Objective To investigate the role and molecular mechanism of SOX4 in Helicobacter pylori(H.pylori)-mediated gastric mucosal epithelial dysplasia.Methods The expression of SOX4 in gastric tissues and cells was analyzed with reverse transcription-polymerase chain reaction(RT-PCR),Western blotting,and immunohistochemical staining.The effects of SOX4 on gastric epithelial cell proliferation and colony formation were determined with CCK-8 and colony formation assays.A PCR array was used to screen downstream target genes involved in H.pylori-induced dysplasia mediated by SOX4.The transcriptional regulation and binding sites of the target gene MLH3 by SOX4 were elucidated with luciferase reporter assay,promoter truncation assay,and chromatin immunoprecipitation(ChIP).Results SOX4 expression was significantly increased in H.pylori-infected gastric tissues(P<0.05).Overexpression of SOX4 markedly enhanced the proliferation and colony formation abilities of normal gastric epithelial cells(P<0.05).Elevated SOX4 led to the dysregulation of MLH3 and other DNA damage repair-related molecules after H.pylori infection in gastric epithelial cells(|logFC|>1,P<0.05).H.pylori promoted MLH3 expression in gastric epithelial cells through SOX4.SOX4 transcriptionally activated MLH3 expression by binding to the 5th site of the MLH3 promoter.The increased expression of SOX4 and MLH3 is associated with poor prognosis of gastric cancer patients.Conclusion SOX4 is closely associated with H.pylori-induced dysplasia in gastric epithelial cells.Upregulation of SOX4 promotes H.pylori-related dysplasia by transcriptionally activating MLH3,leading to the imbalance of proliferation and colony formation in gastric epithelial cells.

Objective To investigate the mechanism by which Yangmai Tongluo formula improves microcirculation disorders induced by high homocysteine(Hcy)levels via regulation of the acid sphingomyelinase(ASM)and endoplasmic reticulum(ER)stress pathways.Methods Fifty male C57BL/6J mice were divided randomly into a control group,Hcy model group,Yangmai Tongluo formula low-/high dose groups(5.3,10.4 g/kg,respectively),and a folic acid group(0.08 g/kg).Except for the control group,microcirculation disorders were induced in all mice using drinking water containing 1.8 g/L Hcy for 6 weeks.After modeling for 2 weeks,mice were administered the corresponding treatments by gavage for 4 weeks.Serum Hey concentrations and the blood perfusion volume of the lower extremity microvessels were measured.Protein expression levels of zonula occludens ZO-1,ZO-2,intercellular adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1),ASM,glucose-regulated protein 78(GRP78),and C/EBP homologous protein(CHOP)in the cardiac microvascular endothelium were analyzed using immunofluorescence.Results Serum Hcy levels were significantly increased in the Hcy model group compared with the control group(P＜0.05).Yangmai Tongluo formula did not significantly reduce Hey levels compared with the Hey model group,but blood perfusion in the lower extremities was significantly increased(P＜0.01)and expression levels of ZO-1 and ZO-2 in the cardiac microvascular endothelium were restored(P＜0.001)in the Yangmai Tongluo formula high dose group.It also inhibited the expression of ICAM-1,VCAM-1,ASM,GRP78,and CHOP(P＜0.05),with comparable effects to folic acid.Conclusions Yangmai Tongluo formula improves Hcy-induced microcirculation disorders and endothelial dysfunction by inhibiting ASM activity and alleviating ER stress,via a mechanism closely related to the regulation of endothelial inflammation and barrier stability.These result provide experimental evidence to support the use of traditional Chinese medicine to treat microvascular diseases.

Objective To investigate the role and molecular mechanism of SOX4 in Helicobacter pylori(H.pylori)-mediated gastric mucosal epithelial dysplasia.Methods The expression of SOX4 in gastric tissues and cells was analyzed with reverse transcription-polymerase chain reaction(RT-PCR),Western blotting,and immunohistochemical staining.The effects of SOX4 on gastric epithelial cell proliferation and colony formation were determined with CCK-8 and colony formation assays.A PCR array was used to screen downstream target genes involved in H.pylori-induced dysplasia mediated by SOX4.The transcriptional regulation and binding sites of the target gene MLH3 by SOX4 were elucidated with luciferase reporter assay,promoter truncation assay,and chromatin immunoprecipitation(ChIP).Results SOX4 expression was significantly increased in H.pylori-infected gastric tissues(P<0.05).Overexpression of SOX4 markedly enhanced the proliferation and colony formation abilities of normal gastric epithelial cells(P<0.05).Elevated SOX4 led to the dysregulation of MLH3 and other DNA damage repair-related molecules after H.pylori infection in gastric epithelial cells(|logFC|>1,P<0.05).H.pylori promoted MLH3 expression in gastric epithelial cells through SOX4.SOX4 transcriptionally activated MLH3 expression by binding to the 5th site of the MLH3 promoter.The increased expression of SOX4 and MLH3 is associated with poor prognosis of gastric cancer patients.Conclusion SOX4 is closely associated with H.pylori-induced dysplasia in gastric epithelial cells.Upregulation of SOX4 promotes H.pylori-related dysplasia by transcriptionally activating MLH3,leading to the imbalance of proliferation and colony formation in gastric epithelial cells.

Objective To investigate the mechanism by which Yangmai Tongluo formula improves microcirculation disorders induced by high homocysteine(Hcy)levels via regulation of the acid sphingomyelinase(ASM)and endoplasmic reticulum(ER)stress pathways.Methods Fifty male C57BL/6J mice were divided randomly into a control group,Hcy model group,Yangmai Tongluo formula low-/high dose groups(5.3,10.4 g/kg,respectively),and a folic acid group(0.08 g/kg).Except for the control group,microcirculation disorders were induced in all mice using drinking water containing 1.8 g/L Hcy for 6 weeks.After modeling for 2 weeks,mice were administered the corresponding treatments by gavage for 4 weeks.Serum Hey concentrations and the blood perfusion volume of the lower extremity microvessels were measured.Protein expression levels of zonula occludens ZO-1,ZO-2,intercellular adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1),ASM,glucose-regulated protein 78(GRP78),and C/EBP homologous protein(CHOP)in the cardiac microvascular endothelium were analyzed using immunofluorescence.Results Serum Hcy levels were significantly increased in the Hcy model group compared with the control group(P＜0.05).Yangmai Tongluo formula did not significantly reduce Hey levels compared with the Hey model group,but blood perfusion in the lower extremities was significantly increased(P＜0.01)and expression levels of ZO-1 and ZO-2 in the cardiac microvascular endothelium were restored(P＜0.001)in the Yangmai Tongluo formula high dose group.It also inhibited the expression of ICAM-1,VCAM-1,ASM,GRP78,and CHOP(P＜0.05),with comparable effects to folic acid.Conclusions Yangmai Tongluo formula improves Hcy-induced microcirculation disorders and endothelial dysfunction by inhibiting ASM activity and alleviating ER stress,via a mechanism closely related to the regulation of endothelial inflammation and barrier stability.These result provide experimental evidence to support the use of traditional Chinese medicine to treat microvascular diseases.

Objective To explore the mechanism by which puerarin alleviates the cardiotoxicity induced by doxorubicin in myocardial cells. Methods Cells in the logarithmic growth phase were divided into normal control group,model group,low-(20 mmol·L-1),medium-(40 mmol·L-1) and high-(80 mmol·L-1) dose puerarin groups,and positive control group(captopril,1 mmol·L-1). Except for the normal control group,the other groups were co-incubated with 5 mmol·L-1 doxorubicin. Cell viability was assessed using CCK-8 and lactate dehydrogenase (LDH) assays. ROS levels were detected using a ROS probe. Autophagy flux was detected by transfection with HBAD-mcherry-EGFP-LC3 adenovirus. Western Blot was used to measure the protein expression levels of Beclin-1,LC3,p62,p-AMPKα,and AMPKα. Lysosomal function was assessed using a lysosomal probe. Immunofluorescence was used to detect the relative intensity and co-localization of ASMase and LAMP1. Molecular docking analysis was performed to predict the binding capacity of PUE with ASMase. Differential gene expression was analyzed by gene set enrichment analysis. Results Compared to the normal control group,the model group showed reduced cell viability (P<0.01),increased release levels of LDH and ROS (P<0.05,P<0.01),increased number of autophagosomes (P<0.01),and decreased number of autophagic lysosomes (P<0.05). Beclin-1 protein expression and LC3-II/LC3-I ratio decreased(P<0.01),but p62 protein expression increased(P<0.01). Fluorescence intensity of lysosome decreased(P<0.01),whereas fluorescence intensity of ASMase increased(P<0.01). Immunofluorescence co-localization of ASMase and LAMP1 increased (P<0.01),the ratio of p-AMPKα/AMPKα decreased(P<0.05). Compared to the model group,the high-dose puerarin group showed a rebound in cell viability (P<0.05). The medium-and high-dose puerarin groups showed a decreasing trend in LDH level (P<0.05),and all puerarin groups showed a decreasing trend in ROS level (P<0.01). The number of autophagosomes in high-dose puerarin group reduced (P<0.01). The number of autophagic lysosomes in all puerarin groups increased (P<0.05,P<0.01). The high-dose puerarin group showed increased expression of Beclin-1 (P<0.05) and LC3-II/LC3-I ratio,and decreased p62 expression (P<0.01). All puerarin groups showed increased lysosomal fluorescence intensity (P<0.05,P<0.01). The medium-and high-dose puerarin groups showed a decrease in ASMase fluorescence intensity(P<0.05),a reduction in the immunofluorescence co-localization of ASMase with LAMP1 (P<0.01),and an increase in the p-AMPKα/AMPKα ratio (P<0.01). Molecular docking analysis discovered puerarin showed a binding energy of-8.6 kcal·mol-1 with ASMase. Gene enrichment analysis indicated that the differentially expressed genes in the doxorubicin cardiotoxicity model were related to apoptosis,autophagy,and lysosomal function. Conclusion Puerarin can alleviate doxorubicin-induced cardiotoxicity in myocardial cells and protect myocardial cells by regulating autophagy through AMPK/ASMase,as well as restoring autophagic flux.

Acute myeloid leukaemia (AML) is the most common form of acute leukaemia in adults, with increasing incidence with age and a generally poor prognosis. Almost 20% of AML patients express mutant isocitrate dehydrogenase 2 (mIDH2), which leads to the accumulation of the carcinogenic metabolite 2-hydroxyglutarate (2-HG), resulting in poor prognosis. Thus, global institutions have been working to develop mIDH2 inhibitors. SH1573 is a novel mIDH2 inhibitor that we independently designed and synthesised. We have conducted a comprehensive study on its pharmacodynamics, pharmacokinetics and safety. First, SH1573 exhibited a strong selective inhibition of mIDH2 R140Q protein, which could effectively reduce the production of 2-HG in cell lines, serum and tumors of an animal model. It could also promote the differentiation of mutant AML cell lines and granulocytes in PDX models. Then, it was confirmed that SH1573 possessed characteristics of high bioavailability, good metabolic stability and wide tissue distribution. Finally, toxicological data showed that SH1573 had no effects on the respiratory system, cardiovascular system and nervous system, and was genetically safe. This research successfully promoted the approval of SH1573 for clinical trials (CTR20200247). All experiments demonstrated that, as a potential drug against mIDH2 R140Q acute myeloid leukaemia, SH1573 was effective and safe.

Objective:To compare the early and late predictive values of critical illness score (CIS) and procalcitonin (PCT) in septic patients with blood stream infection (BSI) induced by intra-abdominal infection (IAI), and to identify the value of PCT in etiological diagnosis.Methods:The clinical data of patients with at least one positive blood culture within 24 hours admission to the emergency department of China-Japan Friendship Hospital from January 2014 to December 2019 and with final diagnosis of IAI induced sepsis were enrolled. Sequential organ failure assessment (SOFA), mortality in emergency department sepsis (MEDS), Logistic organ dysfunction system (LODS), and acute physiology and chronic health evaluation Ⅱ (APACHEⅡ) scores were calculated based on the parameters on the day of admission. Differences in various indicators among different Gram-stained bacterial infections and among patients with different prognosis at 28 days or 60 days were compared. Receiver operator characteristic curve (ROC curve) was used to analyze the value of PCT in differential etiological diagnosis of IAI induced sepsis caused by single bacterial infection, and the predictive value of CIS and PCT on 28-day and 60-day death of septic patients with BSI induced by IAI.Results:A total of 221 septic patients with IAI caused by single bacterial infection were enrolled. The 28-day mortality was 19.9% (44/221), and the 60-day mortality was 25.8% (57/221). Mortality caused by Gram-positive (G +) bacterial infection of patients was significantly higher than that caused by Gram-negative (G -) bacterial infection (28 days: 34.6% vs. 11.4%, 60 days: 42.0% vs. 16.4%, both P < 0.01). Compared with patients with G + bacterial infection, the PCT value of patients with G - bacterial infection was higher [μg/L: 4.31 (0.71, 25.71) vs. 1.29 (0.32, 10.83), P < 0.05]. Compared with survival group, the values of CIS and PCT in death group were higher, either in 28 days or in 60 days [death group vs. survival group in 28 days: SOFA score was 6.0 (4.0, 10.0) vs. 3.0 (2.0, 5.0), MEDS score: 11 (9, 14) vs. 6 (6, 9), LODS score: 4.0 (2.0, 6.0) vs. 1.0 (0, 2.0), APACHEⅡ score: 17.0 (15.0, 24.0) vs. 12.0 (8.0, 15.0), PCT (μg/L): 3.48 (1.01, 26.70) vs. 2.45 (0.32, 15.65); death group vs. survival group in 60 days: SOFA score: 6.0 (4.0, 10.0) vs. 3.0 (2.0, 5.0), MEDS score: 9 (6, 14) vs. 6 (6, 9), LODS score: 4.0 (1.0, 5.0) vs. 1.0 (0, 2.0), APACHEⅡ score: 16.5 (12.0, 20.0) vs. 12.0 (8.0, 15.0), PCT (μg/L): 2.67 (0.98, 17.73) vs. 2.22 (0.31, 16.75); all P < 0.05]. ROC curve showed that: ① the area under ROC curve (AUC) of PCT in the diagnosis of IAI induced sepsis with single bacterial infection was 0.740 [95% confidence interval (95% CI) was 0.648-0.833]. When the optimal cut-off value of PCT was 1.82 μg/L, the sensitivity of diagnosis of G - bacterial infection was 74.0%, and the specificity was 68.2%. When PCT value was higher than 10.92 μg/L, the specificity of diagnosis of G - bacterial infection could reach 81.8%. ② In the prediction of 28-day and 60-day mortality for septic patients with BSI induced by IAI, the APACHEⅡ score achieved the highest AUC [28 days: 0.791 (95% CI was 0.680-0.902), 60 days: 0.748 (95% CI was 0.645-0.851)]. APACHEⅡ score higher than 14.5 could help to predict 28-day and 60-day mortality for IAI patients with negative predictive values of 94.9% and 88.5%. However, the predictive value of PCT for septic patients with BSI induced by IAI was relatively lower [28-day AUC: 0.610 (95% CI was 0.495-0.725), 60-day AUC: 0.558 (95% CI was 0.450-0.667)]. Conclusion:PCT is more reliable in the identification of pathogen type among IAI induced sepsis with BSI, while APACHEⅡ score may perform better in predicting early and late mortality.

Objective:To compare the early and late predictive value of several critical illness scores (CISs) and biomarkers in patients with bloodstream infection (BSI)-associated pneumonia, and to identify the value of procalcitonin (PCT) in etiological diagnosis.Methods:Patients with at least one positive blood culture within 24 hours admission to department of emergency of China-Japan Friendship Hospital from January 2014 to December 2018 and with final diagnosis of pneumonia were enrolled. Sequential organ failure assessment (SOFA), mortality in emergency department sepsis (MEDS), Logistic organ dysfunction system (LODS), and acute physiology and chronic health evaluationⅡ (APACHEⅡ) scores were calculated based on the first parameters on the day of admission. Differences of various indicators among different Gram-stained bacterial infections and among patients with different prognosis at 28-day or 60-day were compared. Receiver operating characteristic (ROC) curve was used to analyze the value of biomarkers in differential diagnosis of pneumonia caused by single bacterial infection, and the predictive value of several CISs and biomarkers on 28-day and 60-day death of patients with pneumonia.Results:Among 540 patients with pneumonia caused by single bacterial infection, 256 (47.4%) patients with Gram-positive bacteria (GPB) infection and 284 (52.6%) with Gram-negative bacteria (GNB) infection. The 28-day mortality was 29.4% (159/540) and the 60-day mortality was 36.3% (196/540). PCT level was significantly higher in patients with GNB infection than that in GPB infected patients [μg/L: 1.99 (0.32, 13.19) vs. 0.45 (0.13, 3.53), P < 0.01]. There were significant differences of CISs and biomarkers between death group and survival group in predicting 28-day and 60-day mortality in BSI-associated pneumonia. ROC curve analysis showed that: ① the optimal cut-off value of PCT in the diagnosis of single bacterial infection was 0.48 μg/L, with the area under ROC curve (AUC) was 0.739 [95% confidence interval (95% CI) was 0.686-0.793]. When PCT value was greater than 4.49 μg/L, the specificity of diagnostic of GNB infection could reach 81.8%, and the positive predictive value (PPV) was 75.0%. When PCT value was greater than 10.16 μg/L, the diagnostic specificity could reach 91.2%. ② In the prediction of 28-day and 60-day mortality, the SOFA score showed highest AUC [28-day: 0.818 (95% CI was 0.768-0.867), 60-day: 0.800 (95% CI was 0.751-0.849)]. SOFA score greater than 8.5 points could help to predict 28-day and 60-day mortality for pneumonia patients with specificity of 90.5% and 91.6%, respectively. AUC of PCT for predicting 28-day and 60-day mortality in patients with BSI associated with pneumonia was 0.637 (95% CI was 0.575-0.700) and 0.628 (95% CI was 0.569-0.688), respectively. When PCT value was greater than 8.15 μg/L, the specificity and negative predictive value (NPV) were 80.2% and 75.1% respectively, and they could reach 80.2% and 68.7% when PCT value was greater than 7.46 μg/L. Conclusion:PCT is more reliable in the identification of pathogen type in BSI-associated pneumonia, while CISs may be more advantageous in the assessment of early and late prognosis.

Objective To investigate the characteristics and the risk factors of pulmonary function in patients with chronic obstructive pulmonary disease (COPD) for a 3 year follow-up.Methods Subjects diagnosed as COPD were followed up for 3 years in the Management Center of Chronic Respiratory Disease at XINQIAO Hospital from September 2009 to June 2012.This was a retrospective study.Parameters related to respiratory function mainly first second forced expiratory volume (FEV1),COPD assessment test (CAT),6 minutes walking distance (6MWD) and acute exacerbation were recorded during follow-up.Results Although the majority of patients were treated with drugs such as inhaled corticosteroid combined with longterm bronchial dilatation during the three years,FEV1 decreased progressively.The average annual decline of FEV1 was(31.80 ± 61.99) ml,translating into a mean annual decline of(3.74 ± 6.18) ％.However,there were significant differences in changes of FEV1.Approximately,FEV1 in 78.3％ (47/60) patients decreased,only 21.7％ (13/60) patients kept stable FEV1.There was a correlation between decrease of FEV1,FEV1 ％ predicted and the exacerbation (r =0.298,0.361,0.273;P ＜ 0.05).Logistic regression showed that the positive bronchodilator reversibility and the initial FEV1 were the independent factors associated with significant changes in FEV1 (respectively,OR =5.54,95％ CI 1.55-19.73;OR =8.28,95％ CI 1.42-48.32).Conclusion The changes of pulmonary function in patients with COPD are heterogeneous,although most patients are treated in a standard way.Nearly 80％ patients still represent deterioration of pulmonary function.Decline of FEV1 is closely related to the initial pulmonary function and bronchodilator reversibility.

Background contamination is a major problem in the analysis of organophosphate esters (OPEs). In this study, the possible sources of OPEs pollution were screened and several different ways were applied to minimize the blank contamination. Under the strict quality control measures, the cleanup efficiency of different solid phase extraction (SPE) was investigated for OPEs in different environmental matrices. A method was developed for the detection of 7 OPEs in dust, soil and sediment samples by gas chromatograph coupled with mass spectrometry ( GC / MS). Target compounds were extracted by hexane:dichloromethane (1 : 1, V/ V) followed by aminopropyl silica gel SPE column cleanup for dust, and target compounds in soil and sediment were Soxhlet extracted and cleanuped by two-step SPE. The results showed that the aminopropyl silica gel SPE column displayed the best purification performance among the three employed columns. Instrumental detection limits among the 7 OPEs ranged from 2. 5 to 25. 8 μg / L, and the method limits of quantification (MLOQs) in dust and soil sample ranged from 1. 4 to 15. 7 ng / g and 0. 3 to 2. 9 ng / g, respectively. The average recoveries of 7 OPEs in different matrices ( dust and soil) at two spiked concentration levels ranged from 67. 9% to 117. 4% . The proposed method was successfully applied to detect OPEs in different environmental matrices collected in Shanghai.