The role of TGF-ß in the electrotactic reaction of mouse 3T3 fibroblasts in vitro.

Endogenous electric fields (EFs) serve as a crucial signal to guide cell movement in processes such as wound healing, embryonic development, and cancer metastasis. However, the mechanism underlying cell electrotaxis remains poorly understood. A plausible hypothesis suggests that electrophoretic or electroosmotic forces may rearrange charged components of the cell membrane, including receptors for chemoattractants which induce asymmetric signaling and directional motility. This study aimed to explore the role of Transforming Growth Factor Beta (TGFß) signaling in the electrotactic reaction of 3T3 fibroblasts. Our findings indicate that inhibiting canonical and several non-canonical signaling pathways originating from the activated TGF-ß receptor does not hinder the directed migration of 3T3 cells to the cathode. Furthermore, suppression of TGF-ß receptor expression does not eliminate the directional migration effect of 3T3 cells in the electric field. Additionally, there is no observed redistribution of the TGF-ß receptor in the electric field. However, our studies affirm the significant involvement of Phosphoinositide 3-Kinase (PI3K) in electrotaxis, suggesting that in our model, its activation is likely associated with factors independent of TGFß action.

3D Melt-Extrusion Printing of Medium Chain Length Polyhydroxyalkanoates and Their Application as Antibiotic-Free Antibacterial Scaffolds for Bone Regeneration.

In this work, we investigated, for the first time, the possibility of developing scaffolds for bone tissue engineering through three-dimensional (3D) melt-extrusion printing of medium chain length polyhydroxyalkanoate (mcl-PHA) (i.e., poly(3-hydroxyoctanoate-co-hydroxydecanoate-co-hydroxydodecanoate), P(3HO-co-3HD-co-3HDD)). The process parameters were successfully optimized to produce well-defined and reproducible 3D P(3HO-co-3HD-co-3HDD) scaffolds, showing high cell viability (100%) toward both undifferentiated and differentiated MC3T3-E1 cells. To introduce antibacterial features in the developed scaffolds, two strategies were investigated. For the first strategy, P(3HO-co-3HD-co-3HDD) was combined with PHAs containing thioester groups in their side chains (i.e., PHACOS), inherently antibacterial PHAs. The 3D blend scaffolds were able to induce a 70% reduction of Staphylococcus aureus 6538P cells by direct contact testing, confirming their antibacterial properties. Additionally, the scaffolds were able to support the growth of MC3T3-E1 cells, showing the potential for bone regeneration. For the second strategy, composite materials were produced by the combination of P(3HO-co-3HD-co-HDD) with a novel antibacterial hydroxyapatite doped with selenium and strontium ions (Se-Sr-HA). The composite material with 10 wt % Se-Sr-HA as a filler showed high antibacterial activity against both Gram-positive (S. aureus 6538P) and Gram-negative bacteria (Escherichia coli 8739), through a dual mechanism: by direct contact (inducing 80% reduction of both bacterial strains) and through the release of active ions (leading to a 54% bacterial cell count reduction for S. aureus 6538P and 30% for E. coli 8739 after 24 h). Moreover, the composite scaffolds showed high viability of MC3T3-E1 cells through both indirect and direct testing, showing promising results for their application in bone tissue engineering.

The adaptation of 3T3 cells to simulated microgravity by retrieving the major cell cycle-related protein expression during long-term in vitro proliferation.

The present study aimed to assess the effects of simulated microgravity (SMG) on 3T3 cell proliferation and the expression of cell cycle regulators. 3T3 cells were induced to SMG by Gravite® for 8 days, while the control group was treated with 1G condition. The result showed that the SMG condition causes a decrease in proliferative activity in 3T3 cells. In the SMG group, the expression of cell cycle-related proteins was lower than the control on day 3. However, these proteins were upregulated in 3T3 cells of the SMG group on day 5, suggesting that these cells were rescued from the arrest and retrieved a higher proliferation. A down-regulation of cell cycle-related proteins was observed in 3T3 cells of both SMG and control groups on day 7. In conclusion, SMG results in the attenuation of cell proliferation during the initial exposure to SMG, but the cells will adapt to this condition and retrieve normal proliferation by increasing the expression of cell cycle regulators.

The SPI1/SMAD5 cascade in the promoting effect of icariin on osteogenic differentiation of MC3T3-E1 cells: a mechanism study.

BACKGROUND: Dysregulation of osteogenic differentiation is a crucial event during osteoporosis. The bioactive phytochemical icariin has become an anti-osteoporosis candidate. Here, we elucidated the mechanisms underlying the promoting function of icariin in osteogenic differentiation. METHODS: Murine pre-osteoblast MC3T3-E1 cells were stimulated with dexamethasone (DEX) to induce osteogenic differentiation, which was evaluated by an Alizarin Red staining assay and ALP activity measurement. The mRNA amounts of SPI1 and SMAD5 were detected by real-time quantitative PCR. Expression analysis of proteins, including osteogenic markers (OPN, OCN and RUNX2) and autophagy-associated proteins (LC3, Beclin-1, and ATG5), was performed by immunoblotting. The binding of SPI1 and the SMAD5 promoter was predicted by the Jaspar2024 algorithm and confirmed by chromatin immunoprecipitation (ChIP) experiments. The regulation of SPI1 in SMAD5 was examined by luciferase assays. RESULTS: During osteogenic differentiation of MC3T3-E1 cells, SPI1 and SMAD5 were upregulated. Functionally, SPI1 overexpression enhanced autophagy and osteogenic differentiation of MC3T3-E1 cells, while SMAD5 downregulation exhibited opposite effects. Mechanistically, SPI1 could enhance SMAD5 transcription and expression. Downregulation of SMAD5 also reversed SPI1 overexpression-induced autophagy and osteogenic differentiation in MC3T3-E1 cells. In MC3T3-E1 cells under DEX stimulation, icariin increased SMAD5 expression by upregulating SPI1. Furthermore, icariin could attenuate SPI1 depletion-imposed inhibition of autophagy and osteogenic differentiation of MC3T3-E1 cells. CONCLUSION: Our findings demonstrate that the SPI1/SMAD5 cascade, with the ability to enhance osteogenic differentiation, underlies the promoting effect of icariin on osteogenic differentiation of MC3T3-E1 cells.

Comparison of two biological systems used for phototoxicity testing: Cellular and tissue.

The OECD has approved two similar methods for testing the phototoxic potency of chemicals. The first method, OECD 432, is based on the cytotoxicity properties of materials to the mouse 3T3 (clone A31) cell line (fibroblasts) after exposure to light. The second method, OECD 498, is based on the same properties but using reconstructed human epidermis - EpiDerm (stratified keratinocytes). The aim of this study was to compare these two methods using statistical tests (specificity, sensitivity, negative predictive value, positive predictive value and accuracy) and non-statistical characteristics (e.g. price and experimental duration, amount of material, level of complications, cell type, irradiation dose). Both tests were performed according to the relevant guidelines using the same 11 control substances. Higher performance values were observed for OECD 432 in both phototoxic and non-phototoxic classifications. The accuracy of OECD 432 was 90.9%, while that of OECD 498 was 72.7%. OECD 432 was also shorter and less expensive. On the other hand, OECD 498 was less complicated, and used human cells with stratum corneum, which better reflects real skin. This method can also be used with oily substances that are poorly soluble in water. However, both methods are important for testing the phototoxic properties of materials, and can be used alone or in a tiered strategy.

A promising eco-sustainable wound dressing based on cellulose extracted from Spartium junceum L. and impregnated with Glycyrrhiza glabra L extract: Design, production and biological properties.

Glycyrrhiza glabra extract is widely known for its antioxidant and anti-inflammatory properties and can improve the wound healing process. The aim of this work was to shorten the time of the healing process by using an eco-sustainable wound dressing based on Spanish broom flexible cellulosic fabric by impregnation with G. glabra extract-loaded ethosomes. Chemical analysis of G. glabra extract was performed by LC-DAD-MS/MS and its encapsulation into ethosomes was obtained using the ethanol injection method. Lipid vesicles were characterized in terms of size, polydispersity index, entrapment efficiency, zeta potential, and stability. In vitro release studies, biocompatibility, and scratch test on 3T3 fibroblasts were performed. Moreover, the structure of Spanish broom dressing and its ability to absorb wound exudate was characterized by Synchrotron X-ray phase contrast microtomography (SR-PCmicroCT). Ethosomes showed a good entrapment efficiency, nanometric size, good stability over time and a slow release of polyphenols compared to the free extract, and were not cytotoxic. Lastly, the results revealed that Spanish broom wound dressing loaded with G. glabra ethosomes is able to accelerate wound closure by reducing wound healing time. To sum up, Spanish broom wound dressing could be a potential new green tool for biomedical applications.

FFAR-mediated signaling drives migration of pancreatic cancer cells in hypoxic fibroblast co-cultures.

The tumor microenvironment (TME) comprises cancer and non-cancerous stromal cells, including fibroblasts. Free fatty acids (FFAs) regulate various biological responses by binding to G protein-coupled FFA receptors (FFARs). In this study, we examined the impact of FFAR1 and FFAR4 on the cell migration of pancreatic cancer PANC-1 cells co-cultured with 3T3 fibroblast cells under hypoxic conditions. PANC-1 cells cultured at 1 % O2 exhibited elevated FFAR1 expression and decreased FFAR4 expression compared to those at 21 % O2. Cell migration of PANC-1 cells was reduced under 1 % O2 conditions. FFAR1 knockdown enhanced PANC-1 cell migration, whereas FFAR4 knockdown inhibited it. Co-culture of PANC-1 cells with 3T3 cells at 1 % O2 significantly increased FFAR4 expression, while FFAR1 expression remained unchanged. To evaluate the effects of FFAR1 and FFAR4 on PANC-1 cell migration in co-culture with 3T3 cells, we conducted a wound healing assay using the Culture-Insert 2 Well. PANC-1 and 3T3 cells were individually seeded into the two wells and incubated at both 21 % and 1 % O2 for 13 h. The cell migration of PANC-1 cells co-cultured with 3T3 cells at 1 % O2 was notably higher compared to 21 % O2. TUG-770 reduced and TUG-891 enhanced the cell migration of PANC-1 cells co-cultured with 3T3 cells under both 21 % and 1 % O2 conditions. These findings suggest that FFAR1 and FFAR4 play important roles in regulating the cell migration of PANC-1 cells co-cultured with 3T3 cells under hypoxic conditions.

Harnessing oriented arrangement of collagen fibers by 3D printing for enhancing mechanical and osteogenic properties of mineralized collagen scaffolds.

As the structural basis of connective and load-bearing tissues, collagen fibers with orientation play an important role in the mechanical properties and physiological and biochemical functions of the tissues, but viable methods for preparing scaffolds with highly oriented collagenous structure still need to be further studied. In this study, pure collagen was used as printing ink to 3D printing. Harnessing oriented collagen fiber structure by 3D printing for promoting mechanical and osteogenic properties of scaffolds. The scaffolds with different printed angles and thicknesses were prepared to fit the bone defect site and realize personalized customization. The orientation assembly of collagen fibers was promoted by shear force action of 3D printing, the regular arrangement of collagen fibers and stabilization of fiber structure were promoted by pH adjustment and glutaraldehyde cross-linking, and the collagen fibers were mineralized by cyclic mineralization method. The microscopic morphology of fiber arrangement in the scaffolds were investigated by scanning electron microscopy. Results demonstrated that collagen fibers were changed from non-oriented to oriented after 3D printing. And the tensile modulus of the scaffolds with oriented collagen fibers was nine times higher than that of the scaffolds with non-oriented fibers. Moreover, the effects of oriented collagen fibers on the proliferation, differentiation and mineralization of MC3T3-E1 cells were studied by CCK-8 assay, live/dead cell staining, alkaline phosphatase activity test, and Alizarin red staining. The results indicated that cell proliferation, differentiation and mineralization were significantly promoted by oriented collagen fibers, and the cells proliferated directionally in the direction of the fibers. Taken together, mineralized collagen fiber scaffolds with oriented collagen fibers have great potential in bone tissue engineering applications.

Gallein increases the fibroblast growth factor 2-elicited osteoprotegerin synthesis in osteoblasts.

Gallein is known as an inhibitor of Gßγ subunits, but roles of gallein in bone metabolism have not been reported. Fibroblast growth factor 2 (FGF-2) increases angiogenesis and promotes bone regeneration during the early stages of fracture healing. Osteoprotegerin (OPG) secreted by osteoblasts, binds to the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) as a decoy receptor and prevents RANKL from binding to RANK, resulting in the suppression of bone resorption. Our previous report demonstrated that FGF-2 activates the phosphorylation of p38 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (JNK), and p44/p42 MAPK in osteoblast-like MC3T3-E1 cells. Additionally, FGF-2-activated phosphorylation of p38 MAPK and JNK but not p44/p42 MAPK is positively involved in OPG synthesis in these cells. This work aimed to investigate the effects of gallein on the FGF-2-elicited OPG synthesis in osteoblast-like MC3T3-E1 cells and the mechanism. Our findings demonstrated that gallein significantly increased the FGF-2-elicited OPG synthesis in MC3T3-E1 cells. By contrast, fluorescein, gallein-like compound that does not bind Gßγ, did not affect the FGF-2-elicited OPG synthesis. Gallein significantly enhanced the FGF-2-induced OPG mRNA expression levels. Gallein did not affect the FGF-2-activated phosphorylation of p38 MAPK and p44/p42 MAPK, but significantly increased the FGF-2-activated phosphorylation of JNK, while fluorescein did not affect JNK phosphorylation. SP600125, a specific JNK inhibitor, strongly inhibited gallein-induced enhancement of FGF-2-induced OPG synthesis and mRNA expression levels. Our results indicated that gallein increases the FGF-2-induced OPG synthesis due to the JNK activation in the osteoblast.

Effect of Peptide-Polymer Host-Guest Electrostatic Interactions on Self-Assembling Peptide Hydrogels Structural and Mechanical Properties and Polymer Diffusivity.

Peptide-based supramolecular hydrogels are an attractive class of soft materials for biomedical applications when biocompatibility is a key requirement as they exploit the physical self-assembly of short self-assembling peptides avoiding the need for chemical cross-linking. Based on the knowledge developed through our previous work, we designed two novel peptides, E(FKFE)2 and K(FEFK)2, that form transparent hydrogels at pH 7. We characterized the phase behavior of these peptides and showed the clear link that exists between the charge carried by the peptides and the physical state of the samples. We subsequently demonstrate the cytocompatibility of the hydrogel and its suitability for 3D cell culture using 3T3 fibroblasts and human mesenchymal stem cells. We then loaded the hydrogels with two polymers, poly-l-lysine and dextran. When polymer and peptide fibers carry opposite charges, the size of the elemental fibril formed decreases, while the overall level of fiber aggregation and fiber bundle formation increases. This overall network topology change, and increase in cross-link stability and density, leads to an overall increase in the hydrogel mechanical properties and stability, i.e., resistance to swelling when placed in excess media. Finally, we investigate the diffusion of the polymers out of the hydrogels and show how electrostatic interactions can be used to control the release of large molecules. The work clearly shows how polymers can be used to tailor the properties of peptide hydrogels through guided intermolecular interactions and demonstrates the potential of these new soft hydrogels for use in the biomedical field in particular for delivery or large molecular payloads and cells as well as scaffolds for 3D cell culture.

Loading rutin on surfaces by the layer-by-layer assembly technique to improve the oxidation resistance and osteogenesis of titanium implants in osteoporotic rats.

The purpose of this study was to construct a rutin-controlled release system on the surface of Ti substrates and investigate its effects on osteogenesis and osseointegration on the surface of implants. The base layer, polyethylenimine (PEI), was immobilised on a titanium substrate. Then, hyaluronic acid (HA)/chitosan (CS)-rutin (RT) multilayer films were assembled on the PEI using layer-by-layer (LBL) assembly technology. We used scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and contact angle measurements to examine all Ti samples. The drug release test of rutin was also carried out to detect the slow-release performance. The osteogenic abilities of the samples were evaluated by experiments on an osteoporosis rat model and MC3T3-E1 cells. The results (SEM, FTIR and contact angle measurements) all confirmed that the PEI substrate layer and HA/CS-RT multilayer film were effectively immobilised on titanium. The drug release test revealed that a rutin controlled release mechanism had been successfully established. Furthermore, thein vitrodata revealed that osteoblasts on the coated titanium matrix had greater adhesion, proliferation, and differentiation capacity than the osteoblasts on the pure titanium surface. When MC3T3-E1 cells were exposed to H2O2-induced oxidative stressin vitro, cell-based tests revealed great tolerance and increased osteogenic potential on HA/CS-RT substrates. We also found that the HA/CS-RT coating significantly increased the new bone mass around the implant. The LBL-deposited HA/CS-RT multilayer coating on the titanium base surface established an excellent rutin-controlled release system, which significantly improved osseointegration and promoted osteogenesis under oxidative stress conditions, suggesting a new implant therapy strategy for patients with osteoporosis.

Amine functionalised graphene embedded polyvinyl alcohol (PVA) and PVA-chitosan hydrogel composites.

A novel amine-functionalized graphene oxide (AFG) doped polyvinyl alcohol (PVA)/chitosan (PVA-Ch) composite film was developed using an eco-synthesis approach, eliminating the need for halogenated compounds. The resulting AFG-doped PVA/Chitosan (PVA-Ch/AFG) polymer film exhibited promising properties for controlled delivery and biosensing applications. The investigation included assessing the swelling behaviour, dissolution percent, gel fraction, and mechanical properties of the polymer film. The swelling characteristics of PVA-Ch and PVA-Ch/AFG were found to be pH and temperature-dependent across various pH ranges (3, 5, 7, and 9). Interestingly, PVA-Ch/AFG demonstrated a stable swelling pattern at pH 5 and 7, unaffected by changes in chitosan concentration, indicating enhanced stability compared to PVA-Ch. The study also explored the use of PVA-Ch/AFG in a drug delivery system, revealing controlled release of the model antibiotic amphicillin, emphasizing its potential in medical applications. Furthermore, the eco-friendly synthesis route underscored the safety of PVA-Ch/AFG for use in food and medical applications. Biocompatibility assessments, including biodegradability studies and cytotoxicity tests on fibroblasts (3T3 cells), confirmed the safety profile of PVA-Ch/AFG. In conclusion, the study suggests that PVA-Ch/AFG holds promise for bio-sensing applications, offering a flexible and colorimetric platform capable of encapsulating, adsorbing, and desorbing biomolecules such as drugs and sensing compounds.

Nanoemulsion containing Jatropha gossypiifolia leaf extract reduces dermonecrosis induced by Bothrops erythromelas venom and accelerates wound closure.

ETHNOPHARMACOLOGICAL RELEVANCE: The species Jatropha gossypiifolia, popularly known as "pinhão-roxo", is distributed throughout Brazil, is commonly employed for topical or oral administration in treating wounds, inflammations, and snake bites. Given the significant impact of snakebites on public health and the limitations of antivenom, coupled with the diverse molecular composition of this plant species, investigating its healing and antidermonecrotic capacities is relevant. AIM OF THE STUDY: This study aimed to develop a topical nanoemulsion incorporating the hydroethanolic extract of J. gossypiifolia leaves, to evaluate its therapeutic potential, particularly in terms of its efficacy in wound healing and inhibition of dermonecrosis induced by B. erythromelas venom (BeV). MATERIAL AND METHODS: The extract of J. gossypiifolia (JgE) leaves was obtained by maceration and remaceration. The phytochemical analysis was conducted and J. gossypiifolia nanoemulsion (JgNe) was obtained, characterized and assessed for stability. The cytotoxicity was determined in normal cells (erythrocytes and 3T3) using hemolytic assay and cell viability assay using crystal violet staining. The antioxidant activity was evaluated by the reduction of ABTS and DPPH radicals. The evaluation of wound healing was conducted in vivo following treatment with JgNe, wherein the percentage of wound closure and inflammatory mediators. The skin irritation test was assessed in vivo by applying JgNe directly to the animal's skin. In vitro, the antivenom capacity was evaluated through enzymatic inhibition assays (phospholipase A2 and hyaluronidase) of BeV. Additionally, the in vivo antidermonecrotic activity of JgNe was evaluated by measuring the reduction of the dermonecrotic halo. RESULTS: The HPLC-DAD analysis identified flavonoids, specifically vitexin, luteolin derivatives and apigenin derivatives. In addition, 95.08 ± 5.46 mg of gallic acid/g of extract and 137.92 ± 0.99 mg quercetin/g extract, was quantified. JgNe maintained stability over a 4-week period. Moreover, JgE and JgNe demonstrated no cytotoxicity in human erythrocytes and murine fibroblasts at tested concentrations (32.25-250 µg/mL). Additionally, exhibited significant antioxidant activity by reducing ABTS and DPPH radicals. The treatment with JgNe did not induce skin irritation and accelerated wound healing, with significant wound closure observed from 5th day and reduction in nitrite levels, myeloperoxidase activity, and cytokine. Both JgE and JgNe demonstrated in vitro inhibition of the phospholipase and hyaluronidase enzymes of BeV. Moreover, JgNe exhibited antidermonecrotic activity by reducing the dermonecrotic halo caused by BeV after 24 h. CONCLUSIONS: JgNe and JgE exhibited no cytotoxicity at the tested concentrations. Additionally, our findings demonstrate that JgNe has the ability to accelerate wound closure and reduce dermonecrosis caused by BeV, indicating to be promising formulation for complementary therapy to antivenom treatment.

Mineral matrix deposition of MC3T3-E1 pre-osteoblastic cells exposed to silicocarnotite and nagelschmidtite bioceramics: In vitro comparison to hydroxyapatite.

This work presents the effect of the silicocarnotite (SC) and nagelschmidtite (Nagel) phases on in vitro osteogenesis. The known hydroxyapatite of biological origin (BHAp) was used as a standard of osteoconductive characteristics. The evaluation was carried out in conventional and osteogenic media for comparative purposes to assess the osteogenic ability of the bioceramics. First, the effect of the material on cell viability at 24 h, 7 and 14 days of incubation was evaluated. In addition, cell morphology and attachment on dense bioceramic surfaces were observed by fluorescence microscopy. Specifically, alkaline phosphatase (ALP) activity was evaluated as an osteogenic marker of the early stages of bone cell differentiation. Mineralized extracellular matrix was observed by calcium phosphate deposits and extracellular vesicle formation. Furthermore, cell phenotype determination was confirmed by scanning electron microscope. The results provided relevant information on the cell attachment, proliferation, and osteogenic differentiation processes after 7 and 14 days of incubation. Finally, it was demonstrated that SC and Nagel phases promote cell proliferation and differentiation, while the Nagel phase exhibited a superior osteoconductive behavior and could promote MC3T3-E1 cell differentiation to a higher extent than SC and BHAp, which was reflected in a higher number of deposits in a shorter period for both conventional and osteogenic media.

Morphological Changes of 3T3 Cells under Simulated Microgravity.

BACKGROUND: Cells are sensitive to changes in gravity, especially the cytoskeletal structures that determine cell morphology. The aim of this study was to assess the effects of simulated microgravity (SMG) on 3T3 cell morphology, as demonstrated by a characterization of the morphology of cells and nuclei, alterations of microfilaments and microtubules, and changes in cycle progression. METHODS: 3T3 cells underwent induced SMG for 72 h with Gravite®, while the control group was under 1G. Fluorescent staining was applied to estimate the morphology of cells and nuclei and the cytoskeleton distribution of 3T3 cells. Cell cycle progression was assessed by using the cell cycle app of the Cytell microscope, and Western blot was conducted to determine the expression of the major structural proteins and main cell cycle regulators. RESULTS: The results show that SMG led to decreased nuclear intensity, nuclear area, and nuclear shape and increased cell diameter in 3T3 cells. The 3T3 cells in the SMG group appeared to have a flat form and diminished microvillus formation, while cells in the control group displayed an apical shape and abundant microvilli. The 3T3 cells under SMG exhibited microtubule distribution surrounding the nucleus, compared to the perinuclear accumulation in control cells. Irregular forms of the contractile ring and polar spindle were observed in 3T3 cells under SMG. The changes in cytoskeleton structure were caused by alterations in the expression of major cytoskeletal proteins, including ß-actin and α-tubulin 3. Moreover, SMG induced 3T3 cells into the arrest phase by reducing main cell cycle related genes, which also affected the formation of cytoskeleton structures such as microfilaments and microtubules. CONCLUSIONS: These results reveal that SMG generated morphological changes in 3T3 cells by remodeling the cytoskeleton structure and downregulating major structural proteins and cell cycle regulators.

Regulator of G protein signalling 18 promotes osteocyte proliferation by activating the extracellular signal­regulated kinase signalling pathway.

Osteocyte function is critical for metabolism, remodelling and regeneration of bone tissue. In the present study, the roles of regulator of G protein signalling 18 (RGS18) were assessed in the regulation of osteocyte proliferation and bone formation. Target genes and signalling pathways were screened using the Gene Expression Omnibus (GEO) database and Gene Set Enrichment Analysis (GSEA). The function of RGS18 and the associated mechanisms were analysed by Cell Counting Kit 8 assay, 5­ethynyl­2'­deoxyuridine assay, flow cytometry, reverse transcription­quantitative PCR, western blotting and immunostaining. Overlap analysis of acutely injured subjects (AIS) and healthy volunteers (HVs) from the GSE93138 and GSE93215 datasets of the GEO database identified four genes: KIAA0825, ANXA3, RGS18 and LIPN. Notably, RGS18 was more highly expressed in peripheral blood samples from AIS than in those from HVs. Furthermore, RGS18 overexpression promoted MLO­Y4 and MC3T3­E1 cell viability, proliferation and S­phase arrest, but inhibited apoptosis by suppressing caspase­3/9 cleavage. Silencing RGS18 exerted the opposite effects. GSEA of GSE93138 revealed that RGS18 has the ability to regulate MAPK signalling. Treatment with the MEK1/2 inhibitor PD98059 reversed the RGS18 overexpression­induced osteocyte proliferation, and treatment with the ERK1/2 activator 12­O­tetradecanoylphorbol­13­acetate reversed the effects of RGS18 silencing on osteocyte proliferation. In conclusion, RGS18 may contribute to osteocyte proliferation and bone fracture healing via activation of ERK signalling.

Cytocompatibility and cell proliferation evaluation of calcium phosphate-based root canal sealers

OBJECTIVES: This study aimed to evaluate the cell viability and migration of Endosequence Bioceramic Root Canal Sealer (BC Sealer) compared to MTA Fillapex and AH Plus.MATERIALS AND METHODS: BC Sealer, MTA Fillapex, and AH Plus were placed in contact with culture medium to obtain sealers extracts in dilution 1:1, 1:2 and 1:4. 3T3 cells were plated and exposed to the extracts. Cell viability and migration were assessed by 3-(4,5-dimethyl-thiazoyl)-2,5-diphenyl-tetrazolium bromide (MTT) and Scratch assay, respectively. Data were analyzed by Kruskal-Wallis and Dunn's test (p < 0.05).RESULTS: The MTT assay revealed greater cytotoxicity for AH Plus and MTA Fillapex at 1:1 dilution when compared to control (p < 0.05). At 1:2 and 1:4 dilutions, all sealers were similar to control (p > 0.05) and MTA Fillapex was more cytotoxic than BC Sealer (p < 0.05). Scratch assay demonstrated the continuous closure of the wound according to time. At 30 hours, the control group presented closure of the wound (p < 0.05). At 36 hours, only BC Sealer presented the closure when compared to AH Plus and MTA Fillapex (p < 0.05). At 42 hours, AH Plus and MTA Fillapex showed a wound healing (p > 0.05).CONCLUSIONS: All tested sealers demonstrated cell viability highlighting BC Sealer, which showed increased cell migration capacity suggesting that this sealer may achieve better tissue repair when compared to other tested sealers.

The molecular mechanism of fibroblast growth factor 21-inhibited leptin expression in adipocytes / 生理学报

The present study was aimed to clarify the signaling molecular mechanism by which fibroblast growth factor 21 (FGF21) regulates leptin gene expression in adipocytes. Differentiated 3T3-F442A adipocytes were used as study object. The mRNA expression level of leptin was detected by fluorescence quantitative RT-PCR. The phosphorylation levels of proteins of signal transduction pathways were detected by Western blot. The results showed that FGF21 significantly down-regulated the mRNA expression level of leptin in adipocytes, and FGF21 receptor inhibitor BGJ-398 could completely block this effect. FGF21 up-regulated the phosphorylation levels of ERK1/2 and AMPK in adipocytes. Either ERK1/2 inhibitor SCH772984 or AMPK inhibitor Compound C could partially block the inhibitory effect of FGF21, and the combined application of these two inhibitors completely blocked the effect of FGF21. Neither PI3K inhibitor LY294002 nor Akt inhibitor AZD5363 affected the inhibitory effect of FGF21 on leptin gene expression. These results suggest that FGF21 may inhibit leptin gene expression by activating ERK1/2 and AMPK signaling pathways in adipocytes.

Supplementation of Fermented Barley Extracts with Lactobacillus Plantarum dy-1 Inhibits Obesity via a UCP1-dependent Mechanism / 生物医学与环境科学（英文）

OBJECTIVE@#We aimed to explore how fermented barley extracts with Lactobacillus plantarum dy-1 (LFBE) affected the browning in adipocytes and obese rats.@*METHODS@#In vitro, 3T3-L1 cells were induced by LFBE, raw barley extraction (RBE) and polyphenol compounds (PC) from LFBE to evaluate the adipocyte differentiation. In vivo, obese SD rats induced by high fat diet (HFD) were randomly divided into three groups treated with oral gavage: (a) normal control diet with distilled water, (b) HFD with distilled water, (c) HFD with 800 mg LFBE/kg body weight (bw).@*RESULTS@#In vitro, LFBE and the PC in the extraction significantly inhibited adipogenesis and potentiated browning of 3T3-L1 preadipocytes, rather than RBE. In vivo, we observed remarkable decreases in the body weight, serum lipid levels, white adipose tissue (WAT) weights and cell sizes of brown adipose tissues (BAT) in the LFBE group after 10 weeks. LFBE group could gain more mass of interscapular BAT (IBAT) and promote the dehydrogenase activity in the mitochondria. And LFBE may potentiate process of the IBAT thermogenesis and epididymis adipose tissue (EAT) browning via activating the uncoupling protein 1 (UCP1)-dependent mechanism to suppress the obesity.@*CONCLUSION@#These results demonstrated that LFBE decreased obesity partly by increasing the BAT mass and the energy expenditure by activating BAT thermogenesis and WAT browning in a UCP1-dependent mechanism.

Interaction between necroptosis and apoptosis in MC3T3-E1 cell death induced by dexamethasone / 南方医科大学学报

OBJECTIVE@#To investigate the relationship between necroptosis and apoptosis in MCET3-E1 cell death induced by glucocorticoids.@*METHODS@#MC3T3-E1 cells were incubated with 10-6 mol/L dexamethasone followed by treatment with the apoptosis inhibitor z-VAD-fmk (40 μmol/L) or the necroptosis inhibitor necrostatin-1 (40 μmol/L) for 2 h. At 72 h after incubation with dexamethasone, the cells were harvested to determine the cell viability using WST-1 assay and the rate of necrotic cells using annexin V/PI double staining; the percentage of apoptotic cells was determined using Hoechst staining. The mitochondrial membrane potential and the level of ATP in the cells were also evaluated. Transmission electron microscopy was used to observe the microstructural changes of the cells. The expressions of RIP-1 and RIP-3 in the cells were detected by Western blotting.@*RESULTS@#At a concentration of 10-6 mol/L, dexamethasone induced both apoptosis and necroptosis in MC3T3- E1 cells. Annexin V/PI double staining showed that inhibition of cell apoptosis caused an increase in cell necrosis manifested by such changes as mitochondrial swelling and plasma membrane disruption, as shown by electron microscopy; Hoechst staining showed that the percentage of apoptotic cells was significantly reduced. When necroptosis was inhibited by necrostatin-1, MC3T3-E1 cells showed significantly increased apoptosis as shown by both AV/PI and Hoechst staining, and such changes were accompanied by changes in mitochondrial membrane potential and ATP level in the cells.@*CONCLUSIONS@#In the process of dexamethasone-induced cell death, necroptosis and apoptosis can transform reciprocally accompanied by functional changes of the mitochondria.