This study aims to establish a method to determine the serum acetaminophen concentration based on diazo reaction, and apply it in the gastric emptying evaluation. Theoretically, acetaminophen could take hydrolysis reaction in hydrochloric acid solution to produce p-aminophenol, which could then take diazo reaction resulting in a product with special absorption peak at 312 nm. Then the serum acetaminophen concentration and recovery rate were calculated according to the standard curve drawn with absorbance at 312 nm. ICR mice were given a dose of acetaminophen (500 mg x kg(-1)) by gavage and the serum acetaminophen was dynamically measured through the diazo reaction. Besides, ICR mice were subcutaneously injected with the long-acting GLP-1 analog GW002 before the gavage of acetaminophen, and serum acetaminophen concentration was measured as above to study how GW002 could influence the gastric emptying. The data showed acetaminophen ranging from 0 to 160 μg x mL(-1) could take diazo reaction with excellent linear relationship, and the regression equation was y = 0.0181 x +0.0104, R2 = 0.9997. The serum acetaminophen was also measured with good linear relationship (y = 0.0045 x + 0.0462, R = 0.9982) and the recovery rate was 97.4%-116.7%. The serum concentration of acetaminophen reached peak at about 0.5 h after gavage, and then gradually decreased. GW002 could significantly lower the serum acetaminophen concentration and make the area under the concentration-time curve (AUC) decrease by 28.4%. In conclusion, a method for the determination of serum acetaminophen based on the diazo reaction was established with good accuracy and could be used in the evaluation of gastric emptying.
Acetaminophen ; blood ; pharmacokinetics ; Aminophenols ; Animals ; Gastric Emptying ; Mice ; Mice, Inbred ICR

Objective To apply RNA interference technique for reducing the expression of MDC1 gene in esophageal carcinoma cell line ECA109, observe the changes in cell cycle and radiosensitivity after radiation, and discuss related mechanisms. Methods Three pairs of effective interference sequences and negative control sequences were synthesized for MDC1 mRNA sequence, and a recombinant plasmid was constructed with the vector pSIH1?H1?copGFP. RT?PCR and Western blot were used to determine the expression levels of MDC1 mRNA and protein. Colony?forming assay was applied to measure radiosensitivity, flow cytometry to determine cell cycle, Western blot to determine the expression of CHK1 and CHK2 proteins, and laser scanning confocal microscope to observe the number of MDC1 blotches inside the nucleus. One?way analysis of variance was used to analyze the differences between groups. Results The pSIH1?H1?copGFP plasmid was constructed successfully and ECA109 cells were infected to obtain ECA109M cells with stable transfection. The expression levels of MDC1 mRNA and protein in ECA109M cells were lower than those in ECA109N and ECA109 cells ( P= 0. 032 and 0. 041, respectively ) . After 5?Gy radiation, ECA109M cells had a lower proportion of G2+M cells than ECA109N and ECA109 cells ( P=0. 026) . After 5?Gy radiation, ECA109, ECA109N, and ECA109M cells had similar expression levels of CHK1 and CHK2 proteins ( P= 0. 345 and 0. 451, respectively ) , and ECA109M cells had a lower expression level of CHK2 T68 protein than ECA109 and ECA109N cells ( P=0. 012) . ECA109 cells had a D0 value of 3. 06 Gy and an SF2 value of 0. 91;the D0 values for ECA109N and ECA109M cells were 2. 90 Gy and 1. 88 Gy, respectively, and the SF2 values for them were 0. 89 and 0. 84, respectively ( P=0. 021 and 0. 037, respectively ) . Conclusions RNA interference can reduce the expression levels of MDC1 protein and cell cycle?related proteins, release cell cycle arrest, and enhance radiosensitivity in esophageal carcinoma ECA109 cells.

Objective To investigate the value of prophylactic irradiation to the lymphatic drainage area in radical three-dimensional conformal radiotherapy (3DCRT) and to evaluate the efficacy and adverse effects of 3DCRT with different clinical target volumes.Methods A retrospective analysis was performed on the records of 219 esophageal cancer patients without distant metastasis who received 3DCRT from January 2005 to December 2010.One hundred and five patients received involved-field irradiation (IFI) with a total dose of 54-66 Gy;114 patients received elective nodal irradiation (ENI) with a total dose of 46-52 Gy; the prescribed dose to the primary lesion was 56-70 Gy.The Kaplan-Meier method was used to calculate local control (LC) and overall survival (OS) rates,and the log-rank test was used for univariate prognostic analysis.Results The 1-,3-,and 5-year sample sizes were 219,172 and 67,respectively.The 1-,3-,and 5-year LC rates for IFI group were 63.0％,39.1％,and 27.2％,respectively,versus 70.5％,53.3％,and 51.7％ for ENI group (x2 =6.22,P =0.013) ;the 1-,3-,and 5-year OS rates for IFI group were 67.6％,24.9％,and 15.0％,respectively,versus 73.7％,45.1％,and 26.0％ for ENI group (x2=5.04,P =0.025).The univariate stratified analysis showed that the LC and OS rates were significantly higher in the ENI group than in the IFI group for patients with middle-or lower-thoracic primary lesion or N0 disease (P=0.007,0.015;P=0.054,0.013).Conclusions For esophageal cancer patients with middle-or lower-thoracic primary lesion or without lymph node metastasis,prophylactic irradiation to the lymphatic drainage area can increase LC and OS rates.

Objective To analyze the outcomes and prognostic factors of esophageal carcinoma treated with three-dimensional eonformal radiotherapy (3DCRT). Methods From January 2001 to August 2007, 209 patients with esophageal carcinoma treated with 3DCRT were retrospectively analyzed. The local control rotes, the survival rates and the related prognostic factors were evaluated with SPSS 11.5 software. Results The follow-up rate was 98. 1% by December 2008. The number of patients followed up for 1,3, 4 and 5 years was 209,131,95 and 56, respectively. The 1-, 3- and 4-year local control rates were 74. 9%, 50. 4% and 45. 8%, respectively. The 1-, 3-and 4-year overall survival rates were 64. 6%, 30. 8% and 23.6%, respectively, with a median survival time of 18 months. Univariate analysis showed that the significant prognostic factors included the degree of dysphagia, tumor site, lesion length in barium esophagogram and CT image, the largest diameter of lesion in CT image, T stage, N stage, clinical TNM stage, short term effect, and degree of acute esophagitis. Multivariate analysis revealed that the degree of dysphagia, primary tumor site, clinical stage, and radiotherapy technique (3DCRT or late half course 3DCRT) were independent prognostic factors. Conclusions Three-dimensional conformai radiotherapy is effective and feasible in the treatment of esophageal cancer. The degree of dysphagia, primary tumor site, and clinical stage are independent prognostic factors for survival of patients treated with 3DCRT.

Objective To investigate the effects of inhibition of MDC1 protein expression on xenografted tumors in nude mice,and to observe the histopathological and cellular changes in nude mice.Methods Three pairs of effective and control short hairpin RNA targeting MDC1 mRNA were designed and cloned into the pSIH1-H1-copGFP vector.Real-time PCR and Western blot were used to determine the mRNA and protein expression of MDC1.After selection by copGFP reporter gene,cells were divided into negative transfection group (ECA109-N) and MDC1 transfection group (ECA109-M).The transfected cells were injected into nude mice.The mice were divided into ECA109 group,ECA109-N group,and ECA109-M group.Each group was divided into irradiation subgroup and non-irradiation subgroup.The changes in tumor size after irradiation were evaluated in each group.Western blot was used to measure the expression of CHK1,CHK2,and CHK2T68 in xenografted tumors.Flow cytometry was used to analyze the cell cycle distribution and apoptosis of tumor cells in nude mice.The variance analysis was used to compare the mean of multiple groups,and the SNK-q test was used in the two two groups.Results The pMDC1-shRNA plasmid was successfully constructed and used to transfect ECA109 cells.ECA109-M cells were obtained by stable transfection with the recombinant plasmid.All inoculated nude mice survived with visible xenografted tumors at the underside of the paw in about one week.There was no swelling and wound in inoculation sites.There was no significant difference in tumor size between different groups (P＞0.05).The tumor growth in the ECA109 group and the ECA109-N group significantly slowed down after irradiation with a dose of 15 Gy (P＜0.05).Compared with the other two groups,the ECA109-M group had a significant smaller tumor size,significantly slower relative tumor growth,and significantly higher growth inhibition (all P＜0.05).The q value of the ECA109-M group was 1.36.In the ECA109-M group,there were no significant changes in the protein expression of CHK1 and CHK2 after irradiation (P＞ 0.05);however,the phosphorylation of CHK2T68 protein was significantly reduced after irradiation (P＜0.05).There were no significant differences in cell cycle distribution or the proportion of apoptotic cells in tumor tissue between the three groups (P＞0.05).Conclusions Inhibition of MDC1 protein expression by RNA interference can effectively inhibit the growth of xenografted tumors after irradiation in the nude mice by increasing their radiosensitivity.

OBJECTIVETo study the effect of Mudan Granule (MD) on the glucose metabolism and beta cell function in monosodium glutamate (MSG) induced obese mice with insulin resistance (IR).

METHODSMSG obese mice were induced by subcutaneous injecting MSG (4 g/kg for 7 successive days in neonatal ICR mice). Forty MSG mice with IR features were recruited and divided into four groups according to body weight, fasting blood glucose, triglyceride (TG), total cholesterol (TC), and the percentage of blood glucose decreased within 40 min in the IR test, i.e., the model group (Con), the low dose MD group, the high dose MD group, and the Metformin group (Met). Besides, another 10 ICR mice were recruited as the normal control group (Nor). The water solvent of 2.5 g/kg MD or 5 g/kg MD was respectively administered to mice in the low dose MD group and the high dose MD group. Metformin hydrochloride was given to mice in the Met group at 0.2 g/kg body weight. Equal dose solvent distilled water was administered to mice in the Nor group and the Con group by gastrogavage, once per day. All medication was lasted for 15 weeks. Insulin tolerance test (ITT) and oral glucose tolerance test (OGTT) were performed after 6 weeks of treatment. Beta cell function was assessed by hyperglycemic clamp technique. The morphological changes in the pancreas were evaluated by hematoxylin-eosin (HE) staining. Changes of iNOS, NF-kappaB p65, and p-NF-kappaB p65 in the pancreas were tested.

RESULTSCompared with the Nor group, the blood glucose level, AUC, and fasting blood insulin, ONOO-contents, iNOS activities, and the expression of iNOS, NF-kappaB p65 subunit, pNF-kappaB p65 subunit obviously increased; decreased percentage of blood glucose within 40 min in ITT, glucose infusion rate (GIR), Clamp 1 min insulin, and Max-Insulin obviously decreased in the Con group (P < 0.05, P < 0.01). Compared with the Con group, the aforesaid indices could be improved in the Met group (P < 0.05, P < 0.01). In the low dose MD group, AUC, iNOS activities, and the expression of iNOS and p-NF-kappaB p65 subunit obviously decreased; percentage of blood glucose within 40 min in ITT and GIR obviously increased (P < 0.05, P < 0.01). In the high dose MD group, AUC, ONOO-contents, iNOS activities, and the expression of iNOS, NF-kappaB p65 subunit, and p-NF-KB p65 subunit obviously decreased; percentage of blood glucose within 40 min in ITT, Max-Insulin, and GIR obviously increased (P < 0.05, P < 0.01).

CONCLUSIONMD could significantly improve IR and functional disorder of 3 cells in MSG obese mice, which might be associated with lowering inflammatory reaction in the pancreas.

Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Female ; Insulin Resistance ; Insulin-Secreting Cells ; drug effects ; metabolism ; Male ; Metformin ; pharmacology ; Mice ; Mice, Inbred ICR ; Mice, Obese ; Obesity ; chemically induced ; metabolism ; Pancreas ; cytology ; drug effects ; Sodium Glutamate

This study is to evaluate the effects of the metformin (Met) on β cell function of diabetic KKAy mice. Female diabetic KKAy mice selected by insulin tolerance test (ITT) were divided randomly into two groups. Con group was orally administered by gavage with water, Met group with metformin hydrochloride at a dose of 0.2 g x kg(-1) for about 12 weeks. ITT and glucose tolerance tests (OGTT) were determined. Beta cell function was assessed by hyperglycemic clamp. Pancreatic biochemical indicators were tested. The changes of gene and protein expression in the pancreas and islets were also analyzed by Real-Time-PCR and immunostaining. Met significantly improved glucose intolerance and insulin resistance in KKAy mice. Fasting plasma glucose and insulin levels were also decreased. In addition, Met markedly increased glucose infusion rate (GIR) and elevated the Ist phase and maximum insulin secretion during clamp. It showed that Met decreased TG content and iNOS activities and increased Ca(2+) -Mg(2+)-ATPase activity in pancreas. Islets periphery was improved, and down-regulation of glucagon and up-regulated insulin protein expressions were found after Met treatment. Pancreatic mRNA expressions of inflammation factors including TLR4, NF-κB, JNK, IL-6 and TNF-α were down-regulated, p-NF-κB p65 protein levels also down-regulated by Met. And mRNA expressions of ion homeostasis involved in insulin secretion including SERCA2 and Kir6.2 were up-regulated by Met. Met increased SIRT5 expression level in pancreas of KKAy mice under the hyperglycemic clamp. These results indicated that chronic administration of Met regulated pancreatic inflammation generation, ion and hormone homeostasis and improved β cell function of diabetic KKAy mice.
Animals ; Blood Glucose ; Diabetes Mellitus, Experimental ; drug therapy ; Down-Regulation ; Female ; Glucose Tolerance Test ; Homeostasis ; Inflammation ; drug therapy ; Insulin ; secretion ; Insulin Resistance ; Insulin-Secreting Cells ; drug effects ; Interleukin-6 ; metabolism ; Metformin ; pharmacology ; Mice ; NF-kappa B ; metabolism ; Pancreas ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism

Objective To explore the value of elective nodal prophylactic irradiation with intensity modulated radiotherapy(IMRT) for esophageal carcinoma.Screening patients who are suitable for elective nodal prophylactic irradiation (ENI),in order to improve locoregional control and overall survival.Methods The concurrent control study was conducted to esophageal cancer patients who were treated by definitive radiotherapy.A total of 148 patients finished treatment were identified.Seventy-four patients received ENI,while the other seventy-four patients received involved-field irradiation (IFI).Kaplan-Meier method was used for calculation of locoregional control rates and overall survival rates.The univariate and multivariate analysis of prognostic factors were also tested.Results The 1-,3-,and 5-year locoregional control rates of ENI group and IFI group were 72.5％,52.8％,50.6％ and 58.4％,35.8％,21.9％ (x2 =7.881,P ＜0.05),respectively.The 1,3,and 5 years survival rates of the ENI group and IFI group were 74.3％,44.2％,24.5％ and 68.9％,27.6％,15.9％ (x2 =1.903,P ＜ 0.05),respectively.In Cox multivariate analysis,clinical T stage,tumor location,different radiotherapy region were independent factors for the locoregional control of all patients,and clinical T,N stage,the length of esophageal barium meal and chemotherapy were independent factors for the overall survival of all patients.Conclusions Esophageal carcinoma patients treated with ENI could achieve better locoregional control than those treated with IFI.Esophageal carcinoma patients with early stage or middle thoracic lesion could benefit from ENI for local control and overall survival.

Objective To compare the efficacy of elective nodal prophylactic irradiation ( ENI) and involved?field irradiation ( IFI) in radical radiotherapy for early?stage esophageal cancer and to determine the appropriate irradiation range for early?stage esophageal cancer. Methods The clinical data of 121 patients with early?stage esophageal cancer receiving radical radiotherapy in our hospital from January 2006 to December 2011 were collected and respectively analyzed. Sixty?one patients received ENI, and the other 60 patients received IFI. The Kaplan?Meier method was used to calculate local control ( LC) and overall survival ( OS) rates;the log?rank test was used for survival difference analysis and univariate prognostic analysis;the Cox regression model was used for multivariate prognostic analysis. Results The 1?, 3?, and 5?year LC rates in ENI group and IFI group were 81. 1%, 60. 1%, and 57. 5% vs. 64. 5%, 43. 9%, and 27. 2%, respectively ( P=0. 003 ) . The 1?, 3?, and 5?year OS rates in ENI group and IFI group were 86. 9%, 56. 8%, and 34. 8% vs. 86. 7%, 34. 3%, and 19. 1%, respectively ( P=0. 019) . The 1?, 3?,and 5?year overall failure rates in ENI group and IFI group were 22. 3%, 53. 8%, and 63. 2% vs. 43. 3%, 65. 8%, and 78. 8%, respectively ( P=0. 023) . Multivariate analysis showed that irradiation range was the influencing factor for LC and OS. Conclusions As for the radical radiotherapy for early?stage esophageal cancer, ENI can significantly increase LC and reduce locoregional failure, and therefore improve long?term OS.

This study aims to determine a reasonable clinical staging standard for patients with esophageal carcinoma who were receiving non-surgical treatment. The patients were staged on the basis of the (2004 and 2009 editions of clinical staging stan-dards. The prognosis of patients with different staging standards, as well as the effect of gross tumor volume-tumor (GTV-T) on clinical T stage and prognosis, was observed. Methods:Data on 219 patients with esophageal carcinoma who were receiving radical radiothera-py were retrospectively analyzed. Prior to radiotherapy, all patients underwent examinations, including esophageal barium meal and po-sitioning CT scan, for use in the radiation treatment planning system to outline the target range and to calculate the volume of GTV-T. All patients were staged with the use of the aforementioned clinical staging standards. Prognostic outcomes of the patients were ob-served. Results:For all patients, the one-, three-, and five-year overall survival rates were 70.8%, 35.6%, and 20.7%, respectively. The survival curve resolution of patients who were staged with the use of the 2009 edition of clinical staging standards was better than that of the patients who were staged with the use of the 2004 edition. Survival difference was significant (χ2=29.497, P<0.001). The clinical T stage positively correlated with GTV-T (r=0.615, P<0.001). GTV-T could thus affect prognosis at different T stages. Conclusion:Both esophageal carcinoma clinical staging standards could reflect the prognosis of patients undergoing radiotherapy, but the 2009 edi-tion appeared more accurate than the 2004 edition.