins in immune-stained lung sections, as well as fibrotic markers measured in serum samples. They also managed to alleviate tissue levels of oxidative stress and inflammatory and pro-fibrotic mediators. para-Hydroxycinnamic acid enhanced the expression of crucial genes associated with oxidative stress, inflammation and fibrosis in vivo. para-Hydroxycinnamic acid has demonstrated similar effectiveness to pirfenidone, suggesting it could be a promising treatment for fibrotic lung conditions by inhibiting the TGF-ß1/Smad3 pathway or through its anti-inflammatory and antioxidant properties.

Exploring Anti-Fibrotic Effects of Adipose-Derived Stem Cells: Transcriptome Analysis upon Fibrotic, Inflammatory, and Hypoxic Conditioning.

Autologous fat transfers show promise in treating fibrotic skin diseases, reversing scarring and stiffness, and improving quality of life. Adipose-derived stem cells (ADSCs) within these grafts are believed to be crucial for this effect, particularly their secreted factors, though the specific mechanisms remain unclear. This study investigates transcriptomic changes in ADSCs after in vitro fibrotic, inflammatory, and hypoxic conditioning. High-throughput gene expression assays were conducted on ADSCs exposed to IL1-ß, TGF-ß1, and hypoxia and in media with fetal bovine serum (FBS). Flow cytometry characterized the ADSCs. RNA-Seq analysis revealed distinct gene expression patterns between the conditions. FBS upregulated pathways were related to the cell cycle, replication, wound healing, and ossification. IL1-ß induced immunomodulatory pathways, including granulocyte chemotaxis and cytokine production. TGF-ß1 treatment upregulated wound healing and muscle tissue development pathways. Hypoxia led to the downregulation of mitochondria and cellular activity.

Effect of simultaneous and sequential use of TGF-ß1 and TGF-ß3 with FGF-2 on teno/ligamentogenic differentiation of periodontal ligament stem cells.

OBJECTIVE: The periodontal ligament is a crucial part of the periodontium, and its regeneration is challenging. This study compares the effect of simultaneous and sequential use of FGF-2 and TGF-ß1 with FGF-2 and TGF-ß3 on the periodontal ligament stem cells (PDLSCs) teno/ligamentogenic differentiation. DESIGN: This study comprises ten different groups. A control group with only PDLSCs; FGF-2 group containing PDLSCs with a medium culture supplemented with FGF-2 (50 ng/mL). In other experimental groups, different concentrations (5 ng/mL or 10 ng/mL) of TGF-ß1&-ß3 simultaneously or sequentially were combined with FGF-2 on the cultured PDLSCs. TGF-ß was added to the medium after day 3 in the sequential groups. Methyl Thiazolyl Tetrazolium (MTT) assay on days 3, 5, and 7 and Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) analysis after day 7 were conducted to investigate PLAP1, SCX, and COL3A1, RUNX2 genes. All experiments were conducted in a triplicate. The One-way and Two-way ANOVA with Tukey post hoc were utilized to analyze the results of the MTT and RT-qPCR tests, respectively. A p-value less than 0.05 is considered significant. RESULTS: The proliferation of cells on days 3, 5, and 7 was not significantly different among different experimental groups (P > 0.05). A higher expression of the PLAP1, SCX, and COL3A1 have been seen in groups with sequential use of growth factors; among these groups, the group using 5 ng/mL of TGF-ß3 led other groups with the most amount of significant upregulation in PLAP1(17.69 ± 1.11 fold; P < 0.0001), SCX (5.71 ± 0.38 fold; P < 0.0001), and COL1A3 (6.35 ± 0.39 fold; P < 0.0001) expression, compared to the control group. The expression of the RUNX2 decreased in all groups compared to the control group; this reduction was more in groups with sequential use of growth factors. CONCLUSION: The sequential use of growth factors can be more effective than simultaneous use in teno/ligamentogenic differentiation of PDLSCs. Moreover, treatment with 5 ng/mL TGF-ß3 after FGF-2 was more effective than TGF-ß1.

Clinical and biochemical assessment of the effect of glutamine in management of radiation induced oral mucositis in patients with head and neck cancer: Randomized controlled clinical trial.

BACKGROUND: This study aimed to evaluate the effect of oral glutamine suspension on salivary levels of transforming growth factor beta 1 (TGF-ß1), a cytokine involved in inflammation and Tumor progression, and the severity of radiation-induced oral mucositis (RIOM) in head and neck cancer patients. This is the first study to investigate the impact of glutamine on TGF-ß1 levels in head and neck cancer patients with radiation induced oral mucositis (RIOM). METHODS: In this randomized controlled clinical trial, 50 HNC patients were enrolled and received either glutamine oral suspension or maltodextrin as a placebo from the baseline of RIOM to the end of radiotherapy. Salivary TGF-ß1 levels were measured at baseline and after treatment. Also, RIOM was assessed using the World Health Organization (WHO) Oral Toxicity Scale, the Oral Mucositis Assessment Scale (OMAS), the Pain Visual Analog Scale (Pain-VAS), the incidence of opioid use, and body mass index (BMI). RESULTS: Glutamine significantly reduced salivary TGF-ß1 levels and improved RIOM symptoms, such as pain, opioid use, and weight loss. The reduction of TGF-ß1 levels was associated with the improvement of RIOM severity. CONCLUSION: Glutamine may modulate the inflammatory response and enhance wound healing in RIOM by decreasing salivary TGF-ß1 levels. These findings support the use of glutamine as a potential intervention for RIOM and nutritional support for improving radiation sensitivity. TRIAL REGISTRATION: This study was registered on clinicalTrials.gov with identifier no. NCT05856188.

Network pharmacology and experimental validation to reveal the pharmacological mechanisms of Sini decoction against renal fibrosis.

OBJECTIVE: To investigate the mechanism by which Sini decoction (, SND) improves renal fibrosis (Rf) in rats based on transforming growth factor ß1/Smad (TGF-ß1/Smad) signaling pathway. METHODS: Network pharmacology was applied to obtain potentially involved signaling pathways in SND's improving effects on Rf. The targets of SND drug components and the targets of Rf were obtained by searching databases, such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCSMP) and GeenCard. The intersection targets of two searches were obtained and underwent signaling pathway analysis using a Venn diagram. Then experimental pharmacology was utilized to prove and investigate the effects of SND on target proteins in the TGF-ß1/Smad signaling pathway. The Rf rat model was established by unilateral ureteral occlusion (UUO). The expression levels of transforming growth factor, matrix metalloproteinase-9 (MMP-9), matrix metal protease-2 (MMP-2), connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were determined by Masson staining of rat renal tissue, and immunohistochemical methods. The expression levels of Smad3, Smad2, and Smad7 in renal tissue were determined by Western blotting (WB). The mechanism of the improving effects of SND on Rf was investigated based on TGF-ß1/Smad signaling pathway. RESULTS: A total of 12 drug components of Fuzi (Radix Aconiti Lateralis Preparata), 5 drug components of Ganjiang (Rhizoma Zingiber), and 9 drug components of Gancao (Radix Glycy et Rhizoma) were obtained from the database search, and 207 shared targets were found. A total of 1063 Rf targets were found in the database search. According to the Venn diagram, in total, 96 intersection targets were found in two database searches. The metabolic pathways involved included TGF-ß signaling pathway, phosphatidylinositol-3-kinase/serine-threonine protein kinase signaling (PI3K/Akt) pathway, and hypoxia-inducible factor-1 (HIF-1) signaling pathway. Masson staining analysis showed that compared with the model group, the renal interstitial collagen deposition levels in the SSN and SND groups were significantly lower (P < 0.05). Immunohistochemical analysis, compared with the control group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the model group were significantly decreased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-ß1 were significantly increased (P < 0.01). Compared with the model group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the SSN and SND groups were significantly increased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-ß1 in the kidney tissue were significantly decreased (P < 0.05, P < 0.01). WB results showed that the SSN group and the SND group could reduce the expression of Smad2 and Smad3 (P < 0.05) and increase the expression of Smad7 (P < 0.05).

microRNA-141-3p Suppressed the Progression of the Clear Cell Renal Cell Carcinoma by Targeting Transforming Growth Factor Beta 2 Gene Expression.

Clear cell renal cell carcinoma (ccRCC) is a malignant tumor of kidney epithelial cells, one of the most common tumors in the world. Transforming growth factor beta (TGFß)1 is a crucial factor that induces epithelial-mesenchymal transition (EMT) in cancer cells. microRNA-141-3p (miR-141-3p) is a microRNA that is considered a tumor suppressor. However, the role and mechanism of miR-141-3p in TGFß1-induced ccRCC cells are not fully understood. This study investigated the roles of miR-141-3p and its target gene in regulating EMT in ccRCC development. 786-0 and Caki-1cells were treated with TGFß1 to induce EMT. The levels of miR-141-3p and TGFß2 were determined by quantitative real-time polymerase chain reaction and Western blotting. The progression of EMT was evaluated by E-cadherin detection by immunofluorescence, and E-cadherin, N-cadherin, and vimentin detection by Western blotting. Furthermore, migration and invasion capacities were assessed using a Transwell system. The direct binding of miR-141-3p with the target gene TGFß2 was confirmed by dual luciferase reporter gene assay. Results indicated that TGFß1 treatment decreased the protein abundance of E-cadherin while increasing the protein expression of N-cadherin and vimentin, indicating TGFß1-induced EMT was constructed successfully. Moreover, TGFß1 treatment repressed the expression of miR-141-3p. miR-141-3p mimics reversed the effect of TGFß1 on the migration, invasion, and expression of E-cadherin, N-cadherin, and vimentin. The miR-141-3p directly binds with the 3' untranslated region of TGFß2 mRNA and suppresses its expression. Furthermore, TGFß2 overexpression abrogated the above changes regulated by miR-141-3p mimics. Taken together, miR-141-3p inhibited TGFß1-induced EMT by suppressing the migration and invasion of ccRCC cells via directly targeting TGFß2 gene expression.

The Effect of Rubus idaeus Polyphenols Extract in Induced Endometriosis in Rats.

Endometriosis is a common gynecological condition with a complex physio-pathological background. This study aimed to assess the role of Rubus idaeus leaf extract (RiDE) as a potential therapeutic agent in reducing the size of the endometriotic lesions and modulate the plasma expression of MMP-2, MMP-9, and TGF-ß1. The endometriotic lesions were induced in a rat model by the autologous transplant of endometrium. Thirty-six female rats, Wistar breed, with induced endometriosis, were divided into four groups and underwent treatment for 28 days. The CTRL group received 0.5 mL/day of the vehicle; the DG group received 1 mg/kg b.w./day dienogest; the RiDG group received 0.25 mL/kg b.w./day RiDE and the D+RiDG group received 1 mg/kg b.w./day dienogest and 0.25 mL/kg b.w./day RiDE, respectively. Rats' weight, endometriotic lesion diameter and grade, and plasma levels of MMP-2, MMP-9, and TGF-ß1 were assessed before and after treatment. The administration of RiDE in association with dienogest vs. dienogest determined a lower weight gain and a reduction in diameter of the endometriotic lesions. RiDE administration restored MMP2 and MMP9 plasma levels to initial conditions. Rubus idaeus extract may help in reducing dienogest-associated weight gain, lower the size of endometriotic lesions, and have anti-inflammatory effects through MMP2 and MMP9 reduction.