Corticotomy is a clinical procedure to accelerate orthodontic tooth movement characterized by the regional acceleratory phenomenon (RAP). Despite its therapeutic effects, the surgical risk and unclear mechanism hamper the clinical application. Numerous evidences support macrophages as the key immune cells during bone remodeling. Our study discovered that the monocyte-derived macrophages primarily exhibited a pro-inflammatory phenotype that dominated bone remodeling in corticotomy by CX3CR1^CreERT2; R26^GFP lineage tracing system. Fluorescence staining, flow cytometry analysis, and western blot determined the significantly enhanced expression of binding immunoglobulin protein (BiP) and emphasized the activation of sensor activating transcription factor 6 (ATF6) in macrophages. Then, we verified that macrophage specific ATF6 deletion (ATF6^f/f; CX3CR1^CreERT2 mice) decreased the proportion of pro-inflammatory macrophages and therefore blocked the acceleration effect of corticotomy. In contrast, macrophage ATF6 overexpression exaggerated the acceleration of orthodontic tooth movement. In vitro experiments also proved that higher proportion of pro-inflammatory macrophages was positively correlated with higher expression of ATF6. At the mechanism level, RNA-seq and CUT&Tag analysis demonstrated that ATF6 modulated the macrophage-orchestrated inflammation through interacting with Tnfα promotor and augmenting its transcription. Additionally, molecular docking simulation and dual-luciferase reporter system indicated the possible binding sites outside of the traditional endoplasmic reticulum-stress response element (ERSE). Taken together, ATF6 may aggravate orthodontic bone remodeling by promoting Tnfα transcription in macrophages, suggesting that ATF6 may represent a promising therapeutic target for non-invasive accelerated orthodontics.
Animals ; Mice ; Macrophages/metabolism* ; Tumor Necrosis Factor-alpha/genetics* ; Tooth Movement Techniques/methods* ; Activating Transcription Factor 6/metabolism* ; Bone Remodeling ; Flow Cytometry ; Blotting, Western

OBJECTIVE: To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation. METHODS: Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 ^-/-, arhgef10 ^+/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively. RESULTS: WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 ^-/- zebrafish had more severe symptoms than arhgef10 ^+/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis. CONCLUSION Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.
Animals ; Annexin A5 ; Apoptosis ; Blotting, Western ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line ; Cell Proliferation ; Cells, Cultured ; Flow Cytometry ; Genotype ; Humans ; In Situ Hybridization ; Larva/physiology* ; Phenotype ; RNA/isolation & purification* ; Real-Time Polymerase Chain Reaction/standards* ; Rho Guanine Nucleotide Exchange Factors/metabolism* ; Sincalide/analysis* ; Spectrophotometry/methods* ; Zebrafish/physiology*

METHODS: For primary human cavernous EC culture, cavernous tissues were implanted into Matrigel in dishes. For primary human cavernous pericyte culture, cavernous tissues were settled by gravity into dishes. We performed immunocytochemistry and Western blot to determine phenotype and morphologic changes from passage 1 to 5. The primary cultured cells were exposed to a normal-glucose (5 mmol/L) or a high-glucose (30 mmol/L) condition, and then tube formation assay was done.RESULTS: We successfully isolated high-purity EC and pericytes from human corpus cavernosum tissue. Primary cultured EC showed highly positive staining for von Willebrand factor, and pericyte revealed positive staining for NG2 and platelet-derived growth factor receptor-β. Primary cultured EC and pericytes maintained their cellular characteristics up to passage 2 or 3. However, we observed significant changes in their typical phenotype from the passage 4 and morphological characteristics from the passage 3. Human cavernous EC or pericytes formed well-organized capillary-like structures in normal-glucose condition, whereas severely impaired tube formation was detected in high-glucose condition.CONCLUSIONS: This study provides a simple and nonenzymatic method for primary culture of human cavernous EC and pericytes. Our study will aid us to understand the pathophysiology of diabetic erectile dysfunction, and also be a valuable tool for determining the efficacy of candidate therapeutic targets.]]>
Blotting, Western ; Cells, Cultured ; Diabetes Mellitus ; Endothelial Cells ; Erectile Dysfunction ; Gravitation ; Humans ; Immunohistochemistry ; Male ; Methods ; Pericytes ; Phenotype ; Platelet-Derived Growth Factor ; von Willebrand Factor

BACKGROUND: With the popularity of laparoscopic cholecystectomy, common bile duct injury has been reported more frequently. There is no perfect method for repairing porcine biliary segmental defects.METHODS: After the decellularization of human arterial blood vessels, the cells were cultured with GFP⁺ (carry green fluorescent protein) porcine bile duct epithelial cells. The growth and proliferation of porcine bile duct epithelial cells on the human acellular arterial matrix (HAAM) were observed by hematoxylin-eosin (HE) staining, electron microscopy, and immunofluorescence. Then, the recellularized human acellular arterial matrix (RHAAM) was used to repair biliary segmental defects in the pig. The feasibility of it was detected by magnetic resonance cholangiopancreatography, liver function and blood routine changes, HE staining, immunofluorescence, real-time quantitative PCR (RT-qPCR), and western blot.RESULTS: After 4 weeks (w) of co-culture of HAAM and GFP? porcine bile duct epithelial cells, GFP⁺ porcine bile duct epithelial cells grew stably, proliferated, and fused on HAAM. Bile was successfully drained into the duodenum without bile leakage or biliary obstruction. Immunofluorescence detection showed that GFP-positive bile duct cells could still be detected after GFP-containing bile duct cells were implanted into the acellular arterial matrix for 8 w. The implanted bile duct cells can successfully resist bile invasion and protect the acellular arterial matrix until the newborn bile duct is formed.CONCLUSION: The RHAAM can be used to repair biliary segmental defects in pigs, which provides a new idea for the clinical treatment of common bile duct injury.
Bile ; Bile Ducts ; Blood Vessels ; Blotting, Western ; Cholangiopancreatography, Magnetic Resonance ; Cholecystectomy, Laparoscopic ; Coculture Techniques ; Common Bile Duct ; Duodenum ; Epithelial Cells ; Fluorescent Antibody Technique ; Humans ; Infant, Newborn ; Liver ; Methods ; Microscopy, Electron ; Polymerase Chain Reaction ; Swine ; Tissue Engineering

BACKGROUND: Spinal cord injury (SCI) is a worldwide medical concern. This study aimed to elucidate the mechanism underlying the protective effect of hyperbaric oxygen (HBO) against SCI-induced neurologic defects in rats via exploring the stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) axis and expression of brain-derived neurotrophic factor (BDNF). METHODS: An acute SCI rat model was established in Sprague-Dawley rats using the Allen method. Sixty rats were divided into four groups (n = 15 in each group): sham-operated, SCI, SCI treated with HBO (SCI + HBO), and SCI treated with both HBO and AMD3100 (an antagonist of CXCR4; SCI + HBO + AMD) groups. The rats were treated with HBO twice a day for 3 days and thereafter once a day after the surgery for up to 28 days. Following the surgery, neurologic assessments were performed with the Basso-Bettie-Bresnahan (BBB) scoring system on postoperative day (POD) 7, 14, 21, and 28. Spinal cord tissues were harvested to assess the expression of SDF-1, CXCR4, and BDNF at mRNA and protein levels, using quantitative real-time polymerase chain reaction, Western blot analysis, and histopathologic analysis. RESULTS: HBO treatment recovered SCI-induced descent of BBB scores on POD 14, (1.25 ± 0.75 vs. 1.03 ± 0.66, P < 0.05), 21 (5.27 ± 0.89 vs. 2.56 ± 1.24, P < 0.05), and 28 (11.35 ± 0.56 vs. 4.23 ± 1.20, P < 0.05) compared with the SCI group. Significant differences were found in the mRNA levels of SDF-1 (mRNA: day 21, SCI + HBO vs. SCI + HBO + AMD, 2.89 ± 1.60 vs. 1.56 ± 0.98, P < 0.05), CXCR4 (mRNA: day 7, SCI + HBO vs. SCI, 2.99 ± 1.60 vs.1.31 ± 0.98, P < 0.05; day 14, SCI + HBO vs. SCI + HBO + AMD, 4.18 ± 1.60 vs. 0.80 ± 0.34, P < 0.05; day 21, SCI + HBO vs. SCI, 2.10 ± 1.01 vs.1.15 ± 0.03, P < 0.05), and BDNF (mRNA: day 7, SCI + HBO vs. SCI, 3.04 ± 0.41 vs. 2.75 ± 0.31, P < 0.05; day 14, SCI + HBO vs. SCI, 3.88 ± 1.59 vs. 1.11 ± 0.40, P < 0.05), indicating the involvement of SDF-1/CXCR4 axis in the protective effect of HBO. CONCLUSIONS HBO might promote the recovery of neurologic function after SCI in rats via activating the SDF-1/CXCR4 axis and promoting BDNF expression.
Animals ; Blotting, Western ; Brain-Derived Neurotrophic Factor ; metabolism ; Disease Models, Animal ; Hyperbaric Oxygenation ; methods ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, CXCR4 ; metabolism ; Receptors, Interleukin-8A ; metabolism ; Spinal Cord Injuries ; metabolism ; therapy

BACKGROUND AND OBJECTIVES: Manuka honey has anti-microbial, anti-inflammatory, and anti-proliferative action with a high concentration of methylglyoxal compound. It is also effective in killing Staphylococcus aureus biofilm and effective for the acute exacerbation of chronic rhinosinusitis. The aim of this study was to determine the anti-fibrotic effect of manuka honey in nasal polyp fibroblasts. MATERIALS AND METHOD: Primary nasal fibroblasts were isolated from nasal polyps and treated with transforming growth factor-beta 1 (TGF-β1). To determine the anti-fibrotic effect of manuka honey, fibroblasts were pre-treated with various concentration of the honey. Reverse transcription-polymerase chain reaction and western blot analysis were then performed to determine α-smooth muscle actin (α-SMA), collagen type I, and matrix metalloproteinase-9 (MMP-9) messenger ribonucleic acid (mRNA) expression and protein production in nasal polyp fibroblasts. Phosphorylated Smad (pSmad) 2/3 and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) were then determined by western blotting. RESULTS: TGF-β1 stimulation increased α-SMA, collagen type I, and MMP-9 mRNA expression and protein production in nasal polyp fibroblasts. Manuka honey effectively suppressed α-SMA, collagen type I, and MMP-9 mRNA expression and protein production. Its inhibitory role on TGF-β1 induced myofibroblast differentiation and its extracellular matrix production was associated with Smad2/3 and AMPK pathway. CONCLUSION: Manuka honey can inhibit TGF-β1 induced myofibroblast differentiation, collagen type I, and MMP-9 production in nasal fibroblasts. These results suggest that manuka honey might be a useful candidate for the inhibition of nasal polyp formation if further studies in vivo were accompanied.
Actins ; Adenosine ; AMP-Activated Protein Kinases ; Biofilms ; Blotting, Western ; Collagen Type I ; Extracellular Matrix ; Fibroblasts ; Homicide ; Honey ; Matrix Metalloproteinase 9 ; Methods ; Myofibroblasts ; Nasal Polyps ; Protein Kinases ; Pyruvaldehyde ; RNA ; RNA, Messenger ; Staphylococcus aureus ; Transforming Growth Factor beta ; Transforming Growth Factors

Among various vaccines against Actinobacillus pleuropneumoniae, subunit vaccines using recombinant proteins of ApxI, ApxII, and ApxIII as vaccine antigens have shown good efficacy in terms of safety and protection. Therefore, subunit vaccines are being applied worldwide and the development of new subunit vaccines is actively being conducted. To evaluate the efficacy of the subunit vaccines, it is important to measure immune responses to each Apx toxin separately. However, the cross-reactivity of antibodies makes it difficult to measure specific immune reactivity to each toxin. In the present study, specific antigen regions among the toxins were identified and cloned to solve this problem. The antigenicity of each recombinant protein was demonstrated by Western blot. Using the recombinant proteins, we developed enzyme-linked immunosorbent assay (ELISA) methods that can detect specific immune responses to each Apx toxin in laboratory guinea pigs. We suggest that the ELISA method developed in this study can be an important tool in the evaluation of vaccine efficiency and vaccine development.
Actinobacillus pleuropneumoniae ; Actinobacillus ; Animals ; Antibodies ; Blotting, Western ; Clone Cells ; Enzyme-Linked Immunosorbent Assay ; Guinea Pigs ; Methods ; Recombinant Proteins ; Vaccines ; Vaccines, Subunit

PURPOSE: Family with sequence similarity 83 member H (FAM83H) plays key roles in tumorigenesis. However, the specific roles of FAM83H in cervical cancer (CC) have not been well studied. MATERIALS AND METHODS: The RNA-seq data of 306 CC tissues and three normal samples downloaded from The Cancer Genome Atlas were used to analyze the expression of FAM83H. The Kaplan-Meier method was used to draw survival curves. Associations between FAM83H expression and clinicopathological factors were analyzed by chi-square test. Cox proportional hazards model was used to analyze prognostic factors. Loss-of-function assays were conducted to discover the biological functions of FAM83H in cell proliferation, colony formation, invasion, and migration. Real-time Quantitative Reverse Transcription PCR (qRT-PCR) and Western blotting were used to measure the expression levels of FAM83H in CC cell lines. RESULTS: Our results demonstrated that FAM83H is overexpressed in CC tissues and that high FAM83H expression is associated with worse overall survival (OS). High FAM83H expression in CC was associated with clinical stage, pathologic tumor, and pathologic node. Univariate analysis suggested that FAM83H expression was significantly related to the OS of CC patients. Although multivariate analysis showed that FAM83H expression was not an independent prognostic factor for the OS of CC patients, the effects of FAM83H on CC cell growth and motility was significant. Loss-of-function experiments demonstrated that knockdown of FAM83H inhibited proliferation, colony formation, migration, and invasion of CC cells by inactivating PI3K/AKT pathway. CONCLUSION: FAM83H might play a crucial role in CC progression and could act as a novel therapeutic target in CC.
Blotting, Western ; Carcinogenesis ; Cell Line ; Cell Proliferation ; Genome ; Humans ; Methods ; Multivariate Analysis ; Neoplasm Metastasis ; Polymerase Chain Reaction ; Prognosis ; Proportional Hazards Models ; Reverse Transcription ; Uterine Cervical Neoplasms

BACKGROUND: Vernix caseosa (VC), which is known as a unique human substance, is a biofilm that covers the skin of most human newborns. VC has many biological functions including anti-infective, skin cleansing and skin barrier repair. OBJECTIVE: In the study, we purpose to investigate the novel effect of lipids extracted from VC on the regulation of filaggrin (FLG) expression and anti-inflammation in normal human epidermal keratinocyte (NHEK) cells. METHODS: The lipids were extracted by chloroform/methanol (Folch method) and the major properties of fatty acid methyl esters were determined with gas chromatography-mass spectrometer. The relative viability of NHEK cells was evaluated by Cell Counting Kit 8 assay. The related expression of skin barrier protein was accessed with real-time quantitative polymerase chain reaction, Western blot and Immunofluorescence in NHEK cells with or without poly (I:C). Meanwhile, the changes of thymic stromal lymphopoietin (TSLP) and tumor necrosis factor alpha (TNF-α) are analyzed by enzyme-linked immunosorbent assay. RESULTS: VC lipids mostly contained saturated and branched chains fatty acids. The expression of mRNA and protein of FLG were significantly increased after the supplement with lipid in NHEK cells. Meanwhile, lipids reversed the inhibition of poly (I:C) on FLG. Moreover, lipids suppressed the over secretion of TSLP and TNF-α induced by poly (I:C). CONCLUSION: These results indicate that lipids extracted from VC has positive effects on the expression of FLG and anti-inflammation, suggesting that lipids of VC may be used for a reference for novel therapeutic method in reducing and remedying skin disease like atopic disease.
Biofilms ; Blotting, Western ; Cell Count ; Enzyme-Linked Immunosorbent Assay ; Esters ; Fatty Acids ; Fluorescent Antibody Technique ; Humans ; Infant, Newborn ; Inflammation ; Keratinocytes ; Methods ; Polymerase Chain Reaction ; RNA, Messenger ; Skin ; Skin Diseases ; Tumor Necrosis Factor-alpha ; Vernix Caseosa

BACKGROUND/AIMS: The current study aims to investigate the protective effects of Bifidobacterium infantis on the abnormal immune response to inflammatory bowel disease (IBD) in dextran sodium sulfate (DSS)-induced colitis. METHODS: Eight-week-old BALB/c mice were separated into five groups at random (control, DSS, DSS+B9 [B. infantis 1×10⁹ CFU], DSS+B8 [B. infantis 1×10⁸ CFU], and DSS+B7 [B. infantis 1×10⁷ CFU]). Colitis was induced by 5% DSS ad libitum for 7 days, at which time we assessed weight, the disease activity index (DAI) score, and the histological damage score. The nuclear transcription factor Foxp3 (a marker of Treg cells), cytokines interleukin-10 (IL-10) and transforming growth factor β1 (TGF-β1), and related proteins (programmed cell death ligand 1 [PD-L1] and programmed cell death 1 [PD-1]) were detected by an immunohistochemical method and Western blot. RESULTS: B. infantis increased weight, decreased DAI scores and histological damage scores, increased the protein expression of Foxp3 (p<0.05) and cytokines IL-10 and TGF-β1 in mouse colon tissue (p<0.05), and increased the expression of PD-L1 in the treatment groups relative to that in the DSS group (p<0.05). The effect of B. infantis on Foxp3 and PD-L1 was dose dependent in the treatment groups (p<0.05). PD-L1 was positively correlated with Foxp3, IL-10, and TGF-β1. CONCLUSIONS: In a mouse model of IBD, B. infantis can alleviate intestinal epithelial injury and maintain intestinal immune tolerance and thus may have potential therapeutic value for the treatment of immune damage in IBD.
Animals ; Bifidobacterium ; Blotting, Western ; Cell Death ; Colitis ; Colon ; Cytokines ; Dextrans ; Immune Tolerance ; Inflammatory Bowel Diseases ; Interleukin-10 ; Methods ; Mice ; Models, Theoretical ; Sodium ; T-Lymphocytes, Regulatory ; Transcription Factors ; Transforming Growth Factors