Objective: To analyze the chemical constituents of Polygonum multiflorum extract which may cause human liver cell damage and to explore the mechanism. Methods: Raw and processed Polygonum multiflorum were extracted by 70% ethanol, then raw and processed Polygonum multiflorum water-eluted material (RW and PW), 50% ethanol-eluted material (R50 and P50) and 95% ethanol-eluted material (R95 and P95) were obtained by absorbing through AB-8 macroporous resin, followed by water, 50% ethanol and 95% ethanol elution in order. The water extracts of raw and processed Polygonum multiflorum (RWE or PWE) were obtained by boiling them in water as usual. Normal human liver L02 cells were treated by different concentrations of eluted Polygonum multiflorum materials for different time, and the cell growth inhibition of each group was determined by methylthiazolyldiphenyl-tetrazolium bromide method. The chemical constituents which had a significant cytotoxicity to L02 cells were analyzed by high-performance liquid chromatography (HPLC). Morphological changes of L02 cells were observed by Giemsa staining and cell cycle distribution was observed by flow cytometry. Results: It was found that 95% ethanol-eluted extracts of raw and processed Polygonum multiflorum showed significant growth inhibition on normal human liver L02 cells, while the other components showed no significant inhibition on cell growth. HPLC analysis showed that the main component in 95% ethanol-eluted extract of raw and processed Polygonum multiflorum was emodin at content of (18.53+/-2.96)% and (10.28+/-1.34)% respectively. Cell cycle analysis showed that 95% ethanol-eluted material of Polygonum multiflorum and emodin had a similar significant effect of S phase arrest and all could induce L02 cell apoptosis. Conclusion: The main part of Polygonum multiflorum causing liver cell damage is the 95% ethanol-eluted extract, and emodin is one of the important chemical constituents leading to liver cell damage.

ObjectiveTo investigate the clinical efficacy of points Shenmai(BL62)and Zhaohai(KI6)in treating cervical vertigo. MethodSixty-eight patients meeting the diagnostic criteria were randomly allocated to treatment and controlgroups, 34 cases each. The treatment group received conventional acupuncture at heel vessel points Shenmai and Zhaohai plus cervical Huatuo jiaji points and the control group, conventional acupuncture at cervical Huatuo jiaji points alone. In the two groups, the vertigo symptom score was recorded, and the clinical therapeutic effects and pre-/post-treatment change in the score were observed.ResultThe total efficacy rate was 94.1% in the treatment group and 79.4% in the control group. The therapeutic effect was significantly better in the treatment group than in the control group (P<0.05). The vertigo symptom score decreased significantly in the two groups after the course of treatment compared with before (P<0.01). Vertigo improved significantly more inthe treatment group than in the control group (P<0.05).ConclusionAcupuncture at points Shenmai and Zhaohai is significantly effective by comparison with conventional acupuncture in clinicaltreatment of cervical vertigo.

Objective:To observed the effects of electroacupuncture frequency on the nerve function and postsynaptic destination-95 (PSD-95), growth associated protein-43 (GAP-43) in brain tissue of rats with cerebral ischemia were observed.Methods:Healthy adult male SD rats were randomly divided into the blank group, sham operation group, model group and 2 Hz, 50 Hz, 100 Hz electroacupuncture groups. MCAO models were prepared in each group, except blank group and sham operation group. After modeling, Qiansanli and Waiguan acupoints were given stimulation with 2 Hz, 50 Hz and 100 Hz respectively in 2 Hz electroacupuncture group, 50 Hz electroacupuncture group and 100 Hz electroacupuncture group, 1 time/d and 20 min/time for 21 consecutive days. All groups were scored by Bederson score on the 1st, 3rd, 7th, 14th and 21th day after operation. The ultrastructure of synapses was observed by transmission electron microscope, and the positive expression of PSD-95 and GAP-43 was detected by immunohistochemistry. Results:After modeling 7, 14 and 21 days, compared with the model group, the Bederson behavioral score (1.70 ± 0.52 vs. 2.40 ± 0.56, 1.20 ± 0.65 vs. 2.30 ± 0.46, 0.70 ± 0.72 vs. 2.00 ± 0.86) in 50 Hz electroacupuncture group significantly decreased ( P<0.01 or P<0.05). The expression of PSD-95 (58.67 ± 1.72, 55.22 ± 2.45, 60.10 ± 2.49 vs. 70.87 ± 2.34) intergral optical density of rats’ cortex in 2 Hz, 50 Hz, 100 Hz electroacupuncture groups significantly decreased ( P<0.01), the expression of GAP-43 in 2 Hz and 50 Hz electroacupuncture groups (58.89 ± 1.28, 64.76 ± 3.94 vs. 53.24 ± 2.58) significantly increased ( P<0.01). Conclusions:Electroacupuncture with different frequency can improve motor nerve disfunction of cerebral ischemia rats, and 50 Hz electroacupuncture group showed the most obvious effect. It can regulate PSD-95 and GAP-43 expressions of ischemic area, lower neural excitatory toxicity, increase neuronal axon regeneration and exert the neural protect function.

Objective To investigate the changes in the expression of mitochondrial transcription factor A (mtTFA) during one-lung ventilation (OLV)-induced lung injury in rabbits.Methods Sixty healthy male New Zealand white rabbits,weighing 2.5-3.0 kg,were randomized into 2 groups (n =8 each):two-lung ventilation (TLV) group and OLV group.The animals were anesthetized with iv 3％ pentobarbital sodium 30 mg/kg and tracheostomized.A self-made double lumen catheter was then intubated.Bilateral lungs were ventilated for 3 h in group TLN.In group OLV the left lung was ventilated for 2 h followed by 1 h TLV.Arterial blood samples were taken for blood gas analysis immediately after the beginning of ventilation,at 1 and 2 h of ventilation,and immediately after the end of ventilation.The oxygenation index was calculated.The animals were sacrificed after the end of ventilation and the apex of the left lung was removed and then cut and stained with HE for microscopic examination.The pathological changes of the lung were scored.The expression of mtTFA in lung tissues was measured by Western blot.Results Oxygenation index was significantly decreased,lung injury score was increased,the expression of mtTFA was down-regulated in group OLV compared with group TLV (P ＜ 0.05).The pathological changes of the lung were aggravated in group OLV.Conclusion OLV induces lung injury by down-regulation of mtTFA expression in rabbit lung tissues.

Objective To explore the role of intracellular reactive oxygen species (ROS) in the senescence of bone marrow mesenchymal stem cells(BMSCs)in patients with systemic lupus erythematosus(SLE) and the underlying mechanisms that controls the intracellular ROS levels in vitro. Methods Human bone marrow aspirates were collected from iliac of eight donors and eight SLE patients and cultured in vitro.Morphological appearance of BMSCs at different passages was examined by inverted microscope. Nuclear size was measured by fluorescence microscope. BMSCs were monitored using the senescence associated β-galacto-sidase (SAβ-gal) assay to characterize senescence in vitro. The quantification of intracellular ROS production was detected by flow cytometry. Real-time PCR technique was used to determine the gene expressions of PI3K, KRas, NRas and FoxO3 at transcription level. The expression of FoxO3, phospho-FoxO3 (p-FoxO3),AKT and phospho-AKT (p-AKT) protein were determined by Western blot analysis. Statistical analysis was conducted with t-test and Mann-Whitney rank test.Results There were no differences in morphology and nuclear size[(31.4±4.5) vs (28.2±4.8) μm, P=0.628] of BMSCs between SLE patients and normal controls.The percentage of SA β-gal positive BMSCs from SLE patients was higher than that from healthy controls [(31.8±9.0)% vs (12.4±0.7)%, P＜0.05]. Intracellular ROS levels of BMSCs from SLE patients increased more significantly than healthy donors in vitro (34600±9600 vs 17 958±5400, P＜0.05). No significant differences in the expression of PI3K, NRas, KRas and FoxO3 from SLE subjects were observed at mRNA levels compared with normal controls, though all showed a similar upward trend. The expression of p-FoxO3 and p-AKT of BMSCs from SLE patients increased significantly compared with healthy controls at protein levels.Conclusion These data suggest that BMSCs from SLE patients aged more quickly, with high SA β-gal activity and up-regulation of intracellular ROS, which is associated with up-regulation of p-FoxO3 and pAKT at protein levels. These results indicate that bone marrow mesenchymal cell senescence may be associated with the pathogcnesis of SLE by maintaining the lifespan of BMSCs.

OBJECTIVE To investigate the proliferation effect of di(2-ethylhexyl) phthalate (DEHP) on prostate in aged rats at the environmental exposure dose and the possible mechanism.METHODS Thirty-two male Sprague-Dawley rats,aged 1.5 years,were randomly divided into 4 groups (8 rats per group) and treated with DEHP (30,90 and 270 μg· kg-1,ig) and vehicle once daily respectively for 4 weeks.All the animals were anesthetized with pentobarbital sodium and sacrificed on the day subsequent to the last treatment.① Abdominal aortic blood samples were collected,and serum estradiol (E2),testosterone (T) and prolactin (PRL) levels were assayed by ELISA.② The prostate tissues were dissected and categorized into different lobes,weighed and measured.The prostate relative mass was calculated.③ The morphological changes were detected by HE staining and prostate epithelial height was analyzed with microscopic image analysis software.RESULTS Compared with vehicle control group,the prostate relative mass,dorsolateral prostate mass,and dorsolateral prostate index in DEHP 270 μg· kg-1 group were significantly higher (P＜0.05).The height of the ventral prostate epithelium in DEHP 30,90 and 270 μg· kg-1 groups was increased significantly (P＜0.01),so was the height of dorsal prostate epithelium in DEHP 270 μg· kg-1 group (P＜0.01).There were no significant changes in levels of E2,PRL or T in DEHP 30,90 and 270 μg· kg-1 groups,but the ratios of E2/T in DEHP 30 and 270 μμg· kg-1 groups were increased significantly (P＜0.05).CONCLUSION Low-dose DEHP could promote the proliferation of prostatic hyperplasia in the aged rats,which might be associated with the relative levels of endogenous hormone.

Objective To investigate the apoptosis of bone marrow mesenchymal stem cells(BMSCs)from systemic lupus erythematosus(SLE)patients and the expression of apoptotic molecules.Methods BMSCs were isolated from bone marrow of SLE patients and normal controls by density eentrifugation and adhesive culture in vitro.The apoptosis of BMSCs were evaluated by TUNEL assay.The expressions of Fas.Bcl-2 and the activity of Caspase 8 were detected by flow cytometry.Real-time PCR technique was used to determine the gene expressions of Fas,Bcl-2,Bax,Bcl-w,Caspase 8 and Apaf-1.Meanwhile,cytochrome C was detected by immunocytochemistry.Statistical analysis was conducted with t-test and Mann-Whitney rank test.Results The percentage of apoptotic BMSCs increased in SLE patients compared with healthy donors[(64±10)%vs [14±9)%,U=0,P<0.05 ].The expression of Bcl-2 in BMSCs of SLE patients was lower than the normal controls at protein leve[(11±9)%vs(56±18)%,U=0,P<0.05 ],and mRNA level(0.2±0.2vs 2.4±0.7,U=24,P<0.05).More cytochrome C positive pellets in the cytosolic fraction could be detected in BMSCs from SLE patients compared with healthy controls [(56±21)%vs (16±16)%,U=1,P<0.05].The activity of Caspase 8 was enhanced[(49±14)%vs(16±12)%,U=0.P<0.05 ],although with no significant difierence at mRNA Ievel.Both groups expressed Fas but with no significant difference (U=19,P>0.05).Conclusion BMSCs from SLE patients undergo more apoptosis,the mechanisms may be associated with the down regulation of Bcl-2,up-regulation of Cytoehrome C in cytoplasm and the activation of Caspase 8,which directs the intrinsic and extrinsic apoptosis pathways.

Objective To investigate the expression of FcγRⅡb on peripheral B cells and its clinical significance in primary Sj(o)gren's syndrome (pSS).Methods FcγRⅡb expression on peripheral B cells from 19 pSS patients and 15 healthy controls was examined by flow cytometry.The levels of serum anti-SSA and SSB antibodies were determined using enzyme-linked immunosorbent assay (ELISA).Data were analyzed with t test,one-way ANOVA,SNK-q test and Pearson's correlation test.Results The percentage of memory CD19+CD27+ B cell subpopulation was significantly lower in pSS patients [ (20.8±2.7)％] when compared to normal controls [(37.8±2.2)％ ](t=-4.002,P＜0.01).The level of expression of FcγRⅡb in active pSS memory CD19+CD27+ B cells [ MFI(74±8)] was significantly reduced when compared to inactive [ MFI( 132±11)] and normal controls [ MFI (139±12)] (F=10.699,P＜0.01).The level of expression of FcγRⅡb on memory CD19+CD27+ B cells from pSS patients was inversely correlated with Sj(o)gren's syndrome disease activity index (SSDAI) (r=-0.744,P=0.0003 ).pSS patients with the serum anti-SSA/SSB antibodies positive group [ MFI(75+3),(48±7)] displayed a lower expression of FcγRⅡb on memory CD19+CD27+ B cell than in patients with the serum anti-SSA/SSB antibodies negative group [MFI( 122±11),(108±9)] (t=-4.336 and -3.776 respectively,the P value of both tests were less than 0.01).The level of expression of FcγRⅡb in the memory CD19+CD27+ B cells of patients with active pSS was inversely correlated with anti-SSA antibody titers (r=-0.685,P=0.014).Conclusion The expression of FcγRⅡb on peripheral memory B cells from active pSS patients is inversely correlated with SSDAI and is also inversely associated with anti-SSA antibody levels.Decreased expression of FcγRⅡb might play an important role in the pathogenesis of pSS.

Objective To study the influence of multidrug resistance gene (MDR)1 C3435T genetic polymorphism on the eradication of gastric ulcer with Helicobacter pylori (Hp) infection.Methods A total of 106 gastric ulcer patients with positive Hp were randomly divided into two groups by lot with 53 cases each.One group was assigned with 20 mg esomeprazole,0.5 g clarithromycin,1.0 g amoxicillin twice one day(EAC group),and the other group was assigned with 20 mg omerprazole,0.5 g clarithromycin,1.0 g amoxicillin twice one day (OAC group).The therapy of two groups was one week.Hp was detected at least 4 weeks after the end of treatment.MDR1 C3435T genetic polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism assay.The influence of MDR1 C3435T genetic polymorphism on the eradication of Hp was recorded and analyzed.Results The Hp eradication rate was 84.9％ (45/53) and 77.4％ (41/53) in EAC group and OAC group,and there was no significant difference between two groups (P ＞ 0.05).There was no significant difference in the Hp eradication rate in patients with different MDR1 C3435T genotypes in two groups (P ＞ 0.05).The Hp eradication rate was 66.7％(16/24),86.3％(44/51),83.9％(26/31) in TT,CT,CC genotype,and there was significant difference(P＜ 0.05).The Hp eradication rate in patients with TT genotype was lower than that in patients with CT,CC genotype,and there was significant difference (P＜ 0.05).Conclusion There is significant relationship between the effect of gastric ulcer with Hp eradication and MDR1 C3435T genetic polymorphism,and the Hp eradication rates of patients with TT genotype are more lower.

Objective To assess the correlation of promoter methylation of DAPK1,RAR-β and MGMT with cervical lesions from cytology to histology,and to reveal the clinical value of DNA methylation in diagnosis of cervical intraepithelial neoplasia (CIN).Methods A total of 103 random-selected cervical samples were collected from residual liquid-based cytology specimens after clinical use in cytopathological diagnosis in outpatient clinic of obstetrics and gynecology,Peking Union Medical Collage Hospital from March 2010 to October 2010.Informed consent was obtained from each woman before the initiation of the study.The methylation seusitive-high resolution melt (MS-HRM) assay was used to evaluate promoter methylation of three genes ( DAPKI,RAR-β and MGMT) in 103 biopsy-confirmed liquid-based cervical cytology samples.Methylation levels and high-risk HPV DNA loading ( HC Ⅱ values) were analyzed in relation to both cytological and histological diagnosis.Results The methylation level of all three genes showed significant difference among the different cytological groups ( P =0.000,0.011 and 0.002,respectively).The methylation level of DAPK1 and RAR-β showed significant difference among the different histological groups ( P =0.000 and 0.021 ),while there was no significant difference for MGMT.DAPK1 methylation levels was 1.47％ in the CIN Ⅱ/high-grade precancerous lesions group,and 20.98％ in the normal/CIN I groups ( P =0.000 ),but there was no significant difference between CIN I/high-grade precancerous lesions and normal/CIN Ⅰ groups for RAR-β and MGMT.The combination of DAPK1/HR-HPV loading showed a sensitivity of 0.825 and an area under the receiver operating characteristic curve (ROC) curve (AUC) of 0.695 as diagnostic methods for detecting CIN Ⅱ/high-grade precancerous lesions.Conclusions DNA methylation such as DAPK1 and RAR-β,in combination with HR-HPV detection,may serve as biomarkers to detect CIN Ⅱ/high-grade precancerous lesions.Detection of methylated DNA from liquid-based cervical cytology specimens is technically feasible with the MS-HRM assay.