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

OBJECTIVES: To investigate the effects of hyperoxia on the expression of N-methyl-D-aspartate receptor 1 (NMDAR1) and its synapse-associated molecules, including cannabinoid receptor 1 (CB1R), postsynaptic density 95 (PSD95), and synapsin (SYN), in the hippocampus of neonatal rats. METHODS: One-day-old Sprague-Dawley neonatal rats were randomly divided into a hyperoxia group and a control group (n=8 per group). The hyperoxia group was exposed to 80% ± 5% oxygen continuously, while the control group was exposed to room air, for 7 days. At 1, 3, and 7 days after hyperoxia exposure, hematoxylin and eosin (HE) staining was used to observe histopathological changes in the brain. The expression levels of NMDAR1, CB1R, PSD95, and SYN proteins and mRNAs in the hippocampus were detected by immunohistochemistry, Western blotting, and quantitative real-time PCR. RESULTS: After 7 days of hyperoxia exposure, the hyperoxia group showed decreased neuronal density and disordered arrangement in brain tissue. Compared with the control group, after 1 day of hyperoxia exposure, CB1R mRNA and both NMDAR1 and CB1R protein expression in the hyperoxia group were significantly downregulated, while SYN protein expression was significantly upregulated (P<0.05). After 3 days, mRNA expression of NMDAR1, CB1R, and SYN was significantly decreased (P<0.05); NMDAR1 and CB1R protein expression was significantly downregulated (P<0.05), while PSD95 and SYN protein expression was significantly upregulated (P<0.05). After 7 days of hyperoxia, the protein expression of NMDAR1 and CB1R was significantly upregulated (P<0.05). CONCLUSIONS Continuous hyperoxia exposure induces time-dependent changes in the expression levels of NMDAR1 and its synapse-associated molecules in the hippocampus of neonatal rats.
Animals ; Receptors, N-Methyl-D-Aspartate/genetics* ; Rats, Sprague-Dawley ; Hippocampus/pathology* ; Rats ; Animals, Newborn ; Receptor, Cannabinoid, CB1/genetics* ; Hyperoxia/metabolism* ; Disks Large Homolog 4 Protein/genetics* ; Synapsins/genetics* ; Synapses ; Male ; Female ; RNA, Messenger/analysis*

Transcriptome analysis based on high-throughput sequencing of a cDNA library has been widely applied to functional genomic studies. However, the cDNA dependence of most RNA sequencing techniques constrains their ability to detect base modifications on RNA, which is an important element for the post-transcriptional regulation of gene expression. To comprehensively profile the N⁶-methyladenosine (m⁶A) and N⁵-methylcytosine (m⁵C) modifications on RNA, direct RNA sequencing (DRS) using the latest Oxford Nanopore Technology was applied to analyze the transcriptome of six tissues in rice. Approximately 94 million reads were generated, with an average length ranging from 619 nt to 1013 nt, and a total of 45,707 transcripts across 34,763 genes were detected. Expression profiles of transcripts at the isoform level were quantified among tissues. Transcriptome-wide mapping of m⁶A and m⁵C demonstrated that both modifications exhibited tissue-specific characteristics. The transcripts with m⁶A modifications tended to be modified by m⁵C, and the transcripts with modifications presented higher expression levels along with shorter poly(A) tails than transcripts without modifications, suggesting the complexity of gene expression regulation. Gene Ontology analysis demonstrated that m⁶A- and m⁵C-modified transcripts were involved in central metabolic pathways related to the life cycle, with modifications on the target genes selected in a tissue-specific manner. Furthermore, most modified sites were located within quantitative trait loci that control important agronomic traits, highlighting the value of cloning functional loci. The results provide new insights into the expression regulation complexity and data resource of the transcriptome and epitranscriptome, improving our understanding of the rice genome.
RNA ; Oryza/genetics* ; RNA, Messenger ; Gene Expression Profiling ; Transcriptome ; Sequence Analysis, RNA ; High-Throughput Nucleotide Sequencing/methods* ; RNA Processing, Post-Transcriptional

Objective: To explore the changes of γ-GCS mRNA expression and GSH-PX in serum of workers exposed to manganese in order to provide scientific basis for early diagnosis of manganese poisoning. Methods: In June 2017, a total of 180 workers from a motorcycle manufacturer were selected by stratified random sampling, including 115 welders as the exposure group and 65 administrative office workers as the Control Group, the exposure group was divided into high exposure group (43 persons) and low exposure group (72 persons) according to whether the exposure group exceeded the standard limit. The levels of γ-gcs Mrna expression and GSH-Px activity in serum were determined by Occupational Health Survey, and the differences of γ-gcs Mrna expression and GSH-Px activity among different groups were analyzed. Results: Compared with the control group, the serum GSH-Px activity was lower and the serum γ-GCS mRNA expression level was higher in the exposed group (F=370.52, 275.95, P<0.01) . Compared with the control group, there was significant difference in γ-GCS mRNA expression level and GSH-Px activity (F=0.475、1.06, P<0.01; F=48.53、111.70, P<0.01) . The concentrations of manganese in air, welding dust and urine were positively correlated with the level of γ-GCS mRNA (r=0.71, 0.50, 0.31, P<0.01) The serum GSH-Px activity was negatively correlated with the concentrations of manganese in air, welding dust and urine (r=-0.80, -0.52, -0.30, P< 0.01) , There was no correlation between Serum γ-GSH-Px activity and age and years of exposure (P>0.05) . Conclusion: Serum γ-GCS mRNA expression level and GSH-Px activity level can be used as early biomarkers of manganese poisoning. The concentrations of manganese in workplace air, welding dust and urine manganese in workers are the influencing factors.
Air Pollutants, Occupational ; Dust ; Humans ; Ions ; Manganese ; Manganese Poisoning ; Occupational Exposure/analysis* ; RNA, Messenger/genetics* ; Welding

In this study, we aimed to evaluate the toxic effects, changes in life span, and expression of various metabolism-related genes in Caenorhabditis elegans, using RNA interference (RNAi) and mutant strains, after 3-bromopyruvate (3-BrPA) treatment. C. elegans was treated with various concentrations of 3-BrPA on nematode growth medium (NGM) plates, and their survival was monitored every 24 h. The expression of genes related to metabolism was measured by the real-time fluorescent quantitative polymerase chain reaction (qPCR). Nematode survival in the presence of 3-BrPA was also studied after silencing three hexokinase (HK) genes. The average life span of C. elegans cultured on NGM with 3-BrPA was shortened to 5.7 d compared with 7.7 d in the control group. hxk-1, hxk-2, and hxk-3 were overexpressed after the treatment with 3-BrPA. After successfully interfering hxk-1, hxk-2, and hxk-3, the 50% lethal concentration (LC₅₀) of all mutant nematodes decreased with 3-BrPA treatment for 24 h compared with that of the control. All the cyp35 genes tested were overexpressed, except cyp-35B3. The induction of cyp-35A1 expression was most obvious. The LC₅₀ values of the mutant strains cyp-35A1, cyp-35A2, cyp-35A4, cyp-35B3, and cyp-35C1 were lower than that of the control. Thus, the toxicity of 3-BrPA is closely related to its effect on hexokinase metabolism in nematodes, and the cyp-35 family plays a key role in the metabolism of 3-BrPA.
Animals ; Caenorhabditis elegans/metabolism* ; Caenorhabditis elegans Proteins/genetics* ; Cytochrome P-450 Enzyme System/genetics* ; Hexokinase/physiology* ; Pyruvates/toxicity* ; RNA, Messenger/analysis*

OBJECTIVES: To evaluate whether garlicin post-conditioning can attenuate myocardial ischemiareperfusion injury in a catheter-based porcine model of acute myocardial infarction (AMI) by affecting adhesion molecules integrin β1/CD29 and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31). METHODS: Twenty-two swine were devided into 3 groups: 6 in a sham-operation group, and 8 each in the model and garlicin groups. AMI porcine model was established in the model and garlicin groups. The distal parts of the left anterior descending coronary artery in the animals of the model and garlicin groups were occluded by dilated balloon for 2 h, followed by reperfusion for 3 h. Garlicin (1.88 mg/kg) was injected over a period of 1 h, beginning just before reperfusion, in the garlicin group. Real-time polymerase chain reaction, immunohistochemistry and Western blot were carried out to detect mRNA and protein expressions of CD29 and CD31 3 h after reperfusion. RESULTS: Hematoxylin-eosin staining showed a better myocardial structure in the garlicin group after reperfusion. Compared to the model group, garlicin inhibited both the mRNA and protein expression of CD29 and CD31 in reperfusion area and no-reflflow area (P<0.05 respectively). CONCLUSIONS Garlicin post-conditioning induced cardio-protection against myocardial ischemia-reperfusion injury in this catheter-based porcine model of AMI. The cardio-protective effect of garlicin is possibly owing to suppression of production of CD29 and CD31, by inhibition of the mRNA expression of CD29 and CD31.
Allyl Compounds ; pharmacology ; Animals ; Disease Models, Animal ; Disulfides ; pharmacology ; Integrin beta1 ; analysis ; genetics ; physiology ; Ischemic Postconditioning ; Male ; Myocardial Reperfusion Injury ; prevention & control ; Platelet Endothelial Cell Adhesion Molecule-1 ; analysis ; antagonists & inhibitors ; genetics ; RNA, Messenger ; analysis ; Swine

OBJECTIVE: To observe the effect of Quyu Chencuo Formula (, QCF) on renal fibrosis in rats with obstructive nephropathy. METHODS: Twenty-four rats were randomly divided into three groups, 4 for sham operation as the control group, 10 for unilateral ureteral obstruction (UUO) model group, and the rest 10 for QCF treating UUO model group. All rats were sacrificed under 3% pentobarbital (50 mg/kg) anesthesia on the 14th day after surgery, then the right kidney samples of rats were harvested for hematoxylin eosin (HE) staining and Masson staining to observe the renal pathological changes. Immunohistochemistry and Western blotting were used to examine the expression of transforming growth factor β1 (TGF-β1), and real-time polymerase chain reaction (RT-PCR) was employed to examine the expressions of TGF-β1, α-smooth muscle actin (α-SMA) and E-cadherin mRNA. RESULTS: HE and Masson staining showed that the renal interstitial of the rats in the control group had no significant fibrotic lesion; in the model group, there were obvious interstitial fibrosis; for the QCF group, there were epithelial cell necrosis, infiltration of lymphocytes and mononuclear cells, aggravated interstitial fibrosis in varied degrees, but the pathological changes were less in the QCF group than in the model group. The immunohistochemistry and Western blotting results showed that the TGF-β1 expression was increased significantly in the model group, while decreased significantly in the QCF group (P<0.05); RT-PCR showed that the mRNA expression of α-SMA and TGF-β1 increased significantly in the model group, while both were significantly decreased in the QCF group compared with the model group (P<0.05). The mRNA expression of E-cadherin was decreased significantly in the model group, and it was significantly increased in the QCF group as compared with the model group (P<0.05). CONCLUSION QCF may improve renal fibrosis by regulating the expressions of TGF-β1, α-SMA and E-cadherin, and prevent the progress of kidney fibrosis.
Actins ; genetics ; Animals ; Cadherins ; genetics ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Fibrosis ; Kidney ; pathology ; Kidney Diseases ; drug therapy ; metabolism ; pathology ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Transforming Growth Factor beta1 ; genetics

OBJECTIVETo prepare the LINE1-ORF1p polyclonal antibody, and to study the effect of LINE1-ORF1p on the proliferation of nephroblastoma WT_CLS1 cells.

METHODSA genetic engineering method was used to achieve prokaryotic expression of LINE1-ORF1p, and rabbits were immunized with LINE1-ORF1p to prepare polyclonal antibody. Indirect ELISA was used to evaluate antibody titer, and Western blot and immunohistochemistry were used to evaluate the specific ability of antibody to recognize LINE1-ORF1p. The eukaryotic expression vector pEGFP-N1-LINE1-ORF1 was constructed and used to transfect WT_CLS1 cells. Western blot and qRT-PCR were used to measure the protein and mRNA expression of LINE1-ORF1, respectively, and cell proliferation assay and colony-forming assay were used to evaluate the effect of LINE1-ORF1p on the proliferation of WT_CLS1 cells and the formation of tumor cell clone.

RESULTSThe LINE1-ORF1p antibody prepared had a titer of >1:16 000 and could specifically recognize LINE1-ORF1p in cells and tumor tissue. WT_CLS1 cells transfected with pEGFP-N1-LINE1-ORF1 had significant increases in the mRNA and protein expression of LINE1-ORF1 and significantly enhanced cell proliferation ability and colony formation ability (P<0.05).

CONCLUSIONSLINE1-ORF1p can promote the growth of nephroblastoma cells and the formation of tumor cell clone, and may be involved in the pathogenesis of nephroblastoma.

Animals ; Antibodies ; analysis ; Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; Deoxyribonuclease I ; analysis ; genetics ; metabolism ; Humans ; Long Interspersed Nucleotide Elements ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Transfection ; Wilms Tumor ; genetics ; metabolism ; physiopathology

OBJECTIVETo investigate the molecular mechanism of action of BET bromodomain inhibitor JQ1 in treating airway remodeling in asthmatic mice.

METHODSA total of 24 mice were randomly divided into control group, ovalbumin (OVA)-induced asthma group (OVA group), and JQ1 intervention group (JQ1+OVA group), with 8 mice in each group. OVA sensitization/challenge was performed to establish a mouse model of asthma. At 1 hour before challenge, the mice in the JQ1+OVA group were given intraperitoneal injection of JQ1 solution (50 μg/g). Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected at 24 hours after the last challenge, and the total number of cells and percentage of eosinophils in BALF were calculated. Pathological staining was performed to observe histopathological changes in lung tissue. RT-PCR and Western blot were used to measure the mRNA and protein expression of E-cadherin and vimentin during epithelial-mesenchymal transition (EMT).

RESULTSCompared with the control group, the OVA group had marked infiltration of inflammatory cells in the airway, thickening of the airway wall, increased secretion of mucus, and increases in the total number of cells and percentage of eosinophils in BALF (P<0.01). Compared with the OVA group, the JQ1+OVA group had significantly alleviated airway inflammatory response and significant reductions in the total number of cells and percentage of eosinophils in BALF (P<0.01). Compared with the control group, the OVA group had significant reductions in the mRNA and protein expression of E-cadherin and significant increases in the mRNA and protein expression of vimentin (P<0.01); compared with the OVA group, the JQ1+OVA group had significant increases in the mRNA and protein expression of E-cadherin and significant reductions in the mRNA and protein expression of vimentin (P<0.01); there were no significant differences in these indices between the JQ1+OVA group and the control group (P>0.05).

CONCLUSIONSMice with OVA-induced asthma have airway remodeling during EMT. BET bromodomain inhibitor JQ1 can reduce airway inflammation, inhibit EMT, and alleviate airway remodeling, which provides a new direction for the treatment of asthma.

Airway Remodeling ; drug effects ; Animals ; Asthma ; drug therapy ; Azepines ; pharmacology ; Cadherins ; analysis ; genetics ; Epithelial-Mesenchymal Transition ; Female ; Mice ; Nuclear Proteins ; antagonists & inhibitors ; Ovalbumin ; immunology ; RNA, Messenger ; analysis ; Transcription Factors ; antagonists & inhibitors ; Triazoles ; pharmacology ; Vimentin ; analysis ; genetics

OBJECTIVETo investigate the changes in the expression of EphA5 and its ligand ephrinA5 in the hippocampus of rats with epilepsy and their role in the pathogenesis of temporal lobe epilepsy (TLE).

METHODSA total of 240 Sprague-Dawley rats were randomly divided into control group and TLE group, with 120 rats in each group. A rat model of lithium-pilocarpine TLE was established, and then the rats were divided into subgroups at 12 and 24 hours and 7, 15, 30, and 60 days after epilepsy was induced. In-situ hybridization was used to measure the mRNA expression of ephrinA5 in the CA3 region and the dentate gyrus of the hippocampus in 9 rats; immunohistochemistry was used to measure the protein expression of EphA5 in the CA3 region and the dentate gyrus of the hippocampus in 9 rats; Neo-Timm silver staining was used to observe mossy fiber sprouting in the CA3 region of the hippocampus in 2 rats.

RESULTSIn-situ hybridization showed mRNA expression of ephrinA5 in the CA3 region of the hippocampus, but this was not found in the dentate gyrus. Compared with the control group at the same time point, the TLE group had a significant reduction in the mRNA expression of ephrinA5 in the CA3 region of the hippocampus at 7 and 15 days after epilepsy was induced (P<0.05); at 30 and 60 days after epilepsy was induced, the TLE group had a gradual increase in the mRNA expression of ephrinA5 in the CA3 region of the hippocampus, and there was no significant difference between the TLE and control groups (P>0.05). Immunohistochemistry showed that EphA5 protein was expressed in the CA3 region and the dentate gyrus of the hippocampus and had a similar trend of change as ephrinA5 mRNA. Neo-Timm silver staining showed that the TLE group developed marked mossy fiber sprouting in the CA3 region of the hippocampus at 7 and 15 days after epilepsy was induced.

CONCLUSIONSDownregulation of ephrinA5 and EphA5 in the CA3 region of the hippocampus may participate in the mechanism of mossy fiber sprouting and is closely associated with the development and progression of epilepsy.

Animals ; Ephrin-A5 ; analysis ; genetics ; physiology ; Epilepsy, Temporal Lobe ; etiology ; metabolism ; Hippocampus ; chemistry ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptor, EphA5 ; analysis ; genetics ; physiology