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

Adenosine, a critical molecule regulating cellular function both inside and outside cells, is controlled by two human adenosine deaminases: ADA1 and ADA2. While ADA1 primarily resides in the cytoplasm, ADA2 can be transported to lysosomes within cells or secreted outside the cell. Patients with ADA2 deficiency (DADA2) often suffer from systemic vasculitis due to elevated levels of TNF-α in their blood. Monocytes from DADA2 patients exhibit excessive TNF-α secretion and differentiate into pro-inflammatory M1-type macrophages. Our findings demonstrate that ADA2 localizes to endolysosomes within macrophages, and its intracellular concentration decreases in cells secreting TNF-α. This suggests that ADA2 may function as a lysosomal adenosine deaminase, regulating TNF-α expression by the cells. Interestingly, pneumonia patients exhibit higher ADA2 concentrations in their bronchoalveolar lavage (BAL), correlating with elevated pro-inflammatory cytokine levels. Conversely, cord blood has low ADA2 levels, creating a more immunosuppressive environment. Additionally, secreted ADA2 can bind to apoptotic cells, activating immune cells by reducing extracellular adenosine levels. These findings imply that ADA2 release from monocytes during inflammation, triggered by growth factors, may be crucial for cell activation. Targeting intracellular and extracellular ADA2 activities could pave the way for novel therapies in inflammatory and autoimmune disorders.
Humans ; Adenosine Deaminase/deficiency* ; Monocytes/cytology* ; Cell Differentiation ; Intercellular Signaling Peptides and Proteins/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Biomarkers/metabolism* ; Macrophages/metabolism* ; Pneumonia/metabolism*

OBJECTIVE: To investigate whether the G protein-coupled estrogen receptor (GPER) can attenuates acute lung injury in mice with exertional heat stroke (EHS) by inhibiting ferroptosis. METHODS: Sixty SPF-grade male C57BL/6 mice were randomly divided into four groups: normal control group (control group), EHS model group (EHS group), dimethyl sulfoxide (DMSO) solvent group (EHS+DMSO group), and GPER-specific agonist G1 group (EHS+G1 group), with 15 mice in each group. All mice underwent 14 days of adaptive training at 24-26 centigrade before modeling, and the EHS model was established using a high-temperature treadmill device. After successful modeling, the mice were allowed to cool naturally at room temperature. In the EHS+G1 group, 40 μg/kg of the GPER-specific agonist G1 was slowly injected intraperitoneally immediately after modeling. In the EHS+DMSO group, 40 μg/kg of DMSO was slowly injected intraperitoneally immediately after modeling. The control group received no treatment. Five hours after modeling, abdominal aortic blood was collected, and lung tissues were harvested after euthanasia. The lung coefficient was calculated to evaluate lung injury. Lung histopathological changes were observed under a light microscope after hematoxylin-eosin (HE) staining, and a lung histopathological score was assigned. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), malondialdehyde (MDA), and Fe²⁺ in lung tissue. Immunofluorescence was used to detect the expression of glutathione peroxidase 4 (GPX4). Real-time polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of GPX4, ferroportin 1 (FPN1), and ferritin heavy chain 1 (FTH1). Western blotting was performed to detect the protein expression of GPX4, FPN1, and FTH1. RESULTS: Compared with the control group, the lung coefficient and lung histopathological score were significantly increased in the EHS group. HE staining showed significant thickening and unevenness of the alveolar septa and alveolar walls, partial alveolar collapse, and extensive erythrocyte, inflammatory cell, and plasma-like material extravasation in the alveolar spaces. Serum levels of TNF-α, IL-1β, MDA, and Fe²⁺ were significantly elevated. Immunofluorescence staining showed a significant decrease in GPX4-positive expression in lung tissue. Western blotting and RT-PCR showed significantly reduced protein and mRNA expression of GPX4, FPN1, and FTH1 in lung tissue. Compared with the EHS group, the EHS+G1 group showed a significant reduction in lung coefficient and lung histopathological score [lung coefficient (mg/g): 3.9±0.1 vs. 4.6±0.3, lung histopathological score: 4.2±0.2 vs. 6.9±0.2, both P < 0.05]. HE staining revealed reduced severity of lung tissue fluid extravasation, inflammatory infiltration, decreased hemorrhage, and less severe alveolar structural damage. Serum levels of TNF-α, IL-1β, MDA, and Fe²⁺ were significantly reduced [TNF-α (ng/L): 44.3±0.2 vs. 64.6±0.3, IL-1β (ng/L): 69.3±0.4 vs. 97.8±0.2, MDA (nmol/L): 2.8±0.3 vs. 3.6±0.5, Fe²⁺ (nmol/L): 0.021±0.004 vs. 0.028±0.004, all P < 0.05]. Immunofluorescence staining showed a significant decrease in GPX4-positive expression in lung tissue (fluorescence intensity: 35.53±2.41 vs. 16.45±0.31, P < 0.05). RT-PCR and Western blotting showed significantly increased mRNA and protein expression of GPX4, FPN1, and FTH1 in lung tissue [mRNA expression: GPX4 mRNA (2^-ΔΔCt): 0.44±0.05 vs. 0.09±0.01, FPN1 mRNA (2^-ΔΔCt): 0.77±0.17 vs. 0.42±0.14, FTH1 mRNA (2^-ΔΔCt): 0.75±0.04 vs. 0.58±0.01; protein expression: GPX4/β-actin: 0.96±0.11 vs. 0.24±0.04, FPN1/β-actin: 1.26±0.21 vs. 0.44±0.14, FTH1/β-actin: 0.27±0.12 vs. 0.15±0.07; all P < 0.05]. However, there were no statistically significant differences in any of the above indicators between the EHS+DMSO group and the EHS group. CONCLUSION Activation of GPER can attenuate EHS-related lung injury in mice, and its mechanism may be related to the activation of the GPX4 signaling pathway and inhibition of ferroptosis.
Animals ; Mice, Inbred C57BL ; Male ; Mice ; Heat Stroke/metabolism* ; Receptors, G-Protein-Coupled ; Ferroptosis ; Receptors, Estrogen ; Acute Lung Injury/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-1beta/metabolism* ; Lung Injury ; Lung/metabolism*

OBJECTIVE: To explore the protective effect of glycosaminoglycans (GAG) on vascular endothelium in patients with sepsis. METHODS: A prospective study was conducted on adult patients with sepsis admitted to the intensive care unit (ICU) of Hangzhou Normal University Affiliated Hospital from December 2022 to December 2023. Patients were randomly divided into conventional treatment group and GAG intervention group. Both groups were treated according to the 2021 Surviving Sepsis Campaign Guidelines. The GAG intervention group was additionally treated with GAG (2 mL of sulodexide intramuscular injection once daily for 7 days) on the basis of conventional treatment. Venous blood was collected from patients at 0, 6, 24, 48, 72 hours and 7 days after enrollment to detect serum vascular endothelial glycocalyx [heparan sulfate (HS) and syndecan-1 (SDC-1)], inflammatory markers [C-reactive protein (CRP), procalcitonin (PCT), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6)], and coagulation markers [prothrombin time (PT), activated partial thromboplastin time (APTT), antithrombin-III (AT-III), fibrinogen (Fib), D-Dimer], and to perform acute physiology and chronic health evaluation II (APACHE II), sequential organ failure assessment (SOFA), and International Society on Thrombosis and Haemostasis (ISTH) scores. The prognosis of patients (length of hospital stay, ICU and 28-day mortality) was observed. The receiver operator characteristic curve (ROC curve) was drawn to evaluate the value of HS in predicting the prognosis of sepsis patients, and the correlation between endothelial glycocalyx degradation products and various clinical indicators was analyzed. RESULTS: A total of 50 adult patients with sepsis meeting the inclusion criteria were enrolled, with 25 in the conventional treatment group and 25 in the GAG intervention group. In terms of degradation products of endothelial glycocalyx, compared to baseline, both groups showed an increasing trend in HS and SDC-1 levels post-treatment. However, the GAG intervention group exhibited significantly lower HS levels at 72 hours and 7 days, as well as lower SDC-1 levels at 6, 24, 48, 72 hours and 7 days compared to the conventional group. Among the surviving patients, the HS levels at 72 hours and SDC-1 levels at 6 hours of treatment in the GAG intervention group were significantly reduced compared to the conventional treatment group. In terms of severity score, compared with before treatment, the GAG intervention group showed a significant decrease in APACHE II, SOFA, and ISTH scores after 7 days of treatment. The SOFA scores of the GAG intervention group after 48 hours and 7 days of treatment were significantly lower than those of the conventional treatment group. In terms of inflammatory indicators, compared with before treatment, the GAG intervention group showed a significant decrease in IL-6 levels after 48 hours of treatment. With the prolongation of treatment time, the CRP levels of both groups of patients showed a significant downward trend, and at 7 days of treatment, the CRP level in the GAG intervention group was significantly lower than that in the conventional treatment group. In terms of coagulation function, with prolonged treatment time, PT and APTT of both groups of patients showed an increasing trend, while Fib showed a decreasing trend. The GAG intervention group showed a significant prolongation of PT after 72 hours of treatment compared to the conventional treatment group. In terms of prognosis, there were no statistically significant differences in ICU and 28-day mortality rates between the two groups. The GAG intervention group had significantly shorter hospital stays than the conventional treatment group. ROC curve analysis showed that HS, CRP, APTT, IL-6, APACHE II, SOFA, and ISTH scores were predictive factors for the prognosis of sepsis patients (all P < 0.05). Compared to a single indicator, the combined detection of multiple indicators has a higher value in predicting the prognosis of sepsis patients [area under the curve (AUC) = 0.911, 95% confidence interval (95%CI) was 0.817-1.000], with a sensitivity of 76.9% and a specificity of 91.9%. Correlation analysis showed that HS was significantly negatively correlated with Fib, PT, TNF-α, IL-6, and PCT (r values were -0.338, -0.396, -0.288, -0.319, and -0.340, all P < 0.05), while HS was significantly positively correlated with D-Dimer and CRP (r values were 0.347 and 0.354, both P < 0.05); SDC-1 was significantly negatively correlated with Fib, PT, APTT, TNF-α, IL-6, and ISTH scores (r values were -0.314, -0.294, -0.408, -0.353, -0.289, -0.287, all P < 0.05). CONCLUSIONS Early glycocalyx degradation can occur in sepsis patients. GAG have a protective effect on,the vascular endothelium, reducing the severity of sepsis and providing organ protection. HS, CRP, APTT, IL-6, APACHE II score, SOFA score, and ISTH score are independent predictive factors for the prognosis of sepsis patients. The combination of HS and the above indicators can significantly improve the accuracy of prediction.
Humans ; Sepsis/blood* ; Glycocalyx/drug effects* ; Glycosaminoglycans/pharmacology* ; Prospective Studies ; Endothelium, Vascular/metabolism* ; Syndecan-1/blood* ; Male ; Female ; C-Reactive Protein/metabolism* ; Interleukin-6/blood* ; Heparitin Sulfate/blood* ; Middle Aged ; Adult ; Tumor Necrosis Factor-alpha/blood* ; Procalcitonin/blood*

OBJECTIVE: To investigate the interaction between α-amylase (α-AMS) and inflammatory response in patients with ventilator-associated pneumonia (VAP) and their predictive value for prognosis. METHODS: A prospective cohort study was conducted. Patients with mechanical ventilation who were treated in the intensive care unit (ICU) of the Second Hospital of Hebei Medical University from June 2020 to June 2023 were enrolled, and the patients were divided into VAP group and non-VAP group according to whether VAP occurred. VAP patients were stratified into mild [acute physiology and chronic health evaluation II (APACHE II) < 10 scores], moderate (APACHE II were 10-20 scores), and severe (APACHE II > 20 scores) groups based on the APACHE II. All patients were followed up for 28 days. In addition, healthy subjects who underwent health examination in our hospital at the same time were selected as the healthy control group. Baseline data including gender, age, mechanical ventilation mode, mechanical ventilation time, underlying diseases, drug use, and laboratory test indicators were collected. The serum levels of α-AMS, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), C-reactive protein (CRP) and other inflammatory factors were analyzed and compared. Pearson correlation analysis was performed to analyze the correlation between serum α-AMS and inflammatory factors. Logistic regression was used to analyze the influencing factors of poor prognosis in patients with VAP. The receiver operator characteristic curve (ROC curve) was plotted to evaluate the predictive value of α-AMS on the poor prognosis of patients with VAP. RESULTS: A total of 100 mechanically ventilated patients were enrolled, including 60 cases in the VAP group and 40 cases in the non-VAP group. Among the patients with VAP, there were 24 cases in the mild group, 20 cases in the moderate group, and 16 cases in the severe group. A total of 44 patients survived at 28 days, while 16 died. Additionally, 100 healthy individuals were included as the healthy control group. Serum levels of α-AMS, IL-6, TNF-α and CRP in the VAP group were significantly higher than those in the non-VAP group and the healthy control group, while the levels of α-AMS, IL-6, TNF-α and CRP in the non-VAP group were significantly higher than those in the healthy control group. There were statistically significant differences in serum α-AMS, IL-6, TNF-α, CRP levels and APACHE II scores among VAP patients with different disease severities, and the levels of the above indicators in the severe group were significantly higher than those in the moderate group and mild group, and the levels of the above indicators in the moderate VAP group were significantly higher than those in the mild group. Pearson correlation analysis showed that serum α-AMS was positively correlated with IL-6, TNF-α, CRP, and APACHE II scores (r values were 0.404, 0.392 and 0.493, 0.493, all P < 0.01). Univariate analysis showed that age, mechanical ventilation, diabetes mellitus, ventilation time, ventilation position, prophylactic use of antimicrobial drugs, and serum α-AMS, IL-6, TNF-α, CRP, and APACHE II scores were correlated with the prognosis of VAP patients (all P < 0.05). Multivariate Logistic regression analysis identified age [odds ratio (OR) = 1.340, 95% confidence interval (95%CI) was 1.119-1.605], tracheostomy (OR = 3.050, 95%CI was 1.016-9.157), diabetes mellitus (OR = 1.379, 95%CI was 1.102-1.724), and ventilation time ≥ 7 days (OR = 2.557, 95%CI was 1.163-5.623) and serum α-AMS (OR = 1.428, 95%CI was 1.098-1.856), IL-6 (OR = 1.543, 95%CI was 1.005-2.371), TNF-α (OR = 2.228, 95%CI was 1.107-4.485), CRP (OR = 1.252, 95%CI was 1.131-1.387), APACHE II scores (OR = 1.422, 95%CI was 1.033-1.957) were independent influencing factors for the 28-day prognosis of patients with VAP (all P < 0.05). ROC curve analysis demonstrated that serum α-AMS, IL-6, TNF-α and CRP exhibited significant predictive performance on the prognosis of patients with VAP. The best cut-off value for α-AMS had a sensitivity of 81.3%, specificity of 75.0%, and an area under the ROC curve (AUC) of 0.791, which was significantly higher than those of inflammatory markers IL-6, TNF-α, and CRP (P < 0.05). The combined parameter diagnostic performance was significantly better than those of individual parameters (P < 0.05), with the highest diagnostic performance when combined, corresponding to an AUC of 0.868 (95%CI was 0.798-0.938), sensitivity of 87.5%, and specificity of 79.5%. CONCLUSIONS VAP in mechanically ventilated patients can lead to an increase in the levels of peripheral blood α-AMS and inflammatory factors, and there is an interaction between α-AMS and inflammatory markers in severe VAP patients. These markers are closely related to the severity of the disease and prognosis and have significant implications for predicting patient outcomes.
Humans ; Pneumonia, Ventilator-Associated/diagnosis* ; Prognosis ; Prospective Studies ; Respiration, Artificial ; alpha-Amylases/blood* ; Interleukin-6/blood* ; Male ; Female ; C-Reactive Protein/metabolism* ; APACHE ; Inflammation ; Middle Aged ; Intensive Care Units ; Tumor Necrosis Factor-alpha/blood* ; Aged

OBJECTIVE: To investigate the effects of liriodendrin on the intestinal flora and the ferroptosis signaling pathway in renal tissue of rats with sepsis-induced acute kidney injury (AKI). METHODS: Thirty male Sprague-Dawley (SD) rats were randomly divided into sham operation group (Sham group), sepsis model induced by cecal ligation and puncture group (CLP group), and liriodendrin intervention group (CLP+LIR group), with 10 rats in each group. The CLP+LIR group was given 0.2 mL of 100 mg/kg liriodendrin by gavage 2 hours before modeling; Sham group and CLP group were given the same volume of normal saline by gavage. The samples were collected after anesthesia 24 hours after modeling. The pathological changes of renal tissue were observed by hematoxylin-eosin (HE) staining. The levels of inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukins (IL-1β, IL-6) were detected by enzyme linked immunosorbent assay (ELISA). The levels of renal function indicators such as creatinine (Cr), and urea nitrogen (UREA) in peripheral blood, and the content of malondialdehyde (MDA) and Fe²⁺ in renal tissue were detected. Western blotting was used to detect the expressions of nuclear factor E2-related factor 2 (Nrf2), glutathione peroxidase 4 (GPX4) and heme oxygenase-1 (HO-1) in renal tissues. The changes of intestinal flora were detected by 16S rDNA high-throughput sequencing. RESULTS: Compared with the Sham group, the CLP group showed significantly enlarged glomeruli, noticeable renal interstitial edema, disorganized kidney tissue, and significantly increased pathological scores. The contents of TNF-α, IL-1β, IL-6, Cr, and UREA in peripheral blood and the levels of MDA and Fe²⁺ in renal tissue were significantly increased. The protein expressions of Nrf2, GPX4, and HO-1 in renal tissue were significantly down-regulated. The species richness of intestinal flora decreased significantly, and the relative abundances of pathogenic bacteria such as Morganella, Citrobacter, Proteus, Klebsiella, Shigella, Aggregatibacter, and Enterococcus increased significantly, while the relative abundances of beneficial bacteria such as Butyricimonas, Veillonella, Prevotella, Lactobacillus, Bifidobacterium, and Ruminococcus decreased significantly. Compared with the CLP group, CLP+LIR group could significantly reduce the pathological damage of renal tissue, the pathological score significantly decreased (1.80±0.84 vs. 4.20±1.30, P < 0.05), and improve the composition of intestinal flora, reduce the relative abundances of pathogenic bacteria such as Proteus, Klebsiella, Shigella, Aggregatibacter, and Enterococcus, and significantly increase the relative abundances of Lactobacillus, Bifidobacterium, and Ruminococcus, significantly reduce the contents of TNF-α, IL-1β, IL-6, Cr, and UREA in peripheral blood and the levels of MDA and Fe²⁺ in renal tissue [blood TNF-α (ng/L): 191.31±7.23 vs. 254.90±47.89, blood IL-1β (ng/L): 11.15±4.04 vs. 23.06±1.67, blood IL-6 (ng/L): 163.20±17.83 vs. 267.69±20.92, blood Cr (μmol/L): 24.14±4.25 vs. 41.17±5.43, blood UREA (mmol/L): 4.59±0.90 vs. 8.01±1.07, renal MDA (μmol/g): 9.67±0.46 vs. 16.05±0.88, renal Fe²⁺ (mg/g): 0.71±0.07 vs. 0.93±0.04, all P < 0.05], and increase the protein expressions of Nrf2, GPX4, and HO-1 (Nrf2/GAPDH: 1.21±0.01 vs. 0.39±0.01, GPX4/GAPDH: 0.74±0.04 vs. 0.48±0.04, HO-1/GAPDH: 0.91±0.01 vs. 0.41±0.02, all P < 0.05). CONCLUSIONS Liriodendrin has an obvious protective effect on sepsis-induced AKI. The mechanism may involve regulating the intestinal flora, increasing the activation of the Nrf2/HO-1/GPX4 signaling pathway in renal tissue, and reducing ferroptosis.
Animals ; Acute Kidney Injury/microbiology* ; Rats, Sprague-Dawley ; Sepsis/complications* ; Male ; Ferroptosis/drug effects* ; Gastrointestinal Microbiome/drug effects* ; Rats ; Signal Transduction ; Kidney/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

The anti-inflammatory phytochemical investigation of the leaves of Illicium dunnianum (I. dunnianum) resulted in the isolation of five pairs of new lignans (1-5), and 7 known analogs (6-12). The separation of enantiomer mixtures 1-5 to 1a/1b-5a/5b was achieved using a chiral column with acetonitrile-water mixtures as eluents. The planar structures of 1-2 were previously undescribed, and the chiral separation and absolute configurations of 3-5 were reported for the first time. Their structures were determined through comprehensive spectroscopic data analysis [nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass (HR-ESI-MS), infrared (IR), and ultraviolet (UV)] and quantum chemistry calculations (ECD). The new isolates were evaluated by measuring their inhibitory effect on NO in lipopolysaccharide (LPS)-stimulated BV-2 cells. Compounds 1a, 3a, 3b, and 5a demonstrated partial inhibition of NO production in a concentration-dependent manner. Western blot and real-time polymerase chain reaction (PCR) assays revealed that 1a down-regulated the messenger ribonucleic acid (mRNA) levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), COX-2, and iNOS and the protein expressions of COX-2 and iNOS. This research provides guidance and evidence for the further development and utilization of I. dunnianum.
Lignans/isolation & purification* ; Plant Leaves/chemistry* ; Anti-Inflammatory Agents/isolation & purification* ; Mice ; Animals ; Molecular Structure ; Plant Extracts/pharmacology* ; Illicium/chemistry* ; Cyclooxygenase 2/immunology* ; Interleukin-6/immunology* ; Nitric Oxide/metabolism* ; Cell Line ; Tumor Necrosis Factor-alpha/immunology* ; Nitric Oxide Synthase Type II/immunology* ; Lipopolysaccharides

OBJECTIVES: This study aimed to observe the effects of initial periodontal therapy on the level of neutrophil extracellular traps (NETs) in gingival crevicular fluid (GCF) of patients with severe periodontitis and to analyze the factors related to the formation of NETs. METHODS: Thirty-one patients with stage Ⅲ-Ⅳ periodontitis were recruited. Clinical periodontal parameters, including plaque index (PLI), gingival index (GI), probing depth (PD), and clinical atta-chment loss (CAL), were recorded before and 6-8 weeks after initial periodontal therapy. Levels of NETs in GCF were detected by immunofluorescence staining. Quantities of total bacteria, Porphyromonas gingivalis (P. gingivalis), Aggregatibacter actinomycetemcomitans (A. actionomycetemcomitans) and Prevotella intermedia (P. intermedia)in unattached subgingival plaque were determined by real-time quantitative PCR, and levels of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) in GCF were explored by enzyme-linked immunosorbent assay. In addition, the correlations between the level of NETs and the above indicators were analyzed. RESULTS: After initial periodontal therapy, the level of NETs in GCF, PLI, GI, PD, and CAL; quantities of total bacteria, P. gingivalis, A. actinomycetemcomitans, and P. itermedia; and levels of IL-8 and TNF-α significantly decreased (P<0.05). We observed strong positive correlations between the level of NETs and PLI, GI, PD, CAL, the amount of total bacteria, P. gingivalis, TNF-α, and IL-8 (P<0.05). CONCLUSIONS Initial periodontal therapy might decrease the level of NETs in GCF from patients with severe periodontitis, which might be positively correlated with the quantities of P. gingivalis andthe levels of TNF-α and IL-8 in GCF.
Humans ; Gingival Crevicular Fluid ; Extracellular Traps/metabolism* ; Porphyromonas gingivalis/isolation & purification* ; Aggregatibacter actinomycetemcomitans/isolation & purification* ; Periodontitis/metabolism* ; Tumor Necrosis Factor-alpha/analysis* ; Prevotella intermedia/isolation & purification* ; Interleukin-8/analysis* ; Male ; Female ; Middle Aged ; Periodontal Index ; Adult

OBJECTIVES: This study aims to explore the potential relationships of cell-free DNA (cfDNA) in gingival crevicular fluid (GCF) with periodontal clinical indicators and the expression of DNA receptor pathway cyclic guanosine phosphate-adenosine phosphate synthase (cGAS)-stimulator of interferon genes (STING) in gingival tissues and human gingival fibroblasts (HGFs). METHODS: GCF and gingival tissue samples were collected from periodontally healthy individuals and patients diagnosed with periodontitis. Periodontal clinical indicators were recorded, including plaque index (PLT), bleeding index (BI), probing depth (PD), and clinical attachment level (CAL). The concentration of cfDNA in GCF was quantified, and the correlation between GCF and periodontal clinical indicators was analyzed. Immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to assess the distribution of cGAS, STING, and p-STING in gingival tissues. Additionally, the mRNA expression levels of the key components of the cGAS-STING signaling pathway, namely, cGAS, STING, inhibitory of kappa-B kinase (IKK), nuclear factor kappa-B p65 (NF-κB p65), interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), were measured. Furthermore, cfDNA extracted from GCF was employed to stimulate HGFs in the healthy control and periodontitis groups, and the mRNA expression levels of the key molecules of cGAS-STING signaling pathway were detected through Western blot and RT-qPCR. RESULTS: The concentration of cfDNA in GCF was found to be significantly elevated in the periodontitis group compared with the control group. Moreover, cfDNA concentration demonstrated a strong positive correlation with the periodontal clinical indicators. Immunofluorescence analysis revealed considerably increased percentage of fluorescence co-localization of cGAS, STING, and p-STING with the gingival fibroblast FSP-1 marker in the gingival tissues of the periodontitis group. The mRNA expression levels of cGAS, STING, IKK, NF-κB p65, IL-1β, IL-6,and TNF-α were significantly higher in the periodontitis group. In vitro stimulation of HGFs with GCF-derived cfDNA resulted in increased protein expression of cGAS and p-STING and considerably upregulated the mRNA expression levels of cGAS, STING, IKK, NF-κB p65, IL-1β, IL-6, and TNF-α in the healthy and periodontitis groups compared with the blank group. Correlation analysis showed that the concentration of cfDNA at the sampling site was positively correlated with the mRNA expression levels of cGAS, STING, NF-κB p65, and IL-6 in gingival tissues. CONCLUSIONS cfDNA concentrations in the GCF of patients with periodontitis are considerably elevated, and are associated with the activation of the cGAS-STING signaling pathway in HGFs. These findings suggest that cfDNA contributes to the progression of periodontitis.
Humans ; Gingival Crevicular Fluid/metabolism* ; Signal Transduction ; Gingiva/cytology* ; Nucleotidyltransferases/genetics* ; Membrane Proteins/genetics* ; Cell-Free Nucleic Acids/analysis* ; Fibroblasts/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Periodontitis/metabolism* ; Interleukin-1beta/metabolism* ; Interleukin-6/metabolism* ; Adult ; RNA, Messenger/metabolism* ; Male ; Female

Objective To explore the effect of rehmanniae radix extract(RRE)on chondrocyte apoptosis in the rabbit model of knee osteoarthritis(KOA)by regulating the miR-485-5p/heat shock protein 90 beta family member 1(Hsp90b1)axis.Methods New Zealand rabbits were randomly assigned into control,KOA,low-dose RRE,medium-dose RRE,high-dose RRE,celecoxib,high-dose RRE+antagonist control,and high-dose RRE+miR-485-5p antagonist groups,with 12 rabbits in each group.Rabbits in other groups except the control group were modeled for KOA with the improved Hulth method.After modeling for 8 weeks,the rabbits were administrated with corresponding agents for 4 weeks.The changes in the activity rating of rabbits were recorded.ELISA was employed to measure the levels of tumor necrosis factor-α(TNF-α)and interleukin(IL)-6 in the serum.Safranine O-fast green staining was conducted to reveal the pathological changes in the cartilage tissue and Mankin scoring was performed.TUNEL was employed to detect chondrocyte apoptosis.Real-time fluorescence quantitative PCR was performed to determine the expression of miR-485-5p in the cartilage tissue.Western blot was employed to determine the protein levels of Hsp90b1,cleaved cysteinyl aspartate-specific proteinase-3(Caspase-3),and Bcl2-associated-X(Bax)in the cartilage tissue.The dual-luciferase reporter assay was employed to examine the relationship between miR-485-5p and Hsp90b1.Results Compared with the control group,the KOA group showed down-regulated expression of miR-485-5p,elevated levels of TNF-α and IL-6 in the serum,cartilage erosion and losses,and increases in activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.001).Compared with the KOA group,RRE at low,medium,and high doses,and celecoxib up-regulated the expression of miR-485-5p,lowered the levels of TNF-α and IL-6 in the serum,alleviated the pathological damage to the cartilage tissue,and decreased the activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.05).Compared with the high-dose RRE group and the high-dose RRE+antagonist control group,high-dose RRE+miR-485-5p antagonist down-regulated the expression of miR-485-5p,elevated the levels of TNF-α and IL-6 in the serum,exacerbated the pathological damage to the cartilage tissue,and increased the activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.05).The results indicated that there was a targeted regulatory relationship between miR-485-5p and Hsp90b1.Conclusion RRE may inhibit the expression of Hsp90b1 by up-regulating miR-485-5p,thereby inhibiting chondrocyte apoptosis in the rabbit model of KOA.
Animals ; Rabbits ; Apoptosis/drug effects* ; Chondrocytes/pathology* ; Osteoarthritis, Knee/drug therapy* ; MicroRNAs/metabolism* ; Rehmannia/chemistry* ; Disease Models, Animal ; Tumor Necrosis Factor-alpha/blood* ; Plant Extracts/pharmacology* ; Interleukin-6/blood* ; HSP90 Heat-Shock Proteins/metabolism* ; Male ; Drugs, Chinese Herbal/pharmacology*