Purpose To investigate the expression of bone sialoprotein(BSP)in the tissues and cells of endome-trial cancer(EC)and its effects on the proliferation and invasion of EC cells.Methods The expression of BSP was assessed by immunohistochemistry in 235 EC tissues and 88 normal endometrial tissues,and its correlation with clinico-pathological features was analyzed.Western blot was used to compare BSP levels between human endometrial carcinoma cell line(HHUA)and normal human endumetial epithelial cells(HEEC).BSP was knocked down in HHUA cells via transient transfection,and the cells were divided into blank control group and BSP-knockdown group.The effects of BSP knockdown on cell cycle,proliferation,migration,invasion,and apoptosis were evaluated using PI staining,CCK-8,scratch,Transwell,and Annexin V-FITC assays,respectively.Protein levels of TGF-β signaling pathway compo-nents were analyzed by Western blot.Results BSP expression was significantly higher in EC tissues than in normal endometrium(P＜0.001)and correlated with lymph node metastasis and advanced FIGO stage(P＜0.05).BSP pro-tein level was also significantly elevated in HHUA cells(2.455 8±0.008 9)compared to HEECs(1.571 2±0.005 4)(P＜0.01).After knockdown,compared with the control group,the proliferation index(74.4±3.33),migration rate(0.48±0.03),and invasion ability(0.36±0.11)of the cells were increased,and the apoptosis rate(25.97％)of the cells was increased(P＜0.05).Furthermore,the expression levels of TGF-β signaling pathway downstream proteins TGF-β1(0.290 4±0.002 3)、TGF-β2(0.292 9±0.001 6)、Smad2(0.469 3±0.001 1)、Smad3(0.247 0±0.001 7)、pAKT(0.382 1±0.001 9)、ATK(0.119 6±0.001 6)and MEK1(0.258 9±0.000 3)in the BSP-knockdown group of EC cells decreased(P＜0.01).Conclusion BSP is highly expressed in endometrial cancer and promotes cancer cell proliferation,invasion,and metastasis by activating the TGF-β signaling pathway.

Purpose To investigate the expression of bone sialoprotein(BSP)in the tissues and cells of endome-trial cancer(EC)and its effects on the proliferation and invasion of EC cells.Methods The expression of BSP was assessed by immunohistochemistry in 235 EC tissues and 88 normal endometrial tissues,and its correlation with clinico-pathological features was analyzed.Western blot was used to compare BSP levels between human endometrial carcinoma cell line(HHUA)and normal human endumetial epithelial cells(HEEC).BSP was knocked down in HHUA cells via transient transfection,and the cells were divided into blank control group and BSP-knockdown group.The effects of BSP knockdown on cell cycle,proliferation,migration,invasion,and apoptosis were evaluated using PI staining,CCK-8,scratch,Transwell,and Annexin V-FITC assays,respectively.Protein levels of TGF-β signaling pathway compo-nents were analyzed by Western blot.Results BSP expression was significantly higher in EC tissues than in normal endometrium(P＜0.001)and correlated with lymph node metastasis and advanced FIGO stage(P＜0.05).BSP pro-tein level was also significantly elevated in HHUA cells(2.455 8±0.008 9)compared to HEECs(1.571 2±0.005 4)(P＜0.01).After knockdown,compared with the control group,the proliferation index(74.4±3.33),migration rate(0.48±0.03),and invasion ability(0.36±0.11)of the cells were increased,and the apoptosis rate(25.97％)of the cells was increased(P＜0.05).Furthermore,the expression levels of TGF-β signaling pathway downstream proteins TGF-β1(0.290 4±0.002 3)、TGF-β2(0.292 9±0.001 6)、Smad2(0.469 3±0.001 1)、Smad3(0.247 0±0.001 7)、pAKT(0.382 1±0.001 9)、ATK(0.119 6±0.001 6)and MEK1(0.258 9±0.000 3)in the BSP-knockdown group of EC cells decreased(P＜0.01).Conclusion BSP is highly expressed in endometrial cancer and promotes cancer cell proliferation,invasion,and metastasis by activating the TGF-β signaling pathway.

Objective:To investigate the clinical characteristics of patients with aspergillus spondylitis, and to provide reference for timely diagnosis and treatment.Methods:The clinical manifestations, imaging performance, laboratory examination results, diagnosis and treatment outcomes of six patients with confirmed aspergillus spondylitis in Department of Infectious Diseases, Henan Provincial People′s Hospital during April 30, 2015 and May 1, 2020 were retrospectively analyzed.Results:The main manifestations of six patients were fever and neck pain or low back pain. The time from the onset of clinical manifestations to diagnosis was more than two months to 14 months. Spine magnetic resonance imaging (MRI) showed long T1 and T2 signals on vertebral body, high pressure lipid signal, obvious enhanced scan enhancement, and paravertebral abscess formation might be presented. Among the six patients, C-reactive protein increased in four patients, erythrocyte sedimentation rate increased in five patients, β-D-glucan test (G test) increased in three patients, galactomannan antigen test (GM test) increased in four patients. Six patients with aspergillus spondylitis were all confirmed by biopsy of diseased tissue for fungal smear, tissue culture or metagenomics next generation sequencing. After treatment with voriconazole or itraconazole, five patients recovered and one patient was still under treatment.Conclusions:The clinical manifestations and imaging examination of patients with aspergillus spondylitis are nonspecific. Peripheral blood G test and GM test need to be combined for diagnosis. The diagnosis depends on tissue puncture pathology examination, and the metagenomics next generation sequencing is needed if necessary.

Objective:To evaluate the short-term prognostic value of model for end-stage liver disease (MELD) combined with high density lipoprotein-cholesterol (HDL-C) in patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF).Methods:From December 2015 to December 2018, 182 patients with HBV-ACLF who were treated in Henan Provincial People′s Hospital were included. Prognosis and clinical data including HDL-C, total bilirubin, international standardized ratio (INR), creatinine of patients within 24 hours after admission were collected and analyzed retrospectively.The values of MELD were calculated. The binary logistic regression analysis was used to analyze the independent risk factors affecting 90-day mortality in HBV-ACLF patients.The receiver operator characteristic curve (ROC) and MedCalc 15.2 software were used to assess the predictive value of MELD, HDL-C and MELD-HDL-C model for prognosis. Kaplan-Meier survival curve was performed to analyze the prognosis of patients in different groups.Results:Sixty patients were divided into the death group and 122 patients were divided into the survival group according to the prognosis during hospitalization and 90 days after discharge. The MELD score of patients in the survival group was 21(19, 24), which was significantly lower than that in the death group (29(25, 34)), and the HDL-C value of patients in the survival group was significantly higher than that in the death group (0.3 (0.1, 0.6) mmol/L vs 0.2(0.1, 0.5) mmol/L). The differences were both statistically significant ( Z=-6.290 and -4.087, respectively, both P<0.01). Multivariate logistic regression analysis showed that MELD score and HDL-C value were the independent risk factors for 90-day mortality in patients with HBV-ACLF(odds ratio ( OR)=1.432, 95% confidence interval ( CI)1.271-1.613; OR=0.584, 95% CI 0.487-0.700, respectively; both P<0.01). Areas under the ROC of MELD, HDL-C and MELD-HDL-C scoring models were 0.775, 0.782 and 0.878, respectively. MELD-HDL-C scoring model was superior to both MELD and HDL-C , and the differences were both statistically significant ( Z=3.944 and 3.104, respectively, both P<0.01). When the MELD-HDL-C Youden′s index was set at 0.72, the optimal threshold was 24.69. Patients with MELD-HDL-C score≥24.69 had lower survival rate than patients with MELD-HDL-C score<24.69, and the difference was statistically significant ( χ2=142.900, P<0.01). Conclusion:MELD, HDL-C and MELD-HDL-C scoring systems could predict the short-term prognosis in patients with HBV-ACLF, and the predictive value of MELD-HDL-C has the superiority.

Objective:To screen the differential proteomic of plasma exosomes before and after magnesium isoglycyrrhizinate (MgIG) treatment in chronic hepatitis B patients.Methods:Plasma samples were collected from 36 cases with chronic hepatitis B before and after MgIG treatment (2 ml/case). Plasma exosomes were extracted by ultracentrifugation. Exosomal particles concentration and inner diameter were detected by Nanosight NS300 particle size analyzer. Three cases of plasma exosomes were randomly selected before and after MgIG treatment. Proteins were extracted after lysis and digested with trypsin. Label-free differential proteomics analysis was performed by liquid chromatography-tandem mass spectrometry to screen out differential proteins that changed more than 1.5 times. Enzyme linked immunosorbent assay (ELISA) was used to verify the quantitative differential protein expression ( n = 30). Measurement data were compared by paired sample t-test. Results:The average particle concentration of the extracted exosomes was 2.2×10 9/ml, and the average size was (107 ± 52) nm, which was consistent with the theoretical value of plasma exosome size, proving that the plasma exosomes were successfully extracted. Proteomics results showed that before and after MgIG treatment in chronic hepatitis B patients, a total of 153 differentially expressed proteins were screened, including 85 up-regulated and 68 down-regulated proteins. Enzyme-linked immunosorbent assay results showed that compared with the MgIG before and after treatment group of chronic hepatitis B patients, the differences in the concentrations of hepatocyte growth factor activator and hepatocyte growth factor like protein in plasma exosomes were statistically significant ( P < 0.05). Hepatocyte growth factor activator concentration in the plasma exosomes before and after MgIG treatment group was (45.9 ± 9.4) μg/ml and (13.9 ± 2.0) μg/ml, respectively, and it was down-regulated by about 3 times. Hepatocyte growth factor-like protein concentration in the plasma exosomes before and after MgIG treatment group was (23.4 ± 4.9) μg/ml and (13.8 ± 2.2) μg/ml, respectively, and it was down-regulated by about 2 times. Enzyme-linked immunosorbent assay results had consistency with the proteomics results. Conclusion:This study successfully screened the differential proteomic of plasma exosomes before and after MgIG treatment in chronic hepatitis B, and provided experimental basis for studying the molecular mechanism of MgIG treatment for chronic hepatitis B.

ObjectiveTo investigate the mechanism of action of miR-196b in regulating the growth and apoptosis of hepatoma cells by targeting nuclear apoptosis-inducing factor 1 (NAIF1). MethodsReal-time PCR was used to measure the expression of miR-196b in hepatoma HuH-7, SNU-449, HepG2, and SMCC7721 cells versus normal human HL7702 hepatocytes. The hepatoma HepG2 cells were collected and divided into Control group (blank control), Anti-NC group (transfected with inhibitor control), Anti-miR-196b group (transfected with miR-196b inhibitor), si-NC group (transfected with siRNA control), si-NAIF1 group (transfected with NAIF1 siRNA), Anti-miR-196b+si-NAIF1 group (co-transfected with miR-196b inhibitor and NAIF1 siRNA), and Anti-miR-196b+si-NC group (co-transfected with miR-196b inhibitor and siRNA control). MTT assay was used to measure the change in proliferation, plate colony formation assay was used to measure colony formation ability, flow cytometry was used to measure cell apoptosis, and Western blot was used to measure the protein expression of Bax and C-caspase-3. Target gene prediction software predicted that NAIF1 might be a target gene of miR-196b, and the luciferase reporting system was used to identify the targeting relationship. The t-test was used for comparison of continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the SNK-q test was used for further comparison between two groups. ResultsThere was a significant difference in the expression level of miR-196b between hepatoma HuH-7, SNU-449, HepG2, and SMCC7721 cells and normal human HL7702 hepatocytes (1.85±0.16/1.63±012/2.36±0.25/1.92±0.13 vs 1.00±0.09, F=29.05, P＜0.001). Compared with the Anti-NC group, the Anti-miR-196b group had significant reductions in the expression level of miR-196b (0.42±0.03 vs 1.02±0.10, P＜0.05), cell proliferation (0.20±0.02 vs 0.30±0.05, P＜0.05), and colony formation ability (64.35±6.97 vs 119.54±11.82, P＜0.05) and significant increases in apoptosis rate (22.30%±2.09% vs 4.26%±0.35%, P＜0.05) and relative protein expression of Bax (0.69±0.08 vs 0.30±0.05, P＜0.05) and C-caspase-3 (0.63±0.05 vs 0.21±0.04, P＜0.05). Compared with the si-NC group, the si-NAIF1 group had significant increases in proliferation ability (0.46±0.05 vs 0.31±0.04, P＜0.05) and colony formation ability (138.92±9.66 vs 118.47±838, P＜0.05) and significant reductions in apoptosis rate (4.12%±0.40% vs 1.23%±0.12%, P＜0.05), NAIF1 (0.10±0.01 vs 0.17±0.02, P＜0.05), and protein expression of Bax (0.18±0.02 vs 0.29±0.03, P＜0.05) and C-caspase-3 (0.12±0.01 vs 020±0.03, P＜0.05). Compared with the Anti-miR-196b+si-NC group, the Anti-miR-196b+si-NAIF1 group had significant increases in proliferation ability (0.28±0.02 vs 0.21±0.03, P＜0.05) and colony formation ability (97.12±8.23 vs 66.35±5.20, P＜0.05) and significant reductions in apoptosis rate (9.60%±1.11% vs 21.14%±1.32%, P＜0.05), NAIF1 (0.30±0.04 vs 0.52±0.06, P＜0.05), and protein expression of Bax (0.28±0.03 vs 0.67±0.06, P＜0.05) and C-caspase-3 (0.22±0.05 vs 0.60±004, P＜0.05). ConclusionDownregulation of miR-196b can inhibit the growth and induce the apoptosis of hepatoma cells via negative regulation of NAIF1.

Objective To construct a radiomics model for predicting hematoma enlargement in early hypertensive intracerebral hemorrhage and explore its predictive value.Methods A retrospective collection of 212 patients with hypertensive intracerebral hemorrhage within 6 h of onset,admitted to our hospital from February 2010 to August 2018,was performed.CT examination was performed within half an hour of admission.CT re-examination was performed 24 h after admission to determine whether there was hematoma enlargement.The regions of interest were delineated on the first CT,and 431 image indicators were extracted from the Matlab software.The LASSO regression model was used to screen out the most predictive imaging features,and the selected features and support vector machine classifier (SVM) were used to build the prediction model.Receiver operating characteristic (ROC) curve was used to evaluate the predicted effect of the model.Results After 24 h of admission,the incidence of hematoma enlargement was 18.9％ (40/212).Eighteen imaging ensemble features (including 4 first-order statistics features:standard deviation,kurtosis,uniformity,and variance;one shape-and size-based feature:surface to volume ratio;7 textual features:long run low grey level emphasis,inertia,correlation-angle 90,short run emphasis,correlation-all direction,long run emphasis,and inverse difference moment;6 wavelet features:autocorrelation-3,informational measure of correlation2-3,long run high gray level emphasis-4,short run high gray level emphasis-4,short run low gray level emphasis-7,and sum variance-3) were combined with SVM to establish a prediction model by LASSO regression model.The area under ROC curve was 0.928,enjoying sensitivity and specificity of 92.5％ and 83.5％,respectively.Conclusion The constructed radiomics model is helpful in predicting the expansion of hypertensive cerebral hemorrhage.

Objective: To investigate the influence of hepatitis B virus X gene (HBx) on apoptosis of hepatic cells mediated by Fas in HePG2 cells. Methods: HBx eukaryotic vector pcDNA3.1(+)-X was transfected into HEPG2 cells with lipofectamine, and the null vector pcDNA3.1(+) and untransfected HEPG2 were used as normal controls. The cells were collected 72 h after transfection, and the expression of HBx mRNA and protein was determined using RT-PCR and Western blot, respectively. The mRNA expression of apoptosis-related genes Bcl-2 and Bax mRNA was also determined using RT-PCR. Cytotoxicity and apoptosis were evaluated using CCK-8 and flow cytometry, respectively, after HepG2-HBx and HepG2-3.1 cells were treated with stimulatory monoclonal antibody anti-Fas CH11. The t test was used for pairwise comparison. Results: The cell line HepG2-HBx was successfully established, as confirmed by RT-PCR and Western blot, and RT-PCR results showed that HepG2-HBx cells had significantly higher expression of Bcl-2 mRNA than HepG2-3.1 and HepG2 cells (P < 0.05), but had significantly lower expression of Bax mRNA than HepG2-3.1 and HepG2 cells (P < 0.05); CCK-8 and flow cytometry showed that anti-Fas CH11 had a lower cytotoxicity to HepG2-HBx cells and allowed for a lower apoptosis rate of HepG2-HBx cells compared with HepG2-3.1 and HepG2 cells. Conclusions HBx can inhibit apoptosis of hepatic cells mediated by the Fas pathway.

Objective To explore the molecular mechanisms of hepatic stimulator substance (HSS) gene knockout in promoting the development and progression of nonalcoholic steatohepatitis (NASH).Methods NASH model mice (n=20) with HSS wild-type (HSS+/+) or HSS gene knockout (HSS-/-) were constructed using modified choline-deficient diet (CD-diet),untreated C57BL6-HSS-/-and C57BL6-HSS+/+ mice (n=20) were considered as control.Ten mice of each group were killed at month 1 and 2,respectively.The levels of triglyceride (TG) and total cholesterol (TC) in liver were measured using ELISA method.Histopathology and collagen deposition in liver tissue were observed using HE staining and Masson staining,respectively.Lipid content in liver tissue was observed and calculated using oil red O staining.The levels of mRNA and proteins of peroxisome proliferators activated receptor gama coactivator 1 alpha (PGC-1α),mitochondrial transcription factor A (TFAM),transcription factor-E2 related factor α (Nrf2),[-loop,dynamin-related protein 1 (Drp1),mitochondrial fission 1 protein (Fis1),mitofusins 1 (Mfn1),autophagy related gene 3 (Atg3) in liver tissue were detected using Real-time PCR and Western blot,respectively.Content of malonaldehyde (MDA),cyclooxygenase Ⅳ (COX Ⅳ) and adenosine tirphosphate (ATP) were measured using kits,and the activity of respiratory chain complex Ⅴ and cytochrome C oxidase in liver tissue were measured using spectrophotometry.the comparison between groups was done by t test.Results The levels of HSS mRNA and protein in mice-HSS-/-were 0.154± 0.04 and 0.08± 0.01,respectively,which were both significantly lower than those in mice-HSS+/+ (0.952 ± 0.08 and 1.362±-0.130,respectively),and t he differences had statistical significance (t =10.244 and 10.375,respectively,both P＜0.05).One month and 2 months after NASH modeled,TC contents in mice-HSS-/ were (248.6±21.5) μmol/g and (217.4±18.0) μmol/g,respectively,which were both remarkably higher than those in mice-HSS+/+ [(153.5 ± 11.2) μmol/g and (140.8 ±7.5) μmol/g,respectively],and the differences had statistical significance (t=15.270 and 10.524,respectively,both P＜0.05).The results form HE staining,oil red O staining and Masson staining indicated that fat deposition,collage deposition and inflammation in liver tissues of mice-HSS-/-were severer than those in mice-HSS+/+.One month after NASH modeled,protein levels of Drp1,Fis1,Mfn1 and Atg3 in liver tissues of mice-HSS-/ were all significantly decreased compared with those in mice-HSS+/+,and the differences had statistical significance (t=10.705,24.072,9.892 and 17.540,respectively,all P＜ 0.05).Two months after NASH modeled,protein levels of Drp1,Fis1,Mfn1and Atg3 in liver tissues of mice-HSS-/ were all significantly decreased compared with those in mice-HSS+/+,and the differences had statistical significance (t=125.378,15.926,34.330 and 13.437,respectively,all P＜0.05).One month after NASH modeled,mRNA levels of Drp1,Fis1,Mfn1 and Atg3 in liver tissues of mice-HSS-/-were all significantly decreased compared with those in mice-HSS+/+,the differences had statistical significance (t=36.337,40.825,33.508 and 28.104,respectively,all P＜0.05).Two months after NASH modeled,mRNA levels of Drp1,Fis1,Mfn1 and Atg3 in liver tissues of mice-HSS-/-were all significantly decreased compared with those in mice-HSS+/+,and the differences had statistical significance (t=35.210,42.375,27.753 and 20.560,respectively,all P＜0.05).The protein levels of PGC-1α,TFAM,Nrf2 and D-loop in liver of C57BL6-HSS-/-group were lower than those in liver of C57BL6-HSS+/+ group,and the differences had statistical significance (one month:t=20.548,31.036,19.445 and 10.974,respectively;two months:t=9.887,13.330,22.375 and 18.903,respectively,all P＜0.05).The mRNA levels of PGC-1α,TFAM,Nrf2 and D-loop in liver of C57BL6-HSS-/-group were all lower than those in C57BL6-HSS+/+ group,and the differences had statistical significance (one month:t=9.087,12.582,21.451 and 7.774,respectively;two months:t=23.758,17.924,9.924 and 15.209,respectively,all P＜0.05).One month and 2 months after NASH modeled,the levels of ATP mRNA in liver of C57BL6-HSS / group were both significantly lower than those in C57BL6-HSS+/+,and the differences had statistical significance 0=43.775 and 28.375,respectively,both P＜0.05);the levels of COXⅣ mRNA in liver of C57BL6-HSS / group were 0.142 ± 0.06 and 0.068± 0.001,respectively,which were both significantly lower than those in C57BL6-HSS+/+ group (0.255± 0.08 and 0.172 ±0.06,respectively),and the differences had statistical significance (t=28.337 and 19.782,respectively,both P＜0.05);the levels of MDA mRNA in liver of C57BL6-HSS-/-group were 0.973 ±0.112 and 1.253±0.054,respectively,which were both significantly lower than those in C57BL6-HSS+/+ group (0.366±0.02 and 0.872±0.05,respectively),and the differences had statistical significance (t=8.357 and 6.582,respectively,both P＜0.05).Conclusion Deletion of HSS accelerates NASH progression via inhibiting mitochondrial fusion,which leads to dysfunction of mitochondrial respiratory chain and inhibition of fatty acid oxidation.

Objective To investigate the role of serum and glucocorticoid regulated protein kinase (SGK) 1 in the inflammatory responses mediated by toll like receptors.Methods Mice were injected with lipopolysaccharide (LPS,1 mg/kg) 2 h after the pretreatment of EMD638683 (10 mg/kg) or phosphate buffered saline (PBS) as control.At the time points of 3 and 24 h,pro-inflammatory cytokines (interleukin [IL]-6,IL-12 and tumor necrosis factor [TNF]-α) in serum were measured using enzymelinked immunosorbent assay (ELISA).Livers and lung were harvested at 6 h and 24 h after the injection of LPS,embedded by optimum cutting temperature (OCT) and then stained with hematoxylin and eosin (HE).Peripheral blood mononuelear cell (PBMC) were isolated and stimulated by LPS with or without the pretreatment of EMD or LY294002.Cytokines (IL-6,IL-12 and TNF-α) were measured using ELISA.IKKα/β,IKBα and nuclear factor (NF)-κB p65 were detected by Western bolt.Data were analyzed by one way analysis of variance.Results In the model of LPS-induced endotoxin sepsis,inhibition of SGK1 induced secretion of pro-inflammatory cytokine (IL-6 [t=3.007,P＜0.05],IL-12[t=4.413,P＜0.05] and TNF-α[t=5.403,P＜0.05]),increased inflammatory cells infikration into the liver and lung within 6 h,and induced serious multiple organ damage with collapse of alveoli and fatty degeneration of liver.After 24 h,pharmacological inhibition of SGK1 with EMD638683 increased proinflammatory cytokine (IL-6 [t=18.540,P＜0.01],IL-12[t=16.520,P＜0.01] and TNF-α[t=34.880,P＜0.01]) production in human PBMC upon LPS stimulation and inhibited the phosphorylation of IKKα/ β/IKBα and nuclear factor (NF)-κB p65.Conclusions SGK1 suppresses the toll like receptor 4 mediated inflammatory responses via NF-κB.