BACKGROUND: Currently, lung cancer is one of the malignant tumors with a high morbidity and mortality all over the world. However, the exact mechanisms underlying lung cancer progression remain unclear. The tumor necrosis factor receptor associated factor (TRAF) family members are cytoplasmic adaptor proteins, which function as both adaptor proteins and ubiquitin ligases to regulate diverse receptor signalings, leading to the activation of nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase (MAPK) and interferon regulatory factor (IRF) signaling. The aim of this study was to investigate the expression of TRAFs in different tissues and cancer types, as well as its mRNA expression, protein expression, prognostic significance and functional enrichment analysis in non-small cell lung cancer (NSCLC), in order to provide new strategies for the diagnosis and treatment of NSCLC. METHODS: RNA sequencing data from the The Genotype-Tissue Expression database was used to analyze the expression patterns of TRAF family members in different human tissues. RNA sequencing data from the Cancer Cell Line Encyclopedia database was used to analyze the expression patterns of TRAF family members in different types of cancer cell lines. RNA sequencing data from the The Cancer Genome Atlas (TCGA) database was used to analyze the mRNA levels of TRAF family members across different types of human cancers. Immunohistochemistry (IHC) analyses from HPA database were used to analyze the TRAF protein levels in NSCLC [lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC)]. Overall survival analysis was performed by Log-rank test using original data from Kaplan-Meier Plotter database to evaluate the correlation between TRAF expressions and prognosis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed on the TRAF family-related genes using RNA sequencing data from the TCGA database for NSCLC. The correlation between the expression levels of TRAF family members and the tumor immune microenvironment was analyzed using the ESTIMATE algorithm based on RNA sequencing data from the TCGA database. RESULTS: The TRAF family members exhibited significant tissue-specific expression heterogeneity. TRAF2, TRAF3, TRAF6 and TRAF7 were widely expressed in most tissues, while the expressions of TRAF1, TRAF4 and TRAF5 were restricted to specific tissues. The expressions of TRAF family members were highly specific among different types of cancer cell lines. In mRNA database of LUAD and LUSC, the expressions of TRAF2, TRAF4, TRAF5 and TRAF7 were significantly upregulated; while TRAF6 did the opposite; moveover, TRAF1 and TRAF3 only displayed a significant upregulation in LUAD and LUSC, respectively. Except for TRAF3, TRAF4 and TRAF7, other TRAF proteins displayed an obviously deeper IHC staining in LUAD and LUSC tissues compared with normal tissues. Additionally, patients with higher expression levels of TRAF2, TRAF4 and TRAF7 had shorter overall survival; while patients with higher expression levels of TRAF3, TRAF5 and TRAF6 had significantly longer overall survival; however, no significant difference had been observed between TRAF1 expression and the overall survival. TRAF family members differentially regulated multiple pathways, including NF-κB, immune response, cell adhesion and RNA splicing. The expression levels of TRAF family members were closely associated with immune cell infiltration and stromal cell content in the tumor immune microenvironment, with varying positive and negative correlations among different members. CONCLUSIONS TRAF family members exhibit highly specific expression differences across different tissues and cancer types. Most TRAF proteins exhibit upregulation at both mRNA and protein levels in NSCLC, whereas, only upregulated expressions of TRAF2, TRAF4 and TRAF7 predict worse prognosis. The TRAF family members regulate processes such as inflammation, immunity, adhesion and splicing, and influence the tumor immune microenvironment.
Humans ; Carcinoma, Non-Small-Cell Lung/pathology* ; Lung Neoplasms/mortality* ; Prognosis ; Gene Expression Regulation, Neoplastic ; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/metabolism*

Objective:To discuss the expression and biological function of gap junction protein β2(GJB2)in the lung adenocarcinoma(LUAD)A549 cells,and to procide the basis for the treatment of LUAD.Methods:The TIMER database was used to analyze the differential expression of GJB2 gene in various tumors;the GEPIA database was used to detect the mRNA expression of GJB2 in LUAD and analyze its correlation with different clinical stages of LUAD;the Kaplan-Meier plotter database was used to analyze the correlation between GJB2 protein and the prognosis of the LUAD patients.A total of 111 pairs of cancer tissues and adjacent normal lung tissues of LUAD patients were collected,and immunohistochemical staining was used to observe the expression of GJB2 protein in LUAD tissues and adjacent normal lung tissues.The human LUAD A549 cells were cultured in vitro and divided into GJB2 overexpression group(OE-GJB2 group,transfected with GJB2 overexpression plasmid)and its negative control group(OE-NC group,transfected with GJB2 empty vector plasmid),small interfering RNA(siRNA)-GJB2 group(si-GJB2 group,transfected with GJB2-siRNA)and its negative control group(si-NC group,transfected with control siRNA).Western blotting method was used to verify the transfection efficiency of the cells;cell couting kit-8(CCK-8)method was used to detect the proliferation activities of the A549 cells in various groups;5-ethynyl-2'-deoxyuridine(EdU)staining was used to detect the positive expression rates of the A549 cells in various groups;colony formation assay was used to detect the number of colony formation of the A549 cells in various groups;cell scratch assay was used to detect the migration rate of the A549 cells in various groups;Transwell chamber assay was used to detect the number of the invasion A549 cells in various groups;Western blotting method was used to detect the expression levels of GJB2 protein and epithelial-mesenchymal transition(EMT)-related proteins in the A549 cells in various groups.Results:The RT-PCR results showed that GJB2 gene was abnormally expressed in various tumors;compared with adjacent normal lung tissue,the mRNA expression level of GJB2 in cancer tissue of LUAD patients was significantly increased(P<0.05),and the high expression of GJB2 was correlated with the clinicopathological stage of LUAD(P<0.05).The Kaplan-Meier plotter results showed that compared with patients with low GJB2 expression,the overall survival of the patients with high GJB2 expression was significantly decreased[hazard ratio(HR)=1.71,95%CI:1.34-2.18,P<0.05].GJB2 protein was negatively expressed in adjacent normal alveolar and bronchial epithelial cells;compared with adjacent normal lung tissue,the expression of GJB2 protein in cancer tissue of LUAD patients was significantly enhanced.In LUAD,the positive expression of GJB2 protein was significantly associated with lymphnode metastasis(P<0.05)but not with gender(P=0.626),age(P=0.639),or TNM stage(P=0.837)(P>0.05).The CCK-8 results showed that compared with OE-NC group,the proliferation activity of the A549 cells in OE-GJB2 group was significantly increased(P<0.05 or P<0.01);compared with si-NC group,the proliferation activity of the A549 cells in si-GJB2 group was significantly decreased(P<0.05 or P<0.01).The EdU staining results showed that compared with OE-NC group,the positive expression rate of EdU in the A549 cells in OE-GJB2 group was significantly increased(P<0.01);compared with si-NC group,the positive expression rate of EdU in the A549 cells in si-GJB2 group was significantly decreased(P<0.01).The colony formation assay results showed that compared with OE-NC group,the number of colony formation of the A549 cells in OE-GJB2 group was significantly increased(P<0.01);compared with si-NC group,the number of colony formation of the A549 cells in si-GJB2 group was significantly decreased(P<0.01).The Transwell chamber assay results showed that compared with OE-NC group,the number of invasion A549 cells in OE-GJB2 group was significantly increased(P<0.01);compared with si-NC group,the number of invasion A549 cells in si-GJB2 group was significantly decreased(P<0.01).The cell scratch assay results showed that 24 h after scratching,compared with OE-NC group,the migration rate of the A549 cells in OE-GJB2 group was significantly increased(P<0.01);compared with si-NC group,the migration rate of the A549 cells in si-GJB2 group was significantly decreased(P<0.01).The Western blotting results showed that compared with OE-NC group,the expression levels of GJB2 and N-cadherin proteins in the A549 cells in OE-GJB2 group were significantly increased(P<0.01),while the expression level of E-cadherin protein was significantly decreased(P<0.05);compared with si-NC group,the expression levels of GJB2 and N-cadherin proteins in the A549 cells in si-GJB2 group were significantly decreased(P<0.01),while the expression level of E-cadherin protein was significantly increased(P<0.01).Conclusion:GJB2 is highly expressed in cancer tissue of LUAD patients and is associated with the poor prognosis in LUAD patients.GJB2 promotes the proliferation,invasion,migration,and EMT of the A549 cells.

Objective To establish a dynamic model of lipopolysaccharide-induced acute lung injury in mice based on the NLRP3/Caspase-1/gasdermin D(GSDMD)pyroptosis pathway,and observe the result ing lung injury at different time points.We aimed to identify the optimal time for modelling according to the injury at different time points and the expression of pyroptosis pathway-related proteins,to lay the foundation for animal models for subsequent experiments.Methods Fifty-four 6～8 weeks old male SPF BALB/c mice were divided randomly into nine groups,including Con group and model groups at 1,3,6,12,18,24,48,and 72 h.Body weight and lung tissue were detected by general and pathological observations and semi-quantitative scoring,including lung index,lung water content,and wet and dry weight ratio.The white blood cell count and concentrations of tumor necrosis factor-α,interleukin(IL)-6,IL-1β,IL-18,and BCA protein were detected in bronchoalveolar lavage fluid(BALF).The classic pyroptosis pathway-related proteins NLRP3,pro-Caspase 1,Caspase 1,and GSDMD were detected by Western Blot.Results Body weight decreased in all experimental groups,with the most significant weight loss in the 24 and 48 h groups.Gross observation and pathological examination of lung tissue showed that the most severe lung injury occurred at 24～72 h,with significant differences between each group and the control group.The lung index,lung water content,and wet/dry weight ratio were also significantly increased at 24～72 h.White blood cells in BALF started to increase from 6 h after model initiation,48 h can reach a peak,72 h all keep increasing.IL-18 in BALF began to increase at 24 h and continued to increase at 72 h.The inflammatory factors tumor necrosis factor-α,IL-1β,IL-6 were highest at 6 h and significantly reduced at 48 h.Protein concentrations in BALF were significantly increased within 24,48,and 72 h compared with those in the control group.The pyroptosis pathway proteins NLRP3,pro-Caspase-1,Caspase-1,and GSDMD were significantly enhanced in each time series,and channel protein expression was significantly enhanced at 24～72 h compared with that in the Con group.Conclusions Comprehensive analysis of experimental indicators,inflammatory factors,and pathway proteins at different times showed that the mechanism of pyroptosis was closely related to the occurrence and progression of acute lung injury.Expression of the pyroptosis pathway was most obvious and lung injury was most serious at 24～48 h.This study provides a model reference and experimental basis for subsequent studies of the specific mechanism and intervention targets of acute lung injury.

The inflammatory response is a systemic process produced by the body in response to stimulation such as infection or injury.The inflammatory response is closely related to the pathogenesis of various diseases,and plays an important role in the progression and outcome of diseases.Under normal conditions,the inflammatory response can maintain tissue homeostasis when the body is not seriously damaged.However,an uncontrolled inflammatory response that occurs when the body is subjected to more severe stimuli may often cause serious harm to the body.The termination of the ideal inflammatory response occurs after the elimination of the inflammatory stimulus.Therefore,it is particularly important to effectively modulate the extent and scope of the inflammatory response.β-arrestin 1(ARRB1)is a multifunctional regulatory protein that plays a key role in G protein-coupled receptor(GPCR)and non-GPCR-mediated signal transduction.Meanwhile,this protein also participates in the regulation of some immune cell development,differentiation and other functions.Although ARRB1 was originally thought to be a terminator of GPCR signal transduction and can inhibit the inflammatory response,recent studies suggest that its role in inflammatory responses is complex,with dual anti-inflammatory and pro-inflammatory effects.Based on this,this article reviews the relevant research on ARRB1 and the inflammatory response in recent years,focusing on the regulatory role of ARRB1 in signaling pathway transduction and immune cell development and differentiation function,as well as its mechanism of action in regulating the progression of inflammatory diseases,in order to provide new insights for the clinical precision treatment of inflammatory diseases and the screening of drug effect targets.

Objective:To explore the characteristics of endothelial function in children with incomplete Kawasaki disease (IKD) of different ages and its relationship with coronary artery lesion (CAL).Methods:A total of 200 children with IKD admitted to the Affiliated Hospital of Jining Medical University from February 2020 to May 2022 were selected as the IKD group, and another 200 healthy children who underwent physical examinations during the same period were selected as the control group. According to the age of children with IKD, infants ( n=78) were classified as those under 1 year old, infants ( n=62) were classified as those between 1-3 years old, and preschool children ( n=60) were classified as those over 3-6 years old. The endothelial function characteristics of IKD children in different age groups and normal control group children were analyzed and compared. In addition, IKD patients were divided into CAL group ( n=110) and nCAL group ( n=90) based on whether CAL was merged. The age, carotid intima-media thickness (IMT), flow-mediated-dilation (FMD), carotid stiffness index (SI), nitroglycerin-mediated dilation (NMD), tumor necrosis factor α (TNF-α), C-reactive protein (CRP), brachial artery endothelial dependent dilation function (EDD) and other clinical data were compared between the two groups. Cox proportional hazards model was used for univariate and multivariate analysis to identify the risk factors for CAL in IKD patients. A column chart prediction model was constructed and evaluated. Results:There were statistically significant differences in FMD, NMD, SI, IMT, reactive congestion index (RHI), and EDD between IKD patients and normal control group children (all P<0.05). There were statistically significant differences in FMD, NMD, SI, IMT, RHI, and EDD among three groups of IKD children of different ages (all P<0.05). There were statistically significant differences in the proportion of children aged ≤3 years, TNF-α, CRP, SI, IMT, EDD, FMD, and NMD between the nCAL and CAL groups (all P<0.05). Age ≤3 years, elevated levels of TNF-α, CRP, SI, IMT, and EDD, as well as decreased levels of FMD and NMD, were independent risk factors for CAL in children with IKD (all P<0.05). The column chart prediction model had high discrimination, and the area under the curve for predicting CAL occurrence in children with IKD was 0.868 and 0.830, respectively. Conclusions:There are significant differences in endothelial function among children with IKD of different ages, and endothelial function is closely related to the occurrence of CAL. When endothelial function is abnormal, CAL is more likely to occur.

The inflammatory response is a systemic process produced by the body in response to stimulation such as infection or injury.The inflammatory response is closely related to the pathogenesis of various diseases,and plays an important role in the progression and outcome of diseases.Under normal conditions,the inflammatory response can maintain tissue homeostasis when the body is not seriously damaged.However,an uncontrolled inflammatory response that occurs when the body is subjected to more severe stimuli may often cause serious harm to the body.The termination of the ideal inflammatory response occurs after the elimination of the inflammatory stimulus.Therefore,it is particularly important to effectively modulate the extent and scope of the inflammatory response.β-arrestin 1(ARRB1)is a multifunctional regulatory protein that plays a key role in G protein-coupled receptor(GPCR)and non-GPCR-mediated signal transduction.Meanwhile,this protein also participates in the regulation of some immune cell development,differentiation and other functions.Although ARRB1 was originally thought to be a terminator of GPCR signal transduction and can inhibit the inflammatory response,recent studies suggest that its role in inflammatory responses is complex,with dual anti-inflammatory and pro-inflammatory effects.Based on this,this article reviews the relevant research on ARRB1 and the inflammatory response in recent years,focusing on the regulatory role of ARRB1 in signaling pathway transduction and immune cell development and differentiation function,as well as its mechanism of action in regulating the progression of inflammatory diseases,in order to provide new insights for the clinical precision treatment of inflammatory diseases and the screening of drug effect targets.

Background/Aims: Epstein-Barr virus-associated gastric cancers (EBVaGCs) have unique molecular and clinicopathological characteristics. The cyclic GMP-AMP synthase-stimulator of interferon genes (STING) pathway is recently recognized as the critical innate immunity against pathogens and tumors. STING is also a master regulator in the cancer-immunity cycle and targeting STING could synergize with existing immune-checkpoint therapies. However, the role of STING in GC, especially in EBVaGC, and its correlation with programmed death-ligand 1 (PD-L1) remain largely unclear. Methods: We collected 78 cases of EBVaGCs and 210 cases of EBV-negative GC (EBVnGC) from a total of 1,443 cases of GC analyzed by EBV-encoded small RNA in situ hybridization. We investigated STING and PD-L1 expression and their concomitant prognostic value in EBVaGCs and EBVnGCs using tissue microarray and immunohistochemistry. The effects of STING and PD-L1 expression on the overall survival of patients with EBVaGC or EBVnGC were assessed by univariate and multivariate analysis. Results: We found that both STING and PD-L1 exhibited significantly higher expression in the EBVaGCs than that in the EBVnGCs. The expression of STING was positively correlated with that of PD-L1 in EBVaGCs. Simultaneous negative expression of STING and PD-L1, and positive expression of STING were independent prognostic risk factors for EBVaGC and EBVnGC, respectively. Conclusions This is the first prognostic retrospective study of STING and PD-L1 expression and the prognosis among EBVaGC and EBVnGC. The expression and prognostic value of STING and PD-L1 are different in the two types of GCs. STING and PD-L1 are promising prognostic biomarkers and therapeutic targets for EBVaGC and EBVnGC.

The co-existence of different neurodegenerative diseases in the same case has received increasing attention and reports. A rare comorbidity of amyotrophic lateral sclerosis (ALS) and progressive supranuclear palsy (PSP) in a sporadic patient was reported. The patient presented with pseudobulbar palsy, freezing gait, and developed muscle weakness and fasciculation. Brain magnetic resonance imaging, positron emission computed tomography and whole exomesequencing showed no evident abnormality. The patient had no response towards the treatment of levodopa, and received supportive treatment and gastrostomy. He is in stable condition by now. Totally 23 cases of ALS with PSP were reviewed, and found that the clinical manifestations were related to the main distribution of pathological inclusion bodies and the location of neuron loss, and the deposition of the same pathological protein in multiple systems may lead to the coexistence of different neurodegenerative diseases. Traditional treatment is generally ineffective, thus support treatment is pivotal. The genetic background and pathogenesis of this comorbidity should be studied in the future.

The aim of the present study was to investigate the effects and mechanisms of quercetin on plantar incision-induced matrix metalloproteinase (MMP)-9 and MMP-2 activity,microglia activation and the analgesic effect of quercetin on plantar incision surgery-treated mice.Postoperative pain model was mediated by plantar incision surgery and Von Frey Hairs was used to test the mechanical pain threshold.The activity of MMP-9 and MMP-2 in spinal cord was evaluated by gelatin zymography.The marker of microglia ionized calcium binding adapter molecule 1 (IBA-1),phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) was detected by Western blot.Results showed that quercetin (20,40,80 mg/kg,ip) significantly inhibited plantar incisioninduced mechanical allodynia and suppressed the activity of MMP-9 and MMP-2 in the spinal cord.Moreover,quercetin also markedly inhibited plantar incision-induced up-regulation of IBA-1 and p-p38 in spinal cord.In conclusion,quercetin may alleviate postoperative pain by suppressing MMP-9 and MMP-2 activity in microglia.