Objective To investigate the role of ASPP2 (apoptosis stimulating protein 2 of p53,ASPP2) in starvation-induced autophagy and apoptosis of colorectal cancer HCT116 p53-/-(p53 gene deletion) cell line.Methods The study included three experiment groups:green fluorescent protein adenovirus (rAd-GFP) infection group,autophagy inhibitor LY294002 treatment group and ASPP2 adenovirus (rAd-ASPP2) infection group.Celluar autophagy and apoptosis were induced by coculturing with serum-free medium for 0 h,24 h,48 h.Apoptosis level was detected by Calcein/PI uptaking test.Autophagy level was observed under the fluorescence microscope via transfection with cerise fluorescent protein autophagy plasmid CFP-Lc3.Results In control group,starvation for 24 hours significantly promoted autophagy of HCT116 cells (0 h:1.04 ±0.24; 24 h:12.17 ±0.86,P ＜0.05),while apoptosis was not increased (0 h:2.01％ ±0.06％; 24 h:3.23％ ±0.34％,P ＞0.05).With 48 h starvation,autophagy(0 h:1.04 ±0.24; 48 h:21.09 ±3.32) and apoptosis(0 h:2.01％ ±0.06％ ; 48 h:30.20％ ±3.18％)of HCT116 increased (P ＜ 0.05).With the use of LY294002 apoptosis induced by 24 h starvation significantly increased (rAd-GFP group:3.23％ ± 0.34％ ; LY294002 group:15.68％ ± 1.24％,P ＜0.01),but aopotosis under 48 h starvation decreased (rAd-GFP group:30.20％ ± 3.18％; LY294002group:25.44％ ± 3.01％,P ＜ 0.05).With ASPP2 transfection,autophagy under 24 h starvation significantly declined (rAd-GFP group:12.17 ± 0.86,ASPP2 group:1.45 ± 0.45,P ＜ 0.01),and apoptosis increased(rAd-GFP group:3.23％ ± 0.34％ ; ASPP2 group:10.45％ ± 0.81％,P ＜ 0.05).Both autophagy (rAd-GFP group:21.09 ± 3.32; ASPP2 group:29.93 ± 3.48) and apoptosis (rAd-GFP group:30.20％ ±3.18％ ; ASPP2 group:36.72％ ±2.74％) were higher than that in controls under 48 h starvation (P ＜ 0.05).Conclusions ASPP2 probably promotes apoptosis of colorectal cancer cells by two-way regulated autophagy.

Objective To analyze the current situation and developing trend of utilizaion of medical insurance drugs.Methods Data were collected from Suqian Hospital.Drug Utilization Analysis System and DDD were taken as the basic unit of measurement.Results The consumption of cost,DDDs and variety of type A medical insurance drugs were rising year by year,but the proportion in total was in declining trend.When analyzed according to DDDc,the type A,non medical insurance drugs and the type B ranked the first,the second and the third place during the 2009-2010,the DDDc of type A was lower than the non medical insurancedrugs and the type B.For example,the DDDc of type A was 3.32 yuan/day,the DDDc of the non medical insurance drugs and the type B was 15.04 and 15.97 yuan/day respectively.Conclusion Analyzed in respect to DDDs,the protortion of type A was in high level and the type B was in low.But analyzed in respect to the comsumption cost,the protortion of type B was in high level and the type A was in low.

Objective To investigate the role of apoptosis stimulating protein 2 of p53 (ASPP2)in the apoptosis,cell cycle and autophagy of starvation-induced colorectal cancer HCT116 p53 +/+ (p53 wild-type) cell line.Methods Six groups were included:(1) control group; (2) green fluorescent protein adenovirus (rAd-GFP) infection group; (3)ASPP2 adenovirus (rAd-ASPP2) infection group; (4)starvation group; (5)rAd-GFP + starvation group; (6) rAd-ASPP2 + starvation group.HCT116 cells were infected with ASPP2 adenovirus (rAd-ASPP2),resulting ASPP2 gene over-expression.The apoptosis,autophagy and cell cycle changes were induced by culturing with serum-free medium for 24 h.Apoptosis was evaluated by Calcein/PI uptaking test,and autophagy was observed by counting the red fluorescent protein autophagy plasmid CFP-Lc3 which was transfected into cytoplasm.Cell cycle was detected by flow cytometry.Statistical analysis was performed by one-way analysis of variance (ANOVA).Results Over-expressed ASPP2 was found to significantly promote starvation-induced HCT116 apoptosis and autophagy.The cell apoptosis rate in rAd-GFP + starvation group was 10.00％ ± 1.42％,and 18.44％ ±2.06％ in rAd-ASPP2 + starvation group(q =9.548,P =0.000).The cell autophagy rate in rAd-GFP+ starvation group and rAd-ASPP2 + starvation group was 35.00％ ± 5.34％ and 57.61％ ± 6.06％ respectively(q =7.657,P =0.000).Over-expressed ASPP2 accelerated HCT116 G2/M arrest under starvation,but resulted in both G0/G1 and G2/M arrest without starvation.Conclusion These results suggest that ASPP2 can promote starvation-induced HCT116 p53 +/+ cells apoptosis and autophagy,and affect the cell cycle.

Objective To investigate the central neurotoxicity induced by nucleoside analog reverse transcriptase inhibitors (NRTIs)-stavudine (D4T).Methods Mouse primary cortical neurons were cultured and treated with different concentrations of stavudine.Neuron apoptosis was analyzed by calcein/acetomethoxy/propidium iodide (AM/PI) staining. Morphological change of neuron was confirmed by immunofluorescence.Mitochondrial DNA copies which were usually evaluated through Cycloxygenase 2 (COX-2) and thymidine kinase2 (TK2) mRNA were determined by real-time quantitative polymerase chain reaction.Chi-square test,student t test and Wilcoxon nonparameter test were used to analyze the data.Results Neuronal apoptosis observed in 50 μmol/L D4T treatment group was more significant than that in 0μmol/L D4T treatment group and 25 μmol/L D4T treatment group (51.3％±12.4％ vs 24.9％±8.2％ and 26.5％±10.6％,respectively; x2 =7.25 and 6.93,respectively; both P＜0.01).The average neurite numbers of each neuron were 11.2±3.6 in 0μmol/L D4T treatment group,8.6±2.8 in 25 μmol/L D4T treatment group and 4.3±2.4 in 50 μmol/L D4T treatment group.The difference was statistically significant between 25 μmol/L D4T treatment group and 0 μmol/L D4T treatment group (t=4.06,P＜0.01) and between 50 μmol/L D4T treatment group and 25 μmol/L D4T treatment group (t =4.35,P＜ 0.01). Furthermore,the average lengths of neuritis were (319.9±100.2) μm in 0 μmol/L D4T treatment group,(298.3±83.9) μm in 25 μmol/L D4T treatment group and (258.4±82.2) μm in 50 μmol/L D4T treatment group.The difference was statistically significant between 25 μmol/L D4T treatment group and 0 μmol/L D4T treatment group (t=4.58,P＜0.01) and between 50 μmol/L D4T treatment group and 25 μmol/L D4T treatment group (t=4.65,P＜0.01).TK2 mRNA expression dramatically decreased along with the increasing D4T concentration.The fold changes were 0.34 in 25 μmol/L D4T treatment group and 0.08 in 50 μmol/L D4T treatment group. The difference was statistically significant between 25 μmol/L D4T treatment group and 0μmol/L D4T treatment group (Z=- 3.28,P＜0.01) and between 50 μmol/L D4T treatment group and 25 μmol/L D4T treatment group (Z=-4.25,P＜0.01).Compared with 0μmol/L D4T treatment group,the relative fold changes of COX-2 copies were 1.01 in 25 μmol/L D4T treatment group and 1.12 in 50 μmol/L D4T treatment group.The differences were not significant among the three groups (Z=0.98 and 1.24,respectively; both P＞0.05).Conclsion It suggests that short-term exposure to D4T may result in neuron apoptosis,neurite shrink and down-regulated expression of TK2,but the level of mitochondrial DNA copies keeps stable.

OBJECTIVETo determine the differential protein and mRNA expressions of mitochondria fusion protein-2 (Mfn2) in hepatic tissues in conditions of cirrhosis and acute on chronic liver failure using rat model systems,and to determine the correlative effects on production of adenosine triphosphate (ATP) and reactive oxygen species (ROS).

METHODSA liver cirrhotic rat model (LC rats) was established by intraperitoneal injection of carbon tetrachloride (CCl4,in vegetable oil),and these mice were subsequently used (10 weeks later) to establish the acute on chronic liver failure rat model (LF rats) by injecting lipopolysaccharide and D-amino-galactose.Control groups (normal controls,NC rats) were established for each model by intraperitoneal injection of vegetable oil only.Protein expression of Mfn2 in liver was quantified by western blotting with fluorescence densitometry and immunofluorescence staining,and mRNA expression was measured by real-time fluorescence quantitative PCR.ROS levels in liver were measured by fluorescence spectrophotometry,and ATP content was measured by bioluminescence assay.Significance of inter-group differences was assessed by one-way ANOVA,and correlations were determined using bivariate statistical modeling.

RESULTSMfn2 protein expression was significantly lower in the liver tissues from modeled rats than that from the control rats (LC:0.051+/-0.004 and LF:0.037+/-0.007 vs.NC:0.254+/-0.008;F=444.98,P less than 0.05).The mRNA expression followed the same trend of lower expression (LC:21.21+/-0.93 and LF:24.35+/-0.85 vs.NC:19.09+/-0.69; F=66.941,P less than 0.05).The ATP content in liver tissues was also significantly lower in the modeled rats (LC:2.07+/-0.05 mol/L and LF:1.81+/-0.11 mol/L vs.NC:3.24+/-0.08 mol/L; F =574.21,P less than 0.05).Lower Mfn2 expression was correlated with lower ATP content (r =0.982) and higher ROS content (r =0.803).

CONCLUSIONReduced Mfn2 expression in liver tissue may cause a decrease in ATP synthesis and increase in ROS generation,thereby disrupting metabolism and increasing oxidative stress in the liver under conditions of cirrhosis and liver failure.

Acute-On-Chronic Liver Failure ; metabolism ; Animals ; Carbon Tetrachloride ; GTP Phosphohydrolases ; metabolism ; Liver Cirrhosis ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Mitochondrial Proteins ; metabolism ; Rats

Objective:To evaluate the efficacy and safety of simultaneous integrated boost intensity-modulated radiation therapy (SIB-IMRT) for rectal cancer with lateral lymph node metastasis (LLNM).Methods:From January 2016 to December 2022, 103 rectal cancer patients with LLNM were enrolled. The patients were divided into SIB-IMRT group (52 cases) and conventional chemoradiotherapy (CRT) group (51 cases) using the random number table method. The dose was 50 Gy for the pelvis with 60 Gy of SIB-IMRT for the LLNM in the SIB-IMRT group. The dose was 50 Gy for the pelvis in the CRT group. The primary endpoint was the lateral recurrence rate. The efficacy and adverse reactions of the two groups were compared.Results:The adverse reactions and surgical complications after neoadjuvant radiotherapy were comparable between the two groups. The response rates of LLNM treatment were 76.9% and 56.9%, respectively, in the two groups ( χ2=4.69, P=0.03). The SIB-IMRT group and CRT group had a local recurrence rate of 7.7% and 25.5% ( χ2=5.92, P=0.015), respectively, and a lateral recurrence rate of 3.8% and 23.5% ( χ2=8.49, P=0.004), respectively. Univariate analysis showed that the SIB-IMRT, short axis of lateral lymph nodes <5 mm after radiotherapy, and negative result in the postoperative lymph node pathological examination were factors associated with lateral recurrence. Multivariable regression analysis demonstrated that the SIB-IMRT ( HR=6.42, 95% CI: 1.40-29.49) and short axis of lateral lymph nodes <5 mm after radiotherapy ( HR=0.17, 95% CI: 0.04-0.66) were independent factors associated with lateral recurrence. The two groups had a 3-year disease-free survival of 73.25% and 62.6% ( P>0.05), respectively, and a 3-year overall survival of 87% and 82.5% ( P>0.05), respectively. Conclusions:The SIB-IMRT is safe and effective for rectal cancer with LLNM. The short axis of lateral lymph nodes <5 mm after neoadjuvant radiotherapy and SIB-IMRT is an independent risk factor for lateral recurrence.