Objective To investigate the effects of 3-methyladenine(3-MA)on mouse mesangial cell line MES-13 cultured in high glucose,and on the kidney of streptozotocin(STZ)-induced mouse model of diabetes and the po-tential mechanism.Methods MES-13 cells were divided into low glucose control group(LG),hyper osmotic pres-sure control group(HOP),high glucose group(HG),3-methyladenine+high glucose group(HG+3-MA)and chloroquine+high glucose group(HG+CQ).The groups were respectively incubated with low glucose DMEM,30 mmol/L mannitol hypertonic control medium,30 mmol/L high glucose medium,5 mmol/L 3-MA+30 mmol/L high glucose medium and 10 mmol/L CQ+30 mmol/L high glucose medium for 24 hours.CCK-8 assay and Western blot were performed.In vivo experiment:Male C57BL/6J mice were induced diabetes for model development by in-tra-peritoneal injection of STZ 60 mg/kg for five consecutive days.After two weeks of injection,the blood glucose was measured.Animals with blood glucose level higher than 16.7 mmol/L(250 mg/dL)were randomly divided in-to diabetes control group(DM),3-MA intervention group(DM+3-MA)and CQ intervention group(DM+CQ),then were fed under the same conditions as normal control group(NC)mice.The DM+3-MA group was given 10 mg/kg of 3-MA aqueous solution by gavage every day,the DM+CQ group was given 50 mg/kg of CQ by intrap-eritoneal injection every three days,the NC group and DM group were given the same amount of normal saline and killed after 6 weeks.The kidneys were stripped for kidney/body weight ratio determination,periodic acid-schiff staining(PAS),MASSON staining microscopy and Western blot.Results In vitro experiment:Compared to the LG group,the cell viability,PCNA expression,ratio of phosphorylated Akt to total Akt(p-Akt/Akt)and ratio of phosphorylated rpS6 to total rpS6(p-rpS6/rpS6)were significantly increased in the HG group(P＜0.05).Com-pared with HG group,the cell viability,PCNA and p-Akt/Akt ratio of HG+3-MA group and HG+CQ group were significantly decreased and p-rpS6/rpS6 ratio of HG+3-MA group was significantly decreased(P＜0.01).In vivo experiment:Compared to NC group,the kidney/body weight ratio,glomerular volume,renal tubular injury index,PCNA,fibronectin,COL1A1,p-Akt/Akt,p-rpS6/rpS6 in DM group were all significantly up-regulated(P＜0.05).Compared with DM group,the kidney/body weight ratio,glomerular volume,renal tubular injury in-dex,PCNA,fibronectin,COL1A1,p-Akt/Akt,p-rpS6/rpS6 of DM+3-MA group mice were all significantly de-creased(P＜0.05).Conclusions 3-MA can improve glomerular mesangial cell proliferation and renal ECM deposi-tion in early diabetes nephropathy(DN).The improvement of 3-MA in early DN may be related to the inhibition of Akt/rpS6 signaling pathway.

Objective The objective of this study is to examine the expression level of the S100A7A protein in both gastric cancer tissues and cells,as well as to evaluate its impact on the malignant phenotype of gastric cancer(GC)cells.Methods Immunohistochemical assay was used to detect the expression characteristics of S100A7A in 21 gastric cancer tissues and their corresponding paracancerous tissues,as well as to investigate its correlation with gastric cancer clinicopathological factors.Gastric cancer cells were genetically modified to overex-press S100A7A through plasmid transfection.Subsequently,the impact of S100A7A on the proliferation,migra-tion,and invasion capacities of gastric cancer cells was assessed using cell proliferation assays(EdU assay and plate cloning assay)as well as cell migration and invasion assays(Transwell assay and scratch assay).Results The expression of S100A7A protein was higher in GC tissues than in paracancerous tissues;Overexpression of S100A7A may increase gastric cancer cell proliferation,migration,and invasion.Conclusion S100A7A is a possible oncogene in GC and is predicted to serve as a new diagnostic and therapeutic target for the disease.

Objective To investigate the relationship between serum levels of nuclear factor E2-related fac-tor 2(Nrf2),advanced oxidation protein products(AOPP)and blood lipid,liver fibrosis in patients with non-alcoholic steatohepatitis(NASH).Methods A total of 104 patients with NASH in Bishan Hospital Affiliated to Chongqing Medical University were selected as the study group,and 90 healthy people were selected as the control group.The serum levels of Nrf2 and AOPP in each group were detected and compared.Pearson or Spearman correlation analysis was used to analyze the relationship between serum Nrf2,AOPP levels and blood lipid,liver fibrosis in patients with NASH.Receiver operating characteristic(ROC)curve was used to e-valuate the diagnostic value of serum Nrf2,AOPP levels for NASH.Results The levels of triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),Nrf2 and AOPP in the study group were higher than those in the control group(P＜0.05),and the level of high density lipoprotein cholesterol(HDL-C)was significantly lower than that in the control group(P＜0.05).The serum levels of Nrf2 and AOPP in severe group were higher than those in moderate group and mild group(P＜0.05),and the serum levels of Nrf2 and AOPP in moderate group were higher than those in mild group(P＜0.05).Correlation analysis showed that serum Nrf2 and AOPP levels in NASH patients were positively correlated with TG,TC,LDL-C and the degree of liver fibrosis(P＜0.05),and negatively correlated with HDL-C(P＜0.05).The area under the curve(AUC)of serum Nrf2 for NASH diagnosis was 0.830(95％CI 0.780-0.880).The AUC of serum AOPP in the diagnosis of NASH was 0.866(95％CI 0.816-0.916).The AUC of the combined diagnosis of NASH was 0.925(95％CI 0.875-0.975).Conclusion The serum levels of Nrf2 and AOPP are increased in NASH patients,and they are closely related to blood lipids and liver fibrosis,which are expected to be effective indicators for the diagnosis of NASH.

OBJECTIVETo prepare the polyclonal antibody against methyl-accepting chemotaxis signal transduction protein (MCP) of Helicobacter hepaticus (H.hepaticus).

METHODSThe recombinant plasmid pET22b+/MCP was transformed into E.coli BL2l(DE3) to express the fusion protein His-rhMCP under the induction of IPTG. The fusion protein was purified and the antibody was obtained by immunizing rabbits. The titer of the polyclonal antibody was tested by indirect ELISA, and the specificity of the antibody was identified based on Western blotting using the prepared cell surface proteins (CSPs) of the bacteria.

RESULTSThe fusion protein was successfully expressed, and the titer of the antibody reached 1:32 000. Western blotting indicated that the antibody could specifically bind to CSPs and His-rhMCP.

CONCLUSIONThe antibody with a high titer and specificity was prepared to facilitate further study of the pathogenicity and epidemiology of H.hepaticus in human.

Animals ; Antibodies, Bacterial ; biosynthesis ; genetics ; Antibody Specificity ; Bacterial Proteins ; immunology ; Helicobacter hepaticus ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Signal Transduction

Objective To prepare the polyclonal antibody against methyl-accepting chemotaxis signal transduction protein (MCP) of Helicobacter hepaticus (H.hepaticus). Methods The recombinant plasmid pET22b+/MCP was transformed into E.coli BL2l(DE3) to express the fusion protein His-rhMCP under the induction of IPTG. The fusion protein was purified and the antibody was obtained by immunizing rabbits. The titer of the polyclonal antibody was tested by indirect ELISA, and the specificity of the antibody was identified based on Western blotting using the prepared cell surface proteins (CSPs) of the bacteria. Results The fusion protein was successfully expressed, and the titer of the antibody reached 1∶32 000. Western blotting indicated that the antibody could specifically bind to CSPs and His-rhMCP. Conclusion The antibody with a high titer and specificity was prepared to facilitate further study of the pathogenicity and epidemiology of H.hepaticus in human.

Objective To prepare the polyclonal antibody against methyl-accepting chemotaxis signal transduction protein (MCP) of Helicobacter hepaticus (H.hepaticus). Methods The recombinant plasmid pET22b+/MCP was transformed into E.coli BL2l(DE3) to express the fusion protein His-rhMCP under the induction of IPTG. The fusion protein was purified and the antibody was obtained by immunizing rabbits. The titer of the polyclonal antibody was tested by indirect ELISA, and the specificity of the antibody was identified based on Western blotting using the prepared cell surface proteins (CSPs) of the bacteria. Results The fusion protein was successfully expressed, and the titer of the antibody reached 1∶32 000. Western blotting indicated that the antibody could specifically bind to CSPs and His-rhMCP. Conclusion The antibody with a high titer and specificity was prepared to facilitate further study of the pathogenicity and epidemiology of H.hepaticus in human.

Objective To understand the ethics conflict situations between the pre-hospital patients and ambulance staff's determinations. Methods Taking a survey among the pre-hospital emergency physicians(80 people)and nurses(248 people)by Questionnaire of ethics conflicts during pre-hospital emergeney service,to investigate the ethics conflict situations between the pre-hospital patients and ambulance staff's determinations. Resulls (8.046±6.990)%of the patients who needed treatments refused to be treated completely,and(14.544±10.558)%of them refused partially.(14.451±14.747)% of the patients who needed ambulance transport refused to be delivered.In the patients who refused treatments and transportation.payment problem accounted for(23.52±19.79)%,(22.22±20.84)%of them did not believe they needed.(5.77±4.47)%of them wished to die,(19.44.4±18.65)%of them were hard to be idenfified.Other reasons accounted for(30.08±25.78)%.(20.31.4±16.66)% of the patients refused the ambulance crews' judge for some state.(29.66.4±24.02)%of the patients who got the pre-hospital emergency service were not necessary to call an ambulance.(22.1 l±19.52)%of the patients' demand conflicted with pre-hospital emergency services network management system.Conclusions There exists some conflicts between the pre-hospital patients and ambulance crews' determinations.

To find novel antihepatitis drugs, a series of nitrate-oleanolic acid (OA) hybrids (10a, 10b, 11a-11e and 12a-12c) were designed and synthesized on the basis of previous studies using OA as lead compound, which is widely found in natural plants and liver-specific metabolism. In the present study, ten novel NO-releasing derivatives of OA were synthesized by connecting nitrate to the OA-3-OH through varying lengths of linkers containing antioxidants which were designed to increase the ability of these target compounds to scavenge free radicals. The structures of these objective compounds were determined by IR, MS, 1H NMR and elemental analysis. Their protective effects on anti-Fas mediated HepG2 cell apoptosis were in vitro evaluated by LDH assay. Compound 12a is the most potent inhibitor. Its effect on anti-Fas mediated HepG2 cell apoptosis and amount of NO-releasing in vitro are similar to those of positive control NCX-1000.

Objective To explore the effects of polymorphisms of Crohn's disease related NOD2 gene and human beta-defensin 2 (hBD-2) on transcription of hBD-2 gene and its mechanism. Methods HEK293T cells were transfected with hBD-2 gene and NOD2 eukaryotic expression plasmid, and were then stimulated with LPS, TNF-α, or BAY 11-7082 (antagonist of NF-κB), respectively. Transcriptional activity of hBD-2 was detected afterwards. Results LPS could suppress transcription of hBD-2 (P=0. 020), which was increased by TNF-α in a dose-dependent manner (P =0. 004). In the presence of LPS, there was sig-nificant difference in transcriptional activity of hBD-2 between wild-NOD2 transfected group and mutated NOD2 (P268S) transfected group (P=0. 008), but there was no significant difference between wild hBD-2 transfected group and mutated hBD-2 transfected group (P=0. 053). With the stimulation of TNF-α (5 ng/ml), there was a significant difference between mutated hBD-2 transfected group and wild hBD-2 transfected group (P=0. 006), but no significant difference between wild-NOD2 transfected and mutated NOD2 transfected group was defected (P = 0. 064). Pretreatment with BAY 11-7082 before TNF-α (5 ng/ml) significantly inhibited the transcriptional activity of hBD-2 (P < 0. 001). Conclusion The poly-morphism of NOD2 affects the innate expression of hBD-2, the polymorphism of site in hBD-2 promoter (-233) may lead to significant decline of the inducible expression of hBD-2, and NF-κB might be a key pathway that NOD2 protein mediates the expression of defensin.

Objective To evaluate the clinical manifestations, endoscopic features and the clinical pathological characteristics of H. heilmannii-associated gastritis, and to compare these variables with those of H. pylori-ussociated gastritis. Methods The clinical data, endoscopic findings and pathologic characteristics of 3107 patients, who underwent endoscopy from 2005 to 2007, were retrospectively analyzed. Results Twenty-five cases of H. heilmannii infection were identified, the infection rates of H. heilmannii and H. pylori were 0.80% (25/3107) and 4.12% (1060/3107) respectively. Three cases were mixed infections. Of 25 patients, 20 showed such gastroenterologic symptoms to a greater or less extent as abdominal distending pain,nausea and anorexia, and other 5 cases were asymptomatic. All 25 patients showed chronic gastritis by en-doscopy, including chronic superficial gastritis (7/25, 28% ), erosion ( 3/25, 12% ), chronic atrophic gastritis (4/25, 16%), bile reflux(1/25, 4%), ulcer (1/25, 4%), polyp (1/25, 4%) and duodenal bulbar inflammation (2/25, 8% ). In rapid urease test, 3 cases were hyper-positive, 3 cases positive, 7 ca-ses mild-positive and 12 cases negative. According to histological observation, H. heilmannii scattered or ac-cumulated within the gastric pits, glandular lumen or mucus. The organism was observed in parietal cells with cell damage in one case. Sporadic lymphatic and plasmic infiltration were found in all patients with H.heilmannii infection, infiltration of neutrophils (12/25), gland atrophy and intestinal metaplasia (4/25)and lymphoid follicles (6/25) were also observed. Compared with H. pylori-associated gastritis, H. heilman-nii-associated gastritis showed less inflammation, less helicobacter density, mononuclear cell infiltration and neutrophilic activity ( P < 0.05 ). Conclusion H. heilmanaii mainly induces chronic gastritis, which is less severe than H. pylori-associated gastritis.