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

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

The role of oxymatrine in regulating TGF-ß1 in rats with hepatic fibrosis

Abstract Purpose: To investigate whether oxymatrine (OMT) prevents hepatic fibrosis in rats by regulating liver transforming growth factor β1 (TGF-β1) level. Methods: Hepatic fibrosis was induced in rats by thioacetamide (TAA). Blood was collected at the end of week 12 to determine the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutathione (GSH). Changes in liver tissue were observed after hematoxylin-eosin (HE) staining. Results: Fibrosis was confirmed by Masson's collagen staining. Liver TGF-β1 level was determined by ELISA. OMT significantly reduced serum ALT and AST but increased GSH levels in rats with hepatic fibrosis. Moreover, it significantly improved liver histology in rats with TAA-induced hepatic fibrosis. It significantly decreased liver TGF-β1 level compared to that in the untreated group. It also significantly reduced collagen deposition in rats. Conclusion: Oxymatrine is effective in protecting rats from thioacetamide-induced hepatic fibrosis by regulating TGF-β1 expression.

Immunohistochemical expression of TGF-ß1 and osteonectin in engineered and Ca(OH)2-repaired human pulp tissues

Abstract The aim of the present study was to evaluate the expression of transforming growth factor-β1 (TGF-β1) and osteonectin (ON) in pulp-like tissues developed by tissue engineering and to compare it with the expression of these proteins in pulps treated with Ca(OH)2 therapy. Tooth slices were obtained from non-carious human third molars under sterile procedures. The residual periodontal and pulp soft tissues were removed. Empty pulp spaces of the tooth slice were filled with sodium chloride particles (250-425 µm). PLLA solubilized in 5% chloroform was applied over the salt particles. The tooth slice/scaffold (TS/S) set was stored overnight and then rinsed thoroughly to wash out the salt. Scaffolds were previously sterilized with ethanol (100-70°) and washed with phosphate-buffered saline (PBS). TS/S was treated with 10% EDTA and seeded with dental pulp stem cells (DPSC). Then, TS/S was implanted into the dorsum of immunodeficient mice for 28 days. Human third molars previously treated with Ca(OH)2 for 90 days were also evaluated. Samples were prepared and submitted to histological and immunohistochemical (with anti-TGF-β1, 1:100 and anti-ON, 1:350) analyses. After 28 days, TS/S showed morphological characteristics similar to those observed in dental pulp treated with Ca(OH)2. Ca(OH)2-treated pulps showed the usual repaired pulp characteristics. In TS/S, newly formed tissues and pre-dentin was colored, which elucidated the expression of TGF-β1 and ON. Immunohistochemistry staining of Ca(OH)2-treated pulps showed the same expression patterns. The extracellular matrix displayed a fibrillar pattern under both conditions. Regenerative events in the pulp seem to follow a similar pattern of TGF-β1 and ON expression as the repair processes.

Effect of EDTA on TGF-ß1 released from the dentin matrix and its influence on dental pulp stem cell migration

Abstract: Bioactive molecules stored in dentin, such as transforming growth factor beta1 (TGF-b1), may be involved in the signaling events related to dental tissue repair. The authors conducted an in vitro evaluation of the amount of TGF-b1 released from dentin slices after treatment with 10% ethylenediaminetetraacetic acid (EDTA), 2.5% sodium hypochlorite (NaOCl) or phosphate-buffered saline (PBS), and the effect of this growth factor on stem cell migration from human exfoliated deciduous teeth (SHED). Sixty 1-mm-thick tooth slices were prepared with or without the predentin layer, and treated with either 10% EDTA for 1 minute, 2.5% NaOCl for 5 days or kept in PBS. Tooth slice conditioned media were prepared and used for TGF-b1 ELISA and migration assays. Culture medium with different concentrations of recombinant human TGF-b1 (0.5, 1.0, 5.0 or 10.0 ng/mL) was also tested by migration assay. The data were evaluated by ANOVA and Tukey's test. Optical density values corresponding to media conditioned by tooth slices either containing or not containing the predentin layer and treated with 10% EDTA were statistically greater than the other groups and close to 1 ng/mL. Increased rates of migration toward media conditioned by tooth slices containing the predentin layer and treated with PBS, 10% EDTA or 2.5% NaOCl were observed. Recombinant human TGF-b1 also stimulated migration of SHED, irrespective of the concentration used. EDTA may be considered an effective extractant of TGF-b1 from the dentin matrix. However, it does not impact SHED migration, suggesting that other components may account for the cell migration.

Protective roles of pulmonary rehabilitation mixture in experimental pulmonary fibrosis in vitro and in vivo

Abnormal high mobility group protein B1 (HMGB1) activation is involved in the pathogenesis of pulmonary fibrosis. Pulmonary rehabilitation mixture (PRM), which combines extracts from eight traditional Chinese medicines, has very good lung protection in clinical use. However, it is not known if PRM has anti-fibrotic activity. In this study, we investigated the effects of PRM on transforming growth factor-β1 (TGF-β1)-mediated and bleomycin (BLM)-induced pulmonary fibrosis in vitro and in vivo. The effects of PRM on TGF-β1-mediated epithelial-mesenchymal transition (EMT) in A549 cells, on the proliferation of human lung fibroblasts (HLF-1) in vitro, and on BLM-induced pulmonary fibrosis in vivo were investigated. PRM treatment resulted in a reduction of EMT in A549 cells that was associated with attenuating an increase of vimentin and a decrease of E-cadherin. PRM inhibited the proliferation of HLF-1 at an IC50 of 0.51 µg/mL. PRM ameliorated BLM-induced pulmonary fibrosis in rats, with reduction of histopathological scores and collagen deposition, and a decrease in α-smooth muscle actin (α-SMA) and HMGB1 expression. An increase in receptor for advanced glycation end-product (RAGE) expression was found in BLM-instilled lungs. PRM significantly decreased EMT and prevented pulmonary fibrosis through decreasing HMGB1 and regulating RAGE in vitro and in vivo. PRM inhibited TGF-β1-induced EMT via decreased HMGB1 and vimentin and increased RAGE and E-cadherin levels. In summary, PRM prevented experimental pulmonary fibrosis by modulating the HMGB1/RAGE pathway.

Resveratrol ameliorates diabetes-induced renal damage through regulating the expression of TGF-beta 1, collagen IV and Th17/Treg-related cytokines in rats / El resveratrol mejora el daño renal inducido por la diabetes a través de la regulación de la expresión de TGF-beta 1, colágeno IV y las citocinas relacionados con Th17/Treg en ratas

OBJECTIVES: This study aimed at detecting the protective effects of resveratrol on diabetes-induced renal damage and on the expression of transforming growth factor-beta 1 (TGF-β1), collagen IV and Th17/Tregrelated cytokines in streptozotocin-induced diabetic rats. METHODS: Twenty diabetic rats were further randomly divided into diabetic model group (DM group) and resveratrol group with 10 animals in each group. Another 10 non-diabetic rats served as control. The dia-betic rats in the resveratrol group were administered resveratrol for eight consecutive weeks (via gavage, 50 mg/kg daily, dissolved in saline). Rats in the control group and DM group received the same volume of saline only (via gavage). Renal function was measured. Histopathology changes of the kidney tissue were observed using haematoxylin and eosin staining. Levels of TGF-β1 and collagen IV in kidney homogenate were measured with enzyme-linked immunosorbent assay (ELISA). The level of Th17-related cytokines (IL-17A, IL-25) and Treg-related cytokines (IL-35, IL-10) in serum and in the supernatant of the kidney homogenate were determined using ELISA. RESULTS: Diabetic rats had damaged renal function, higher levels of TGF-β1, collagen IV, IL-17A and IL-25, as well as lower levels of IL-35 and IL-10, when compared to the control rats. Compared to the diabeticrats without resveratrol treatment, application of resveratrol to the diabetic rats ameliorated the renal function, inhibited the expression of TGF-β1, collagen IV, IL-17A and IL-25, and increased the expression IL-35 and IL-10. CONCLUSION: Resveratrol might ameliorate diabetes-induced renal damage through mediating the balance of Th17/Treg-related cytokines and inhibiting the expression of TGF-β1 and collagen IV.

OBJETIVOS: Este estudio estuvo encaminado a detectar los efectos protectores del resveratrol en el daño renal inducido por diabetes y en la expresión del factor de crecimiento transformante beta-1 (TGF-β1), el colágeno IV, y las citocinas relacionados con Th17/Treg en ratas con diabetes inducida por estreptozotocina. MÉTODOS: Veinte ratas diabéticas fueron divididas aleatoriamente en un grupo modelo diabético (Grupo MD) y un grupo de resveratrol, con 10 animales en cada grupo. A las ratas diabéticas en el grupo de resveratrol se les administró resveratrol durante ocho semanas consecutivas (mediante sonda nasogástrica, 50 mg/kg diarios, disuelto en suero salino). Las ratas en el grupo control y el grupo MD recibieron el mismo volumen de solución salina solamente (vía sonda nasogástrica). Se midió la función renal. Se observaron cambios en la histopatología del tejido del riñón usando tinción con hematoxilina y eo-sina. Se midieron los niveles de TGF-β1 y colágeno IV en un homogeneizado de riñón con ensayo por inmunoabsorción ligado a enzimas (ELISA). El nivel de las citocinas de Th17 (IL-17A, IL-25) y las citocinas de Treg (IL-35, IL-10) en suero y en el sobrenadante del homogeneizado de riñón, se determinaron mediante ELISA. RESULTADOS: Las ratas diabéticas tuvieron daño de la función renal, niveles más altos de TGF-β1, colágeno IV, IL-17A y IL-25, así como niveles más bajos de IL-35 e IL-10, en comparación con las ratas control. En comparación con las ratas diabéticas sin tratamiento con resveratrol, la aplicación de resveratrol en las ratas diabéticas mejoró la función renal, inhibió la expresión de TGF-β1, colágeno IV, IL-17A y IL-25 y aumentó la expresión de IL-35 y IL-10. CONCLUSIÓN: El resveratrol podría mitigar el daño renal inducido por la diabetes mediante la mediación con el equilibrio de las citocinas relacionados con Th17/Treg, e inhibiendo la expresión de TGF-β1 y colágeno IV.