mRNA Expression Levels Of Type Research Articles

A novel combination against skin aging via promoting the synthesis of biological collagen

Supplementing collagen is considered to contribute to delaying skin aging. In this work, we developed a novel combination for improving skin aging by stimulating the biosynthesis of collagen. By screening a library of Chinese herbal medicines (CHM), we found that Angelicae Dahuricae Radix (Baizhi), Lilii bulbus (Baihe), Glycyrrhizae radix et rhizoma (Gancao), and Jujubae fructus (Dazao) substantially increased the mRNA expression levels of type I collagen, suggesting their potential anti-skin-aging activity. To keep the structural integrity of collagen, prolyl 4-hydroxylase (P4H), a key enzyme in collagen synthesis, was recombinantly expressed in Escherichia coli. In addition, the transfersome with added P-4H was prepared to improve the transdermal absorption of combination. Vitamin C (VC), the substrate required for the activity of P4H, was also incorporated into the combination. Eventually, an optimal combination, consisting of Baizhi, Baihe, Gancao, P4H transfersome and VC, was obtained by a series of combination experiments. Based on traditional CHM and modern biological agents, we developed a novel combination against skin aging by promoting the synthesis of collagen. Collectively, the combination show the high potential of application to delay skin aging

Granulocyte-macrophage colony-stimulating factor suppresses induction of type I interferon in infants with severe pneumonia.

The underlying mechanisms for infantile bronchopneumonia development remain unknown. Peripheral blood mononuclear cell (PBMCs) and serum derived from severe and mild infantile bronchopneumonia were obtained, and the expression of various molecules was detected with enzyme-linked immunosorbent assay and quantitative PCR. Such molecules were also detected in granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced bone marrow-derived NFκB2-/- dendritic cells (DCs) or NIK SMI1 (NF-κB-inducing kinase inhibitor) administrated DCs. The relative mRNA expression levels of type I interferons (IFNs) (IFN-α4, IFN-β), Th17 cell-associated markers (interleukin-17A, retinoic-acid-receptor-related orphan nuclear receptor gamma, and GM-CSF), and non-canonical NF-κB member (NFκB2) were significantly up-regulated in PBMCs and DCs derived from infantile bronchopneumonia compared with healthy controls. However, compared with Th17 cell-associated markers and non-canonical NF-κB molecules, the expression of IFN-α4 and IFN-β was significantly inhibited in severe infantile bronchopneumonia compared with mild infantile bronchopneumonia. The relative protein expression of the above molecules also showed a similar expression pattern in the PBMCs or serum. NF-κB2 knockout or NIK SMI1 administration could reverse the diminished expression of IFN-β in GM-CSF-induced bone marrow-derived DCs. GM-CSF-dependent non-canonical NF-κB pathway-mediated inhibition of type I IFNs production in DCs contributes to the development of severe bronchopneumonia in infant. Granulocyte-macrophage colony-stimulating factor-dependent non-canonical NF-κB pathway-mediated inhibition of type I IFNs production in dendritic cells is critical for the development of infantile bronchopneumonia. Our findings reveal a possible mechanism underlying the development of severe infantile bronchopneumonia. The results could provide therapeutic molecular target for the treatment of such disease.

Synergistic effect of UV/chlorine in bacterial inactivation, resistance gene removal, and gene conjugative transfer blocking

Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) were investigated from effluent of two hospital and two municipal wastewater treatment plants (WWTPs) before and after disinfection. The results of network analysis showed that 8 genera were identified to be the main potential hosts of ARGs, including Mycobacterium, Ferruginibacter, Thermomonas, Morganella, Enterococcus, Bacteroides, Myroides and Romboutsia. The removal of ARGs and their possible bacterialhosts were synchronous and consistent by chlorine or ultraviolet (UV) disinfection in WWTPs. The mechanisms of ARB and ARGs removal, and conjugation transfer of RP4 plasmids by UV, chlorine and synergistic UV/chlorine disinfection was revealed. Compared to UV alone, ARB inactivation was improved 1.4 log and photoreactivation was overcomeeffectively by UV/chlorine combination (8 mJ/cm2, chlorine 2 mg/L). However, ARGs degradation was more difficult than ARB inactivation. Until UV dosage enhanced to 320 mJ/cm2, ARGs achieved 0.58–1.60 log removal. Meanwhile, when 2 mg/L of chlorine was combined with UV combination, ARGs removal enhanced 1–1.5 log. The synergistic effect of adding low-dose chlorine (1–2 mg/L) during UV radiation effectively improved ARB and ARGs removal simultaneously. The same synergistic effect also occurred in the horizontal gene transfer (HGT). Non-lethal dose chlorine (0.5 mg/L) increased the conjugation transfer frequency,which confirmed that the mRNA expression levels of type IV secretion system (T4SS) proteins vir4D, vir5B and vir10B were significantly enhanced. The risk of RP4 plasmid conjugation transfer was significantly reduced with UV/chlorine (UV ≥ 4 mJ/cm2, chlorine ≥ 1 mg/L). These findings may serve as valuable implications for assessing and controlling the risk of ARGs transfer and propagation in the environment.

Acetyl-CoA carboxylase 1 and 2 inhibition ameliorates steatosis and hepatic fibrosis in a MC4R knockout murine model of nonalcoholic steatohepatitis.

Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in de novo lipogenesis, which is increased in the livers of patients with nonalcoholic steatohepatitis. GS-0976 (firsocostat), an inhibitor of isoforms ACC1 and ACC2, reduced hepatic steatosis and serum fibrosis biomarkers such as tissue inhibitor of metalloproteinase 1 in patients with nonalcoholic steatohepatitis in a randomized controlled trial, although the impact of this improvement on fibrosis has not fully been evaluated in preclinical models. Here, we used Western diet-fed melanocortin 4 receptor-deficient mice that have similar phenotypes to nonalcoholic steatohepatitis patients including progressively developed hepatic steatosis as well as fibrosis. We evaluated the effects of ACC1/2 inhibition on hepatic fibrosis. After the confirmation of significant hepatic fibrosis with a 13-week pre-feeding, GS-0976 (4 and 16 mg/kg/day) treatment for 9 weeks lowered malonyl-CoA and triglyceride content in the liver and improved steatosis, histologically. Furthermore, GS-0976 reduced the histological area of hepatic fibrosis, hydroxyproline content, mRNA expression level of type I collagen in the liver, and plasma tissue metalloproteinase inhibitor 1, suggesting an improvement of hepatic fibrosis. The treatment with GS-0976 was also accompanied by reductions of plasma ALT and AST levels. These data demonstrate that improvement of hepatic lipid metabolism by ACC1/2 inhibition could be a new option to suppress fibrosis progression as well as to improve hepatic steatosis in nonalcoholic steatohepatitis.

A developed subunit vaccine based on fiber protein VP56 of grass carp reovirus providing immune protection against grass carp hemorrhagic disease

Grass carp reovirus (GCRV) is the main viral pathogen that endangers grass carp seriously. Application of vaccine has been considered to be the most effective way to prevent virus infection. VP56 is a protein encoded by gene segment 7 of grass carp reovirus, and is predicted to share homology with fiber protein of mammalian reovirus (MRV). In our study, the immunogenicity of VP56 was evaluated by neutralization test. GCRV was incubated with mouse anti-VP56 antibody, and then was injected into grass carp. Results showed that disease progress and death occurrence was hindered in the experimental group compared with the control group. For further study, the recombinant VP56 protein (rVP56) expressed by pET-32a (+) vector was purified, and was used as subunit vaccine to immunize grass carp. After each fish (15 ± 1.5 g) was injected with 30 μg purified rVP56 intraperitoneally, the immune protective efficacy of recombinant VP56 protein was assessed by a series of immune parameters. The population of red blood cells in immunized fish increased significantly after 5 d post injection (dpi), and reached a peak with (2.98 ± 0.17) × 109/ml at 7 dpi (p < 0.05). The numbers of white blood cells peaked with (8.42 ± 1.01) × 107/ml at 7 dpi (p < 0.05). Additionally, the percentage of monocytes and neutrophils rose to a peak with (9.05 ± 0.92)% and (25.93 ± 2.60)% respectively at 5 dpi (p < 0.05 or p < 0.01), whereas lymphocytes reached the highest value of (85.81 ± 2.73) % at 14 dpi (p < 0.01). Serum antibody titer in the vaccinated fish increased significantly and reached a peak at 21 dpi (p < 0.01). The mRNA expression levels of type I interferon (IFN1), major histocompatibility complex class I (MHC I), Toll-like receptor 22 (TLR22), and immunoglobulin M (IgM) were significantly up-regulated in head kidney and spleen (p < 0.05 or p < 0.01). The GCRV challenge test showed that the relative survival rate in immunized group was 71%–75%. Collectively, the results indicated that rVP56 protein can induce immune protection in grass carp, and can be consider as a candidate vaccine against GCRV infection.

Activin A, a Potential Key Factor of the Malignant Bone Marrow Niche, Enhances B-Cell Precursor-Acute Lymphoblastic Leukemic Cell Migratory and Invasive Properties

The bone marrow (BM) represents a peculiar microenvironment characterized by a high concentration of growth factors and cytokines necessary for hematopoiesis, that make it a sanctuary for leukemic cell homing, survival and proliferation. B-cell precursor-Acute Lymphoblastic Leukemia (BCP-ALL) reprogram the BM stroma to create a leukemia-supporting and chemoprotective niche. Strategies to modulate the microenvironment could offer new approaches for anti-leukemia therapies. We identified ActivinA, a TGF-β family member, with a well-described promoting role in several solid malignancies, as a new potentially targetable leukemia-favoring factor.ActivinA resulted overexpressed in the BM plasma of 108 BCP-ALL pediatric patients compared to 44 Healthy Donors (HDs), as evaluated by ELISA assay. Upon in vitro culture, primary BCP-ALL cells significantly increased ActivinA secretion by mesenchymal stromal cells (MSCs) derived from HD BM. Interestingly this effect was achieved both by cell-to-cell contact-mediated mechanisms (direct contact) and by soluble factor release (transwell-mediated co-culture). Interestingly, MSCs isolated from the BM of leukemic patients showed an intrinsic ability to secrete higher amounts of ActivinA (mean=983.6±362.9 pg/mL), compared to their normal counterpart (mean=218.5±31.99 pg/mL). In addition, we demonstrated that the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α are increased in the leukemic BM and that they were able to synergize with leukemic blasts in inducing ActivinA release by MSCs (>100 fold increase compared to basal production).Both type I and type II Activin receptors were found to be expressed by leukemic cells as demonstrated by flow cytometry analysis of ALK4, ACVR2A and ACVR2B receptors and western blot analysis of ALK2. Of note, ActivinA exposure could increase the mRNA expression levels of type I receptors ALK2 and ALK4, thus suggesting a possible self-reinforcing mechanism.Gene expression analysis of ActivinA-treated BCP-ALL cells showed that this protein was able to significantly influence motility-associated molecular pathways. Accordingly, time lapse microscopy analyses revealed that ActivinA significantly increased random motility of leukemic cells (p<0.0001). In addition, ActivinA was able to almost double the migration of BCP-ALL primary cells in response to CXCL12, as demonstrated by in vitro chemotaxis assays. The specificity of the observed effect was demonstrated by using the ALK4 specific inhibitor SB431542.CXCL12 reduction is one of the typical microenvironmental alterations occurring in the leukemic BM. In line with literature data, we showed 6-fold decrese of CXCL12 levels in the BM of BCP-ALL patients compared to HDs (p<0.0001). Dose-responses chemotaxis experiments revealed that ActivinA was able to sensitize leukemic cells to suboptimal CXCL12 concentrations. On the other site, ActivinA exerted an opposite effect on CD34+ cells isolated both from HD cord blood or BM. In detail, in HD-CD34+ cells ActivinA severely impaired CXCL12-induced migration (p<0.0001). This opposite effect could be explained by ActivinA ability to increase free cytosolic calcium only in leukemic cells, both basally and after addition of CXCL12 (flow cytometry analysis of Fluo-4 NW stained cells). Of note, calcium levels of HD-CD34+ cells resulted unaffected or even decreased (in 2 out of 3 experiments performed) by ActivinA treatment. Since calcium is critically involved in boosting cytoskeleton dynamics, we analysed, by flow cytometry, actin polymerization in phalloidin stained BCP-ALL cells. Interestingly, ActivinA treatment of BCP-ALL cells significantly increased the rate of conversion of globular into filamentous actin, which is a prerequisite of site-directed migration, as soon as CXCL12 was added. Moreover, ActivinA resulted a leukemia-promoting factor: protein treatment significantly increased the in vitro migration of BCP-ALL cells through Matrigel-coated transwells in response to CXCL12, thus stimulating leukemic cell invasiveness.Overall, ActivinA resulted a key factor conferring a migratory advantage to leukemic cells over healthy hematopoiesis within the leukemic niche. Indeed, our in vitro findings provide the biological rationale for designing novel therapeutical approaches targeting the leukemia-stroma interplay. DisclosuresLocatelli:bluebird bio: Consultancy; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

Plasmid pcDNA3.1-s11 constructed based on the S11 segment of grass carp reovirus as DNA vaccine provides immune protection

Although some commercial vaccines against grass carp reovirus (GCRV) are available, given the many varieties of GCRV and limited types of vaccines, the disease caused by GCRV remains a major problem, which leads to economic losses in grass carp aquaculture. A reovirus strain (GCRV-HN14) was recently isolated from local diseased fish in our laboratory. The S11 segment of GCRV-HN14 was speculated to encode the virus capsid protein VP35. In our study, the S11 segment was cloned into the eukaryotic expression vector pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1-s11, which was then transfected into CIK cells, and the VP35 protein was successfully expressed. Grass carp was immunized with pcDNA3.1-s11, and the in vivo distribution and expression of the pcDNA3.1-s11 plasmids were analyzed by PCR and Western blot. Recombinant plasmids were detected in the blood, liver, spleen, kidney, and muscle. However, protein expression could only be detected in the muscle. The immune protection of the pcDNA3.1-s11 plasmid in grass carp was evaluated using a series of experiments. Results showed that the population of white blood cells significantly increased at 1, 7, and 14 days post-immunization (dpi) and reached a peak with (9.58 ± 0.72) × 107/ml at 7 dpi (P < 0.01 or P < 0.05). The percentage of neutrophils reached a peak with (24.13 ± 2.38)% at 7 dpi (P < 0.01), whereas the lymphocytes peaked with (93.30 ± 4.71)% at 14 dpi (P < 0.05). Serum antibody levels were significantly enhanced in immunized fish at 14, 21, and 28 dpi (P < 0.01). The mRNA expression levels of type I interferon, immunoglobulin M, Toll-like receptor 22, and major histocompatibility complex class I were significantly up-regulated in the head kidney and spleen of immunized fish (P < 0.05). Grass carp immunized with pcDNA3.1-s11 exhibited a higher survival percentage (70.4%–73.3%) than the controls (5%–13%). Overall, as a DNA vaccine, the pcDNA3.1-s11 plasmid could induce immune protection against GCRV.

Novel subunit vaccine based on grass carp reovirus VP35 protein provides protective immunity against grass carp hemorrhagic disease

The grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus (GCRV), is one of the most severe infectious diseases in aquaculture. Given that antiviral therapies are currently limitedly available, vaccination remains the most effective means for the prevention of viral diseases, such as GCRV. A reovirus strain, which was temporarily named GCRV-HN14, was recently isolated from grass carp in Henan province, China. The S11 gene fragment of GCRV-HN14 was speculated to encode viral structural protein VP35, which has no equivalent gene in other aquareviruses but has antigenic epitopes. In this study, the recombinant plasmid pET-32a-vp35 was constructed to express recombinant VP35 proteins in prokaryotic cells, which was used to create a novel subunit vaccine. The immune protection of recombinant VP35 protein was evaluated by a series of experiments in grass carp. Results showed that the number of white blood cells (WBC) in the peripheral blood increased significantly to 7.92 ± 0.72 × 107/ml 5 days after vaccination (P < 0.05). The number of neutrophils and monocytes in WBC were significantly higher than those of the control 3 days after vaccination (P < 0.05) and maximally got to 12.22 ± 1.28% and 18.70 ± 1.78%, respectively. Owing to the significant increase in the number of lymphocytes (92.37 ± 2.10%; P < 0.01), the percentages of neutrophils and monocytes declined significantly (14 dpi; P < 0.01). Serum antibody levels induced by recombinant VP35 protein significantly increased 7 days post immunization and continued to increase until 5 weeks post vaccination. The mRNA expression levels of type I interferon (designated as IFN1), immunoglobulin M, Toll-like receptor 22 and major histocompatibility complex class I were up-regulated significantly in the head kidneys and spleens of immunized fish (P < 0.01). Grass carp immunized by recombinant VP35 protein showed that the relative percentage of survival was about 60% after it was challenged with GCRV. Overall, the results suggested that recombinant VP35 protein can induce immunity and protect grass carp against GCRV infection. Thus, it can be used as a subunit vaccine.

Oral epithelial cells inhibit the proliferation, mRNA expressions of collagen type I, RUNX2 and BGP, and ALP activity of osteoblast-like cells in coculture

Cell-to-cell interactions play important roles in regulating cell proliferation and differentiation, tissue production, organ construction and ontogenesis. The purpose of this study was to investigate the effects of oral epithelial (OE) cells on osteoblasts’ behavior for proliferation and osteogenic differentiation. Osteoblast-like cells were co-cultured in 6-well-dishes with or without OE cells growing on cell culture inserts. After 1, 3, 7 and 14 d, the characteristics of osteoblast-like cells were evaluated regarding cell proliferation, mRNA expression levels of collagen type I, RUNX2 and bone Gla protein (BGP), and alkaline phosphate (ALP) activity. In cell proliferation assays, the numbers of osteoblast-like cells cultured with or without OE cells increased over time for 2 w, but the proliferation of osteoblast-like cells co-cultured with OE cells plateaued after 7 d. The mRNA expression levels of type I collagen in osteoblast-like cells cultured with or without OE cells tended to decrease over time, but the decrease in osteoblast-like cells co-cultured with OE cells was more dramatic. The mRNA expression levels of RUNX2 and BGP in osteoblast-like cells cultured without OE cells tended to increase over time, but those increases in cells cultured without OE cells were more dramatic than in osteoblast-like cells cocultured with OE cells. The ALP activity of osteoblast-like cells cultured without OE cells increased until 7 d and then decreased at 14 d, but osteoblast-like cells cultured with OE cells underwent little change in ALP activity throughout the 14 d of culture. The ALP activity of osteoblast-like cells cultured without OE cells was significantly higher at 7 and 14 d than in osteoblast-like cells cultured with OE cells. In conclusion, these results indicate that OE cells inhibit the osteogenic differentiation and function of osteoblast-like cells.

Regulation of type II collagen, matrix metalloproteinase-13 and cell proliferation by interleukin-1β is mediated by curcumin via inhibition of NF-κB signaling in rat chondrocytes.

Curcumin possesses strong anti-inflammatory, anti-rheumatoid and anti-oxidative activities, and has the potential to inhibit nuclear factor-κB (NF-κB) signaling. Cartilage damage in osteoarthritis (OA) is largely mediated by interleukin-1β (IL-1β) via activation of various transcription factors, including NF-κB and activator protein-1. The aim of the present study was to determine whether IL-1β induces matrix metalloproteinase-13 (MMP-13) expression and inhibits type II collagen expression, as well as to examine whether cell proliferation may be inhibited by curcumin through the inhibition of NF-κB signaling. The effects of curcumin were investigated in rat articular chondrocyte cell cultures treated with IL-1β in the presence or absence of curcumin or the NF-κB inhibitor pyrrolidine dithiocarbamate. Western blotting and reverse transcription-quantitative polymerase chain reaction were conducted to evaluate protein and mRNA expression levels of type II collagen, MMP-13, NF-κB inhibitor α (IκBα), phosphorylated-IκBα and NF-κB subunit p65/RelA. Western blotting and immunofluorescence were performed to examine the effects of curcumin on the expression, phosphorylation and nuclear translocation of NF-κB-associated proteins. The effects of curcumin on cell proliferation were evaluated by Cell Counting Kit-8 (CCK-8). Curcumin was demonstrated to inhibit the IL-1β-induced activation of NF-κB by suppressing IκBα phosphorylation and p65/RelA nuclear translocation. These events were associated with the downregulation of MMP-13 expression and the upregulation of type II collagen expression, both of which are considered to be NF-κB targets. CCK-8 assays revealed that co-treatment with curcumin resulted in increased proliferation in IL-1β-treated chondrocytes. These findings implicated curcumin as a naturally occurring anti-inflammatory agent for the treatment of OA via inhibition of NF-κB signaling.

Effect of three wavelength lasers on mRNA expression of types I and III procollagen in fibroblasts

Objective To investigate the effects of different wavelength (595 nm, 755 nm and 1064 nm) lasers on the mRNA expression of types Ⅰ and Ⅲ procollagen in cultured fibroblasts. Methods Fibroblasts from Kunming mice were cultured in vitro. They were divided into 595 nm laser group, 755 nm laser group, 1064 nm laser group and no laser irra diating group. The mRNA expression of the types Ⅰ and Ⅲ procollagen was detected by RT-PCR. Results The mRNA expression level of type Ⅰ procollagen in 1064 nm group was higher than that of 755 nm group, 595 nm group and control group (P 0.05). The mRNA expression level of type Ⅲ procollagen in 1064 nm group was higher than that in 755 nm group, 595 nm group and control group (P 0.05). Conclusions Three wavelength lasers can directly promote mRNA expression of types Ⅰ and Ⅲ procollagen in fibroblasts. 595 nm laser mainly promotes mRNA expression of type Ⅰ procollagen, and 755 nm laser promotes more mRNA expression of type Ⅲ procollagen than 595 nm laser. The most mRNA expression of types Ⅰ and Ⅲ procollagen is promoted by 1064 nm laser. Key words: Laser; Fibroblasts; Procollagen; Collagen type Ⅰ; Collagen type Ⅲ

Inhibitory effects of SRT1720 on the apoptosis of rabbit chondrocytes by activating SIRT1 via p53/bax and NF-κB/PGC-1α pathways.

SRT1720, a new discovered drug, was reported to activate silent information regulator 1 (SIRT1) and inhibit the chondrocyte apoptosis. However, the underlying mechanism remains elusive. In the present study, the chondrocytes were extracted from the cartilage tissues of New Zealand white rabbits, cultured in the presence of sodium nitroprusside (SNP) (2.5 mmol/L) and divided into five groups: 1, 5, 10, and 20 μmol/L SRT1720 groups and blank control group (0 μmol/L SRT1720). MTT assay was used to detect the chondrocyte viability and proliferation, and DAPI staining and flow cytometry to measure the chondrocyte apoptosis. The expression levels of SIRT1, p53, NF-κB/p65, Bax, and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) were detected by Western blotting and the expression levels of SIRT1, type II collagen, and aggrecan mRNA by RT-PCR. The results showed that in the SRT1720-treated groups, the nuclei of chondrocytes were morphologically intact and had uniform chromatin. In the blank control group, nuclear rupture into debris was observed in chondrocytes. With the SRT1720 concentration increasing, the chondrocyte viability increased, the apoptosis rate decreased, the protein expression levels of SIRT1 and PGC-1α and the mRNA expression levels of type II collagen and aggrecan increased ({ptP}<0.05), and the expression levels of p53, NF-κB and bax decreased (P<0.05). It was suggested that SRT1720 inhibits chondrocyte apoptosis by activating the expression of SIRT1 via p53/bax and NF-κB/PGC-1α pathways.

Enhancement of osseointegration of artificial ligament by nano-hydroxyapatite and bone morphogenic protein-2 into the rabbit femur.

The MTT assay showed that the cell proliferation on hydroxyapatite (HAp) and HAp/bone morphogenic protein (BMP) coated group was better than the control and BMP coated groups at 5 days. And after 7 days of culture, the mRNA expression levels of type I collagen, osteonectin, osteopontin, bonesialoprotein, BMP-2, alkaline phosphatase (ALP) and Runx-2 in the HAp/BMP coated group were significantly higher than the other groups. Also, in this group showed the most significant induction of osteogenic gene expression compared to mesenchymal stem cells (MSCs) grown on the other groups. In addition, the cells in the HAp/BMP coated group delivered higher levels of ALP than the other three groups. Also, silk scaffolds were implanted as artificial ligaments in knees of rabbits, and they were harvested 1 and 3 months after implantation. On gross examination, HE staining showed that new bone tissue formation was more observed in the HAp/BMP coated group 3 weeks postoperatively. And masson staining showed that in the HAp/BMP coated group, the silk fibers were encircled by osteoblast, chondrocyte, and collagen. Furthermore, the analysis showed that the width of the graft-bone interface in the HAp and HAp/BMP coated group was narrower than that in the other two groups 3 weeks postoperatively. So, it is concluded that BMP incorporated HAp coated silk scaffold can be enhanced osseointegration and osteogenesis in bone tunnel. As a result, these experimental designs have been demonstrated to be effective in the acceleration of graft-to-bone healing by increasing new bone or fibrocartilage formation at the interface between graft and bone.

Atelocollagen Enhanced Osteogenesis in a Geometric Structured Beta-TCP Scaffold by VEGF Induction

Abstract In order to establish the convertibility of a host for bone augmentation, we herein developed a new honeycombshaped β-tricalcium phosphate (37H) using atelocollagen as a scaffold, which exhibited unique geometric properties for in vitro and in vivo studies. Human mesenchymal stem cells (MSC) were cultured with 37H or atelocollagen-coated honeycomb-shaped β-tricalcium phosphate (Col37H), and their osteoblastic differentiation was then analyzed. Atelocollagen promoted cell adhesion and formation of vessel-like structures in the tunnels of scaffolds of cultured MSC. The mRNA expression levels of type I collagen, osteocalcin, and VEGF were greater in MSC cultured with Col37H than with 37H. Bone generation with Col37H in the rat calvaria was greater than with 37H, and this was attributed to early vessel construction. A large number of blood vessels invaded tunnels from the periosteum and existing bone surface. A strong VEGF signal was detected immediately before the new bone surface in the tunnels of Col37H. These results indicate that the addition of atelocollagen to Col37H has potential in the construction of functional artificial bone.

Identification of orange-spotted grouper (Epinephelus coioides) interferon regulatory factor 3 involved in antiviral immune response against fish RNA virus

Interferon regulatory factor 3 (IRF3) is an important transcription factor which regulates the expression of interferon (IFN) and IFN-stimulated genes (ISGs) following virus recognition. In this study, a novel IRF3 gene was cloned from grouper Epinephelus coioides (EcIRF3) and its effects against Singapore grouper iridovirus (SGIV) and red spotted grouper nervous necrosis virus (RGNNV) was investigated. The full-length of EcIRF3 cDNA was composed of 2513 bp and encoded a polypeptide of 458 amino acids which shared 82% identity with European seabass (Dicentrarchus labrax). EcIRF3 contained three conserved domains including a DNA-binding domain (DBD), an IRF associated domain (IAD) and a serine-rich domain. Expression profile analysis revealed that EcIRF3 was abundant in head kidney, kidney, spleen and gill. Upon different stimuli in vitro, the transcript of EcIRF3 was significantly up-regulated after RGNNV infection or treatment with polyinosin-polycytidylic acid (poly I:C). During SGIV infection, the increase of the EcIRF3 transcription was only detected at the late stage, suggesting that EcIRF3 was differently regulated by different stimuli. Immune fluorescence assay indicated that the fluorescence signal of EcIRF3 was increased significantly after infection with RGNNV or treatment with poly I:C, but moderately at the late stage of SGIV infection. Reporter gene assay showed that EcIRF3 activated zebrafish type I IFN and type III IFN promoter in vitro. The viral gene transcription and virus production of RGNNV were significantly decreased in EcIRF3 overexpressing cells. However, the ectopic expression of EcIRF3 did not affect the gene transcription and virus production of SGIV. Moreover, the mRNA expression levels of type I IFN and IFN-inducible genes (MxI, ISG15 and ISG56) were increased in RGNNV infected EcIRF3 overexpressing cells compared to empty vector transfected cells. Together, our results demonstrated that IFN immune response mediated by grouper IRF3 was exerted crucial roles for fish RNA virus, but not for DNA virus replication.

Rabbiteye blueberry prevents osteoporosis in ovariectomized rats.

ObjectiveIt has been forecasted that the rabbiteye blueberry could inhibit osteoporosis. However, the inhibition and prevention of osteoporosis via rabbiteye blueberry are still elusive. This study was aim to evaluate the anti-osteoporosis effects of rabbiteye blueberry in ovariectomized rats.MethodsThirty rats were randomly divided into three groups of ten rats each as follows: sham-operated group (SG), ovariectomized model control group (OMG), and ovariectomized rabbiteye blueberry treatment group (OBG). The blood mineral levels, the alkaline phosphatase (ALP) activity, and osteoprotegerin (OPG) level were determined. The expression analyses of type I collagen, integrin-β1, and focal adhesion kinase (FAK) were performed. Besides, the bone mineral density (BMD) and bone histomorphometry (BH) were measured.ResultsThe ALP activity in SG and OBG was significantly lower than that in OMG. For the OPG level, the significant increase of OPG level in OBG was indicated compared with the other groups. The mRNA expression levels of type I collagen, integrin-β1, and FAK in OMG were significantly lower than those in other groups. The BMD in OMG were all significantly lower than those in SG and OBG. For BH, blueberry significantly improved the trabecular bone volume fraction, trabecular thickness, mean trabecular bone number, and bone formation rate, and decreased the trabecular separation, the percent of bone resorption perimeter, and mean osteoclast number in OBG compared with OMG.ConclusionsThe rabbiteye blueberries had an effective inhibition in bone resorption, bone loss, and reduction of bone strength of ovariectomized rats and could improve the BMD, osteogenic activity, and trabecular bone structure.

N-acetylcysteine alleviated silica-induced lung fibrosis in rats by down-regulation of ROS and mitochondrial apoptosis signaling

Reactive oxygen species (ROS) is a normal metabolic product of cellular respiration, but too much ROS can induce cell apoptosis. Here, we used N-acetylcysteine (NAC) to inhibit ROS activity to explore the effects of NAC on silica-induced pulmonary fibrosis in rats and provide evidence for study on the mechanism of silicosis. 24 adult male Sprague–Dawley rats weighing 180–220 g were randomly divided into three groups with eight rats in each group. Silicosis model group and NAC group were adopted non-tracheal exposure method of disposable intrapulmonary injection of 50 g/L, silica suspension 1 mL to establish animal silicosis model, NAC group treated with 600 mg/kg NAC by gavage from the right day of modeling, all animals were sacrificed after 28 days. The level of ROS contents and mitochondrial transmembrane potential changes of AM, the mRNA expression level of type I and type III procollagen, cytochrome C, cysteinyl aspartate specific protease-9 and caspase-3 were detected. The severity of pathological changes and pulmonary fibrosis were observed by pathologic specimens. It was showed that ROS contents and MTP changes were lower in the NAC group compared with the silicosis model group, other indexes were lower in the NAC group than the model group, but higher than those of the control group, the degree of lung fibrotic lesions observed from the pathological slices showed the same trend. These data indicated that NAC can reduce ROS content of AM in silica exposure rats, the mitochondrial apoptosis pathway can also be inhibited, the severity of pulmonary fibrosis alleviated as a result.

Resveratrol-Mediated Reduction of Collagen by Inhibiting Proliferation and Producing Apoptosis in Human Hypertrophic Scar Fibroblasts

Hypertrophic scar (HS) is a dermal fibroproliferative disorder characterized by excessive deposition of extracellular matrix. Here, to investigate the regulative effects of resveratrol, a natural antioxidant compound, on fibroblasts from human skin HS tissue, a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the inhibitory effect of resveratrol on cells. Cellcycle progression and apoptosis were measured by flow cytometry and Hoechst 33258 staining respectively. The hydroxyproline content and mRNA expression levels of type I and III procollagen were measured separately by ELISA and reverse transcription-polymerase chain reaction (RT-PCR). The results indicated that resveratrol significantly inhibited cell growth, arresting the cell cycle at the G1 phase and inducing apoptosis in the fibroblasts, decreasing hydroxyproline (or collagen) levels, and downregulating the expression levels of type I and III procollagen mRNA. Taken together, these data indicate that resveratrol-mediated reduction of collagen in fibroblasts is at least partially effected by causing inhibitory cell growth, cellcycle arrest, and apoptosis, and they suggest that resveratrol is a potential agent for HS treatment.

English

Donkey (Equus asinus) is a good donor for the collagen production. However, the information on mRNA and protein of donkey collagen has never been reported. In this work, the cDNA sequences coding pro&alpha;1 and pro&alpha;2 chains of donkey type I procollagen were determined from six and seven overlapping RT-PCR products, respectively. Further characterization of deduced amino acid sequences detailed the propeptides, telopeptides and triple-helical regions in donkey type I procollagen and collagen chains. Two pro&alpha; chains of donkey type I procollagen share high similarities with corresponding sequences in mammalian species observed in this study. Considering the significance of lysine and proline in the structure and function of collagen, the distribution patterns of these two characteristic residues in &alpha; chains of donkey type I collagen were observed. The mRNA expression levels of type I collagen in donkey tissues were evaluated by quantitative real-time PCR. Key words:&nbsp;Collagen, Col1a1, Col1a2, donkey, complementary DNA.

Impact of intense pulsed light irradiation on cultured primary fibroblasts and a vascular endothelial cell line

The aim of this study was to determine the effects of intense pulsed light (IPL) on cell proliferation and the secretion of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) in human fibroblasts and vascular endothelial cell lines, and to investigate the effects of IPL on the mRNA expression levels of type I and III procollagens in cultured human fibroblasts. Foreskin fibroblasts and a vascular endothelial cell line (ECV034) were cultured and treated with various wavelengths and doses of IPL irradiation. After culture for 1, 12, 24 and 48 h following IPL irradiation, fibroblasts and the vascular endothelial cell line were harvested for investigation of morphological changes by light microscopy, cell proliferation viability by MTT assay, and VEGF and MMP secretions by ELISA. The mRNA expression levels of type I and III procollagens in the fibroblasts were detected by RT-PCR. No marked morphological changes were observed in the cultured fibroblasts compared with the control. Cell growth and cellular viability were increased in fibroblasts 24 and 48 h after IPL irradiation. The levels of type I and III procollagen mRNA expression in fibroblasts increased in a time-dependent manner. However, the IPL management had no impact on VEGF and MMP secretion levels in fibroblasts and the ECV034 cell line at any time-point after irradiation as well as cell morphology and cellular proliferation. IPL irradiation may induce cellular proliferation and promote the expression of procollagen mRNAs directly in cultured primary fibroblasts, which may primarily contribute to photorejuvenation.