AbstractBACKGROUND: Fas and P53 are important regulator and control gene which can promote apoptosis. They belong to the receptor family part of tumor necrotic factor/nerve growth factor. Their expression products have effects on apoptosis signal transmission, and can regulate and control cell apoptosis in cerebral ischemia-reperfusion injury. And puerarin can alleviate the level of cell apoptosis.OBJECTIVE: To observe the effect of puerarin on Fas and P53, the apoptosis-related gene of nerve cell in hippocampns CA1 region of rats af ter cerebral resuscitation.DESIGN: Randomized controlled trial. SETTING: Department of Emergency, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: The experiment was carried out at Emergency Department, Tongji Hospital, Tongji Medical College, Huazhong University ofScience and Technology from September 2001 to Februray 2002. Totally 45 of 3 months old Wistar rats of clean grade were selected, and randomly divided into 3 groups: sham operation group, model control group and puerarin treatment group with 15 rats in each group.METHODS: Acute global brain ischemia-reperfusion models were established in rats of puerarin treatment group and model control group. In rats of sham operation group, stigmata of both flanks of the first cervical vertebrae were isolated, but bilateral vertebral arteries were not electric coagulated, and biolateral common carotid arteries were only isolatedwithout clamping close. Rats in puerarin treatment group were given puerarin injection 100 mg/kg, 1 hour before ischemia, and model control group were given normal saline in equivalence while rats in sham operation group were not given medicine. Death of rats in each group was performed separately in the 3rd, 6th, 12th, 24th and 48th hours after cerebral ischemia-reperfusion with 3 rats per group in each time. Hippocampus tissues of rats were isolated, and tissue slices were preparated. And the changes of- the protein expression levels and the number of apoptosis cells of rats in each group at different time point after cerebral ischemia-reperfusion were detected in immuno-histochemical method and end labelling in situ method. MAIN OUTCOME MEASURES: ① The number of positive cells inprotein expression of Fas and P53 in hippocampus CA1 region of rats in each group at different time point after cerebral ischemia-reperfusion was studied. ② Comparison of the number of apoptosis cells in hippocampus CA1 region of rats between groups at different time point after cerebral ischemia-reperfusion were studied, too.RESULTS: All the 45 rats enrolled in research were entered the stage of result analysis: ① The number of positive cells in protein expression of Fas in hippocampus CA1 region of rats in each group at different time point after cerebral ischemia-reperfusion: Obvious gene expression of Fas was not found in sham operation group. In contrast with model control group, obvious decrease was found at all time points after cerebral ischemi a-reperfusion in puerarin treatment group, and in the 6th, 12th, 24th and 48th hour the differences were significant [(15.0±4.3), (13.5±4.9); (40.7±3.4), (27.2±3.1); (37.0±4.8), (22.0±2.1); (24.7±4.1), (18.9±5.3)/mm; P < 0.05,P < 0.01]. ② The number of positive cells in protein expression of P53 in hippocanpus CA1 region of rats in each group at different time point after cerebral ischemia-reperfusion: Obvious gene expression of P53 was not found in sham operation group. In contrast with model control group, obvious decrease was found in the 24th and 48th hour after cerebral ischemiareperfusion in puerarin treatment group [(25.3±4.4), (12.8±2.7); (24.3±3.6), (10.9±3.0)/mm; P < 0.01]. ③ Comparison of the number of apoptosis cells in hippocampus CA1 region of rats between groups at different time point after cerebral ischemia-reperfusion :In contrast with model control group,obvious decrease was found in the 12th, 24th and 48th hour after cerebral is chemia-reperfusion in puerarin treatment group [(34.0±3.7), (21.0±3.7); (41.0±4.2), (33.0±4.8); (71.0±5.5), (41.0±3.4)/mm; P < 0.01].CONCLUSION: In rats which were given puerarin treatment, the expression of Fas decrease obviously in 6 to 48 hours after cerebral ischemiareperfusion, and the expression of P53 decreased obviously in the 24th to 48th hour after cerebral ischemia-reperfusion, and a descent tendency could be found in the number of apoptosis cells. These can further prove the cerebral protective effect of puerarin, and indicate that the inhibition of puerarin to cell apotosis after cerebral resuscitation is related to its effect on the decrease in protein expression of apoptosis-promoting gene, Fas and P53.Puerarin has a protective effect on cerebral ischemia-reperfusion injury of rats. In comparison with model control group, the expression of Fas in puerarin treatment group has an obvious decrease inthe 6th to 48th hour after cerebral ischemia-reperfusion, the expression of P53 has an obvious decrease in the 24th to 48th hour after cerebral ischemia-reperfusion, and the number of apoptosis cells decrease obviously, too, which further improves the cerebral protective effect of puerarin and indicates that the inhibition of puerarin to cell apoptosis after cerebral resuscitation is related to its effect on the decrease in protein expression of apoptosis-promoting gene Fas and P53.

Objective To investigate the genotype distribution of CTX-M-type extended-spectrum β-1actamase(ESBLs) by denaturing high-performance liquid chromatography(DHPLC) in Hunan Province and the accuracy of DHPLC assay. Methods The blaCTX-M genes of standard strains and clinical ESBLs-producing Enterobacteriaceae were amplified by multiplex PCR followed by DHPLC and genotype determination. 25 isolates randomly selected were sequenced to assess the accuracy of DHPLC method. Results Among 142 ESBLs-producing isolates, 109 isolates carried blaCTX-M gene (76. 8% ). Four different CTX-M genotypes were detected by DHPLC, including CTX-M-3 (33 isolates), CTX-M-15 (19 isolates), CTX-M-14 (52 isolates) and CTX-M-9 (5 isolates). The DHPLC typing of 25 isolates suggested that 24 isolates were verified uniformly by the sequencing, but one CTX-M-15 isolate typed by DHPLC was shown to be CTX-M-82 by sequencing. Conclusion DHPLC is a powerful tool for genotyping of the resistance gene and is worth being applied in the clinical and scientific research with accurate, rapid and economic advantages.