Actin filament-associated protein 1-antisense RNA1 promotes the development and invasion of tongue squamous cell carcinoma via the AFAP1-AS1/miR-133a-5p/ZIC2 axis.

BACKGROUND: The present study aimed to explore the biological role and underlying mechanism of the long non-coding RNA actin filament-associated protein 1-antisense RNA1 (lncRNA AFAP1-AS1) in the progression of tongue squamous cell carcinoma (TSCC). METHODS: A quantitative reverse transcriptase-PCR (RT-qPCR) was conducted to assess relative levels of the miR-133a-5p, lncRNAs AFAP1-AS1 and zinc finger family member 2 (ZIC2) in TSCC cell lines and specimens, whereas ZIC2 protein levels were measured using western blotting. After modifying the levels of expression of lncRNA AFP1-AS1, miR-133a-5p and ZIC2 using lentivirus or plasmid transfection, we examined AKT/epithelial-mesenchymal transition signaling pathway alterations, in vivo carcinogenesis of TSCC in nude mice and in vitro malignant phenotypes. A dual-luciferase reporter assay was conducted to confirm the targeting relationship between ZIC2 and miR-133a-5p, as well as between miR-133a-5p and lncRNA AFAP1-AS1. Based on The Cancer Genome Atlas (TCGA) database, we additionally validated AFP1-AS1. The potential biological pathway for AFP1-AS1 was investigated using gene set enrichment analysis (GSEA). We also evaluated the clinical diagnostic capacities of AFP1-AS1 and clustered the most potential biomarkers with the Mfuzz expression pattern. Finally, we also made relevant drug predictions for AFP1-AS1. RESULTS: In TSCC cell lines and specimens, lncRNA AFAP1-AS1 was upregulated. ZIC2 was upregulated in TSCC cells as a result of lncRNA AFAP1-AS1 overexpression, which also promoted TSCC cell migration, invasion, viability, and proliferation. Via the microRNA sponge effect, it was found that lncRNA AFAP1-AS1 could upregulate ZIC2 by competitively inhibiting miR-133a-5p. Interestingly, knockdown of ZIC2 reversed the biological roles of lncRNA AFAP1-AS1 with respect to inducing malignant phenotypes in TSCC cells. In addition, in vivo overexpression of lncRNA AFAP1-AS1 triggered subcutaneous tumor growth in nude mice implanted with TSCC cells and upregulated ZIC2 in the tumors. The TCGA database findings revealed that AFAP1-AS1 was significantly upregulated in TSCC specimens and had good clinical diagnostic value. The results of GSEA showed that peroxisome proliferator-activated receptor signaling pathway was significantly correlated with low expression of AFP1-AS1. Finally, the results of drug prediction indicated that the group with high AFAP1-AS1 expression was more sensitive to docetaxel, AZD4547, AZD7762 and nilotinib. CONCLUSIONS: The upregulation of lncRNA AFAP1-AS1, which increases TSCC cell viability, migration, proliferation and invasion via the AFAP1-AS1/miR-133a-5p/ZIC2 axis, aids in the progression of TSCC.

Evaluating the Bonding Performance of a Novel Dual-curing Composite Cement to Zirconia.

PURPOSE: To investigate the bond strength and durability of a novel dual-curing composite cement to zirconia under different curing conditions. MATERIALS AND METHODS: Zirconia plates of different thickness (0.5, 1, and 2 mm) were bonded with either a novel dual-curing composite cement (Panavia V5, PV5, Kuraray Noritake) or a traditional one (RelyX Ultimate, RUL, 3M Oral Care; Multilink Automix, MLA, Ivoclar Vivadent), in light-, self-, or dual-curing mode. Bonded specimens were subjected to shear bond strength (SBS) tests after 24 h of water storage or after artificially aging by 20,000 thermal cycles plus 150 days of water storage. The degree of conversion (DC) of the composite cements under different curing conditions was measured by Fourier transform infrared (FTIR) spectroscopy. The irradiance and translucency of the zirconia plates of different thickness were also investigated. RESULTS: The irradiance and translucency of zirconia decreased significantly with increasing thickness (p = 0.00). Both before and after aging, SBS of PV5 in self-curing mode was significantly higher than that of RUL (p = 0.07 before aging and 0.02 after aging) and MLA (p = 0.00 both before and after aging). However, for the three composite cements, light- and dual curing yielding the same SBSs for a constant Y-TZP thickness (p > 0.05). The FTIR analysis showed that, for all three dual-curing composite cements examined in this study, the mean DC values obtained in dual-curing mode were lower than those achieved in light-curing mode (p = 0.00 for PV5, RUL, and MLA). For RUL and MLA, lower mean DC values were obtained in self-curing than dual-curing mode (p = 0.00 for both RUL and MLA), while the DC values of PV5 showed no significant difference between self-curing and dual-curing mode (p = 0.33). CONCLUSION: When the photoactivation time is 60 s and the thickness of the zirconia restoration is less than 2 mm, it is safe to use the two traditional dual-curing composite cements RUL and MLA and PV5 for bonding zirconia. However, when the light exposure time is insufficient, PV5 provides improved bond strength and durability to zirconia.

Effects of Tribochemical Silica Coating and Alumina-Particle Air Abrasion on 3Y-TZP and 5Y-TZP: Evaluation of Surface Hardness, Roughness, Bonding, and Phase Transformation.

PURPOSE: To determine and compare the effects of tribochemical silica coating and alumina-particle air abrasion on 3 mol% and 5 mol% yttria-stabilized tetragonal zirconia polycrystals (Y-TZP). MATERIALS AND METHODS: Two different 3Y-TZP samples (Lava Plus, 3M Oral Care; Ceramill Zolid, Amann Girrbach) and one 5Y-TZP sample (Katana Zirconia UTML, Kuraray Noritake) were prepared and treated with alumina-particle air abrasion and a 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP)-containing self-adhesive composite cement or with tribochemical silica coating followed by silanization (n = 30). Resin columns were cemented onto the treated ceramic surfaces to form specimens. After 24-h water storage or aging with 10,000 thermocycles plus 60-day water storage, shear bond strength (SBS) testing was conducted. Surface roughness, surface Vickers hardness, and crystallographic phase analyses were also performed. RESULTS: The SBS of tribochemically silica-coated 5Y-TZP before and after aging were 13.8 ± 1.4 and 13.2 ± 1.5 MPa, resp., for Lava Plus (3Y-TZP) 14.4 ± 1.4 and 13.9 ± 1.6 MPa, respectively, and for Ceramill Zolid (3Y-TZP) 14.8 ± 1.1 and 13.9 ± 1.5 MPa, respectively. There was no statistical difference between tribochemical silica coating and alumina air abrasion treatments (p = 0.21) on the bonding performance (SBS) of the 3Y-TZPs and 5Y-TZP (p = 0.25) before and after aging (p = 0.50). After alumina air abrasion, 5Y-TZP showed higher surface roughness (Ra = 1.7 ± 0.1) than did the 3Y-TZPs (Ra = 1.2 ± 0.1 for Lava Plus; Ra = 1.2 ± 0.1 for Ceramill Zolid), while the Vickers hardness was similar among the three materials (p = 0.70). Monoclinic zirconia was not detected in 5Y-TZP irrespective of treatment, with the zirconia being mainly cubic phase. However, the 3Y-TZPs were mainly tetragonal phase with some monoclinic zirconia; the latter increased after being alumina-particle air abraded. CONCLUSION: The bond strength to 5Y-TZP is similar to those of the 3Y-TZPs under the same bonding strategies. Durable bonding can be achieved both by alumina air abrasion combined with a 10-MDP-containing self-adhesive composite cement and by tribochemical silica coating followed by silanization for both the 3Y-TZPs and 5Y-TZP.

Effects of hydrothermal aging, thermal cycling, and water storage on the mechanical properties of a machinable resin-based composite containing nano-zirconia fillers.

OBJECTIVES: This study aimed to investigate the effects of hydrothermal aging, thermal cycling, and water storage on the mechanical properties of a machinable resin-based composite containing nano-zirconia fillers. MATERIALS AND METHODS: A machinable resin-based composite containing nano-zirconia fillers (Lava Ultimate, LU) and a resin-based composite with a similar resin matrix-to-filler ratio but without zirconia fillers (Tetric N-Ceram, TNC) were prepared into bars and assigned into four groups based on the type of aging treatment (hydrothermal aging, thermal cycling, water storage, or no aging). The phase transformations of the zirconia fillers in LU after aging were evaluated by X-ray diffraction. The flexural strength, Weibull modulus, flexural modulus, and Vickers hardness of each group were investigated. The fracture surface morphologies of both resin-based composites before and after aging were observed by a scanning electron microscopy (SEM). RESULTS: Only Tetragonal zirconia was detected in the LU samples. Both before and after aging, the flexural strength, flexural modulus, and Vickers hardness values of LU were significantly higher than those of TNC (p < 0.05) with the exception of the flexural modulus of LU, which showed no difference with that of TNC after water storage (p = 0.68). Hydrothermal aging, thermal cycling, and water storage had no significant effects on the surface Vickers hardnesses of LU or TNC (p > 0.05). Hydrothermal aging significantly improved the flexural strength of LU (p = 0.00). Thermal cycling (p = 0.00) and water storage (p = 0.00) significantly decreased the flexural strength of LU. The flexural strength of TNC was not decreased by hydrothermal aging (p = 0.82) or water storage (p = 0.36), while it was decreased by thermal cycling (p = 0.00). The hydrothermal aging group of LU exhibited the highest Weibull modulus. CONCLUSIONS: The machinable resin-based composite containing nano-zirconia fillers provides superior flexural strength, flexural modulus, and Vickers hardness compared to the direct-filling resin-based composite with a similar resin matrix-to-filler ratio, although it fails to provide better aging resistance.

Bond durability when applying phosphate ester monomer-containing primers vs. self-adhesive resin cements to zirconia: Evaluation after different aging conditions.

PURPOSE: This study aimed to evaluate bond durability when applying 2 phosphate ester monomer-containing self-adhesive resin cements alone, versus a combination of phosphate ester monomer-containing primer conditioning plus 2 conventional resin cements requiring primers, to zirconia after different artificial aging methods. METHODS: We cemented air-abraded zirconia plates to composite resin cylinders with self-adhesive resin cements (MS; RU) alone or cemented them with traditional resin cements (ZRV; ZVN) after pre-conditioning with a zirconia primer. A shear bond strength (SBS) test were performed after subjecting them to 19 different aging conditions (n = 15) comprising 30,000× thermocycles, air storage at room temperature (RT), water storage at RT, or at 37 °C for 24 h, 1 week, 1, 3, 6, and 12 months. Zirconia powders mixed with zirconia primer or 2 self-adhesive resin cements were characterized by X-ray photoelectron spectroscopy. RESULTS: Groups MS and ZVN obtained the highest SBS after all of aging methods. SBS after 6 months of storage was similar to SBS after 24 h of storage, while both were higher than SBS after 1 year of storage. Water storage at 37 °C provided higher SBS than RT water storage did. We detected a Zr-O-P bond in both self-adhesive resin cement/zirconia powder mixtures. CONCLUSIONS: Application of self-adhesive resin cements alone could be an alternative to pre-conditioning with a zirconia primer followed by the application of conventional resin cements. Formation of Zr-O-P bonds contributed to the bonding improvement of self-adhesive resin cements. Different aging conditions affected SBS values.

Effectiveness of pre-silanization in improving bond performance of universal adhesives or self-adhesive resin cements to silica-based ceramics: Chemical and in vitro evidences.

OBJECTIVES: To examine effectiveness of pre-silanization in improving bond performance of multipurpose products such as universal adhesives or self-adhesive resin cements to silica-based ceramics. METHODS: The present study investigated reactions between silanol groups of γ-methacryloxypropyltrimethoxysilane (γ-MPS) and silica, dehydration self-condensation of γ-MPS, and condensation polymerization between γ-MPS and 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP) by using thermodynamic calculations. Shear bond strength (SBS) tests were used to evaluate the influence of pre-silanization on resin bonding when a silane-containing universal adhesive, a silane-unknown universal adhesive, or two self-adhesive resin cements were applied for bonding lithium disilicate to resin. In addition, reactions between silane and lithium disilicate were analyzed using X-ray photoelectric spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). RESULTS: Acquired thermodynamic data indicated formation of siloxane between γ-MPS and silica. However, self-condensation of γ-MPS and reaction between γ-MPS and 10-MDP consumed the silanol. Pre-silanization enhanced SBS for self-adhesive resin cements or universal adhesives when applied for bonding silica-based ceramics. Thermocycling and aging decreased SBS in most groups. XPS and FTIR supported formation of siloxane between the employed silane coupling agent and two universal adhesives and lithium disilicate. SIGNIFICANCE: Pre-silanization is beneficial in further enhancing bond performance of universal adhesives or self-adhesive resin cements to silica-based ceramics.

Effects of Luting Cements and Surface Conditioning on Composite Bonding Performance to Zirconia.

PURPOSE: This study compared bond strength, durability, and mechanical properties of luting cements for bonding zirconia: a resin-modified glass ionomer cement (RMGIC), a conventional composite cement, and two self-adhesive composite cements. MATERIALS AND METHODS: The air-abraded zirconia specimens were assigned to 12 groups (n = 30) to prepare bonded specimens. Classification was based on the pre-conditioning selection: none; primers containing 10-methacryloyloxydecyl dihydrogen phosphate (MDP); universal adhesives containing MDP. Further classification was done according to the type of luting cement: RMGIC; conventional composite cement; MDP-free self-adhesive composite cement; MDP-containing self-adhesive composite cement. The shear bond strength (SBS) was measured after 24 h of water storage or aging with 20,000 thermocycles plus an additional 60 days of water storage at 37°C. The elastic modulus and three-point bending strength were determined, and a related Weibull analysis of the four luting cements was performed. RESULTS: The choice of luting cement and aging significantly affected the SBS. Aging decreased the SBS in most groups, except for those that used MDP-containing primers plus conventional composite cements. RMGIC had SBS (before and after aging) that were similar to the MDP-free self-adhesive composite cement. Use of MDP-containing products prior to conventional composite cements provided the highest initial SBS. However, pre-conditioning with MDP-containing products failed to increase the SBS of RMGIC. RMGIC showed lower elastic modulus and three-point bending strength than did the three composite cements. CONCLUSIONS: RMGIC is an alternative to composite cements for luting zirconia restorations. Conditioning with a primer containing MDP combined with conventional composite cement is more reliable.

Effects of multiple firings on mechanical properties and resin bonding of lithium disilicate glass-ceramic.

OBJECTIVES: This study aimed to evaluate the effects of different firing cycles on surface hardness, fracture toughness, and roughness of lithium disilicate glass-ceramic, as well as their bond strength to resin. MATERIALS AND METHODS: A total of 320 polished lithium disilicate glass-ceramic plates were assigned to four main groups (n = 60) to receive one, two, three, or four firing cycles, respectively. Ceramic plates of the four groups were conditioned with HF acid followed by silanization. The pre-treated ceramic plates were cemented with composite resin cylinders using conventional or self-adhesive resin cements to build bonded specimens, and submitted to shear-bond-strength (SBS) testing after water storage for 24 h or 3 mo at 37 °C. The ceramic received different firing cycles after polishing or HF etching was observed using a scanning electron microscope, and their surface roughnesses were determined by a profilometer. The surface Vickers hardness, fracture toughness, and related Weibull analysis results of the polished ceramics after undergoing different firing-cycle times were compared. RESULTS: One sintering significantly increased fracture toughness of lithium disilicate glass-ceramic; however, multiple firing cycles failed to increase it further. Weibull analysis revealed a significant difference in terms of structural reliability among the specimens receiving 0-4 firing cycles. Specimens that received no firing cycle showed the highest surface hardness. Multiple firing cycles had no significant influence on the surface Vickers hardness and surface roughness. HF etching increased surface roughness, and the roughened surface improved the resin SBS of lithium disilicate glass-ceramic. Multiple firing cycles had no significant effect on surface roughness. Furthermore, multiple firing cycles and 3-mo water storage had no significant effect on the SBS. CONCLUSIONS: The mechanical properties of lithium disilicate glass-ceramics would be partially affected by multiple firing cycles, while their resin bonding would not be.

[Effects of nano-zirconium hydroxide coating on resin bonding of 10-methacryloxy decyldihydrogen phosphate-conditioned zirconia].

OBJECTIVE: This study evaluated the influence of alkaline coating of nano-zirconium hydroxide on resin bonding of 10-methacryloxy decyldihydrogen phosphate (MDP)-conditioned zirconia. METHODS: A total of 140 yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) plates were prepared and sandblasted with alumina particles, and then subjected to different surface treatment. Resin bonding specimens were prepared with a MDP-free resin cement and tested for shear bond strength (SBS). X-ray photoelectron spectroscopy (XPS) was used to investigate the formation of chemical bond on the surface of Y-TZP treated successively with nano-zirconium hydroxide coating and MDP containing primer. RESULTS: The two alkaline coatings increased the 24 h SBS compared to control groups, and groups using nano-zirconium hydroxide coating yielded higher SBS. After thermocycling, no statistical difference was observed between groups, but SBS decreased significantly compared to the 24 h SBS (P<0.05). XPS analysis detected -OH bond on the surface of Y-TZP treated with nano-zirconium hydroxide coating, and -P-O-Zr bond was detected on the surface of Y-TZP treated with nano-zirconium hydroxide coating and MDP-containing primer. CONCLUSIONS: Alkaline coatings improved the bonding of resin to zirconia conditioned with MDP-containing primers.

Chemical affinity of 10-methacryloyloxydecyl dihydrogen phosphate to dental zirconia: Effects of molecular structure and solvents.

OBJECTIVES: To examine whether solvents and changing the molecular structure of 10-Methacryloyloxydecyl dihydrogen phosphate (10-MDP) affect its chemical affinity to Yttria-stabilized tetragonal zirconia polycrystals (Y-TZP). METHODS: The present work investigated the chemical affinity between Y-TZP and 10-MDP dissolved in different solvents (acetone/ethanol/water or mixture) using X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, and thermodynamic calculations. Shear bond strength (SBS) tests were used to evaluate the influence of different solvents on 10-MDP bonding. In addition, several phosphate ester monomer variants were created by changing the 10-MDP molecular structure. Changes included extending/shortening the spacer chain-length, and installing hydroxyl or carboxyl groups as side chains at different positions along the spacer chain. The thermodynamic parameters of the complexes formed between the 10-MDP variants and tetragonal zirconia were evaluated. RESULTS: The acquired data indicated that solvent is necessary for the formation of Zr-O-P bonds between 10-MDP and Y-TZP. Solvents affected the chemical affinity of 10-MDP to Y-TZP; acetone facilitated the best bonding, followed by ethanol. Changing the molecular structure of 10-MDP affected its chemical affinity to Y-TZP. The variants 15-MPDP, 12-MDDP, 6-hydroxyl-10-MDP and 6-carboxy-10-MDP all exhibited higher thermodynamic stability than 10-MDP when coordinated with tetragonal zirconia. In contrast, 2-MEP, 5-MPP, 10-hydroxyl-MDP, 10-carboxy-MDP, 5,6-dihydroxyl-10-MDP and 5,6-dicarboxy-10-MDP exhibited lower thermodynamic stability. SIGNIFICANCE: 10-MDP coordinates with zirconia through dissociating in solvents. Changing the molecular structure of 10-MDP theoretically affects its chemical affinity to Y-TZP.

Effect of 10-Methacryloxydecyl Dihydrogen Phosphate Concentration on Chemical Coupling of Methacrylate Resin to Yttria-stabilized Zirconia.

PURPOSE: To test the hypothesis that the concentration of 10-methacryloxydecyl dihydrogen phosphate (MDP) in zirconia primers has no effect on the chemical bonding efficacy of methacrylate resins to yttria-stabilized tetragonal zirconia (Y-TZP). MATERIALS AND METHODS: Shear bond strength testing was performed to evaluate the efficacy of experimental primers containing 5, 10, 15, 20 or 30 wt% MDP (5M, 10M, 15M, 20M, 30M) in improving composite-zirconia bond strength. Bonding without use of MDP-containing primer served as the negative control (Ctr0). Bonding with a commercially available MDP-containing primer served as the positive control (CtrM). Thermogravimetric analysis (TGA), inductively coupled plasma-mass spectrometry (ICP-MS), x-ray photoelectron spectroscopy (XPS), Fourier transform-infrared spectroscopy (FT-IR), and computational simulation of infrared spectra were used to confirm the formation of Zr-O-P bond between MDP and Y-TZP. RESULTS: Results derived from TGA, ICP-MS, XPS, and FT-IR suggested that MDP chemically bonded with Y-TZP. Simulation of IR data supported the FT-IR results. There was a higher concentration of phosphorus on the 10M-conditioned Y-TZP surface when compared with the other groups, suggesting bettter formation of Zr-O-P bond in the 10M group. Shear bond strengths were significantly lower for group 5M (p < 0.05), compared to groups 10M to 30M, which were not significantly different from one another (p > 0.05). CONCLUSIONS: MDP improves resin bonding of zirconia through the formation of Zr-O-P bonds with zirconia. 10 wt% MDP appears to be the most optimal concentration for synthesizing zirconia primers for resin bonding.

Dipentaerythritol penta-acrylate phosphate - an alternative phosphate ester monomer for bonding of methacrylates to zirconia.

The present work examined the effects of dipentaerythritol penta-acrylate phosphate (PENTA) as an alternative phosphate ester monomer for bonding of methacrylate-based resins to yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) and further investigated the potential bonding mechanism involved. Shear bond strength testing was performed to evaluate the efficacy of experimental PENTA-containing primers (5, 10, 15, 20 or 30 wt% PENTA in acetone) in improving resin-Y-TZP bond strength. Bonding without the use of a PENTA-containing served as the negative control, and a Methacryloyloxidecyl dihydrogenphosphate(MDP)-containing primer was used as the positive control. Inductively coupled plasma-mass spectrometry (ICP-MS), X-ray photoelectron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FTIR) were used to investigate the potential existence of chemical affinity between PENTA and Y-TZP. Shear bond strengths were significant higher in the 15 and 20 wt% PENTA groups. The ICP-MS, XPS and FTIR data indicated that the P content on the Y-TZP surface increased as the concentration of PENTA increased in the experimental primers, via the formation of Zr-O-P bond. Taken together, the results attest that PENTA improves resin bonding of Y-TZP through chemical reaction with Y-TZP. Increasing the concentration of PENTA augments its binding affinity but not its bonding efficacy with zirconia.

Alkaline nanoparticle coatings improve resin bonding of 10-methacryloyloxydecyldihydrogenphosphate-conditioned zirconia.

Creating an alkaline environment prior to 10-methacryloyloxydecyldihydrogenphosphate (MDP) conditioning improves the resin bonding of zirconia. The present study evaluated the effects of four alkaline coatings with different water solubilities and pH values on resin bonding of MDP-conditioned zirconia. Two alkaline nanoparticle coatings were studied in particular. Thermodynamics calculations were performed to evaluate the strengths of MDP-tetragonal phase zirconia chemical bonds at different pH values. Zirconia surfaces with and without alkaline coatings were characterized by scanning electron microscope (SEM)/energy dispersive spectrometer and Fourier transform infrared spectroscopy; alkaline coatings included NaOH, Ca(OH)2, nano-MgO, and nano-Zr(OH)4. A shear bond strength (SBS) test was performed to evaluate the effects of the four alkaline coatings on bonding; the alkaline coatings were applied to the surfaces prior to conditioning the zirconia with MDP-containing primers. Gibbs free energies of the MDP-tetragonal zirconia crystal model coordination reaction in different pH environments were -583.892 (NaOH), -569.048 [Ca(OH)2], -547.393 (MgO), and -530.279 kJ/mol [Zr(OH)4]. Thermodynamic calculations indicated that the alkaline coatings improved bonding in the following order: NaOH > Ca(OH)2 > MgO > Zr(OH)4. Statistical analysis of SBS tests showed a different result. SBSs were significantly different in groups that had different alkaline coatings, but it was not influenced by different primers. All four alkaline coatings increased SBS compared to control groups. Of the four coatings, nano-Zr(OH)4 and -MgO showed higher SBS. Therefore, preparing nano-Zr(OH)4 or -MgO coatings prior to conditioning with MDP-containing primers may potentially improve resin bonding of zirconia in the clinic.

Protective Effect of Spironolactone on Endothelial-to-Mesenchymal Transition in HUVECs via Notch Pathway.

BACKGROUND: Fibrosis results in excessive buildup of extracellular matrix proteins along with abnormalities in structure and is partly derived by a process involving transforming growth factor ß (TGF-ß) called endothelial-to-mesenchymal transition (EndMT). We investigated whether the aldosterone receptor-blocker spironolactone could abrogate TGF-ß-induced fibrosis in EndMT and the underlying mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) were divided into 5 groups for treatment: blank; vehicle control; TGF-ß (10 ng/ml); spironolactone (1 µM)+TGF-ß; and spironolactone+TGF-ß+DAPT (10 µM). Cell chemotaxis was assayed by transwell assay. The expression of CD31 and vimentin was determined by Immunofluorescence staining and western blot analysis. Notch1 protein level was detected by western blot analysis. RESULTS: Spironolactone significantly prevented TGF-ß-stimulated EndMT by down-regulate vimentin and up-regulate CD31 in HUVECs (p<0.01).It inhibited cell migration during EndMT (p<0.01). The protective effect of spironolactone against EndMT could be attenuated by blocking the Notch signal pathway with DAPT (p<0.01). Notch signaling was activated and cross-interacted with TGF-ß and spironolactone in regulating EndMT in HUVECs and reversed the spironolactone-related signaling by abrogating the antifibrotic actions with decreased Notch1 protein expression (p<0.01). CONCLUSION: Spironolactone may have a protective role in TGF-ß-induced EndMT in HUVECs mediated by the Notch signal pathway.

Anti-fibrosis effect of scutellarin via inhibition of endothelial-mesenchymal transition on isoprenaline-induced myocardial fibrosis in rats.

Scutellarin (SCU) is the major active component of breviscapine and has been reported to be capable of decreasing myocardial fibrosis. The aim of the present study is to investigate whether SCU treatment attenuates isoprenaline-induced myocardial fibrosis and the mechanisms of its action. Rats were injected subcutaneously with isoprenaline (Iso) to induce myocardial fibrosis and rats in the SCU treatment groups were intraperitoneally infused with SCU (10 mg·kg-1·d-1 or 20 mg·kg-1·d-1, for 14 days). Post-treatment, cardiac functional measurements and the left and right ventricular weight indices (LVWI and RVWI, respectively) were analysed. Pathological alteration, expression of type I and III collagen, Von Willebrand factor, α-smooth muscle actin, cluster of differentiation-31 (CD31), and the Notch signalling proteins (Notch1, Jagged1 and Hes1) were examined. The administration of SCU resulted in a significant improvement in cardiac function and decrease in the cardiac weight indices; reduced fibrous tissue proliferation; reduced levels of type I and III collagen; increased microvascular density; and decreased expression of α-smooth muscle actin and increased expression of CD31, Notch1, Jagged1 and Hes1 in isoprenaline-induced myocardial fibrosis in rats. Our results suggest that SCU prevents isoprenaline-induced myocardial fibrosis via inhibition of cardiac endothelial-mesenchymal transition potentially, which may be associated with the Notch pathway.

[Effect of crucumin on vascular endothelial function in atherosclerotic rabbits].

OBJECTIVE: To observe the effect of curcumin on nitric oxide (NO) in plasma of atherosclerotic rabbits, activity of constitutive nitric oxide synthase (cNOS) and asymmetric dimethylarginine (ADMA), and discuss curcumin's effect against AS and its correlation with ADMA. METHOD: Thirty-eight male Japanese white rabbits were randomly divided into four groups: the control group (eight rabbits fed with standard diets), the model group (ten rabbits fed with high-fat diets), the low dose curcumin group (ten rabbits fed with high-fat diets and 100 mg . kg-1 d -1 ) and the high dose curcumin group (ten rabbits fed with high-fat diets and 200 mg kg-1 d-1 curcumin). At the end of the 12th week, their plasmas were tested for TC, LDL-C, NO, endothelin (ET) , ADMA and activity of aortic cNOS. Aortic tissues were collected for histological examinations. RESULT: The three groups fed with high-fat diets showed higher plasma ADMA and ET than the control group (P <0. 01) , but with decrease in plasma NO concentration and arterial cNOS activity (P <0. 01). Compared with the model group (P <0. 05) , the curcumin groups showed lower plasma ADMA and ET (P <0. 05), but higher plasma NO concentration and arterial cNOS activity than the model group (P <0. 01). There was no significant difference between the two curcumin groups. CONCLUSION: Curcumin may play an important protective role in AS process by reducing plasma ADMA level. [Key words] atherosclerosis; asymmetric dimethylarginine; crucumin; nitric oxide; nitric oxide synthase

[Association between polymorphisms of inflammatory factor genes and coronary heart disease].

OBJECTIVE: To investigate the association between genetic polymorphisms of inflammatory factors and susceptibility to coronary heart disease(CHD) in southern Chinese Han population. METHODS: Using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) method, the genotypes of five inflammatory factors (BRCA1-associated protein, a disintegrin and metalloproteinase 8, inter-alpha-trypsin inhibitor H3, interleukin-15, cyclooxygenase-2) were anaylzed in 283 CHD patients diagnosed by angiography and 176 controls. RESULTS: In these inflammatory factors, the 270T/C and 90A/G polymorphisms of the BRAP gene showed a significant association with CHD. The allele and genotype frequencies of BRAP gene were consistent with those predicted by Hardy-Weinberg equilibrium (chi-square=0.878, P> 0.05; chi-square=0.776, P> 0.05, respectively). The frequencecies of 270C and 90G alleles in CHD patients was significantly higher than those of the control group (29.51% vs. 21.31%, P=0.006; 30.04% vs. 21.31%, P=0.004, respectively). Compared with 270TT and 90AA, 270CC and 90GG genotypes had a significantly increased CHD risk by Logistic regression analysis (OR=4.51, 95%CI: 1.41-14.45, P=0.011; OR=5.09, 95%CI: 1.60-16.26, P=0.006, respectively). This association was still signifcant after adjustment for the sex, age, smoke, hypertension, diabetes, plasma total cholesterol and low density lipoprotein levels. No evidence of association was found for other single nucleotide polymorphisms. CONCLUSION: The 270T/C and 90A/G polymorphisms in the BRAP gene may contribute to an increased risk of CHD among southern Chinese Han population.

[Ryanodine downregulates the expression of p-eNOS (Thr495) and improves the functions of rapamycin treated endothelial outgrowth cells].

OBJECTIVE: To observe the effects of ryanodine on rapamycin treated endothelial outgrowth cells (EOCs). METHODS: The mononuclear cells were harvested from umbilical cord blood by Ficoll density gradient centrifugation, then induced into EOCs and expanded in vitro. The endothelial characteristics of EOCs were identified by immunostaining and fluorescent staining. The EOCs were pretreated with or without ryanodine (10 µmol/L) for 1 h, and then treated with or without rapamycin (10 nmol/L) for 24 h. Proliferation was evaluated by CCK8 and migration was measured by Transwell. The protein expression of EOCs was evaluated by immunobloting technique with total eNOS antibody and phospho-eNOS (Thr495) antibody. RESULTS: Compared with control group, the proliferation and migration capacities of EOCs were significantly reduced while the phosphorylation of eNOS (Thr495) protein was significantly upregulated in rapamycin group (P < 0.05), expression of total eNOS was not affected by rapamycin (P > 0.05). Compared with rapamycin group, the proliferation and migration capacities of EOCs were significantly increased and the phosphorylation of eNOS (Thr495) protein was significantly downregulated in ryanodine + rapamycin group (P < 0.05). The proliferation and migration capacities, the phosphorylation of eNOS (Thr495) protein and the expression of total eNOS were not affected by ryanodine alone (P > 0.05). CONCLUSIONS: Rapamycin reduced proliferation and migration capacities while upregulated the phosphorylation of eNOS (Thr495) protein of EOCs and these effects could be partly reversed by cotreatment with ryanodine.

[Effect of surface pretreatment with chemical etchants on bond strength between a silicone-based resilient liner and denture base resin].

OBJECTIVE: To evaluate the effect of denture base resin surface pretreatment with chemical etchants on microleakage and bond strength between silicone-based resilient liner and denture base resin. The initial bending strength of denture base resin after surface pretreatment was also examined. METHODS: Thirty-six polymethyl methacrylate (PMMA) denture base resin blocks (30 mm × 30 mm × 2 mm) were prepared and divided into three groups: group acetone, group methyl methy acrylate (MMA) and group control. Subsequently, a 2 mm silicone-based resilient liner was applied between every two blocks. After 5000 cycles in the thermal cycler (5 and 55°C), they were immersed in the (131) I solution for 24 hours and γ-ray counts were measured. Another 36 PMMA resin blocks (30 mm × 10 mm × 7.5 mm) were prepared. The blocks were divided into three groups and treated as mentioned above. A 3 mm silicone-based resilient liner was applied between every two blocks. After 5000 thermal cycles, tensile bond strength of the sample was measured in a universal testing machine. Another 18 PMMA resin blocks (65 mm × 10 mm × 3.3 mm) were prepared. They were divided into 3 groups and treated in the same way. After an adhesive was applied, the bending strength was measured with three-piont bending test. RESULTS: Two experimental groups showed lower microleakage (520.0 ± 562.2 and 493.5 ± 447.9) and higher tensile bond strength [(1.5 ± 0.4) and (1.4 ± 0.5) MPa] than the group control [microleakage: (1369.5 ± 590.2); tensile bond strength: (0.9 ± 0.2) MPa, P < 0.05]. There was no statistically significant difference between group acetone and MMA in microleakage and tensile bond strength (P > 0.05). There was no statistically significant difference in bending strength among the three groups (P > 0.05). CONCLUSIONS: Treating the denture base resin surface with acetone and MMA decreased the microleakage, increased the tensile bond strength between the two materials and did not make the initial bending strength of denture base resin decline.

Effect of oxygen plasma treatment on the bonding of a soft liner to an acrylic resin denture material.

Mechanical roughening reportedly had a weakening effect on bond strength. Therefore, the purpose of this study was to evaluate the effect of an alternative surface roughening method, namely oxygen plasma treatment, on the tensile bond strength between denture base resin and soft liner. The soft liner used in this study was Soft Reverse, whilst the denture base material was Zi Ran. Three groups of specimens were prepared, comprising untreated specimens and oxygen plasma-treated specimens with exposure to air for 1 day and 2 days. All specimens were subjected to surface composition analysis and tensile bond strength testing. All data were analyzed using one-way ANOVA, and their mean values were compared using Tukey's HSD test (p<0.01). Highest tensile bond strength was observed in the 1-day exposure group (5.2 MPa), whilst the lowest in the control group of untreated specimens (2.8 MPa). Hence, results of this study clearly indicated that oxygen plasma treatment was effective in enhancing tensile bond strength.