Objective To explore the expression and significance of Rac1 and WAVE2 protein in glomerulus of high-
fat diet induced C57BL/6J model mice. Methods Thirty-two male C57BL/6J mice (3-week old) were randomly assigned into two groups(16 in each group). The control group was fed with basic diet (10%fat) for 4 weeks. The high-fat diet group was fed with high-fat diet (60%fat) for 4 weeks. The kidney morphological changes were examined by HE and PAS staining. The expressions of Rac1 and WAVE2 protein were examined by Western blot and immunohistochemistry analysis. Results HE and PAS results showed that there were glomeruli mesangial matrix hyperplasia and exudation in high-fat diet group compared with control group. The immunohistochemistry and Western blotting results showed that expressions of Rac 1 and WAVE2 in glomerulus were both increased in high-fat diet group compared with those of control group. Conclusion Rac1 and WAVE2 protein may be involved in glomerular injuries induced by high-fat diet.

ObjectiveTo evaluate the reliability and clinical value for detecting paraquat (PQ)concentration in serum by spectrometry. MethodsThe determinations of wave length for detecting serum PQ concentration by ordinary spectrometry and second-derivative spectrometry were carried out. When the second-derivative spectrometry was used for detecting PQ in serum, the linear range and precision for PQ concentration were well defined. The results of serum PQ concentration determined by second-derivative spectrometry and by HPLC (high performance liquid chromatography) were compared in 8 patient with PQ poisoning. A total of 21 patients with acute poisoning after PQ ingestion over 4 hours admitted from October 2008 through September 2010 were retrospectively studied. Patients were divided into two groups as per the serum concentrations more than 1.8 μg/mL or less than that by second-derivative spectrometry on the day of admission. The severity of clinical manifestations between two groups was analyzed with t-test or Fisher's exact probabilities analysis. Results ( 1 ) The absorption peak of 257 nm could not be found by using ordinary spectrometry to detect the PQ concentration in serum. (2) The calibration curve in the 0. 4 ～ 8.0μg/mL range for detecting PQ concentration by second-derivative spectrometry observed the Beer's law (r =0. 996) . The average retrieval rate of PQ was within the range of 95.0％～ 99. 5％ with relative standard deviation (RSD) within 1.35％～ 5.41％ ( n = 6), and the lowest detection limit was 0. 05μg/mL. (3) The results of PQ concentrations from 8 patients with PQ poisoning detected by second-derivative spectrometry were consistent with those of the quantitative determinations by HPLC ( r = 0. 995,P＜0. 01 ) . (4) The survival rate of patients with serum PQ concentration more than 1.8 μg/mL was 22. 2％ ,and the incidences of acidosis, oligouria and pneumomediastium in these patients were 55.6％,55. 6％ and 77.8％, respectively. These clinical manifestations were significantly different from those in patients with serum PQ concentration less than 1.8 μg/mL ( P ＜ 0. 05 ) . Conclusions ( 1 ) It was inappropriate to take 257 nm as the determination wave length for detecting serum PQ concentration by ordinary spectrometry. (2) The method of second-derivative spectrometry was reliable for detecting serum PQ concentration. (3) Serum PQ concentration detected by second derivative spectrometry could be used to predict the severity of clinical manifestations of patients with PQ poisoning and was an important predictive factor for poor prognosis if the serum PQ concentration more than 1.8 μg/mL after PQ ingestion over 4 hours.

Objective:A novel tuberculosis DNA vaccine integrating siRNA,fusional antigen and hIL-12 triple expression units was constructed in our laboratory.Methods:To evaluate the independent expression of the three expression units,two eukaryotic expression plasmid pVAX1-siRNA-PVAE[EGFP]-hIL12 with TB fusional gene Ag85B-ESAT6(PVAE) and enhanced green fluorescent protein(EGFP),and pVAX1-siEGFP-PVAE[EGFP]-hIL12 with siRNA coding sequence targeting EGFP instead of Mcl-1L were constructed.Then two plasmids were used to transfer human embryonic kidney 293 cells.Based on EGFP report gene,it was demonstrated that siRNA expression unit and fusional antigen gene were independently expressed.Results:The hIL12 expression at 48h and 72h post transfection were also detected by ELISA analysis up to 1571.63pg/ml and 2392.25pg/ml respectively in the cell culture fluid.Conclusion:The results demonstrated that the novel DNA vaccine with siRNA,TB fusional antigen and hIL12 three expression units in the same plasmid frame is successfully constructed and independently expressed in eukaryotic cells.It laid a foundation for further animal model study on anti-tuberculosis effects of this novel DNA vaccine.

Objective To study the changes and significance of Bid gene expression in hypoxic-ischemic brain damage (HIBD) in neonatal rats.Methods The animal models of HIBD were made. Total RNA from ipsilateral cerebral hemisph ere was extracted.RT-PCR was used to evaluate the levels of Bid gene expression at different time points after hypoxic-ischemic. Results The levels of Bid mRNA were higher in HIBD rats than those in normal rats.The ex pression of Bid mRNA in ipsilateral hemiphere following cerebral hypoxic-ischem ic began to increase at 6 h, increased to the highest level at 24 h, and then gr adually returned.Conclusion The upregulation of Bid mRNA can play a role in the induction of apoptosis fol lowing cerebral hypoxic-ischemic. J Appl Clin Pediatr,2005,20(2):121-122

OBJECTIVE:To establish a method for simultaneous determination of paeoniflorin,berberine hydrochloride and ammonium glycyrrhizinate in Xiaoer huadu powder.METHODS:HPLC method was adopted.The separation was performed on Waters SunFireTM-C18 column with mobile phase consisted of acetonitrile-0.1％ phosphoric acid (gradient elution) at the flow rate of 1.0 mL/min.The detection wavelength was set at 238 nm,and column temperature was 25 ℃.The sample size was 10 μL.RESULTS:The linear ranges of paeoniflorin,berberine hydrochloride and ammonium glycyrrhizinate were 8.808-88.08 μg/mL(r=0.999 8),1.778-17.78 μg/mL(r=0.999 6),2.533-25.33 μg/mL(r=0.999 9),respectively.LOQ were 4.404,0.889,2.533 μg/mL;LOD were 1.101,0.445,1.267 μg/mL.RSDs of precision,stability and reproducibility tests were all lower than 2％.The recoveries were 95.08％-99.61％ (RSD=1.77％,n =9),96.93％-99.94％ (RSD=0.92％,n=9),98.33％-102.05％ (RSD=1.27％,n=9).CONCLUSIONS:The method is simple,accurate and reproducible,and can be used for simultaneous determination of paeoniflorin,berberine hydrochloride and ammonium glycyrrhizinate in Xiaoer huadu powder.

Objective To investigate the bid gene expression and cell death in brain after cerebral hypoxia-ischemia in neonatal rats and the effects of dexamethasone(DEX)on bid gene expression,so as to elucidate the possible mechanism of the neuroprotective effect of DEX pretreatment on rats following cerebral hypoxia-ischemia.Methods Twenty-four SD neonatal rats were divided randomly into hypoxia-ischemia brain damage(HIBD),normal,dexamethasone-pretreated and 9 g/L NaCl(NS)control group.The animal models of HIBD were made.Total RNA from ipsilateral cerebral hemisphere was extracted.Reverse transcription polymerase chain reaction(RT-PCR)was used to evaluate the level of bid gene expression after hypoxia-ischemia.Cerebral apoptosis was determined by terminal-deoxynucleotidy transferase mediated d-UTP nick end labeling(TUNEL).Results The levels of bid mRNA were higher in HIBD rats than those in normal rats.The number of positive apoptosis cells significantly increased in HIBD group(P

Objective To explore the cellular inhibitor of apoptosis protein 1(cIAP1)gene expression and Caspase-3 activity in brain after cerebral hypoxia-ischemia in neonatal rats and the influence of dexamethasone(DEX)on cIAP1 gene expression and Caspase-3 activity,so as to elucidate the possible mechanism of the neuro-protective effect of DEX pretreatment on rats following cerebral hypoxia-ischemia.Methods Twenty-four SD neonatal rats were divided randomly into hypoxic-ischemic brain damage group(HIBD group),normal group(NS group),dexamethasone-pretreated group(DEX group)and 9 g/L NaCl control group(NS group).The animal models of HIBD were made.Total RNA from ipsilateral cerebral hemisphere was extracted.Reverse transcription polymerase chain reaction(RT-PCR)was used to evaluate the level of cIAP1 gene expression after hypoxia-ischemia.Caspase-3 relative activity of brain tissue was determined by colorimetric assay.Results The levels of cIAP1 mRNA were lower in HIBD group than those in NS group.Caspase-3 relative activity significantly increased in HIBD group(P

Background Inherited retinal degeneration,one of the major causes of blindness worldwide,comprises a large number of disorders characterized by a slow and progressive retinal degeneration.Interleukin-1 (IL-1)was recognized to be involved in inherited retinal degeneration.Whether IL-1 receptor antagonist (IL-1ra) can arrest retinal degeneration is worthy of investigation.Objective This study was to investigate the effects of IL-1ra on photoreceptor apoptosis in Royal College of Surgeons (RCS) rats.Methods The use of the animals complied with the Regulations for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission.The SPF RCS rats aged 9,15,16,25,30,35,40,50 and 60 postnatal days were collected,with 9 rats for each age group.Retinal sections were used for the TdT-mediated biotin-dUTP nick-end labeling (TUNEL) cell apoptosis assay.1 μl of IL-1ra (1.8 g/L) was intravitreally injected in the right eyes of 9 RCS rats aged 15 postnatal days and PBS was used in the same way in the fellow eyes.The injection procedure was repeated on the 20 th and 25 th day,respectively.The rats were sacrificed on the 30 th day and retinal sections were prepared for the TUNEL assay.The differences in the percentage of the positive cellular nuclei area among different ages of rats were compared by one-way ANOVA,and the differences in retinal layer thickness between IL-1ra injection group and PBS injection group were assessed by paired t test.Results Photoreceptor apoptosis appeared in 20-day-old RCS rats and progressed and peaked in 30 and 35-day-old rats and then reduced,showing a significant difference among rat of various age groups (F=28.020,P＜0.01).Images from TUNEL assay showed a weaker and less TUNEL staining in the IL-1ra injected eyes than the PBS injected eyes in 30-day-old rats.The total area and relative area of TUNEL positive nuclei were (223.052±153.092) μm2 and (2.206±1.531) ％ in the IL-1ra injected group,and those in PBS injected group were (1235.050±359.767) μm2 and (7.269± 1.624) ％,with a significant difference between them (t =4.370,t=3.250,P＜0.01).The cone and rod thickness was (15.324±9.035) μm in the IL-1ra injected group and (49.566±4.605)μm in the PBS injected group,showing a significant difference (t =22.674,P＜0.01).However,no significant difference was seen in the outer nuclear layer thickness between the two groups (t =0.268,P＞0.05).Conclusions IL-1 participates in the pathogenesis and development of inherited retinal degeneration of RCS rats.The use of IL-1ra might be a potential approach in the treatment of inherited retinal degeneration.

Objective To study the gene expression profile in mice kidney with acute paraquat (PQ) poisoning and identify key genes related to renal injury.Methods A total of 20 mice (C57BL/6) were randomly (random number) divided into four groups, namely control group (group A, n =5) and poisoned groups (groups B, C, D, n =5/group).In group A, mice were administrated with distilled water (0.01 mL/g weight) while in groups B, C, D were administered with equivalent volume of PQ solution (diluted from 20％ to 0.05％ with distilled water) dissolved in distilled water via a gastric tube.Mice of group A were sacrificed immediately and mice of groups B and C at 6 h and 24 h after administration of PQ.The gene expression profile changes of kidney tissue were measured by cDNA Arraychip technology.Mice of group D were observed for mortality rate 48 h later.Results The body weights of mice decreased significantly after administration of PQ.The mortality in group D at 48 h after PQ poisoning was 100％.Compared with the control group, totally 1 792 genes with differential expression variations were identified in 6 h group and 24 h group.There were 8 key genes selected through bioinformatics analysis and they were arranged in real-time PCR: Nlrc5 , Serpinb9 , Cd40 , Rnf135 , Dhx58 , Spl 10 , Fcgrl , and Arhgef12.And then, Nlrc5 , Serpinb9 and Rnf135 were under Western blot investigation.The results of PCR and Western blot showed no significant difference to those from bioinformatics genetic analysis.Conclusions The investigation based on genome wide chip in researching related genes of PQ kidney has offered a novel idea in studying pathogenesis of acute PQ intoxication.

BACKGROUND: Paraquat (PQ) is a world-wide used herbicide and also a type of common poison for suicide and accidental poisoning. Numerous studies have proved that the concentration of serum PQ plays an important role in prognosis. Spectrophotometry, including common spectrophotometry and second-derivative spectrophotometry, is commonly used for PQ detection in primary hospitals. So far, lack of systematic research on the reliability of the method and the correlation between clinical features of patients with PQ poisoning and the test results has restricted the clinical use of spectrophotometry. This study aimed to evaluate the reliability and value of spectrophotometry in detecting the concentration of serum PQ. METHODS: The wavelengths for detecting the concentration of serum PQ by common and second-derivative spectrophotometry were determined. Second-derivative spectrophotometry was applied to detect the concentration of serum PQ. The linear range and precision for detection of PQ concentration by this method were confirmed. The concentration of serum PQ shown by second-derivative spectrophotometry and HPLC were compared in 8 patients with PQ poisoning. Altogether 21 patients with acute poisoning 4 hours after PQ ingestion treated in the period of October 2008 to September 2010 were retrospectively reviewed. The patients were divided into higher and lower than 1.8 μg/mL groups based on their concentrations of serum PQ measured by second-derivative spectrophotometry on admission. The severity of clinical manifestations between the two groups were analyzed with Student's t test or Fisher's exact test. RESULTS: The absorption peak of 257 nm could not be found when common spectrophotometry was used to detect the PQ concentration in serum. The calibration curve in the 0.4–8.0 μg/mL range for PQ concentration shown by second-derivative spectrophotometry obeyed Beer's law with r=0.996. The average recovery rates of PQ were within a range of 95.0％ to 99.5％, relative standard deviation (RSD) was within 1.35％ to 5.41％ (n=6), and the lower detection limit was 0.05 μg/mL. The PQ concentrations in serum of 8 patients with PQ poisoning shown by second-derivative spectrophotometry were consistent with the quantitative determinations by HPLC (r=0.995, P<0.0001). The survival rate was 22.2％ in patients whose PQ concentration in serum was more than 1.8 μg/mL, and the incidences of acidosis, oliguria and pneumomediastinum in these patients were 55.6％, 55.6％and 77.8％, respectively. These clinical manifestations were different significantly from those of the patients whose PQ concentration in serum was less than 1.8 μg/mL (P<0.05). CONCLUSIONS: For common spectrophotometry, the wavelength at 257 nm was not suitable for detecting serum PQ as no absorbance was shown. Second-derivative spectrophotometry was reliable for detecting serum paraquat concentration. Serum PQ concentration detected by second-derivative spectrophotometry could be used to predict the severity of clinical manifestations of patients with PQ poisoning, and PQ content higher than 1.8 μg/mL 4 hours after ingestion could be an important predictive factor for poor prognosis.