The immune regulation and therapeutic potential of the SMAD gene family in breast cancer.

Breast cancer is a serious threat to human health. The transforming growth factor-ß signaling pathway is an important pathway involved in the occurrence and development of cancer. The SMAD family genes are responsible for the TGF-ß signaling pathway. However, the mechanism by which genes of the SMAD family are involved in breast cancer is still unclear. Therefore, it is necessary to investigate the biological roles of the SMAD family genes in breast cancer. We downloaded the gene expression data, gene mutation data, and clinical pathological data of breast cancer patients from the UCSC Xena database. We used the Wilcox test to estimate the expression of genes of the SMAD family in cancers. And the biological functions of SMAD family genes using the DAVID website. The Pearson correlation method was used to explore the immune cell infiltration and drug response of SMAD family genes. We conducted in biological experiments vitro and vivo. In this study, we integrated the multi-omics data from TCGA breast cancer patients for analysis. The expression of genes of SMAD family was significantly dysregulated in patients with breast cancer. Except for SMAD6, the expression of other SMAD family genes was positively correlated. We also found that genes of the SMAD family were significantly enriched in the TGF-ß signaling pathway, Hippo signaling pathway, cell cycle, and cancer-related pathways. In addition, SMAD3, SMAD6, and SMAD7 were lowly expressed in stage II breast cancer, while SMAD4 and SMAD2 were lowly expressed in stage III cancer. Furthermore, the expression of genes of the SMAD family was significantly correlated with immune cell infiltration scores. Constructing a xenograft tumor mouse model, we found that SMAD3 knockdown significantly inhibited tumorigenesis. Finally, we analyzed the association between these genes and the IC50 value of drugs. Interestingly, patients with high expression of SMAD3 exhibited significant resistance to dasatinib and staurosporine, while high sensitivity to tamoxifen and auranofin. In addition, SMAD3 knockdown promoted the apoptosis of BT-549 cells and decreased cell activity, and BAY-1161909 and XK-469 increased drug efficacy. In conclusion, genes of the SMAD family play a crucial role in the development of breast cancer.

Gypenoside XLIX loaded nanoparticles targeting therapy for renal fibrosis and its mechanism.

Renal fibrosis is the main pathological feature of the occurrence and development of chronic nephropathy. At present, there is no effective treatment, except for renal transplantation and dialysis. Previous studies have shown that nano-preparations can be used as a therapeutic tool to target organs. In this study, we studied the therapeutic effect and mechanism of Chinese medicine monomer Gypenoside (Gyp) XLIX on renal fibrosis and explored the targeting and therapeutic effects of polylactic acid-co-glycoside (PLGA)-Gyp XLIX nanoparticles in unilateral ureteral occlusion (UUO) kidney. Gyp XLIX and PLGA-Gyp XLIX nanoparticles were used to treat UUO mice and Human renal tubular epithelial (HK2) cells stimulated by transforming growth factor-ß (TGF-ß). Histopathological and molecular biological techniques were used to detect the expression of type I collagen and alpha-smooth muscle actin (α-SMA). To investigate the in vivo targeting of PLGA nanoparticles, they were loaded with 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide and injected into UUO mice. We evaluated the effect of Gyp XLIX nanoparticles on TGF-ß/Smad3 pathway, a central driver for renal fibrosis in Smad-deficient HK2 cells. Fluorescence imaging showed that the PLGA nanoparticles around 120 nm could be targeted to the UUO kidney. Compared with Gyp XLIX, PLGA-Gyp XLIX nanoparticles could effectively inhibit renal fibrosis and reduce collagen deposition and reduce renal tubular necrosis. Gyp XLIX decreased the phosphorylation of Smad3, but could not further reduce the levels of type I collagen and α-SMA in Smad-deficient cells. This study opens a promising way for targeted drug treatment of renal fibrosis.

Drug-containing serum of rhubarb-astragalus capsule inhibits the epithelial-mesenchymal transformation of HK-2 by downregulating TGF-ß1/p38MAPK/Smad2/3 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheum palmatum L; Astragalus membranaceus (Fisch.), is referred to as 'Dahuang, Huangqi' in China. As an important medicinal plant, the rhizome of rhubarb and astragalus is traditionally used in the treatment of kidney diseases associated with renal failure, inflammation and tumors. AIM OF THE STUDY: This study aimed to investigate the effect of a drug-containing serum of rhubarb-astragalus capsules (composed of rhubarb and astragalus) and to elucidate its mechanism in the epithelial-mesenchymal transformation of renal tubular epithelial cells. MATERIALS AND METHODS: Epithelial-mesenchymal transformation (EMT) of HK-2 cells was induced by TGF-ß1, and rhubarb-astragalus and losartan drug-containing serum from rats, as well as SB203580 (a specific inhibitor of p38 MAPK), were used. High-performance liquid chromatography analysis was performed to determine the main components of the drug-containing serum of rhubarb-astragalus from rats. Western blotting and immunofluorescence analysis were used to determine the levels of protein expression, and real-time quantitative PCR analysis was used to detect the levels of gene expression. RESULTS: The drug-containing serum of rhubarb-astragalus contained emodin (0.36 µg/ml) and danthraquinone (0.96 µg/ml). Rhubarb-astragalus significantly decreased the protein expression levels of α-SMA, FN, vimentin and N-cadherin in HK-2 cells that were increased by TGF-ß1, while it significantly increased the E-cadherin protein expression level that was decreased by TGF-ß1. Rhubarb-astragalus also significantly decreased the protein expression levels of TGF-ß1 and p38 MAPK and the mRNA expression levels of α-SMA, vimentin, TGF-ß1, p38 MAPK, Smad2 and Smad3 in HK-2 cells that were increased by TGF-ß1. It is worth noting that SB203580 (a p38 MAPK inhibitor) had similar effects as rhubarb-astragalus in this study. CONCLUSION: The drug-containing serum of rhubarb-astragalus can inhibit EMT in HK-2 cells by downregulating the TGF-ß1/p38 MAPK/Smad2/3 pathway.

Natural flavonol fisetin attenuated hyperuricemic nephropathy via inhibiting IL-6/JAK2/STAT3 and TGF-ß/SMAD3 signaling.

BACKGROUND: The naturally occurring flavonol fisetin (3,3',4',7-tetrahydroxyflavone), widely dispersed in fruits, vegetables and nuts, has been reported to exert anti-inflammatory, antioxidant and anti-angiogenic effects. Our previous study indicated fisetin ameliorated inflammation and apoptosis in septic kidneys. However, the potential nephroprotective effect of fisetin in hyperuricemic mice remains unknown. PURPOSE: The current study was designed to investigate the effect of fisetin on hyperuricemic nephropathy (HN) and explore the underlying mechanisms. METHODS: The HN was induced in mice by mixing of potassium oxonate (2400 mg/kg) and adenine (160 mg/kg) in male C57BL/6J mice. Fisetin (50 or 100 mg/kg) was orally administrated either simultaneously with the establishment of HN or after HN was induced. As a positive control, allopurinol of 10 mg/kg was included. Uric acid levels in the serum and urine as well as renal function parameters were measured. Renal histological changes were measured by periodic acid-Schiff (PAS) and Masson's trichrome stainings. The expression of gene/protein in relation to inflammation, fibrosis, and uric acid excretion in the kidneys of HN mice or uric acid-treated mouse tubular epithelial (TCMK-1) cells were measured by RNA-seq, RT-PCR, western blot and immunohistochemical analysis. RESULTS: Treatment with fisetin, regardless of administration regimen, dose-dependently attenuated hyperuricemia-induced kidney injury as indicated by the improved renal function, preserved tissue architecture, and decreased urinary albumin-to-creatinine ratio. Additionally, fisetin lowered uricemia by modulating the expression of kidney urate transporters including urate transporter 1(URAT1), organic anion transporter 1 (OAT1), organic anion transporter 3 (OAT3) and ATP binding cassette subfamily G member 2 (ABCG2). Moreover, hyperuricemia-induced secretions of proinflammatory factors including tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and monocyte chemoattractant protein-1(MCP-1) in HN mice and uric acid-stimulated TCMK-1 cells were mitigated by fisetin treatment. Meanwhile, fisetin attenuated kidney fibrosis in HN mice with restored expressions of alpha-smooth muscle actin (α-SMA), collagen I and fibronectin. Mechanistically, fisetin regulated the aberrant activation of signal transducer and activator of transcription-3 (STAT3) signaling and transforming growth factor-ß (TGF-ß) signaling in the HN kidneys and uric acid-stimulated TCMK-1 cells. CONCLUSION: Fisetin lowered uricemia, suppressed renal inflammatory response, and improved kidney fibrosis to protect against hyperuricemic nephropathy via modulation of STAT3 and TGF-ß signaling pathways. The results highlighted that fisetin might represent a potential therapeutic strategy against hyperuricemic nephropathy.

Inhibitory role of ginsenoside Rb2 in endothelial senescence and inflammation mediated by microRNA­216a.

Targeting microRNAs (miRs) using small chemical molecules has become a promising strategy for disease treatment. miR­216a has been reported to be a potential therapeutic target in endothelial senescence and atherosclerosis via the Smad3/NF­κB signaling pathway. Ginsenoside Rb2 (Rb2) is the main bioactive component extracted from the plant Panax ginseng, and is a widely used traditional Chinese medicine. In the present study, Rb2 was identified to have a high score for miR­216a via bioinformatics analysis based on its sequence and structural features. The microscale thermophoresis experiment further demonstrated that Rb2 had a specific binding affinity for miR­216a and the dissociation constant was 17.6 µM. In both young and senescent human umbilical vein endothelial cells (HUVECs), as well as human aortic endothelial cells, Rb2 decreased the expression of endogenous miR­216a. Next, a replicative endothelial senescence model of HUVECs was established by infection with pre­miR­216a recombinant lentiviruses (Lv­miR­216a) and the number of population­doubling level (PDL) was calculated. Stable overexpression of miR­216a induced a premature senescent­like phenotype, whereas the senescent features and increased activity of senescence­associated ß­galactosidase (SA­ß­gal) were reversed after Rb2 treatment. The percentage of SA­ß­gal­positive cells in senescent PDL25 cells transfected with Lv­miR­216a was decreased 76% by Rb2 treatment compared with the Lv­miR­216a group without Rb2 treatment (P=0.01). Mechanistically, miR­216a inhibited Smad3 protein expression, promoted IκBα degradation and activated NF­κB­responsive genes, such as vascular cell adhesion molecule 1 (VCAM1), which promoted the adhesiveness of endothelial cells to monocytes. These pro­inflammatory effects of miR­216a were significantly suppressed by Rb2 treatment. When Smad3 was suppressed by small interfering RNA, the elevated expression levels of intercellular adhesion molecule 1 and VCAM1 induced by miR­216a were significantly reversed. Collectively, to the best of our knowledge, the present study demonstrated for the first time that Rb2 exerted an anti­inflammation effect on the process of endothelial cell senescence and could be a potential therapeutic drug by targeting miR­216a.

Inhibitory effects of alkaline extract from the pericarp of Citrus reticulata Blanco on collagen behavior in bleomycin-induced pulmonary fibrosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Peel of Citrus reticulata, a Chinese herbal drug with functions of regulating Qi and expelling phlegm, has been used for the treatment of lung related diseases in Chinese medicine for a long time. Its detailed effects on collagen in anti-idiopathic pulmonary fibrosis (IPF) is still unclear. AIM OF THE STUDY: To explore the effects of citrus alkaline extract (CAE) on collagen synthesis, crosslinking and deposition in pulmonary fibrosis and understand the possible signal pathways involved in the activity. MATERIALS AND METHODS: CAE was prepared from C. reticulata. Bleomycin-induced pulmonary fibrosis mouse model was applied. Pulmonary fibrosis of lung was estimated with histopathology analysis, and collagen deposition was evaluated with immunohistochemistry. Collagen crosslinking related biomarkers and enzymes were analyzed with chemical methods, immunohistochemical and western blot analyses. RESULTS: CAE oral administration lowered hydroxyproline content, inhibited the collagen deposition including expressions of collagen I and III, and relieved bleomycin-induced pulmonary fibrosis in mice model. The productions of a collagen crosslink pyridinoline and crosslinking related enzymes including lysyl oxidase (LOX), lysyl oxidase-like protein 1 (LOXL1) in lung were suppressed by CAE treatment. Furthermore, the protein expressions of TGF-ß1 and Smad3 levels in lungs were also downregulated by CAE. CONCLUSIONS: This study demonstrated that CAE inhibited collagen synthesis, crosslinking and deposition, and ameliorated bleomycin-induced pulmonary fibrosis. Preliminary mechanism study revealed that CAE exerted its bioactivity at least via downregulation of TGF-ß1/Smad3 pathway. Our findings provided a great potential in fighting IPF based on CAE.

Knockdown of dual oxidase 1 suppresses activin A-induced fibrosis in cardiomyocytes via the reactive oxygen species-dependent pyroptotic pathway.

Fibrotic diseases account for more than 8 million deaths worldwide annually. Reactive oxygen species (ROS) has been shown to activate pyroptosis and promote the production of interleukin (IL)-1ß and IL-18, leading to fibrosis development. However, the role of dual oxidase 1 (DUOX1)-induced ROS production and pyroptosis in cardiac fibrosis remains largely unknown. Activin A was used to induce ROS and pyroptosis in cardiomyocytes. ROS level, pyroptosis, and cytokine production were detected using Active Oxygen Detection Kit, flow cytometry, and enzyme-linked immunosorbent assay, respectively. Western blotting analysis was used to measure expression changes of proteins. DUOX1 was silenced or overexpressed to investigate its role in fibrosis. We found that activin A induced ROS production and pyroptosis in cardiomyocytes, which was blocked by the ROS scavenger, N-acetyl-L-cysteine (NAC). Knockdown of DUOX1 reversed activin A-induced ROS production, pyroptosis, cytokine release, and the upregulation of proinflammatory proteins. Overexpression of DUOX1 resulted in opposite effects of knockdown DUOX1. Administration of an ROS scavenger blocked the effect of DUOX1 overexpression. Supplementation of IL-1ß and IL-18 caused significant fibrosis in human cardiac fibroblasts (hCFs). The knockdown of DUOX1 protected cardiomyocytes against activin A-induced fibrosis via the inhibition of ROS, cytokine release, and pyroptosis.

Involvement of eIF2α in halofuginone-driven inhibition of TGF-ß1-induced EMT.

Halofuginone (HF) is an extract from the widely used traditional Chinese medicine (TCM) Dichroa febrifuga that facilitates the recovery of wounds and attenuates hepatic fibrosis. However, the role of HF in the epithelial-mesenchymal transition (EMT) of IPEC-J2 cells remains unclear. The current study explored the anti-EMT effect of HF in IPEC-J2 cells and illustrates its molecular mechanism. Transforming growth factor ß1 (TGF-ß1), as a recognized profibrogenic cytokine, decreased the level of the epithelial marker E-cadherin and increased the level of the mesenchymal markers, such as N-cadherin, fibronectin (FN), vimentin (Vim), and α-smooth muscle actin (α-SMA), in IPEC-J2 cells depending on the exposure time and dose. HF markedly prevented the EMT induced by TGF-ß1. Dissection of the mechanism revealed that HF inhibited IPEC-J2 cell EMT via modulating the phosphorylation of SMAD2/3 and the SMAD2/3-SMAD4 complex nuclear translocation. Furthermore, HF could promote the phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), which modulates the SMAD signaling pathway. These results suggested that HF inhibits TGF-ß1-induced EMT in IPEC-J2 cells through the eIF2α/SMAD signaling pathway. Our findings suggest that HF can serve as a potential anti-EMT agent in intestinal fibrosis therapy.

Poricoic acid A activates AMPK to attenuate fibroblast activation and abnormal extracellular matrix remodelling in renal fibrosis.

BACKGROUND: In chronic kidney disease, although fibrosis prevention is beneficial, few interventions are available that specifically target fibrogenesis. Poricoic acid A (PAA) isolated from Poria cocos exhibits anti-fibrotic effects in the kidney, however the underlying mechanisms remain obscure. PURPOSE: We isolated PAA and investigated its effects and the underlying mechanisms in renal fibrosis. STUDY DESIGN: Unilateral ureteral obstruction (UUO) and 5/6 nephrectomy (Nx) animal models and TGF-ß1-induced renal fibroblasts (NRK-49F) were used to investigate the anti-fibrotic activity of PAA and its underlying mechanisms. METHODS: Western blots, qRT-PCR, immunofluorescence staining, co-immunoprecipitation and molecular docking methods were used. Knock-down and knock-in of adenosine monophosphate-activated protein kinase (AMPK) in the UUO model and cultured NRK-49F cells were employed to verify the mechanisms of action of PAA. RESULTS: PAA improved renal function and alleviated fibrosis by stimulating AMPK and inhibiting Smad3 specifically in Nx and UUO models. Reduced AMPK activity was associated with Smad3 induction, fibroblast activation, and the accumulation and aberrant remodelling of extracellular matrix (ECM) in human renal puncture samples and cultured NRK-49F cells. PAA stimulated AMPK activity and decreased fibrosis in a dose-dependent manner, thus showing that AMPK was essential for PAA to exert its anti-fibrotic effects. AMPK deficiency reduced the anti-fibrotic effects of PAA, while AMPK overexpression enhanced its effect. CONCLUSION: PAA activated AMPK and further inhibited Smad3 specifically to suppress fibrosis by preventing aberrant ECM accumulation and remodelling and facilitating the deactivation of fibroblasts.

Danggui Buxue Tang ameliorates bleomycin-induced pulmonary fibrosis in rats through inhibiting transforming growth factor-ß1/Smad3/ plasminogen activator inhibitor-1 signaling pathway.

OBJECTIVE: To investigate the effect of Danggui Buxue Tang (DBT), a decoction from Traditional Chinese Medicine, on bleomycin-induced pulmonary fibrosis in rats, and to propose the possible underlying mechanism. METHODS: Forty male Sprague-Dawley rats were randomly divided into sham group, model group, prednisone group and DBT group. Pulmonary fibrosis rat model was established by intratracheal injection with bleomycin. Body weight and lung index were monitored. Histopathologic examination and collagen deposition were determined using Hematoxylin and eosin (HE) and Masson's trichrome staining. Immunohistochemistry staining was applied to observe the expression of alpha-smooth muscle actin (α-SMA). mRNA expression of α-SMA, collagen Ⅰ and collagen Ⅲ were measured by real-time fluorescence quantitative PCR (RT-qPCR). Inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and IL-1ß in serum were detected by Enzyme-linked immunosorbent assay. Alkali hydrolysis method was conducted to investigate the content of hydroxyproline (HYP). Transforming growth factor-ß1 (TGF-ß1), Smad3 and plasminogen activator inhibitor-1 (PAI-1) protein level were examined by Western blot assay. RESULTS: DBT significantly reduced the severity of bleomycin-induced pulmonary fibrosis and inflammation as indicated by minimizing the lost of weight, and by lowering the levels of lung index, inflammation score, Ashcroft score, collagen volume fraction (%), HYP, α-SMA, collagen Ⅰ, collagen Ⅲ, TNF-α, IL-6, IL-1ß, TGF-ß1, Smad3 and PAI-1, consistent with the effect of prednisone. CONCLUSION: Our findings suggest that DBT is able to ameliorate the pulmonary fibrosis, the possible mechanism may involve inhibition of pulmonary inflammation and collagen deposition, possibly via suppressing TGF-ß1/Smad3/PAI-1 signaling pathway.

Cyanidin-3-glucoside from Black Rice Ameliorates Diabetic Nephropathy via Reducing Blood Glucose, Suppressing Oxidative Stress and Inflammation, and Regulating Transforming Growth Factor ß1/Smad Expression.

Diabetic nephropathy (DN) is one of the serious complications in diabetes. Cyanidin-3-glucoside (C3G) from black rice was reported to have hypoglycemic effects and an anti-osteoporosis effect in diabetic rats. Whether it has preventive effects on DN has not been reported. In this study, we established a rat model of DN, and C3G at two doses (10 and 20 mg kg-1 day-1) were administered to see its anti-DN effect. A total of 8 weeks of C3G supplementation decreased blood glucose and serum insulin, improved the renal function, and relieved renal glomerular sclerosis and interstitial fibrosis of DN rats. Also, the kidneys of DN rats had improved the oxidative defense system. Pro-inflammatory mediators were markedly reduced in serum and kidneys of the C3G-treated groups. Transforming growth factor ß1 (TGF-ß1), phosphor-Smad2, and phosphor-Smad3 protein expression levels were significantly decreased in the kidney of the C3G-treated group, whereas the Smad7 expression level was upregulated by C3G. Our results indicate that C3G can ameliorate DN via antioxidative stress and anti-inflammation and regulate the TGF-ß1/Smad2/3 pathway. Our results suggest that C3G from black rice might be used as a renal-protective nutrient in DN.

Raw and vinegar processed Curcuma wenyujin regulates hepatic fibrosis via bloking TGF-ß/Smad signaling pathways and up-regulation of MMP-2/TIMP-1 ratio.

ETHNOPHARMACOLOGICAL RELEVANCE: Curcuma wenyujin Y.H. (CW), a variety of Curumae Rhizoma, which documented in China Pharmacopeia, has long been used as plant medicine for its traditional effect on promoting Qi, activating blood stagnation and expelling blood stasis. Nowadays, it is often used in clinic for extraordinary effect on liver diseases. It is worthy to be noted that CW processed with vinegar has been applied in clinic for 1500 years which started in the northern and southern dynasties. AIM OF STUDY: Liver fibrosis is a worldwide clinical issue. It is worth developing a multi-target and multicellular approach which is high efficiency and low side effects for the treatment of hepatic fibrosis. The anti-hepatic fibrosis molecular mechanisms of CW and vinegar Curcuma wenyujin (VCW) need to be explored and elucidated. Furthermore, the study aimed to discuss the efficiency and mechanism differences between CW and VCW in hepatic fibrosis. METHODS AND RESULTS: Biochemical assays and histopathology were adopted to evaluate the anti-hepatic fibrosis effect of CW and VCW. The TGF-ß/Smad signaling involving TGF-ß1, TGF-ßRⅠ, TGF-ßRⅡ and Smad2, Smad3, Smad7 in fibrosis is examined, which is a critical step towards the evaluation of anti-hepatic fibrosis agents. Meanwhile, the MMP/TIMP balance is a potential therapy target by modulating extracellular matrix, which is also examined. Both CW and VCW inhibit the activation and proliferation of hepatic stellate cells and induce apoptosis via blocking TGF-ß/Smad signaling pathways. Additionally, the level of MMP-2/TIMP-1 regulated significantly, which suggest CW and VCW participate in the degradation process, and maintain the formation and production of extracellular matrix. CONCLUSION: Raw and vinegar processed Curcuma wenyujin regulates hepatic fibrosis via bloking TGF-ß/Smad signaling pathways and up-regulation of MMP-2/TIMP-1 ratio. And VCW has more exhibition than CW.

Hepatoprotective effect of a fucoidan extract from Sargassum fluitans Borgesen against CCl4-induced toxicity in rats.

The in vivo antifibrotic effect of a fucoidan extract (FE) from Sargassum fluitans Borgesen was evaluated in a carbon tetrachloride-induced liver damage model in rats over twelve weeks. Chemical analysis showed the FE to contain carbohydrates, sulfates, uronic acids, protein, phenols, and to have a molecular weight of ~60 kDa. Physiological, biochemical, histological and genetic assays were done. Daily oral administration of FE (50 mg/kg) reduced liver enzymatic activity, liver infiltration of inflammatory cells, collagen fiber deposition and gene expression cytokines such as interleukin beta 1 (IL-ß1), tumor necrosis factor alpha (TNF-α), transforming growth factor beta 1 (TGF-ß1), Smad-3, Smad-2, collagen 1 alpha 1 (col1α1) and tissue inhibitor of metalloproteinase 1 (TIMP-1). It also increased RNA expression of Smad-7 and metalloproteinase 2 and 9 (MMP2 and MMP9). The fucoidan extract exhibited an antifibrotic effect mediated by the inhibiting TGF-ß1/Smad pathway, as well as anti-inflammatory effects.

Long-Term Intake of Glucoraphanin-Enriched Kale Suppresses Skin Aging via Activating Nrf2 and the TßRII/Smad Pathway in SAMP1 Mice.

Sulforaphane, a potent antioxidant compound, is unstable at ambient temperature, whereas its precursor glucoraphanin is stable and metabolized to sulforaphane. Thus, we hypothesized that glucoraphanin-rich diet could effectively induce antioxidant enzyme activities and investigated the protective effects of long-term intake of a glucoraphanin-enriched kale (GEK) diet on skin aging in senescence-accelerated mouse prone 1 (SAMP1) mice. The senescence grading score was significantly lower after treatment with GEK for 39 weeks than that of the control mice. GEK also suppressed the thinning of the dorsal skin layer. Moreover, the GEK treatment enhanced the collagen production and increased the nuclear translocation of Nrf2 and HO-1 expression level in the skin tissue. TßRII and Smad3 expressions were clearly higher in the GEK-treated group than in the control group. Thus, GEK suppressed senescence in SAMP1 mice by enhancing the antioxidant activity and collagen production via the TßRII/Smad3 pathway, suggesting its practical applications for protection against skin aging.

A high-fat diet rich in corn oil induces cardiac fibrosis in rats by activating JAK2/STAT3 and subsequent activation of ANG II/TGF-1ß/Smad3 pathway: The role of ROS and IL-6 trans-signaling.

This study compared the effect of low-fat diet (LFD) and high-fat diet rich in corn oil (HFD-CO) on left ventricular (LV) fibrosis in rats and examined their effect of angiotensin II (ANG II), JAK/STAT, and TGF-1ß/smad3 pathways. As compared to LFD which didn't affect any of the measured parameters, HFD-CO-induced type 2 diabetes phenotype and increased LV collagen synthesis. Mechanistically, it increased LV levels of ROS, ANG II, ACE, IL-6, s-IL-6Rα, TGF-ß1, Smad-3, and activities of JAK1/2 and STAT1/3. AG490, a JAK2 inhibitor, partially ameliorated these effect while Losartan, an AT1 inhibitor completely abolished collagen synthesis. However, with both treatments, levels of ANG II, IL-6, and s-IL-6Rα, and activity of JAK1/STAT3 remained high, all of which were normalized by co-administration of NAC or IL-6 neutralizing antibody. In conclusion: HFD-CO enhances LV collage synthesis by activation of JAK1/STAT3/ANG II/TGF-1ß/smad3 pathway. PRACTICAL APPLICATIONS: We report that chronic consumption of a high-fat diet rich in corn oil (HFD-CO) induces diabetes mellitus phenotype 2 associated with left ventricular (LV) cardiac fibrosis in rats. The findings of this study show that HFD-CO, and through the increasing generation of ROS and IL-6 levels and shedding, could activate LV JAK1/2-STAT1/3  and  renin-angiotensin system (RAS) signaling pathways, thus creating a positive feedback between the two which ultimately leads to activation of TGF-1ß/Smad3 fibrotic pathway. Herein, we also report a beneficial effect of the antioxidant, NAC, or IL-6 neutralizing antibody in preventing such adverse effects of such HFD-CO. However, this presents a warning message to the current sudden increase in idiopathic cardiac disorders, especially with the big shift in our diets toward n-6 PUFA.

Activity and molecular targets of pioglitazone via blockade of proliferation, invasiveness and bioenergetics in human NSCLC.

BACKGROUND: Pioglitazone, a synthetic peroxisome proliferator activated receptor (PPAR-γ) ligand, is known as an antidiabetic drug included in the thiazolidinediones (TZDs) class. It regulates the lipid and glucose cell metabolism and recently a role in the inhibition of numerous cancer cell processes has been described. METHODS: In our work we investigate the anti-tumor effects of pioglitazone in in vitro models of non small cell lung cancer (NSCLC) and also, we generated ex-vivo three-dimensional (3D) cultures from human lung adenocarcinoma (ADK) as a model to test drug efficacy observed in vitro. The inhibitory effect of pioglitazone on cell proliferation, apoptosis and cell invasion in a panel of human NSCLC cell lines was evaluated by multiple assays. RESULTS: Pioglitazone reduced proliferative and invasive abilities with an IC50 ranging between 5 and 10 µM and induced apoptosis of NSCLC cells. mRNA microarray expression profiling showed a down regulation of MAPK, Myc and Ras genes after treatment with pioglitazone; altered gene expression was confirmed by protein analysis in a dose-related reduction of survivin and phosphorylated proteins levels of MAPK pathway. Interestingly mRNA microarray analysis showed also that pioglitazone affects TGFß pathway, which is important in the epithelial-to-mesenchimal transition (EMT) process, by down-regulating TGFßR1 and SMAD3 mRNA expression. In addition, extracellular acidification rate (ECAR) and a proportional reduction of markers of altered glucose metabolism in treated cells demonstrated also cell bioenergetics modulation by pioglitazone. CONCLUSIONS: Data indicate that PPAR-γ agonists represent an attractive treatment tool and by suppression of cell growth (in vitro and ex vivo models) and of invasion via blockade of MAPK cascade and TGFß/SMADs signaling, respectively, and its role in cancer bioenergetics and metabolism indicate that PPAR-γ agonists represent an attractive treatment tool for NSCLC.

Antifibrotic Effects of 1,25(OH)2D3 on Nonalcoholic Steatohepatitis in Female Mice.

BACKGROUND: Postmenopausal women have a higher risk of nonalcoholic steatohepatitis (NASH) along with an increase in age, and vitamin D deficiency occurs in some patients with NASH. AIM: We performed ovariectomy (OVX) surgery on female mice to mimic menopause, fed them a choline-deficient high-fat (CDHF) diet to induce NASH, and then investigated the effects of treatment with 1,25(OH)2D3. METHODS: Seven-week-old C57BL/6J female mice were separated into five experimental groups: SHAM, OVX, and OVX + intraperitoneal (i.p.) injection of 1,25(OH)2D3 (0.0008, 0.004, and 0.02 µg/kg). All groups were fed a CDHF diet for 8 weeks. Injections took place twice per week throughout the experimental period. Blood samples and liver tissue were collected for analyzing liver histological changes, serum biochemical indicators of hepatic function, and hepatic genes associated with fibrosis. RESULTS: Supplementation of 1,25(OH)2D3 in CDHF-diet mice showed decreased serum levels of ALT, AST, indicating the improvement in overall liver function, and suppressed histological NASH regarding fibrosis stage, lobular inflammation, and steatosis compared to the OVX group. Primary fibrotic markers of TGF-ß, TIMP-1, α-SMA, and COL1A1 were significantly lower in the 1,25(OH)2D3 groups than in the OVX group. Furthermore, down-regulated levels of SMAD2 and SMAD3 were also observed in 1,25(OH)2D3 groups. CONCLUSION: Supplementation of 1,25(OH)2D3 may ameliorate liver fibrosis and improve liver function in OVX mice with NASH induced by a CDHF diet, suggesting the therapeutic effects on postmenopause with NASH.

Compound Astragalus and Salvia miltiorrhiza extract inhibits hepatocellular carcinoma progression via miR-145/miR-21 mediated Smad3 phosphorylation.

ETHNOPHARMACOLOGICAL RELEVANCE: Compound Astragalus and Salvia miltiorrhiza extract (CASE), containing astragalosides, astragalus polysaccharide extracted from Astragalus membranaceus (Fisch.) Bge. and salvianolic acids from Salvia miltiorhiza Bge., has been found to inhibit hepatocarcinogenesis via mediating transforming growth factor-ß (TGF-ß)/Smad signaling, especially Smad3 phosphorylation. The crucial interaction between microRNA-145/microRNA-21 (miR-145/miR-21) and Smad3 phosphorylation is implicated in the pathogenesis and progression of hepatocellular carcinoma (HCC). However, effects of CASE on HCC progression involved in the expression of miR-145/miR-21 and their interaction with Smad3 phosphorylation downstream of TGF-ß/MAPK/Smad pathway remain unclear. This study addressed above questions using in vitro (HepG2 cells) and in vivo (Xenografts of nude mice) models of HCC. MATERIALS AND METHODS: In vivo [Diethylnitrosamine (DEN)-induced HCC in rats] and in vitro [TGF-ß1-stimulated HepG2 cells] models of HCC were established and co-administrated using graded doses/concentrations CASE (60, 120, 240 mg/kg used in rats; 20, 40, 80 µg/ml used in HepG2 cells), miR-145 and miR-21 were measured. HepG2 cells were transfected with miR-145 antagomir, miR-21 agomir and Smad3C/L plasmids (Smad3 EPSM, Smad3 3S-A and Smad3 WT related to up-regulated expression of pSmad3C, pSmad3L and pSmad3C/3L respectively) and then treated by CASE (80 µg/ml). Similarly, HepG2 cell xenografted nude mice were administered with miR-145 antagomir, miR-21 agomir and CASE (310 mg/kg); Smad3 WT, Smad3 EPSM and Smad3 3S-A plasmids stably transfected HepG2 cell lines were constructed respectively and their xenografted nude mice were established, and then treated by CASE (310 mg/kg). Cell proliferation, migration, apoptosis, tumor growth and histopathologic characteristics of xenografts were assessed; also, domain-specific Smad3 phosphorylation isoforms (pSmad3C/pSmad3L), activated MAPKs (pERK1/2, pJNK1/2, pp38) and miR-145, miR-21 were measured. RESULTS: CASE up-regulated miR-145 while down-regulated miR-21 expression in both rats with DEN-induced HCC and TGF-ß1-stimulated HepG2 cells; CASE inhibited cell migration, proliferation and tumor growth while facilitated cell apoptosis in TGF-ß1-stimulated HepG2 cells and xenografts of nude mice with miR-145 antagomir/miR-21 agomir treatment via increasing miR-145 and facilitating miR-145 modulated pSmad3L→pSmad3C signaling switch while decreasing miR-21 and inhibiting miR-21 modulated MAPK-dependent Smad3L phosphorylation. Also, up-regulated pSmad3C enhanced inhibited effect of CASE on tumor growth and facilitated effect of CASE on cell apoptosis involved in increased miR-145 while decreased miR-21 expression, however, inverse phenomena were observed when up-regulated pSmad3L. CONCLUSION: Our results suggest that CASE inhibits HCC progression via mediating the interaction of miR-145/miR-21 and Smad3 phosphorylation, especially miR-145/miR-21 mediated Smad3 phosphorylation, which maybe provides an important theoretical foundation for CASE's anti-HCC therapy used for patients in a near future.

[Influence of nourishing yin and tonifying yang sequential therapy combined with Western medicine on TGF-ß1/Smads signaling pathway in anovulatory infertility rats with diminished ovarian reserve].

OBJECTIVE: To explore the influence for combination of nourishing yin and tonifying yang sequential therapy (NYTYST) with Western medicine in treating anovulatory infertility rats with diminished ovarian reserve (DOR) based on TGF-ß1/Smads signaling pathway. 
 Methods: A total of 40 female rats were randomly divided into 5 groups, a normal control group, a model group, a Western medicine group, a NYTYST group and a combination group (n=8 in each group). The DOR model was established through orally taking tripterygium pill for continuous 2 weeks. The normal control group and the model group were treated with saline for 10 days. The Western medicine group was treated with hormone replacement therapy (HRT) and ovarian stimulation. The NYTYST group was treated with nourishing yin herbs in proestrus and tonifying yang herbs in late estrus and the combination group was treated with Chinese herb and Western drugs for 10 days. HE staining was used to observe histopathologic changes in ovary. Expression levels of transforming growth factor ß1 receptor (TGF-ß1R) in rats ovarian were detected by immunohistochemistry. Expression levels of Smad2, Smad3 and Smad7 protein in rat ovarian were detected by Western blot.
 Results: Compared with the control group, the numbers of developing follicles, mature follicles and corpus luteum were decreased , while atrefic follicles were increased significantly in the model group (P<0.01); the levels of TGF-ß1R, Smad2 and Smad3 were decreased significantly, while Smad7 was increased significantly (P<0.01). Compared with the model group, the numbers of developing follicles, mature follicles and corpus luteum, Smad2 and Smad3 expression were increased, while atrefic follicles and Smad7 were decreased significantly in the treatment group (P<0.05 or P<0.01). The numbers of developing follicles and corpus luteum in the combination group was superior to the Western medicine group (P<0.05). Compared with the Western medicine group, the levels of TGF-ß1R, Smad2 and Smad3 were increased significantly, while Smad7 was decreased significantly in the combination group (P<0.05 or P<0.01). 
 Conclusion: NYTYST combined with Western medicine can improve the function of ovaries reserve by up-regulation of TGF-ß1R, Smad2 and Smad3 while down-regulation of Smad7 in DOR rats.

Anthocyanins from black soybean seed coat prevent radiation-induced skin fibrosis by downregulating TGF-ß and Smad3 expression.

The aim of this study was to evaluate the protective effects of anthocyanins from the black soybean seed coat against radiation injury in dermal fibroblasts and mouse skin. Dermal fibroblasts treated with 50 and 100 µg/mL anthocyanins were irradiated with single doses of 20 Gy. Cell viability, intracellular reactive oxygen species (ROS) production, and mRNA expression were measured. A total of 60 mice were used for an in vivo study. A dose of 100 µg/mL anthocyanins was administered daily for 5 days before or after radiation therapy. Following irradiation (45 Gy), mice were inspected for gross pathology twice per wk for 8 weeks. At 4 and 8 weeks post-irradiation, dorsal skin was harvested for histopathologic examination and protein isolation. In dermal fibroblasts, treatment with 50 and 100 µg/mL anthocyanins significantly reduced radiation-induced apoptosis at 72 h and intracellular reactive oxygen species generation at 48 h. Furthermore, 100 µg/mL anthocyanins markedly decreased Smad3 mRNA expression and increased Smad7 mRNA expression at 72 h post-irradiation. In mice, treatment with 100 µg/mL anthocyanins resulted in a significant reduction in the level of skin injury, epidermal thickness, and collagen deposition after irradiation. Treatment with 100 µg/mL anthocyanins significantly decreased the number of α-SMA-, TGF-ß-, and Smad3-positive cells after irradiation. Our study demonstrated that black soybean anthocyanins inhibited radiation-induced fibrosis by downregulating TGF-ß and Smad3 expression. Therefore, anthocyanins may be a safe and effective candidate for the prevention of radiation-induced skin fibrosis.