A Multi-Omics Approach Identifies Key Regulatory Pathways Induced by Long-Term Zinc Supplementation in Human Primary Retinal Pigment Epithelium.

In age-related macular degeneration (AMD), both systemic and local zinc levels decline. Elevation of zinc in clinical studies delayed the progression to end-stage AMD. However, the molecular pathways underpinning this beneficial effect are not yet identified. In this study, we used differentiated primary human fetal retinal pigment epithelium (RPE) cultures and long-term zinc supplementation to carry out a combined transcriptome, proteome and secretome analysis from three genetically different human donors. After combining significant differences, we identified the complex molecular networks using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). The cell cultures from the three donors showed extensive pigmentation, development of microvilli and basal infoldings and responded to zinc supplementation with an increase in transepithelial electrical resistance (TEER) (apical supplementation: 443.2 ± 79.3%, basal supplementation: 424.9 ± 116.8%, compared to control: 317.5 ± 98.2%). Significant changes were observed in the expression of 1044 genes, 151 cellular proteins and 124 secreted proteins. Gene set enrichment analysis revealed changes in specific molecular pathways related to cell adhesion/polarity, extracellular matrix organization, protein processing/transport, and oxidative stress response by zinc and identified a key upstream regulator effect similar to that of TGFB1.

Si-Miao-Yong-An decoction attenuates cardiac fibrosis via suppressing TGF-ß1 pathway and interfering with MMP-TIMPs expression.

BACKGROUND: Myocardial fibrosis is an important pathological feature of pressure overload cardiac remodeling. Si-Miao-Yong-An decoction (SMYAD), a traditional Chinese formula, is now clinically used in the treatment of cardiovascular diseases in China. However, its mechanisms in the prevention of heart failure are not fully revealed. PURPOSE: To determine whether treatment with SMYAD for 4 weeks would lead to changes in collagen metabolism and ventricular remodeling in a mice model of heart failure. METHODS: Mice were subjected to transverse aorta constriction to generate pressure overload induced cardiac remodeling and then were administered SMYAD (14.85 g/kg/day) or captopril (16.5 mg/kg/day) intragastrically for 4 weeks after surgery. Echocardiography and immunohistochemical examination were used to evaluate the effects of SMYAD. The mRNA of collagen metabolism biomarkers were detected. Protein expression of TGF-ß1/Smad and TGF-ß1/TAK1/p38 pathway were assessed by Western blot. RESULTS: SMYAD significantly improved cardiac function, increased left ventricle ejection fraction, and decreased fibrosis area and αSMA expression. Moreover, SMYAD reduced proteins expression related to collagen metabolism, including Col1, Col3, TIMP2 and CTGF. The increased levels of TGF-ß1, Smad2, and Smad3 phosphorylation were attenuated in SMYAD group. In addition, SMYAD reduced the levels of TGF-ß1, p-TAK1 and p-p38 compared with TAC group. CONCLUSIONS: SMYAD improved cardiac fibrosis and heart failure by inhibition of TGF-ß1/Smad and TGF-ß1/TAK1/p38 pathway. SMYAD protected against cardiac fibrosis and maintained collagen metabolism balance by regulating MMP-TIMP expression. Taken together, these results indicate that SMYAD might be a promising therapeutic agent against cardiac fibrosis.

Effects of Acupoint Application Therapy with TianGui Powder on Osteoporosis in Ovariectomized Rats through TGF-ß1 and Smad2/3 Signaling Pathway.

OBJECTIVES: To explore the effects of acupoint application therapy (AAT) with TianGui Powder (TGP) on the expressions of the transforming growth factor ß1 (TGF-ß1) and Smad-2/3 signaling pathway in ovariectomized osteoporosis rats. METHODS: Sixty rats were randomly divided into four groups: normal group (group A), model group (group B), TGP group (group C), and Western medicine group (group D). Group A had only the corresponding amount of adipose tissue around the ovary removed; rats in the other groups had bilateral ovariectomies. After 1 week, groups A and B were given 1 mL/100 mg normal saline solution by gavage, group C was treated with AAT with TGP on ShenQue acupoint (0.2 piece/rat, 6 h/time, 1 time/d) and group D was given calcium carbonate vitamin D3 (36 mg/kg/d) and alfacalcidol (0.05 µg/kg/d) tablet suspension. In this study, the bone mineral density (BMD) , the levels of BALP, TRAP-5b, and BGP in serum and the changes in bone histomorphology was detected. For acquiring lumbar experimental data, the expression of TGF-ß1, Smad-2/3 proteins and mRNA of TGF-ß1and Smad-2/3 were assessed. After 12 weeks, the data were collected for analysis. RESULTS: Compared with group A, the bone trabecula was thinner and significantly reduced in other groups. The result of BMD improved significantly in both groups C and D compared to group B after intervention (P < 0.05). In contrast, compared to group B, the levels of BALP, TRAP-5b, and BGP significantly declined in both groups C and D. In group C, the results of protein expressions in TGF-ß1, Smad-2/3 were 2.870 ± 0.270, 1.552 ± 0.111, and 1.420 ± 0.079, respectively. In groups C and D, those indications significantly declined compared to group B (P < 0.01). In group C, the level of mRNA expressions of TGF-ß1, Smad-2/3 were 1.872 ± 0.177, 1.672 ± 0.086, and 1.790 ± 0.136, respectively. Compared with group B, those indications had significant difference in groups C and D (P < 0.05). CONCLUSION: Acupoint application therapy with TGP could significantly improve the BMD. The TGF-ß1 and Smad-2/3 signaling pathway could be a therapeutic target of TGP in postmenopausal osteoporosis rats.

Regulation of Atrial Fibrosis by the Bone.

MR (mineralocorticoid receptor) antagonists have been demonstrated to provide beneficial effects on preventing atrial fibrosis. However, the underlying cellular and molecular mechanisms remain unclear. We aim to determine the role of osteoblast MR in atrial fibrosis and to explore the underlying mechanism. Using osteoblast MR knockout mouse in combination with mutant TGF (transforming growth factor)-ß1 transgenic mouse, we demonstrated that MR deficiency in osteoblasts significantly attenuated atrial fibrosis. Mechanistically, MR directly regulated expression of OCN (osteocalcin) in osteoblasts. Both carboxylated and undercarboxylated OCNs (ucOC) were less secreted in osteoblast MR knockout mice. Mutant TGF-ß1 transgenic mice supplemented with recombinant ucOC showed aggravated atrial fibrosis. In cultured atrial fibroblasts, ucOC treatment promoted proliferation and migration of atrial fibroblasts, whereas cotreatment with an antagonist for a GPRC6A (G-protein-coupled receptor, family C, group 6, member A) abolished these effects. Western blotting analysis revealed upregulation of PKA (protein kinase A) and CREB (cAMP-response element-binding protein) phosphorylation after ucOC treatment. Inhibition of PKA with its antagonist reduced ucOC-induced proliferation and migration of atrial fibroblasts. Finally, the impact of osteoblast MR deficiency on atrial fibrosis was abolished by ucOC administration in mutant TGF-ß1 transgenic mice. Taken together, MR deficiency in osteoblasts attenuated atrial fibrosis by downregulation of OCN to promote proliferation and migration of atrial fibroblasts.

Article Effect and Mechanism of Ganoderma lucidum Polysaccharides on Human Fibroblasts and Skin Wound Healing in Mice.

OBJECTIVE: To investigate the effects of Ganoderma lucidum polysaccharides (GL-PS) on human fibroblasts and skin wound healing in Kunming male mice and to explore the putative molecular mechanism. METHODS: Primary human skin fibroblasts were cultured. The viability of fibroblasts treated with 0, 10, 20, 40, 80, and 160 µg/mL of GL-PS, respectively were detected by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-Htetrazolium bromide (MTT). The migration ability of fibroblasts treated with 0, 10, 20, and 40 µg/mL of GL-PS were measured by transwell assay. The secretion of the C-terminal peptide of procollagen type I (CICP) and transforming growth factor-ß1 (TGF-ß1) in the cell supernatant was tested by enzyme-linked immunosorbent assay. The expression of ß-catenin was detected by Western blot. Furthermore, the Kunming mouse model with full-layer skin resection trauma was established, and was treated with 10, 20, and 40 mg/mL of GL-PS, respectively as external use. The size of the wound was measured daily, complete healing time in each group was recorded and the percentage of wound contraction was calculated. RESULTS: Compared with the control group, 10, 20, and 40 µg/mL of GL-PS significantly increased the viability of fibroblasts, promoted the migration ability of fibroblasts, and up-regulated the expressions of CICP and TGF-ß1 in fibroblasts (Plt;0.05 or Plt;0.01). The expression of ß-catenin in fibroblasts treated with 20 and 40 µg/mL of GL-PS was significantly higher than that of the control group (Plt;0.01). Furthermore, after external use of 10, 20, and 40 mg/mL of GL-PS, the rates of wound healing in mice were significantly higher and the wound healing time was significantly less than the control group (Plt;0.05 or Plt;0.01). CONCLUSION: A certain concentration of GL-PS may promote wound healing via activation of the Wnt/ß-catenin signaling pathway and up-regulation of TGF-ß1, which might serve as a promising source of skin wound healing.

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

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

Glaucocalyxin A Attenuates the Activation of Hepatic Stellate Cells Through the TGF-ß1/Smad Signaling Pathway.

Glaucocalyxin A (GLA) is a biologically active ent-kauranoid diterpenoid isolated from Rabdosia japonica var. glaucocalyx. A large number of studies have shown that GLA possesses important pharmacological activities, such as anti-inflammatory, antitumor, antifibrosis, and antiplatelet activities. However, the role of GLA in the pathogenesis of liver fibrosis remains undefined. Therefore, the aim of this study was to investigate the effects of GLA on hepatic stellate cells (HSCs) activation/proliferation and migration in vitro and its possible mechanism in liver fibrosis. HSCs were incubated with different concentrations of GLA in the presence or absence of TGF-ß1 for 24 h. Cell proliferation and migration were evaluated by the MTT assay and Transwell migration assay, respectively. Intracellular reactive oxygen species (ROS) production was measured using 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA). Western blot was used to detect the expression levels of α-smooth-muscle actin (α-SMA), collagen-I, p-Smad2, Smad2, p-Smad3, and Smad3. Our results demonstrated that GLA significantly inhibited the proliferation and migration of HSCs, and suppressed the expression of extracellular matrix in TGF-ß1-stimulated HSC-T6 cells. In addition, pretreatment with GLA markedly suppressed TGF-ß1-induced ROS level in HSC-T6 cells. Furthermore, GLA greatly inhibited the phosphorylation levels of Smad2 and Smad3 in TGF-ß1-stimulated HSC-T6 cells. Taken together, these findings indicated that GLA inhibits the proliferation and activation of HSC-T6 cells, at least in part, through the TGF-ß1/Smad signaling pathway. Therefore, GLA may be a promising potential therapeutic agent for liver fibrosis.

Effects of calcium Ionophore A23187 on the apoptosis of hepatic stellate cells stimulated by transforming growth factor-ß1.

BACKGROUND: Our previous study showed that during in vitro experiments changes in calcium concentration were associated with apoptosis. We presumed that the calcium ion might play a role as intermediate messenger for apoptosis-related genes. No such evidence has been reported in the literature. Here, we investigate the effect of calcium ionophore A23187 on the apoptosis of rat hepatic stellate cells (HSCs) stimulated by transforming growth factor-ß1 (TGF-ß1) to explore the mechanism of apoptosis through the endoplasmic reticulum stress pathway. METHODS: The apoptotic rate was determined using flow cytometry. The changes in Ca2+ level in HSCs were examined with laser confocal microscopy. The expressions of caspase-12 GRP78 and caspase-9 were assayed via western blot. RESULTS: The respective apoptosis rates for the blank group, the TGF-ß1 group and the TGF-ß1 + low, medium and high dose calcium ionophore A23187 groups were 3.40 ± 0.10%, 1.76 ± 0.12%, 5.86 ± 0.31%, 11.20 ± 0.48% and 15.08 ± 0.75%, with significant differences between the groups (p < 0.05). The concentration of Ca2+and the expression of the GRP78, caspase-9 and caspase-12 proteins significantly increased with increasing calcium ionophore A23187 doses (p < 0.05). CONCLUSION: Calcium ionophore A23187 increased intracellular Ca2+ and activated endoplasmic reticulum stress, which promoted HSC apoptosis.

Hypaconitine inhibits TGF-ß1-induced epithelial-mesenchymal transition and suppresses adhesion, migration, and invasion of lung cancer A549 cells.

Epithelial-mesenchymal transition (EMT) has been implicated in tumor invasion and metastasis and provides novel strategies for cancer therapy. Hypaconitine (HpA), a diester-diterpenoid alkaloid isolated from the root of the Aconitum species, exhibits anti-inflammatory, analgesic, and especially, cardiotoxic activities. Here, we reported the anti-metastatic potentials of HpA in transforming growth factor-ß1 (TGF-ß1)-induced EMT in lung cancer A549 cells. The cytotoxic effect of HpA was determined by MTT assay. A549 cells were treated with TGF-ß1 with or without HpA co-treatment, and the morphological alterations were observed with a microscopy. The expression of E-cadherin, N-cadherin, and NF-κB was determined by both Western blotting and immunofluorescence analyses. The adhesion, migration, and invasion were detected with Matrigel, wound-healing, and transwell assays, respectively. The expression of Snail was determined by Western blotting. The expression of NF-κB p65, IκBα, and p-IκBα in nuclear and cytosolic extracts was assessed by Western blotting. The results showed that low concentration of HpA (<16 µmol·L-1) had no obvious cytotoxicity to A549 cells. Morphologically, TGF-ß1 treatment induced spindle-shaped alteration in the cells. The upregulation of N-cadherin, NF-κB, and Snail and the downregulation of E-cadherin were detected after TGF-ß1 treatment. The adhesion, migration and invasion abilities were also increased by TGF-ß1. Besides, TGF-ß1 induced expression of Snail in a time-dependent manner. Furthermore, TGF-ß1 induced nuclear translocation of NF-κB p65. All these alterations were dramatically inhibited by HpA co-treatment. In addition, the NF-κB inhibitor PDTC showed similar inhibitory effect. In conclusion, these results showed that HpA inhibited TGF-ß1-induced EMT in A549 cells, which was possibly mediated by the inactivation of the NF-κB signaling pathway, providing an evidence for anti-cancer effect of HpA.

Combined treatment with zingerone and its novel derivative synergistically inhibits TGF-ß1 induced epithelial-mesenchymal transition, migration and invasion of human hepatocellular carcinoma cells.

The epithelial-mesenchymal transition (EMT) is an important cellular process during which polarized epithelial cells become motile mesenchymal cells, which promote cancer metastasis. Ginger, the rhizome of Zingiber officinale, is extensively used in cooking worldwide and also as a traditional medicinal herb with antioxidant, anti-inflammatory and anticancer properties. Several pungent compounds have been identified in ginger, including zingerone, which has anticancer potential. However, the role of zingerone in EMT is unclear. We investigated the synergistic effect of zingerone and its derivative on EMT. Transforming growth factor-beta 1 (TGF-ß1) induces the EMT to promote hepatocellular carcinoma metastasis, including migration and invasion. To understand the repressive role of the combination of zingerone and its derivative (ZD 2) in hepatocellular carcinoma metastasis, we investigated the potential use of each compound of ginger, such as zingerone, ZD 2 and 6-shogaol, or the mixture of zingerone and ZD 2 (ZD 2-1) as inhibitors of TGF-ß1 induced EMT development in SNU182 hepatocellular carcinoma cells in vitro. We show that ZD 2-1, but not zingerone, ZD 2 and 6-shogaol significantly increased expression of the epithelial marker E-cadherin and repressed Snail upregulation and expression of the mesenchymal marker N-cadherin during initiation of the TGF-ß1 induced EMT. In addition, ZD 2-1 inhibited the TGF-ß1 induced increase in cell migration and invasion of SNU182 hepatocellular carcinoma cells. Furthermore, ZD 2-1 significantly inhibited TGF-ß1 regulated matrix metalloproteinase-2/9 and activation of Smad2/3. We also found that ZD 2-1 inhibited nuclear translocation of NF-κB, activation of p42/44 MAPK/AP1 signaling pathway in the TGF-ß1 induced EMT. Our findings provide new evidence that combined treatment with ZD 2, novel zingerone derivative, and zingerone synergistically suppresses hepatocellular carcinoma metastasis in vitro by inhibiting the TGF-ß1 induced EMT.

QiShenYiQi Attenuates Renal Interstitial Fibrosis by Blocking the Activation of ß-Catenin.

Chronic kidney disease (CKD) is becoming a worldwide problem. However, current treatment options are limited. In the current study we showed that QiShenYiQi (QSYQ), a water-ethanol extract from several Chinese medicines, is a potent inhibitor of renal interstitial fibrosis. QSYQ inhibited transforming growth factor-ß1 (TGF-ß1)-responsive α-smooth muscle actin (α-SMA), collagen I, and fibronectin up-regulation in obstructive nephropathy and cultured cells. Administration of QSYQ also inhibited the established renal interstitial fibrosis in obstructive nephropathy. Interestingly, QSYQ selectively inhibited TGF-ß1-induced ß-catenin up-regulation and downstream gene transcription. Taken together, our study suggests that QSYQ selectively inhibits TGF-ß1-induced ß-catenin up-regulation and might have significant therapeutic potential for the treatment of renal fibrosis.

Pentoxifylline and its active metabolite lisofylline attenuate transforming growth factor ß1-induced asthmatic bronchial fibroblast-to-myofibroblast transition.

Bronchial asthma is characterized by persistent airway inflammation and airway wall remodeling. Among many different cells and growth factors triggering changes in bronchi structure, transforming growth factor ß1-induced fibroblast to myofibroblast transition is believed to be very important. The aim of this study was to evaluate whether theophylline (used in asthma therapy) and two other methylxanthines (pentoxifylline and its active metabolite lisofylline), may affect transforming growth factor ß1-induced fibroblast to myofibroblast transition in bronchial fibroblasts derived from asthmatic patients. We show here for the first time that selected methylxanthines effectively reduce transforming growth factor ß1-induced myofibroblast formation in asthmatic bronchial fibroblast populations. PTX was found to be the most effective methylxanthine. The number of differentiated myofibroblasts after PTX, LSF and THEO administration was reduced at least twofold. Studies on the use of methylxanthines opens a new perspective in the development of novel strategies in asthma therapy through their two-pronged, anti-inflammatory and anti-fibrotic action. In the future they can be considered as promising anti-fibrotic drugs.

A network pharmacology approach to discover active compounds and action mechanisms of San-Cao Granule for treatment of liver fibrosis.

ETHNOPHARMACOLOGICAL RELEVANCE: San-Cao Granule (SCG) has been used in patients with liver fibrosis for many years and has shown good effect. However, its mechanism of therapeutic action is not clear because of its complex chemical system. The purpose of our study is to establish a comprehensive and systemic method that can predict the mechanism of action of SCG in antihepatic fibrosis. MATERIALS AND METHODS: In this study, a "compound-target-disease" network was constructed by combining the SCG-specific and liver fibrosis-specific target proteins with protein-protein interactions, and network pharmacology was used to screen out the underlying targets and mechanisms of SCG for treatment of liver fibrosis. Then, some key molecules of the enriched pathway were chosen to verify the effects of SCG on liver fibrosis induced by thioacetamide (TAA). RESULTS: This systematic approach had successfully revealed that 16 targets related to 11 SCG compounds were closely associated with liver fibrosis therapy. The pathway-enrichment analysis of them showed that the TGF-ß1/Smad signaling pathway is relatively important. Animal experiments also proved that SCG could significantly ameliorate liver fibrosis by inhibiting the TGF-ß1/Smad pathway. CONCLUSION: SCG could alleviate liver fibrosis through the molecular mechanisms predicted by network pharmacology. Furthermore, network pharmacology could provide deep insight into the pharmacological mechanisms of Chinese herbal formulas.

Molecular Mechanisms Responsible for Neuron-Derived Conditioned Medium (NCM)-Mediated Protection of Ischemic Brain.

The protective value of neuron-derived conditioned medium (NCM) in cerebral ischemia and the underlying mechanism(s) responsible for NCM-mediated brain protection against cerebral ischemia were investigated in the study. NCM was first collected from the neuronal culture growing under the in vitro ischemic condition (glucose-, oxygen- and serum-deprivation or GOSD) for 2, 4 or 6 h. Through the focal cerebral ischemia (bilateral CCAO/unilateral MCAO) animal model, we discovered that ischemia/reperfusion (I/R)-induced brain infarction was significantly reduced by NCM, given directly into the cistern magna at the end of 90 min of CCAO/MCAO. Immunoblocking and chemical blocking strategies were applied in the in vitro ischemic studies to show that NCM supplement could protect microglia, astrocytes and neurons from GOSD-induced cell death, in a growth factor (TGFß1, NT-3 and GDNF) and p-ERK dependent manner. Brain injection with TGFß1, NT3, GDNF and ERK agonist (DADS) alone or in combination, therefore also significantly decreased the infarct volume of ischemic brain. Moreover, NCM could inhibit ROS but stimulate IL-1ß release from GOSD-treated microglia and limit the infiltration of IL-ß-positive microglia into the core area of ischemic brain, revealing the anti-oxidant and anti-inflammatory activities of NCM. In overall, NCM-mediated brain protection against cerebral ischemia has been demonstrated for the first time in S.D. rats, due to its anti-apoptotic, anti-oxidant and potentially anti-glutamate activities (NCM-induced IL-1ß can inhibit the glutamate-mediated neurotoxicity) and restriction upon the infiltration of inflammatory microglia into the core area of ischemic brain. The therapeutic potentials of NCM, TGFß1, GDNF, NT-3 and DADS in the control of cerebral ischemia in human therefore have been suggested and require further investigation.

Effect of hesperidin on TGF-beta1/Smad signaling pathway in HSC.

Liver fibrosis is a common pathological process for chronic liver injury caused by multiple etiological factors and an inevitable phase leading to liver cirrhosis. According to the previous studies, hesperidin (HDN) shows a very good protective effect on CCl4-induced chemical hepatic fibrosis in rats. In this experiment, based on the findings of the previous studies, a platelet-derived growth factor (PDGF)-induced HSC-T6 model was established to observe the inhibitory effect of HDN on HSC-T6 proliferation. The ELISA method was adopted to detect the content of collagen I in HSC-T6 supernatant. Transforming growth factor (TGF)-beta1, Smad2, Smad3, Smad7 and connective tissue growth factor (CTGF) mRNA expressions were measured by RT-PCR; TGF-beta1 and CT-GF protein expressions in HSC-T6 were determined by Western blot, in order to study HDN's effect on TGF-beta1 signaling pathway in HSC and its potential action mechanism. The results demonstrated that HDN could notably improve HSC-T6 proliferation, Collagen I growth and TGF-beta1, Smad2, Smad3 and CTGF mRNA.expressions. After being intervened with HDN, it could notably inhibit HSC-T6 proliferation and Collagen I growth, reduce TGF-beta1, Smad2, Smad3 and CTGF mRNA and TGF-beta1, CTGF protein expressions and increase Smad7 mRNA expression. HDN's antihepatic fibrosis effect may be related to the inhibition of HSC proliferation and activation by modulating TGF-beta/Smad signaling pathway.

Triptolide alleviates isoprenaline-induced cardiac remodeling in rats via TGF-ß1/Smad3 and p38 MAPK signaling pathway.

Triptolide (TPL) is a diterpene triepoxide with potent immunosuppressive and anti-inflammatory properties. It is the main effective component of the traditional Chinese herb Tripterygium wilfordii Hook F and has been used in China for centuries to treat immune-related disorders. The present study was conducted to investigate the effects of TPL on cardiac remodeling in rats. Age matched male Wistar rats were used in this study. Cardiac remodeling rat model was established by hypodermic injection of isoprenaline for ten days. The rats were treated with TPL (20 or 100 µg/kg/d) for six consecutive weeks. At the end of the study, the cardiac function, collagen volume fraction, perivascular collagen area and hydroxyproline concentration were studied. Echocardiography, Masson staining, immunohistochemistry, western blot and real-time polymerase chain reaction were performed. The protein and mRNA expression of transforming growth factor-ß1 (TGF-ß1), drosophila mothers against decapentaplegic protein 3 (Smad3) and p38 mitogen activated protein kinase (p38 MAPK) were analyzed. The results indicated that TPL treatment significantly reduced the collagen volume fraction, perivascular collagen area, ventricular weight/body weight ratio and hydroxyproline concentration in myocardial tissue compared with the model group. In addition, it also improved the cardiac function. TPL attenuated cardiac remodeling in rats by down-regulating the p38 MAPK and TGF-ß1/Smad3 signaling pathways. TPL treatment significantly attenuated cardiac fibrosis and improved cardiac function through suppressing the p38 MAPK and TGF-ß1/Smad3 signaling pathway in isoprenaline-induced cardiac remodeling rats. Our findings suggested that TPL might be a novel complementary medicine in the treatment of chronic heart failure.

Diosmectite-zinc oxide composite improves intestinal barrier restoration and modulates TGF-ß1, ERK1/2, and Akt in piglets after acetic acid challenge.

The present study evaluated the beneficial effect of diosmectite-zinc oxide composite (DS-ZnO) on improving intestinal barrier restoration in piglets after acetic acid challenge and explored the underlying mechanisms. Twenty-four 35-d-old piglets (Duroc × Landrace × Yorkshire), with an average weight of 8.1 kg, were allocated to 4 treatment groups. On d 1 of the trial, colitis was induced via intrarectal injection of acetic acid (10 mL of 10% acetic acid [ACA] solution for ACA, DS-ZnO, and mixture of diosmectite [DS] and ZnO [DS+ZnO] groups) and the control group was infused with saline. Twenty-four hours after challenged, piglets were fed with the following diets: 1) control group (basal diet), 2) ACA group (basal diet), 3) DS-ZnO group (basal diet supplemented with DS-ZnO), and 4) DS+ZnO group (mixture of 1.5 g diosmectite [DS]/kg and 500 mg Zn/kg from ZnO [equal amount of DS and ZnO in the DS-ZnO treatment group]). On d 8 of the trial, piglets were sacrificed. The results showed that DS-ZnO supplementation improved (P < 0.05) ADG, ADFI, and transepithelial electrical resistance and decreased (P < 0.05) fecal scores, crypt depth, and fluorescein isothiocyanate-dextran 4 kDa (FD4) influx as compared with ACA group. Moreover, DS-ZnO increased (P < 0.05) occludin, claudin-1, and zonula occluden-1 expressions; reduced (P < 0.05) caspase-9 and caspase-3 activity and Bax expression; and improved (P < 0.05) Bcl2, XIAP, and PCNA expression. Diosmectite-zinc oxide composite supplementation also increased (P < 0.05) TGF-ß1 expression and ERK1/2 and Akt activation. These results suggest that DS-ZnO attenuates the acetic acid-induced colitis by improving mucosa barrier restoration, inhibiting apoptosis, and improving intestinal epithelial cells proliferation and modulation of TGF-ß1 and ERK1/2 and Akt signaling pathway.

Herbal supplement attenuation of cardiac fibrosis in rats with CCl4-induced liver cirrhosis.

Previously we found carbon tetrachloride (CCl4) induced cirrhosis associated cardiac hypertrophy and apoptosis. The purpose of this study is to determine whether further CCl4 treatment would induce cardiac cell fibrosis. The cardiac tissues were analyzed by H&E. histological staining, Trichrome Masson staining and Western blotting. The results showed that the CCl4-treated-only group exhibits more trichrome staining, meaning that more fibrosis is present. Moreover, CCl4 could further induce cardiac-fibrosis via TGF-ß-p-Smad2/3-CTGF pathway. However, our data showed that the CCl4- indcued cardiac abnormalities were attenuated by Ocimum gratissimum extract (OGE) and silymarin co- treatments. In conclusion, our results indicated that the OGE and silymarin may be a potential traditional herb for the protection of cardiac tissues from the CCl4 induced cirrhosis associated cardiac fibrosis through modulating the TGF-ß signaling pathway.

Generation of human induced pluripotent stem (Ips) cells in serum- and feeder-free defined culture and TGF-Β1 regulation of pluripotency.

Human Embryonic Stem cells (hESCs) and human induced Pluripotent Stem cells (hiPSCs) are commonly maintained on inactivated mouse embryonic fibroblast as feeder cells in medium supplemented with FBS or proprietary replacements. Use of culture medium containing undefined or unknown components has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. In addition, there is no consensus as to the optimal formulation, or the nature of the cytokine requirements of the cells to promote their self-renewal and inhibit their differentiation. In this study, we successfully generated hiPSCs from human dental pulp cells (DPCs) using Yamanaka's factors (Oct3/4, Sox2, Klf4, and c-Myc) with retroviral vectors in serum- and feeder-free defined culture conditions. These hiPSCs retained the property of self-renewal as evaluated by the expression of self-renewal marker genes and proteins, morphology, cell growth rates, and pluripotency evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo. In this study, we found that TGF-ß1 increased the expression levels of pluripotency markers in a dose-dependent manner. However, increasing doses of TGF-ß1 suppressed the growth rate of hiPSCs cultured under the defined conditions. Furthermore, over short time periods the hiPSCs cultured in hESF9 or hESF9T exhibited similar morphology, but hiPSCs maintained in hESF9 could not survive beyond 30 passages. This result clearly confirmed that hiPSCs cultured in hESF9 medium absolutely required TGF-ß1 to maintain pluripotency. This simple serum-free adherent monoculture system will allow us to elucidate the cell responses to growth factors under defined conditions and can eliminate the risk might be brought by undefined pathogens.

NADPH oxidase-dependent formation of reactive oxygen species contributes to transforming growth factor ß1-induced epithelial-mesenchymal transition in rat peritoneal mesothelial cells, and the role of astragalus intervention.

OBJECTIVE: To investigate the role of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidasedependent formation of reactive oxygen species (ROS) in the transforming growth factor ß1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) in rat peritoneal mesothelial cells (RPMCs), and the effect of Astragalus injection (AGI) intervention. METHODS: Primary RPMCs were cultured to the second generation in vitro. After synchronization for 24 h, the cells were randomly assigned to the following groups: control (Group A), AGI (2 g/mL; Group B), TGF-ß1 (10 ng/mL; Group C), TGF-ß1 (10 ng/mL) + AGI (2 g/mL; Group D; pretreated for 1 h with AGI before TGF-ß1 stimulation). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were employed to evaluate the mRNA and protein expression of the NADPH oxidase subunit p67phox, α-smooth muscle actin (α-SMA) and E-cadherin. The dichlorofluorescein-sensitive cellular ROS levels were measured by a fluorometric assay and confocal microscopy. RESULTS: TGF-ß1 significantly induced NADPH oxidase subunit p67phox mRNA and protein expression in RPMCs, as well as inducing the production of intracellular ROS. AGI inhibited this TGF-ß1-induced up-regulation by 39.3% and 47.8%, respectively (P<0.05), as well as inhibiting the TGF-ß1-induced ROS generation by 56.3% (P<0.05). TGF-ß1 also induced α-SMA mRNA and protein expression, and down-regulated E-cadherin mRNA and protein expression (P<0.05). This effect was suppressed by AGI (P<0.05). CONCLUSIONS: NADPH oxidase-dependent formation of ROS may mediate the TGF-ß1-dependent EMT in RPMCs. AGI could inhibit this process, providing a theoretical basis for AGI in the prevention of peritoneal fibrosis.