Gut microbiota regulates the ALK5/NOX1 axis by altering glutamine metabolism to inhibit ferroptosis of intrahepatic cholangiocarcinoma cells.

Intrahepatic cholangiocarcinoma (ICC) is a kind of hepatobiliary tumor that is increasing in incidence and mortality. The gut microbiota plays a role in the onset and progression of cancer, however, the specific mechanism by which the gut microbiota acts on ICC remains unclear. In this study, feces and plasma from healthy controls and ICC patients were collected for 16S rRNA sequencing or metabolomics analysis. Gut microbiota analysis showed that gut microbiota abundance and biodiversity were altered in ICC patients compared with controls. Plasma metabolism analysis showed that the metabolite glutamine content of the ICC patient was significantly higher than that of the controls. KEGG pathway analysis showed that glutamine plays a vital role in ICC. In addition, the use of antibiotics in ICC animals further confirmed that changes in gut microbiota affect changes in glutamine. Further experiments showed that supplementation with glutamine inhibited ferroptosis and downregulated ALK5 and NOX1 expression in HuCCT1 cells. ALK5 overexpression or NOX1 overexpression increased NOX1, p53, PTGS2, ACSL4, LPCAT3, ROS, MDA and Fe2+ and decreased FTH1, SLC7A11 and GSH. Knockdown of NOX1 suppressed FIN56-induced ferroptosis. In vivo, supplementation with glutamine promoted tumor growth. Overexpression of ALK5 repressed tumor growth and induced ferroptosis in nude mice, which could be reversed by the addition of glutamine. Our results suggested that the gut microbiota altered glutamine metabolism to inhibit ferroptosis in ICC by regulating the ALK5/NOX1 axis.

Ethyl ferulate suppresses post-myocardial infarction myocardial fibrosis by inhibiting transforming growth factor receptor 1.

BACKGROUND: With an increasing number of myocardial infarction (MI) patients, myocardial fibrosis is becoming a widespread health concern. It's becoming more and more urgent to conduct additional research and investigations into efficient treatments. Ethyl ferulate (EF) is a naturally occurring substance with cardioprotective properties. However, the extent of its impact and the underlying mechanism of its treatment for myocardial fibrosis after MI remain unknown. PURPOSE: The goal of this study was to look into how EF affected the signaling of the TGF-receptor 1 (TGFBR1) in myocardial fibrosis after MI. METHODS: Echocardiography, hematoxylin-eosin (HE) and Masson trichrome staining were employed to assess the impact of EF on heart structure and function in MI-affected mice in vivo. Cell proliferation assay (MTS), 5-Ethynyl-2'-deoxyuridine (EdU), and western blot techniques were employed to examine the influence of EF on native cardiac fibroblast (CFs) proliferation and collagen deposition. Molecular simulation and surface plasmon resonance imaging (SPRi) were utilized to explore TGFBR1 and EF interaction. Cardiac-specific Tgfbr1 knockout mice (Tgfbr1ΔMCK) were utilized to testify to the impact of EF. RESULTS: In vivo experiments revealed that EF alleviated myocardial fibrosis, improved cardiac dysfunction after MI and downregulated the TGFBR1 signaling in a dose-dependent manner. Moreover, in vitro experiments revealed that EF significantly inhibited CFs proliferation, collagen deposition and TGFBR1 signaling followed by TGF-ß1 stimulation. More specifically, molecular simulation, molecular dynamics, and SPRi collectively showed that EF directly targeted TGFBR1. Lastly, knocking down of Tgfbr1 partially reversed the inhibitory activity of EF on myocardial fibrosis in MI mice. CONCLUSION: EF attenuated myocardial fibrosis post-MI by directly suppressing TGFBR1 and its downstream signaling pathway.

Gramine protects against pressure overload-induced pathological cardiac hypertrophy through Runx1-TGFBR1 signaling.

BACKGROUND: Gramine, also named 3-(N,N-dimethylaminomethyl) indole, is a indole alkaloid. It is mainly extracted from various natural raw plants. Despite being the simplest 3-aminomethylindole, Gramine has broad pharmaceutical and therapeutic effects, such as vasodilatation, antioxidation, mitochondrial bioenergetics-related effects, and angiogenesis via modulation of TGFß signaling. However, there is little information available about Gramine's role in heart disease, especially pathological cardiac hypertrophy. PURPOSE: To investigate Gramine's effect on pathological cardiac hypertrophy and clarify the mechanisms behind its action. METHODS: In the in vitro experiment, Gramine (25 µM or 50 µM) was used to investigate its role in Angiotensin II-induced primary neonatal rat cardiomyocytes (NRCMs) hypertrophy. In the in vivo experiment, Gramine (50 mg/kg or 100 mg/kg) was administrated to investigate its role in transverse aortic constriction (TAC) surgery mice. Additionally, we explored the mechanisms underlying these roles through Western blot, Real-time PCR, genome-wide transcriptomic analysis, chromatin immunoprecipitation and molecular docking studies. RESULTS: The in vitro data demonstrated that Gramine treatment obviously improved primary cardiomyocyte hypertrophy induced by Angiotensin II, but had few effects on the activation of fibroblasts. The in vivo experiments indicated that Gramine significantly mitigated TAC-induced myocardial hypertrophy, interstitial fibrosis and cardiac dysfunction. Mechanistically, RNA sequencing and further bioinformatics analysis demonstrated that transforming growth factor ß (TGFß)-related signaling pathway was enriched significantly and preferentially in Gramine-treated mice as opposed to vehicle-treated mice during pathological cardiac hypertrophy. Moreover, this cardio-protection of Gramine was found to mainly involved in TGFß receptor 1 (TGFBR1)- TGFß activated kinase 1 (TAK1)-p38 MAPK signal cascade. Further exploration showed that Gramine restrained the up-regulation of TGFBR1 by binding to Runt-related transcription factor 1 (Runx1), thereby alleviating pathological cardiac hypertrophy. CONCLUSION: Our findings provided a substantial body of evidence that Gramine possessed a potential druggability in pathological cardiac hypertrophy via suppressing the TGFBR1-TAK1-p38 MAPK signaling axis through interaction with transcription factor Runx1.

Effects of Dietary Koumine on Growth Performance, Intestinal Morphology, Microbiota, and Intestinal Transcriptional Responses of Cyprinus carpio.

Gelsemium elegans Benth. (GEB) is a traditional medicinal plant in China, and acts as a growth promoter in pigs and goats. Koumine (KM) is the most abundant alkaloid in GEB and produces analgesic, anti-cancer, and immunomodulatory effects. KM can be used as an aquatic immune stimulant, but its growth-promoting effects and transcriptional mechanisms have not been investigated. Diets containing KM at 0, 0.2, 2, and 20 mg/kg were fed to Cyprinus carpio for 71 days to investigate its effects on growth performance, intestinal morphology, microflora, biochemical indicators, and transcriptional mechanisms. Cyprinus carpio fed with KM as the growth promoter, and the number of intestinal crypts and intestinal microbial populations were influenced by KM concentration. KM increased the abundance of colonies of Afipia, Phyllobacterium, Mesorhizobium, and Labrys, which were associated with compound decomposition and proliferation, and decreased the abundance of colonies of pathogenic bacteria Methylobacterium-Methylorubrum. A total of 376 differentially-expressed genes (DEGs) among the four experimental groups were enriched for transforming growth factor-ß1 and small mother against decapentaplegic (TGF-ß1/Smad), mitogen-activated protein kinase (MAPK), and janus kinases and signal transducers and activators of transcription (Jak/Stat) signaling pathways. In particular, tgfbr1, acvr1l, rreb-1, stat5b, smad4, cbp, and c-fos were up-regulated and positively correlated with KM dose. KM had a growth-promoting effect that was related to cell proliferation driven by the TGF-ß1/Smad, MAPK, and Jak/Stat signaling pathways. KM at 0.2 mg/kg optimized the growth performance of C. carpio, while higher concentrations of KM (2 and 20 mg/kg) may induce apoptosis without significantly damaging the fish intestinal structure. Therefore, KM at low concentration has great potential for development as an aquatic growth promotion additive.

Diterpenoid alkaloids isolated from Delphinium trichophorum alleviate pulmonary fibrosis via the TGF-ß/Smad pathway in 3T6 and HFL-1 cells.

Delphinium trichophorum Franch (DTF), a species endemic to China, has been widely used for centuries in Tibet as an indigenous medicine for treating cough, pneumonia, and pulmonary fibrosis. Hetisine-type C20-diterpenoid alkaloids have been reported to be characteristic and active ingredients. Herein, five ones with relatively high contents in D. trichophorum, including 2α,11α,13ß-triacetylhetisine (DTF1), trichodelphinine A (DTF2), trichodelphinine D (DTF3), 2α-acetyl-11α,13ß-dihydroxyhetisine (DTF4), and trichodelphinine C (DTF5), were investigated for anti-fibrosis effects using fibroblasts induced by TGF-ß1 or LPS for the first time. The results showed that all five tested compounds decreased hydroxyproline (HYP) levels and inhibited the abnormal proliferation of 3T6 and HFL-1 cells induced by either TGF-ß1 or LPS. Moreover, DTF1 and DTF2 attenuated the production of collagen (Col-1 and Col-3) at relatively low doses, suggesting their higher efficiency among the five alkaloids. Based on large-scale ligand-based pharmacophore modeling, TGFBR1 was screened as a potential target for these tested alkaloids. The molecular docking results also exhibited high-affinity interactions between TGFBR1 and five alkaloids, especially DTF1 and DTF2. Further experiments revealed that DTF1 and DTF2 could inhibit the expression of TGF-ß1 and α-SMA and the phosphorylation of Smad3 and Smad4 while restoring the expression of Smad7 protein. Overall, DTF1 and DTF2 may reduce collagen generation and delay the development of pulmonary fibrosis by inhibiting the activation of the TGF-ß/Smad signaling pathway. Our results provide experimental and theoretical evidence for DTF1 and DTF2 as superior candidates for further development of anti-fibrotic drugs.

Calycosin reduces myocardial fibrosis and improves cardiac function in post-myocardial infarction mice by suppressing TGFBR1 signaling pathways.

BACKGROUND: Excessive myocardial fibrosis is the pathological basis of heart failure following myocardial infarction (MI). Although calycosin improves cardiac function, its effect on cardiac fibrosis and cardiac function after MI in mice and its precise mechanism remain unclear. PURPOSE: Here, we firstly investigated the effects of calycosin on cardiac fibrosis and ventricular function in mice after MI and the role of transforming growth factor-beta receptor 1 (TGFBR1) signaling in the amelioration of cardiac fibrosis and ventricular function. METHODS: In vivo effects of calycosin on cardiac structure and function in mice with MI induced by left anterior descending coronary artery ligation were determined by hematoxylin and eosin staining, Masson trichrome staining, and echocardiography. The molecular mechanism of the interaction between TGFBR1 and calycosin was investigated using molecular docking, molecular dynamics (MD) simulation, surface plasmon resonance imaging (SPRi), immunohistochemistry, and western blotting (WB). Subsequently, cardiac-specific Tgfbr1 knockout mice were used to verify the effects of calycosin. The effect of calycosin on primary cardiac fibroblasts (CFs) proliferation and collagen deposition was detected using cell counting (CCK-8), EdU assay, and WB in vitro. CFs infected with an adenovirus that encodes TGFBR1 were used to verify the effects of calycosin. RESULTS: In vivo, calycosin attenuated myocardial fibrosis and cardiac dysfunction following MI in a dose-dependent pattern. Calycosin-TGFBR1 complex was found to have a binding energy of -9.04 kcal/mol based on molecular docking. In addition, calycosin bound steadily in the cavity of TGFBR1 during the MD simulation. Based on SPRi results, the solution equilibrium dissociation constant for calycosin and TGFBR1 was 5.11 × 10-5 M. Calycosin inhibited the expression of TGFBR1, Smad2/3, collagen I, and collagen III. The deletion of TGFBR1 partially counteracted these effects. In vitro, calycosin suppressed CFs proliferation and collagen deposition after TGF-ß1 stimulation by suppressing the TGFBR1 signaling pathway. The suppressive effects of calycosin were partially rescued by overexpression of TGFBR1. CONCLUSION: Calycosin attenuates myocardial fibrosis and cardiac dysfunction following MI in mice in vivo via suppressing the TGFBR1 signaling pathway. Calycosin suppresses CFs proliferation and collagen deposition induced by TGF-ß1 via inhibition of the TGFBR1 signaling pathway in vitro.

Differentiation of Microencapsulated Neonatal Porcine Pancreatic Cell Clusters in Vitro Improves Transplant Efficacy in Type 1 Diabetes Mellitus Mice.

BACKGROUND: Neonatal porcine pancreatic cell clusters (NPCCs) have been proposed as an alternative source of ß cells for islet transplantation because of their low cost and growth potential after transplantation. However, the delayed glucose lowering effect due to the immaturity of NPCCs and immunologic rejection remain as a barrier to NPCC's clinical application. Here, we demonstrate accelerated differentiation and immune-tolerant NPCCs by in vitro chemical treatment and microencapsulation. METHODS: NPCCs isolated from 3-day-old piglets were cultured in F-10 media and then microencapsulated with alginate on day 5. Differentiation of NPCCs is facilitated by media supplemented with activin receptor-like kinase 5 inhibitor II, triiodothyronine and exendin-4 for 2 weeks. Marginal number of microencapsulated NPCCs to cure diabetes with and without differentiation were transplanted into diabetic mice and observed for 8 weeks. RESULTS: The proportion of insulin-positive cells and insulin mRNA levels of NPCCs were significantly increased in vitro in the differentiated group compared with the undifferentiated group. Blood glucose levels decreased eventually after transplantation of microencapsulated NPCCs in diabetic mice and normalized after 7 weeks in the differentiated group. In addition, the differentiated group showed nearly normal glucose tolerance at 8 weeks after transplantation. In contrast, neither blood glucose levels nor glucose tolerance were improved in the undifferentiated group. Retrieved graft in the differentiated group showed greater insulin response to high glucose compared with the undifferentiated group. CONCLUSION: in vitro differentiation of microencapsulated immature NPCCs increased the proportion of insulin-positive cells and improved transplant efficacy in diabetic mice without immune rejection.

Kaempferol 3-O-gentiobioside, an ALK5 inhibitor, affects the proliferation, migration, and invasion of tumor cells via blockade of the TGF-ß/ALK5/Smad signaling pathway.

Overactivation of TGF-ß/ALK5/Smad signaling pathway has been observed in the advanced stage of various human malignancies. As a key component of TGF-ß/ALK5/Smad signaling pathway transduction, TGF-ß type I receptor (also known as ALK5) has emerged as a promising therapeutic target for cancer treatment. In this study, to discover a novel ALK5 inhibitor, a commercial natural products library was screened using docking-based virtual screening, followed by luciferase reporter assay. A flavonoid glycoside kaempferol 3-O-gentiobioside (KPF 3-O-G) was identified as a potent ALK5 inhibitor through directly bound to the ATP-site of ALK5, resulting in the inhibitory effects on phosphorylation and translocation of Smad2 and expression of Smad4. Additionally, we found that KPF 3-O-G reduced cell proliferation and inhibited TGF-ß-induced cell migration and invasion. Moreover, western blotting and immunofluorescent analysis showed that KPF 3-O-G significantly reversed the TGF-ß-induced EMT biomarkers, including upregulation of E-cadherin and downregulation of N-cadherin, vimentin, and snail. In vivo study showed that KPF 3-O-G administration reduced tumor growth in human ovarian cancer xenograft mouse model, without obvious toxic effect. This study provided novel insight into the anticancer effects of KPF-3-O-G and indicated that KPF-3-O-G might be developed as potential therapeutics for cancer treatment after further validation.

Myricetin suppresses the proliferation and migration of vascular smooth muscle cells and inhibits neointimal hyperplasia via suppressing TGFBR1 signaling pathways.

BACKGROUND: Neointimal formation, mediated by the proliferation and migration of vascular smooth muscle cells (VSMCs), is a common pathological basis for atherosclerosis and restenosis. Myricetin, a natural flavonoid, reportedly exerts anti-atherosclerotic effects. However, the effect and mechanism of myricetin on VSMCs proliferation and migration and neointimal hyperplasia (NIH) remain unknown. PURPOSE: We investigated myricetin's effect on NIH, as well as the potential involvement of transforming growth factor-beta receptor 1 (TGFBR1) signaling in mediating myricetin's anti-atherosclerotic and anti-restenotic actions. METHODS: Myricetin's effects on the proliferation and migration of HASMCs and A7R5 cells were determined by CCK-8, EdU assays, wound healing, Transwell assays, and western blotting (WB).Molecular docking, molecular dynamics (MD) simulation, surface plasmon resonance (SPR) and TGFBR1 kinase activity assays were employed to investigate the interaction between myricetin and TGFBR1. An adenovirus vector encoding TGFBR1 was used to verify the effects of myricetin. In vivo, the left common carotid artery (LCCA) ligation mouse model was adopted to determine the impacts of myricetin on neointimal formation and TGFBR1 activation. RESULTS: Myricetin dose-dependently inhibited the migration and proliferation in VSMCs, suppressed the expression of CDK4, cyclin D3, MMP2, and MMP9. Molecular docking revealed that myricetin binds to key regions for TGFBR1 antagonist binding, and the binding energy was -9.61 kcal/mol. MD simulation indicated stable binding between TGFBR1 and myricetin. Additionally, SPR revealed an equilibrium dissociation constant of 4.35 × 10-5 M between myricetin and TGFBR1. According to the TGFBR1 kinase activity assay, myricetin directly inhibited TGFBR1 kinase activity (IC50 = 8.551 µM). Furthermore, myricetin suppressed the phosphorylation level of TGFBR1, Smad2, and Smad3 in a dose-dependent pattern, which was partially inhibited by TGFBR1 overexpression. Consistently, TGFBR1 overexpression partially rescued the suppressive roles of myricetin on VSMCs migration and proliferation. Moreover, myricetin dramatically inhibited NIH and reduced TGFBR1, Smad2, and Smad3 phosphorylation in the LCCA. CONCLUSION: This is the first study to demonstrate that myricetin suppresses NIH and VSMC proliferation and migration via inhibiting TGFBR1 signaling. Myricetin can be developed as a potential therapeutic candidate for treating atherosclerosis and vascular restenosis.

Teucrium polium Extract Enhances the Anti-Angiogenic Effect of Tranilast in a Three-Dimensional Fibrin Matrix Model.

OBJECTIVE: Angiogenesis plays a dominant role in many pathophysiologic disorders, including cancer. Tranilast, which is an anti-fibrotic drug, is also suggested as an anti-angiogenesis agent. As Teucrium polium (TP) is known as an herbal medicine with antitumor properties, this study aimed to investigate the effects of TP and Tranilast on human umbilical vein endothelial cells (HUVECs), in vitro model of angiogenesis, as well as rat's aortic ring ex vivo model. METHODS: In this study, The HUVECs were treated with various doses of TP and Tranilast each one alone or in combination together. Cell survival test, aortic ring ex-vivo assay, and evaluating mRNA expressions of VEGFA and TGF-ß ligands and receptors were performed. RESULTS: The survival rate of HUVECs has significantly (p <0.05) reduced by TP and Tranilast. The combination of both TP and Tranilast significantly reduced cell viability as compared to the administration of TP or Tranilast alone. As well, the treatment of HUVECs with TP and/or Tranilast significantly (p <0.05) decreased TGF-ß1, TGF-ß 2, TGF-ßRI, and TGF-ßRII mRNA expression levels, but not the expression of TGF-ß3 and TGF-ßRIII in the TP-treated cells. Image analysis showed that TP and/or Tranilast inhibited vascular growth in the aortic ring assay. CONCLUSION: Our results strongly support the anti-angiogenic effects of the TP and Tranilast combination on both in vitro and ex vivo models of angiogenesis. However, further investigations in in vivo models and human studies are needed before human use.

Anti-Fibrosis Effects of Magnesium Lithospermate B in Experimental Pulmonary Fibrosis: By Inhibiting TGF-ßRI/Smad Signaling.

Pulmonary fibrosis is a severe and irreversible interstitial pulmonary disease with high mortality and few treatments. Magnesium lithospermate B (MLB) is a hydrosoluble component of Salvia miltiorrhiza and has been reported to have antifibrotic effects in other forms of tissue fibrosis. In this research, we studied the effects of MLB on pulmonary fibrosis and the underlying mechanisms. Our results indicated that MLB treatment (50 mg/kg) for seven days could attenuate bleomycin (BLM)-induced pulmonary fibrosis by reducing the alveolar structure disruption and collagen deposition in the C57 mouse model. MLB was also found to inhibit transforming growth factor-beta (TGF-ß)-stimulated myofibroblastic transdifferentiation of human lung fibroblast cell line (MRC-5) cells and collagen production by human type II alveolar epithelial cell line (A549) cells, mainly by decreasing the expression of TGF-ß receptor I (TGF-ßRI) and regulating the TGF-ß/Smad pathway. Further studies confirmed that the molecular mechanisms of MLB in BLM-induced pulmonary fibrosis mice were similar to those observed in vitro. In summary, our results demonstrated that MLB could alleviate experimental pulmonary fibrosis both in vivo and in vitro, suggesting that MLB has great potential for pulmonary fibrosis treatment.

Evaluating Deep Learning models for predicting ALK-5 inhibition.

Computational methods have been widely used in drug design. The recent developments in machine learning techniques and the ever-growing chemical and biological databases are fertile ground for discoveries in this area. In this study, we evaluated the performance of Deep Learning models in comparison to Random Forest, and Support Vector Regression for predicting the biological activity (pIC50) of ALK-5 inhibitors as candidates to treat cancer. The generalization power of the models was assessed by internal and external validation procedures. A deep neural network model obtained the best performance in this comparative study, achieving a coefficient of determination of 0.658 on the external validation set with mean square error and mean absolute error of 0.373 and 0.450, respectively. Additionally, the relevance of the chemical descriptors for the prediction of biological activity was estimated using Permutation Importance. We can conclude that the forecast model obtained by the deep neural network is suitable for the problem and can be employed to predict the biological activity of new ALK-5 inhibitors.

Taohong siwu decoction attenuates myocardial fibrosis by inhibiting fibrosis proliferation and collagen deposition via TGFBR1 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Myocardial fibrosis after myocardial infarction (MI) leads to cardiac remodeling and loss of function. Taohong siwu decoction (THSWD), a well-known traditional Chinese medicinal prescription, has been clinically used to treat various cardiovascular and cerebrovascular diseases, but its potential functions in myocardial fibrosis after MI remain uncharacterized. AIM OF THE STUDY: The purpose of current study was to explore the potential mechanism action and anti-myocardial fibrosis effects of treatment with THSWD in vivo and in vitro. MATERIALS AND METHODS: Mouse underwent ligation of coronary artery to induce MI and divided equally into the sham group, model group and THSWD treatment groups. After 4 weeks, the effects of THSWD treatment on cardiac function were estimated by echocardiography. HE staining was used to detect the pathologic changes and Masson trichrome staining was used to estimate tissue fibrosis. To further explore the regulatory molecular mechanisms of THSWD, transcriptome analysis was performed. Furthermore, in vitro, we investigated the effect of THSWD on cell proliferation and collagen deposition in primary cardiac fibrosis cells and its possible mechanism of action. Overexpression of TGFBR1 was achieved by infection with an adenovirus vector encoding TGFBR1. RESULTS: Treatment with THSWD significantly decreased myocardial fibrosis and recovered cardiac function in the post-MI mouse. The transcriptomics data imply that the TGF-ß pathway might be a target in the anti-fibrosis effect of THSWD. THSWD inhibits TGF-ß1-induced proliferation of primary cardiac fibroblasts. THSWD decreased collagen expression and TGFBR1 and Smad2/3 phosphorylation. Moreover, the inhibitory effect of THSWD on CFs proliferation and collagen deposition, as well as TGFBR1 signaling pathway-associated proteins expression was partially abrogated by overexpression of TGFBR1. CONCLUSION: Collectively, the results implicate that THSWD attenuates myocardial fibrosis by inhibiting fibrosis proliferation and collagen deposition via inhibiting TGFBR1, and might be a potential therapeutic agent for treatment of myocardial fibrosis post-MI.

Functional targeting of the TGF-ßR1 kinase domain and downstream signaling: A role for the galloyl moiety of green tea-derived catechins in ES-2 ovarian clear cell carcinoma.

The galloyl moiety is a specific structural feature which dictates, in part, the chemopreventive properties of diet-derived catechins. In ovarian cancer cells, galloylated catechins were recently demonstrated to target the transforming growth factor (TGF)-ß-mediated control of the epithelial-mesenchymal transition process. The specific impact of the galloyl moiety on such signaling, however, remains poorly understood. Here, we questioned whether the sole galloyl moiety interacted with TGF-ß-receptors to alter signal transduction and chemotactic migratory response in an ES-2 serous carcinoma-derived ovarian cancer cell model. In line with the LogP and LogS values of the tested molecules, we found that TGF-ß-induced Smad-3 phosphorylation and cell migration were optimally inhibited, provided that the lateral aliphatic chain of the galloyl moiety reached 8-10 carbons. Functional inhibition of the TGF-ß receptor (TGF-ßR1) kinase activity was supported by surface plasmon resonance assays showing direct physical interaction between TGF-ßR1 and the galloyl moiety. In silico molecular docking analysis predicted a model where galloylated catechins may bind TGF-ßR1 within its adenosine triphosphate binding cleft in a site analogous to that of Galunisertib, a selective adenosine triphosphate-mimetic competitive inhibitor of TGF-ßR1. In conclusion, our data suggest that the galloyl moiety of the diet-derived catechins provides specificity of action to galloylated catechins by positioning them within the kinase domain of the TGF-ßR1 in order to antagonize TGF-ß-mediated signaling that is required for ovarian cancer cell invasion and metastasis.

Design and synthesis of novel tranilast analogs: Docking, antiproliferative evaluation and in-silico screening of TGFßR1 inhibitors.

The discovery of the antiproliferative potential of tranilast prompted additional studies directed at understanding the mechanisms of tranilast action. Its inhibitory effect on cell proliferation depends principally on the capacity of tranilast to interfere with transforming growth factor beta (TGFßR1) signaling. This work summarizes design, synthesis and biological evaluation of sixteen novel tranilast analogs on different tumors such as PC-3, HepG-2 and MCF-7 cell lines. The in vitro cytotoxicity was evaluated using MTT assay showed that, twelve compounds out of sixteen showed higher cytotoxic activities (IC50's 1.1-6.29 µM), than that of the reference standard, 5-FU (IC50 7.53 µM). The promising cytotoxic hits (4b, 7a, b and 14c-e), proved to be selective to cancer cells when their cytotoxicity's are examined on human normal cell line (WI-38). Then they are investigated for their possible mode of action as TGFßR1 inhibitors; remarkable inhibition of TGFßR1 by these hits was observed at the range of IC50 0.087-3.276 µM. The cell cycle analysis of the most potent TGFßR1 inhibitor, 4b revealed cell cycle arrest at G2/M phase on prostate cancer cells. Additionally, it is clearly indicated apoptosis induction at Pre-G1 phase, this is substantiated by significant increase in the expression on the tumor suppressor gene, p53 and up regulation the level of apoptosis mediator, caspase-3. In addition, in silico study was performed for validating the physicochemical and ADME properties which revealed that, all compounds are orally bioavailable with no side effects complying with Lipinski rule. The proposed mode of action can be further explored on the light of molecular modeling simulation of the most potent compounds, 4b and 14e which were docked into the active sites of TGFßR1 to predict their affinities toward the receptor.

Astragaloside IV protects human cardiomyocytes from hypoxia/reoxygenation injury by regulating miR-101a.

Astragaloside IV (AS/IV) is one of the extracted components from the traditional Chinese medicine Astragalus which has been demonstrated to have potential capacity for anti-inflammation activity and for treating cardiovascular disease. Our purpose was to determine the function and underlying molecular mechanism of AS/IV in hypoxia/reoxygenation (H/R) injured in cardiomyocytes. Differentially expressed genes (DEGs) were screened using bioinformatic analysis, and the molecular targeting relationship was verified by the dual-luciferase report system. H/R injured cardiomyocytes were employed to explore the effect of AS/IV. QRT-PCR and Western blot analysis were applied to detect the expression of mRNA and proteins, respectively. Additionally, superoxide dismutase (SOD), lactic dehydrogenase (LDH) and MDA (malondialdehyde) levels were detected to determine the oxidative damage. Cell viability was assessed by CCK-8, and flow cytometry was used to evaluate cell apoptosis ratio. TGFBR1 and TLR2 were selected as DEGs. Additionally, AS/IV could enhance cell proliferation and upregulated miR-101a expression, which suppressed TGFBR1 and TLR2 expression in H/R injured cardiomyocytes. Moreover, the results of Western blot exhibited that the downstream genes (p-ERK and p-p38) in the MAPK signaling pathway were suppressed, which meant AS/IV could inhibit this pathway in H/R injured cardiomyocytes. Overall, this study demonstrated AS/IV could attenuate H/R injury in human cardiomyocytes via the miR-101a/TGFBR1/TLR2/MAPK signaling pathway axis, which means that it could serve as a possible alternate for H/R treatment.

miR-196b-5p controls adipocyte differentiation and lipogenesis through regulating mTORC1 and TGF-ß signaling.

MicroRNAs have been reported to play a role in adipogenesis and obesity. This study was performed to investigate the role of miR-196b-5p in adipogenesis and the mechanism involved. The data revealed that miR-196b-5p expression increased in primary or established marrow stromal progenitor cells after adipogenic treatment. Supplementing miR-196b-5p in the progenitor cells stimulated adipogenic differentiation and lipogenesis, along with the induction of adipogenic and lipogenic factors. Conversely, inhibition of endogenous miR-196b-5p blocked adipogenesis and lipogenesis. Tuberous sclerosis 1 (Tsc1) and transforming growth factor-ß receptor 1 (TGFBR1) were demonstrated to be the direct target genes of miR-196b-5p. Supplementing miR-196b-5p activity in progenitor cells reduced the protein level of TSC1 and activated mammalian target of rapamycin complex 1 (mTORC1) signaling. We further demonstrated that the perturbation of TSC1 in progenitor cells altered the trend of adipogenic differentiation and lipogenesis. Overexpression of Tsc1 or inactivation of mTORC1 signaling attenuated the stimulation of adipogenic differentiation and lipogenesis by miR-196b-5p. Overexpression of Tgfbr1 also partially blocked the adipogenic effect of miR-196b-5p. Further investigations demonstrated that zinc finger E-box-binding homeobox 1 (ZEB1) transcriptionally upregulated miR-196b-5p expression. The current study suggests that miR-196b-5p promotes adipogenic differentiation and lipogenesis in progenitor cells through targeting TSC1 and TGFBR1 and therefore regulating mTORC1 and TGF-ß signaling.

The Role of Th17/Treg Axis in the Traditional Chinese Medicine Intervention on Immune-Mediated Inflammatory Diseases: A Systematic Review.

The Th17/Treg axis plays a crucial role in immune-mediated inflammatory diseases (IMID) and might represent an interesting drug target of treatment strategy for these diseases. Accumulating evidence suggests a role for traditional Chinese medicine (TCM) in the modulation of Th17/Treg axis, but a comprehensive overview which summarizes this field hitherto is lacked. This paper performs a systematic literature review of the regulatory effects of TCM on the imbalance of Th17/Treg axis and its potential mechanisms. In addition, the frequency analysis and network pharmacology for the collected TCM herbs from clinical trial data were performed. The studies reported the changes in the ratio of Th17 and/or Treg cells as well as their transcription factor and related cytokines were included. Frequency analysis of composition of the 39 assessed TCM prescriptions showed that Astragalus membranaceus var.mongholicus (5.20%), Glycyrrhiza uralensis (3.67%), Paeonia obovate (3.06%), Salvia digitaloides (3.06%), and Angelica sinensis (2.75%) were the top five herbal components, which were closely associated to the treatment of IMID. Network pharmacology showed that six target proteins (transforming growth factor (TGF)-beta receptor type-1, TGF-beta receptor type-2, retineic-acid-receptor-related orphan nuclear receptor gamma (ROR-gamma), TGFB2, IL-17 and IL-2, respectively) might be involved in the regulatory effects of TCM on Th17/Treg axis. Moreover, there were nine active ingredients (including Oxymatrine, Baicalin, Triptolide, Paeoniflorin, Sinomenine, Celastrol, Emodin, Diosgenin and Chlorogenic acid) originating from TCM reported to have an immunological regulation effect on the Th17/Treg axis. The highlight of this systematic review is to reveal the pharmacological basis of TCM treating IMID and is helpful for supporting future pharmacologic-driven studies. Further research elucidates the immune-modulating mechanisms on Th17/Treg axis by TCM might provide a broader insight for the treatment of IMID.

The α7 Nicotinic Acetylcholine Receptor: A Promising Target for the Treatment of Fibrotic Skin Disorders.

Targeting neuroendocrine receptors can be considered as another interesting approach to treating fibrotic disorders. Previously, we could demonstrate that tropisetron, a classical serotonin receptor blocker, can modulate collagen synthesis and acts in vitro through the α7 nicotinic acetylcholine receptor (α7nAchR). Here, we used a pharmacologic approach with specific α7nAchR agonists to validate this hypothesis. PHA-543613, an α7nAchR-specific agonist, not only prevented but also reversed established skin fibrosis of mice injected with bleomycin. Interestingly, agonistic stimulation of α7nAchR also attenuated experimental skin fibrosis in the non-inflammation driven adenovirus coding for TGFß receptor Iact mouse model, indicating fibroblast-mediated and not only anti-inflammatory effects of such agents. The fibroblast-mediated effects were confirmed in vitro using human dermal fibroblasts, in which the α7nAchR-specific agonists strongly reduced the impact of TGFß1-mediated expression on collagen and myofibroblast marker expression. These actions were linked to modulation of the redox-sensitive transcription factor JunB and impairment of the mitochondrial respiratory system. Our results indicate that pharmacologic stimulation of the α7nAchR could be a promising target for treatment of patients with skin fibrotic diseases. Moreover, our results suggest a mechanistic axis of collagen synthesis regulation through the mitochondrial respiratory system.

Herring roe oil supplementation alters microglial cell gene expression and reduces peripheral inflammation after immune activation in a neonatal piglet model.

Neonatal brain development can be disrupted by infection that results in microglial cell activation and neuroinflammation. Studies indicate that polyunsaturated fatty acids (PUFAs) and their metabolites can resolve inflammation. It is not known if dietary PUFA increases lipid metabolites in brain or reduces neuroinflammation in neonates. We hypothesized that dietary PUFAs might suppress neuroinflammation by inhibiting pro-inflammatory cytokine over-production and promoting inflammatory resolution in the periphery and brain. Piglets were obtained on postnatal day (PD) 2 and randomly assigned to herring roe oil (HRO) or control (CON) diet. HRO was included at 2 g/kg powdered diet. HRO increased DHA levels in occipital lobe and the DHA to arachidonic acid (ARA) ratio in hippocampal tissue. HRO decreased ARA metabolites in occipital lobe. HRO failed to attenuate microglial pro-inflammatory cytokine production ex vivo. HRO did not affect fever or circulating resolvin D1 levels. HRO decreased circulating neutrophils and liver inflammatory gene expression, but increased resolution marker gene expression in liver post LPS. HRO upregulated CXCL16, TGFBR1, and C1QA in microglial cells. HRO supplementation exerted beneficial effects on inflammation in the periphery, but further studies are needed to evaluate the specific effects of omega-3 supplementation on microglial cell physiology in the neonate.