Theacrine alleviates chronic inflammation by enhancing TGF-ß-mediated shifts via TGF-ß/SMAD pathway in Freund's incomplete adjuvant-induced rats.

Rheumatoid arthritis is a chronic and systemic autoimmune disease, which affects approximately 1% of the adult population worldwide. The present study investigated the therapeutic effect of theacrine (TC) on arthritis and its mechanisms in Freund's incomplete adjuvant (FIA)-induced SD rats. Rats were randomly divided into 5 groups: i) healthy control; ii) model; iii) positive control with methotrexate (MTX); iv) treatment with 12.5 mg/kg TC; and v) treatment with 25.0 mg/kg TC. The apparent scores, including changes in body weights, degree of paw swelling and arthritis indicators, were analyzed to evaluate the anti-chronic inflammatory effect of TC. The levels of interleukin (IL)-6 and transforming growth factor-ß (TGF-ß) in serum were measured by enzyme-linked immunosorbent assay. The protein and RNA expression levels of the critical factors in rats were measured to elucidate the mechanisms responsible for chronic inflammation and to verify molecular indexes of chronic inflammatory conditions. TC notably suppressed the severity of FIA-induced rat by attenuating the apparent scores, animal weight and inflammatory indexes in the 25 mg/kg TC group compared with the FIA rat model. Furthermore, TC significantly decreased the levels of IL-6 and increased the levels of TGF-ß. Histopathological examinations indicated that TC rescued the synovial hyperplasia and inflammatory cell infiltration in joint tissues. In addition, TC enhanced TGF-ß-mediated shifts in inflammatory marker expression in joint tissue. Overall, the present study demonstrated that TC exerted a superior anti-arthritic effect via the suppression of IL-6 and the activation of TGF-ß by the TGF-ß/SMAD pathway.

[Effect of acupuncture on TGF-ß1/Smads pathway in mice with airway remodeling mic].

OBJECTIVE: To investigate the effect of acupuncture on TGF-ß1/Smads signaling pathway in the lung tissue of mice with airway remodeling. METHODS: Thirty specific pathogen-free mice were randomly divided into blank group, model group and acupuncture group (n=10). Mouse models of asthma were established in the model group and the acupuncture group, and the mice in the latter group received 7 acupuncture therapies (at bilateral Fei Shu, Da Zhui and Zu Sanli, 20 min each time) every other day, starting on the 10th day after the modeling. At 24 h after the last acupuncture, the mice were subjected to inhalation of 1% OVA for 3 days, and 24 h after the last challenge, the mice were given methacholine chloride (Mch) inhalation at different concentrations for measurement of lung resistance using a noninvasive stroke volume meter. HE staining was used to observe the pathological changes in the lung tissues, and TGF-ß1 levels in the the bronchoalveolar lavage fluid (BALF) and serum were detected using ELISA; Western blotting was used to detect the differential protein expressions in the airway smooth muscles between the two groups. The airway smooth muscle cells were isolated from the mice in the acupuncture group and treated with a TGF- ß1 inhibitor (LY2157299), and the relative expressions of type-Ⅰ and Smads proteins were detected using Western blotting. RESULTS: The mice in the model showed obvious tracheal fistula with airway pathologies including lumen narrowing, bronchial mucosa thickening, dissociation of the epithelial cells, and thickening of the alveolar septum and airway smooth muscles. These pathological changes were obviously milder in the acupuncture group. The asthmatic mice exhibited significantly increased lung resistance in positive correlation with Mch concentration. Serum TGF-ß1 level was significantly elevated in asthmatic mice (P < 0.05); TGF-ß1 levels in the serum and BALF were significantly lower in the acupuncture group than in the model group (P < 0.05). In the model group, the expressions of α-SMA, TGF-ß1 and Smads in the airway smooth muscles were significantly higher than those in the other two groups (both P < 0.05). In cultured airway smooth muscle cells, the expressions of type-Ⅰ and Smads were significantly higher in cells treated with LY2157299 than in the control cells (P>0.05). CONCLUSIONS: Acupuncture can inhibit airway remodeling by inhibiting the expression of airway TGF-ß1 and down-regulating the expression of Smads and α-SMA to reduce airway inflammatory response. Airway expressions of type-Ⅰ and Smads proteins remain high after inhibiting TGF-ß1. Acupuncture may control asthma progression through the TGF-ß1/Smads pathway.

Herbal compound 861 prevents hepatic fibrosis by inhibiting the TGF-ß1/Smad/SnoN pathway in bile duct-ligated rats.

BACKGROUND: This study was to evaluate the effects of herbal compound 861 (Cpd861) on ski-related novel protein N (SnoN) and transforming growth factor-ß1 (TGF-ß1) /Smad signaling in rats with bile duct ligation (BDL)-induced hepatic fibrosis, and to explore the mechanisms of Cpd861 on hepatic fibrosis. METHODS: Thirty Wistar male rats were randomly divided into three groups: sham operation, BDL, and Cpd861. To induce hepatic fibrosis, BDL and Cpd861 group rats underwent bile duct ligation. Cpd861 at 9 g/kg/d or an equal volume of normal saline was administered intragastrically for 28 days. Liver injury was assessed biochemically and histologically. Protein and mRNA changes for SnoN and TGF-ß1/Smad signaling (TGF-ß1, Smad2, phosphorylated Smad2 [p-Smad2], phosphorylated Smad3 [p-Smad3], fibronectin, and collagen III) were determined by Western blotting and quantitative real-time PCR. RESULTS: BDL rats treated with Cpd861 had significantly alleviated hepatic fibrosis compared to BDL rats not receiving Cpd861 treatment. Moreover, Cpd861 decreased the expression of fibrosis-associated proteins fibronectin and collagen III in liver tissue. Cpd861 administration increased the expression of SnoN protein, did not change SnoN mRNA level, and decreased TGF-ß1, p-Smad2, and p-Smad3 protein expression compared to BDL without Cpd861 treatment. CONCLUSIONS: Cpd861 attenuates hepatic fibrosis by increasing SnoN protein expression and inhibiting the TGF-ß1/Smad signaling pathway.

Oxymatrine inhibits aldosterone-induced rat cardiac fibroblast proliferation and differentiation by attenuating smad-2,-3 and-4 expression: an in vitro study.

BACKGROUND: We previously demonstrated oxymatrine, an alkaloid from the Chinese medicine radix Sophorae flavescentis, ameliorates hemodynamic disturbances and cardiac fibrosis; however, the underlying mechanisms are unclear. Here, we investigated the effect and mechanism of action of oxymatrine on aldosterone-induced cardiac fibroblast to myofibroblast differentiation in vitro. METHODS: Cardiac fibroblasts were isolated purified from neonatal Sprague Dawley rats. The optimal concentration of aldosterone to stimulate cardiac fibroblast proliferation was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cardiac fibroblasts were pretreated with 7.57 × 10(-4) mol/L or 3.78 × 10(-4) mol/L oxymatrine or without oxymatrine for 2 h, and then coincubated with 1 × 10(-8) mol/L aldosterone for 48 h. The MTT assay and Masson staining were used to detect the cardiac fibroblast proliferation and myofibroblast differentiation. The secretion of type I and III collagen was measured by commercial ELISA kits, and the hydroxyproline content was determined by the colorimetric assay. Western blotting assayed the Smad-2, Smad-3, and Smad-4 protein expression in cardiac fibroblasts. RESULTS: The present results confirmed that aldosterone induced cardiac fibroblast to myofibroblast proliferation and differentiation. The MTT assay and Masson staining indicated oxymatrine significantly inhibited aldosterone-induced cardiac fibroblast proliferation and myofibroblast differentiation. Oxymatrine significantly inhibited aldosterone-induced secretion of type I and III collagen, as indicated by commercial ELISA kits, and aldosterone-induced increase in hydroxyproline content, as indicated by a colorimetric assay. Western blotting revealed oxymatrine attenuated aldosterone-induced Smad-2, Smad-3, and Smad-4 expression in cardiac fibroblasts. CONCLUSION: Oxymatrine can inhibit cardiac fibroblast proliferation and differentiation into myofibroblasts via a mechanism linked to attenuation of the Smad signaling pathway.

Development and validation of a phosphorylated SMAD ex vivo stimulation assay.

Assessing the pharmacodynamics (PD) of a potential therapeutic through the use of a downstream biomarker is essential. This is traditionally performed in the target tissue but limited volume and invasiveness of sampling pose challenges with solid tumours. Currently, there are several small molecule receptor kinase inhibitors and large molecule therapeutic antibodies in clinical trials that interfere with TGFbeta signalling to treat various forms of cancer. With the advent of these new therapies, there is a need for a surrogate tissue that is easily accessible and indicative of tumour response. We propose the use of an ex vivo TGFbeta1 stimulation of peripheral blood mononuclear cells (PBMCs) coupled with the measurement of phosphorylated SMAD2 (Sma/Mothers Against dpp, a downstream transcriptional activator) using a sandwich ELISA. TGFbeta is involved in many different cellular responses, such as proliferation, angiogenesis, migration, invasion and immunomodulation. SMAD2 and SMAD3 are phosphorylated as a result of the canonical cascade through ligand binding and receptor kinase activation. These phosphorylated SMADs (pSMAD) associate with SMAD4, a co-SMAD, and transcriptionally activate TGFbeta-mediated genes. This paper describes the novel method for measuring the downstream effects of inhibiting canonical TGFbeta signalling using ex vivo stimulation of surrogate tissue to predict tumour response. In addition, we present the assay validation rationale and data. This novel, validated assay can be used to gain insight into clinical trials regarding TGFbeta signal modulation by multiple inhibitor platforms for both large and small molecules.