BACKGROUND: Senescent human skin primary fibroblast (FB) models have been established for studying aging-related, proliferative, and inflammatory skin diseases. The aim of this study was to compare the transcriptome characteristics of human primary dermal FBs from children and the elderly with four senescence models. METHODS: Human skin primary FBs were obtained from healthy children (FB-C) and elderly donors (FB-E). Senescence models were generated by ultraviolet B irradiation (FB-UVB), D-galactose stimulation (FB-D-gal), atazanavir treatment (FB-ATV), and replication exhaustion induction (FB-P30). Flow cytometry, immunofluorescence staining, real-time quantitative polymerase chain reaction, co-culturing with immune cells, and bulk RNA sequencing were used for systematic comparisons of the models. RESULTS: In comparison with FB-C, FB-E showed elevated expression of senescence-related genes related to the skin barrier and extracellular matrix, proinflammatory factors, chemokines, oxidative stress, and complement factors. In comparison with FB-E, FB-UVB and FB-ATV showed higher levels of senescence and expression of the genes related to the senescence-associated secretory phenotype (SASP), and their shaped immune microenvironment highly facilitated the activation of downstream immune cells, including T cells, macrophages, and natural killer cells. FB-P30 was most similar to FB-E in terms of general transcriptome features, such as FB migration and proliferation, and aging-related characteristics. FB-D-gal showed the lowest expression levels of senescence-related genes. In comparisons with the single-cell RNA sequencing results, FB-E showed almost complete simulation of the transcriptional spectrum of FBs in elderly patients with atopic dermatitis, followed by FB-P30 and FB-UVB. FB-E and FB-P30 showed higher similarity with the FBs in keloids. CONCLUSIONS Each senescent FB model exhibited different characteristics. In addition to showing upregulated expression of natural senescence features, FB-UVB and FB-ATV showed high expression levels of senescence-related genes, including those involved in the SASP, and FB-P30 showed the greatest similarity with FB-E. However, D-galactose-stimulated FBs did not clearly present aging characteristics.
Humans ; Fibroblasts/drug effects* ; Cellular Senescence/physiology* ; Skin/metabolism* ; Child ; Transcriptome/genetics* ; Aged ; Ultraviolet Rays ; Cells, Cultured ; Galactose/pharmacology*

ObjectiveTo investigate the potential mechanism of Liangxue Tuizi Formula （凉血退紫方， LXTZF） in treating Henoch-Schönlein Purpura （HSP） by examining its regulatory effect on neutrophil extracellular trap （NETs） dysregulation via the rapidly accelerated fibrosarcoma kinase （RAF）/mitogen-activated protein kinase （MEK）/extracellular signal-regulated kinase （ERK） signaling pathway. MethodsSeventy Wistar rats were randomly allocated into a blank control group （n=14） and a modeling group （n=56）. Rats in the modelling group underwent an eight-week modelling period to establish HSP rat models with blood-heat syndrome via modified ovalbumin （OVA） induction method combined with oral administration of heat-property Chinese herbal medicine. Fifty successfully modeled rats were subsequently randomly divided into five groups （n=10 per group）， model group， compound glycyrrhizin group， LXTZF group， RAF inhibitor group， and LXTZF + RAF agonist group. Additionally， 10 rats were selected from the original blank control group for the final experiment. From the 11th week of modelling， rats in the blank control group and the model group received 1 ml/（100 g·d） ultrapure water via oral administration， in addition to 0.5 ml/（kg·d） 0.9% sodium chloride solution via intraperitoneal injection. The LXTZF group and the compound glycyrrhizin group received 7.5 g/（kg·d） LXTZF granule suspension via gavage， 13.5 mg/（kg·d） compound glycyrrhizin suspension via gavage， respectively. The RAF inhibitor group received 1 mg/（kg·d） GW5074 suspension via intraperitoneal injection and ultrapure water via oral administration； the LXTZF + RAF agonist group received 7.5 g/（kg·d） LXTZF granule suspension via gavage and 1 mg/（kg·d） paclitaxel suspension via intraperitoneal injection. All administrations were performed once daily for 4 weeks. After intervention， skin tissue histopathology was examined by hematoxylin and eosin （H&E） staining， immunoglobulin A （IgA） deposition was assessed via immunofluorescence， serum levels of neutrophil elastase （NE）， tumor necrosis factor-α （TNF-α）， and vascular cell adhesion molecule-1 （VCAM-1） were measured using enzyme-linked immunosorbent assay （ELISA）， serum myeloperoxidase （MPO） level was determined by a colorimetric assay； the mRNA expression levels of RAF， MEK， and ERK in skin tissue were detected by real-time quantitative polymerase chain reaction （RT-qPCR）； and the protein expression of RAF， MEK， ERK， as well as phosphorylated MEK （p-MEK） and phosphorylated ERK （p-ERK）， were analyzed by Western Blot. ResultsSkin tissue in the blank control group rats remained normal， whereas the model group exhibited neutrophil infiltration and haemorrhage with red blood cell rupture. In all drug intervention groups， neutrophil infiltration and haemorrhagic exudation reduced markedly， with LXTZF group demonstrating the most pronounced improvement. Compared with the blank control group， rats in the model group exhibited enhanced IgA fluorescence intensity in skin tissue， elevated serum levels of NE， MPO， TNF-α and VCAM-1， increased mRNA expression of RAF， MEK， ERK1 and ERK2， as well as heightened RAF protein levels and p-MEK/MEK and p-ERK/ERK ratios （P＜0.05）. Compared with the model group， the drug intervention groups exhibited reduced IgA fluorescence intensity in skin tissue， along with decreased serum levels of NE， MPO， TNF-α， and VCAM-1 （P＜0.05）. In LXTZF group and RAF inhibition groups， reduced mRNA expression of RAF， MEK， ERK1， and ERK2 was observed in rat skin tissue， alongside decreased RAF protein levels and reduced p-MEK/MEK and p-ERK/ERK ratios （P＜0.05）. Compared with LXTZF + RAF agonist group， the compound glycyrrhizin group， LXTZF group， and RAF inhibitior group exhibited reduced IgA fluorescence intensity in skin tissue， decreased serum NE， MPO， TNF-α， and VCAM-1 levels， and decreased MEK mRNA expression and p-MEK/MEK ratio （P＜0.05）. ConclusionThe potential mechanism by which LXTZF treats Henoch-Schönlein purpura with blood heat syndrome may involve blocking the RAF/MEK/ERK signaling pathway in skin tissue， and suppressing excessive formation of NETs， thereby reducing IgA deposition in dermal microvessels and attenuating systemic inflammatory responses.

Objective To explore whether renal denervation (RDN) could improve the left ventricular ejection fraction (LVEF) of patients with heart failure (HF) on the basis of guideline-directed management and therapy (GDMT). Methods From January 1, 2023 to August 31, 2024, HF patients diagnosed as dilated cardiomyopathy (DCM) who underwent RDN in the Second Affiliated Hospital of Chongqing Medical University were retrospectively enrolled, all patients had received GDMT for at least three months but the LVEF remained below 55%. Parameters of transthoracic echocardiography (TTE) at baseline, during GDMT, and after RDN were compared to analyze whether RDN can further improve the LVEF of patients on the basis of GDMT. Results A total of 7 HF patients diagnosed with DCM were enrolled, the mean age was (52.86±9.86) years old, and 5(71.4%) were male. After an average of (9.29±8.06) months of GDMT, LVEF significantly increased from baseline (34.86%±10.22%) to (44.57%±5.59%, P=0.024).Three months after RDN, LVEF was further significantly improved (54.43%±9.05%, P=0.026). The average follow-up after RDN was (11.00±4.12) months. The LVEF remained stable (54.86%±7.10%, P=0.805), and no adverse events occurred in the patients. Conclusions RDN can further enhance the LVEF of HF patients on the basis of GDMT.

OBJECTIVES: To investigate the effects of periodontitis induced by Prevotella nigrescens (Pn) combined with ligation on cognitive functions in mice. METHODS: Twenty-four C57BL/6J mice were randomly divided into control group, ligation group, and ligation + Pn treatment (P+Pn) group. Experimental periodontitis was induced by silk ligation of the first molars followed by topical application of Pn for 6 weeks. After modeling, alveolar bone resorption was assessed using micro-CT and histological analysis. Learning and memory abilities of the mice were evaluated using open field test (OFT), novel object recognition test (NORT), and Morris water maze test (MWM). Seven weeks after the start of modeling, the mice were sacrificed for examining histopathological changes in the hippocampus using HE and Nissl staining. RESULTS: After 6 weeks of molar ligation, micro-CT revealed horizontal alveolar bone resorption and furcation exposure in the mice, and histological analysis showed apical migration of the junctional epithelium, epithelial ridge hyperplasia, and lymphocyte infiltration, and these changes were obviously worsened in P+Pn group. Alveolar bone height decreased significantly in both ligation groups compared to the control group. Cognitive tests showed that the mice in both of the ligation groups traveled shorter distances in OFT, showed reduced novel object preference in NORT, and exhibited longer escape latencies in MWM, and the mice in P+Pn group had significantly poorer performances in the tests. Histologically, obvious neuronal cytoplasmic degeneration, necrosis, nuclear pyknosis, vacuolation, and reduced Nissl bodies and viable neurons were observed in the hippocampal regions of the mice in the two ligation groups. CONCLUSIONS Pn infection aggravates alveolar bone destruction, accelerates necrosis and causes morphological abnormalities of neuronal cells in the hippocampus to reduce cognitive functions of mice with periodontitis.
Animals ; Periodontitis/microbiology* ; Mice ; Mice, Inbred C57BL ; Cognition ; Alveolar Bone Loss ; Hippocampus/pathology* ; Male ; Inflammation ; Maze Learning

OBJECTIVE To investigate the efficacy and safety of tirofiban for early neurological deterioration in patients with acute ischemic stroke. METHODS A total of 126 patients with early neurological deterioration of acute ischemic stroke who were admitted to the Department of Neurology， Heji Hospital Affiliated to Changzhi Medical College from January 2022 to December 2023 were selected and divided into observation group and control group according to random number table method， with 63 cases in each group. All patients received standardized treatment such as lipid-lowering and blood pressure-lowering therapy. Based on the standard treatment， patients in the control group additionally took Aspirin enteric-coated tablets 100 mg+Clopidogrel bisulfate tablets 75 mg orally （once a day， for 14 consecutive days）. The patients in the observation group received Tirofiban hydrochloride and sodium chloride injection based on the standardized treatment [first intravenous infusion of 0.40 μg/（kg·min） for 30 min， and then continuous intravenous infusion of 0.10 μg/（kg·min） for 47.5 h]； subsequently， patients were given Aspirin enteric-coated tablets （100 mg） and Clopidogrel bisulfate tablets （75 mg） once a day for 14 consecutive days. The clinical efficacy， the National Institutes of Health Stroke Scale （NIHSS） score， modified Rankin Scale （mRS） score， and hemorheological indexes before and after treatment were compared between the two groups， and the adverse reactions were recorded. RESULTS The total effective rate （87.30%） of the observation group was significantly higher than that of the control group （71.43%） （P＜0.05）. NIHSS scores of the two groups at 1st， 7th and 14th day after treatment， the mRS score at 90th day after treatment， and the platelet aggregation rate， whole blood viscosity， plasma viscosity and fibrinogen at 14th day after treatment were significantly lower than those before treatment in the same group， and the observation group was significantly lower than the control group at the same period （P＜0.05）. The total incidences of adverse reactions such as nausea， headache， fever， gastrointestinal bleeding， oral and nasal mucosal bleeding and thrombocytopenia in both groups of patients were 28.57% respectively， with no statistically significant difference （P＞0.05）. CONCLUSIONS For patients with early neurological deterioration in acute ischemic stroke， the addition of tirofiban can accelerate the recovery of neurological function， improve blood hyperviscosity and platelet aggregation， and improve the prognosis of patients with good safety.

OBJECTIVE To investigate the roles and mechanisms of γ-bungarotoxin(γ-BGT)in inducing respiratory distress in mice.METHODS Six male Kunming mice were selected and anesthe-tized before tracheal intubation and respiratory recording.After stabilizing respiration,the mice were intraperitoneally injected with γ-BGT at a dose of 6 mg·kg-1.Once a decrease in respiratory frequency was observed,the mice were intravenously injected with nikethamide at a dose of 12.5 mg·kg-1.Respi-ratory frequency was monitored using the BL420 signal acquisition and processing system.Male Kunming mice were randomly divided into the normal control group(saline,ip),γ-BGT group(6 mg·kg-1,ip),and γ-BGT+nikethamide group(γ-BGT 6 mg·kg-1,ip,followed by nikethamide 12.5 mg·kg-1,ip,when shal-low breathing and enhanced abdominal respiration were observed).The levels of Glu and GABA in the medulla oblongata were measured using ELISA.The protein expression levels of GAD65 and GAD67 in the medulla oblongata were determined by Western blotting.Primary mouse medullary neurons were cultured in vitro and divided into the following groups:cell control group,γ-BGT group,carbachol group,gallamine group,γ-BGT+H-89 group,and γ-BGT+Y-27632 group.The γ-BGT group,carbachol group,and gallamine group were incubated with γ-BGT(40 mg·L-1),carbachol(100 mmol·L-1),and gallamine(100 mmol·L-1),respectively,for 4 h.The γ-BGT+H-89 and γ-BGT+Y-27632 groups were pretreated with γ-BGT(40 mg·L-1)for 4 h,followed by incubation with the protein kinase A(PKA)inhibitor H-89(50 mmol·L-1)and the Ca2+channel inhibitor Y-27632(50 mmol·L-1)for another 2 h,respectively.ELISA was used to measure the levels of Glu,GABA,cAMP,and calpain in the primary mouse medul-lary neurons.Western blotting was employed to assess the protein expression levels of GAD65 and GAD67,and PKA phosphorylation levels.Fluo-4 fluorescent probe was used to detect the intracellular Ca2+level.RESULTS The respiratory rate of mice significantly decreased after iv administration of γ-BGT(γ-BGT group)(P<0.05).After treatment with nikethamide(nikethamide group),the respiratory rate significantly recovered(P<0.05).Compared with the normal control group,the γ-BGT group exhib-ited a significant decrease in Glu content(P<0.05),a significant increase in GABA content(P<0.05),and a significant decrease in the Glu/GABA ratio.Additionally,the protein expression levels of GAD65 and GAD67 were significantly elevated(P<0.05).Compared with the γ-BGT group,the γ-BGT+niketh-amide group showed a significant increase in Glu content(P<0.05),a significant decrease in GABA content(P<0.05),a significant increase in the Glu/GABA ratio,and a significant reduction in GAD65 and GAD67 protein expression levels(P<0.05).Compared to the cell control group,the γ-BGT group demonstrated a significant decrease in Glu content(P<0.05),a significant increase in GABA content(P<0.05),and a significant reduction in the Glu/GABA ratio.Furthermore,the protein expression levels of GAD65 and GAD67 were significantly elevated(P<0.05).Additionally,cAMP content,PKA phosphor-ylation levels,Ca2+levels,and calpain activity were all significantly increased(all P<0.05).Glu,GABA,Glu/GABA ratio,and GAD expression levels in the γ-BGT group changed in the same way as in the gallamine group;In the γ-BGT+Y-27632 group,calpain activity and expression levels of GAD65 and GAD67 were all significantly decreased(all P<0.05).In the γ-BGT+H-89 group,Ca2+levels and calpain activity were significantly reduced(all P<0.05).CONCLUSION γ-BGT-induced poisoning can lead to respiratory distress in mice,possibly through the antagonism of M2 muscarinic acetylcholine receptors in medullary neurons,activation of the cAMP/PKA signaling pathway,elevation of intracellular Ca2+levels,and increased expression and activity of GAD,resulting in an imbalance of Glu and GABA in the medulla.

Objective To investigate the effects of periodontitis induced by Prevotella nigrescens(Pn)combined with ligation on cognitive functions in mice.Methods Twenty-four C57BL/6J mice were randomly divided into control group,ligation group,and ligation+Pn treatment(P+Pn)group.Experimental periodontitis was induced by silk ligation of the first molars followed by topical application of Pn for 6 weeks.After modeling,alveolar bone resorption was assessed using micro-CT and histological analysis.Learning and memory abilities of the mice were evaluated using open field test(OFT),novel object recognition test(NORT),and Morris water maze test(MWM).Seven weeks after the start of modeling,the mice were sacrificed for examining histopathological changes in the hippocampus using HE and Nissl staining.Results After 6 weeks of molar ligation,micro-CT revealed horizontal alveolar bone resorption and furcation exposure in the mice,and histological analysis showed apical migration of the junctional epithelium,epithelial ridge hyperplasia,and lymphocyte infiltration,and these changes were obviously worsened in P+Pn group.Alveolar bone height decreased significantly in both ligation groups compared to the control group.Cognitive tests showed that the mice in both of the ligation groups traveled shorter distances in OFT,showed reduced novel object preference in NORT,and exhibited longer escape latencies in MWM,and the mice in P+Pn group had significantly poorer performances in the tests.Histologically,obvious neuronal cytoplasmic degeneration,necrosis,nuclear pyknosis,vacuolation,and reduced Nissl bodies and viable neurons were observed in the hippocampal regions of the mice in the two ligation groups.Conclusion Pn infection aggravates alveolar bone destruction,accelerates necrosis and causes morphological abnormalities of neuronal cells in the hippocampus to reduce cognitive functions of mice with periodontitis.

Objective To develop a rapid and accurate ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)method for the detection of etomidate,metomidate,propoxate,and isopropoxate in human hair.Methods Hair samples containing etomidate,metomidate,propoxate,and isopropoxate were extracted with methanol containing the internal standard orthoxine,filtered with a 0.22 μm organic filter membrane and detected vio UPLC-MS/MS.All components were separated by using a gradient elution procedure consisting of 0.01％formic acid(1 mmol/L ammonium acetate)and acetonitrile.Positive electrospray ionization was performed using multiple reaction monitoring(MRM)mode.Results The linear relationships of etomidate,metomidate,propoxate,and isopropoxate were good in the range of 0.01～1 ng/mg(r ≥ 0.997 9),with recovery rates ranging from 87.9％to 101.5％.The accuracy was between 80.0％and 110.0％.The intra-day and inter-day relative standard deviations(RSD)were 2.9％～9.6％and 3.6％～19.9％.Conclusion This method is easy to operate and has high recovery efficiencies.It is sufficiently simple and sensitive to be applied to detect etomidate,metomidate,propoxate,and isopropoxate in hair.

Objectives:QT interval prolongation during treadmill test exercise is one of the clinical feature of patients with long QT syndrome(LQTS).This study aimed to explore the feasibility and efficacy of treadmill exercise testing as an auxiliary diagnosis tool for LQTS in clinical practice.Methods:We enrolled normal healthy individuals,common cardiovascular disease patients,and clinically diagnosed or suspected LQTS patients,who underwent treadmill exercise test from July 2023 to July 2024 at Fuwai Hospital.A special treadmill exercise testing procedure was designed to record the QT interval correction(QTc)intervals of the twelve lead electrocardiogram at 6 time points when performing the exercise tablet,including supine,sitting,standing,peak exercise,and recovery at 1-minute and 4-minute.The differences in QTc intervals among healthy group,cardiovascular diseases group,and suspected LQTS group were compared.Results:A total of 80 cases were consecutively enrolled,including 37 normal healthy controls,25 patients with common cardiovascular disease,and 18 patients with suspected LQTS.The QTc intervals at 6 points did not differ significantly between normal healthy controls and patients with cardiovascular disease,with QTc intervals less than 480 ms at all measurement.For patients with suspected LQTS,67.7%(12/18)of these patients presented a QTc interval≥480 ms at the 4-minute during recovery period.Among them,5 cases were confirmed to have pathogenic gene mutations of LQTS by genetic testing(including 1 case with a lying electrocardiogram QTc interval of 489 ms diagnosed with LQTS 1 type and a QTc interval of 636 ms during the 4-minute recovery period after exercise);5 clinically diagnosed patients(negative or undetectable in genetic testing)with a Schwartz score≥4,and the remaining 2 patients had a Schwartz score of 3.The remaining 5/18 patients,include 2 patients with clinical Schwartz scores≥4 and 3 patients with clinical suspicion(Schwartz scores 2-3)had a 4 min QTc interval of 445-480 ms during exercise recovery.Another patient with clinical suspicion(Schwartz score 3)had a 4 min QTc interval of<445 ms during exercise recovery and a negative genetic test at a later stage.Receiver operating characteristic curve analysis showed a sensitivity of 83.3%and specificity of 98.4%for QTc interval≥482 ms during the 4-minute recovery period of exercise as the LQTS diagnostic cutoff.Conclusions:This study results suggest that this special treadmill exercise testing protocol is effective in identifying LQTS and has strong feasibility and generalizability for clinical practice.

Objective To investigate the diagnostic accuracy and clinical applicability of the Linknet-VGG16 deep learning algorithm for measuring the effective optical zone(EOZ)after corneal refractive surgery.Methods This single-center retrospective cohort study included 69 patients(69 eyes)who underwent femtosecond laser-assisted in situ kerato-mileusis(FS-LASIK)(34 eyes)or small incision lenticule extraction(SMILE)(35 eyes)at the Refractive Surgery Center of Affiliated Zhengzhou Aier Eye Hospital of Henan University from June 2023 to June 2024.Data from the right eyes of all patients were selected for statistical analysis.During the surgery,patients in the FS-LASIK group adopted the VisuMax fem-tosecond laser system combined with the Amaris 750S excimer laser system,while those in the SMILE group only used the VisuMax femtosecond laser system.A total of 276 Pentacam images were re-examined postoperatively.A Linknet segmenta-tion model based on the VGG16 encoder was constructed,and image normalization techniques were applied to accelerate model convergence.Model performance was assessed using accuracy,intersection over union(IoU),and the Dice coeffi-cient.The traditional EOZ measurement method based on corneal tangential curvature served as the reference standard.Bland-Altman analysis was conducted to evaluate consistency across all images and within each group,and the time effi-ciency of both methods was compared.Results Six representative medical image segmentation architectures(U-Net,U-Net++,DeepLabv3-ResNet50,DeepLabv3+-ResNet50,Unet-Densenet169,and Linknet-VGG16)were systematically evaluated.The Linknet-VGG16 model demonstrated superior performance over the other 5 models in pixel-level accuracy,IoU and Dice coefficient,which were 99.83％,99.48％and 99.74％,respectively.Although there was no significant differ-ence in accuracy and Dice coefficient between Linknet-VGG16 and U-Net models(whose accuracy was 99.82％and Dice coefficient was 99.72％),the inference speed of the U-Net model(62.46 ms)was 31.76％slower than that of the Linknet-VGG16 model(42.62 ms).The evaluation results of a clinically applicable comprehensive scoring model(weights:accura-cy 20％,IoU 20％,Dice coefficient 20％,speed 25％,model size 15％)showed that the Linknet-VGG16 model achieved a score of 88.01,surpassing other architectures(U-Net:86.29;DeepLabv3+-ResNet50:80.41;DeepLabv3-ResNet50:73.82;U-Net++:73.22;Unet-Densenet169:66.66).Bland-Altman analysis revealed that the mean difference of the 136 images in the FS-LASIK group was 0.01 mm[95％limits of agreement(LoA):-0.36 to 0.35 mm],with 96.3％of data points falling within the LoA.The mean difference of the 140 images in the SMILE group was-0.01 mm(95％LoA:-0.36 to 0.33 mum),with 95.7％of data points falling within the LoA.The mean difference of all 276 images was 0.00 mm(95％LoA:-0.36 to 0.34 mm),with 96.4％of data points falling within the LoA.These results indicated excellent consistency.The average measurement time per image using the traditional EOZ measurement method was 13.00 minutes,whereas the deep learning model required only 3.22 seconds.Conclusion The traditional EOZ measurement method based on corne-al tangential curvature exhibits good consistency with the fully automatic EOZ measurement method based on deep learning algorithms,achieving high image recognition accuracy.Additionally,the deep learning algorithm significantly reduces measurement time,compared with the traditional method based on corneal tangential curvature.