TGF-β1 Gene Expression Research Articles

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.

Abstract PO-095: TGF-β carrying exosomes in plasma of HNSCC as potential biomarkers of disease progression in patients with HNSCC

Abstract Background: The contribution of TGFβ+ exosomes, now referred to as small extracellular vesicles (sEV), in plasma of cancer patients to disease progression has been unclear. Levels of TGFβ in plasma often do not correlate with clinicopathological data, and TGFβ can exert dual (yin-yang) effects as a tumor promoter and a tumor inhibitor. We evaluated the potential of tumor-derived sEV dubbed TEX to serve as liquid tumor biopsy in patients with HNSCC. Methods: Plasma was obtained from 3 different cohorts of patients with HNSCC (n=97) and HDs (n=22). Clinicopathology/demographic data were available for all patients. sEV were isolated by ultrafiltration/size exclusion chromatography (SEC) from plasma or cell supernatants. TGF-β levels in plasma were assessed by ELISA; TGF-β expression in tissues was studied by IHC and immunoblots; immunoblots, flow cytometry and LC-MS/MS were used to measure TGF-β in isolated sEV. TGFB1 gene expression was analyzed using the TCGA Head and Neck Cancer database (n=520 cases and 44 NC). TGF-β signaling and activity of EVs were quantified in the reporter cell (MFB-F11) proliferation assays. Changes of TGF-β expression levels in plasma, tissues and sEV during carcinogenesis were studied in an orthotopic 4NQO mouse model. Results: Using the TCGA we found high expression levels of the vesiculation genes regulating EV production/release and of the TGFB1 gene expression in HNSCC. In the 4NQO orthotopic model of OSCC, cancer progression correlated with TGF-β accumulations in tumor tissues and a significant increase in circulating TGF-β+ sEV. In HNSCC patients, TGF-β levels and activity in plasma and TGF-β expression levels in tumor tissues were significantly elevated relative to controls but did not correlate with clinicopathologic data, tumor burden or disease outcome. TEX produced by HNSCC cell lines carried TGF-β and initiated Smad3 signaling in reporter cells. TGF-β inhibitors blocked this signaling. High TGFβ abundance in sEV was confirmed by proteomics and by RNAseq using the TCGA data base. Levels of TGF-β expression in sEV, sEV numbers and TGF-β activity of sEV were significantly elevated in HNSCC patients vs HDs’ sEV and correlated with tumor stage and LN involvement. Conclusions: TEX and sEV from HNSCC patients’ plasma carry TGF-β which reflects disease progression in HNSCC patients and in a mouse model of oral carcinogenesis. Neither plasma levels of TGF-β (ELISA) nor levels of TGF-β expression in the tumor (IHC) correlated with disease progression. Only sEV-associated TGF-β correlates with tumor burden and shows promise as a non-invasive biomarker of HNSCC progression and response to therapy. Citation Format: Nils Ludwig, Saigopalakrishna S. Yerneni, Theresa L. Whiteside. TGF-β carrying exosomes in plasma of HNSCC as potential biomarkers of disease progression in patients with HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-095.

Effect of adlay seed extract on inflammation and fibrogenesis in human corneal activated keratocytes at transcriptional level

Corneal activated keratocytes (CAKs) -representing the injured phenotype of corneal stromal cells- are associated with several corneal diseases. Inflammatory cytokines are the key drivers of CAK formation subsequently leading to fibrogenesis. This study aimed to investigate the effect of adlay seed extract on the expression of genes involved in inflammation (IL-6, IL-1b, LIF) and fibrogenesis (TGF-β) in CAK cells. CAKs (106 cells/10 cm2) were exposed to methanolic (MeOH) and residual (Res) extract of adlay seed (1 mg/ml, 24 h). The control group received the vehicle solution without extract at the same time and condition. Then, RNA extraction, cDNA synthesis, and real-time PCR were performed to quantify the relative expression of IL-6, IL-1b, LIF, and TGF-β in the treated vs. control cells. This study showed that the MeOH extract of adlay seed could significantly downregulate the expression of IL-6 and IL-1b in the CAKs, while the Res extract led to a significant decrease in TGF-β gene expression. We showed that CAK treatment with adlay seed extract could decrease the expression of genes related to inflammation and fibrogenesis. However, the genes to be targeted depended on the method of extraction. This proof-of-concept study could provide groundwork for the treatment of corneal stromal diseases and ocular regenerative medicine in the future.

Positive effects of dietary Clostridium butyricum supplementation on growth performance, antioxidant capacity, immunity and viability against hypoxic stress in largemouth bass.

The effects of dietary supplementation of Clostridium butyricum (CB) on growth performance, serum biochemistry, antioxidant activity, mRNA levels of immune-related genes and resistance to hypoxia stress were studied in largemouth bass. Feed with CB0 (control, 0 CFU/kg), CB1 (4.3×108 CFU/kg), CB2 (7.5×108 CFU/kg), CB3 (1.5×109 CFU/kg) and CB4 (3.2×109 CFU/kg) CB for 56 days, and then a 3h hypoxic stress experiment was performed. The results showed that dietary CB significantly increased the WGR (weight gain rate), SGR (specific growth rate), PDR (protein deposition rate) and ISI (Intestosomatic index) of largemouth bass (P<0.05). Hepatic GH (growth hormone)/IGF-1 (insulin-like growth factor-1) gene expression was significantly upregulated in the CB3 and CB4 groups compared with the CB0 group (P<0.05), while the FC (feed conversion) was significantly decreased (P<0.05). Serum TP (total protein) and GLU (glucose) levels were significantly higher in the CB4 group than in the CB0 group (P<0.05), while the contents of serum AST (aspartate transaminase), ALT (alanine transaminase), AKP (alkline phosphatase) and UN (urea nitrogen) in CB4 were significantly lower than those in CB0 (P<0.05). T-AOC (total antioxidant capacity), SOD (superoxide dismutase), CAT (catalase), POD (peroxidase) and GSH-Px (glutathione peroxidase) activities were significantly higher in CB3 and CB4 groups than in CB0 group (P<0. 05). The liver MDA (malondialdehyde) content of CB1, CB2, CB3 and CB4 groups was significantly higher than that of CB0 group (P<0. 05). The relative expressions of IL-1β (interleukin 1β), TNF-α (tumor necrosis factor α) and TLR22 (toll-like receptor-22) genes in CB2, CB3 and CB4 groups were significantly lower than those in CB0 group (P<0.05). The relative expression of IL-8 (malondialdehyde) and MyD88 (Myeloid differentiation factor 88) genes in the CB4 group was significantly lower than that in the CB0 group (P<0.05). The liver LZM (lysozyme) content of CB2, CB3 and CB4 groups was significantly higher than that of CB0 group (P<0. 05). The relative expression of IL-10 (interleukin 10) and TGF-β (transforming growth factor β) genes in the CB4 group was significantly higher than that in the CB0 group (P<0.05). Under hypoxic stress for 3h, the CMR of CB0 group was significantly higher than that of CB1, CB2, CB3 and CB4 groups (P<0.05). Dietary CB can improve the growth performance and resistance to hypoxic stress of largemouth bass by regulating the expression of GH/IGF-1 gene and inflammatory factors and inhibiting TLR22/MyD88 signaling pathway.

Exosomes derived from human adipose mesenchymal stem cells loaded bioengineered three-dimensional amniotic membrane-scaffold-accelerated diabetic wound healing.

The occurrence of wounds and defects in the healing process is one of the main challenges in diabetic patients. Herein, we investigated whether adipose-derived stem cells (ADSCs)-derived exosomes loaded bioengineered micro-porous three-dimensional amniotic membrane-scaffold (AMS) could promote healing in diabetic rats. Sixty diabetic rats were randomly allocated into the control group, exosome group, AMS group, and AMS + Exo group. On days 7, 14, and 21, five rats from each group were sampled for stereological, immunohistochemical, molecular, and tensiometrical assessments. Our results indicated that the wound closure rate, the total volumes of newly formed epidermis and dermis, the numerical densities of fibroblasts and proliferating cells, the length density blood vessels, collagen density as well as tensiometrical parameters of the healed wounds were considerably greater in the treated groups than in the control group, and these changes were more obvious in the AMS + Exo ones. Furthermore, the expression of TGF-β, bFGF, and VEGF genes was meaningfully upregulated in all treated groups compared to the control group and were greater in the AMS + Exo group. This is while expression of TNF-α and IL-1β, as well as cell numerical densities of neutrophils, M1 macrophages, and mast cells decreased more considerably in the AMS + Exo group in comparison with the other groups. Generally, it was found that using both AMS transplantation and ADSCs-derived exosomes has more effect on diabetic wound healing.

Cx43 regulates mechanotransduction mechanisms in human preterm amniotic membrane defects.

The effects of mechanical stimulation in preterm amniotic membrane (AM) defects were explored. Preterm AM was collected from women undergoing planned preterm caesarean section (CS) due to fetal growth restriction or emergency CS after spontaneous preterm prelabour rupture of the membranes (sPPROM). AM explants near the cervix or placenta were subjected to trauma and/or mechanical stimulation with the Cx43 antisense. Markers for nuclear morphology (DAPI), myofibroblasts (αSMA), migration (Cx43), inflammation (PGE2 ) and repair (collagen, elastin and transforming growth factor β [TGFβ1 ]) were examined by confocal microscopy, second harmonic generation, qPCR and biochemical assays. In preterm AM defects, myofibroblast nuclei were highly deformed and contractile and expressed αSMA and Cx43. Mechanical stimulation increased collagen fibre polarisation and the effects on matrix markers were dependent on tissue region, disease state, gestational age and the number of fetuses. PGE2 levels were broadly similar but reduced after co-treatment with Cx43 antisense in late sPPROM AM defects. TGFβ1 and Cx43 gene expression were significantly increased after trauma and mechanical stimulation but this response dependent on gestational age. Mechanical stimulation affects Cx43 signalling and cell/collagen mechanics in preterm AM defects. Establishing how Cx43 regulates mechanosignalling could be an approach to repair tissue integrity after trauma.

The effects of the differentiated macrophages by dexamethasone on the immune responses

The characteristics of M2 macrophages suggest immunotherapeutic approaches for inducing immunological tolerance. The current study aimed to evaluate the effect of Dexamethasone (Dex) treatment on monocytes polarization and its impact on immune responses. The monocytes were extracted from the rat's blood samples. The effects of Dex concentration and treatment duration on monocyte viability, phagocytosis of rabbit red blood cell (RRBC) antigens, and cytokine gene expression were evaluated using MTT, ELISA, and Real-Time PCR analysis, respectively. The monocytes treated with Dex were injected into the rats as an autograft. The effects of the grafted cells were assessed on immune responses, monocyte differentiation, and pathological lesions, in comparison to the control groups.Treatment of monocytes with 10-5 M of Dex for 48 h increased the expression of IL-10 and TGF-β genes, while reducing the expression of TNF-α and IL-1 genes. The monocytes treated with antigen and Dex showed higher CD206 gene expression compared to CD80. The cells that were treated with Dex had the highest concentration of antigens after five days. Administration of the grafted cells to the animals has some significant effects on innate immune responses and no impact on pathological lesions. The group that received cells treated with Dex and antigen experienced a significant decrease in anti-RBC antibody titers. Additionally, there was a significant difference in the expression of cytokine genes and M2 differentiation markers among the groups that were evaluated. The effects of Dex on the viability and differentiation of monocytes depend on the dosage, timing, and duration of the treatment.

Zika virus co-opts microRNA networks to persist in placental niches detected by spatial transcriptomics

Zika virus congenital infection evades double-stranded RNA detection and may persist in the placenta for the duration of pregnancy without accompanying overt histopathologic inflammation. Understanding how viruses can persist and replicate in the placenta without causing overt cellular or tissue damage is fundamental to deciphering mechanisms of maternal-fetal vertical transmission. Placenta-specific microRNAs are believed to be a tenet of viral resistance at the maternal-fetal interface. We aimed to test the hypothesis that the Zika virus functionally disrupts placental microRNAs, enabling viral persistence and fetal pathogenesis. To test this hypothesis, we used orthogonal approaches in human and murine experimental models. In primary human trophoblast cultures (n=5 donor placentae), we performed Argonaute high-throughput sequencing ultraviolet-crosslinking and immunoprecipitation to identify any significant alterations in the functional loading ofmicroRNAs and their targets onto the RNA-induced silencing complex.Trophoblasts from same-donors were split and infected with a contemporary first-passage Zika virus strain HN16 (multiplicity of infection=1 plaque forming unit per cell) or mock infected. To functionally cross-validate microRNA-messenger RNA interactions, we compared our Argonaute high-throughput sequencing ultraviolet-crosslinking and immunoprecipitation results with an independent analysis of published bulk RNA-sequencing data from human placental disk specimens (n=3 subjects; Zika virus positive in first, second, or third trimester, CD45- cells sorted by flow cytometry) and compared it with uninfected controls (n=2 subjects). To investigate the importance of these microRNA and RNA interference networks in Zika virus pathogenesis, we used a gnotobiotic mouse model uniquely susceptible to the Zika virus. We evaluated if small-molecule enhancement of microRNA and RNA interference pathways with enoxacin influenced Zika viruspathogenesis (n=20 dams total yielding 187 fetal specimens). Lastly, placentae (n=14 total) from this mouse model were analyzed with Visium spatial transcriptomics (9743 spatial transcriptomes) to identify potential Zika virus-associated alterations in immune microenvironments. We found that Zika virus infection of primary human trophoblast cells led to an unexpected disruption of placental microRNA regulation networks. When compared with uninfected controls, Zika virus-infected placentae had significantly altered SLC12A8, SDK1, andVLDLR RNA-induced silencing complex loading and transcript levels (-2<log2(fold-change)>2; adjusted P value <.05; Wald-test with false discovery rate correction q<0.05). In silico microRNA target analyses revealed that 26 of 119 transcripts (22%) in the transforming growth factor-β signaling pathway were targeted by microRNAs that were found to be dysregulated following Zika virus infection in trophoblasts. In gnotobiotic mice, relative to mock controls, Zika virus-associated fetal pathogenesis included fetal growth restriction (P=.036) and viral persistence in placental tissue (P=.011). Moreover, spatial transcriptomics of murine placentae revealed that Zika virus-specific placental niches were defined by significant up-regulation of complement cascade components and coordinated changes in transforming growth factor-β gene expression. Finally, treatment of Zika virus-infected mice with enoxacin abolished placental Zika virus persistence, rescued the associated fetal growth restriction, and the Zika virus-associated transcriptional changes in placental immune microenvironments were no longer observed. These results collectively suggest that (1) Zika virus infection and persistence is associated with functionally perturbed microRNA and RNA interference pathways specifically related to immune regulation in placental microenvironments and (2) enhancement of placental microRNA and RNA interference pathways in mice rescued Zika virus-associated pathogenesis, specifically persistence of viral transcripts in placental microenvironments and fetal growth restriction.

The effects of anti-PD-L1 monoclonal antibody on the expression of angiogenesis and invasion-related genes.

The role of PD-L1 in regulating the immunosuppressive tumor microenvironment via its binding on PD-1 receptors is extensively studied. The PD-1/PD-L1 axis is a significant way of cancer immune escape, and PD-L1 expression on tumor cells is suggested as a predictive marker for anti-PD-1/PD-L1 monoclonal antibodies (MoAbs). However, the tumor-intrinsic role of PD-L1 is not known well. Therefore, we aimed to investigate the effects of anti-PD-L1 antibodies on the expression of angiogenesis and metastasis-related genes in tumor cells. The experiments were done with prostate cancer and melanoma cells with low PD-L1 expression (<5%) and prostate and breast cancer cells with high PD-L1 expression (>50%). The gene and protein expressions of VEGFA, E-cadherin, TGFβ1, EGFR, and bFGF in tumor cells were assayed at the 3 different doses of the anti-PD-L1 antibody. We found that VEGFA, E-cadherin and TGFβ1 expressions increased in PD-L1 high cells but decreased in PD-L1 low cells after anti-PD-L1 treatment. EGFR expression levels were variable in PD-L1 high cells, while decreased in PD-L1 low cells upon treatment. Also, the anti-PD-L1 antibody was found to increase bFGF expression in the prostate cancer cell line with high PD-L1 expression. Our results suggest that the binding of PD-L1 on tumor cells by an anti-PD-L1 monoclonal antibody may affect tumor-intrinsic mechanisms. The activation of angiogenesis and metastasis-related pathways by anti-PD-L1 treatment in PD-L1 high tumors might be a tumor-promoting mechanism. The decrease of VEGFA, TGFβ1 and EGFR upon anti-PD-L1 treatment in PD-L1 low tumor cells provides a rationale for the use of those antibodies in PD-L1 low tumors.

Resveratrol mitigates miR-212-3p mediated progression of diesel exhaust-induced pulmonary fibrosis by regulating SIRT1/FoxO3

BackgroundDiesel exhaust (DE) exposure contributes to the progression of chronic respiratory diseases and is associated with dysregulation of microRNA expression. The present study aims to investigate the involvement of miRNAs and target genes in DE-induced lung fibrosis. MethodsC57BL/6 mice were divided into three groups. Group 1 mice were exposed to filtered air (Control). Group 2 mice were exposed to DE for 30 min per day, 5 days per week, for 8 weeks (DE). Group 3 mice received DE exposure along with resveratrol on alternate days for the last 2 weeks (DE + RES). Mice were sacrificed to isolate RNA from lung tissue for miRNA microarray profiling. Bronchoalveolar lavage fluid and lung tissues were collected for cell count and biochemical analysis. ResultsDE exposure resulted in differential expression of 28 miRNAs with fold change >2 (p < 0.05). The upregulated miR-212-3p was selected for further analysis. Consensus analysis revealed enrichment of SIRT1 in the FoxO pathway, along with a co-annotation of reduced body weight (p < 0.05). A549 cells transfected with a miR-212-3p inhibitor showed a dose-dependent increase in SIRT1 expression, indicating SIRT1 as a direct target. Treatment with resveratrol restored SIRT1 and miR-212-3p expression and led to a reduction in inflammatory cytokines (p < 0.05). The modulation of SIRT1 correlated negatively with macrophage infiltration, confirming its role in regulating cellular infiltration and lung inflammation. Fibronectin, alpha-SMA, and collagen levels were significantly decreased in DE + RES compared to DE group suggesting modulation of cellular functions and resolution of lung fibrosis. Furthermore, a significant decrease in FoxO3a and TGF-β gene expressions was observed upon resveratrol administration thereby downregulating pro-fibrotic pathway. ConclusionsThe present study demonstrates resveratrol treatment stabilizes SIRT1 gene expression by attenuating miR-212-3p in DE-exposed mice, leading to downregulation of TGF-β and FoxO3a expressions. The study highlights the therapeutic role of resveratrol in the treatment of DE-induced pulmonary fibrosis.

Anti-inflammatory effect of Alpinia galanga extract on acute inflammatory cell model of peripheral blood mononuclear cells stimulated with TNF-α.

Aim Studies have shown that SARS-Cov-2 has the ability to activate proinflammatory cytokine leading to acute inflammation. During the SARS-Cov-2 infection, an increase of the secretion of production TNF-α in seen in COVID-19 patients along with a decrease in anti-inflammatory cytokine IL-10, and growth factor TGF-β caused cytokine storm and damaged tissues. Alpinia galanga extract contains several secondary metabolites with strong antiinflammation and antioxidant effect. The aim of this study was to evaluate the effect of Alpinia galanga extract on peripheral blood mononuclear cells (PMBC) acute inflammation cells model stimulated with TNF-α. Methods Alpinia galanga was extracted under maceration methods on ethanol 96%. The PMBC was collected from three healthy humans and isolated using ficol reagent and cultured with the TNF-α 100pg/mL for 72 h. The TNF-α levels were evaluated under ELISA reader. Furthermore, the IL-10 and TGF-β gene expression was analysed using qRT-PCR after 24 h treatment with Alpinia galanga extract. Results Alpinia galanga extract has no cytotoxic effect on Vero cells with IC50 value of >1000µg/mL. The PBMC acute inflammation cells model stimulated by TNF-α 100pg/mL, after 72 h induction the PBMC cells significantly expressed a high level of TNF-α up to 341±10.87 pg/mL. Furthermore, the treatment of Alpinia galanga significantly increased the anti-inflammatory cytokine IL-10 and growth factor TGF-β in dose dependent manner. Conclusion These findings suggested that Alpinia galanga extract has strong antiinflammation activity.

Comparison of Clinical Outcomes, Pathologic Characteristics, and Immune-Related Features of Postradiation vs Sporadic Oral Cavity Squamous Cell Carcinoma

Postradiation oral cavity squamous cell carcinoma (OCSCC) is a common secondary malignant neoplasm affecting survivors of head and neck cancer who underwent radiotherapy. The clinical, pathologic, and immune-related features of postradiation OCSCC are poorly characterized, and treatment options are limited because of surgical difficulty and high morbidity associated with reirradiation. To determine whether postradiation OCSCC has distinctive clinical, pathologic, and immune-related features compared with demographic-matched sporadic OCSCC. This retrospective matched cohort study was conducted at a single tertiary oncology center in Hong Kong. Participants included consecutive patients with OCSCC diagnosed between 2000 and 2020. Patients with postradiation OCSCC were matched with patients with sporadic OCSCC using age, year of diagnosis, sex, and anatomic subsites. Data analysis was performed from July to December 2022. Head and neck irradiation involving the oral cavity before the diagnosis of OCSCC. The primary outcomes were relapse pattern, survival, and causes of death. Pathologic features; immunohistochemical staining for programmed death-ligand 1, PD-1, MSH6, PMS2, FOXP3, and Ki67; and mRNA expression of 31 immune-related genes were also analyzed. A total of 173 patients, 60 with postradiation OCSCC (median [IQR] age, 63.8 [53.0-71.7] years; 43 men [71.7%]) and 113 with sporadic OCSCC (median [IQR] age, 64.4 [52.8-70.6] years; 83 men [73.5%]), were included. Patients with postradiation OCSCC had a higher proportion of N0 disease than those with sporadic OCSCC (50 patients [83.3%] vs 56 patients [49.6%]). With a median (IQR) follow-up of 10.2 (1.2-20.5) years, the 10-year relapse-free survival rates were lower in patients with postradiation OCSCC than sporadic OCSCC (29.6% [95% CI, 17.1%-43.2%] vs 52.4% [95% CI, 41.8%-62.0%]; P = .04), and the same was true for overall survival (30.5% [95% CI, 17.6%-44.4%] vs 52.3% [95% CI, 41.4%-62.1%]; P = .03). All relapses in patients with postradiation OCSCC were locoregional, whereas 35.2% of relapses (12 of 34 patients) in patients with sporadic OCSCC were distant. Despite similar 10-year disease-specific survival rates between the 2 groups (68.8% [95% CI, 55.8%-81.0%] vs 67.1% [95% CI, 57.5%-76.5%]; P = .91), patients with postradiation OCSCC had excess mortality due to pneumonia and cerebrovascular events. Postradiation OCSCC exhibited more adverse pathologic features (perineural invasion, worse pattern of invasion, and tumor budding), higher PD-1 expression, and higher gene expression of CD4 and TGF-β compared with sporadic OCSCC. This retrospective matched cohort study found distinctive pathologic characteristics and relapse patterns of postradiation OCSCC compared with sporadic OCSCC, which may be attributable to the lack of adjuvant radiotherapy, aggressive biologic phenotype, and different host immune response. Further exploration of the role of immune checkpoint therapy may be justified.

Amelioration of CCl4-induced oxidative stress and hepatotoxicity by Ganoderma lucidum in Long Evans rats

Liver disease is a serious health problem affecting people worldwide at an alarming rate. The present study aimed to investigate the protective effects of Ganoderma lucidum against CCl4-induced liver toxicity in rats. The experimental Long Evans rats were divided into five groups, of which four groups were treated with carbon tetrachloride (CCl4). Among the CCl4 treated groups, one of the groups was treated with silymarin and two of them with ethanolic extract of G. lucidum at 100 and 200 mg/Kg body weight. The oxidative stress parameters and endogenous antioxidant enzyme concentrations were assessed by biochemical tests. Liver enzymes ALT, AST, and ALP were determined spectrophotometrically. Histopathological examinations were carried out to assess hepatic tissue damage and fibrosis. Reverse transcription PCR (RT-PCR) was performed to determine the expression of IL-1β, IL-6, IL-10, TNF-α, and TGF-β genes. Gas Chromatography-Mass Spectroscopy (GC–MS) analysis revealed that G. lucidum is rich in several phytochemicals including 6-Octadecanoic acid (55.81%), l-( +)-Ascorbic acid 2,6-dihexadecanoate (18.72%), Cis-11-Eicosenamide (5.76%), and Octadecanoic acid (5.26%). Treatment with the G. lucidum extract reduced the elevated ALT, AST, ALP levels, and cellular oxidative stress markers and increased the endogenous antioxidant levels. Histopathology observations revealed that the inflammation, infiltration of immune cells, and aberration of collagen fibers in the hepatocytes were altered by the G. lucidum treatment. The increased expression of inflammatory cytokines TNF-α, TGF-β, IL-1 β, and IL-6 were markedly suppressed by G. lucidum extract treatment. G. lucidum also prevented the suppression of protective IL-10 expression by CCl4. This study strongly suggests that G. lucidum extract possesses significant hepatoprotective activity as evidenced by reduced oxidative stress and inflammation mediated by suppression in inflammatory cytokine expression and increased protective IL-10 cytokine expression.

Investigation of the roles of TGFβ1, CUG2, TGFBI genes, and thiol-disulfide balance on prostate cancer and metastasis

Abstract Objectives Transforming growth factor-beta (TGFβ1) is involved in tumorigenesis and metastasis. It provides this effect both by disrupting the thiol-disulfide balance and through the cancer-upregulated gene (CUG2) and transforming growth factor beta-induced (TGFBI) genes in the signaling pathway. In this study, the roles of TGFβ1 and related genes, as well as thiol-disulfide balance, in the formation of prostate cancer and metastasis were investigated. Methods Tissue samples were taken from 33 benign prostatic hyperplasia (BPH) and 35 prostate cancer (PC) patients to determine the Gleason score and metastasis. TGFβ1, CUG2, and TGFBI gene expression levels were measured by RT-PCR. Serum prostate specific antigen (PSA) levels were measured in patients, and PSA density (PSAD) was calculated. Total thiol and native thiol measurements in serum were performed spectrophotometrically, and disulfide was calculated. Results In patients with prostate cancer and metastases, PSA and PSAD levels were high, while total thiol and native thiol were significantly lower (p&lt;0.05). TGFβ1, CUG2 and TGFBI gene expression levels were higher in patients with prostate cancer and metastases and were negatively correlated with total thiol and native thiol (p&lt;0.001). Conclusions As a result of our study, we determined that the increase in TGFβ1, CUG 2 and TGFBI in prostate cancer plays an important role in cancer formation and metastasis by disrupting the thiol-disulfide balance.

In ovo injection of silver nanoparticles modulates some productive traits and hepatic gene expression of broilers exposed to lipopolysaccharide challenge.

This study aimed to evaluate the effectiveness of the embryonic injection of silver nanoparticles (SilNPs) on some productive traits and hepatic gene expression of lipopolysaccharide (LPS)-challenged broilers after a 42d rearing period. 560 fertile eggs were randomly allocated to four groups and received either of the following treatments at d 7 of incubation, control (no injection), placebo (1mL saline), SilNP20 (20mg/kg silver nanoparticles), or SilNP40 (40mg/kg silver nanoparticles). After the incubation, 320 hatchlings experienced a 42d standard rearing period. Live body weight (LBW), feed intake (FI), and feed conversion ratio (FCR) were weekly recorded. At the end of the experiment, two birds from each replicate (n = 8 per treatment) were exposed to LPS intraperitoneal injection at 48, 24, and 12h before slaughter time. They were also used for blood, intestinal, and microbial evaluations. The hepatic mRNA levels of tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), transforming growth factor beta (TGF-β), and insulin-like growth factor I (IGF-I) were assessed at d 1 and 42 of the experiment. Adminstration of SiLNPs improved LBW, FI, and FCR and also enhanced liver and spleen weights (P < 0.05). SilNP20 birds had significantly lower bursa of Fabricius weight (P < 0.05). SilNP20 had lower total cholesterol levels than others. There was a significant difference (P < 0.05) between SliNP40 and SilNP20 in the ratio of villus height to crypt width. Compared to control groups, chicks of SilNP20, but not SilNP40, showed a significant increase in the relative expression of TNF-α, IL-6, TGF-β, and IFG-I genes at d 1. On d 42, however, both SilNP20 and SilNP40 had significantly higher TNF-α and TGF-β levels than both controls. Silver nanoparticles did not significantly affect the microflora of the ileum and cecum in the current study. In summary, SilNPs administration to chick embryos showed a long-term positive effect on their productive performance.

The Effects of Mesenchymal Stem Cells on the Gene Expression of TGF-beta and IFN-gamma in Patients with Rheumatoid Arthritis.

The therapeutic and immunomodulatory potential of mesenchymal stem cells (MSCs) in rheumatoid arthritis (RA) has attracted considerable scientific attention in recent decades. This study aimed to evaluate the expression of genes encoding interleukin (IL)4 and IL10, as well as interferon-gamma (IFNG) and transforming growth factor beta (TGFB1) in refractory RA patients following intravenous injection of autologous bone marrow-derived MSCs (BM-MSCs). This study was registered in Iranian Registry of Clinical Trials (IRCT) (2015102824760N1) and ClinicalTrials.gov (identifier: NCT03333681). Blood samples were taken from 13 patients before and 1 and 6 months after the MSC injection to evaluate the clinical manifestations, paraclinical factors, and expression of IL4, IL10, IFNG, and TGFB1 genes employing the SYBR Green real-time reverse-transcriptase polymerase chain reaction (RT-PCR) technique. There was a significant increase in the expression of TGFB1 at 1 and 6 months after the MSC injection compared to that in the baseline, while the expression of IL4 and IL10 did not change significantly. On the other hand, the expression of IFNG increased significantly after 1 month but decreased significantly at 6 months compared to 1 month after the intervention. Nevertheless, it showed no significant decrease compared to the baseline. A significant decrease was observed for the expression of IFNG 6 months after the injection compared to that after 1 month, which was in concordance with the rise in the expression of the TGFB1 gene. A significant change in the gene expression of TGFB1 and IFNG in our study wasconsistent with the amelioration of clinical manifestations, suggesting a mechanism of action for MSCs in the treatment of RA.

Assessment of the Influence of 5-Fluorouracil on SMAD4 and TGFB1 Gene Expression, Apoptosis Induction and DNA Damage in Human Cell Lines.

Suppressor of mothers against decapentaplegic homolog 4 (SMAD family member 4, SMAD4) is involved in the adenoma-carcinoma pathway, leading to the development of colon cancer. The encoded protein is a key downstream signaling mediator in the TGFβ pathway. This pathway has tumor-suppressor functions, including cell-cycle arrest and apoptosis. Its activation in late-stage cancer can promote tumorigenesis, including metastasis and chemoresistance. Most colorectal cancer patients receive chemotherapy based on 5-FU as an adjuvant treatment. However, the success of therapy is hampered by multidrug resistance by neoplastic cells. In colorectal cancer, resistance to 5-FU-based therapy is influenced by SMAD4 gene expression, as patients with decreased SMAD4 gene expression probably have a higher risk of developing 5-FU-induced resistance. The mechanism leading to the development of this phenomenon is not fully understood. Therefore, the present study assesses the possible influence of 5-FU on changes in the expression of the SMAD4 and TGFB1 genes. The effect of 5-FU on the expression of SMAD4 and TGFB1 in colorectal cancer cells derived from the CACO-2, SW480 and SW620 cell lines was evaluated using real-time PCR. The cytotoxicity of 5-FU on colon cancer cells was assessed by the MTT method, and its effect on the induction of cell apoptosis and the initiation of DNA damage using a flow cytometer. Significant changes in the level of SMAD4 and TGFB1 gene expression were noted in the CACO-2, SW480 and SW620 cells treated with 5-FU at various concentrations during 24 h and 48 h exposure. The use of 5-FU at a concentration of 5 µmol/L resulted in a decrease in the expression of the SMAD4 gene in all cell lines at both exposure times, while the concentration of 100 µmol/L increased the expression of the SMAD4 gene in CACO-2 cells. The level of expression of the TGFB1 gene was higher for all cells treated with 5-FU at the highest concentrations, while the exposure time was extended to 48 h. The observed in vitro changes in CACO-2 cells caused by 5-FU may be of clinical relevance when choosing the drug concentration for treating patients with colorectal cancer. It is possible that 5-FU has a stronger effect on colorectal cancer cells at the higher concentrations. Low concentrations of 5-FU may not have a therapeutic effect and may also influence drug resistance in cancer cells. Higher concentrations and prolonged exposure time may affect SMAD4 gene expression, which may increase the effectiveness of therapy.

Clitoria ternatea Flower Extract-Based Gel Elevates TGF-β1 Gene Expression and Collagen Density in UVB-Induced Collagen Loss Rat Skin

Ultraviolet B (UVB) irradiation on the skin induces collagen loss by the ROS pathways and pro-collagen factors, mainly the TGF-β family. Many studies of antioxidant compounds in Clitoria ternatea have shown to induce decreasing ROS production, in the collagen loss process. However, the molecular mechanism of action is still not clearly studied, inducing the mechanism of TGF-β1 activation and regulation of collagen loss need to be explored. This study aimed to investigate the effect of Clitoria ternatea flower extract-based gel on TGF-β1 gene expression and collagen density in UVB-induced rat skin. A randomized control group post-test-only design was conducted on 20 male Wistar rats aged 8-10 weeks (150-250 grams). Rats were divided into four groups: Untreated group (Healthy Control), UVB+Based gel group (Negative Control), 5% Clitoria ternatea flower extract-based gel group (T-5%), and 10% Clitoria ternatea flower extract-based gel group (T-10%). Rats exposed to UVB for 5 consecutive days and treated with gel every day for two weeks. At week 3, rat skins were isolated for TGF-β1 gene expression using qRT-PCR, and Masson Trichrome-collagen specific staining. Comparative analysis of qRT-PCR showed that the lowest mean TGF-β1 expression was seen in the Negative Control group (0.01±0.00), followed by T-5% (0.11±0.22), Healthy Control group (1.01±0.01) and T-10% (2.32±2.46). The highest amount of collagen is in the Healthy Control group (43.83±5.3), followed by T-10% (38.39±3.1), T-5% (29.04±3.2), and the Negative Control group. (13.87±2.7). Clitoria ternatea flower extract-based gel may increase TGF-β1 gene expression and collagen deposition in the UVB-induced collagen loss rat skin.

Salivary transforming growth factor beta in oral submucous fibrosis: A diagnostic and predictive marker.

Growth factors and cytokines like transforming growth factor beta (TGF-β) play a key role in the pathogenesis of oral submucous fibrosis. To elucidate the role of Salivary TGF-β isoforms as a predictive and diagnostic marker for oral submucous fibrosis. A total of 30 OSMF and 10 control patients were included in this study, and their clinic-epidemiological data was recorded. The expression of TGF-β genes-TGF-β1, TGF-β2, TGF-β3-was studied by a real-time polymerase chain reaction in tissue and saliva. Patients were given medicinal intervention for 12 weeks along with jaw-opening exercises. Expression of salivary TGF-β genes was studied at 12 weeks. SPSS software version 20. Expression of salivary TGF beta isoforms in OSMF was more than in the control group. There was an increase in salivary TGF-β1, β2, β3 expressions with increasing clinical grades of OSMF and advancing the stage of the disease. Expression of all the TGF beta isoforms was decreased after treatment with statistically significant results. Statistically significant correlations were found between the mean difference of TGF-β1 and the mean difference between mouth opening and tongue protrusion. Salivary TGF-β isoforms may be used in diagnosis, risk assessment, and screening of the entire population at risk of OSMF after its clinical validation. However, adequate sample size and segmental assessment of the expression of TGF-β isoforms are needed for further evaluation.

In Vitro Study of the Anti-inflammatory and Antifibrotic Activity of Tannic Acid-Coated Curcumin-Loaded Nanoparticles in Human Tenocytes.

Tendinitis is a tendon disorder related to inflammation and pain, due to an injury or overuse of the tissue, which is hypocellular and hypovascular, leading to limited repair which occurs in a disorganized deposition of extracellular matrix that leads to scar formation and fibrosis, ultimately resulting in impaired tendon integrity. Current conventional treatments are limited and often ineffective, highlighting the need for new therapeutic strategies. In this work, acetalated-dextran nanoparticles (AcDEX NPs) loaded with curcumin and coated with tannic acid (TA) are developed to exploit the anti-inflammatory and anti-fibrotic properties of the two compounds. For this purpose, a microfluidic technique was used in order to obtain particles with a precise size distribution, aiming to decrease the batch-to-batch variability for possible future clinical translation. Coating with TA increased not only the stability of the nanosystem in different media but also enhanced the interaction and the cell-uptake in primary human tenocytes and KG-1 macrophages. The nanosystem exhibited good biocompatibility toward these cell types and a good release profile in an inflammatory environment. The efficacy was demonstrated by real-time quantitative polymerase chain reaction, in which the curcumin loaded in the particles showed good anti-inflammatory properties by decreasing the expression of NF-κb and TA-coated NPs showing anti-fibrotic effect, decreasing the gene expression of TGF-β. Overall, due to the loading of curcumin and TA in the AcDEX NPs, and their synergistic activity, this nanosystem has promising properties for future application in tendinitis.