In this paper, a qualitative and quantitative analysis method was established for the scopoletin in herba Artemisiae hedinii hy HPLC. The chromatographic column was Agilent TC-C18 (4.6 mm×250 mm, 5μm);the tem-perature of column was 30℃, eluted with a mobile phase consisted of methanol and 0.3%phosphoric acid solution and gradient elution at a flow rate of 1.0 mL·min-1. The detection wavelength was 344 nm. The results showed that the mass concentration and peak area of scopoletin had a good linear relationship within the range of measurement (r=0.999 9). The reproducibility was good;and the RSD was 1.51%. The average sample recovery rate was 101.42%. It was concluded that the established method was stable, reliable and simple. It provided theoretical evidences for the quality evaluation and quality control of herba A. hedinii.

OBJECTIVETo observe the effects of ginsenoside Rb1 (GRb1) on the oxidative stress in the skeletal muscles of rats with postoperative fatigue syndrome (POFS) and to study its anti-fatigue mechanisms.

METHODSThe POFS model was established using resection of 70% of mid-small intestine. Ninety-six Sprague-Dawley (SD) rats were screened using grasping test. The rats were randomly divided into the control group, the model group, and the GRb1 treated group (at 10 mg/kg) by the body weight. The maximum grip strength of rats was detected on the 1st, 3rd, 7th, and 10th day after operation, respectively. The contents of malondialdehyde (MDA), the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) were detected.

RESULTSCompared with the model group, the maximum grip strength was obviously enhanced on the postoperative day 7 and 10 (P < 0. 05), the MDA content obviously decreased on the postoperative day 3 and 7 (P < 0.05), the SOD activity obviously increased in the GRb1 treated group (P < 0.05). There was no obvious change in the activities of CAT and GSH-PX among the three groups at each time point (P > 0.05).

CONCLUSIONGRb1 could reduce the oxidative stress injury in the skeletal muscles, improve the activities of antioxidant enzymes, and enhance the functions of the skeletal muscles in POFS rats, which may be important reasons for fighting against POFS.

Animals ; Catalase ; metabolism ; Fatigue ; etiology ; metabolism ; Ginsenosides ; pharmacology ; Glutathione Peroxidase ; metabolism ; Male ; Malondialdehyde ; analysis ; Muscle, Skeletal ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Postoperative Period ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism

BACKGROUNDBoth p16(INK4) and p21(Waf1) are tumor suppressors with similar biological functions in the regulation of cellular senescence. Previous reports showed that p16(INK4) could be activated by p21(Waf1) through transcriptional factor Sp1 in HeLa cells. This study was undertaken to determine the effects of p16(INK4) on the expression and functions of p21(Waf1).

METHODSHuman diploid fibroblast 2BS cells were stably transfected with sense (2BS/p16(INK4)), antisense p16(INK4) (2BS/asp16(INK4)) or empty vector (2BS/neo). Then they were assayed by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence activated cell sorting (FACS) and Western blot.

RESULTS2BS/p16(INK4) cells exhibited cell cycle arrest in both G1 and G2/M phases. Endogenous p21(Waf1) protein levels increased twofold in the 2BS/p16(INK4) cells, but not decreased in the 2BS/asp16(INK4) cells. p21(Waf1) mRNA levels were not affected in neither 2BS/p16(INK4) nor 2BS/asp16(INK4) cells.

CONCLUSIONp16(INK4) may play an important role in the regulation of cellular senescence by modulating the p21(Waf1) protein level via the posttranscriptional mechanism.

Cell Cycle ; Cells, Cultured ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; physiology ; Cyclin-Dependent Kinase Inhibitor p21 ; physiology ; Fibroblasts ; metabolism ; Humans ; Transcription, Genetic

BACKGROUNDThe accumulation of free radicals and advanced glycation end products (AGEs) in cell plays a very important role in replicative senescence. Aminoguanidine (AG) has potential antioxidant effects and decreases AGE levels. This study aimed to investigate its effect on replicative senescence in vitro.

METHODSThe effects of aminoguanidine on morphology, replicative lifespan, cell growth and proliferation, AGEs, DNA damage, DNA repair ability and telomere length were observed in human fetal lung diploid fibroblasts (2BS).

RESULTSAminoguanidine maintained the non-senescent phenotype of 2BS cells even at late population doubling (PD) and increased cumulative population doublings by at least 17 - 21 PDs. Aminoguanidine also improved the potentials of growth and proliferation of 2BS cells as detected by the MTT assay. The AGE levels of late PD cells grown from early PD in DMEM containing aminiguanidine decreased significantly compared with those of late PD control cells and were similar to those of young control cells. In addition, the cells pretreated with aminoguanidine had a significant reduction in DNA strand breaks when they were exposed to 200 micromol/L H(2)O(2) for 5 minutes which indicated that the compound had a strong potential to protect genomic DNA against oxidative stress. And most of the cells exposed to 100 micromol/L H(2)O(2) had much shorter comet tails and smaller tail areas after incubation with aminoguanidine-supplemented DMEM, which indicated that the compound strongly improved the DNA repair abilities of 2BS cells. Moreover, PD55 cells grown from PD28 in 2 mmol/L or 4 mmol/L aminoguanidine retain telomere lengths of 7.94 kb or 8.12 kb, which was 0.83 kb or 1.11 kb longer than that of the control cells.

CONCLUSIONAminoguanidine delays replicative senescence of 2BS cells and the senescence-delaying effect of aminoguanidine appear to be due to its many biological properties including its potential for proliferation improvement, its inhibitory effect of AGE formation, antioxidant effect, improvement of DNA repair ability and the slowdown of telomere shortening.

Cell Proliferation ; drug effects ; Cells, Cultured ; Cellular Senescence ; drug effects ; DNA Damage ; DNA Repair ; Diploidy ; Dose-Response Relationship, Drug ; Female ; Fibroblasts ; drug effects ; Glycation End Products, Advanced ; analysis ; Guanidines ; pharmacology ; Humans ; Hydrogen Peroxide ; toxicity ; Telomere

BACKGROUNDAstragali Radix, the root of Astragalus membranceus (Fish) Bunge Var. mongholicus (Bge), is a crude drug considered as one of the effective traditional Chinese anti-ageing material. The two isomers of 4-hydroxy-5-hydroxymethyl-[1, 3] dioxolan-2, 6'-spirane-5', 6', 7', 8'-tetrahydro-indolizine-3'-carbaldehyde (HDTIC), HDTIC-1 and HDTIC-2, were first extracted from the herb in 2002. We demonstrated previously that 0.1 micromol/L HDTIC-1 or 1.0 micromol/L HDTIC-2 strongly delay replicative senescence of human fetal lung diploid fibroblasts (2BS). In this study, we chose them to investigate their effects on the expression of senescence-associated genes to explore the mechanism of how HDTIC delays replicative senescence.

METHODSThe effects of HDTIC-1 and HDTIC-2 on the expression of p16 and p21 were observed in vitro by RT-PCR and Western blot. The anti-oxidative activities of the compounds were also observed by phenotype alteration after treatment with antioxidants.

RESULTSThere was an obvious expression of p16 in the control senescent cells. However, in the 2BS cells, after 56 population doublings (PDs) grown from PD28 in 0.1 micromol/L HDTIC-1 or 1.0 micromol/L HDTIC-2, there was a weak mRNA expression of p16 and no protein expression of p16 was observed. The expression level of p21 increased with cell ageing. Moreover, there was no difference between the expression level of p21 in the control cells and that in the same PD cells cultured with HDTIC compounds. The results also showed that 2BS cells exposed to 100 micromol/L H2O2 for 5 minutes return to their non-senescent phenotype and continue to be confluent after incubating the damaged cells with HDTIC-1 (1.0 micromol/L ) or HDTIC-2 (10 micromol/L ) for 1 hour.

CONCLUSIONSExpression of p16 by 2BS cells was strongly inhibited by HDTIC compounds, which could contribute to their delayed replicative senescence by the way of p16(INK4a)/Rb/MAPK. The anti-oxidative activities of HDTIC-1 and HDTIC-2, described in this study for the first time, might be indirectly related to their inhibition of p16 expression.

Antioxidants ; pharmacology ; Astragalus Plant ; chemistry ; Cells, Cultured ; Cellular Senescence ; drug effects ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; analysis ; genetics ; Dioxolanes ; pharmacology ; Female ; Fibroblasts ; chemistry ; drug effects ; metabolism ; Humans ; Indolizines ; pharmacology ; Plant Roots ; chemistry ; RNA, Messenger ; analysis

BACKGROUNDPromoter analysis is currently applied to detect the expression of the targeted gene in studies of signal transduction and transcriptional regulation. As a reporter gene, luciferase plays an important role and has been used widely in the promoter assay.

METHODSHuman embryonic lung fibroblast cells (2BS), HeLa cells and MCF-7 cells were transfected with various genes embedded by lipofectamine. This study determined various factors that affect promoter activity determination, such as the selection of the reporter genes and internal references, the dose and the type of the vectors carrying the transcription factors, the host cells and the instruments.

RESULTSThe sensitivity of the luciferase assay was much higher than that of enhanced green fluorescence protein (EGFP). Moreover, promoter activity is increased in a dose-related manner only in certain ranges outside of which the results may be reversed and the promoter activity is related to the expression vector which is carrying the cDNA. Otherwise, the length of the promoter, internal references and the host cell can also influence the promoter activity.

CONCLUSIONSTo detect the promoter activity accurately, a few factors including dose, vector, length and host cell which influence reporter gene assay aforementioned should be considered.

Cells, Cultured ; Genes, Reporter ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Luciferases ; genetics ; Plasmids ; Promoter Regions, Genetic ; Transfection

Objective To investigate the clinical characteristics of primary gastrointestinal lymphoma (PGIL) on different origin site in order to improve its diagnosis.Methods The clinical data from 202 patients with PGIL diagnosed by histology from January 1999 to June 2007 were identified from the clinical databases of 8 hospitals in Wuhan area and retrospectively analyzed.The patients were divided into gastric,small intestinal and large intestinal lymphoma groups according to the site of origin and there clinical characteristics were compared.Results The PGIL localization was gastric in 113 (56.0%) cases, small intestine in 37(18.3%) cases and large intestine in 52 (25.7%) cases.One hundred and thirty (64.4%) were males and 72 (35.6%) were females.The male patients were predominant.The median duration of symptoms in gastric lymphoma group was longer than small intestinal lymphoma group (3.0 months vs.1.0 month,P=0.013).The most common symptoms were abdominal pain and anemia. The clinical stage was Ⅰ E and Ⅱ E in 71.3% of cases.The large intestinal lymphoma group presented more advanced-stage disease compared with gastric lymphoma group (P = 0.014).The frequent histological type was mucosa-associated lymphoid tissue lymphoma (MALT),diffuse large B-cell lymphoma and T-cell lymphoma.Gastric,small intestinal and large intestinal lymphomas presented more frequently as low-grade MALT lymphoma (56.9%),T-cell lymphoma (34.4%) and high-grade B-cell lymphoma (51.1%),respectively (all P value ＜0.05).The common macroscopic type of PGIL were nodular protruding and ulcerative type.Compared with gastric lymphoma,nodular protruding type was more common and ulcerative type was less common in large intestinal lymphoma (P = 0.000).The diagnosis confirmed by endoscopic biopsy were 58.7% (61/104),25.0% (4/16),48.2% (13/27) in gastric,small intestinal and large intestinal lymphoma groups,respectively.Conclusions The clinical characteristics are different in patients with different localization of PGIL including patient characters, initial symptoms,histological classification,clinical stage,macroscopic feature,endoscopic findings. Analysis of these clinical characteristics is helpful to improve its diagnosis.

OBJECTIVETo study the etiology, diagnosis and treatment of transverse testicular ectopia (TTE).

METHODSA case of TTE was treated with orchidopexy.

RESULTSSix months after the operation, both of the two testes were in proper positions with normal vascular supply.

CONCLUSIONTTE is a rare congenital abnormality of the male reproductive system with unknown etiology, and surgical correction remains the best option for treatment.

Child ; Humans ; Male ; Testicular Diseases ; diagnosis ; therapy ; Testis ; abnormalities

OBJECTIVETo explore the mechanism of postoperative fatigue syndrome(POFS) by detecting the change of central monoamine neurotransmitters in a rat model after major abdominal surgery.

METHODEighty-four rats were randomly divided into fatigue assessment groups (including model group and sham group) and experimental groups (including postoperative day 1, 3, 5, 7, and 14 recovery groups and the corresponding control groups). Postoperative fatigue was evaluated after surgery. The brains were removed thereafter to detect the levels of 5-hydroxytryptamine (5-HT), norepinephrine (NE), dopamine (DA) in the hippocampus, midbrain, hypothalamus by high performance liquid chromatography. Serum free tryptophan (f-Trp) and branched-chain amino acids (BCAA) were measured.

RESULTSThe level of 5-HT increased to the highest at postoperative day 3, but reduced rapidly to the minimum at postoperative day 5, and then gradually recovered to the preoperative level. There was significant difference of 5-HT among experimental groups (P<0.05), also between the postoperative 24 hrs group and control groups (P<0.05). f-Trp and the ratio of f-Trp/BCAA increased in the early postoperative period, reduced to minimum at postoperative day 5. f-Trp was still lower compared to the control group at postoperative day 14, while the ratio of f-Trp/BCAA and BCAA restored to control level. Both of them were significantly different among experimental groups (P<0.05), also between the experimental groups and control groups (P<0.05).

CONCLUSIONChanges of f-Trp, the ratio of f-Trp/BCAA, and central 5-HT may play an important role in the development of POFS.

Abdomen ; surgery ; Amino Acids ; analysis ; Animals ; Fatigue ; blood ; Postoperative Period ; Rats ; Serotonin ; analysis

Objective To study the contents of puerarin in decoction with different compatibility ratios of Astragali Radix and Puerariae Lobatae Radix; To verify the rationality of compatibility ratios of Huangqi Gegen Decoction and Yu y e Decoction; To provide references for clinical medication. Methods Different compatibility ratios of Astragali Radix and Puerariae Lobatae Radix were set as 0:1, 1:1, 2:1, 3:1, 4:1, 1:2, 1:3, and 1:4. SinoChrom ODS-BP (4.6 mm × 250 mm, 5 μm) was used as the chromatographic column to detect the contents of puerarin;methanol-0.1% phosphoric acid (35:65) was used as the mobile phase with 0.5 mL/min flow rate; sample volume was 20 μL; the wavelength was 250 nm. Results The regression equation of puerarin was Y=66 449.269 1X-175 665.663 1 (r=0.999 6) in the range of 5 to 300 μg/mL, showing a good linear relationship. The repeatability, stability and recovery rate were fine as well. The contents of puerarin were (2.506 7±0.025 8)%, (2.526 7±0.071 2)%, (2.863 3± 0.086 4)%, (2.956 7±0.119 6)%, (2.835 0±0.078 7)%, (2.480 0±0.072 4)%, (2.530 0±0.064 8)%, and (2.183 3±0.128 9)% in different compatibility ratios of Astragali Radix and Puerariae Lobatae Radix with 0:1, 1:1, 2:1, 3:1, 4:1, 1:2, 1:3, and 1:4, respectively. Conclusion When the compatibility ratios of Astragali Radix and Puerariae Lobatae Radix are 2:1. 3:1, and 4:1, the contents of puerarin in decoction are the highest. This study verifies that the compatibility ratios of Astragali Radix and Puerariae Lobatae Radix in classical prescriptions are rational, which can be the optimal compatibility ratios in clinic.