Objective To investigate necroptosis-related diagnostic biomarkers of bronchopulmo-nary dysplasia(BPD)and their relationships with the immune microenvironment through the analysis of necroptosis-related genes(NRGs)in BPD.Methods The dataset GSE32472 was downloaded from the Gene Expression Omnibus(GEO)database,and NRGs were obtained from the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway and Gene Cards databases.Differentially expressed necroptosis-related genes(DE-NRGs)were screened,and their biological functions and pathways were explored through functional enrichment analysis.Machine learning algorithms,inclu-ding least absolute shrinkage and selection operator(LASSO)and support vector machine-recursive feature elimination(SVM-RFE),were applied to screen feature genes.The Cell-type Ⅰdentification By Estimating Relative Subsets of RNA Transcripts(CIBERSORT)algorithm and the Estimation of Stromal and Immune Cells in Malignant Tumor Tissues using Expression Data(ESTIMATE)algo-rithm were used to explore the immune infiltration characteristics of BPD.Spearman correlation anal-ysis between feature genes and immune cells was performed using the"corrplot"package in R lan-guage.Results A total of 19 DE-NRGs were identified.The main biological functions and path-ways of DE-NRGs included the regulation of necroptosis and inflammatory responses.Three feature genes,namely flotillin-2(FLOT2),CASP8 and FADD-like apoptosis regulators(CFLAR),and charged multivesicular body protein 7(CHMP7),were further screened to construct a nomogram.In the validation sets GSE8586 and GSE188944,the area under the curve(AUC)values were all greater than 0.7.CIBERSORT analysis revealed that BPD group presented a higher proportion of naive B cells,neutrophils,eosinophils and resting mast cells compared to control group(P＜0.05).Meanwhile,the proportion of CD8+T cells,CD4+naive T cells,CD4+resting memory T cells,regulatory T cells,resting natural killer(NK)cells,M0 macrophages,M2 macrophages and activated dendritic cells was lower than that in the control group(P＜0.05).ESTIMATE analysis showed that the stromal score in the BPD group was higher than that in the control group,while the immune score was lower,with statistically significant differences(P＜0.05).Correlation analysis between the three feature genes and ESTIMATE scores indicated that FLOT2 and CFLAR were posi-tively correlated with the stromal score and negatively correlated with the immune score,whereas CHMP7 was positively correlated with the immune score and negatively correlated with the stromal score.Conclusion The three necroptosis-related feature genes can serve as diagnostic biomarkers for BPD-related necroptosis,with high diagnostic efficacy.They may play an important roles through immune mechanisms,providing new insights and theoretical references for the early diagnosis and immune intervention of BPD.

Objective:To investigate whether herb cake-insulated moxibustion affects the expression of cholesterol metabolism-related protein 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR)and inhibits the development of atherosclerosis by regulating the exosomal miR-223 expression.Methods:Thirty-six male New Zealand rabbits were randomly assigned to a normal group,a model group,and an herb cake-insulated moxibustion group,with 12 rabbits in each group.The model and the herb cake-insulated moxibustion groups were fed a high-fat diet for 8 weeks to induce an atherosclerosis model.Following successful modeling,the herb cake-insulated moxibustion group was subjected to bundling and herb cake-insulated moxibustion intervention,while the other two groups were subjected only to bundling without moxibustion.After 8 weeks of intervention,hematoxylin-eosin(HE)staining was used to observe aortic morphology;the serum levels of total cholesterol(TC),triglyceride(TG),and low-density lipoprotein cholesterol(LDL-C)were detected using an automatic biochemical analyzer in each group.Exosome morphology was observed using the transmission electron microscope;Western blotting(WB)was used to detect the protein levels of serum exosomal CD63 and CD9 markers,as well as liver HMGR;additionally,real-time fluorescence quantitative polymerase chain reaction was used to quantify serum exosomal miR-223.Results:HE staining showed thickened aortic intima,lipid infiltration,foam cell aggregation,and structural damage to the arterial wall in the model group.Meanwhile,after modeling,the serum levels of LDL-C,TC,and TG increased significantly in the model and herb cake-insulated moxibustion groups compared to the normal group(P<0.05),suggesting successful atherosclerosis rabbit model preparation.The serum exosomes of rabbits in the model group exhibited a saucer-like or semi-concave spherical shape with diameters of 120-150 nm.WB detection results showed positive expression of the exosomal markers CD63 and CD9.After 8 weeks of intervention,the miR-223 level in the model group was significantly lower than that in the normal group(P<0.01).In contrast,the herb cake-insulated moxibustion group demonstrated significantly reduced serum levels of TC,TG,and LDL-C(P<0.05),increased miR-223 expression(P<0.01),and decreased relative liver HMGR protein expression(P<0.05)compared to the model group.Conclusion:Herb cake-insulated moxibustion may alleviate the progression of atherosclerosis by up-regulating exosomal miR-223 expression and down-regulating HMGR protein expression,thereby inhibiting cholesterol anabolic metabolism.

Objective:To investigate whether herb cake-insulated moxibustion affects the expression of cholesterol metabolism-related protein 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR)and inhibits the development of atherosclerosis by regulating the exosomal miR-223 expression.Methods:Thirty-six male New Zealand rabbits were randomly assigned to a normal group,a model group,and an herb cake-insulated moxibustion group,with 12 rabbits in each group.The model and the herb cake-insulated moxibustion groups were fed a high-fat diet for 8 weeks to induce an atherosclerosis model.Following successful modeling,the herb cake-insulated moxibustion group was subjected to bundling and herb cake-insulated moxibustion intervention,while the other two groups were subjected only to bundling without moxibustion.After 8 weeks of intervention,hematoxylin-eosin(HE)staining was used to observe aortic morphology;the serum levels of total cholesterol(TC),triglyceride(TG),and low-density lipoprotein cholesterol(LDL-C)were detected using an automatic biochemical analyzer in each group.Exosome morphology was observed using the transmission electron microscope;Western blotting(WB)was used to detect the protein levels of serum exosomal CD63 and CD9 markers,as well as liver HMGR;additionally,real-time fluorescence quantitative polymerase chain reaction was used to quantify serum exosomal miR-223.Results:HE staining showed thickened aortic intima,lipid infiltration,foam cell aggregation,and structural damage to the arterial wall in the model group.Meanwhile,after modeling,the serum levels of LDL-C,TC,and TG increased significantly in the model and herb cake-insulated moxibustion groups compared to the normal group(P<0.05),suggesting successful atherosclerosis rabbit model preparation.The serum exosomes of rabbits in the model group exhibited a saucer-like or semi-concave spherical shape with diameters of 120-150 nm.WB detection results showed positive expression of the exosomal markers CD63 and CD9.After 8 weeks of intervention,the miR-223 level in the model group was significantly lower than that in the normal group(P<0.01).In contrast,the herb cake-insulated moxibustion group demonstrated significantly reduced serum levels of TC,TG,and LDL-C(P<0.05),increased miR-223 expression(P<0.01),and decreased relative liver HMGR protein expression(P<0.05)compared to the model group.Conclusion:Herb cake-insulated moxibustion may alleviate the progression of atherosclerosis by up-regulating exosomal miR-223 expression and down-regulating HMGR protein expression,thereby inhibiting cholesterol anabolic metabolism.

Objective: Tumor markers have been widely used clinically. Detection of serum CA125 is one of the commonly used clinical methods for early screening and early diagnosis of epithelial ovarian cancer, but it is difficult to diagnose epithelial ovarian cancer with a single specific tumor marker. In this study, the combinatorial tumor marker detection method was used to compare the value of each tumor marker alone and different combinations in the diagnosis of epithelial ovarian cancer. Methods: The clinical data of patients with epithelial ovarian cancer (n=65) and ovarian benign disease (n=29) were collected. Multiple tumor marker protein chip was used to detect cancer antigen 125 (CA125), carbohydrate antigen 242 (CA242), alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (β-HCG), carcinoembryonic antigen (CEA), cancer antigen 199 (CA199), neuron-specific enolase (NSE), Ferritin, cancer antigen 153 (CA153), and human growth hormone (HGH) serum levels, and to compare the differences between the benign and malignant ovarian tumors. The correlation between tumor markers and clinicopathologic features for ovarian epithelial carcinoma was analyzed by χ2 test. Spearman rank analysis showed the correlation between CA125 expression level and other tumor markers in epithelial ovarian cancer and the correlation between age and the above 10 tumor markers. Sensitivity, specificity, positive predictive value, negative predictive value, Youden index, and diagnostic efficiency were used to evaluate the diagnostic value of single tumor marker and the combination of tumor markers. Results: The levels of β-HCG, NSE, CA153, and CA125 in the epithelial ovarian cancer group were higher than those in the ovarian benign disease group. The level of NSE in the serum of patients with epithelial ovarian cancer was related to the clinical stage of patients. In addition, the levels of CA242, β -HCG, CEA, NSE, Ferritin, CA153 in the serum of patients with epithelial ovarian cancer were positively correlated with CA125 (rs=0.497, P< 0.001; rs=0.612, P<0.001; rs=0.358, P=0.003; rs=0.680, P<0.001; rs=0.322, P=0.009; rs= 0.609, P<0.001, respectively), and the levels of β-HCG, Ferritin, CA153 were positively correlated with the patient's age (rs=0.256, P=0.040; rs=0.325, P=0.008; rs=0.249, P=0.046, respectively). In the diagnosis of epithelial ovarian cancer, the sensitivity, Youden index, and diagnostic efficiency of CA125 detection alone were higher than the results of the other 9 separate detections. When CA153, CA199, CA242, Ferritin, and CEA were combined with CA125, the sensitivity of the combined detection of different combinations was higher than that of CA125 alone. The combined detection sensitivities of CA125+CEA and CA125+Ferritin+CEA were 89.2％ and 90.8％, respectively, and the diagnostic efficiencies were both 84.1％, which were higher than those of other combinations. The Youden index of CA125+CEA joint detection was 0.616, which was higher than those of other combinations. Conclusion: CA125 has a high diagnostic value in the diagnosis of epithelial ovarian cancer. The detection of combined tumor markers in serum has higher sensitivity and specificity in epithelial ovarian cancer.

Objective:Hepatocellular carcinoma(HCC)prognosis involves multiple clinical factors.Although nomogram models targeting various clinical factors have been reported in early and locally advanced HCC,there are currently few studies on complete and effective prognostic nomogram models for stage Ⅳ HCC patients.This study aims to creat nomograms for cancer-specific survival(CSS)in patients at stage Ⅳ of HCC and developing a web predictive nomogram model to predict patient prognosis and guide individualized treatment. Methods:Clinicopathological information on stage Ⅳ of HCC between January,2010 and December,2015 was collected from the Surveillance,Epidemiology,and End Results(SEER)database.The patients at stage Ⅳ of HCC were categorized into ⅣA(without distant metastases)and ⅣB(with distant metastases)subgroups based on the presence of distant metastasis,and then the patients from both ⅣA and ⅣB subgroups were randomly divided into the training and validation cohorts in a 7:3 ratio.Univariate and multivariate Cox regression analyses were used to analyze the independent risk factors that significantly affected CSS in the training cohort,and constructed nomogram models separately for stage ⅣA and stage ⅣB patients based on relevant independent risk factors.Two nomogram's accuracy and discrimination were evaluated by receiver operator characteristic(ROC)curves and calibration curves.Furthermore,web-based nomogram models were developed specifically for stage ⅣA and stage ⅣB HCC patients by R software.A decision analysis curve(DCA)was used to evaluate the clinical utility of the web-based nomogram models. Results:A total of 3 060 patients were included in this study,of which 883 were in stage ⅣA,and 2 177 were in stage ⅣB.Based on multivariate analysis results,tumor size,alpha-fetoprotein(AFP),T stage,histological grade,surgery,radiotherapy,and chemotherapy were independent prognostic factors for patients with stage ⅣA of HCC;and tumor size,AFP,T stage,N stage,histological grade,lung metastasis,surgery,radiotherapy,and chemotherapy were independent prognostic factors for patients with stage ⅣB HCC.In stage ⅣA patients,the 3-,6-,9-,12-,15-,and 18-month areas under the ROC curves for the training cohort were 0.823,0.800,0.772,0.784,0.784,and 0.786,respectively;and the 3-,6-,9-,12-,15-,and 18-month areas under the ROC curves for the validation cohort were 0.793,0.764,0.739,0.773,0.798,and 0.799,respectively.In stage ⅣB patients,the 3-,6-,9-,and 12-month areas under the ROC curves for the training cohort were 0.756,0.750,0.755,and 0.743,respectively;and the 3-,6-,9-,and 12-month areas under the ROC curves for the validation cohort were 0.744,0.747,0.775,and 0.779,respectively;showing that the nomograms had an excellent predictive ability.The calibration curves showed a good consistency between the predictions and actual observations. Conclusion:Predictive nomogram models for CSS in stage ⅣA and ⅣB HCC patients are developed and validated based on the SEER database,which might be used for clinicians to predict the prognosis,implement individualized treatment,and follow up those patients.

Objective:Hepatocellular carcinoma(HCC)patients at the same stage exhibit different prognosis,and the underlying molecular mechanism remains unclear.This study aims to identify the key genes impacting the prognosis of HCC patients. Methods:Differentially expressed gene analyses were performed between HCC samples and normal ones,and between patients with long overall survival(OS)and those with short OS,in TCGA-LIHC and GSE14520 datasets.The Kaplan-Meier method with log-rank test was used to evaluate the role of secreted phosphoprotein 2(SPP2)in the prognosis of HCC patients.Gene set enrichment analysis(GSEA)was used to understand the difference of enriched signaling pathways between SPP2-stratified HCC subgroups.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses were performed to predict the potential functional pathways in which SPP2 might participate. Results:SPP2 was significantly down-regulated in tumors when compared with normal tissues,or in tumor samples with short OS when compared with those with long OS[fold change(FC)>2 and false discovery rate(FDR)<0.05].Low expression of SPP2 was associated with worse clinicopathological features like vascular invasion(P=1.6e-05),poor cancer status(with tumor,P=0.021),advanced T stage(T3 or T4,P=4.5e-04),advanced TNM stage(stage Ⅲ or Ⅳ,P=3.1e-04),and with unfavorable prognosis(shorter OS,P= 0.002).Gene enrichment analyses revealed that SPP2 might involve in the metabolic homeostasis of HCC and in the development of liver fibrosis and cirrhosis. Conclusion:SPP2 might inhibit the development of liver fibrosis and cirrhosis and the tumorigenesis of HCC,and analogs of SPP2 might be potential drugs in the prevention of these diseases.

Objective: To compare the treatment outcomes for locally advanced hypopharyngeal carcinoma between surgery plus radio(chemo) therapy(SRT) and non-surgery chemoradiotherapy(CRT). Methods: A total of 119 patients diagnosed with advanced hypopharyngeal carcinoma without distant metastases between 2010 and 2014 were identified in the Chinese People′s Liberation Army General Hospital, and they were divided into 2 groups: 42 cases in SRT group and 77 cases in CRT group. Patients′ clinical information was collected. Survival rates and prognostic factors were analyzed by the Kaplan-Meier method with SPSS 23.0 software. The survival rates, laryngeal preservation rates and complication rates were compared between the two groups using the chi-square test.Among the 119 patients, 112 were males and 7 were females. Age ranged from 27 to 78 years, with an average age of 57 years. Results: There were no significant difference between the SRT and CRT group for five-year disease-free survival (DFS, 53.9% vs. 45.1%, χ²=1.251, P=0.263) and overall survival (OS, 54.9% vs. 45.6%, χ²=1.749, P=0.186). Compared to SRT group, CRT group did not showed the significant increase of treatment complications (χ²=0.858, P=0.354), with a higher laryngeal preservation rate (50.0% vs. 71.4%, χ²=6.493, P=0.011). Conclusions Advanced hypopharyngeal carcinoma is of high malignancy and poor prognosis. Combined modality treatment is a main approach for advanced hypopharyngeal cancer. SRT offers disease-free survival and overall survival rates equivalent to CRT, but with a higher laryngeal preservation rate.

Objective There are few studies on the application of low molecular weight heparin (LMWH) in the therapy of women with recurrent spontaneous abortion (RSA) pregnant by assisted reproductive technology (ART).The article aimed to explore the clinical research of LMWH in therapy of women with RSA pregnant by ART.Methods 126 women with RSA pregnant by ART were enrolled and they were diagnosed and treated in Reproductive Medicine Center in the Affiliated Hospital of Guangdong Medical University from January 2010 to February 2016.According to the patient′s agreement on LMWH treatment, 60 patients in agreement with LMWH treatment were divided into 2 groups: LMWH+IUI group(n=30) and LMWH+ IVF-ET group(n=30).66 patients in disagreement with LMWH treatment were also divided into 2 groups: IUI group(n=32) and IVF-ET group(n=34).Comparison was made in patients from 4 groups concerning pregnancy success rate, live birth rate, pregnancy time involving RSA and incidence of pregnancy complications.At the same time, the occurrence of adverse reactions during the use of LMWH was also observed.Results The pregnancy time involving RSA in LMWH+IUI group significantly increased compared with IUI group([82.67±9.10]d vs [48.17±8.68]d)(P<0.05).The pregnancy success rate and live birth rate in LMWH+ IVF-ET group were both higher than those of IVF-ET group (66.7% vs 35.29%, 85.00% vs 50.00%)(P<0.05) and significant difference was also found in the incidence of RSA, the pregnancy time involving RSA, and the morbidity of gestational hypertension between groups(P<0.05).The results of D2D at 4 weeks of pregnancy in LMWH+IUI group([0.65±0.07]mg/L) and LMWH+ IVF-ET group([0.625±0.06]mg/L) were lower than those of LMWH group ([0.76±0.12]mg/L) and LMWH group([0.77±0.06]mg/L).The result of D2D at 6 weeks of pregnancy in LMWH+IUI group was lower than those of IUI group and IVF-ET group, and D2D in LMWH+ IVF-ET group was lower compared with IVF-ET group(P<0.05).The results of D2D in all the four groups increased with the pregnancy weeks(P<0.05).The prothrombin time(PT) at 4 weeks of pregnancy in LMWH+IUI group, IUI group and LMWH+ IVF-ET group ([12.53±0.38]s, [12.38±0.65]s, [12.47±0.58]s) was significantly higher at pre-pregnancy([12.33±0.52]s, [12.30±0.68]s, [12.22±0.64]s) and 6 weeks of pregnancy([12.13±0.62]s, [12.05±0.60]s, [12.03±0.54]s) (P<0.05).Among 60 cases treated with LMWH, small area ecchymoma were found in 11 cases and the incidence was 18.33%(11/60), only two cases reported with uncomfortable light pain in the location of subcutaneous injection.Conclusion Low-dose LMWH is safe and effective in the therapy of pregnant women with RSA through ART.

OBJECTIVETo determine the effect of AQP1 gene on facial nerve edema following injury through investigation of the relationship between the expression of AQP1 gene and Schwann cells swelling.

METHODSThe AQP1 expression in Schwann cells of mouse facial nerve tissues was detected by immunofluorescent staining. The transgenic protocol by lentivirus transduction was used to specifically upregulate AQP1 expression in Schwann cells. Lenti-AQP1 and CTRL (empty vector) transduced cells were observed during gene overexpression every 24 h for 6 days by using phase contrast microscopy. Cell volume of CTRL and Lenti-AQP1 treated cells was measured daily from the day of treatment, through day 6.

RESULTSSchwann cell primary cultures maintained a high level of AQP1 water channels, representing an ideal cell model to study the role of AQP1 in the facial nerve. The expression of AQP1 mRNA and protein in Schwann cells infected with the Lenti-AQP1 was increased significantly compared with CTRL lentivirus (P < 0.05). Lenti-AQP1 caused cell swelling in cultured Schwann cells, as validated by cell volume determinations (P < 0.01).

CONCLUSIONSAQP1 is an important factor responsible for the fast water transport of cultured Schwann cells. It plays an important role in facial nerve edema.

Animals ; Aquaporin 1 ; genetics ; metabolism ; Cell Size ; Edema ; etiology ; Facial Nerve ; metabolism ; Facial Nerve Diseases ; etiology ; Lentivirus ; Mice ; RNA, Messenger ; metabolism ; Schwann Cells ; cytology ; metabolism ; virology ; Time Factors ; Transduction, Genetic ; methods ; Up-Regulation