LncRNA MALAT1 regulates inflammatory cytokine production in lipopolysaccharide-stimulated human gingival fibroblasts through sponging miR-20a and activating TLR4 pathway.

BACKGROUND AND OBJECTIVE: It has been reported that long non-coding RNAs (lncRNAs), such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), act as key regulators of the development of inflammatory diseases. However, it is unclear whether MALAT1 regulates the function of human gingival fibroblasts (HGFs) in periodontitis. This study is to explore the role of MALAT1 on inflammatory cytokine production of HGFs. MATERIAL AND METHODS: Primary HGFs were harvested from human gingiva. MALAT1 was detected in inflammatory and healthy gingival tissues via quantitative real-time PCR (qRT-PCR). Bioinformatics analysis, dual-luciferase reporter assay, and RNA-binding protein immunoprecipitation (RIP) were used to detect the relationship among MALAT1, toll-like receptor 4 (TLR4), and microRNA (miR) -20a. After transfection LPS-treated HGFs with MALAT1 siRNA (si-MALAT1), miR-20a mimic or overexpression MALAT1 plasmid (sno-MALAT1), the levels of MALAT1, miR-20a, TLR4, IL-6 and IL-8 were analyzed by qRT-PCR, enzyme-linked immunosorbent assay, or western blot assay. RESULTS: MALAT1 up-regulated in inflammatory gingival tissues of chronic periodontitis. MiR-20a was bound with MALAT1 and TLR4 3'-UTR in RNA-protein complex with Ago2, respectively. Moreover, MALAT1, TLR4, IL-6, and IL-8 increased while miR-20a decreased after 1 µg/mL Porphyromonas gingivalis lipopolysaccharide (LPS) or Escherichia coli LPS stimulation. MiR-20a inhibited the expression of proinflammatory cytokines via binding to TLR4 3'-UTR. In addition, MALAT1 increased TLR4 level and the secretion of inflammatory cytokines. CONCLUSION: MALAT1 enhances inflammatory cytokine production through sponging miR-20a and releasing TLR4, indicating a regulatory role of MALAT1 in periodontal inflammation.

miR-146a regulates inflammatory cytokine production in Porphyromonas gingivalis lipopolysaccharide-stimulated B cells by targeting IRAK1 but not TRAF6.

It has been suggested that microRNAs (miRs) are involved in the immune regulation of periodontitis. However, it is unclear whether and how miRs regulate the function of B cells in the context of periodontitis. This study is to explore the role of miR-146a on the inflammatory cytokine production of B cells challenged by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). Primary B cells were harvested from mouse spleen. Quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of inflammatory cytokines in B cells in the presence or absence of P. gingivalis LPS and/or miR-146a. Bioinformatics, luciferase reporter assay and overexpression assay were used to explore the binding target of miR-146a. Our results showed that miR-146a level in B cells was elevated by P. gingivalis LPS stimulation, and the mRNA expressions of interleukin (IL)-1ß, 6 and 10, and IL-1 receptor associated kinase-1 (IRAK1), but not TNF receptor associated factor 6 (TRAF6), were also upregulated. The expression levels of IL-1ß, 6, 10 and IRAK1 were reduced in the presence of miR-146a mimic, but were elevated by the addition of miR-146a inhibitor. MiR-146a could bind with IRAK1 3' untranslated region (UTR) but not TRAF6 3'-UTR. Overexpression of IRAK1 reversed the inhibitory effects of miR-146a on IL-1ß, 6 and 10. In summary, miR-146a inhibits inflammatory cytokine production in B cells through directly targeting IRAK1, suggesting a regulatory role of miR-146a in B cell-mediated periodontal inflammation.

Relationship between the Pathogenesis of Glaucoma and miRNA.

Small RNA (microRNA or miRNA) is a kind of small noncoding single-stranded RNA that regulates complementary mRNA at the posttranscriptional level in eukaryotic organisms. As important regulatory factors, miRNAs play an important role in the occurrence and development of glaucoma and widely participate in regulating biological processes of glaucoma-related genes. This article reviews the connection between the aqueous humor, trabecular meshwork, the apoptosis of retinal ganglion cells, and miRNA.

Sex Hormones Enhance Gingival Inflammation without Affecting IL-1ß and TNF-α in Periodontally Healthy Women during Pregnancy.

Hormones (progesterone and estradiol) change greatly during pregnancy; however, the mechanism of hormonal changes on gingival inflammation is still unclear. This study is to evaluate the effects of hormonal changes during pregnancy on gingival inflammation and interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in gingival crevicular fluid (GCF). 30 periodontally healthy pregnant women were evaluated in the first, second, and third trimesters. 20 periodontally healthy nonpregnant women were evaluated twice (once per subsequent month). Clinical parameters including probing pocket depth (PPD), bleeding index (BI), gingival index (GI), clinical attachment level (CAL), and plaque index (PLI) were recorded. GCF levels of IL-1ß and TNF-α and serum levels of progesterone and estradiol were measured. From the data, despite low PLI, BI and GI increased significantly during pregnancy; however, no significant changes in PLI, CAL, IL-1ß, or TNF-α GCF levels were observed. Although IL-1ß, not TNF-α, was higher in pregnant group than in nonpregnant group, they showed no correlation with serum hormone levels during pregnancy. GI and BI showed significant positive correlation with serum hormone levels during pregnancy. This study suggests that sex hormone increase during pregnancy might have an effect on inflammatory status of gingiva, independent of IL-1ß and TNF-α in GCF.

The negative feedback regulation of microRNA-146a in human periodontal ligament cells after Porphyromonas gingivalis lipopolysaccharide stimulation.

OBJECTIVE: Toll-like receptors (TLRs) pathway has been demonstrated to play an important role in periodontitis. However, the regulatory mechanism of microRNAs (miRNAs) on TLRs pathway is still unclear. Hence, this study is to explore the function of miRNA-146a in inflammatory reaction induced by Porphyromonas gingivalis lipopolysaccharide (LPS) in human periodontal ligament cells (hPDLCs). METHODS: Cells were treated with 1 or 10 µg/ml P. gingivalis LPS. The expression of TLR2, TLR4 and miRNA-146a were measured by real-time polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was applied to detect nuclear factor (NF)-κ B p65 nuclear activity, interleukin-1ß (IL-1ß), IL-6, IL-8 and tumor necrosis factor-α (TNF-α). To examine the underlying mechanisms, cells were exposed to anti-TLR2/4 mAb or miRNA-146a inhibitor/mimic and evaluated by real-time PCR and ELISA. RESULTS: 10 µg/ml P. gingivalis LPS increased the expressions of TLR2 (3.79 ± 0.31), TLR4 (2.21 ± 0.31), and miRNA-146a (4.91 ± 0.87), NF-κ B p65 nuclear activity (6.51 ± 0.77 fold) (p < 0.05). 1 µg/ml P. gingivalis LPS induced TLR2 (3.05 ± 0.23), miRNA-146a (3.66 ± 0.83) and NF-κ B p65 nuclear activity (4.06 ± 0.78 fold) (p < 0.05), except TLR4 (1.11 ± 0.30, p > 0.05). Also, cytokines production increased (p < 0.05). The up-regulation of miRNA-146a could be blocked by anti-TLR2/4 mAb (p < 0.05). After the blockage of miRNA-146a, TLR2, TLR4, NF-κ B p65 nuclear activity and proinflammatory cytokines increased. However, after application of miRNA-146a mimic, the levels of these indexes decreased obviously (p < 0.05). CONCLUSION: MiRNA-146a functions as a negative feedback regulator via down-regulating proinflammatory cytokine secretion and blocking TLRs signaling pathway in hPDLCs after P. gingivalis LPS stimulation.

Relationship between gingival inflammation and pregnancy.

An increase in the prevalence and severity of gingival inflammation during pregnancy has been reported since the 1960s. Though the etiology is not fully known, it is believed that increasing plasma sex steroid hormone levels during pregnancy have a dramatic effect on the periodontium. Current works of research have shown that estrogen and progesterone increasing during pregnancy are supposed to be responsible for gingivitis progression. This review is focused not only on epidemiological studies, but also on the effects of progesterone and estrogen on the change of subgingival microbiota and immunologic physiological mediators in periodontal tissue (gingiva and periodontal ligament), which provides current information about the effects of pregnancy on gingival inflammation.

Nel-like Molecule Type 1 Combined with Gold Nanoparticles Modulates Macrophage Polarization, Osteoclastogenesis, and Oral Microbiota in Periodontitis.

The disruption of host-microbe homeostasis and uncontrolled inflammatory response have been considered as vital causes for developing periodontitis, subsequently leading to an imbalance between the bone and immune system and the collapse of bone homeostasis. Consequently, strategies to modulate the immune response and bone metabolization have become a promising approach to prevent and treat periodontitis. In this study, we investigated the cooperative effects of Nel-like molecule type 1 (Nell-1) and gold nanoparticles (AuNPs) on macrophage polarization, osteoclast differentiation, and the corresponding functions in an experimental model of periodontitis in rats. Nell-1-combined AuNPs in in vitro studies were found to reduce the production of inflammatory factors (TNF-α, p < 0.0001; IL-6, p = 0.0012), modulate the ratio of M2/M1 macrophages by inducing macrophage polarization into the M2 phenotype, and inhibit cell fusion, maturation, and activity of osteoclasts. Furthermore, the local application of Nell-1-combined AuNPs in in vivo studies resulted in alleviation of damages to the periodontal and bone tissues, modulation of macrophage polarization and the activity of osteoclasts, and alteration of the periodontal microbiota, in which the relative abundance of the probiotic Bifidobacterium increased (p < 0.05). These findings reveal that Nell-1-combined AuNPs could be a promising drug candidate for the prevention and treatment of periodontitis. However, Nell-1-combined AuNPs did not show organ toxicity or impair the integrity of intestinal epithelium but alter the gut microbiota, leading to the dysbiosis of gut microbiota. The adverse impact of changes in gut microbiota needs to be further investigated. Nonetheless, this study provides a novel perspective and direction for the biological safety assessment of biomaterials in oral clinical applications.

MicroRNA-126 regulates macrophage polarization to prevent the resorption of alveolar bone in diabetic periodontitis.

OBJECTIVE: This study aims to investigate the effects of microRNA-126 (miR-126) on the macrophage polarization in vitro and alveolar bone resorption in vivo. DESIGN: The relationship between miR-126 and MEK/ERK kinase 2 (MEKK2) was confirmed by dual-luciferase reporter assay. Real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay or Western blot was used to detect the changes of miR-126, inducible nitric oxide synthase (iNOS), arginase-1 (Arg-1), tumor necrosis factor (TNF)-α, interleukin (IL)-10, MEKK2 and MEKK2-related pathways: mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) in RAW264.7 macrophages challenged with Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) and/or high glucose and/or miR-126 mimic. In mice with diabetic periodontitis, the expressions of iNOS and Arg-1 in gingiva, and alveolar bone level were detected after miR-126 mimic injection. RESULTS: MiR-126 could directly bind with MEKK2 3'-untranslated region (UTR). MEKK2, phosphorylation of NF-κB and MAPK signaling proteins, TNF-α and iNOS increased (P < 0.05), while miR-126, Arg-1 and IL-10 were inhibited (P < 0.05) in macrophage challenged with high glucose and/or P. gingivalis LPS, however, miR-126 mimic reversed these effects (P < 0.05). The expressions of iNOS in gingiva and alveolar bone resorption were elevated (P < 0.05), the expression of Arg-1 in gingiva decreased (P < 0.05) in mice with diabetic periodontitis, which could be inhibited by miR-126 mimic. CONCLUSIONS: miR-126 might prevent alveolar bone resorption in diabetic periodontitis and inhibit macrophage M1 polarization via regulating MEKK2 signaling pathway.

Mussel-inspired "plug-and-play" hydrogel glue for postoperative tumor recurrence and wound infection inhibition.

Currently, clinical tumor resection is faced with two options: open and minimally invasive surgery. Open surgery is easy to completely remove the lesion but is prone to infection, while minimally invasive surgery recovers faster but may cause tumor recurrence. To fill the shortcomings of the two surgical modes and make the choice for tumor resection more effortlessly, we developed a postoperative black phosphorus-Ag nanocomposites-loaded dopamine-modified hyaluronic acid-Pluronic® F127 (BP-Ag@HA-DA-Plu) hydrogel implantation system that can prevent tumor recurrence and wound infection simultaneously. Experiments have shown that the hydrogel system combined with 808 nm near-infrared (NIR) irradiation has excellent anti-tumor, antibacterial, and wound healing abilities. Additionally, unlike existing surgical hydrogel products that require inconvenient in-situ cross-linking, the BP-Ag@HA-DA-Plu hydrogel system offers "plug-and-play" functionality during surgery due to its thermo-responsiveness, injectability, and adhesion, thereby greatly improving the efficiency of surgery.

High glucose induces inflammatory cytokine through protein kinase C-induced toll-like receptor 2 pathway in gingival fibroblasts.

Toll-like receptors (TLRs) play a key role in innate immune response and inflammation, especially in periodontitis. Meanwhile, hyperglycemia can induce inflammation in diabetes complications. However, the activity of TLRs in periodontitis complicated with hyperglycemia is still unclear. In the present study, high glucose (25 mmol/l) significantly induced TLR2 expression in gingival fibroblasts (p<0.05). Also, high glucose increased nuclear factor kappa B (NF-κB) p65 nuclear activity, tumor necrosis factor-α (TNF-α) and interleukin-lß (IL-1ß) levels. Protein kinase C (PKC)-α and δ knockdown with siRNA significantly decreased TLR2 and NF-κB p65 expression (p<0.05), whereas inhibition of PKC-ß had no effect on TLR2 and NF-κB p65 under high glucose (p<0.05). Additional studies revealed that TLR2 knockdown significantly abrogated high-glucose-induced NF-κB expression and inflammatory cytokine secretion. Collectively, these data suggest that high glucose stimulates TNF-α and IL-1ß secretion via inducing TLR2 through PKC-α and PKC-δ in human gingival fibroblasts.

p38 mitogen-activated protein kinase-induced nuclear factor kappa-light-chain-enhancer of activated B cell activity is required for neuroprotection in retinal ischemia/reperfusion injury.

PURPOSE: In our previous study, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) played a neuroprotective role in retinal ischemia/reperfusion (I/R) injury in rats. However, the mechanism of NF-κB neuroprotection is still unclear. We hypothesize that p38 mitogen-activated protein kinase (MAPK) is expressed and NF-κB activity induced by p38 MAPK plays a neuroprotective role through antiapoptotic genes (B-cell lymphoma [Bcl]-2 and Bcl-XL) in retinal cells in retinal I/R injury. METHODS: Retinal ischemia was induced by elevating intraocular pressure in rats. After retinal I/R, the p38 MAPK, NF-κB p65, Bcl-2, and Bcl-XL mRNA levels were measured with real-time polymerase chain reaction. NF-κB p65 activity was assessed with NF-κB enzyme-linked immunosorbent assay in retinal I/R injury and after application of the p38 MAPK inhibitor (SB203580). Furthermore, SB203580 and NF-κB p65 short interfering RNA (siRNA) were used in retinal I/R injury to examine the effects on Bcl-2 and Bcl-XL levels and nucleosome release in the retina and cell survival in the ganglion cell layer. RESULTS: The mRNA levels of NF-κB p65 and p38 MAPK reached a peak at 6 h after retinal I/R and then decreased gradually. The mRNA levels of Bcl-2 and Bcl-XL significantly increased at 2, 4, and 6 h, peaked at 8 h, and decreased gradually, but remained at a higher level compared with the normal control, which was accompanied by an increase in NF-κB p65 in nuclear extracts. After application of SB203580, the increase in the NF-κB p65 levels in the nucleus induced with I/R was completely abolished, and the mRNA expression of Bcl-2 and Bcl-XL decreased significantly compared with the I/R controls. In addition, NF-κB p65 siRNA inhibited Bcl-2 and Bcl-XL expression. Inhibition of the p38 MAPK-NF-κB pathway (using SB203580 or NF-κB p65 siRNA) increased retinal nucleosome release and decreased the number of ganglion cells. CONCLUSIONS: These findings provide evidence of crosstalk between p38 MAPK and NF-κB p65 and demonstrate a possible neuroprotective role for the p38 MAPK-NF-κB pathway through Bcl-2 and Bcl-XL in retinal I/R injury in rats.

A Nested Case-Control Study of the Relationship between Salivary Inflammatory Mediators, Periodontal Parameters, and Preterm Birth in a Chinese Population.

Background: To explore whether salivary inflammatory mediators and periodontal indices at different gestational stages can be taken as indicators of preterm birth (PTB). Methods: This nested case-control study enrolled systemically healthy pregnant women at 9 to 36 weeks of gestation. Periodontal indices were measured at the enrollment date, and interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor (TNF-α), prostaglandin E2 (PGE2), and 8-hydroxy-deoxyguanosine (8-OHdG) in the saliva were determined by enzyme-linked immunosorbent assay (ELISA). The birth outcome was recorded. Results: PTB occurred in 26 women. A total of 104 matched women with full-term birth (FTB) were used as controls. The PTB women enrolled at 24-28 gestational weeks displayed a significantly greater bleeding index (BI), probing pocket depth (PD), PD ≥ 4 mm sites (%), saliva-TNF-α, and saliva-PGE2 (P < 0.05). BI and PGE2 in the saliva were found to be positively associated with PTB (OR = 4.79, P = 0.048, 95%CI = 1.014 to 22.628; OR = 1.07, P = 0.04, 95%CI = 1.004 to 1.135, respectively). The areas under the receiver operating characteristic curve (ROC) of BI and saliva-PGE2 were 0.82 and 0.78, respectively, and that of the combined detection was 0.91, which was larger than either marker alone, although the differences were not significant (P > 0.05). Conclusions: The combination of BI and PGE2 in saliva at 24-28 gestational weeks could be a predictor of PTB in asymptomatic women. However, the results should be further explored with larger sample size.

Levels and patterns of physical activity and sedentary behavior in adults with and without visual impairment.

BACKGROUND: Limited data are available on objectively measured physical activity (PA) and sedentary behavior (SB) among adults with and without visual impairment (VI). OBJECTIVE: To compare PA and SB levels and patterns in adults with and without VI and to examine how these differ based on sex and day of the week. METHODS: Thirty-two participants with VI and 32 participants without VI participated in this cross-sectional study. PA and SB were assessed using GT3X ActiGraph accelerometers during waking hours for 7 days, and variables were examined in terms of disability group, sex, and day of the week. Nonparametric Mann-Whitney U test and Wilcoxon signed-rank test were used, and significance was set at p < 0.05. RESULTS: PA did not differ in terms of sex or day of the week in participants with VI. The PA of participants without VI was significantly higher for men than it was for women and was significantly higher during weekdays than on weekend days. Total sedentary time and the duration of SB breaks were significantly longer for female participants with VI than for those without VI. The number of sedentary bouts lasting ≥10 min during weekend days was significantly higher for participants with VI than for those without VI. CONCLUSIONS: Most adults with and without VI did not meet the recommended levels of daily PA and spend a large portion of the day being sedentary. Interventions to enhance PA and reduce sedentary time in adults with and without VI are required.

Role of amyloid ß-peptide in the pathogenesis of age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common eye disease in elderly patients, which could lead to irreversible vision loss and blindness. Increasing evidence indicates that amyloid ß-peptide (Aß) might be associated with the pathogenesis of AMD. In this review, we would like to summarise the current findings in this field. The literature search was done from 1995 to Feb, 2021 with following keywords, 'Amyloid ß-peptide and age-related macular degeneration', 'Inflammation and age-related macular degeneration', 'Angiogenesis and age-related macular degeneration', 'Actin cytoskeleton and amyloid ß-peptide', 'Mitochondrial dysfunction and amyloid ß-peptide', 'Ribosomal dysregulation and amyloid ß-peptide' using search engines Pubmed, Google Scholar and Web of Science. Aß congregates in subretinal drusen of patients with AMD and participates in the pathogenesis of AMD through enhancing inflammatory activity, inducing mitochondrial dysfunction, altering ribosomal function, regulating the lysosomal pathway, affecting RNA splicing, modulating angiogenesis and modifying cell structure in AMD. The methods targeting Aß are shown to inhibit inflammatory signalling pathway and restore the function of retinal pigment epithelium cells and photoreceptor cells in the subretinal region. Targeting Aß may provide a novel therapeutic strategy for AMD.

Building an aprismatic enamel-like layer on a demineralized enamel surface by using carboxymethyl chitosan and lysozyme-encapsulated amorphous calcium phosphate nanogels.

OBJECTIVES: The purpose of this study was to prepare carboxymethyl chitosan (CMC) and lysozyme nanogels that could encapsulate amorphous calcium phosphate (ACP) for achieving its controlled delivery, thus forming an aprismatic enamel-like layer on the demineralized enamel surface. METHODS: CMC/LYZ-ACP nanogels were developed, and the controlled delivery of ACP from the nanogels was induced by the presence of NaCl. The nanogel morphologies at various NaCl concentrations was measured by transmission electron microscopy (TEM). The particle sizes and zeta potentials (ζ-potential) of the samples were determined using a combined dynamic light scattering/particle electrophoresis instrument. Comparing the remineralization effect of the CMC/LYZ-ACP nanogels on the demineralized enamel surface with that of a fluoride treatment, the remineralization effect was examined by nanoindentation tests, X-ray diffraction (XRD), confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM). RESULTS: CMC/LYZ-ACP nanogels were negatively charged spherical structures with a particle size of approximately 300 nm. At high concentrations of NaCl (0.15 M), ACP was dissociated from the disassembled nanogels and transformed into hydroxyapatite (HAP). Groups treated with the CMC/LYZ-ACP nanogels showed the regeneration of an aprismatic enamel-like layer on an acid-etched enamel surface, which provided increased mechanical properties (P < 0.05) and a high impermeability (P < 0.01) compared to those of the fluoride-treated group. CONCLUSIONS: This research provides a new idea for the stable and controllable delivery of ACP from CMC/LYZ-ACP nanogels, which can form an aprismatic enamel-like layer in situ on the surface of demineralized enamel. In regard to further clinical development, this material and method may be promising for treating early enamel caries.

MicroRNA-146a regulates the production of cytokines in lymphocytes stimulated by Porphyromonas gingivalis lipopolysaccharide.

OBJECTIVES: This study aimed to investigate the effects of microRNA-146a (miR-146a) on the production of cytokines in lymphocytes stimulated by Porphyromonas gingivalis (P.gingivalis) lipopolysaccharide (LPS). METHODS: Lymphocytes were harvested from mouse spleen and cultured in vitro. The cells were treated with P. gingivalis LPS, miR-146a mimic, or miR-146a inhibitor. Scramble RNA served as the negative control of mimic and inhibitor. The production of inflammatory cytokines was detected by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Compared with non-LPS-stimulated group, P. gingivalis LPS could increase the levels of interleukin (IL)-1ß, IL-6, receptor activator NF-κB ligand (RANKL), and IL-10 (P<0.05) and decrease the mRNA level of osteoprotectin (OPG) (P<0.05). However, it did not significantly change the secretion of OPG. Compared with the negative control group, miR-146a mimic upregulated the levels of IL-10 and OPG (P<0.05), downregulated IL-1ß, IL-6, and RANKL (P<0.05). Meanwhile, miR-146a inhibitor had a reverse effect on these cytokines (P<0.05) in P.gingivalis LPS-treated-lymphocytes. CONCLUSIONS: MiR-146a can provide a suitable microenvironment for bone formation by preventing the inflammatory effects of P.gingivalis LPS through the inhibition of IL-1ß, IL-6, and RNAKL, thereby enhancing IL-10 and OPG.

Msx2 alters the timing of retinal ganglion cells fate commitment and differentiation.

Timing of cell fate commitment determines distinct retinal cell types, which is believed to be controlled by a tightly coordinated regulatory program of proliferation, cell cycle exit and differentiation. Although homeobox protein Msx2 could induce apoptosis of optic vesicle, it is unclear whether Msx2 regulates differentiation and cell fate commitment of retinal progenitor cells (RPCs) to retinal ganglion cells (RGCs). In this study, we show that overexpression of Msx2 transiently suppressed the expression of Cyclin D1 and blocked cell proliferation. Meanwhile, overexpression of Msx2 delayed the expression of RGC-specific differentiation markers (Math5 and Brn3b), which showed that Msx2 could affect the timing of RGCs fate commitment and differentiation by delaying the timing of cell cycle exit of retinal progenitors. These results indicate Msx2 possesses dual regulatory functions in controlling cell cycle progression of retinal RPCs and timing of RGCs differentiation.

Different effects of 25-kDa amelogenin on the proliferation, attachment and migration of various periodontal cells.

Previous studies have assumed that amelogenin is responsible for the therapeutic effect of the enamel matrix derivative (EMD) in periodontal tissue healing and regeneration. However, it is difficult to confirm this hypothesis because both the EMD and the amelogenins are complex mixtures of multiple proteins. Further adding to the difficulties is the fact that periodontal tissue regeneration involves various types of cells and a sequence of associated cellular events including the attachment, migration and proliferation of various cells. In this study, we investigated the potential effect of a 25-kDa recombinant porcine amelogenin (rPAm) on primarily cultured periodontal ligament fibroblasts (PDLF), gingival fibroblasts (GF) and gingival epithelial cells (GEC). The cells were treated with 25-kDa recombinant porcine amelogenin at a concentration of 10 microg/mL. We found that rPAm significantly promoted the proliferation and migration of PDLF, but not their adhesion. Similarly, the proliferation and adhesion of GF were significantly enhanced by treatment with rPAm, while migration was greatly inhibited. Interestingly, this recombinant protein inhibited the growth rate, cell adhesion and migration of GEC. These data suggest that rPAm may play an essential role in periodontal regeneration through the activation of periodontal fibroblasts and inhibition of the cellular behaviors of gingival epithelial cells.

The interaction between serum amyloid A and Toll-like receptor 2 pathway regulates inflammatory cytokine secretion in human gingival fibroblasts.

BACKGROUND: Serum amyloid A (SAA) has been identified to trigger inflammation response, and play a crucial role in chronic inflammatory diseases. However, the regulatory mechanism of SAA still remains unclear during the development of periodontitis METHODS: SAA mRNA and protein expression were detected in healthy and inflammatory gingival tissues using real-time polymerase chain reaction (PCR) and immunohistochemistry. Human recombinant SAA (Apo-SAA), Pam3CSK4 (a Toll-like receptor (TLR) 2 ligand), siRNA-SAA, or TLR2 neutralizing antibody was applied to treat human gingival fibroblasts, respectively, or combined. SAA, TLRs, and inflammatory cytokines interleukin (IL)-6 and IL-8 were analyzed by real-time PCR, western blotting, or enzyme-linked immunosorbent assay. RESULTS: SAA expression increased in human inflammatory gingival tissues from patients with periodontitis (P <0.05). Apo-SAA could increase not only the mRNA expression of TLR2 (P <0.05), but also IL-6 and IL-8 mRNA and protein levels (P <0.05) which was suppressed by TLR2 antibody in human gingival fibroblasts. Pam3CSK4 increased SAA, IL-6, and IL-8 levels (P <0.05). However, the expression of SAA, IL-6, and IL-8 decreased after transfection of siRNA-SAA (P <0.05). CONCLUSION: SAA not only increases in inflammatory gingiva, but also triggers inflammatory cytokine secretion via interacting with TLR2 pathway in human gingival fibroblasts, which indicates that SAA is involved in periodontal inflammation.

MicroRNA-126 Regulates Inflammatory Cytokine Secretion in Human Gingival Fibroblasts Under High Glucose via Targeting Tumor Necrosis Factor Receptor Associated Factor 6.

BACKGROUND: MicroRNAs (miRs) play a crucial role in inflammatory diseases, including periodontitis. Meanwhile, miRs act as biomarkers for predicting diabetes mellitus (DM). However, the regulatory mechanism of miR-126 on development of periodontitis in patients with DM still remains unclear. METHODS: Human gingival fibroblasts were cultured with low (5.5 mmol/L), medium (15 mmol/L), and high (25 mmol/L) glucose, respectively. Expressions of miR-126, tumor necrosis factor (TNF) receptor associated factor (TRAF) 6, and related cytokines were analyzed by real-time polymerase chain reaction (PCR). After transfection with miR-126 mimic, PCR and western blot were performed to detect level of TRAF6, and luciferase reporter assay confirmed if TRAF6 is the direct target of miR-126. Production of cytokines was measured using enzyme-linked immunosorbent assay. RESULTS: Increased glucose significantly suppressed miR-126 expression in human gingival fibroblasts (P <0.05). Also, high glucose increased TRAF6, interleukin (IL)-6, TNF-α, and chemical chemokine ligand (CCL) 2 levels, whereas it decreased IL-10 level. MiR-126 mimic significantly decreased TRAF6 mRNA and protein levels under high glucose (P <0.05). Also, miR-126 directly targeted TRAF6 through binding to its 3' untranslated region in human gingival fibroblasts. Overexpression of miR-126 significantly abrogated high glucose-induced secretion of proinflammatory cytokines such as IL-6, TNF-α, and CCL2 and promoted production of IL-10. CONCLUSION: These data suggest that miR-126 inhibits inflammation of human gingival fibroblasts under high glucose through targeting TRAF6, which may be a potential therapeutic target for periodontitis concomitant with DM.