Objective To investigate the effect of midazolam on neuronal damage in ischemic stroke （IS） rats and its regulatory effect on PTEN-induced putative kinase 1 （PINK1）/E3 ubiquitin ligase （PARKIN） signaling pathway. Methods An IS rat model was established using arterial occlusion method. The rats with successful model were randomly divided into IS group, drug-low, medium, high-dose （drug-L, M, H, 30, 60, 90 mg/kg midazolam） groups, drug-H+autophagy inhibitor 3-MA group （90 mg/kg midazolam+30 mg/kg 3-MA）, and rats with only isolated blood vessels were used as sham surgery groups. Each group received corresponding doses of drugs or physiological saline intervention, and the neurological function scoring, brain histopathology, neuronal apoptosis, ultrastructure, and expression of PINK1, PARKIN, microtubule-associated protein 1 light chain 3 （LC3）, and P62 protein in mitochondria were detected. Results Compared with the IS group, the pathological damage of the drug-L group, drug-M group, and drug-H group was improved, and autophagosomes showed an increasing trend, the expression of PINK1, PARKIN, and LC3 proteins increased, the neurological function score, neuronal apoptosis rate, and P62 protein obviously decreased in a dose-dependent manner （P<0.01 or P<0.001）; compared with the drug-H group, the pathological damage in the drug-H+3-MA group increased and autophagosomes decreased, the expression of PINK1, PARKIN, and LC3 proteins decreased, the neurological function score, neuronal apoptosis rate, and P62 protein obviously increased （P<0.001）. Conclusion Midazolam induced mitochondrial autophagy in IS rats by activating the PINK1/PARKIN signaling pathway, neuronal apoptosis was reduced and neuronal damage were improved in IS rats.

OBJECTIVE To establish a content determination method for multiple components in Sophora flavescens from different origins and to evaluate its quality by combining with chemometrics. METHODS Thirteen batches （No. K1-K13） of S. flavescens from different origins were selected as test samples. A high-performance liquid chromatography-tandem triple quadrupole mass spectrometry （HPLC-MS/MS） method was established to determine the contents of 12 components， including matrine， oxymatrine， betaine， cytisine， N-methylcytisine， sophoridine， genistein， sophoricoside， sophorone， formononetin， sophorolone Ⅰ and norkurarinone in S. flavescens. Chromatographic separation was performed on a Shim-pack GIST-HP C18 column with a mobile phase consisting of methanol （A） and water containing 0.1% formic acid （B）， using gradient elution at a flow rate of 0.25 mL/min， column temperature of 35 ℃， and an injection volume of 3 μL. Mass spectrometry was conducted using an electrospray ionization source with positive and negative ion scanning. Data were collected in segments using the multiple reaction monitoring mode. Technique for order preference by similarity to ideal solution （TOPSIS） and grey relational analysis （GRA）methods were employed to compare and comprehensively evaluate the 13 batches of S. flavescens from different origins. RESULTS The methodological validation for the content determination met the relevant regulatory requirements. The contents of the 12 components were 490.66-1 231.00， 11 088.10- 18 021.50， 7.91-25.38， 903.97-1 713.64， 336.08-1 485.54，1 065.33-2 075.50， 27.52-71.80， 109.36-517.83， 6 034.55-10 632.73， 21.26-145.35， 814.84-1 911.32， 1 040.87-3 446.37 μg/g）， respectively. TOPSIS results showed that the top 7 samples in Euclidean distance ranking were K6， K12， K11， K3， K5， K10， K13. The GRA results showed that the top 7 samples in the relative correlation ranking were K12， K11， K10， K6， K13， K5， K3. CONCLUSIONS The established HPLC-MS/MS method is rapid， accurate， highly sensitive， stable and reliable. Combined with chemometrics methods， it can be used for the quality control and evaluation of S. flavescens. The comprehensive quality of samples K3， K5， K6（ from Hebei）， K10（ from Sichuan）， K11-K13（ from Shanxi）， etc. is relatively superior.

Objective To investigate the gene expression and regulatory mechanisms of mouse embryonic fibroblasts (MEFs) under inflammatory conditions, aiming to elucidate the role of MEFs in inflammatory responses and provide a foundation for discovering anti-inflammatory drugs that act by modulating MEF function. Methods MEFs cultured in vitro were divided into the following groups: lipopolysaccharides (LPS)-treated group, inflammatory conditioned medium (CM)-treated group, and control group, which were treated with LPS, CM, and equal volume solvent, respectively. Transcriptome sequencing was used to analyze the effects of two stimuli on gene expression profile of MEFs. Real time fluorescence quantitative PCR (RT-qPCR) was employed to verify the transcription levels of highly expressed genes of MEFs induced by CM. ELISA was performed to determine the concentrations of cytokines in cell supernatants. Finally, the regulatory effects of CM on the activation of signaling pathways in MEFs were analyzed by immunoblotting. Results Transcriptome analysis showed that both LPS and CM induced the transcription of a large number of genes in MEFs. Compared with LPS, CM potentiated the mRNA transcription of some acute phase proteins, inflammatory cytokines, chemokines, matrix metalloproteinases (MMP), prostaglandin synthetases, and colony-stimulating factors. The transcriptome analysis was verified by RT-qPCR. The results of ELISA showed that CM treatment significantly increased the secretion of interleukin 6 (IL-6), C-C motif chemokine ligand (CCL2), and C-X-C motif chemokine ligand (CXCL1) by MEFs compared with LPS. Mechanism study showed that both LPS and CM induced the phosphorylation of nuclear factor-κB p65 (NF-κB p65), p38 mitogen-activated protein kinase (p38 MAPK), extracellular regulated protein kinases 1/2 (ERK1/2), and TANK-binding kinase (TBK) in MEFs, and CM strongly stimulated the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in MEFs. Conclusion Both LPS and CM can induce transcription and protein secretion of various inflammation-related genes in MEFs. CM can partly enhance LPS-induced activation of MEFs, and the mechanism may be related to the enhancement effect of CM on the activation STAT3 signaling pathway.
Animals ; Fibroblasts/immunology* ; Mice ; Lipopolysaccharides/pharmacology* ; Inflammation/metabolism* ; Signal Transduction/drug effects* ; Gene Expression Regulation/drug effects* ; Cytokines/genetics* ; Culture Media, Conditioned/pharmacology* ; Cells, Cultured

Although a single nucleotide polymorphism for N-acetyltransferase 10 (NAT10) has been identified in patients with early-onset stroke, the role of NAT10 in ischemic injury and the related underlying mechanisms remains elusive. Here, we provide evidence that NAT10, the only known RNA N4-acetylcytidine (ac4C) modification "writer", is increased in the damaged cortex of patients with acute ischemic stroke and the peri-infarct cortex of mice subjected to photothrombotic (PT) stroke. Pharmacological inhibition of NAT10 with remodelin on Days 3-7 post-stroke or astrocytic depletion of NAT10 via targeted virus attenuates ischemia-induced infarction and improves functional recovery in PT mice. Mechanistically, NAT10 enhances ac4C acetylation of the inflammatory cytokine tissue inhibitor of metalloproteinase 1 (Timp1) mRNA transcript, which increases TIMP1 expression and results in the accumulation of microtubule-associated protein 1 light chain 3 (LC3) and progression of astrocyte autophagy. These findings demonstrate that NAT10 regulates astrocyte autophagy by targeting Timp1 ac4C after stroke. This study highlights the critical role of ac4C in the regulation of astrocyte autophagy and proposes a promising strategy to improve post-stroke outcomes via NAT10 inhibition.

Objective To study the effect of low concentrations of sodium fluoride on the osteogenic/odontogenic differentiation of human dental pulp cells(hDPCs)in vitro.Methods This study was reviewed and approved by the Ethics Committee.hDPCs were cultured using a modified tissue explant technique in vitro.The effects of different con-centrations of sodium fluoride on the proliferation of hDPCs were measured by methylthiazol tetrazolium(MTT)assay.Appropriate concentrations were added to the osteogenic/odontogenic differentiation induction medium,and the cells were induced in vitro.Alizarin red S staining was used to detect the osteoblastic/odontogenic differentiation ability of the cells,and the mRNA expression of the key differentiation factors was detected by RT-qPCR.Moreover,the expres-sion of key molecules of endoplasmic reticulum stress(ERS)was detected by RT-qPCR and Western blot.The data were analyzed with the SPSS 18.0 software package.Results Low concentration of NaF(0.1 mmol/L)could stimulate cell proliferation in vitro,while a high concentration(5-10 mmol/L)could inhibit cell proliferation(P＜0.05).According to the literature and the experimental data,0.1 mmol/L NaF was selected as the following experimental concentration.The levels of alizarin red S staining were increased after NaF induction of mixed osteogenic/odontogenic differentiation in vi-tro.The mRNA expression levels of key molecules for osteogenic/odontogenic differentiation,dentin sialophosphoprotein(DSPP),bone sialoprotein(BSP)and osteocalcin(OCN),were increased(P＜0.05).The mRNA levels of ERS markers(splicing x-box binding protein-1(sXBP1),glucose-regulated protein 78(GRP78)and activating transcription Factor 4(ATF4)were increased in NaF-treated cells.The protein expression levels of key ER stress molecules(phosphorylated RNA-activated protein kinase-like ER-resident kinase(p-PERK),phosphorylated eukaryotic initiation factor-2α(p-eIF2α)and ATF4)were higher in NaF-treated cells.Conclusion A low concentration of NaF promotes the osteogenic/odontogenic differentiation of hDPCs and increases the level of ER stress.

Objective:To explore the potential relationship between sugar metabolism,acid production and cariogenicity of Prevotella denticola.Methods:Morphological features of Prevotella denticola were observed and respectively cultured under incubation conditions with and without sugar and at different pH values.The growth characteristics of Prevotella denticola were detected by UV-Vis spectro-photometer and pH meter,the organic acid content in the culture supernatants of the cultures was detected by HPLC.Dentin slices were divided into control group,phosphoric acid group and the Prevotella denticola group and cultured in the corresponding mediu for 1 and 2 weeks respectively,the degree of demineralization of the samples was examined SEM and VHM.Results:Prevotella denticola fermen-ted sucrose and glucose,produced acids with its final pH values as low as 4.7,Succinic acid and acetic acid were its main metabolites.Prevotella denticola was moderately acid-tolerant.Furthermore,Prevotella denticola was able to cause dentin demineralization,and the Vickers hardness value of dentin samples in the Prevotella denticola group was significantly decreased compared with the control group(P＜0.05).Conclusion:The cariogenic capacity of Prevotella denticola may be related to its sugar metabolism and acid production.

OBJECTIVE To study and analyze the relationship between serum levels of Endocan,Cyclophilic A (CyPA),and asprosin with the condition severity and cognitive function in patients with obstructive sleep apnea hypopnea syndrome(OSAHS). METHODS From November 2021 to December 2023,99 OSAHS patients who visited our hospital were collected and separated into mild group(n=29),moderate group(n=45),and severe group(n=25) based on the apnea hypopnea index(AHI). ELISA method was applied to detect serum levels of Endocan,CyPA,and asprosin. Multivariate logistic regression was used to analyze the influencing factors of severity of condition. ROC curve was plotted to analyze the diagnostic value of serum Endocan,CyPA,and asprosin for cognitive function in OSAHS patients. Spearman rank correlation was applied to analyze the correlation between serum Endocan,CyPA,asprosin,with severity of condition,cognitive function. RESULTS Compared with the mild group,the serum levels of Endocan,CyPA,and asprosin were obviously increased in the moderate and severe groups(P<0.05),and the serum levels of Endocan,CyPA,and asprosin in the severe group were obviously increased compared to the moderate group(P<0.05). There were statistically obvious differences in AHI,Epworth sleepiness scale(ESS) scores,and some sleep related parameters among the three groups(P<0.05). Further analysis revealed that mean blood oxygen saturation(MSaO2) was a protective factor affecting the severity of condition(P<0.05),while AHI,serum Endocan,CyPA,and asprosin levels were all risk factors affecting the severity of condition(P<0.05). The serum levels of Endocan,CyPA,and asprosin in patients with cognitive impaired were obviously higher than those in patients with normal cognition,and the MoCA score was lower than that in patients with normal cognition(P<0.05). The AUC of serum Endocan,CyPA,asprosin,and combined diagnosis for cognitive impairment in patients was 0.889,0.893,0.861,and 0.974,respectively,and the combined diagnosis was obviously better than individual diagnosis of Endocan(Z=2.832,P=0.005),CyPA(Z=2.907,P=0.004),and asprosin(Z=3.225,P=0.001). Serum Endocan,CyPA,and asprosin were positively correlated with AHI and negatively correlated with MoCA score(P<0.05). CONCLUSION In patients with severe OSAHS and cognitive impairment,serum Endocan,CyPA,and asprosin levels are all increased,and they have certain auxiliary diagnostic value for the severity of condition and cognitive function.

Objective:To assess the performance of machine learning(ML),and integrate the clinical parameters with coronary artery calcium(CAC)score of computed tomography(CT)and quantification of automated epicardial adipose tissue(EAT),so as to predict the long-term risk of myocardial infarction(MI)and cardiogenic death in asymptomatic patients.Methods:A total of 1 058 subjects with cardiovascular risk factors and without symptoms of coronary heart disease who underwent physical examination at the Fifth Medical Center of Chinese PLA General Hospital from January 2013 to October 2015 were selected as this study subjects.A long-term follow-up was conducted on them after CAC score.EAT volume and density were quantified using a fully automated deep learning method.ML extreme gradient boosting was trained by using clinical data,risk score of atherosclerotic cardiovascular disease,CAC score and automated EAT measure,and the repeated 10-fold cross validation was used to verify the model.Results:During the 8-year follow-up period,61 cases of 1 058 subjects occurred events of MI and(or)cardiac death.The area under curve(AUC)value of ML was significantly higher than that of the atherosclerotic cardiovascular disease(ASCVD)risk and the predicting events of CAC score(ML:0.82,ASCVD:0.77,CAC:0.77).Compared with ML with only clinical variable,machine learning based on ASCVD,CAC and EAT had more predictive ability for MI and cardiac death[AUC 0.82(95%CI:77-87)vs.0.78(95%CI:0.72-0.84),P=0.02].The survival rate of subjects with high ML scores had a greater decline degree with the increasing of time,therefore,the subjects with higher ML scores were more likely to experience events.Conclusion:ML,which integrated clinical and quantitative imaging variables,can provide long-term risk prediction for patients with cardiovascular risk factors.

Objective:To evaluate the therapeutic effects of Yufu ointment for acute radiation-induced skin injury. Methods:We enrolled a total of 104 patients with malignant tumors who developed acute skin injury of grade 2-3 during or after the first course of radiotherapy from June 2019 to February 2023. The observation group ( n=53) and control group ( n=51) received external application of Yufu ointment and Shirun Shaoshang ointment, respectively. We recorded the changes in the grade of skin injury and associated symptoms (such as pain, itching, and burning sensation) and healing speed and time for the two groups. Results:A total of 102 patients completed the study, with 53 cases in the observation group and 49 cases in the control group. The incidence rate of moisture skin lesions in the observation group was significantly lower than that in the control group (62.26% vs. 83.67%, P<0.05). The healing speed of moisture skin ulcerations was significantly faster in the observation group than in the control group (1.67 cm 2/d vs. 0.55 cm 2/d, P<0.05). The observation group had a significantly shorter time to skin injury healing than the control group (10.40 d vs. 14.41 d, P<0.05), significantly different for skin injury of grade 2 (10.21 d vs. 17.57 d, P<0.05) but not for skin injury of grades 2.5 and 3 ( P>0.05). Regarding clinical symptoms, both groups experienced significant reductions in pain and burning sensation scores after treatment ( P<0.05); the grade of itching was significantly decreased for the observation group ( P<0.05), but had no significant change for the control group after treatment ( P>0.05). Conclusions:Yufu ointment can significantly alleviate secondary damage for patients with acute radiation-induced skin injury, by accelerating the healing process and relieving symptoms such as pain, itching, and burning sensation with good tolerability, which deserves further promotion.

90Y microspheres radioembolization therapy is an emerging interventional technique used for the treatment and palliative care of intermediate and advanced liver cancer, which was registered by National Medical Products Administration for imported drugs in 2022. In order to improve the tumor response rate and reduce toxic effects, the dose assessment for the tumor and healthy tissues is implemented throughout treatment. The efficiency and accuracy of estimation method are the keys to improving the therapeutic effect. This article investigated and summarized six evaluation method and their latest progress, discussed the unique advantages and applicable scenarios of each methods, and explored the possible challenges faced in clinical applications. This study would support the development and standardization of dose prediction and evaluation in radioembolization with 90Y microspheres.