Boldine protects against carbon tetrachloride-induced chronic liver injury by regulating NF-κB signaling pathway.

Sustained liver injuries predominantly promote oxidative stress and inflammation that lead to the progression of chronic liver disease (CLD), including fibrosis, cirrhosis, and hepatocellular carcinoma. Boldine, an alkaloid isolated from Peumus boldus, has been shown to have antioxidant and anti-inflammatory effects. Currently, there is no definitive treatment option available for CLD. Therefore, we investigated the hepatoprotective effect of boldine against carbon tetrachloride (CCl4 )-induced chronic liver injury in rats. CCl4 (2 mL/kg., b.w., i.p.) was administered twice weekly for 5 weeks to induce chronic liver injury in rats. Separate groups of rats were given boldine (20 mg/kg b.w., and 40 mg/kg b.w.) and silymarin (100 mg/kg b.w.) orally, daily. Serum transaminases, lipid peroxidation, and antioxidant levels were measured, and nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (cox-2), interleukin-1 ß (IL-1ß), and α-smooth muscle actin (α-SMA) gene and protein expressions were evaluated. CCl4 administration increased liver marker enzymes of hepatotoxicity in serum and oxidative stress markers, inflammatory genes and α-smooth muscle actin expression in liver tissue. Boldine concurrent treatment suppressed CCl4 -induced elevation of transaminase levels in serum, restored enzymic and non-enzymic antioxidants, and downregulated NF-κB, TNF-α, Cox-2 and IL-1ß expressions, thereby suppressing hepatic inflammation. Boldine administration also repressed α-SMA expression. The results of this study demonstrate the antioxidant, anti-inflammatory, and antifibrotic properties of boldine, and it can be a potential therapeutic candidate in the treatment of CLD.

Vascular endothelial growth factor induces the migration of human airway smooth muscle cells by activating the RhoA/ROCK pathway.

BACKGROUND: Airway remodeling due to increased airway smooth muscle cell (ASMC) mass, likely due to enhanced proliferation, hypertrophy, and migration, has been proven to be highly correlated with decreased lung function in asthma patients. Vascular endothelial growth factor (VEGF) mediates vascular and extravascular remodeling and inflammation and has been proven to be involved in the progression of asthma. Previous studies have focused on the effects of VEGF on ASMC proliferation, but few researchers have focused on the effects of VEGF on human ASMC migration. The purpose of this study was to explore the effect of VEGF on the migration of ASMCs and its related signaling pathway mechanism to provide evidence for the treatment of airway remodeling. METHODS: We examined the effects of VEGF induction on ASMC migration and explored the mechanisms involved in ASMC migration. RESULTS: We found by wound healing and Transwell assays that VEGF promoted ASMC migration. Through the Cell Counting Kit-8 (CCK-8) experiment, we found that VEGF had no significant effect on the proliferation of ASMCs, which excluded the involvement of cell proliferation in the process of wound healing. Moreover, a cellular immunofluorescence assay showed that VEGF promoted F-actin reorganization, and Western blotting showed that VEGF improved RhoA activation and myosin phosphatase targeting subunit-1 (MYPT1) and myosin light chain (MLC) phosphorylation in ASMCs. Treatment with the ROCK inhibitor Y27632 significantly attenuated the effects of VEGF on MYPT1/MLC activation and cell migration. CONCLUSION: In conclusion, the results suggest that the promigratory function of VEGF activates the RhoA/ROCK pathway, induces F-actin reorganization, improves the migration of ASMCs, and provides a better rationale for targeting the RhoA/ROCK pathway for therapeutic approaches in airway remodeling.

Involvement of transcribed lncRNA uc.291 in hyperproliferative skin disorders.

The uc.291 transcript controls keratinocytes differentiation by physical interaction with ACTL6A and subsequent induction of transcription of the genes belonging to the epidermal differentiation complex (EDC). Uc.291 is also implicated in the dedifferentiation phenotype seen in poorly differentiated cutaneous squamous cell carcinomas. Here, we would like to investigate the contribution of uc.291 to the unbalanced differentiation state of keratinocytes observed in hyperproliferative skin disorders, e. g., psoriasis. Psoriasis is a multifactorial inflammatory disease, caused by alteration of keratinocytes homeostasis. The imbalanced differentiation state, triggered by the infiltration of immune cells, represents one of the events responsible for this pathology. In the present work, we explore the role of uc.291 and its interactor ACTL6A in psoriasis skin, using quantitative real-time PCR (RT-qPCR), immunohistochemistry and bioinformatic analysis of publicly available datasets. Our data suggest that the expression of the uc.291 and of EDC genes loricrin and filaggrin (LOR, FLG) is reduced in lesional skin compared to nonlesional skin of psoriatic patients; conversely, the mRNA and protein level of ACTL6A are up-regulated. Furthermore, we provide evidence that the expression of uc.291, FLG and LOR is reduced, while ACTL6A mRNA is up-regulated, in an in vitro psoriasis-like model obtained by treating differentiated keratinocytes with interleukin 22 (IL-22). Furthermore, analysis of a publicly available dataset of human epidermal keratinocytes treated with IL-22 (GSE7216) confirmed our in vitro results. Taken together, our data reveal a novel role of uc.291 and its functional axis with ACTL6A in psoriasis disorder and a proof of concept that biological inhibition of this molecular axis could have a potential pharmacological effect against psoriasis and, in general, in skin diseases with a suppressed differentiation programme.

[Mitochondrial coenzyme Q attenuates lipopolysaccharide-induced mitochondria-dependent apoptosis in type II alveolar epithelial cells via phosphatidylinositol 3-kinase/Akt pathway].

OBJECTIVE: To investigate the protective effect and potential mechanism of mitochondrial coenzyme Q (MitoQ) on mitochondria-dependent apoptosis in type II alveolar epithelial cells induced by lipopolysaccharide (LPS). METHODS: The type II lung epithelial cell line (A549) were cultured with different concentrations of LPS in vitro, a cell model of acute lung injury (ALI) was reproduced, the optimal concentration of LPS was obtained according to the half maximal inhibitory concentration (IC50). The cells were pretreated with different concentrations of MitoQ to determine the best intervention concentration of MitoQ. The cells were divided into four groups: the cells in blank control group were cultured in DMEM; the cells in LPS group were stimulated with 10 mg/L of LPS for 24 hours; the cells in MitoQ+LPS group were pretreated with 1 µmol/L MitoQ for 60 minutes, and then were co-cultured with 10 mg/L of LPS for 24 hours; and the cells in MitoQ+phosphatidylinositol 3-kinase (PI3K) selective inhibitor LY294002+LPS group were pretreated with 1 µmol/L MitoQ and 20 µmol/L LY294002 for 60 minutes, and then were co-cultured with 10 mg/L of LPS for 24 hours. Cell viability was measured using cell counting kit-8 (CCK-8). The cell apoptosis rate was determined by flow cytometry and TdT-mediated dUTP-nick end labeling (TUNEL) method. The protein expression levels of apoptosis protein Bax, anti-apoptotic protein Bcl-2 and PI3K-serine/threonine kinase (Akt) protein PI3K expression and Akt phosphorylation level were detected by Western blotting. RESULTS: According to the inhibition rate curve, the IC50 of LPS on A549 cells was 11.06 mg/L. Therefore, 10 mg/L was selected as the stimulating concentration of LPS. After stimulation with 10 mg/L LPS, the cell viability first increased and then decreased with the increase in MitoQ pretreatment concentration. According to the cell viability curve, 1 µmol/L was selected as the optimum concentration of MitoQ. Compared with LPS group, after pretreated with 1 µmol/L MitoQ, cell mitochondrial dependent apoptosis was significantly attenuated, which was characterized by the apoptosis rate was significantly decreased [flow cytometry: (8.73±0.25)% vs. (18.10±0.70)%, TUNEL: (12.30±0.82)% vs. (21.43±0.86)%, both P < 0.05], the expression of Bax was significantly down-regulated (Bax/ß-actin: 0.58±0.03 vs. 1.06±0.10, P < 0.05) and Bcl-2 level was significantly up-regulated (Bcl-2/ß-actin: 1.03±0.06 vs. 0.53±0.07, P < 0.05), meanwhile the expression of PI3K and Akt phosphorylation level were significantly increased [PI3K protein (PI3K/ß-actin): 1.20±0.02 vs. 0.96±0.04, phosphorylated Akt (p-Akt) protein (p-Akt/t-Akt): 1.22±0.08 vs. 0.92±0.04, both P < 0.05]. Pretreatment with LY294002 could inhibit the anti-apoptotic effect of MitoQ on cells, it was characterized by the apoptotic rate was significantly increased as compared with MitoQ+LPS group [flow cytometry: (14.50±0.57)% vs. (8.73±0.25)%, TUNEL: (16.50±0.53)% vs. (12.30±0.82)%, both P < 0.05], the expression of Bax was significantly up-regulated (Bax/ß-actin: 0.95±0.03 vs. 0.58±0.03, P < 0.05) and Bcl-2 level was significantly down-regulated (Bcl-2/ß-actin: 0.62±0.03 vs. 1.03±0.06, P < 0.05), meanwhile the expression of PI3K and Akt phosphorylation level were significantly decreased [PI3K protein (PI3K/ß-actin): 0.90±0.05 vs. 1.20±0.02, p-Akt protein (p-Akt/t-Akt): 0.89±0.02 vs. 1.22±0.08, both P < 0.05]. CONCLUSIONS: MitoQ improved LPS induced mitochondria-dependent apoptosis of A549 cells by significantly activating PI3K/Akt signal pathway, which provided a new treatment for LPS induced ALI.

[Effect of Hypoxia Inducible Factor-1α on Chemosensitivity of B-ALL Cells to Vincristine and Its Mechanism].

OBJECTIVE: To explore the effect of hypoxia on the chemosensitivity of B-acute lymphoblastic leukemia （B-ALL） cells to Vincristine (VCR) and the mechanisms. METHODS: B-ALL cells SUP-B15, Nalm-6 and RS4;11 were selected as the research objects. The cells were divided into the control group and the hypoxia mimic group (CoCl2 pretreatment). The two groups were treated with VCR at different concentrations for 24 hours, CCK-8 was used to detect cell viability, flow cytometry was used to detect cell apoptosis, and Western bolt method was used to detect hypoxia inducible factor (HIF-1α), BAX, Bcl-2 and ß-actin protein expression. Quantitative real-time fluorescent PCR (qRT-PCR) was used to detect BAX and ß-actin mRNA levels. RESULTS: CoCl2 could simulate hypoxic environment to induce the expression of HIF-1α. The cells SUP-B15 and RS4;11 of the hypoxia mimic group were lower sensitivity to VCR as compared with the control group; the apoptosis rate of the hypoxia mimic group was lower than that of the control group after 80 nmol/L VCR treatment. The expression levels of BAX protein and mRNA in the hypoxia mimic group were lower than those of the control group, and there was no significant difference in the expression levels of Bcl-2 protein between two groups. CONCLUSION: Under hypoxic conditions, HIF-1α may mediate VCR resistance in B-ALL cells by downregulating the pro-apoptotic protein BAX.

[Effect of Hypoxia Inducible Factor-1α on Chemosensitivity of B-ALL Cells to Vincristine and Its Mechanism].

OBJECTIVE: To explore the effect of hypoxia on the chemosensitivity of B-acute lymphoblastic leukemia （B-ALL） cells to Vincristine (VCR) and the mechanisms. METHODS: B-ALL cells SUP-B15, Nalm-6 and RS4;11 were selected as the research objects. The cells were divided into the control group and the hypoxia mimic group (CoCl2 pretreatment). The two groups were treated with VCR at different concentrations for 24 hours, CCK-8 was used to detect cell viability, flow cytometry was used to detect cell apoptosis, and Western bolt method was used to detect hypoxia inducible factor (HIF-1α), BAX, Bcl-2 and ß-actin protein expression. Quantitative real-time fluorescent PCR (qRT-PCR) was used to detect BAX and ß-actin mRNA levels. RESULTS: CoCl2 could simulate hypoxic environment to induce the expression of HIF-1α. The cells SUP-B15 and RS4;11 of the hypoxia mimic group were lower sensitivity to VCR as compared with the control group; the apoptosis rate of the hypoxia mimic group was lower than that of the control group after 80 nmol/L VCR treatment. The expression levels of BAX protein and mRNA in the hypoxia mimic group were lower than those of the control group, and there was no significant difference in the expression levels of Bcl-2 protein between two groups. CONCLUSION: Under hypoxic conditions, HIF-1α may mediate VCR resistance in B-ALL cells by downregulating the pro-apoptotic protein BAX.

Effect of dimethyl alpha-ketoglutarate supplementation on the in vitro developmental competences of ovine oocytes.

The supplementation of dimethyl alpha-ketoglutarate (DMKG) during the in vitro maturation (IVM) process has been shown to improve the in vitro developmental competences of porcine oocytes. Here, the effects of DMKG supplementation in IVM medium on the development competencies of ovine oocytes were investigated by analyzing the nuclear maturation rate to metaphase II (MII) stage, ATP synthesis, cortical granules (CGs) dynamic, F-actin polymerization, mitochondrial activity, mitochondrial damage, reactive oxygen species (ROS) production, intracellular glutathione (GSH) production, DNA damage, cellular apoptosis, fertilization capacity and blastocyst development potential of ovine oocytes. In addition, the oxidative stress damage model induced by H2O2 treatment was applied to confirm the antioxidative effect of DMKG supplementation on the development of ovine oocytes. The results showed that compared with MII oocytes without DMKG supplementation (Control group), 3 mM DMKG supplementation during IVM significantly (P < 0.05) increased nuclear maturation rate, ATP synthesis, CGs dynamic, F-actin polymerization, mitochondrial activity, GSH production and embryonic developmental competence and decreased ROS production, mitochondrial damage, DNA damage and cellular apoptosis level of ovine MII oocytes. Moreover, the reductions in the developmental competences of ovine MII oocytes caused by H2O2 induced oxidative stress damages were effectively ameliorated by the co-supplementation in IVM of 3 mM DMKG (P < 0.05). Our results demonstrate the promising effect of DMKG supplementation on the in vitro developmental competence of ovine oocytes via the reduction of oxidative stress damages and indicates further research into the clinical applications of DMKG and the development of ovine breeding technologies is warranted.

RHOA inhibits chondrogenic differentiation of mesenchymal stem cells in adolescent idiopathic scoliosis.

PURPOSE: The etiology of adolescent idiopathic scoliosis (AIS) remains unclear. The chondrogenic differentiation of mesenchymal stem cells (MSCs) is important in AIS, and the Ras homolog gene family member A (RHOA) is associated with chondrogenesis. The purpose of this study was to explore the effect of RHOA on the chondrogenic differentiation of MSCs in AIS. METHODS: We isolated MSCs from patients with AIS (AIS MSCs) and individuals without AIS (control MSCs). The inhibitor Y27632 was used to inhibit the function of RHOA/ROCK signaling, and plasmid-based overexpression and siRNA-mediated knockdown were used to manipulate RHOA expression. CCK-8 was used to detect cell viability. The phosphorylation levels of LIMK1, MLC2 and cofilin were detected by Western blotting. The mRNA expression of aggrecan, SOX9, and COL2A1 were confirmed using RT-PCR. Immunofluorescence was used to analyze F-actin and collagen II. Alcian blue staining was performed to assess the secretion of glycosaminoglycans (GAGs). RESULTS: We found that RHOA was significantly upregulated in AIS MSCs, and the phosphorylation levels of LIMK1, MLC2, and cofilin were increased. The mRNA expressions of aggrecan, SOX9, and COL2A1 were notably reduced in AIS MSCs. However, these effects were abolished by Y27632 treatment and RHOA knockdown in AIS MSCs. In addition, RHOA knockdown in AIS MSCs increased the content of collagen II and GAGs. RHOA overexpression in the control MSCs markedly activated the RHOA/ROCK signaling and decreased the expression of aggrecan, SOX9, and COL2A1, F-actin, and GAGs. CONCLUSION: RHOA regulates the chondrogenic differentiation ability of MSCs in AIS via the RHOA/ROCK signaling pathway and this regulation may involve SOX9.

Lidocaine inhibited migration of NSCLCA549 cells via the CXCR4 regulation.

BACKGROUND: Lidocaine is a local anesthetic that wildly used in surgical treatment and postoperative medical care for lung cancers. We hypothesized that lidocaine at clinical plasma concentration can inhibit CXCL12/CXCR4 axis-regulated cytoskeletal remodeling thereby reduce the migration of Non-small-cell lung cancers (NSCLC) cells. METHODS: We determined the effect of lidocaine at clinical plasma concentration on CXCL12-induced cell viability, apoptosis, cell death, monolayer cell wound healing rate, individual cell migration indicators, expression of CXCR4, CD44, and ICAM-1, intracellular Ca2+ level, and filamentous actin level alteration of NSCLC cells A549 and CXCR4-knocked down A549 cells using CCK-8, Bcl-2 ELISA, Cell death ELISA, wound healing assay, chemotaxis assay, western blotting, QPCR, Fura-2-based intracellular Ca2+ assay, and Fluorescein Phalloidin staining respectively. RESULTS: Lidocaine did not affect cell viability, apoptosis, and cell death but inhibited CXCL12-induced migration, intracellular Ca2+ releasing, and filamentous actin increase. Lidocaine decreased expression of CXCR4, increased CD44, but had no effect on ICAM-1. CXCL12 induced the increase of CD44 and ICAM-1 but did not affect CD44 in the presence of lidocaine. The knockdown of CXCR4 eliminated all the effects of lidocaine. The overexpression of CXCR4 promoted migration but the migration was inhibited by lidocaine. CONCLUSION: Lidocaine at clinical plasma concentrations inhibited CXCL12-induced CXCR4 activation, thereby reduced the intracellular Ca2+-dependent cytoskeleton remodeling, resulting in slower migration of A549 cells.

Apelin-13 protects human neuroblastoma SH-SY5Y cells against amyloid-beta induced neurotoxicity: Involvement of anti oxidant and anti apoptotic properties.

OBJECTIVES: We investigated the effect of apelin-13 on the cellular model of AD, amyloid-ß (Aß) treated SH-SY5Y cells in rats. METHODS: The SH-SY5Y cells were pretreated with different doses of apelin-13 (1, 2.5, 5, and 10 µg/mL), half an hour before adding 50% Aß treatment. After 24 h, cells were evaluated for survival, oxidative stress, mitochondrial calcium release, caspase-3, and cytochrome c levels, compared to control group (beta-actin). Statistical analysis was performed by SPSS 16. RESULTS: Apelin-13 at the dose of 2.5 µg/mL protected against IC50 Aß (p<0.001). Apelin-13 at doses of 1, 2.5, and 5 µg/mL showed protective effects against the reactive oxygen species (ROS) produced by Aß (p<0.001). Apelin-13 at doses of 2.5 and 5 µg/mL reduced calcium release, caspase-3, and cytochrome c (all p<0.001). CONCLUSIONS: Apelin-13 prevented apoptosis, oxidative stress, and mitochondrial toxicity and can be a suitable option for treatment of AD. The appropriate treatment strategy for humans has to be investigated in future studies.

[Effect of macrophage-derived exosomes on promoting hepatic stellate cell activation and platelet-derived growth factor expression].

Objective: To explore the effect of macrophage-derived exosomes on the activation of hepatic stellate cells and its possible mechanism. Methods: Differential ultracentrifugation was used to extract macrophage exosomes. The exosomes were co-cultured with the mouse hepatic stellate cell line JS1, and a control group was established with phosphate buffered saline (PBS). Cell immunofluorescence was used to observe the expressional conditions of F-actin. Cell counting kit-8 (CCK8) was used to detect the survival rate of JS1 cells in the two groups. The activation indices of JS1 cells [collagen type Ⅰ (Col Ⅰ) and α-smooth muscle actin (α-SMA)] and its key signal pathway activation index expression level [transforming growth factor (TGF)-ß1/Smads, platelet-derived growth factor (PDGF)] in the two groups were determined using Western blot and RT-PCR. Data comparison between two groups was performed using an independent sample t-test. Results: The membrane structure of exosomes was clearly observed by transmission electron microscopy. The expression of exosome marker proteins CD63 and CD81 was positive, suggesting that exosomes were successfully extracted. Exosomes were co-cultured with JS1 cells. Compared with the PBS control group, there was no statistically significant difference in the proliferation rate of JS1 cells in the exosomes group (P＞0.05). The expression of F-actin was significantly increased in the exosome group. The mRNA and protein expression levels of α-SMA and ColⅠwere significantly increased in exosome group JS1 cells (all P＜0.05). The mRNA relative expression levels of α-SMA in PBS and exosome group were 0.25±0.07 and 1.43±0.19, respectively, while that of ColⅠ was 1.03±0.04 and 1.57±0.06, respectively. The mRNA and protein expressions of PDGF were significantly increased in exosome group JS1 cells (P＜0.05). The mRNA relative expression levels of PDGF in the PBS group and exosome group were 0.27±0.04 and 1.65±0.12, respectively. There were no statistically significant differences in the mRNA and protein expressions of TGF-ß1, Smad2 and Smad3 between the two groups (P＞0.05). Conclusion: Macrophage-derived exosomes significantly promote the activation of hepatic stellate cells. JS1 cells may be the underlying mechanism for the up-regulation of PDGF expression.

ß-Actin Peptide-Based Inhibitors of Histidine Methyltransferase SETD3.

SETD3 was recently identified as the histidine methyltransferase responsible for N3 -methylation of His73 of ß-actin in humans. Overexpression of SETD3 is associated with several diseases, including breast cancer. Here, we report a development of actin-based peptidomimetics as inhibitors of recombinantly expressed human SETD3. Substitution of His73 by simple natural and unnatural amino acids led to selected ß-actin peptides with high potency against SETD3 in MALDI-TOF MS assays. The selenomethionine-containing ß-actin peptide was found to be the most potent SETD3 inhibitor (IC50 =161 nM). Supporting our inhibition assays, a combination of computational docking and molecular dynamics simulations revealed that the His73 binding pocket for ß-actin in SETD3 is rigid and accommodates the inhibitor peptides with similar binding modes. Collectively, our work demonstrates that actin-based peptidomimetics can act as potent SETD3 inhibitors and provide a basis for further development of highly potent and selective inhibitors of SETD3.

Discovery of Matrinic Thiadiazole Derivatives as a Novel Family of Anti-Liver Fibrosis Agents via Repression of the TGFß/Smad Pathway.

A series of novel matrinic thiadiazole derivatives were designed, synthesized and evaluated for their inhibitory effect on COL1A1 promotor. The SAR indicated that: (i) the introduction of a thiadiazole on the 11-side chain was beneficial for activity; (ii) a 12-N-benzyl moiety was favorable for activity. Among them, compound 6n displayed a high activity with an inhibitory rate of 39.7% at a concentration of 40 µM. It also effectively inhibited the expression of two representative collagen proteins (COL1A1 and α-SMA) on both the mRNA and protein levels and showed a high safety profile in vivo, indicating its great promise as an anti-liver fibrosis agent. Further study indicated that it might repress hepatic fibrogenesis via the TGFß/Smad pathway. This study provided powerful information for further strategic optimization and the top compound 6n was selected for further study as an ideal liver fibrosis lead for next investigation.

The role of adrenergic activation on murine luteal cell viability and progesterone production.

Sympathetic innervations exist in mammalian CL. The action of catecholaminergic system on luteal cells has been the focus of a variety of studies. Norepinephrine (NE) increased progesterone secretion of cattle luteal cells by activating ß-adrenoceptors. In this study, murine luteal cells were treated with NE and isoprenaline (ISO). We found that NE increased the viability of murine luteal cells and ISO decreased the viability of luteal cells. Both NE and ISO promoted the progesterone production. Nonselective ß-adrenergic antagonist, propranolol reversed the effect of ISO on cell viability but did not reverse the effect of NE on cell viability. Propranolol blocked the influence of NE and ISO on progesterone production. These results reveal that the increase of luteal cell viability induced by NE is not dependent on ß-adrenergic activation. α-Adrenergic activation possibly contributes to it. Both NE and ISO increased progesterone production through activating ß-adrenergic receptor. Further study showed that CyclinD2 is involved in the increase of luteal cell induced by NE. 3ß-Hydroxysteroid dehydrogenase, LHR, steroidogenic acute regulatory protein (StAR), and PGF2α contribute to the progesterone production induced by NE and ISO.

[INFLUENCE OF INHIBITION OF ACTIN POLYMERIZATION ON ADIPOGENIC DIFFERENTIATION OF RAT Achilles-DERIVED TENDON STEM CELLS IN VITRO].

OBJECTIVE: To investigate the effect of cytoskeleton modification on the adipogenic differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. METHODS: TSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. After the 3rd passage cells were cultured with DMEM medium containing 15% fetal bovine serum and cytochalasin D (CYD) at the concentrations of 0, 50, 100, 500, and 1 000 ng/mL, the cell survival condition and morphology changes were observed by inverted phase contrast microscope, the cytoskeleton was observed through fibrous actin (F-actin) staining, and the ratio of F-actin/ soluble globular actin (G-actin) was detected and calculated through Western blot. According to the above results, the effective concentration of CYD was selected and used for next experiments. After TSCs were cultured for 3 and 7 days respectively with adipogenic induction media (induction group), adipogenic induction media containing CYD (CYD+induction group), ordinary medium (ordinary group), and ordinary medium containing CYD (CYD+ordinary group), the real-time quantitative PCR (qRT-PCR) and Western blot were carried out to measure the mRNA and protein expressions of adipogenic differentiation-related markers, including peroxisome proliferator-activated receptor y (PPARγ), lipoprotein lipase (LPL), and fatty acid binding protein (aP2). RESULTS: The final CYD concentration of 100 ng/mL can inhibit effectively G-actin polymerization into F-actin, but could not affect TSCs survival, which was used for next experiments. qRT-PCR and Western blot suggested that the mRNA expressions of PPARγ, LPL, and aP2 and the protein expressions of PPARγ and aP2 were increased significantly in the CYD+induction group at 3 and 7 days when compared with the induction group (P < 0.05). In the CYD+ordinary group, there still was a significant increase in the mRNA expressions of PPARγ, LPL, and aP2 when compared with the ordinary group (P < 0.05). CONCLUSION: Inhibition of F-actin polymerization can increase adipogenic differentiation of rat Achilles-derived TSCs in vitro, and cytoskeleton modification is a pre-requisite for TSCs differentiation into adipocytes, which might have important implications for the mechanism research of tendinopathy.

CXCL1 gene silencing in skin using liposome-encapsulated siRNA delivered by microprojection array.

The barrier morphology of skin provides major obstacles for the application of siRNA for gene silencing, which current delivery technologies do not effectively overcome. Emerging technologies utilise microprojection array devices to penetrate into the skin epidermis and dermis for delivery of drug payloads. Delivery of siRNA by such devices has been proven in principle, yet requires optimisation for clinical applications. Herein, we demonstrate the use of Nanopatch™ microprojection arrays to deliver liposome-encapsulated siRNA to overcome skin barrier, and in vivo siRNA delivery hurdles. This application provided effective silencing of CXCL1 expression induced by the co-delivery of Fluvax 2012® by microprojection array. Liposomes encapsulating siRNA were dry-coated onto microprojection arrays, and remained intact after elution from arrays in vitro. Microprojection arrays facilitated the delivery of fluorescently-labelled nucleic acids through murine ear stratum corneum to the epidermis and dermis, with diffusion from microprojections into adjacent skin evident within 30s. CXCL1 mRNA, induced by delivery of Fluvax by microprojection array, was reduced by 75% up to 20 h post-treatment by co-delivery of liposome-encapsulated CXCL1-specific siRNA, but not by arrays co-delivering liposome-encapsulated control siRNA. CXCL1 protein expression in explant cultures from skin treated with arrays bearing CXCL1 specific or control siRNA was similarly reduced. These results as a test case have many implications for gene silencing in skin and inflammation, with the benefit of targeted delivery using microprojection arrays to deliver liposome-encapsulated siRNA.

Vitamin D-binding protein controls T cell responses to vitamin D.

BACKGROUND: In vitro studies have shown that the active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), can regulate differentiation of CD4+ T cells by inhibiting Th1 and Th17 cell differentiation and promoting Th2 and Treg cell differentiation. However, the serum concentration of 1,25(OH)2D3 is far below the effective concentration of 1,25(OH)2D3 found in in vitro studies, and it has been suggested that 1,25(OH)2D3 must be produced locally from the inactive precursor 25-hydroxyvitamin D3 (25(OH)D3) to affect ongoing immune responses in vivo. Although it has been reported that activated T cells express the 25(OH)D-1α-hydroxylase CYP27B1 that converts 25(OH)D3 to 1,25(OH)2D3, it is still controversial whether activated T cells have the capacity to produce sufficient amounts of 1,25(OH)2D3 to affect vitamin D-responsive genes. Furthermore, it is not known how the vitamin D-binding protein (DBP) found in high concentrations in serum affects T cell responses to 25(OH)D3. RESULTS: We found that activated T cells express CYP27B1 and have the capacity to produce sufficient 1,25(OH)2D3 to affect vitamin D-responsive genes when cultured with physiological concentrations of 25(OH)D3 in serum-free medium. However, if the medium was supplemented with serum or purified DBP, DBP strictly inhibited the production of 1,25(OH)2D3 and 25(OH)D3-induced T cell responses. In contrast, DBP did not inhibit the effect of exogenous 1,25(OH)2D3. Actin, arachidonic acid and albumin did not affect the sequestration of 25(OH)D3 by DBP, whereas carbonylation of DBP did. CONCLUSIONS: Activated T cells express CYP27B1 and can convert 25(OH)D3 to 1,25(OH)2D3 in sufficiently high concentrations to affect vitamin D-responsive genes when cultured in serum-free medium. However, DBP sequesters 25(OH)D3 and inhibits the production of 1,25(OH)2D3 in T cells. To fully exploit the immune-regulatory potential of vitamin D, future studies of the mechanisms that enable the immune system to exploit 25(OH)D3 and convert it to 1,25(OH)2D3 in vivo are required.

Mapping interactions between myosin relay and converter domains that power muscle function.

Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile(508), Asn(509), and Asp(511)) in communicating with converter domain residue Arg(759). We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle.

Estrogen and its receptor enhance mechanobiological effects in compressed bone mesenchymal stem cells.

Mechanical stimulation and estrogen have been proven to be two important factors in promoting mesenchymal stem cell activity, which is closely associated with bone formation, mass maintenance and remodeling. However, the superposition effects of mechanical stimulation and estrogen on stem cells remain unknown. It is also unclear if the estrogen receptor (ER) plays only a key role in estrogen signaling or if it is also involved in the mechanotransduction of stem cells. To investigate the role of estrogen and its receptors in the mechanobiological effects in bone mesenchymal stem cells (BMSCs), isolated mesenchymal stem cells from bone marrow were exposed to mechanical pressure under additional estrogen treatment or ER blockade. Cell proliferation was examined using an MTT assay and alkaline phosphatase (ALP) activity was determined by a modified enzyme kinetic method. Alignment of the cytoskeleton was observed by Coomassie brilliant blue staining and F-actin fluorescent staining. Cellular ultrastructure was observed under transmission electron microscope. Expression of ERα was investigated using Western blot analysis. Results indicated that mechanical pressure promoted cell proliferation, ALP activity, ERα expression and F-actin stress fiber formation. Overall, this effect was enhanced by the addition of estrogen and inhibited by ER blockade. We concluded that pressure stimulated proliferation and differentiation capability via F-actin transduction in BMSCs. The effects were enhanced by the addition of estrogen, and the ER plays an important role in regulating mechanobiological effects and the mechanotransduction processes of BMSCs.

Functional characteristics of amphioxus troponin in regulation of muscle contraction.

Troponin regulates contraction of vertebrate striated muscle in a Ca(2+)-dependent manner. More specifically, it acts as an inhibitor of actin-myosin interaction in the absence of Ca(2+) during contraction. In vertebrates, this regulatory mechanism is unlike that in some less highly derived taxa. Troponin in the smooth muscle of the protochordate ascidian species Halocynthia roretzi regulates actinmyosin contraction as an activator in the presence of Ca(2+), not as an inhibitor in the absence of Ca(2+) as is the case in vertebrates. In this study, contractile regulation of striated muscle from another protochordate, the amphioxus Branchiostoma belcheri, was analyzed using recombinant troponin components TnT, TnI, and TnC that were produced in an Escherichia coli expression system to further elucidate their roles in Ca(2+)-dependent regulation of the actin-myosin interaction. Combination of these troponin components in an actin-myosin ATPase activity assay showed that troponin in amphioxus striated muscle functions in a similar manner to troponin in vertebrate striated muscle, and differently from ascidian smooth muscle troponin. Thus, troponin function appears to have evolved differently in different protochordate muscles.