Up-regulation of Nrf2/HO-1 and inhibition of TGF-ß1/Smad2/3 signaling axis by daphnetin alleviates transverse aortic constriction-induced cardiac remodeling in mice.

Oxidative damage and accumulation of extracellular matrix (ECM) components play a crucial role in the adverse outcome of cardiac hypertrophy. Evidence suggests that nuclear factor erythroid-derived factor 2 related factor 2 (Nrf2) can modulate oxidative damage and adverse myocardial remodeling. Daphnetin (Daph) is a coumarin obtained from the plant genus Daphne species that exerts anti-oxidative and anti-inflammatory properties. Herein, we investigated the roles of Daph in transverse aortic constriction (TAC)-induced cardiac hypertrophy and fibrosis in mice. TAC-induced alterations in cardiac hypertrophy markers, histopathological changes, and cardiac function were markedly ameliorated by oral administration of Daph in mice. We found that Daph significantly reduced the reactive oxygen species (ROS) generation, increased the nuclear translocation of Nrf2, and consequently, reinstated the protein levels of NAD(P)H quinone dehydrogenase1 (NQO1), heme oxygenase-1 (HO-1), and other antioxidants in the heart. Besides, Daph significantly inhibited the TAC-induced accumulation of ECM components, including α-smooth muscle actin (α-SMA), collagen I, collagen III, and fibronectin, and interfered with the TGF-ß1/Smad2/3 signaling axis. Further studies revealed that TAC-induced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive nuclei and the protein levels of Bax/Bcl2 ratio and cleaved caspase 3 were substantially decreased by Daph treatment. We further characterized the effect of Daph on angiotensin II (Ang-II)-stimulated H9c2 cardiomyoblast cells and observed that Daph markedly decreased the Ang-II induced increase in cell size, production of ROS, and proteins associated with apoptosis and fibrosis. Mechanistically, Daph alone treatment enhanced the protein levels of Nrf2, NQO1, and HO-1 in H9c2 cells. The inhibition of this axis by Si-Nrf2 transfection abolished the protective effect of Daph in H9c2 cells. Taken together, Daph effectively counteracted the TAC-induced cardiac hypertrophy and fibrosis by improving the Nrf2/HO-1 axis and inhibiting the TGF-ß1/Smad2/3 signaling axis.

Chelerythrine inhibits the progression of glioblastoma by suppressing the TGFB1-ERK1/2/Smad2/3-Snail/ZEB1 signaling pathway.

AIMS: Glioblastoma (GBM) is the most common and aggressive intracranial tumor with poor prognosis. A large majority of clinical chemotherapeutic agents cannot achieve the desired therapeutic effect. Chelerythrine (CHE), a natural component with multitudinous pharmacological functions, has been proven to have outstanding antitumor effects in addition to antibacterial, anti-inflammatory, and hypotensive effects. However, the anti-GBM effect of CHE has not been reported to date. The purpose of this paper is to observe the anti-GBM effect of CHE and further explore the related mechanism. MATERIALS AND METHODS: GBM cell lines (U251 and T98G) and BALB/c nude mice were used in the experiments. Methyl thiazolyl tetrazolium (MTT) and clone formation assays were applied to detect the viability, proliferation and stemness of GBM cells. Flow cytometry was utilized to identify the effect of CHE on GBM apoptosis. Scratch and Transwell experiments reflected the migration and invasion of cells. In vivo, xenograft tumors were implanted subcutaneously in nude mice. The progression of tumors was assessed by ultrasound and magnetic resonance imaging. Finally, western blot, bioinformatics, and immunohistochemistry experiments were used to explore the molecular mechanisms in depth. KEY FINDINGS: In vitro tests showed that CHE inhibited the proliferation, stemness, migration, and invasion of GBM cells and induced apoptosis. In vitro, CHE was observed to restrain the progression of xenograft tumors. We eventually proved that the cytotoxicity of CHE was relevant to the TGFB1-ERK1/2/Smad2/3-Snail/ZEB1 signaling pathway. SIGNIFICANCE: CHE inhibited GBM progression by inhibiting the TGFB1-ERK1/2/Smad2/3-Snail/ZEB1 signaling pathway and is a potential chemotherapeutic drug for GBM.

Tetrodecadazinone, a novel tetrodecamycin-pyridazinone hybrid with anti-liver fibrosis activity from Streptomyces sp. HU051.

Tetrodecadazinone (1), a novel tetrodecamycin-pyridazinone hybrid possessing a new 1,2-dimethyl-1-(2-methylnonyl)decahydronaphthalene skeleton, and 4-hydroxydihydrotetrodecamycin (2) were separated from a culture of Streptomyces sp. HU051, together with a known compound, dihydrotetrodecamycin (3). Diverse spectroscopic approaches were applied to assign the structures of 1-3, and the structure of 1 was further confirmed by single crystal X-ray diffraction analysis. Compound 1 is the first example of a pyridazinone-containing natural product. Biosynthetically, 1 is proposed to be derived from a Michael addition reaction of a PKS-derived tetrodecamycin and a piperazic-acid-derived pyridazinone. Biological evaluation revealed 1 could reduce the expressions of extracellular matrix proteins (fibronectin and collagen I) and α-smooth muscle actin (α-SMA) in transforming growth factor-ß (TGF-ß1)-activated LX-2 cells. Preliminary mechanism study showed 1 exerted its anti-liver fibrosis effect by regulating TGF-ß1/Smad2/3 signaling pathway.

Liquiritigenin attenuates isoprenaline­induced myocardial fibrosis in mice through the TGF­ß1/Smad2 and AKT/ERK signaling pathways.

Myocardial fibrosis is a pathological process characterized by excessive accumulation of extracellular matrix in myocardial interstitial spaces. Myocardial fibrosis is a fundamental process in ventricular remodeling and a primary contributor to the progression of heart failure. Liquiritigenin (LQ) is a flavanone compound with anti­oxidative, anti­carcinogenic, anti­inflammatory and estrogenic properties. The present study aimed to investigate the regulatory potential of LQ treatment in a mouse model of isoprenaline (ISO)­induced cardiac fibrosis and in cultured H9C2 cardiomyocytes stimulated with angiotensin II (Ang II). The treatment of ISO­induced mice with LQ significantly decreased the levels of cardiac injury­related proteins in the serum and ECM accumulation in mouse heart tissues. LQ treatment also effectively alleviated cardiac dysfunction in ISO­treated mice. Further analyses revealed that LQ inhibited ISO­induced collagen formation and activation of the transforming growth factor­ß1 (TGF­ß1)/Smad2 and protein kinase B (AKT)/extracellular signal­regulated kinase (ERK) signaling pathways. As a major pathological event in myocardial fibrosis, the apoptosis of cardiomyocytes has been considered a key mechanism contributing to impaired left ventricle performance. The pretreatment of rat cardiomyocytes with LQ significantly reduced the apoptosis of H9C2 cells, and inhibited Ang II­induced activation of the TGF­ß1/Smad2 and AKT/ERK pathways. In conclusion, the present study revealed that LQ ameliorated ISO­induced myocardial fibrosis in mice and inhibited the apoptosis of cardiomyocytes in vitro by inhibiting the TGF­ß1/Smad2 and AKT/ERK signaling pathways. These results suggested the anti­fibrotic and cardioprotective potential of LQ in fibrosis, thus supporting the use of LQ for the management of cardiomyocyte injury and myocardial fibrosis in patients with cardiac diseases.

Demethyleneberberine promotes apoptosis and suppresses TGF-ß/Smads induced EMT in the colon cancer cells HCT-116.

Colorectal cancer (CRC) is one of the most common malignant tumours in the world. Recent reports have revealed natural products displayed inhibition on colon cancer potential by suppressing transforming growth factor-ß/Smads induced epidermal-mesenchymal transition (EMT). In this article, 12 kinds of natural berberine analogues were screened for their effects on the inhibition of the colon cancer cells, the results showed that demethyleneberberine (DM-BBR) exhibited an interesting and potential effect on inducing the apoptosis of HCT-116 cells with drug concentrations of 6, 12 and 18 µM. Particularly, DM-BBR reversed the EMT process by inhibiting the expression of p-Smad2 and p-Smad3 in the transforming growth factor-ß/Smads signal pathway, up-regulated pro-apoptotic protein cleaved caspase-9, and blocked cell cycle at the S phase and increasing the expression of cyclin proteins P27 and P21. Taken together, these findings suggested that DM-BBR could promote apoptosis and suppress TGF-ß/Smads induced EMT in the colon cancer cells HCT-116.

Adipose-Derived Mesenchymal Stem Cells-Derived Exosomes Carry MicroRNA-671 to Alleviate Myocardial Infarction Through Inactivating the TGFBR2/Smad2 Axis.

Mesenchymal stem cells (MSCs) and their derived extracellular vesicles have been reported as promising tools for the management of heart disease. The aim of this study was to explore the function of adipose-derived MSCs (adMSCs)-derived exosomes (Exo) in the progression of myocardial infarction (MI) and the molecules involved. Mouse cardiomyocytes were treated with oxygen-glucose deprivation (OGD) to mimic an MI condition in vitro. The adMSCs-derived Exo were identified and administrated into the OGD-treated cardiomyocytes, and then the viability and apoptosis of cells, and the secretion of fibrosis- and inflammation-related cytokines in cells were determined. Differentially expressed microRNAs (miRNAs) in cells after Exo treatment were screened using a microarray analysis. The downstream molecules regulated by miR-671 were explored through bioinformatic analysis. Involvements of miR-671 and transforming growth factor beta receptor 2 (TGFBR2) in the exosome-mediated events were confirmed by rescue experiments. A murine model with MI was induced and treated with Exo for functional experiments in vivo. Compared to phosphate-buffered saline treatment, the Exo treatment significantly enhanced viability while reduced apoptosis of cardiomyocytes, and in reduced myocardial fibrosis and inflammation both in vitro and in vivo. miR-671 was significantly upregulated in cells after Exo treatment. Downregulation of miR-671 blocked the protective functions of Exo. miR-671 targeted TGFBR2 and suppressed phosphorylation of Smad2. Artificial downregulation of TGFBR2 enhanced viability of the OGD-treated cardiomyocytes. This study suggested that adMSC-derived exosomal miR-671 directly targets TGFBR2 and reduces Smad2 phosphorylation to alleviate MI-like symptoms both in vivo and in vitro.

Protective effects of total flavonoids from Qu Zhi Qiao (fruit of Citrus paradisi cv. Changshanhuyou) on OVA-induced allergic airway inflammation and remodeling through MAPKs and Smad2/3 signaling pathway.

Allergic asthma is one of the inflammatory diseases, which has become a major public health problem. Qu zhi qiao (QZQ), a dry and immature fruit of Citrus paradisi cv. Changshanhuyou, has various flavonoids with pharmacological properties. However, there is a knowledge gap on the pharmacological properties of QZQ on allergic asthma. Therefore, here, we explored the efficacy and mechanism of total flavonoids from QZQ (TFCH) on allergic asthma. We extracted and purified TFCH and conducted animal experiments using an Ovalbumin (OVA)-induced mice model. Bronchoalveolar lavage fluid and Swiss-Giemsa staining were used to count different inflammatory cells in allergic asthma mice. We conducted histopathology and immunohistochemistry to evaluate the changes in the lungs of allergic asthma mice. Moreover, we used ELISA assays to analyze chemokines and inflammatory cytokines. Furthermore, western blot analyses were conducted to elucidate the mechanism of TFCH on allergic asthma. We established that TFCH has anti-inflammatory effects and inhibits airway remodeling, providing a potential therapeutic strategy for allergic asthma.

TGFB1/INHBA Homodimer/Nodal-SMAD2/3 Signaling Network: A Pivotal Molecular Target in PDAC Treatment.

Pancreatic cancer remains a grueling disease that is projected to become the second-deadliest cancer in the next decade. Standard treatment of pancreatic cancer is chemotherapy, which mainly targets the differentiated population of tumor cells; however, it paradoxically sets the roots of tumor relapse by the selective enrichment of intrinsically chemoresistant pancreatic cancer stem cells that are equipped with an indefinite capacity for self-renewal and differentiation, resulting in tumor regeneration and an overall anemic response to chemotherapy. Crosstalk between pancreatic tumor cells and the surrounding stromal microenvironment is also involved in the development of chemoresistance by creating a supportive niche, which enhances the stemness features and tumorigenicity of pancreatic cancer cells. In addition, the desmoplastic nature of the tumor-associated stroma acts as a physical barrier, which limits the intratumoral delivery of chemotherapeutics. In this review, we mainly focus on the transforming growth factor beta 1 (TGFB1)/inhibin subunit beta A (INHBA) homodimer/Nodal-SMAD2/3 signaling network in pancreatic cancer as a pivotal central node that regulates multiple key mechanisms involved in the development of chemoresistance, including enhancement of the stem cell-like properties and tumorigenicity of pancreatic cancer cells, mediating cooperative interactions between pancreatic cancer cells and the surrounding stroma, as well as regulating the deposition of extracellular matrix proteins within the tumor microenvironment.

Inhibition of Smad3 promotes the healing of rotator cuff injury in a rat model.

To investigate the effect of inhibiting transforming growth factor-ß (TGF-ß1)/Smad2/3 signaling on rotator cuff (RC) healing. A bilateral supraspinatus tendon detachment-repair model of Sprague-Dawley (SD) rats was utilized. A total of 120 SD rats were randomly assigned to six groups and each group received the subacromial injection of normal saline, empty vectors, or lentiviral vectors containing small interfering RNA against TGF-ß1, Smad2, Smad3 at the bone-tendon junction. Biomechanical and histological analyses were performed to evaluate bone-tendon junction healing quality at 8 weeks after repair. Histologically, scar healing was found in all surgical groups. Animals with inhibited Smad3 exhibited better bone-tendon junction structures with higher density, parallel orientation, and collagen fiber continuity than other surgical group animals. Immunohistochemistry revealed that the protein expression level of collagen I in animals with inhibited Smad3 was more prominent compared with all other surgical groups. Biomechanically, Animals with inhibited Smad3 showed better results in the maximum load at 4, 6, and 8 weeks after surgery compared with other surgical groups. Besides, C3H10T1/2 (Smad3-) cells increased TT-D6 cell migration and tendon-associated genes expression (scleraxis, tenascin C, collagen I) in coculture system. We conclude that inhibition of Smad3 promotes RC tendon healing in the rat supraspinatus model.

Repurposing mesalazine against cardiac fibrosis in vitro.

Cardiovascular diseases are exacerbated and driven by cardiac fibrosis. TGFß induces fibroblast activation and differentiation into myofibroblasts that secrete excessive extracellular matrix proteins leading to stiffening of the heart, concomitant cardiac dysfunction, and arrhythmias. However, effective pharmacotherapy for preventing or reversing cardiac fibrosis is presently unavailable. Therefore, drug repurposing could be a cost- and time-saving approach to discover antifibrotic interventions. The aim of this study was to investigate the antifibrotic potential of mesalazine in a cardiac fibroblast stress model. TGFß was used to induce a profibrotic phenotype in a human cardiac fibroblast cell line. After induction, cells were treated with mesalazine or solvent control. Fibroblast proliferation, key fibrosis protein expression, extracellular collagen deposition, and mechanical properties were subsequently determined. In response to TGFß treatment, fibroblasts underwent a profound phenoconversion towards myofibroblasts, determined by the expression of fibrillary αSMA. Mesalazine reduced differentiation nearly by half and diminished fibroblast proliferation by a third. Additionally, TGFß led to increased cell stiffness and adhesion, which were reversed by mesalazine treatment. Collagen 1 expression and deposition-key drivers of fibrosis-were significantly increased upon TGFß stimulation and reduced to control levels by mesalazine. SMAD2/3 and ERK1/2 phosphorylation, along with reduced nuclear NFκB translocation, were identified as potential modes of action. The current study provides experimental pre-clinical evidence for antifibrotic effects of mesalazine in an in vitro model of cardiac fibrosis. Furthermore, it sheds light on possible mechanisms of action and suggests further investigation in experimental and clinical settings.

Tranilast Inhibits Pulmonary Fibrosis by Suppressing TGFß/SMAD2 Pathway.

PURPOSE: Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix (ECM) protein in the lungs. Transforming growth factor (TGF) ß-induced ECM protein synthesis contributes to the development of IPF. Tranilast, an anti-allergy drug, suppresses TGFß expression and inhibits interstitial renal fibrosis in animal models. However, the beneficial effects of tranilast or its mechanism as a therapy for pulmonary fibrosis have not been clarified. METHODS: We investigated the in vitro effect of tranilast on ECM production and TGFß/SMAD2 pathway in TGFß2-stimulated A549 human alveolar epithelial cells, using quantitative polymerase chain reaction, Western blotting, and immunofluorescence. In vitro observations were validated in the lungs of a murine pulmonary fibrosis model, which we developed by intravenous injection of bleomycin. RESULTS: Treatment with tranilast suppressed the expression of ECM proteins, such as fibronectin and type IV collagen, and attenuated SMAD2 phosphorylation in TGFß2-stimulated A549 cells. In addition, based on a wound healing assay in these cells, tranilast significantly inhibited cell motility, with foci formation that comprised of ECM proteins. Histological analyses revealed that the administration of tranilast significantly attenuated lung fibrosis in mice. Furthermore, tranilast treatment significantly reduced levels of TGFß, collagen, fibronectin, and phosphorylated SMAD2 in pulmonary fibrotic tissues in mice. CONCLUSION: These findings suggest that tranilast inhibits pulmonary fibrosis by suppressing TGFß/SMAD2-mediated ECM protein production, presenting tranilast as a promising and novel anti-fibrotic agent for the treatment of IPF.

Evogliptin attenuates bleomycin-induced lung fibrosis via inhibiting TGF-ß/Smad signaling in fibroblast.

OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is a pulmonary interstitial fibrosis disease. Excessive activation of fibroblasts in the lung contributes to severe alveoli dysfunction and histological destruction. Evogliptin, a dipeptidyl peptidase IV inhibitor, has been widely used to reduce glucose level in type 2 diabetes, whereas evogliptin treatment to fibrosis process of lung IPF is elusive. The study aimed to investigate the mechanism of evogliptin in transforming growth factor-beta (TGF-ß)-activated lung fibroblasts and evaluate the efficacy of evogliptin in lung fibrosis model. MATERIALS AND METHODS: In lung fibroblast culture, the RNA expression of α-SMA in lung fibroblasts was detected, and α-SMA/COL-1 immunofluorescence co-staining after TGF-ß stimulation and evogliptin administration was displayed. Mechanically, the phosphorylation level of Smad2/3 protein in cells was analyzed using Western blotting, and the scratch assay was used to reflect fibroblast proliferation. Furthermore, bleomycin was employed to induce lung fibrosis in mice, and IHC staining and hematoxylin and eosin (HE) & Masson staining were carried out to examine the extracellular matrix (ECM) expression and tissue fibrosis. RESULTS: The results demonstrated that evogliptin treatment attenuated the activation of fibroblasts and collagen deposition following TGF-ß stimulation. Furthermore, the extracellular matrix expression was descended via evogliptin restraining the TGF-ß/Smad pathway. Besides, it was also found that evogliptin affected the proliferation degree of lung fibroblasts. In vivo, the COL-1 and α-SMA were significantly reduced through evogliptin treatment compared with the bleomycin group, and fibroblasts and collagenous fiber were remarkably decreased. CONCLUSIONS: Evogliptin exerts an anti-fibrosis effect on TGF-ß induced lung fibroblast activation, which restrains ECM formation and decreases cell proliferation level in fibroblasts. Moreover, the fibroblast infiltration and collagen deposition were ameliorated following evogliptin administration. Therefore, evogliptin serves as a potential implication to protect lung fibrosis.

Baihe Wuyao decoction ameliorates CCl4-induced chronic liver injury and liver fibrosis in mice through blocking TGF-ß1/Smad2/3 signaling, anti-inflammation and anti-oxidation effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Baihe Wuyao decoction (BWD), a prescription of Traditional Chinese Medicines, composed of Lilium brownii var. viridulum Baker.(Lilii Bulbus) and Lindera aggregata (Sims) Kosterm. (Linderae Radix), has been used to treat epigastric pain and superficial gastritis for hundreds of years in China. Recently, some compounds obtained from Lilii Bulbus and Linderae Radix had active effects of hepatic protection or liver fibrosis alleviation. Thus, we aim to evaluate the effects of BWD on treatment of chronic liver injury and liver fibrosis induced by carbon tetrachloride (CCl4) and to elucidate the possible molecular mechanism. MATERIALS AND METHODS: Mice were treated with BWD (low, medium and high dose), diammonium glycyrrhizinate or vehicle by oral gavage once daily, simultaneously intraperitoneal injected with a single dose of CCl4 (1 µl/g body weight) twice a week for consecutive 6 weeks. Next, all mice were sacrificed after fasted 12 h, and serums and liver tissues were harvested for analysis. The hepatic injury was detected by serum biomarker assay, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The hepatic histology and collagen were illustrated by hematoxylin-eosin staining and Sirius red staining respectively. The antioxidant capacity of liver tissues was evaluated by the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in liver homogenization. The mRNA gene or protein expressions related to fibrosis, oxidative stress and inflammation molecules were performed by real-time quantitative PCR (RT-PCR) or Western-blot. RESULTS: BWD exhibited a good hepatic protection with ameliorating liver histological changes, decreasing serum AST and ALT contents, and reducing hepatic fibrosis with stimulation ECMs (such as Collagen1 and Collagen3) degradation. BWD inhibited hepatic stellate cells (HSCs) activation, promoted matrix metalloproteinase-2 (MMP2), MMP9, and MMP12 while suppressing tissue inhibitors of matrix metalloproteinase-1 (TIMP1) expression, and blocked traditional fibrosis TGF-ß1/Smad2/3 signal pathway. Moreover, BWD exhibited anti-inflammation effect proved by the reduction of liver Interleukin-1ß (IL-1ß), TNF-α, IL-11 mRNA levels and promoted anti-oxidation effects determined by inhibition of liver MDA and iNOS levels while promoting liver SOD and Mn-SOD. CONCLUSION: BWD ameliorates CCl4-induced CLI and liver fibrosis which is correlated to its blocking TGF-ß1/Smad2/3 signaling, anti-inflammation, and anti-oxidation effects. BWD, as a small traditional prescription, is a promising treatment for CLI and liver fibrosis through multiple pharmacological targets.

Effect of alprostadil on myocardial fibrosis in rats with diabetes mellitus via TGF-ß1/Smad signaling pathway.

OBJECTIVE: To observe the influence of alprostadil on myocardial fibrosis in rats with diabetes mellitus (DM) through the transforming growth factor beta-1 (TGF-ß1)/Smad signaling pathway. MATERIALS AND METHODS: Wistar rats were employed to induce models of DM (DM group), and alprostadil treatment group (ALPR group) and control group (NC group) were set up. After successful modeling, blood and myocardial tissues were collected from rats. Next, blood glucose level, liver function, and myocardial function were detected. In addition, hematoxylin-eosin (HE) assay was performed to determine pathological changes. The enzyme-linked immunosorbent assay (ELISA) was carried out to measure serum interleukin-6 (IL-6) and cardiac function indexes such as ejection fraction (EF), Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting, which were applied to measure the gene and protein expression levels of important molecules in the proliferation and differentiation of myocardial fibroblasts [including checkpoint kinase 1 (Chek1) and alpha-smooth muscle actin (α-SMA)] and the relevant pathway TGF-ß1/Smad2. RESULTS: The blood glucose level was increased in DM group (p<0.01), suggesting that modeling is successful. The tumor necrosis factor-alpha (TNF-α), IL⁃6, and IL-1 levels were higher in DM group than in NC group. DM group had significantly elevated serum content of alkaline phosphatase (ALP), alanine aminotransferase (ALT), and creatine kinase (CK), as well as left ventricular end-diastolic dimension (LVEDd) and left ventricular end-systolic dimension (LVESd), but it clearly decreased fractional shortening (FS) and EF in comparison with NC group. Besides, myocardial cells were orderly arranged in NC group, while myocardial fibrosis was observed in DM group. The results of RT-PCR showed that the levels of Collagen, Chek1, α-SMA, TGF-ß1, and Smad2 in myocardial fibroblasts were notably lowered in ALPR group, but evidently increased in DM group (p<0.05). According to Western blotting, there were evident decreases in the levels of TGF-ß1 and Smad2 in myocardial fibroblasts in ALPR group (p<0.05). The above results suggest that alprostadil represses the expression of the TGF-ß1/Smad2 signaling pathway and its relevant molecules, thus further suppressing the fibrosis of myocardial cells. CONCLUSIONS: Alprostadil treats myocardial fibrosis in DM rats by inhibiting the TGF-ß1/Smad2 signaling pathway.

Knockdown of FOXM1 inhibits activation of keloid fibroblasts and extracellular matrix production via inhibition of TGF-ß1/Smad pathway.

Keloid is characterized by overactive fibroblasts. Forkhead box M1 (FOXM1) is transcription factor that plays important roles in the progression of fibrosis. However, the role of FOXM1 in keloid has not been elucidated. In the present study, we examined the expression levels of FOXM1 in clinical keloid tissue specimens and primary keloid fibroblasts (KFs). The results showed that FOXM1 levels were significantly increased in both keloid tissues and KFs. To further investigate the biological functions of FOXM1, FOXM1 was knocked down in KFs by transfection with small interfering RNA targeting FOXM1 (si-FOXM1). Knockdown of FOXM1 inhibited transforming growth factor-ß1 (TGF-ß1)-induced cell proliferation and migration of KFs. Besides, the increased expressions of collagen (coll I), connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA) in TGF-ß1-induced KFs were suppressed by si-FOXM1 transfection. Furthermore, TGF-ß1-induced increase in p-Smad2 and p-Smad3 expressions was attenuated by FOXM1 knockdown. These data indicated that knockdown of FOXM1 inhibited TGF-ß1-induced KFs activation and extracellular matrix (ECM) accumulation, which was attributed to the inhibition of TGF-ß1/Smad pathway.

Protective effect of DMDD, isolated from the root of Averrhoa carambola L., on high glucose induced EMT in HK-2 cells by inhibiting the TLR4-BAMBI-Smad2/3 signaling pathway.

BACKGROUND: Hyperglycemia stimulated epithelial-mesenchymal transition (EMT) plays a critical role in initiating and progressing renal fibrosis in diabetic kidney disease (DKD). It is crucial to explore novel renal protective drugs for the treatment of DKD. OBJECTIVE: The present study is to confirm our hypothesis and to accumulate the information for the application of DMDD (2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione) as a novel therapeutic agent to potentially inhibit renal fibrogenesis and EMT in the DKD. METHODS: High glucose induced renal proximal tubular epithelial cell line (HK-2 cells) was cultured and treated with DMDD. The cell viability and DMDD cytotoxicity were assessed by CCK8. Immunofluorescence was used for detection of TLR4 and downstream protein in normal and high glucose induced HK-2 cells. HK-2 cells were transfected with lentivirus codifying for BAMBI (BMP and activin membrane bound inhibitor) and interfering RNA for determination of the effect of BAMBI over-expression and silencing, respectively. TLR4-BAMBI-Smad2/3 pathway was analyzed by means of RT-PCR and western blot. RESULTS: A high concentration (60mM) of glucose induced significant EMT process and TLR4 expression was increased obviously in this circumstance. DMDD inhibited high expressions of TLR4 and Smad2/3 in HG induced cells and decreased the expression of BAMBI. In addition, the effects of decreased BAMBI expression and increased Smad2/3 expression in HG cultured cells were reversed in the cells of TAK-242 (TLR4 signaling inhibitor) intervention. BAMBI gene silencing dramatically increased EMT process and the over-expression of BAMBI was opposite in HK-2 cells with HG condition. These observations of EMT were ameliorated when the HK-2 cells were pre-treated with DMDD. CONCLUSIONS: Our study demonstrates that DMDD treatment improves EMT in the HG induced HK-2 cells. In addition, DMDD significantly inhibits EMT by TLR4-BAMBI-Smad2/3 pathway, which hints that DMDD may be an alternative approach in diabetic renal injury.

TGF-ß Upregulated Mitochondria Mass through the SMAD2/3→C/EBPß→PRMT1 Signal Pathway in Primary Human Lung Fibroblasts.

Tissue remodeling of subepithelial mesenchymal cells is a major pathologic condition of chronic obstructive pulmonary disease and asthma. Fibroblasts contribute to fibrotic events and inflammation in both airway diseases. Recent mechanistic studies established a link between mitochondrial dysfunction or aberrant biogenesis leading to tissue remodeling of the airway wall in asthma. Protein arginine methyltransferase-1 (PRMT1) participated in airway wall remodeling in pulmonary inflammation. This study investigated the mechanism by which PRMT1 regulates mitochondrial mass in primary human airway wall fibroblasts. Fibroblasts from control or asthma patients were stimulated with TGF-ß for up to 48 h, and the signaling pathways controlling PRMT1 expression and mitochondrial mass were analyzed. PRMT1 activity was suppressed by the pan-PRMT inhibitor AMI-1. The SMAD2/3 pathway was blocked by SB203580 and C/EBPß by small interference RNA treatment. The data obtained from unstimulated cells showed a significantly higher basal expression of PRMT1 and mitochondrial markers in asthmatic compared with control fibroblasts. In all cells, TGF-ß significantly increased the expression of PRMT1 through SMAD2/3 and C/EBPß. Subsequently, PRMT1 upregulated the expression of the mitochondria regulators PGC-1α and heat shock protein 60. Both the inhibition of the SAMD2/3 pathway or PRMT1 attenuated TGF-ß-induced mitochondrial mass and C/EBPß and α-SMA expression. These findings suggest that the signaling sequence controlling mitochondria in primary human lung fibroblasts is as follows: TGF-ß→SMAD2/3→C/EBPß→PRMT1→PGC-1α. Therefore, PRMT1 and C/EBPß present a novel therapeutic and diagnostic target for airway wall remodeling in chronic lung diseases.

Combination of Ginsenoside Rg1 and Astragaloside IV reduces oxidative stress and inhibits TGF-ß1/Smads signaling cascade on renal fibrosis in rats with diabetic nephropathy.

INTRODUCTION: Anti-oxidative stress and inhibition of TGF-ß1/Smads signaling cascade are essential therapeutic strategies for diabetic nephropathy (DN). In this study, we aimed to explore the effect of combination of Ginsenoside Rg1 and Astragaloside IV on oxidative stress and TGF-ß1/Smads signaling in DN rats. MATERIALS AND METHODS: Wistar rats were divided into five groups: N group, M group (streptozotocin [STZ], intraperitoneally), G group (STZ rats with Ginsenoside Rg1, intragastrically [ig]), A group (STZ rats with Astragaloside IV, ig) and C group (STZ rats with Ginsenoside Rg1 and Astragaloside IV, ig). The levels of methane dicarboxylic aldehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-PX), total anti-oxidative capacity (T-AOC), blood urea nitrogen (BUN), ß2-microglobulin (ß2-MG), serum creatinine (SCr) and urinary creatinine (UCr) were detected in all the groups. The left kidneys of the rats were harvested to detect the expression of TGF-ß1, Smad2/3, Smad7 and CTGF by immunohistochemical staining, while the right kidneys were used to detect the mRNA expression of TGF-ß1, Smad7 and CTGF by real-time PCR. RESULTS: Rats in G group, A group and C group had lower level of MDA but higher levels of CAT, GSH-PX and T-AOC compared with rats in M group. Rats in C group showed the best anti-oxidative stress level. G group, A group and C group treatments significantly decreased the levels of BUN, SCr, ß2-MG and UCr. In addition, C group treatment showed the best kidney protective effect. G group, A group and C group treatments significantly diminish ED both factor and mRNA overexpression of TGF-ß1 and CTGF but increase Smad7 expression in kidney tissue. CONCLUSION: The combination of Ginsenoside Rg1 and Astragaloside IV may potentially protect against DN by reducing oxidative stress and inhibiting TGF-ß1/Smads signaling cascade.

A novel Gallic acid derivative attenuates BLM-induced pulmonary fibrosis in mice.

Idiopathic Pulmonary fibrosis is a disease with high morbidity and mortality. Therefore, the development of new drugs is imperative. Gallic acid derivative is a derivative of Gallic acid that can be extracted from Chinese herbal medicine. In previous experiments, we found that Gallic acid derivative played dual roles in inflammatory and antioxidant activities. Meanwhile, Gallic acid derivative could inhibit the proliferation of lung fibroblast. In the present study, we investigated the function of Gallic acid derivative in inhibiting lung fibrosis. 5 mg/kg of bleomycin was administered to mice by a single intratracheal instillation. Three dosages of Gallic acid derivative (75 mg/kg, 150 mg/kg, 300 mg/kg) and Pirfenidone (80 mg/kg) were given to mice for 21 day. Gallic acid derivative treatment significantly reduced lung histological changes and decreased inflammatory cell infiltration. The content of collagen decreased with the decrease of hydroxyproline level. Analogously, the expression of alpha smooth muscle actin was reduced. Gallic acid derivative enhanced the antioxidant status, but reduced the expression of interleukin 6, NADPH oxidase-4. Our study proved that Gallic acid derivative reduced inflammation activation to some extent and could exert its effects through transforming growth factor ß1/Smad2 signaling pathway and balancing NOX4/Nrf2.

Oridonin prevents epithelial-mesenchymal transition and TGF-ß1-induced epithelial-mesenchymal transition by inhibiting TGF-ß1/Smad2/3 in osteosarcoma.

Osteosarcoma is the most common primary bone tumor with highly invasive characteristic and low long-term survival. Recently, epithelial-mesenchymal transition (EMT) is reported as a key event in cancer invasion and metastasis. Oridonin, a bioactive diterpenoid, has been proved to possess anti-cancer effects. However, the effect of oridonin on EMT and metastasis of osteosarcoma is unclear. In this study, we investigated the underlying mechanism of oridonin on EMT and metastasis of osteosarcoma. We found that oridonin inhibited migration and invasion of MG-63 and 143B cells. Moreover, oridonin increased the protein expression of E-cadherin and decreased that of N-cadherin and Vimentin. Oridonin upregulated the transcription of E-cadherin and downregulated N-cadherin and Vimentin. Oridonin inhibited the protein and mRNA levels of Snail and Slug. Furthermore, oridonin inhibited TGF-ß-induced phosphorylation of Smad 2/3, prevented Smad dimer translocation into the nucleus. Finally, we established metastatic models of osteosarcoma 143B cells, and found that oridonin inhibited lung metastasis in vivo. Oridonin increased the protein expression of E-cadherin and reduced N-cadherin and Vimentin. Oridonin inhibited the protein expression of Snail and Slug as well as Smad 2/3 activation. In conclusion, our study demonstrated that oridonin inhibited EMT and TGF-ß1-induced EMT by inhibiting TGF-ß1/Smad2/3 signaling pathway in osteosarcoma.