Expression and possible role of Smad3 in postnecrotizing enterocolitis stricture

ObjectiveTo investigate the expression of Smad3 (mothers against decapentaplegic homolog 3) protein in postnecrotizing enterocolitis stricture and its possible mechanism of action.MethodsWe used immunohistochemistry to detect the expression characteristics of Smad3 and nuclear factor kappa B (NF-κB) proteins in human postnecrotizing enterocolitis stricture. We cultured IEC-6 (crypt epithelial cells of rat small intestine) in vitro and inhibited the expression of Smad3 using siRNA technique. Quantitative PCR, western blotting, and ELISA were used to detect the changes in transforming growth factor-β1 (TGF-β1), NF-κB, tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), and zonula occludens-1 (ZO-1) messenger RNA (mRNA) and protein expressions in IEC-6 cells. CCK8 kit and Transwell cellular migration were used to detect cell proliferation and migration. Changes in epithelial–mesenchymal transition (EMT) markers (E-cadherin and vimentin) in IEC-6 cells were detected by immunofluorescence technique.ResultsThe results showed that Smad3 protein and NF-κB protein were overexpressed in narrow intestinal tissues and that Smad3 protein expression was positively correlated with NF-κB protein expression. After inhibiting the expression of Smad3 in IEC-6 cells, the mRNA expressions of NF-κB, TGF-β1, ZO-1, and VEGF decreased, whereas the mRNA expression of TNF-α did not significantly change. TGF-β1, NF-κB, and TNF-α protein expressions in IEC-6 cells decreased, whereas ZO-1 and intracellular VEGF protein expressions increased. IEC-6 cell proliferation and migration capacity decreased. There was no significant change in protein expression levels of EMT markers E-cadherin and vimentin and also extracellular VEGF protein expression.ConclusionsWe suspect that the high expression of Smad3 protein in postnecrotizing enterocolitis stricture may promote the occurrence and development of secondary intestinal stenosis. The mechanism may be related to the regulation of TGF-β1, NF-κB, TNF-α, ZO-1, and VEGF mRNA and protein expression. This may also be related to the ability of Smad3 to promote epithelial cell proliferation and migration.

Quercetin Reduces Hepatic Fibrogenesis by Inhibiting TGF-β/Smad3 Signaling Pathway in LX-2 Cell Line

Background: Liver fibrosis has become one of the leading causes of morbidity and mortality in the world. Liver fibrosis progresses to cirrhosis and can eventually lead to hepatocellular carcinoma (HCC). During fibrogenesis, the hepatic stellate cells (HSCs) remain active and continuously produce more extracellular matrix (ECM). Quercetin, one of the main flavonoids in vegetables, has shown hepatoprotective potential, but its effects on liver fibrosis are not apparent. Objectives: In this study, we investigated the antifibrotic activity of quercetin following stimulation of TGF-β in the LX-2 cell line (a type of HSC-derived cell line) and its underlying mechanism in vitro. Methods: The LX-2 cells were treated with TGF-β1 (2 ng/mL) for 24 h. Next, the cells were treated with quercetin for 24 h, and the mRNA expression of α-smooth muscle actin (α-SMA), collagen1α1, and p-Smad3 protein levels were measured. Results: The results showed that the expression of α-SMA, collagen 1α1 (COL1α1) genes, and also the level of p-Smad3 protein in the presence of TGF-β increased significantly compared to the control group. Moreover, quercetin in concentrations of 75 and 100 μM inhibited TGF-β1-induced expression of α-SMA and COL1α1 genes and the p-Smad3 protein in LX-2 cells. Conclusions: We conclude that quercetin inhibits further activation of HSCs by inhibiting the TGF-β/Smad3 signaling pathway and reduces ECM accumulation during liver fibrosis in vitro, and may prevent the progression of liver fibrosis. Thus, the use of quercetin is suggested as a potential therapeutic agent in the treatment of liver fibrosis.

Effects of Simvastatin on Breast Carcinoma Cell Proliferation and Apoptosis via Transforming Growth Factor-β1/Smad3 Pathway

Objective: To investigate the function and causative role of simvastatin (Sim) in breast carcinoma cell apoptosis as well as proliferation. Methods: 20 breast carcinoma patients requiring surgery were treated with Sim (20 days, 30 mg), and samples of pre-treatment (pre) and post-treatment (post) were acquired. We detected tissue cell proliferation and apoptosis changes and used functional experiments to detect cell proliferation and apoptosis changes after treating not only estrogen receptor (ER)-positive (MCF-7) but also ER-negative cells (MDA-MB-231) with Sim or TGF-β1. Detection of p-Smad3 and total Smad3 protein expression changes was conducted, and we finally used in vivo experiments to assess the influence of Sim on breast tumor growth and drug safety. Results: Immunohistochemistry and TUNEL staining results showed that after treatment with Sim, breast carcinoma cell proliferation decreased and apoptosis increased. Functional experiments results showed that Sim notedly promoted the MDA-MB-231 and MCF-7 cell apoptosis, inhibiting migration, proliferation and epithelial mesenchymal transition. Moreover, TGF-β1 protein expression was strikingly lower in Sim group than that in DMSO group. When TGF-β1 and Sim were combined to use, the inhibitory ability of Sim on breast cancer cell proliferation markedly increased and the capability of TGF-β1 protein inducing p-Smad3 protein increased. In addition, after Sim treatment in mice, the tumor volume became smaller, the pathological changes weakened, and there was no significant effect on liver function and kidney function. Conclusion: Sim participates in breast cancer cell apoptosis and proliferation via regulating TGF-β1/Smad3 signal pathway.

Exosomale microRNA-21 Promotes Keloid Fibroblast Proliferation and Collagen Production by inhibiting Smad7

In keloid fibroblasts, microRNA-21 (miR-21) enhances activation of the TGF-β–Smad-signaling pathway by downregulating Smad7 expression, thereby promoting keloid fibroblast proliferation and collagen production. However, it is unclear whether miR-21 performs the above-mentioned functions through exosomal transport. Here, we extracted exosomes from the culture supernatants of keloid and normal skin fibroblasts, and observed that exosomes from both cell types secreted exosomes; however, keloid fibroblasts secreted significantly more exosomal miR-21 than normal skin fibroblasts (P < 0.001). Interestingly, we also observed that exosomal miR-21 could enter target keloid fibroblasts. In addition, inhibiting exosomal miR-21 upregulated Smad7 protein expression and reduced Smad2 and Smad3 protein levels in target keloid fibroblasts. Furthermore, inhibiting exosomal miR-21 downregulated collagen I and collagen III expression in target keloid fibroblasts, increased the proportion of apoptotic cells, and reduced cell proliferation. Taken together, these results show that exosomal miR-21 promoted proliferation and collagen production in keloid fibroblasts by inhibiting Smad7. Thus, we identified regulatory roles for miR-21 in promoting keloid fibroblast proliferation and participating in keloid formation and development. These findings imply that miR-21 may serve as a novel target for controlling the development of keloids.

PDLIM5 inhibits STUB1-mediated degradation of SMAD3 and promotes the migration and invasion of lung cancer cells

Transforming growth factor β (TGFβ) signaling plays an important role in regulating tumor malignancy, including in non–small cell lung cancer (NSCLC). The major biological responses of TGFβ signaling are determined by the effector proteins SMAD2 and SMAD3. However, the regulators of TGFβ–SMAD signaling are not completely revealed yet. Here, we showed that the scaffolding protein PDLIM5 (PDZ and LIM domain protein 5, ENH) critically promotes TGFβ signaling by maintaining SMAD3 stability in NSCLC. First, PDLIM5 was highly expressed in NSCLC compared with that in adjacent normal tissues, and high PDLIM5 expression was associated with poor outcome. Knockdown of PDLIM5 in NSCLC cells decreased migration and invasion in vitro and lung metastasis in vivo. In addition, TGFβ signaling and TGFβ-induced epithelial–mesenchymal transition was repressed by PDLIM5 knockdown. Mechanistically, PDLIM5 knockdown resulted in a reduction of SMAD3 protein levels. Overexpression of SMAD3 reversed the TGFβ-signaling-repressing and anti-migration effects induced by PDLIM5 knockdown. Notably, PDLIM5 interacted with SMAD3 but not SMAD2 and competitively suppressed the interaction between SMAD3 and its E3 ubiquitin ligase STUB1. Therefore, PDLIM5 protected SMAD3 from STUB1-mediated proteasome degradation. STUB1 knockdown restored SMAD3 protein levels, cell migration, and invasion in PDLIM5-knockdown cells. Collectively, our findings indicate that PDLIM5 is a novel regulator of basal SMAD3 stability, with implications for controlling TGFβ signaling and NSCLC progression.

P0030MIR-182 INHIBITS KIDNEY FIBROSIS BY REGULATING TGF-Β1/SMAD3 PATHWAY IN ADPKD

Background and Aims The aim of the present study was to investigate the molecular mechanism of miR-182 in kidney fibrosis in polycystic kidney disease (PKD). Method We measured the expression of miR-182 in kidney tissue of autosomal dominant polycystic kidney disease (ADPKD). Additionally, we investigated the relationship between miR-182 and fibrotic protein by transfecting miR-182 mimics and miR-182 inhibitor into polycystic kidney cyst-lined epithelial cells, respectively. Furthermore, we observed the interaction between TGF-β1 and miR-182 and fibrinogen factors of cyst-lined epithelial cells after TGF-β1 intervention, and measured the expression of Smad2, 3 protein. Results (1) MiR-182 was positively correlated with fibrosis of cyst-lined epithelial cells; (2) TGF-β1 could induce fibrosis of cyst-lined epithelial cells; (3) the expression of miR-182 had an remarkably impact on the fibrosis induced by TGF-β1, but had little effect on the expression of TGF-β1; (4) the expression of Smad3 protein in TGF-β1 induce- cyst-lined epithelial cells were increased. Conclusion TGF-β and miR-182 promoting the fibrosis of polycystic kidney cyst -lined epithelial cells may be mediated by the TGF-β/Smad3 signaling pathway, of which Smad3 was an important regulator.

Inhibitory effect of activin A on IL-9 production by mouse NK cells through Smad3 signaling

Interleukin-9 (IL-9) is a cytokine secreted by T-helper (Th)9 cells, and activin A can enhance Th9 cell differentiation. However, whether activin A affects IL-9 production by natural killer (NK) cells remains unclear. Herein, we found that not only Th cells, but also CD3−CD49b+NKp46+ NK cells of Balb/c mice produced IL-9. Although activin A promoted IL-9 expression in CD4+ Th cells, it inhibited IL-9 production by CD49b+NKp46+ NK cells in mice. Furthermore, the enzyme-linked immunosorbent assay (ELISA) results showed that mouse NK cells could secrete mature IL-9 protein, and activin A inhibited IL-9 release by NK cells. Additionally, activin A inhibited interferon (IFN)-γ production in splenic NK cells in mice, but promoted IL-2 production, and did not alter the production of IL-10. Western blotting results showed that levels of activin type IIA receptor (ActRIIA), Smad3 and phosphorylated-Smad3 (p-SMAD3) protein increased in activin A-treated splenic NK cells, compared with that in control NK cells. The inhibitory effects of activin A on IL-9 production by NK cells were attenuated in the presence of activin antagonist follistatin (FST) or Smad3 knockdown to NK cells. These data suggest that although activin A up-regulates IL-9 expression in Th cells, it inhibits IL-9 production in NK cells through Smad3 signaling.

ANTIFIBROTIC ACTIVITY OF MESNA AGAINST AMIODARONE-ASSOCIATED LUNG INJURY IN WISTAR RATS

Objective: Lung fibrosis is a progressive respiratory disease with a high percentage of mortality. Till now, it had bad prognosis to conventional medications. This study was designed to evaluate the role of mesna, the well-known antioxidant agent, against pulmonary fibrosis.Methods: Pulmonary fibrosis was induced by administration of amiodarone to Wistar rats. Lung indices, leukocytes count, oxidative stress markers, cytokines levels, and hydroxyproline contents, in addition to the histopathological tests, were done for control, amiodarone, and mesna plus amiodarone group.Results: The elevated ratio of lung/body weight and total leukocytes count within bronchoalveolar lavage fluid for amiodarone rats was decline significantly when cotreated with mesna therapy. Furthermore, mesna significantly brought down the lipid peroxidation of amiodarone in lung tissue, represented by decreasing malondialdehyde level and increasing superoxide dismutase (SOD) and catalase activity. In addition, mesna diminished the profibrotic transforming growth factor-β1 level while elevated the antifibrotic interferon-γ level, and the high activity of the enzyme matrix metalloproteinase-7 was restored within mesna group. Meanwhile, mesna counteracts the increment of hydroxyproline contents and Ashcroft grading scale within amiodarone group. Histologically, critical improvement in the inflammatory cell penetration and alveolar septa was seen in the lung tissue of rats within mesna group, contrasted with those received just amiodarone. Trichome staining clarified that collagen deposition was notably diminished in the peri-alveolar and peri-bronchial area within mesna group. Moreover, mesna therapy downregulated SMAD3 protein level, which was overexpressed by amiodarone challenge.Conclusion: This study gives evidence that mesna therapy may act as a protective agent against amiodarone-mediated pulmonary fibrosis.

ANTIFIBROTIC ACTIVITY OF MESNA AGAINST AMIODARONE-ASSOCIATED LUNG INJURY IN WISTAR RATS

Objective: Lung fibrosis is a progressive respiratory disease with a high percentage of mortality. Till now, it had bad prognosis to conventional medications. This study was designed to evaluate the role of mesna, the well-known antioxidant agent, against pulmonary fibrosis.Methods: Pulmonary fibrosis was induced by administration of amiodarone to Wistar rats. Lung indices, leukocytes count, oxidative stress markers, cytokines levels, and hydroxyproline contents, in addition to the histopathological tests, were done for control, amiodarone, and mesna plus amiodarone group.Results: The elevated ratio of lung/body weight and total leukocytes count within bronchoalveolar lavage fluid for amiodarone rats was decline significantly when cotreated with mesna therapy. Furthermore, mesna significantly brought down the lipid peroxidation of amiodarone in lung tissue, represented by decreasing malondialdehyde level and increasing superoxide dismutase (SOD) and catalase activity. In addition, mesna diminished the profibrotic transforming growth factor-β1 level while elevated the antifibrotic interferon-γ level, and the high activity of the enzyme matrix metalloproteinase-7 was restored within mesna group. Meanwhile, mesna counteracts the increment of hydroxyproline contents and Ashcroft grading scale within amiodarone group. Histologically, critical improvement in the inflammatory cell penetration and alveolar septa was seen in the lung tissue of rats within mesna group, contrasted with those received just amiodarone. Trichome staining clarified that collagen deposition was notably diminished in the peri-alveolar and peri-bronchial area within mesna group. Moreover, mesna therapy downregulated SMAD3 protein level, which was overexpressed by amiodarone challenge.Conclusion: This study gives evidence that mesna therapy may act as a protective agent against amiodarone-mediated pulmonary fibrosis.

Effect of Exogenous Fetuin-A on TGF-β/Smad Signaling in Hepatic Stellate Cells

Objective. To explore the effects of low concentration of exogenous fetuin-A intervention on TGF-β1 induced LX2 cells through detection of the expression of mRNA and protein of Smad2, Smad3, and Smad7. Methods. MTT assay was used to detect the LX2 cells proliferation and the regression equation calculating software was applied to determine IC50 of fetuin-A. RT-PCR was used to determine the relative content of Smad2, Smad3, and Smad7 mRNA in LX2 cells. Western blot was used to detect the LX2 cells relative content of Smad2, Smad3, Smad7 protein expression, respectively. Results. The analysis from RT-PCR and western blot showed that when compared with the other groups TGF-β1 + fetuin-A group increased the expression of Smad2 and Smad3 while decreased the expression of Smad7 (P<0.05). Conclusion. Fetuin-A may improve the excessive activation of hepatic stellate cells which is caused by an enhanced positive regulation of Smad2 and Smad3 protein and the deficiency in negative regulation of Smad7 protein. This is through inhibiting the expression of Smad2 and Smad3 gene and promoting the expression of Smad7 gene. As a result, the development of liver fibrosis will be reduced.