The role of inflammatory and remodelling biomarkers in patients with non-small cell lung cancer.

Introduction: Biomarkers play a crucial role in evaluating the prognosis, diagnosis, and monitoring of non-small cell lung cancer (NSCLC). The aim of this study was to compare the levels of inflammatory and remodelling biomarkers among patients with NSCLC and healthy controls (HCs) and to investigate the correlation between these biomarkers. Material and methods: Blood samples were taken from 93 NSCLC and 84 HCs. Each sample was analysed for the inflammatory biomarkers transforming growth factor ß1 (TGF-ß1), mothers against decapentaplegic homolog 2 (SMAD2) and the remodelling biomarkers Wingless-related integration site (Wnt3a) and α-catenin (CTNN-ß1). Results: The patients with NSCLC had significantly higher levels of all the measured biomarkers. In the NSCLC patients, TGF-ß1 correlated significantly with SMAD2 (r = 0.34, p = 0.0008), Wnt3a (r = 0.328, p = 0.0013), and CTNN-ß1 levels (r = 0.30, p = 0.004). SMAD2 correlated significantly with CTNN-ß1 (r = 0.546, p = 0.0001) and Wnt3a (r = 0.598, p = 0.0001). CTNN-ß1 level also correlated with the level of Wnt3a (r = 0.61, p = 0.0001). No correlation was found between biomarkers and symptom scores. Discussion: In this study, patients with NSCLC had higher inflammatory and remodelling biomarker levels than HCs. In the NSCLC, there were significant associations between inflammatory and remodelling biomarkers. This indicates that measuring biomarkers could be valuable in the workup of NSCLC patients. Conclusions: Our investigation showed that inflammatory and remodelling biomarkers might play a role in future immunologic response and pharmacologically targeted NSCLC therapy.

Corosolic acid ameliorates non-alcoholic steatohepatitis induced by high-fat diet and carbon tetrachloride by regulating TGF-ß1/Smad2, NF-κB, and AMPK signaling pathways.

Hawthorn (Crataegus pinnatifida Bunge. var. major) is an edible and medicinal fruit that is very common in food and traditional Chinese medicine. Corosolic acid (CA), a pentacyclic triterpenoid, which is an active component of hawthorn (Crataegus pinnatifida Bunge. var. major), has been exhibiting various pharmacological activities such as antidiabetic, antibacterial, anticancer, antiinflammatory, and antioxidant effects. The study aimed to evaluate the effect of CA on non-alcoholic steatohepatitis (NASH) in mice induced by 60 kcal% high-fat diet (HFD) and carbon tetrachloride (CCl4 ). CA lowered liver index and serum AST, ALT, TG, and TC levels compared to those in the model group. Histological analyses of the liver tissues of mice treated with CA revealed significantly decreased number of lipid droplets and alleviated inflammation and fibrosis. CA inhibited the transcripts of pro-fibrogenic markers (including α-SMA, collagen I, and TIMP-1) and the levels of pro-inflammatory cytokines (including TNF-α, IL-1ß, caspase-1, and IL-6) associated with hepatic fibrosis, and NF-κB translocation and TGF-ß1/Smad2 and AMPK pathways. In addition, CA reduced lipid accumulation via the regulation of AMPK and NF-κB activation in FFA-induced steatotic HepG2 cells. CA also decreased α-SMA, collagen I expressions, and Smad2 phosphorylation, which were reduced by TGF-ß1 treatment in LX2 cells. Our results suggested that CA ameliorated NASH through regulating TGF-ß1/Smad2, NF-κB, and AMPK signaling pathways, and CA could be developed as a potential health functional food or therapeutic agent for NASH patients.

microRNA-141 inhibits TGF-ß1-induced epithelial-to-mesenchymal transition through inhibition of the TGF-ß1/SMAD2 signalling pathway in endometriosis.

PURPOSE: Recent studies have demonstrated the differential expression of micro(mi)RNAs in endometriosis. Previously, we reported the low expression of miR-141 in patients with this disease. Epithelial-to-mesenchymal transition (EMT) and the transforming growth factor-beta1 (TGF-ß1)-induced SMAD2 signalling pathway are central to tumour proliferation and invasion. However, the role of miR-141 in regulating the TGF-ß1/SMAD2 signalling pathway and the associated EMT to be elucidated. METHODS: The levels of TGF-ß1/SMAD2 signalling and EMT markers expression in eutopic and ectopic endometria of endometriosis were determined by immunohistochemistry and western blot analyses. MiR-141 expression was analysed by quantitative reverse-transcription polymerase chain reaction. Cellular invasion and proliferation were determined by transwell and CCK-8 assays, respectively. Functional assay of miR-141 was performed using plasmid and shRNA transfection methods. RESULT: The presence of miR-141, EMT, and TGF-ß1/SMAD2 signalling markers were detected in eutopic and ectopic endometria of endometriosis. TGF-ß1-induced EMT in Ishikawa (ISK) cells by activating the SMAD2 signalling pathway, whereas miR-141 inhibited the TGF-ß1-induced EMT, proliferation and invasion abilities of these cells. CONCLUSION: These data identify miR-141 as a novel driver of EMT in endometriosis, implicates the link between miR-141 and TGF-ß1/SMAD2 signalling pathway in the context of endometriosis, and underscore the role of EMT in the development of endometriosis.

GP73 promotes invasion and metastasis of bladder cancer by regulating the epithelial-mesenchymal transition through the TGF-ß1/Smad2 signalling pathway.

This study investigated the effects of Golgi membrane protein 73 (GP73) on the epithelial-mesenchymal transition (EMT) and on bladder cancer cell invasion and metastasis through the TGF-ß1/Smad2 signalling pathway. Paired bladder cancer and adjacent tissue samples (102) and normal bladder tissue samples (106) were obtained. Bladder cancer cell lines (T24, 5637, RT4, 253J and J82) were selected and assigned to blank, negative control (NC), TGF-ß, thrombospondin-1 (TSP-1), TGF-ß1+ TSP-1, GP73-siRNA-1, GP73-siRNA-2, GP73-siRNA-1+ TSP-1, GP73-siRNA-1+ pcDNA-GP73, WT1-siRNA and WT1-siRNA + GP73-siRNA-1 groups. Expressions of GP73, TGF-ß1, Smad2, p-Smad2, E-cadherin and vimentin were detected using RT-qPCR and Western blotting. Cell proliferation, migration and invasion were determined using MTT assay, scratch testing and Transwell assay, respectively. Compared with the blank and NC groups, levels of GP73, TGF-ß1, Smad2, p-Smad2, N-cadherin and vimentin decreased, and levels of WT1 and E-cadherin increased in the GP73-siRNA-1 and GP73-siRNA-2 groups, while the opposite results were observed in the WT1 siRNA, TGF-ß, TSP-1 and TGF-ß + TSP-1 groups. Cell proliferation, migration and invasion notably decreased in the GP73-siRNA-1 and GP73-siRNA-2 groups in comparison with the blank and NC groups, while in the WT1 siRNA, TGF-ß, TSP-1 and TGF-ß + TSP-1 groups, cell migration, invasion and proliferation showed the reduction after the EMT. These results suggest that GP73 promotes bladder cancer invasion and metastasis by inducing the EMT through down-regulating WT1 levels and activating the TGF-ß1/Smad2 signalling pathway.

YuPingFeng (YPF) upregulates caveolin-1 (CAV1) to alleviate pulmonary fibrosis through the TGF-ß1/Smad2 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) is considered a valuable asset in China's medical tradition. YPF is a classic prescription that has been derived from the "Jiu Yuan Fang" formula and consists of three herbs: Huangqi (Astragalus membranaceus Bunge), Baizhu (Atractylodes rubra Dekker), and Fangfeng (Saposhnikovia divaricata (Turcz.) Schischk). This prescription is widely acclaimed for its exceptional pharmacological properties, including potent antioxidant effects, hormone regulation, and immune modulation effects. AIM OF THE STUDY: Previous research provides evidence suggesting that YPF may have therapeutic effects on pulmonary fibrosis. Further exploration is essential to confirm its effectiveness and elucidate the fundamental processes. MATERIALS AND METHODS: First, the active components and target genes of YPF were extracted from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Next, the GSE53845 dataset, which contains information on pulmonary fibrosis, was downloaded from the GEO database. Network informatics methods was then be utilized to identify target genes associated with pulmonary fibrosis. A YPF-based network of protein-protein interactions was constructed to pinpoint possible target genes for pulmonary fibrosis treatment. Additionally, an in vitro model of pulmonary fibrosis induced by bleomycin (BLM) was established to further investigate and validate the possible mechanisms underlying the effectiveness of YPF. RESULTS: In this study, a total of 24 active ingredients of YPF, along with 178 target genes associated with the treatment, were identified. Additionally, 615 target genes related to pulmonary fibrosis were identified. Functional enrichment analysis revealed that 18 candidate genes (CGs) exhibited significant responses to tumor necrosis factor, NF-kB survival signaling, and positive regulation of apoptosis processes. Among these CGs, CAV1, VCAM1, and TP63 were identified as key target genes. Furthermore, cell experiments confirmed that the expression of CAV1 protein and RNA expression was increased in pulmonary fibrosis, but significantly decreased after treatment with YPF. Additionally, the expression of pSmad2, α-SMA, TGF-ß1, and TNF-α was also decreased following YPF treatment. CONCLUSIONS: Network pharmacology analysis revealed that YPF exhibits significant potential as a therapeutic intervention for pulmonary fibrosis by targeting various compounds and pathways. This study emphasizes that the efficacy of YPF in treating pulmonary fibrosis may be attributed to its ability to up-regulate CAV1 expression and inhibiting pulmonary fibrosis via modulation of the TGF-ß1/Smad2 signaling pathway. These findings underscore the promising role of YPF and its ability to potentially alleviate pulmonary fibrosis.

Portulaca oleracea L. extract relieve mice liver fibrosis by inhibiting TLR-4/NF-κB, Bcl-2/Bax and TGF-ß1/Smad2 signalling transduction.

Portulaca oleracea L. are annual herb, which has various pharmacological effects including hepatoprotective property. However, the effect of Portulaca oleracea L. (POL-1) in mice with carbon tetrachloride (CCl4)-induced liver fibrosis and its mechanism of action have not been clarified. POL-1 ameliorated the CCl4-induced liver fibrosis in mice, as shown by decreased collagen deposition and the decreased expression of liver fibrosis marker collagen I and α-smooth muscle actin (α-SMA) mRNA. In addition, treatment with POL-1 suppressed the proliferation of activated human hepatic stellate cell line (LX-2). POL-1 inhibited the oxidative stress and inflammation in fibrotic livers of mice. Mechanistically, POL-1 inhibited the CCl4-induced expression of toll-like receptor-4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor kappa-B (NF-κBp65) p65, Bcl2-associated X (Bax), transforming growth factor-ß1 (TGF-ß1) and drosophila mothers against decapentaplegic 2 (Smad2) proteins, upregulated B-cell lymphoma -2 (Bcl-2) proteins in livers of mice. These findings suggested that POL-1 attenuated liver fibrosis.

MAP3K19 Promotes the Progression of Tuberculosis-Induced Pulmonary Fibrosis Through Activation of the TGF-ß/Smad2 Signaling Pathway.

Tuberculosis-induced pulmonary fibrosis (PF) is a chronic, irreversible interstitial lung disease, which severely affects lung ventilation and air exchange, leading to respiratory distress, impaired lung function, and ultimately death. As previously reported, epithelial-mesenchymal transition (EMT) and fibrosis in type II alveolar epithelial cells (AEC II) are two critical processes that contributes to the initiation and progression of tuberculosis-related PF, but the underlying pathological mechanisms remain unclear. In this study, through performing Real-Time quantitative PCR (RT-qPCR), Western blot, immunohistochemistry, and immunofluorescence staining assay, we confirmed that the expression levels of EMT and fibrosis-related biomarkers were significantly increased in lung tissues with tuberculosis-associated PF in vivo and Mycobacterium bovis Bacillus Calmette-Guérin (BCG) strain-infected AEC II cells in vitro. Besides, we noticed that the mitogen-activated protein kinase 19 (MAP3K19) was aberrantly overexpressed in PF models, and silencing of MAP3K19 significantly reduced the expression levels of fibronectin, collagen type I, and alpha-smooth muscle actin to decrease fibrosis, and upregulated E-cadherin and downregulated vimentin to suppress EMT in BCG-treated AEC II cells. Then, we uncovered the underlying mechanisms and found that BCG synergized with MAP3K19 to activate the pro-inflammatory transforming growth factor-beta (TGF-ß)/Smad2 signal pathway in AEC II cells, and BCG-induced EMT process and fibrosis in AEC II cells were all abrogated by co-treating cells with TGF-ß/Smad2 signal pathway inhibitor LY2109761. In summary, our results uncovered the underlying mechanisms by which the MAP3K19/TGF-ß/Smad2 signaling pathway regulated EMT and fibrotic phenotypes of AEC II cells to facilitate the development of tuberculosis-associated PF, and these findings will provide new ideas and biomarkers to ameliorate tuberculosis-induced PF in clinic.

Cannabidiol alleviates right ventricular fibrosis by inhibiting the transforming growth factor ß pathway in monocrotaline-induced pulmonary hypertension in rats.

Cannabidiol (CBD) is a non-intoxicating compound of Cannabis with anti-fibrotic properties. Pulmonary hypertension (PH) is a disease that can lead to right ventricular (RV) failure and premature death. There is evidence that CBD reduces monocrotaline (MCT)-induced PH, including reducing right ventricular systolic pressure (RVSP), vasorelaxant effect on pulmonary arteries, and decreasing expression of profibrotic markers in the lungs. The aim of our study was to investigate the effect of chronic administration of CBD (10 mg/kg daily for 21 days) on profibrotic parameters in the RVs of MCT-induced PH rats. In MCT-induced PH, we found an increase in profibrotic parameters and parameters related to RV dysfunction, i.e. plasma pro-B-type natriuretic peptide (NT-proBNP), cardiomyocyte width, interstitial and perivascular fibrosis area, amount of fibroblasts and fibronectin, as well as overexpression of the transforming growth of factor ß1 (TGF-ß1), galectin-3 (Gal-3), suppressor of mothers against decapentaplegic 2 (SMAD2), phosphorylated SMAD2 (pSMAD2) and alpha-smooth muscle actin (α-SMA). In contrast, vascular endothelial cadherin (VE-cadherin) levels were decreased in the RVs of MCT-induced PH rats. Administration of CBD reduced the amount of plasma NT-proBNP, the width of cardiomyocytes, the amount of fibrosis area, fibronectin and fibroblast expression, as well as decreased the expression of TGF-ß1, Gal-3, SMAD2, pSMAD2, and increased the level of VE-cadherin. Overall, CBD has been found to have the anti-fibrotic potential in MCT-induced PH. As such, CBD may act as an adjuvant therapy for PH, however, further detailed investigations are recommended to confirm our promising results.

The miR-124-3p regulates the allergic airway inflammation and remodeling in an ovalbumin-asthmatic mouse model by inhibiting S100A4.

OBJECTIVE: Asthma is a chronic respiratory disease with an increasing incidence every year. microRNAs (miRNAs) have been demonstrated to have implications for asthma. However, limited information is available regarding the effect of miR-124-3p on this disease. Therefore, this study aimed to explore the possible effects of miR-124-3p and S100A4 on inflammation and epithelial-mesenchymal transition (EMT) in asthma using mouse models. METHOD: Ovalbumin was used to induce asthmatic mouse models. Lung injury in mouse models was assessed, and the bronchoalveolar lavage fluid of mice was collected to determine the number of eosinophilic granulocytes and assess inflammation. The expression levels of miR-124-3p, S100A4, E-cadherin, N-cadherin, Snail1, vimentin, and TGF-ß1/Smad2 signaling pathway-related proteins were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. In vitro experiments, cells were transfected with miR-124-3p mimics or inhibitors to test the expression of S100A4 by RT-qPCR and western blot analysis, and the mutual binding of miR-124-3p and S100A4 was validated by dual-luciferase reporter gene assay. RESULTS: Overexpression of miR-124-3p or inhibition of S100A4 expression attenuated bronchial mucus secretion and collagenous fibers and suppressed inflammatory cell infiltration. Additionally, upon miR-124-3p overexpression or S100A4 suppression, eosinophilic granulocytes were decreased, interleukin-4 (IL-4) and IL-13 expression levels were reduced in the bronchoalveolar lavage fluid, serum total IgE level was reduced, and the TGF-ß1/Smad2 signaling pathway was suppressed. Mechanically, a dual-luciferase reporter gene assay verified the binding relationship between miR-124-3p and S100A4. CONCLUSION: miR-124-3p can negatively target S100A4 to attenuate inflammation in asthmatic mouse models by suppressing the EMT process and the TGF-ß/smad2 signaling pathway.

Bone mesenchymal stem cells promote gastric cancer progression through TGF-ß1/Smad2 positive feedback loop.

AIMS: Bone marrow-derived mesenchymal stem cells (BMSCs) have been proven to be recruited into the tumor microenvironment and contribute to gastric cancer (GC) progression, but the underlying mechanism is still unclear. The purpose of this study is to explore the exact role and potential mechanism of BMSCs in the progression of GC. MATERIALS AND METHODS: Bioinformatics analyzed were used to clarify the correlation between TGF-ß1 and prognosis of gastric cancer. Cell co-culture were used to explore the interaction between gastric cancer cells (GCs) and BMSCs. Quantitative real time-PCR and Western blot assay were used to detect gene and protein expression, respectively. The biological characteristics of GCs and BMSCs were detected by immunofluorescence, Transwell migration, Elisa and invasion assay. Xenograft models in nude mice were constructed to evaluate GC development in vivo. KEY FINDINGS: TGF-ß1 was overexpressed in GC cells and tissues, and is positively related to the poor prognosis of patients. TGF-ß1 from GCs activated the Smad2 pathway in BMSCs, promoting their differentiation into carcinoma-associated fibroblasts (CAFs) and TGF-ß1 expression. Concomitantly, TGF-ß1 secreted by CAFs activate Smad2 signaling in GC cells, thus inducing their epithelial-mesenchymal transition (EMT) and TGF-ß1 secretion. BMSCs can dramatically promote the proliferation, migration, and invasion of GCs while blocking TGF-ß1/Smad2 positive feedback loop can reverse these effects. SIGNIFICANCE: The TGF-ß1/Smad2 positive feedback loop between GCs and BMSCs, promotes the CAFs differentiation of BMSCs and the EMT of GCs, resulting in the progression of GC.

Effect of Qiling Jiaogulan Powder on Pulmonary Fibrosis and Pulmonary Arteriole Remodeling in Low-Temperature-Exposed Broilers.

Chinese herbal medicine plays an important role in regulating the nutritional metabolism of poultry and maintaining or improving normal physiological functions and animal health. The present study investigated the effects of dietary supplementation with Qiling Jiaogulan Powder (QLJP) on pulmonary fibrosis and pulmonary arteriole remodeling in low temperature-exposed broilers. Seven-day-old Ross 308 broilers (n = 240) were reared adaptively to 14 days of age. The broilers were randomly divided into six groups: A control group (basal diet and normal feeding temperature); model group (basal diet); low-, medium- and high-dose QLJP groups (basal diet supplemented with 1 g/kg, 2 g/kg, 4 g/kg QLJP); and L-Arg group (basal diet supplemented with 10 g/kg L-arginine). Additionally, all the broilers, except the broilers in the control group, from the age of 14 days old, had a house temperature continuously lowered by 2 °C each day until it reached 12 °C at 21 days of age, and the low temperature was maintained until the end of the experiment. There were four replicates per group and 10 birds per replicate. The results showed that the structure of the lung tissue was clearer and basically intact in the broilers in the QLJP groups, with a small number of collagen fibers formed, and the content of hydroxyproline (HYP) was significantly reduced. QLJP improved pulmonary arteriole lesions, such as tunica media thickening, intimal hyperplasia, arterial wall hypertrophy, and lumen narrowing. QLJP reduced the relative media thickness (%) and relative medial area (%) of the pulmonary arteriole, and significantly decreased the expression level of the alpha-smooth muscle actin (α-SMA) protein in pulmonary arteriole, which alleviated pulmonary arteriole remodeling. The quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) results showed that QLJP treatment significantly reduced the gene and protein levels of transforming growth factor-beta l (TGF-ß1) and Smad2 in the lung and downregulated the gene and protein levels of collagen type I alpha 1 (COL1A1) and matrix metalloproteinase 2 (MMP2). In conclusion, the results of our study suggested that dietary supplementation with QLJP improved pulmonary fibrosis and pulmonary arteriole remodeling by inhibiting the expression of genes related to the TGF-ß1/Smad2 signaling pathway and inhibited the occurrence and development of pulmonary arterial hypertension in low-temperature-exposed broilers.

Melatonin Prevents Chondrocyte Matrix Degradation in Rats with Experimentally Induced Osteoarthritis by Inhibiting Nuclear Factor-κB via SIRT1.

Osteoarthritis (OA) is a common degenerative joint disease characterized by an imbalance of cartilage extracellular matrix (ECM) breakdown and anabolism. Melatonin (MT) is one of the hormones secreted by the pineal gland of the brain and has anti-inflammatory, antioxidant, and anti-aging functions. To explore the role of MT in rats, we established an OA model in rats by anterior cruciate ligament transection (ACLT). Safranin O-fast green staining showed that intraperitoneal injection of MT (30 mg/kg) could alleviate the degeneration of articular cartilage in ACLT rats. Immunohistochemical (IHC) analysis found that MT could up-regulate the expression levels of collagen type II and Aggrecan and inhibit the expression levels of matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), and ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS-4) in ACLT rats. To elucidate the mechanism of MT in protecting the ECM in inflammatory factor-induced rat chondrocytes, we conducted in vitro experiments by co-culturing MT with a culture medium. Western blot (WB) showed that MT could promote the expression levels of transforming growth factor-beta 1 (TGF-ß1)/SMAD family member 2 (Smad2) and sirtuin 2-related enzyme 1 (SIRT1) and inhibit the expression of levels of phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibi-tor (p-p65) and phosphorylated IκB kinase-α (p-IκBα). In addition, WB and real-time PCR (qRT-PCR) results showed that MT could inhibit the expression levels of MMP-3, MMP-13, ADAMTS-4, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in chondrocytes induced by interleukin-1ß (IL-1ß), and up-regulate the expression of chondroprotective protein type II collagen. We found that in vivo, MT treatment protected articular cartilage in the rat ACLT model. In IL-1ß-induced rat chondrocytes, MT could reduce chondrocyte matrix degradation by up-regulating nuclear factor-kB (NF-κB) signaling pathway-dependent expression of SIRT1 and protecting chondrocyte by activating the TGF-ß1/Smad2 pathway.

The application of proteomics and metabolomics to reveal the molecular mechanism of Nutmeg-5 in ameliorating cardiac fibrosis following myocardial infarction.

BACKGROUND: Nutmeg-5, an ancient and classic formula in traditional Mongolian medicine comprising five kinds of traditional Chinese medicine, is widely used in the treatment of myocardial infarction (MI, called heart "Heyi" disease in Mongolian medicine). Cardiac fibrosis plays a critical role in the development and progression of heart failure after MI. However, the material basis and pharmacological mechanisms of the effect of Nutmeg-5 on cardiac fibrosis after MI remain unclear. OBJECTIVE: The aim of this study was to first explore the potential material basis and molecular mechanism of action of Nutmeg-5 in improving cardiac fibrosis after MI via a multiomics approach. METHODS: The constituents in Nutmeg-5 were identified by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). High-performance liquid chromatography (HPLC) and gas chromatography (GC)-based fingerprints of Nutmeg-5 were analysed, and characteristic peaks were identified by comparison to standard samples. A rat MI model was created by permanent ligation of the left anterior descending artery. The protective effect of Nutmeg-5 on cardiac fibrosis after MI was evaluated by tissue histology and measurement of the serum biomarkers of myocardial injury. Cardiac fibrosis levels were evaluated by Sirius red staining. Differentially expressed proteins in the myocardium and metabolites in the serum were explored by proteomic and untargeted metabolome analyses, respectively. Pearson correlation analysis was performed to explore the association between serum metabolites and myocardial proteins. RESULTS: A total of 67 constituents were identified in Nutmeg-5 by UPLC-MS/MS. Sixteen components were identified in the fingerprint of Nutmeg-5 by comparison with a standard sample. Six lactones were isolated from Nutmeg-5 and quantified by HPLC and GC. MI was significantly alleviated in Nutmeg-5-treated rats compared to MI rats, as demonstrated by their decreased mortality, improved cardiac function, and attenuated cardiac fibrosis and myocardial injury. A total of 252 significant differential metabolites were identified in plasma between model and Nutmeg-5-treated rats by untargeted metabolome analysis. Among these, 36 critical metabolites were associated with Nutmeg-5 activity. Proteomic analysis identified 338 differentially expressed proteins in the rat myocardium between MI and Nutmeg-5-treated rats, including 204 upregulated and 134 downregulated proteins. Protein set enrichment analysis revealed that Nutmeg-5 treatment significantly inhibited the extracellular matrix (ECM)-receptor interaction pathway, which was activated in the myocardium of MI rats. A significant decrease in collagen and alpha smooth muscle actin expression levels was found in the myocardium of Nutmeg-5-treated rats compared to MI rats. These results illustrated that Nutmeg-5 had a significant protective effect on cardiac fibrosis after MI. A significant correlation was found between the ECM-receptor interaction pathway in the myocardium and critical metabolites in the serum. In addition, there were positive correlations between the levels of critical metabolites and the expression levels of transforming growth factor (TGF)-ß1 and Smad2 in the rat myocardium. CONCLUSIONS: Nutmeg-5 alleviated cardiac fibrosis after MI in rats by inhibiting the myocardial ECM-receptor interaction pathway and TGF-ß1/Smad2 signalling, which was achieved by regulating plasma metabolites.

Scutellarin-induced A549 cell apoptosis depends on activation of the transforming growth factor-ß1/smad2/ROS/caspase-3 pathway.

Scutellarin plays an anti-tumor role in A549 lung cancer cells, but the underlying mechanism is unclear. In this study, scutellarin was used to treat A549 cells for 12, 24, and 48 h, followed by the addition of Tempo, a selective scavenger of mitochondrial reactive oxygen species (ROS) and SB431542, a transforming growth factor (TGF)-ß1 receptor inhibitor. A dihydroethidium fluorescence probe was used to measure the intracellular ROS level, Cell Counting Kit-8 (CCK-8) was used to detect cell viability, and flow cytometry was performed to examine apoptosis. Western blots were used to detect the total protein level of TGF-ß1, p-smad2, and cleaved caspase-3 in A549 cells. The results showed that scutellarin significantly inhibited cell viability and increased apoptosis. Scutellarin also promoted intracellular ROS production, TGF-ß1/smad2 signaling pathway activation, and cleaved caspase-3 expression, which was partly reversed by Tempo. Moreover, scutellarin-induced intracellular ROS production and cleaved caspase-3 expression were inhibited by blocking the TGF-ß1/smad2 pathway with SB431542. In conclusion, scutellarin promoted apoptosis and intracellular ROS accumulation, which could be abrogated by Tempo and SB431542 treatment in A549 cells. Our study indicated that scutellarin induced A549 cell apoptosis via the TGF-ß1/smad2/ROS/caspase-3 pathway.

Islet transplantation ameliorates diabetes-induced testicular interstitial fibrosis and is associated with inhibition of TGF-ß1/Smad2 pathway in a rat model of type 1 diabetes.

Islet transplantation (IT) is considered the most effective endocrine replacement therapy for diabetes mellitus (DM). Studies have demonstrated that IT can repair testicular structural injury caused by inflammatory and oxidative stress in a diabetic rat model. However, highly effective exogenous antioxidant and anti-inflammatory drugs can achieve this effect. Testicular interstitial fibrosis caused by long-term hyperglycemia is however difficult to reverse or recover. Thus far, there are no effective drugs that prevent or relieve testicular interstitial fibrosis. Therefore, it is necessary to explore the potential benefit of IT on testicular interstitial fibrosis induced by DM and its underlying molecular mechanisms. In the present study, Wistar rats were used to establish a DM model by intraperitoneal injection of streptozotocin. The diabetic models then underwent IT or received insulin treatment after 12 weeks. IT was more effective than insulin treatment in ameliorating diabetic-induced testicular interstitial fibrosis, Leydig cells apoptosis, testosterone deficiency and poor sperm motility. IT and insulin treatment both significantly inhibited the upregulation of TGF-ß1 and phosphorylated Smad2 in DM, with IT being more effective than insulin. The present study's findings proved that IT effectively protects diabetic-induced testicular interstitial fibrosis probably by inhibiting the TGF-ß1/Smad2 signaling pathway, which offers hope in male patients with DM complicating with testicular interstitial fibrosis.

Interleukin-26 promotes the proliferation and activation of hepatic stellate cells to exacerbate liver fibrosis by the TGF-ß1/Smad2 signaling pathway.

Liver fibrosis is a wound-healing process of liver featured by the activation of hepatic stellate cells (HSCs) and the deposition of extra cellular matrix (ECM). Accumulating facts have suggested that interleukin (IL) 26 is involved in the pathogenesis of liver fibrosis by the modulation of HSCs. However, the biological roles of IL-26 in liver fibrosis are still unclear. The present study aimed to determine the effect and mechanism of IL-26 on the proliferation and activation of HSCs in vitro. By cell counting kit (CCK)-8 assay, we observed that IL-26 significantly promoted the proliferation of HSCs by increasing S phase and decreasing G0/G1 phase. Annexin V-FITC/PI double staining showed that IL-26 could suppress the apoptosis of HSCs by inhibition of caspase 3 (CASP3) and Bcl-2 associated X protein (BAX). Furthermore, quantitative real-time PCR (qRT-PCR) assay and western blotting analysis revealed that IL-26 exacerbated the degree of hepatic fibrosis, which was associated with the upregulation of the mRNA levels and protein concentrations of IL-6, IL-10, tumor necrosis factor (TNF)-α, matrix metallopeptidase (MMP)-9, and α-smooth muscle act in (SMA). Mechanistically, western blotting analysis showed that IL-26 upregulated the protein expression levels of transforming growth factor (TGF)-ß1 and SMAD family member 2 (Smad2) in HSCs. In summary, the data demonstrated a key role of IL-26 on the proliferation and activation of HSCs in liver fibrosis and the underlying mechanism might be related to the TGF-ß1/Smad2 signaling pathway. The finding will provide a proof that targeting IL-26 may be developed as therapeutics for liver fibrosis.

Icariside II attenuates myocardial fibrosis by inhibiting nuclear factor-κB and the TGF-ß1/Smad2 signalling pathway in spontaneously hypertensive rats.

Studies have demonstrated that icariin plays important roles in preventing hypertension and improving myocardial hypertrophy, inflammatory and infiltration. Icariside (ICS II) is the main metabolite of icariin, which has anti-inflammatory and anti-oxidant activities and protects against ischaemic brain injury. Whether ICS II improves myocardial fibrosis in spontaneously hypertensive rats (SHRs) and the related mechanism remain unknown. Some studies have suggested that TGF-ß and the nuclear factor κB signalling pathway play a key role in the progression of myocardial fibrosis. Therefore, in the current study, we aimed to evaluate the effects of ICS II on induced myocardial fibrosis in SHRs and explore the mechanism underlying this activity. The SHRs were treated with ICS II (4, 8, and 16 mg/kg) via daily gavage for 12 weeks. Left ventricular function was detected using the Vevo2100 system, and the collagen area was measured by Masson staining. The results indicated that ICS II markedly improved left ventricular function and decreased the left ventricular myocardial collagen area compared with the SHR group. To further investigate the mechanism underlying this activity, we measured the protein expression of interleukin-1ß (IL-1ß), tumour necrosis factor-α (TNF-α), transforming growth factor-ß1 (TGF-ß1), Smad2, inhibitory κB (IκB), and nuclear factor κB (NF-κB) p65 by Western blot. The results showed that ICS II inhibited NF-κB p65 expression and the TGF-ß1/Smad2 signalling pathways. In conclusion, the present results suggest that ICS II suppresses myocardial fibrosis in SHRs, and this effect might be at least partially mediated through suppression of NF-kB signalling and the TGF-ß1/Smad2 signalling pathway.

Effects of miR-340 on hepatocellular carcinoma by targeting the DcR3 gene.

In hepatocellular carcinoma (HCC), miR-340 plays a vital role in the regulation of tumor occurrence and deterioration, while DcR3 gene is involved in cancer cell proliferation and apoptosis. This study analyzed miR-340 in the serum of patients with HCC and healthy controls. Then, miR-340, DcR3, TGF-ß1 and Smad2 expression were measured in HCC tissues and adjacent parts. Relationship between miR-340 and DcR3 was verified. Effects of miR-340 on human HepG2 cell proliferation and apoptosis were explored. miR-340, DcR3, TGF-ß1, Smad2 mRNA and protein expression were also determined after miR-340 transfection. Compared with the control, miR-340 was significantly lower in the serum of the HCC patients (p < 0.01). miR-340 was lower in HCC tissues than in adjacent; however, DcR3, TGF-ß1 and Smad2 were higher (p < 0.01). Furthermore, luciferase activity was significantly lower in the cells co-transfected with miR-340 mimics and DcR3-3'UTR-WT (p < 0.01), indicating that DcR3 was a target gene of miR-340. Moreover, decreased expression in DcR3, TGF-ß1 and Smad2 was detected after miR-340 overexpression (p < 0.01), thus promoting apoptosis and blocking the proliferation of human HepG2 cells (p < 0.05). Furthermore, overexpression of DcR3 could activate the TGF-ß1/Smad2 signal transduction pathway and increase the phosphorylation of Smad2. In conclusion, miR-340 plays a suppressive role in HCC development by targeting DcR3 and silencing the TGF-ß1/Smad2 signaling pathway.

Osthole inhibits intimal hyperplasia by regulating the NF-κB and TGF-ß1/Smad2 signalling pathways in the rat carotid artery after balloon injury.

Osthole (7-methoxy-8-isopentenoxy-coumarin), a compound extracted from Cnidiummonnieri (L.) Cusson seeds, has been found to exhibit potent therapeutic effects in cancer due to its ability to inhibit inflammation and cell proliferation. However, its effects on arterial wall hypertrophy-related diseases remain unclear. Therefore, in this study, we aimed to investigate the effects of Osthole on intimal hyperplasia in a rat model of carotid artery balloon injury. We established the balloon-induced carotid artery injury rat model in male Sprague-Dawley rats, after which we administered Osthole (20mg/kg/day or 40mg/kg/day) or volume-matched normal saline orally by gavage for 14 consecutive days. Intimal hyperplasia and the degree of vascular smooth muscle cell proliferation were then evaluated by histopathological examination of the changes in the carotid artery, as well as by examination of proliferating cell nuclear antigen (PCNA) expression. Tumour necrosis factor-ɑ (TNF-α), interleukin-1ß (IL-1ß), transforming growth factor-beta (TGF-ß1) and PCNA mRNA expression levels were examined by real-time RT-PCR, while nuclear factor-κB (NF-κB (p65)), IκB-α, TGF-ß1 and phospho-Smad2 (p-Smad2) protein expression levels were analysed by immunohistochemistry or western blot analysis. We found that Osthole significantly attenuated neointimal thickness and decreased the elevations in PCNA protein expression induced by balloon injury. Moreover, Osthole down-regulated the pro-inflammatory factors TNF-α and IL-1ß and NF-κB (p65), whose expression had been upregulated after balloon injury. Moreover, IκB-α protein expression levels increased following Osthole treatment. In addition, the elevations in TGF-ß1 and p-Smad2 protein expression induced by balloon injury were both significantly attenuated by Osthole administration. We concluded that Osthole significantly inhibited neointimal hyperplasia in balloon-induced rat carotid artery injury and that the mechanism by which this occurs may involve NF-κB, IL-1ß and TNF-ɑ down-regulation, which alleviates the inflammatory response, and TGF-ß1/Smad2 signalling pathway inhibition.

Synergistic protection of Schizandrin B and Glycyrrhizic acid against bleomycin-induced pulmonary fibrosis by inhibiting TGF-ß1/Smad2 pathways and overexpression of NOX4.

Pulmonary fibrosis, a progressive and lethal lung disease, is a major therapeutic challenge for which new therapeutic strategies are warranted. Schisandrin B (Sch B) and Glycyrrhizic acid (GA) are the principal active ingredients of Schisandra chinensis and Glycyrrhiza glabra respectively, which have been reported to protect against lung injures. The present study was aimed at exploring the combinatorial therapeutic effects on bleomycin-induced pulmonary fibrosis. Lung fibrotic injuries were induced in mice by a single intratracheal instillation of 5mg/kg bleomycin (BLM). Then, these mice were administered with Sch B (100mg/kg) or/and GA (75mg/kg) for 28days. BLM-triggered structure distortion, collagen overproduction, excessive inflammatory infiltration, pro-inflammatory cytokine release, and oxidative stress damages in lung tissues were attenuated to a higher degree by combinatorial treatment than by treatment of the individual agents. The expression of TGF-ß1 and the phosphorylation of its downstream target, Smad2 were enhanced by BLM, but weakened by Sch B or/and GA. Furthermore, the significant overexpression of NADPH oxidase 4 (NOX4) was observed in BLM-induced pulmonary fibrosis, which was inhibited by Sch B or/and GA. Our study reveals that the synergistic protection by Sch B and GA against BLM-induced pulmonary fibrosis is correlated to its anti-inflammatory, anti-oxidative and anti-fibrotic properties, involving inhibition of TGF-ß1/Smad2 signaling pathways and overexpression of NOX4.