Curculigoside attenuates oxidative stress and osteoclastogenesis via modulating Nrf2/NF-κB signaling pathway in RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Curculigo orchioides Gaertn is used for the treatment of impotence, atrophic debility of bones (osteoporosis), limb limpness, and arthritis of the lumbar and knee joints in traditional Chinese medicine and Ayurvedic medical system. Curculigoside (Cur) from Curculigo orchioides Gaertn has been shown to have regulatory effects on bone metabolism via anti-oxidative activities in rats and osteoblasts. However, little is known about the molecular pharmacological activity of Cur in osteoclastic bone resorption. AIM: The aim of this work is to investigate the inhibitory effect of Cur against osteoclasts (OCs) under the oxidative stress status, and explore the possible underlying mechanism. MATERIALS AND METHODS: OCs were induced from RAW264.7 cells using RANKL and H2O2. The number of OCs was measured by tartrate-resistant acid phosphatase (TRAP) staining. F-Actin and nuclear translocation of P65 and Nrf2 were stained with immunofluorescence assay and observed under a laser confocal microscope. The biochemical parameters of OCs were detected with an ELISA kit. The expression of Nrf2 and NF-κB pathway-related proteins was analyzed by Western Blot. RESULTS: Cur inhibited the TRAP activity, release of degrading products from bone slices and the expression of NFATc1, c-Fos, Cathepsin K (Ctsk) and matrix metallopeptidase 9 (MMP9) of OCs induced with RANKL and H2O2. In addition, Cur suppressed the ROS level and NADPH oxidase 1(NOX1) and NADPH oxidase 4 (NOX4) activities of OCS. More importantly, Cur enhanced the expression and nucleus translocation of Nrf2 and activities of its regulatory cytoprotective enzymes, and reduced the NF-κB expression and phosphorylation and nucleus translocation of p65 in OCs. Furthermore, the Nrf2 inhibitor ML385 and NF-κB inhibitor Bay11-7082 counteracted the effect of Cur in OCs. CONCLUSION: Cur mitigated oxidative stress and osteoclastogenesis by activating Nrf2 and inhibiting the NF-κB pathway, suggesting that Cur may prove to be a promising candidate for the treatment of osteoporosis. Our findings may also help partially explain the rationale behind the traditional use of Curculigo orchioides Gaertn.

Actin-binding compounds, previously discovered by FRET-based high-throughput screening, differentially affect skeletal and cardiac muscle.

Actin's interactions with myosin and other actin-binding proteins are essential for cellular viability in numerous cell types, including muscle. In a previous high-throughput time-resolved FRET (TR-FRET) screen, we identified a class of compounds that bind to actin and affect actomyosin structure and function. For clinical utility, it is highly desirable to identify compounds that affect skeletal and cardiac muscle differently. Because actin is more highly conserved than myosin and most other muscle proteins, most such efforts have not targeted actin. Nevertheless, in the current study, we tested the specificity of the previously discovered actin-binding compounds for effects on skeletal and cardiac α-actins as well as on skeletal and cardiac myofibrils. We found that a majority of these compounds affected the transition of monomeric G-actin to filamentous F-actin, and that several of these effects were different for skeletal and cardiac actin isoforms. We also found that several of these compounds affected ATPase activity differently in skeletal and cardiac myofibrils. We conclude that these structural and biochemical assays can be used to identify actin-binding compounds that differentially affect skeletal and cardiac muscles. The results of this study set the stage for screening of large chemical libraries for discovery of novel compounds that act therapeutically and specifically on cardiac or skeletal muscle.

FFNT25 ameliorates unilateral ureteral obstruction-induced renal fibrosis.

Renal fibrosis is a common pathological feature of chronic kidney disease (CKD) patients who progress to end-stage renal disease (ESRD). With the increasing incidence of CKD, it is of importance to develop effective therapies that blunt development of renal fibrosis. FFNT25 is a newly developed molecular compound that could be used to prevent fibrosis. In this study, we administered FFNT25 to rats following unilateral ureteral obstruction (UUO) to investigate its anti-fibrosis mechanism. Thirty-two Sprague-Dawley rats were randomly divided into four groups: (1) control (normal rats), (2) sham-operated, (3) UUO-operated + vehicle, and (4) UUO-operated + FFNT25. Two weeks after UUO, the rats were gavaged with either FFNT25 (20.6 mg/kg/day) or vehicle for two weeks. Serum, urine, and kidney samples were collected at the end of the study. FFNT25 reduced levels of renal fibrosis and decreased mRNA and protein levels of extracellular matrix (ECM) markers α-smooth muscle actin (α-SMA) and plasminogen activator inhibitor-1 (PAI-1) following UUO compared to vehicle treatment (n = 8, p<.05). The current results indicate that FFNT25 can affect both the production and degradation of collagen fibers to reduce fibrosis.

Structure-based virtual screening identifies a small-molecule inhibitor of the profilin 1-actin interaction.

Profilin 1 (Pfn1) is an important regulator of the actin cytoskeleton and plays a vital role in many actin-based cellular processes. Therefore, identification of a small-molecule intervention strategy targeted against the Pfn1-actin interaction could have broad utility in cytoskeletal research and further our understanding of the role of Pfn1 in actin-mediated biological processes. Based on an already resolved Pfn1-actin complex crystal structure, we performed structure-based virtual screening of small-molecule libraries to seek inhibitors of the Pfn1-actin interaction. We identified compounds that match the pharmacophore of the key actin residues of Pfn1-actin interaction and therefore have the potential to act as competitive inhibitors of this interaction. Subsequent biochemical assays identified two candidate compounds with nearly identical structures that can mitigate the effect of Pfn1 on actin polymerization in vitro As a further proof-of-concept test for cellular effects of these compounds, we performed proximity ligation assays in endothelial cells (ECs) to demonstrate compound-induced inhibition of Pfn1-actin interaction. Consistent with the important role of Pfn1 in regulating actin polymerization and various fundamental actin-based cellular activities (migration and proliferation), treatment of these compounds reduced the overall level of cellular filamentous (F) actin, slowed EC migration and proliferation, and inhibited the angiogenic ability of ECs both in vitro and ex vivo In summary, this study provides the first proof of principle of small-molecule-mediated interference with the Pfn1-actin interaction. Our findings may have potential general utility for perturbing actin-mediated cellular activities and biological processes.

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.

Pest control. Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids.

Double-stranded RNAs (dsRNAs) targeted against essential genes can trigger a lethal RNA interference (RNAi) response in insect pests. The application of this concept in plant protection is hampered by the presence of an endogenous plant RNAi pathway that processes dsRNAs into short interfering RNAs. We found that long dsRNAs can be stably produced in chloroplasts, a cellular compartment that appears to lack an RNAi machinery. When expressed from the chloroplast genome, dsRNAs accumulated to as much as 0.4% of the total cellular RNA. Transplastomic potato plants producing dsRNAs targeted against the ß-actin gene of the Colorado potato beetle, a notorious agricultural pest, were protected from herbivory and were lethal to its larvae. Thus, chloroplast expression of long dsRNAs can provide crop protection without chemical pesticides.

Caffeine attenuates liver fibrosis via defective adhesion of hepatic stellate cells in cirrhotic model.

BACKGROUND AND AIM: Several epidemiological studies have shown that coffee intake attenuates the progression of liver fibrosis; however, the mechanism is unclear. AIMS: We investigated the direct effects of caffeine on hepatic stellate cells (HSCs) and assessed whether caffeine attenuated intrahepatic fibrosis in rat model of liver cirrhosis. METHODS: Human hepatic stellate cell line, an immortalized human HSCs line, was used in in vitro assay system. Cell migration and proliferation were assessed in presence of various caffeine concentrations (0, 1, 5, and 10 mmol), and levels of procollagen type Ic and α-smooth muscle actin (α-SMA) were measured by Western blot. Severity of liver inflammation and fibrosis were compared between thioacetamide-treated rats with and without caffeine supplementation. RESULTS: Caffeine increased HSCs apoptosis and intracellular F-actin and cyclic adenosine monophosphate expression. Caffeine also inhibited procollagen type Ic and α-SMA expression in a dose- and time-dependent manner. In rat model, caffeine decreased periportal inflammation, levels of inflammatory cells (1.4 ± 0.52 vs 2.6 ± 0.46, P < 0.05), and fibrosis (2.1 ± 0.35 vs 2.9 ± 0.84, P < 0.05). Transforming growth factor-ß and α-SMA expressions were also reduced by caffeine. CONCLUSION: Caffeine attenuates the progression of liver fibrosis by inhibiting HSCs adhesion and activation.

Cucurbitacin E inhibits breast tumor metastasis by suppressing cell migration and invasion.

Tumor metastasis is the main cause of cancer-related deaths of patients. Breast cancer is highly malignant with considerable metastatic potential, which urges the necessity for developing novel potential drug candidate to prevent tumor metastasis. Here, we report our finding with Cucurbitacin E (CuE, α-elaterin), a tetracyclic triterpenes compound isolated from Cucurbitaceae. The potency of CuE on breast cancer metastasis inhibition was assessed in vivo and in vitro. In our animal experiments, intraperitoneal administrations of CuE significantly inhibited breast tumor metastasis to the lung without affecting apoptosis or proliferation of inoculated 4T1 and MDA-MB-231 breast cancer cells. Treatment of metastatic breast tumor cells with CuE markedly blocked tumor cell migration and invasion in vitro. Subsequent studies showed that CuE impaired Arp2/3-dependent actin polymerization and suppressed Src/FAK/Rac1/MMP involved pathway. Overall, our data demonstrate that CuE blocks breast cancer metastasis by suppressing tumor cell migration and invasion. We provide first evidence of a novel role for CuE as a potential candidate for treating breast cancer metastasis.

Paeoniae radix reduces PDGF-stimulated hepatic stellate cell migration.

Hepatic stellate cells (HSCs) play a key role in the pathogenesis of liver fibrosis. In chronic liver injury, HSCs undergo transdifferentiation to an activated myofibroblastic phenotype and migrate to injured areas in response to chemotactic factors, producing extracellular matrix proteins such as collagen type I to repair the damage as well as overexpression of α-smooth muscle actin (α-SMA). Paeoniae Radix, the root of Paeonia lactiflora Pall, was investigated for PDGF-BB-induced HSC chemotaxis. Rat HSCs and LX-2, a human HSC cell line, were used for the in vitro experiments. Cell migration was analyzed by wound-healing and transwell assays. An ELISA and a Sircol collagen assay kit were used to detect the expressions of α-SMA and of collagen, respectively. Phosphorylations of mitogen-activated protein kinases, including ERK 1/2, p38, and JNK, were evaluated with immunoblotting. Results indicated that PDGF-BB increased migration as well as α-SMA and collagen expression in HSCs. Paeoniae Radix extracts and its active components, paeonol and 1,2,3,4,6-penta- O-galloyl- ß-D-glucose (PGG), inhibited PDGF-BB-induced HSC migration and α-SMA and collagen expressions in a concentration-dependent manner. The inhibitory effects were associated with downregulation of PDGF receptor- α, ERK, p38, and JNK activation. Both paeonol and PGG participate in HSC migration, but via differential mechanisms.

22 : 6n-3 DHA inhibits differentiation of prostate fibroblasts into myofibroblasts and tumorigenesis.

Prostate cancer is one of the most common malignancies in men. Epidemiological and experimental studies have revealed that stromal cells of the tumour microenvironment contribute to the development of prostate cancers, while long-chain n-3 PUFA-enriched diets reduce the risk of this tumour histotype. These findings prompted us to investigate whether DHA, an n-3 PUFA, may abrogate differentiation of prostate fibroblasts into myofibroblasts, the activated form of fibroblasts generally involved in prostate cancer progression. We used the human prostate carcinoma cell line (PC3) as a prostate adenocarcinoma model and found that DHA (1) inhibits α-smooth muscle actin (α-SMA) expression, a typical marker of myofibroblast differentiation, in prostate fibroblasts stimulated in vitro with transforming growth factor-ß (TGF-ß), (2) blocks the matrix metalloproteinase-2-dependent enhanced invasiveness of PC3 prostate adenocarcinoma cells migrated in a medium conditioned by TGF-ß-stimulated prostate fibroblasts, (3) prevents epithelial-mesenchymal transition (EMT) and invasiveness of PC3 cells promoted by a medium conditioned by TGF-ß-stimulated prostate fibroblasts, and (4) reduces the growth rate of tumours obtained in immunodeficient animals injected with PC3 cells plus TGF-ß-stimulated prostate fibroblasts. Moreover, DHA was found to revert α-SMA expression and the invasiveness-promoting activity exerted in PC3 cells by tumoral-activated fibroblasts. Thus, DHA represents a suitable agent to inhibit prostate myofibroblast differentiation, invasiveness and EMT, two most important tumour-promoting activities involved in the progression of prostate cancer cells.

Improved visualization and quantitative analysis of drug effects using micropatterned cells.

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck (1-2). To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Protective effects of Cinnamomum cassia Blume in the fibrogenesis of activated HSC-T6 cells and dimethylnitrosamine-induced acute liver injury in SD rats.

Cinnamomum cassia Blume (CC) is one of the world's oldest natural spices, and is commonly used in traditional oriental medicine. We investigated the protective effect of ethanol extract from Cinnamomum cassia Blume (CCE) on the activation of hepatic stellate cells (HSCs). In addition, we examined the effects of CC powder in Sprague-Dawley rats with acute liver injury induced by dimethylnitrosamine (DMN). In vitro, HSC-T6 cells exhibit an activated phenotype, as reflected in their fibroblast-like morphology. CCE significantly reduced the expression of alpha-smooth muscle actin (alpha-SMA), connective tissue growth factor (CTGF), transforming growth factor beta (TGF-beta1), and tissue inhibitor of metalloproteinase-1 (TIMP-1). In vivo, the results were significantly protected by CC powder in the serum total protein, albumin, total-bilirubin, direct-bilirubin, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and alkaline phosphatase (ALP). We suggest that CC inhibits fibrogenesis, followed by HSC-T6 cell activation and increased restoration of liver function, ultimately resulting in acute liver injury.

Effects of Shugan-Huayu powder, a traditional Chinese medicine, on hepatic fibrosis in rat model.

Shugan-Huayu powder (SHP) has been administered to outpatients with chronic liver disease without clear anti-fibrosis mechanism. To investigate the anti-fibrotic effects of SHP on liver fibrosis in a rat model and in hepatic stellate cells (HSCs) in vitro, rats were gavaged with CCl4 at 1.0 g/kg body weight twice a week for 8 weeks to induce liver fibrosis and the rats were randomly assigned to one of the three groups: -CCl4 alone, low-dose SHP and high-dose SHP. SHP was given by gavages 5 times a week for 8 weeks. Serum, livers and HSCs were assayed for serology, pathology, western blot, zymography and quantitative RT-PCR. Hepatic function improved as decreased serum aspartate aminotransferase and alanine aminotransferase, and collagen deposition and active HSCs were significantly reduced in CCl4-induced liver by SHP treatment. The expression of matrix metalloproteinase-2 (MMP-2) and transforming growth factor-beta1 (TGF-beta1) mRNA in fibrotic liver showed significant downregulation after SHP treatment. In vitro, inhibition of alpha-smooth muscle actin (alpha-SMA) expression and MMP-2 secretion of active HSCs were also noticed by SHP treatment. SHP has an antifibrotic effect on CCl4-induced liver fibrosis in rats. Anti-fibrotic mechanisms were probably inhibiting activation of HSCs and decreased expression of MMP-2 and TGF-beta1.

Curcumin limits the fibrogenic evolution of experimental steatohepatitis.

Nonalcoholic steatohepatitis is characterized by the association of steatosis with hepatic cell injury, lobular inflammation and fibrosis. Curcumin is known for its antioxidant, anti-inflammatory and antifibrotic properties. The aim of this study was to test whether the administration of curcumin limits fibrogenic evolution in a murine model of nonalcoholic steatohepatitis. Male C57BL/6 mice were divided into four groups and fed a diet deficient in methionine and choline (MCD) or the same diet supplemented with methionine and choline for as long as 10 weeks. Curcumin (25 microg per mouse) or its vehicle (DMSO) was administered intraperitoneally every other day. Fibrosis was assessed by Sirius red staining and histomorphometry. Intrahepatic gene expression was measured by quantitative PCR. Hepatic oxidative stress was evaluated by staining for 8-OH deoxyguanosine. Myofibroblastic hepatic stellate cells (HSCs) were isolated from normal human liver tissue. The increase in serum ALT caused by the MCD diet was significantly reduced by curcumin after 4 weeks. Administration of the MCD diet was associated with histological steatosis and necro-inflammation, and this latter was significantly reduced in mice receiving curcumin. Curcumin also inhibited the generation of hepatic oxidative stress. Fibrosis was evident after 8 or 10 weeks of MCD diet and was also significantly reduced by curcumin. Curcumin decreased the intrahepatic gene expression of monocyte chemoattractant protein-1, CD11b, procollagen type I and tissue inhibitor of metalloprotease (TIMP)-1, together with protein levels of alpha-smooth muscle-actin, a marker of fibrogenic cells. In addition, curcumin reduced the generation of reactive oxygen species in cultured HSCs and inhibited the secretion of TIMP-1 both in basal conditions and after the induction of oxidative stress. In conclusion, curcumin administration effectively limits the development and progression of fibrosis in mice with experimental steatohepatitis, and reduces TIMP-1 secretion and oxidative stress in cultured stellate cells.

Differential display proteomic analysis of Picea meyeri pollen germination and pollen-tube growth after inhibition of actin polymerization by latrunculin B.

To investigate roles of the actin cytoskeleton in growth of the pollen tube of Picea meyeri, we used the actin polymerization inhibitor latrunculin B (LATB) under quantitatively controlled conditions. At low concentrations, LATB inhibited polymerization of the actin cytoskeleton in the growing pollen tube, which rapidly inhibited tip growth. The proteomic approach was used to analyse protein expression-profile changes during pollen germination and subsequent pollen-tube development with disturbed organization of the actin cytoskeleton. Two-dimensional electrophoresis and staining with Coomassie Brilliant Blue revealed nearly 600 protein spots. A total of 84 of these were differentially displayed at different hours with varying doses of LATB, and 53 upregulated or downregulated proteins were identified by mass spectrometry. These proteins were grouped into distinct functional categories including signalling, actin cytoskeleton organization, cell expansion and carbohydrate metabolism. Moreover, actin disruption affected the morphology of Golgi stacks, mitochondria and amyloplasts, along with a differential expression of proteins involved in their functions. These findings provide new insights into the multifaceted mechanism of actin cytoskeleton functions and its interaction with signalling, cell-expansion machinery and energy-providing pathways.

(-)-Epigallocatechin-3-gallate, a polyphenolic compound from green tea, inhibits fibroblast adhesion and migration through multiple mechanisms.

It is increasingly evident that the stromal cells are involved in key metastatic processes of melanoma and some malignant solid tumors. (-)-Epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, has been shown to have anti-tumor activity, inhibiting adhesion, migration, and proliferation of tumor cells. However, little attention has been paid on its effects on stromal cells. In the present study, we determined the effects of EGCG on stromal fibroblasts. We showed that fibroblast adhesion to collagen, fibronectin, and fibrinogen were inhibited by EGCG. One of the possible mechanisms is binding of EGCG to fibronectin and fibrinogen but not to collagen. We then focused how EGCG affected fibroblast adhesion to collagen. EGCG treatment attenuated the antibody binding to fibroblast's integrin alpha2beta1, indicating EGCG may affect the expression and affinity of integrin alpha2beta1. Moreover, intracellular H2O2 level was decreased by EGCG treatment, suggesting that the tonic maintenance of intracellular H2O2 may be required for cell adhesion to collagen. In parallel, collagen-induced FAK phosphorylation, actin cytoskeleton reorganization in fibroblasts, migration and matrix metalloproteinase(s) (MMPs) activity were also affected by EGCG. Tubular networks formed by melanoma cells grown on three-dimensional Matrigel were also disrupted when fibroblasts were treated with EGCG in a non-contact coculture system. Taken together, we provided here the first evidence that EGCG is an effective inhibitor on behaviors of the stromal fibroblasts, affecting their adhesion and migration. The inhibitory activity of EGCG may contribute to its anti-tumor activity. The findings and concepts disclosed here may provide important basis for a further experiment towards understanding tumor-stroma interaction.

Polyphenols from Camellia sinenesis attenuate experimental cholestasis-induced liver fibrosis in rats.

Accumulation of hydrophobic bile acids during cholestasis leads to generation of oxygen free radicals in the liver. Accordingly, this study investigated whether polyphenols from green tea Camellia sinenesis, which are potent free radical scavengers, decrease hepatic injury caused by experimental cholestasis. Rats were fed a standard chow or a diet containing 0.1% polyphenolic extracts from C. sinenesis starting 3 days before bile duct ligation. After bile duct ligation, serum alanine transaminase increased to 760 U/l after 1 day in rats fed a control diet. Focal necrosis and bile duct proliferation were also observed after 1-2 days, and fibrosis developed 2-3 wk after bile duct ligation. Additionally, procollagen-alpha1(I) mRNA increased 30-fold 3 wk after bile duct ligation, accompanied by increased expression of alpha-smooth muscle actin and transforming growth factor-beta and the accumulation of 4-hydroxynenonal, an end product of lipid peroxidation. Polyphenol feeding blocked or blunted all of these bile duct ligation-dependent changes by 45-73%. Together, the results indicate that cholestasis due to bile duct ligation causes liver injury by mechanisms involving oxidative stress. Polyphenols from C. sinenesis scavenge oxygen radicals and prevent activation of stellate cells, thereby minimizing liver fibrosis.