AIM: To investigate the relationship between vascular smooth muscle cell (VSMC) injury, organelle stress response and autophagic cell death (autophagy) and ferroptosis induced by the chemical hypoxia inducer cobalt chloride (CoCl2) through the bioinformatics analysis and in vitro cell experimentation. METHODS: The dataset GSE119226 of VSMC treated with cobalt chloride was acquired from the gene expression database (GEO). The R language was used to investigate the relationship between CoCl2 treatment and organelle stress response (Golgi stress, endoplasmic reticulum stress) and two forms of cell death (ferroptosis and autophagic cell death). With primary cultured rat VSMC (rVSMC) and CoCl2-induced anoxia model, the changes in cell viability were detected by CCK-8 method, and reactive oxygen species (ROS) levels were measured using DCFH-DA method. The expression levels of HIF-1α (a key molecule in hypoxia), Golgi stress markers GM130 and p115, endoplasmic reticulum stress markers GRP78 and CHOP, autophagy markers LC3-II / LC3-I and Beclin1, and ferroptosis markers GPx4 and xCT were detected by Western blot. The effect of inducing or inhibiting organelle stress and cell death on the CoCl2-induced cell damage was also observed. RESULTS: Differentially expressed genes analysis of GSE119226 dataset showed that CoCl2 treatment of VSMCs had significant effects on organelle function and stress response, autophagy and ferroptosis-related genes, in which endoplasmic reticulum stress, protein processing in endoplasmic reticulum, regulation of Golgi to plasma membrane protein transport, autophagy / autophagic cell death, and ferroptosis pathways were remarkably enriched. The results of in vitro experiment showed that compared with normal rVSMC, cell viability was significantly decreased after CoCl2 treatment, as well as HIF-1α protein expression and ROS levels in rVSMCs were increased. In rVSMC treated with Co-Cl2, the expression levels of Golgi structural proteins GM130 and p115 (reflecting the occurrence of Golgi stress) were decreased, while the markers GRP78 and CHOP (reflecting the occurrence of endoplasmic reticulum stress) were increased. At the same time, CoCl2 treatment also reduced the expression of autophagy markers LC3-II/LC3-I and Beclin1 (indicating the decrease levels of autophagy), while the expression of ferroptosis markers GPx4 and xCT were decreased (indicating the occurrence of ferroptosis). Compared with CoCl2 treatment group, induced Golgi stress, endoplasmic reticulum stress, or ferroptosis could further reduce cell viability, while inhibition of these processes could improve cell viability. On the other hand, increasing the level of autophagy can improve the cell viability. CONCLUSION: Hypoxia induced by cobalt chloride can lead to VSMC injury. Golgi stress, endoplasmic reticulum stress, ferroptosis, and the reduction of autophagy level play an important role in it. Inhibition of organelle stress response and ferroptosis, or increase of autophagy level can improve VSMC injury caused by cobalt chloride.

This study was designed to explore the transcription level,genome alteration,potential abnormal expression mechanism,prognostic value and the association with immune cells of the tetraspan MS4A6A(membrane-spanning 4A(MS4A)subfamily protein 6A)in pan-cancer.Public databases,such as UCSC Xena,cBioPortal and Timer2,were used to collect relative data,and thenbioinformatic approaches were used to analyze the correlation of MS4A6A genome and MS4A6A expression with immune infiltration,tumor mutation burden(TMB),microsatellite instability(MSI)etc.TMB and MSI were further calculated by R package maftools.Immune score,stromal score and ESTIMATE score were calculated by R package ESTIMATE.The correlation between immune score and MS4A6A expression was conducted based on Spearman correlation coefficient.Data showed that DNA methylation of MS4A6A in most cancer tissues was negatively correlated with its expression level,suggesting the possible relation of differential MS4A6A expression to the methylation level of its promoter region.Univariate Cox analysis revealed that high expression of MS4A6A was the risk factor for LGG,TGCT,UVM and THYM;MS4A6A expression was positively correlated with the immune score in 32 types of cancer;MS4A6A expression was positively correlated with the infiltration levels of various immune cells,such as tumor associated fibroblasts(CAF),regulatory T cells(Treg),B cells,neutrophils,macrophages,monocytes,and CD8+T cells.In conclusion,MS4A6A may participate in the development of cancer,suggesting it is a potential new biomarker for cancer treatment and prevention.

Objective: To investigate the characteristics of fall injuries among primary and middle school students in Yinzhou District, Ningbo City, Zhejiang Province from 2010 to 2022, so as to provide insights into prevention and control of fall injuries among primary and middle school students. Methods: Data of 6 to 17 year-old primary and middle school students with initial diagnosis of fall injury from 2010 to 2022 were collected through Injury Subsystem of Zhejiang Provincial Chronic Disease Surveillance Information Management System. The time, place, activity and clinical characteristics of fall injury were analyzed using a descriptive method. Results: Totally 7 808 fall injury cases among primary and middle school students were reported in Yinzhou District from 2010 to 2022, accounting for 45.72% of the total injury cases in the same age. There were 5 413 boys and 2 395 girls, with a boy/girl ratio of 2.26∶1. Primary school students were the majority, accounting for 55.10%. The incidence of fall injuries among girls, junior high school students and high school students showed increasing trends from 2012 to 2022 (both P<0.05). The primary peak of fall injury was from September to November, and the secondary peak was from May to July, with 4 510 cases (57.76%). The place of fall injury development mainly included schools (2 680 cases, 34.32%), homes (2 343 cases, 30.01%) and streets/urban areas (2 247 cases, 28.78%). The activities at the time of fall injury mainly included leisure time (3 490 cases, 44.70%), sports (2 861 cases, 36.64%) and school activities (1 094 cases, 14.01%). Soft tissue injury was the main characteristics (6 224 cases, 79.71%). Lower limbs (3 101 cases, 39.72%), head (2 419 cases, 30.98%) and upper limbs (1 974 cases, 25.28%) were the main injury sites. Mild injury was predominant (5 896 cases, 75.51%). Conclusions Boys and primary school students are high-risk groups of fall injury in Yinzhou District, schools are high-risk places of fall injury. Schools should be regarded as key intervention places and health education on fall injury prevention should be strengthened.

Objective To establish a human tonsil organoid model and investigate the immunological effects of this tonsil organoid infected by influenza virus.Methods Human tonsil tissue removed by adenoid hypertrophy surgery were subjected to gradient centrifugation,and then the obtain cells were cultured in vitro with using a 2.5D-Transwell chamber in combination with IL-2 and B cell activating factor(Baff)to construct a human tonsil organoid.Single cell transcriptome sequencing(scRNA-seq)was used to analyze the distribution of influenza virus receptors in different cell subsets of the tonsil organoid.Immunofluorescence assay and flow cytometry were applied to detect the immunological effects of the tonsil organoid in 48 h after influenza virus infection.Results The tonsil organoids were established successfully using this 2.5D-Transwell culture technique.scRNA-seq analysis showed that the receptors for influenza virus were extensively distributed in nearly all of cell subsets of tonsil organoids.In 48 h after influenza virus infection,the tonsil organoids could generate a large amount of plasma cells and memory B cells to produce specific IgG antibodies.In addition,direct infection of the virus promoted the production of TNF-α and IL-2 from CD8+T cells and the secretion of TNF-α,IFN-γ and IL-2 by CD4+T cells.Conclusion The tonsil organoids can be established successfully and reach maturity at about the 6th day after culture.The receptors for influenza virus are widely distributed in human tonsil organoids.Influenza virus directly infects human T cells,leading to T cell activation and cytokine releasing.Moreover,influenza virus infection also promotes B cells differentiate into plasma cells and induce the secretion of IgG as well as the formation of memory B cells.

Injury monitoring is an important approach for injury control and is an important part of comprehensive disease monitoring. With the development of medical digitalization, an intelligent injury monitoring system has been created in Yinzhou District, Ningbo City, Zhejiang Province using artificial intelligence techniques based on Ningbo Municipal Health Information Platform and has been applied across the district since 2019. The manual card-reporting mode has been transformed to intelligent card-reporting mode in this system, which achieves functions of epidemiological analyses of the monitoring data, early warning of high-incidence injuries, classified management of injury and quality control of report cards. Nearly 300 thousand cards have been automatically reported since the system was online available since November 2022, and the epidemiological characteristics of injury were preliminarily understood, which provide data supports to early earning and interventions of injury. The intelligent injury monitoring system greatly improves the injury monitoring efficiency and card-reporting quality, and saves a large number of manpower and material resources, which provides a powerful technical support to widespread injury monitoring.

AIM: To compare the results calculated by population pharmacokinetic analysis tools Phoenix NLME, Monolix, R nlmixr package and CPhaMAS cloud platform with the gold standard sofeware NONMEM. METHODS: Fifty sparse sampling data sets based on a one-compartment model and fifty dense sampling data sets based on a two-compartment model were simulated, and the above five analysis tools were used to calculate the population typical value, individual variability and individual pharmacokinetic parameters. RESULTS: The population typical value and individual variability calculated by CPhaMAS and Phoenix NLME had the highest matching degree with NONMEM, followed by nlmixr. Monolix had the lowest matching degree, but Monolix and nlmixr might be more robust. The correspondence between clearance and distribution volume was better than the absorption rate constant. Except the absorption rate constant calculated by Monolix and intercompartmental clearance calculated by nlmixr, the correlation coefficients of individual pharmacokinetic parameters calculated by all analytical tools were greater than 0.99. CONCLUSION: The results calculated by the above four population pharmacokinetic analysis tools are highly correlated with that of NONMEM.

Objective:To evaluate and compare efficacy of intravenous immunoglobulin (IVIG) versus recombinant human tumor necrosis factor-α receptor Ⅱ:IgG Fc fusion protein (rhTNFR:Fc) in the treatment of toxic epidermal necrolysis (TEN) .Methods:Clinical data were collected from patients with TEN treated with IVIG or rhTNFR:Fc in Wuhan No.1 Hospital from 2013 to 2019. There were 11 patients in the IVIG group, including 3 males and 8 females, aged 25-72 years, and the median TEN-specific severity-of-illness score (SCORTEN) was 3 points; there were 10 patients in the rhTNFR:Fc group, including 5 males and 5 females, aged 32-84 years, and the median SCORTEN was 2 points. These patients all showed no response to the 5-day treatment with prednisolone acetate at a dose of 0.6-1.0 mg·kg -1·d -1, and then received IVIG at a dose of 400 mg·kg -1·d -1 for 5 consecutive days, or subcutaneous injection of rhTNFR:Fc at a dose of 25 mg every other day for 4-6 sessions. Changes in skin lesions and adverse events were recorded in the 2 groups. Statistical analysis was carried out by using Mann-Whitney U test. Results:Compared with the rhTNFR:Fc group, the IVIG group showed a significant decrease in the time to onset of reduction of skin lesion exudate (1.73 ± 1.19 days vs. 3.00 ± 1.56 days, P < 0.05) , time to onset of pain relief in the lesion area (1.64 ± 1.28 days vs. 3.70 ± 1.63 days, P < 0.05) , time to lightening of color of the lesion base (2.45 ± 1.12 days vs. 3.90 ± 1.59 days, P < 0.05) , time to onset of new epidermis growth (3.09 ± 1.13 days vs. 5.20 ± 1.22 days, P < 0.05) , and in the time to onset of lesion drying at the intertriginous sites (4.82 ± 2.22 days vs. 7.90 ± 3.14 days, P < 0.05) . However, there was no significant difference in the length of hospital stay between the IVIG group (17.70 ± 8.33 days) and rhTNFR:Fc group (16.70 ± 4.71 days, P > 0.05) . No adverse reactions were observed during the treatment, and no recurrence or complications were found in the 21 patients during the follow-up of 6 months. Conclusion:IVIG and rhTNFR:Fc are both effective in the treatment of TEN, but IVIG is superior to rhTNFR:Fc in terms of the time to onset of pain relief, skin lesion exudate reduction and epidermal growth.

Objective:To evaluate the role of Toll-like receptor 4 (TLR4) in the mechanism by penehyclidine hydrochloride alleviating lipopolysaccharide (LPS)-induced acute lung injury(ALI)in rats.Methods:Twenty-four healthy male Sprague-Dawley rats, aged 10 weeks, weighing 220-250 g, were divided into 4 groups ( n=6 each) according to the random number table method: normal saline group (NS group), endotoxin-induced ALI group (ALI group), penehyclidine hydrochloride + normal saline group (PHC+ NS group) and penehyclidine hydrochloride + endotoxin-induced ALI group (PHC+ ALI group). ALI was induced by intratracheal instillation of LPS 5 mg/kg in anesthetized animals. In group PHC+ ALI, penehyclidine hydrochloride 2 mg/kg was intraperitoneally injected immediately after intratracheal instillation of LPS. The equal volume of normal saline was injected into the airway in group NS, and penehyclidine hydrochloride 2 mg/kg was intraperitoneally injected immediately after intratracheal instillation of normal saline in group PHC+ NS. The rats were sacrificed at 6 h after intratracheal instillation of LPS or normal saline, and lungs were removed. The lung was lavaged and broncho-alveolar lavage fluid (BALF) was collected for determination of concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) by enzyme-linked immunosorbent assay. Lung tissues were obtained for microscopic examination of the pathological changes (with a light microscope) and for determination of wet to dry lung weight ratio (W/D ratio) and TLR4 protein and mRNA expression (by immuno-histochemistry or real-time polymerase chain reaction). Results:Compared with group NS and group PHC+ NS, the W/D ratio and concentrations of TNF-α and IL-1β in BALF were significantly increased, the expression of TLR4 protein and mRNA in lung tissues was up-regulated ( P<0.01), and the pathological changes of lung tissues were aggravated in group ALI . Compared with group ALI, the W/D ratio and concentrations of TNF-α and IL-1β in BALF were significantly decreased, the expression of TLR4 protein and mRNA in lung tissues was down-regulated ( P<0.01), and the pathological changes of lung tissues were significantly mitigated in group PHC+ ALI. Conclusions:The mechanism by which penehyclidine hydrochloride reduces endotoxin-induced lung injury may be related to reduction of TLR4 activity and thus inhibition of pulmonary inflammatory responses in rats.

Objective: To explore the effect of tanshinone IIA on neuronal apoptosis in the compressed spinal cord and its molecular mechanism by establishing a spinal cord compression model. Methods: Twenty-four SD rats weighing 250-300 g were the experimental animals. The spinal cord compression model was established by clamping the spinal cord with arterial clamp. Six rats in each group were randomly selected and randomly divided into sham group (Sham group, given intraperitoneal injection of normal saline at the same dose once a day) and spinal cord compression injury group (SCI group, given normal salts). Water intraperitoneal injection, once a day), tanshinone IIA group (TAN group, 30 mg/kg tanshinone IIA intraperitoneal injection, once a day), LY2904002 group (LY group, 0.3 mg/kg LY294002 intraperitoneal injection, 5 min after 30 mg/kg tanshinone IIA intraperitoneal injection, once 1 d). After 3 d of intervention, the motor function of rats were evaluated by inclined plane test and BBB score. The expression of apoptotic genes and PI3K/AKT signaling molecules in the compressed spinal cord were detected by qPCR, immunohistochemistry and Western blot. Results: The BBB score 3.31±0.45 points, inclined angle 9.31°±1.02°, GSK-3β 0.35±0.06, CyclinD1 0.25±0.06, Bcl-2 0.38±0.06, p-PI3K 0.32±0.05, p-AKT 0.29±0.07 protein expression in SCI group were lower than those in Sham group. The protein expression of Caspase-9 3.27±0.54 and Caspase-3 2.73±0.35 in SCI group was higher than that in Sham group. The BBB score 9.31±1.02 points, inclined angle 24.95°±3.52°, GSK-3β 0.74±0.09, CyclinD1 0.69±0.11, Bcl-2 0.83±0.13, p-PI3K 0.77±0.11, p-AKT 0.69±0.08 in TAN group were higher than those in SCI group BBB score 3.31 ±0.45 points, inclined angle 9.31°±1.02°, GSK-3β 0.35±0.06, CyclinD1 0.25±0.06, Bcl-2 0.38±0.06, p-PI3K 0.32±0.05, p-AKT 0.29±0.07. The protein expression levels of Caspase-9 1.78±0.22 and Caspase-3 1.64±0.2 in TAN group were lower than those in SCI group 3.27±0.54 and Caspase-3 2.73±0.35. The BBB score, oblique angle and the expression of GSK-3β 0.43±0.07, CyclinD1 0.38±0.06, Bcl-2 0.49±0.09 in LY group were lower than those in TAN group. The protein expression of Caspase-9 2.54±0.38 and Caspase-3 2.25±0.37 in LY group were higher than TAN group. Conclusion Tanshinone IIA can inhibit the apoptosis of spinal cord tissue and alleviate the spinal cord injury in spinal cord compression rats by activating the PI3K/AKT signaling pathway.

Objective To discuss the feasibility and value of indocyanine green combined with methylene blue in sentinel lymph node biophy (SLNB) for patients with breast cancer.Methods 196 breast cancer patients who received surgery were studied.SLNB was performed by indocyanine green combined with methylene blue .Frozen section was performed,followed by conventional histopathology .The evaluation criterion of Louisvill University on SLNB was adopted to judge the detection rate ,accuracy rate,sensitivity,false negative rate.Results Among the 196 cases,196 cases were detected with SLN ( detection rate was 100.0%).SLN was all stained in 196 cases. Furthermore,56 cases had ALND metastasis,140 cases had no metastasis,and the sensitivity was 91.8%(56/61). The SLN and ALN fully complied with pathological examination in 191 cases,and the accuracy rate was 97.5%(191/196).Five cases were false negative,and the false negative rate was 8.2%(5 /61).No case was false positive.The total SLNs was 705,and average number of detected SLNs was 3.59 pieces(2-6 pieces).The average ALN was 17.66 pieces(12-26 pieces).Conclusion With the advantages of real-time visual,precise positioning,no radio-active pollution and reduce the operation difficulty ,indocyanine green joint methylene blue as lymph tracer shows high detection rate,accuracy when it is used for SLNB in patients with breast cancer ,it is worthy of clinical application.