Rhodamine-phalloidin Staining Research Articles

Cardioprotective effects of transplanted adipose-derived stem cells under Ang II stress with Danggui administration augments cardiac function through upregulation of insulin-like growth factor 1 receptor in late-stage hypertension rats.

In aging hypertensive conditions, deterioration of insulin-like growth factor 1 receptor (IGF1R) cause a pathological impact on hypertensive hearts with an increased Ang II level. Recovering these adverse conditions through transplanted adipose-derived stem cells is a challenging approach. Moreover, Danggui, a Traditional Chinese medicine (TCM), is used for the treatment of cardioprotective effects. In this study, to evaluate whether the combined effect of MSCs and TCM can recover the cardiac function in late-stage hypertension rats. We observed that lower dose of Danggui crude extract treatment showed an increased level of cell viability with maintained stemness properties and growth rate in rat adipose-derived stem cells (rADSCs). Further, we cocultured the H9c2 cells with rADSCs and the results revealed that Danggui-treated MSCs enhanced the IGF1R expression and attenuated the hypertrophy in H9c2 cells against Ang II challenge by immunoblot and rhodamine-phalloidin staining. In addition, Danggui crude extract was also quantified and characterized by HPLC and LC-MS analysis. Furthermore, the in vivo study was performed by considering 11 months old rats (n = 7). Importantly, the oral administration of Danggui crude extract with stem cells intravenous injection in SHR-D-ADSCs group showed a combination effect to augment the cardiac function through enhancement of ejection fraction, fractional shortening, contractility function in the late-stage hypertension conditions. We have also observed a decreased apoptosis rate in the heart tissue of SHR-D-ADSCs group. Taken together, these results indicate that the combinatorial effects of Danggui crude extract and stem cell therapy enhanced cardiac function in late-stage SHR rats.

The Role of 5-HT on Proplatelet Formation and Thrombopoietin Production

Background: Our previous work confirmed that serotonin (5-HT) promotes the proliferation of hemopoietic stem cells and megakaryocytes (Yang M et al, Stem Cells, 2007; 2014). However, the mechanisms remain indefinite. Methods: Q-PCR, Flow Cytometry, Western Blot, or Immunofluorescence microscope were used in the receptor and TPO study. MTT/CCK-8, Proplatelet assay, and Flow Cytometry were also used in cell proliferation and apoptosis study. The relationship between 5-HT and TPO was studied in a traumatic stress mice model. Results: In-vitro study, there was a stimulating effect of 5-HT on proplatelet formation in human bone marrow megakaryocytes. Human BM MK progenitors cultured in serum-free medium with either 5-HT (200nM) or TPO (100 ng/ml) had more proplatelet bearing MKs than the control group (5-HT (12.3 ± 5.0)% vs. Control (6.2 ± 3.5)%, P=0.025; TPO (15.6 ± 2.5)% vs. Control, P=0.04; n=4). The 5-HT treatment group showed more mature and more in the final stage MK cells as compared to the TPO group. 5-HT2A, 2B, 2C receptors were detected in the surface of megakaryocytes. The effect of 5-HT on proplatelet formation in MK cells was via 5-HT2 receptors and this effect was reduced by 5-HT2 receptor inhibitor ketanserin. 5-HT acted on cytoskeleton reorganization in MKs via 5-HT2 receptors and ERK1/2 pathway. Using an immunofluorescence microscope with F-actin specific binder rhodamine-phalloidin staining, the polymerized actin level was lower in the control group than the 5-HT group and actin distributed diffusely throughout the cytoplasm. In contrast, the polymerization actin level was higher in the 5-HT group. Adding ketanserin and ERK1/2 inhibitor PD98059 to 5-HT treatment, the fluorescence intensity was correspondingly reduced. Our data also demonstrated that ERK1/2 was activated in MKs treated with 5-HT for 30 minutes. In a traumatic stress mice model, both of 5-HT and TPO were increased, but the increasing of TPO is posterior to 5-HT. After added LX1606, the synthesis inhibitor of 5-HT, 5-HT was reduced markedly, as well as TPO. The expression of TPO mRNA and the production of TPO protein were increased as compared with the control in this model. Conclusions: This study suggests that 5-HT promotes thrombopoiesis from two aspects: one is the direct effect on megakaryocytes. 5-HT could promote the proplatelet formation from megakaryocytes. The second is the indirect effect by promoting the production of TPO, which is a paracrine secretion to influence thrombopoiesis. Disclosures No relevant conflicts of interest to declare.

Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells.

We previously showed that in polarized Madin–Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

Cathepsin‐B is a Mediator of Barrier Dysfunction and Hyperpermeability in Blood‐Brain Barrier Endothelial Cells

Cathepsin B belongs to the papain‐like family of cysteine proteases and is an upstream mediator of the NLRP3 inflammasome pathway that leads to interleukin 1β (IL‐1β) secretion. Interleukin 1β is a potent inducer of tight junction disruption and hyperpermeability in blood‐brain barrier (BBB) endothelial cells. However, the involvement of cathepsin B in BBB dysfunction and hyperpermeability is not clearly known. Understanding this pathway is critical to preserving BBB integrity and protection against brain edema. In order to study this relationship, rat brain microvascular endothelial cells were transfected with active cathepsin B. Tight junction organization and changes in permeability were evaluated based on immunofluorescence, immunoblot analysis of zonula occudens‐1 (ZO‐1) and Transwell monolayer permeability assay using FITC‐dextran (10‐kDa). Cathepsin B activity and cell viability were evaluated fluorometrically. F‐actin stress fiber formation was studied using rhodamine phalloidin staining. Active cathepsin B induced ZO‐1 dislocation/tight junction disorganization, F‐actin stress fiber formation and monolayer hyperpermeability significantly. These effects were inhibited by pretreatment with NLRP3 inhibitors isoliquiritigenin and MCC950. Active cathepsin B had no significant effect on ZO‐1 protein expression or cell viability. Furthermore, cathepsin B inhibitor E‐64d attenuated hydrogen peroxide‐induced cathepsin B activity and monolayer hyperpermeability significantly. These results suggest that cathepsin B is a mediator of BBB dysfunction/hyperpermeability. Cathepsin B inhibition has protective effects against barrier dysfunction and hyperpermeability in BBB endothelial cells. One of the mechanisms by which cathepsin B induces barrier dysfunction is via the NLRP3 inflammasome pathway.

Effect of polymer functionalized fullerene soot on C6 glial cells

Fullerene is crafting its dominance in various fields of science. Advanced exploration on the applicability of fullerene and the enhanced chances of exposure has prompted valuation of its biocompatibility. The antioxidant and radical scavenging characteristic of fullerene raises assurance for various biomedical applications. Nevertheless, the hydrophobicity of fullerene lowers its charm in healthcare applications where stable water dispersity is fundamental. In this study, one of the highly exposed forms of fullerenes; fullerene soot was subjected to in vitro toxicity evaluation. For nullify hydrophobic consistency, soot nanoparticles were surface functionalized with dextran polymer. Even though various tissue-specific toxicity evaluations of fullerene are been reported, the idea about its effect on the neuronal tissue still remains vague. In the context, the well-characterized dextran-coated fullerene soot (Dex-FS) nanoparticles were exposed to C6 glial cells to understand the cellular response of Dex-FS. Glial cells are present in plenty in the central nervous system (CNS) and provide support and insulation to neurons. Hence, glial cells turn out to be an ideal choice for in vitro CNS system. Dex-FS showed dose-dependent toxicity. The integrity of cytoskeleton was found to be compromised. Dex-FS was found to trigger ROS production in a dose and time dependent manner. The lysosomal activity and mitochondrial membrane potential during post Dex-FS treatment was evaluated using acridine orange and rhodamine-phalloidin staining. The molecular level toxicity was evaluated by DNA laddering and DAPI staining. The amount of live and dead cells after treating C6 glial cells with Dex-FS was estimated by Calcein AM/ PI flow cytometry. The neurotoxic impact of fullerene soot nanoparticles explored in the present study thus presents a debatable topic regarding vehicle exhaust which contains higher amounts of fullerene soot as active ingredient.

Potential Role of Integrin α₅β₁/Focal Adhesion Kinase (FAK) and Actin Cytoskeleton in the Mechanotransduction and Response of Human Gingival Fibroblasts Cultured on a 3-Dimension Lactide-Co-Glycolide (3D PLGA) Scaffold.

BackgroundThe stability of orthodontic treatment is thought to be significantly affected by the compression and retraction of gingival tissues, but the underlying molecular mechanism is not fully elucidated. The objectives of our study were to explore the effects of mechanical force on the ECM-integrin-cytoskeleton linkage response in human gingival fibroblasts (HGFs) cultured on 3-dimension (3D) lactide-co-glycolide (PLGA) biological scaffold and to further study the mechanotransduction pathways that could be involved.Material/MethodsA compressive force of 25 g/m2 was applied to the HGFs-PLGA 3D co-cultured model. Rhodamine-phalloidin staining was used to evaluate the filamentous actin (F-actin) cytoskeleton. The expression level of type I collagen (COL-1) and the activation of the integrin α5β1/focal adhesion kinase (FAK) signaling pathway were determined by using real-time PCR and Western blotting analysis. The impacts of the applied force on the expression levels of FAK, phosphorylated focal adhesion kinase (p-FAK), and COL-1 were also measured in cells treated with integrin α5β1 inhibitor (Ac-PHSCN-NH 2, ATN-161).ResultsMechanical force increased the expression of integrin α5β1, FAK (p-FAK), and COL-1 in HGFs, and induced the formation of stress fibers. Blocking integrin α5β1 reduced the expression of FAK (p-FAK), while the expression of COL-1 was not fully inhibited.ConclusionsThe integrin α5β1/FAK signaling pathway and actin cytoskeleton appear to be involved in the mechanotransduction of HGFs. There could be other mechanisms involved in the promotion effect of mechanical force on collagen synthesis in addition to the integrin α5β1 pathway.

Chloroxoquinoline inhibits invasion in breast cancer via down-regulating Rho/Rho kinase signaling pathway

To investigate the effect of chloroxoquinoline on cytoskeleton of breast cancer cells and its relation with Rho/Rho kinase signaling pathway. Breast cancer Bcap37 and MDA-MB-453 cells were treated with different concentrations of chloroxoquinoline. Wound healing and Transwell assay were conducted to detect cell migration and invasion, respectively. Rhodamine-phalloidin staining and immunofluorescent staining were used to observe the polymerization state of F-actin and the expression of α-Tublin in breast cancer cells, respectively. Western blot was used to detect the phosphorylation level of key protein in Rho/Rho kinase signaling pathway. Compared with the control group, chloroxoquinoline treatment induced dose-dependent decrease in cell migration and invasion, and Bcap37 and MDA-MB-453 cells treated with chloroxoquinoline showed dose-dependent changes in cell morphology and decrease in cell body. The staining of F-actin and α-Tublin was irregular and clustered. Furthermore, treatment of chloroxoquinoline down-regulated the phosphorylation of the Rho/Rho kinase signaling proteins Cofilin, Limk and Rock2 (all P<0.01). Chloroxoquinoline inhibits the cytoskeleton in breast cancer Bcap37 and MDA-MB-453 cells and inhibits cell migration. This effect may be associated with down-regulation of Rho/Rho kinase signaling pathway.

An osteogenic helioxanthin derivative suppresses the formation of bone-resorbing osteoclasts.

ObjectiveThe helioxanthin derivative 4-(4-methoxyphenyl)thieno[2,3-b:5,4-c′]dipyridine-2-carboxamide (TH) is a low-molecular-weight compound that was identified through screening for osteogenic compounds that enhance the activity of mouse preosteoblastic MC3T3-E1 cells. In the present study, we found that TH suppressed osteoclast differentiation.MethodsUsing the hematopoietic stem cells of ddY mice, TH was added to the culture in the experimental group, and the number of osteoclasts was measured with rhodamine phalloidin staining and TRAP staining. In osteo assay, bone resorption area was compared by the von Kossa staining.ResultsSpecifically, TH inhibited the cyclic guanosine monophosphate (cGMP)-degrading activity of phosphodiesterase (PDE), promoted nitric oxide (NO) production, and dose-dependently suppressed osteoclast differentiation in an osteoclast formation culture of mouse bone marrow cells. The NO-competitive guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) attenuated the suppressive activity of TH on osteoclast differentiation. Conclusion: Given the previously reported suppressive action of cGMP on osteoclastogenesis, our data suggest that TH negatively impacts osteoclast differentiation at least to some extent by stimulating NO production and inhibiting PDE activity, both of which lead to the upregulation of intracellular cGMP. This study supports the potential use of TH as a novel antiosteoporotic reagent that not only stimulates bone formation but also inhibits bone resorption.

Engineering of L-Plastin Peptide-Loaded Biodegradable Nanoparticles for Sustained Delivery and Suppression of Osteoclast Function In Vitro.

We have recently demonstrated that a small molecular weight amino-terminal peptide of L-plastin (10 amino acids; “MARGSVSDEE”) suppressed the phosphorylation of endogenous L-plastin. Therefore, the formation of nascent sealing zones (NSZs) and bone resorption are reduced. The aim of this study was to develop a biodegradable and biocompatible PLGA nanocarrier that could be loaded with the L-plastin peptide of interest and determine the efficacy in vitro in osteoclast cultures. L-plastin MARGSVSDEE (P1) and scrambled control (P3) peptide-loaded PLGA-PEG nanoparticles (NP1 and NP3, respectively) were synthesized by double emulsion technique. The biological effect of nanoparticles on osteoclasts was evaluated by immunoprecipitation, immunoblotting, rhodamine-phalloidin staining of actin filaments, and pit forming assays. Physical characterization of well-dispersed NP1 and NP3 demonstrated ~130-150 nm size, < 0.07 polydispersity index, ~-3 mV ζ-potential, and a sustained release of the peptide for three weeks. Biological characterization in osteoclast cultures demonstrated the following: NP1 significantly reduced (a) endogenous L-plastin phosphorylation; (b) formation of NSZs and sealing rings; (c) resorption. However, the assembly of podosomes which are critical for cell adhesion was not affected. L-plastin peptide-loaded PLGA-PEG nanocarriers have promising potential for the treatment of diseases associated with bone loss. Future studies will use this sustained release of peptide strategy to systematically suppress osteoclast bone resorption activity in vivo in mouse models demonstrating bone loss.

Dopamine Regulates Blood‐Brain Barrier Endothelial Cell Hyperpermeability

Blood‐Brain Barrier (BBB) dysfunction leading to hyperpermeability is one of the major causes of brain edema following conditions such as traumatic and ischemic brain injury. The BBB consists of microvascular endothelial cells that are linked to each other by tight junction and adherens junction proteins and are intracellularly linked to actin cytoskeletal assembly of the cell mainly via zonula occludens‐1 (ZO‐1). Recent studies suggest that the activation of the NLRP3 inflammasome pathway has a major role promoting various pathologies associated with brain injury. An inflammasome is a multiprotein complex that is responsible for the activation of inflammatory responses in the body. Recent studies show that the neurotransmitter, dopamine (DA) is an inhibitor of this pathway. Dopamine regulates bodily functions such as movement, behavior and many systemic functions, but also connects the nervous and immune system in the body. In this study we investigated the effects of DA for its protective effect against oxidative stress‐induced BBB dysfunction and hyperpermeability. In the experiments, hydrogen peroxide (H2O2) was used to induce oxidative stress in rat brain microvascular endothelial cells. Oxidative stress due to increased reactive oxygen species (ROS) formation occurs following trauma and ischemia. Rat brain microvascular endothelial cell monolayers were exposed to H2O2 alone (100μM; 2 hour) or in presence of DA (10μg/ml; 30min) or dopamine D1 receptor agonist (A68930; 10μg/ml; 30min). The changes in tight junction integrity and cytoskeletal assembly were studied using ZO‐1 and β‐catenin immunofluorescence and rhodamine phalloidin staining for F‐actin respectively. Cell viability was studied using Calcein AM cell viability assay. Changes in the formation ROS were studied using 2′, 7′‐dichlorofluorescien diacetate. H2O2 (100μM) treatment resulted in significant increase in monolayer permeability (p&lt;0.05), ROS formation (p&lt;0.05), disruption of the endothelial tight/adherens junctions and induced F‐actin stress fiber formation. Dopamine and A68930 treatment resulted in a decrease in H2O2‐induced monolayer hyperpermeability significantly (p&lt;0.05). Furthermore, DA protected tight junction integrity and actin cytoskeletal assembly. DA and A68930 had no effect on H2O2‐induced ROS formation. The cell viability assay showed that H2O2‐induced barrier disruption is independent of cell death. Our results suggest that one of the major mechanisms for DA‐mediated protection against barrier dysfunction/hyperpermeability may be mediated via inhibiting the NLRP3 inflammasome pathway and not by inhibiting increased ROS formation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Growth-regulated oncogene-α from oral submucous fibrosis fibroblasts promotes malignant transformation of oral precancerous cells.

Oral squamous cell carcinoma (OSCC) is a common human malignancy and is usually preceded by the oral precancerous lesions. Oral submucous fibrosis (OSF) is one of the oral precancerous lesions with high incidence of malignant transformation. In addition to cancer cells, cancer-associated fibroblasts in the tumor microenvironment are correlated with cancer progression, but the role of fibroblasts from OSF in tumorigenesis and progression is still unknown. Growth-regulated oncogene-α (GRO-α), a member of CXC chemokine family, is related to tumorigenesis in several cancers. In this study, we would like to explore the role of GRO-α from OSF-associated fibroblasts in oral cancer progression. We isolated primary culture fibroblasts of normal, precancerous, and tumor tissues from patients with OSCC accompanied with OSF. A cytokine array was used to screen cytokine secretions in the conditioned media of the fibroblasts. A wound healing migration assay, WST-1 cell proliferation assay, rhodamine-phalloidin staining, and soft agar colony formation assay were used to investigate the effects of GRO-α on a dysplastic oral keratinocyte cell line (DOK) cell migration, growth, and anchorage-independent growth. GRO-α was identified to be increased in conditioned media of OSF-associated fibroblasts. GRO-α promotes DOK cells proliferation, migration, and anchorage-independent growth through enhancing the EGFR/ERK signaling pathway, F-actin rearrangement, and stemness properties, respectively. Moreover, GRO-α neutralizing antibodies downregulated the conditioned medium-induced cell proliferation and migration of DOK. GRO-α from OSF-associated fibroblasts paracrinally promotes oral malignant transformation and significantly contributes to OSCC development.

Eriobotrya japonica ameliorates cardiac hypertrophy in H9c2 cardiomyoblast and in spontaneously hypertensive rats

Eriobotrya japonica (EJ) is a traditional Chinese plant with high medicinal value. EJ extracts are reported to exhibit antioxidant and anti-inflammatory biological attributes. The current study aims to evaluate the prospective efficacy of E. japonica leave extract (EJLE) against Angiotensin-II induced cardiac hypertrophy in H9c2 cardiomyoblast and in spontaneously hypertensive rats (SHRs). For the in vitro studies, Angiotensin-II pretreated H9c2 cells were treated with EJLE and analyzed through Western blotting and rhodamine phalloidin staining for their cardio-protective attributes. In the in vivo studies, 12-week-old SHRs were randomly divided into groups: SHRs supplemented with EJLE, control SHR group supplemented with PBS; in addition, a control group of Wistar-Kyoto rats (WKY) was also employed. All rats were supplemented twice a week for 8 week time interval. Finally, echocardiography, morphological, histology, and Western blot analysis were performed to assess their role against cardiac hypertrophy. Interestingly, we could observe that supplementation of EJLE could rescue Ang-II induced cardiac hypertrophy as evident through Western blot, rhodamine phalloidin staining, and Hematoxylin-Eosin staining. Notably, morphological and echocardiography data provided further supports for their ability to ameliorate cardiac characteristics. Cumulatively, the results clearly suggests that supplementation of EJLE promotes cardio-protective effects through amelioration of cardiac hypertrophy in vitro and in vivo.

Abstract LB-211: The cardiac transcription factor Nkx2-5 contributes to SRF-dependent AR action in prostate cancer

Abstract Ligand-activated androgen receptor (AR) drives prostate cancer (CaP) growth. Heterogeneity in AR action exists and AR often remains active when therapies targeting its ligand-activation have failed. Understanding the mechanisms by which AR mediates CaP progression may lead to new treatments. Previously, we identified a mechanism in which AR imparts androgen-responsiveness to the transcription factor Serum Response Factor (SRF). SRF-dependent AR action is enriched in CaP, correlates with CaP recurrence and is maintained in late-stage CaP. SRF binds constitutively at its target genes and becomes transcriptionally active via upstream signaling cascades or SRF cofactors. Our prior work showed that RhoA signaling transduces androgen regulation to a significant fraction, but not all, SRF target genes. Here, we isolate the homeodomain protein Nkx2-5 as a novel regulator of androgen-responsive SRF action. Nkx2-5 co-operates with SRF in transcriptional control of heart development. In CaP, Nkx2-5 promoter hypermethylation is a biomarker that distinguishes CaP from benign prostate, yet consequences of differential Nkx2-5 expression on CaP are unknown. In the CaP cell models LNCaP, C4-2 and VCaP, siRNA-mediated loss of Nkx2-5 increased basal and androgen-dependent expression of exogenously and endogenously expressed SRF target genes, and these effects were reversed after silencing of SRF. Conversely, overexpression of Nkx2-5 decreased androgen-responsiveness of SRF target genes. Co-IP and ChIP assays indicated androgen-stimulated SRF-Nkx2-5 interaction in CaP cell nuclei. Loss of Nkx2-5 resulted in distinct morphological changes in CaP cells, which depended entirely on SRF. In rhodamine phalloidin staining assays, loss of Nkx2-5 increased significantly (p&amp;lt;0.01) cell length, width and circumference, and the number of cell protrusions in cultures with and without androgen stimulation. Changes in cell shape were accompanied by modest decreases in CaP cell proliferation and migration, but significant (p&amp;lt;0.05) decreases in CaP cell-matrix adhesion. Nkx2-5 silencing increased the size of the cell nucleus and led to multilobed and grooved nuclei and enlarged nucleoli. These nuclear shape changes were not associated with alterations in DNA ploidy, but were associated with changed heterochromatin organization as reflected in a reduction in heterochromatin protein 1 immunostaining and nuclear content. Such Nkx2-5-dependent changes in nuclear shape and size and heterochromatin organization occurred also in non-malignant prostate epithelial RWPE2 cells. This work uncovered a novel role for Nkx2-5 in the transition from benign prostate to CaP, showing that Nkx2-5 regulates SRF transcriptional activity in CaP cells and demonstrating that differential Nkx2-5 expression alters cell-matrix adhesion and nuclear morphology, both processes that are associated with aggressive CaP progression. Citation Format: Salma Ben-Salem, Dhirodatta Senapati, Giridhar Mudduluru, Yixue Su, Varadha Balaji Venkadakrishnan, Hannelore V. Heemers. The cardiac transcription factor Nkx2-5 contributes to SRF-dependent AR action in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-211.

The Antioxidant Effect of Rosmarinic Acid by Different Delivery Routes in the Animal Model of Noise-Induced Hearing Loss.

Trans-tympanic Rosmarinic Acid (RA), as compared with the systemic administration, protects against noise-induced auditory hair cell and hearing losses in rats in vivo. ROS production, lipoperoxidative damage, and an imbalance of antioxidant defences play a significant role in noise-induced hearing loss. Several molecules with antioxidant properties have been tested to restore redox homeostasis; however, drug delivery system represents a challenge for their effectiveness. In our model, acute and intense noise exposure induces hearing loss, hair cell death, and oxidative stress, with an increase in superoxide production and over-expression of lipid peroxidation in cochlear structures. RA was administrated in male Wistar rats by trans-tympanic (20 μl) and systemic (10 mg/kg) modality. In systemic administration, RA was injected 1 hour before noise exposure and once daily for the following 3 days. ABRs were measured before and at days 1, 3, 7, and 30 after noise exposure. Rhodamine-phalloidin staining, dihydroethidium and 8-isoprostane immunostainings were performed to assess and quantify outer hair cells loss, superoxide production, and lipid peroxidation in the different experimental groups. Systemic RA administration significantly decreased noise-induced hearing loss and the improvement of auditory function was paralleled by a significant reduction in cochlear oxidative stress. The trans-tympanic modality of drug administration showed a similar degree of protection both at the functional and morphological levels. The effectiveness of RA given via trans-tympanic injection could be interesting for the future application of this minimally-invasive procedure in the treatment of ROS-induced hearing loss.

Effects of denatured collagen type Ⅰ on differentiation of human fibroblasts into myofibroblasts

Objective: To investigate the effects of denatured collagen type Ⅰ on differentiation of human fibroblasts into myofibroblasts. Methods: A small amount of normal skin donated by burn patients undergoing scar surgery was collected. Human fibroblasts were obtained by method of explant culture and then sub-cultured. The fourth passage of cells were used in the following experiments. (1) Fibroblasts were divided into normal collagen group and denatured collagen group according to the random number table, with 10 wells in each group. Fibroblasts in normal collagen group were cultured on normal collagen type Ⅰ coated coverslips. Fibroblasts in denatured collagen group were cultured on denatured type Ⅰ collagen coated coverslips. Expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemical method, and the percentage of PCNA positive cells was calculated. (2) Another batch of fibroblasts were grouped and treated as in (1), with 12 wells in each group. Proliferation activity of cells was determined with methyl-thiazolyl-tetrazolium colorimetry method. (3) Another batch of fibroblasts were grouped and treated as in (1), and the microfilament morphology of cells was observed by rhodamine-phalloidin staining. (4) Another batch of fibroblasts were grouped and treated as in (1). Expression of α smooth muscle actin (α-SMA) of cells was detected by immunohistochemical method, and expression of OB-cadherin of cells was detected by immunofluorescence method. (5) Another batch of fibroblasts were divided into normal collagen, denatured collagen, and common coverslips groups according to the random number table, with 6 wells in each group. Fibroblasts in normal collagen and denatured collagen groups were treated as in (1), while fibroblasts in common coverslips group were cultured on coverslips without collagen coating. Expressions of α-SMA and OB-cadherin of cells were determined with Western blotting. (6) Another batch of fibroblasts were grouped and treated as in (5), and then the mRNA expressions of collagen type Ⅰ, collagen type Ⅲ, and α-SMA of cells were determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with t test, one way analysis of variance, and least-significant difference test. Results: (1) The percentage of PCNA positive cells in denatured collagen group was (83±9)%, significantly higher than (29±9)% in normal collagen group (t=13.53, P<0.01). (2) The proliferation activity of fibroblasts in denatured collagen group was 0.32±0.06, significantly higher than 0.25±0.05 in normal collagen group (t=3.06, P<0.01). (3) The microfilament of fibroblasts in normal collagen group was arranged vertically and in parallel way, paralleling the long axis of cells. The microfilament of fibroblasts in denatured collagen group was denser and thicker. (4) Most fibroblasts in normal collagen group showed long shuttle-like shape typically. Morphology of fibroblasts in denatured collagen group changed, and cells were obviously spreading. Expressions of α-SMA and OB-cadherin of fibroblasts in denatured collagen group were stronger than those in normal collagen group. (5) Expressions of α-SMA of fibroblasts in denatured collagen, normal collagen, and common coverslips groups were respectively 1.69±0.41, 0.89±0.27, and 1.46±0.42. Expression of α-SMA of fibroblasts in denatured collagen group was significantly higher than that in normal collagen group (P<0.01). Expressions of OB-cadherin of fibroblasts in denatured collagen, normal collagen, and common coverslips groups were respectively 5.17±0.28, 2.21±0.10, and 4.01±0.56. Expression of OB-cadherin of fibroblasts in denatured group was significantly higher than that in normal collagen group (P<0.01). (6) There was no significant difference in mRNA expression of collagen type Ⅰ of fibroblasts in denatured collagen, normal collagen, and common coverslips groups (F=2.71, P>0.05). The mRNA expressions of collagen type Ⅲ and α-SMA of fibroblasts in normal collagen group were significantly lower than those in denatured collagen group (P<0.01). Conclusions: Denatured collagen type Ⅰ may influence the activity of fibroblasts, thus inducing fibroblasts differentiating into myofibroblasts.

Pathway Analysis Hints Towards Beneficial Effects of Long-Term Vibration on Human Chondrocytes

Background/Aims: Spaceflight negatively influences the function of cartilage tissue in vivo. In vitro human chondrocytes exhibit an altered gene expression of inflammation markers after a two-hour exposure to vibration. Little is known about the impact of long-term vibration on chondrocytes. Methods: Human cartilage cells were exposed for up to 24 h (VIB) on a specialised vibration platform (Vibraplex) simulating the vibration profile which occurs during parabolic flights and compared to static control conditions (CON). Afterwards, they were investigated by phase-contrast microscopy, rhodamine phalloidin staining, microarray analysis, qPCR and western blot analysis. Results: Morphological investigations revealed no changes between CON and VIB chondrocytes. F-Actin staining showed no alterations of the cytoskeleton in VIB compared with CON cells. DAPI and TUNEL staining did not identify apoptotic cells. ICAM-1 was elevated and vimentin, beta-tubulin and osteopontin proteins were significantly reduced in VIB compared to CON cells. qPCR of cytoskeletal genes, ITGB1, SOX3, SOX5, SOX9 did not reveal differential regulations. Microarray analysis detected 13 differentially expressed genes, mostly indicating unspecific stimulations. Pathway analyses demonstrated interactions of PSMD4 and CNOT7 with ICAM. Conclusions: Long-term vibration did not damage human chondrocytes in vitro. The reduction of osteopontin protein and the down-regulation of PSMD4 and TBX15 gene expression suggest that in vitro long-term vibration might even positively influence cultured chondrocytes.

Effect of different electrical stimulation waves on orientation and alignment of adipose derived mesenchymal stem cells

To investigate the effect of different electrical stimulation waves on orientation and alignment of adipose derived mesenchymal stem cells (ADSCs). ADSCs were isolated from 5-week-old Sprague Dawley rats (weight, 100-150 g) and cultivated. The cells at passages 3-5 were inoculated to prepare cell climbing slices, subsequently was exposed to direct-current electrical stimulations (ES) at electric field strengths of 1, 2, 3, 4, 5, and 6 V/cm on a homemade electric field bioreactor (groups A1, A2, A3, A4, A5, and A6); at electric field strength of 6 V/cm, at 50% duty cycle, and at frequency of 1 and 2 Hz (groups B1 and B2) of square wave ES; at electric field strength of 6 V/cm, at pulse width of 2 ms, and at frequency of 1 and 2 Hz (groups C1 and C2) of biphasic pulse wave ES; and no ES was given as a control (group D). The changes of cellular morphology affected by applied ES were evaluated by time-lapse micropho-tography via inverted microscope. The cell alignment was evaluated via average orientation factor ( OF). The cytoske-leton of electric field treated ADSCs was characterized by rhodamine-phalloidin staining. The cell survival rates were assessed via cell live/dead staining and intracellular calcium activities were detected by calcium ion fluorescent staining. The response of ADSCs to ES was related to the direct-current electric field intensity. The higher the direct-current electric field intensity was, the more cells aligned perpendicular to the direction of electric field. At each time point, there was no obvious cell alignment in groups B1, B2 and C1, C2. The average OF of groups A5 and A6 were significantly higher than that of group D ( P<0.05), but no significant difference was found between other groups and group D ( P>0.05). The cytoskeleton staining showed that the cells of groups A5 and A6 exhibited a compact fascicular structure of cytoskeleton, and tended to be perpendicular to the direction of the electric field vector. The cellular survival rate of groups A4, A5, and A6 were significantly lower than that of group D ( P<0.05), but no significant difference was found between other groups and group D ( P>0.05). Calcium fluorescence staining showed that the fluorescence intensity of calcium ions in groups A4, A5, and A6 was slightly higher than that in group D, and no significant difference was found between other groups and group D. The direct-current electric field stimulations with physiological electric field strength (5 V/cm and 6 V/cm) can induce the alignment of ADSCs, but no cell alignment is found under conditions of less than 5 V/cm direct-current electric field, square wave, and biphasic pulse wave stimulation. The cellular viability is negatively correlated with the electric field intensity.

Biological evaluation of three-dimensional printed co-poly lactic acid/glycolic acid/tri-calcium phosphate scaffold for bone reconstruction

Objective: To biologically evaluate the three-dimensional(3D) printed co-poly lactic acid/glycolic acid/tri-calcium phosphate(PLGA/TCP) scaffold which could be used for repairing oral and maxillofacial bone defects, and to provide experimental evidence for its further research and clinical application. Methods: PLGA/TCP scaffolds were fabricated using low temperature rapid prototyping technique. Micro-CT and scanning electron microscope(SEM) were used to characterize the surface morphology. MC3T3-E1 cells were seeded onto the scaffold and stained with the rhodamine phalloidin and calcein acetomethoxy. After that, confocal laser scanning microscope was exploited to observe the features and viability of the cells. Moreover, the cells were co-cultured with the extract of PLGA/TCP and complete medium, respectively. The proliferation capability of the cells was assessed by the cell counting kit-8 (CCK-8) on the 1st, 2nd, and 3rd day. The PLGA/TCP scaffolds incorporated with recombinant human bone morphogenetic protein-2(rhBMP-2) of 0, 30, 60 μg(i.e. blank control group, low-dose group and high-dose group) were implanted into the latissimus dorsi muscle of the rats, and 6 weeks later, the samples were harvested to estimate the volume and pattern of new bone. Results: The 3D printed PLGA/TCP scaffold possessed a regular and well-defined porous stereo-structure with porosity of (73±3)%. Micro-CT and SEM showed that pore size were (379±32) and (453±29) μm respectively, and distance between layers were (452± 24) and (415±25) μm, and cylinder diameter were (342±24) and (350±28) μm. It also exhibited excellent cell adhesion and growth ability on the exterior and inner surface through rhodamine phalloidin and calcein acetomethoxy staining. The CCK-8 test demonstrated that the absorbance value of extract group on the 1st and 2nd day(0.51±0.08 and 0.63±0.09) were significantly higher than those in the blank control group(0.39± 0.05 and 0.53±0.05)(P<0.05), while there was no significant difference between the extract group(0.67±0.06) and the blank control group(0.68±0.04)(P>0.05) on the 3rd day. For in vivo test, there was obvious ectopic new bone formation on the PLGA/TCP scaffold incorporated with rhBMP-2, and this was demonstrated using the histological examination and micro-CT. The bone formation in the low-dose group was similar to the shape of the pre-implanted 3D printed scaffold, while much diversity was revealed in the high-dose group duo to over osteogenesis which was validated by the examinations of gross observation, histology and micro-CT. Conclusions: Customized PLGA/TCP scaffolds can be manufactured by 3D printing technique. The scaffold showed an excellent biocompatibility and ectopic osteogenesis when incorporated with rhBMP-2. However, further research is needed to validate it's effect on repairment of the oral and maxillofacial bone defects.

RhoA regulates Activin B-induced stress fiber formation and migration of bone marrow-derived mesenchymal stromal cell through distinct signaling

BackgroundIn our previous study, Activin B induced actin stress fiber formation and cell migration in Bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. However, the underlying molecular mechanisms are not well studied. RhoA is recognized to play a critical role in the regulation of actomyosin cytoskeletal organization and cell migration. MethodsPull-down assay was performed to investigate the activity of RhoA. The dominant-negative mutants of RhoA (RhoA(N19)) was used to determine whether RhoA has a role in Activin B-induced cytoskeleton organization and cell migration in BMSCs. Cytoskeleton organization was examined by fluorescence Rhodamine-phalloidin staining, and cell migration by transwell and cell scratching assay. Western blot was carried out to investigate downstream signaling cascade of RhoA. Inhibitor and siRNAs were used to detect the role of downstream signaling in stress fiber formation and/or cell migration. ResultsRhoA was activated by Activin B in BMSCs. RhoA(N19) blocked Activin B-induced stress fiber formation and cell migration. ROCK inhibitor blocked Activin B-induced stress fiber formation but enhanced BMSCs migration. Activin B induced phosphorylation of LIMK2 and Cofilin, which was abolished by ROCK inhibition. Both of siRNA LIMK2 and siRNA Cofilin inhibited Activin B-induced stress fiber formation. ConclusionsRhoA regulates Activin B-induced stress fiber formation and migration of BMSCs. A RhoA-ROCK-LIMK2-Cofilin signaling node exists and regulates actin stress fiber formation. RhoA regulates Activin B-induced cell migration independent of ROCK. General significanceBetter understanding of the molecular mechanisms of BMSCs migration will help optimize therapeutic strategy to target BMSCs at injured tissues.

Effects of transforming growth factor β1 receptor inhibitor SD-208 on human hypertrophic scar

To investigate the effects of transforming growth factor β1 (TGF-β1) receptor inhibitor SD-208 on human hypertrophic scar and its mechanisms. Scar fibroblasts were isolated from deprecated human hypertrophic scar tissue and then sub-cultured. Cells of the fifth passage were used in the following experiments. (1) Cells were divided into blank control group (BC) and 0.5, 1.0, 3.0, and 5.0 μmol/L SD-208 groups according to the random number table (the same grouping method below), with 6 wells in each group. Cells in group BC were added with 1 μL phosphate buffer solution, while cells in the latter four groups were added with 0.5, 1.0, 3.0, and 5.0 μmol/L SD-208, respectively. After being cultured for 12 hours, the proliferation activity of cells was detected by cell counting kit 8 and microplate reader (denoted as absorbance value). Suitable amount of substance concentration of SD-208 according to the results of proliferation activity of cells was chosen for the following experiments. (2) Another batch of cells were divided into group BC and 1, 3 μmol/L SD-208 groups and treated as in (1), with 8 wells in each group. The number of migration cells was detected by transwell method. (3) Another batch of cells were grouped and treated as in (2), and the microfilament morphology of cells was observed by rhodamine-phalloidin staining. (4) Another batch of cells were grouped and treated as in (2), and the protein expression of TGF-β1 was assessed with Western blotting. (5) Forty-eight BALB/c nude mice were divided into normal saline group (NS) and 1 μmol/L SD-208 group, and one longitudinal incision with length of 1 cm was made on their back. Then human hypertrophic scar tissue was embedded into the incision. On post injury day 7, multipoint injection of NS in a volume of 0.05 mL was performed in wounds of rats in group NS, while rats in 1 μmol/L SD-208 group were given 0.05 mL 1 μmol/L SD-208, once a day. On the day 0 (the same day), 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 post first time of injection, the weight of 8 nude mice was weighed by electronic scale, and scar area was measured by vernier caliper and the ratio of rest scar area was calculated. (6) In week 1, 2, and 3 post first time of injection, the protein expression of TGF-β1 of human hypertrophic scar tissue was assessed with Western blotting. Data were processed with one-way analysis of variance and two independent-sample t test. (1) The proliferation activity of cells in group BC, 0.5, 1.0, 3.0, and 5.0 μmol/L SD-208 groups was respectively 1.00±0.03, 0.90±0.08, 0.68±0.11, 0.54±0.04, and 0.42±0.09, and the proliferation activity of cells in 0.5, 1.0, 3.0, and 5.0 μmol/L SD-208 groups was significantly lower than that in group BC (with t values from 2.9 to 22.1, P<0.05 or P<0.01). (2) The number of migration cells in 1, 3 μmol/L SD-208 groups was significantly less than that in group BC (with t values respectively 6.5 and 6.4, P values below 0.01). (3) Compared with that in group BC, fluorescence intensity of microfilaments of cells in 1, 3 μmol/L SD-208 groups was attenuated, and the pseudopod extended less. (4) The protein expressions of TGF-β1 of cells in group BC and 1, 3 μmol/L SD-208 groups were respectively 1.00±0.08, 0.80±0.08, and 0.61±0.05, and the protein expressions of TGF-β1 of cells in 1, 3 μmol/L SD-208 groups were significantly lower than those in group BC (with t values respectively 4.0 and 9.2, P values below 0.01). (5) The weights of nude mice in group NS and 1 μmol/L SD-208 group were similar on each time day (with t values from 0.2 to 1.1, P values above 0.05). The ratios of rest scar area of nude mice in two groups were decreased along with the injection time, and the ratios of rest scar area of nude mice in 1 μmol/L SD-208 group were significantly less than those in group NS from the day 6 to 20 post first time of injection (with t values from 1.8 to 15.9, P<0.05 or P<0.01). In week 1, 2, and 3 post first time of injection, the protein expressions of TGF-β1 of human hypertrophic scar tissue in nude mice in two groups showed a tendency of decrease, and the protein expressions of TGF-β1 of human hypertrophic scar tissue in nude mice in 1 μmol/L SD-208 group were significantly lower than those in group NS (with t values from 6.2 to 19.1, P values below 0.01). SD-208 has significant inhibition effect on human hypertrophic scars, and the mechanism is correlated to the inhibition of protein expression of endogenous TGF-β1.