Synthesis, estrogenic activity, and anti-osteoporosis effects in ovariectomized rats of resveratrol oligomer derivatives.

Three series of resveratrol oligomer derivatives were synthesized, including the indenone-type, indene-type and octahydropentalene-type derivatives, among which ten derivatives were novel compounds. Compounds 2, 14f, and 4d were confirmed as ERß agonists by yeast two-hybrid assay, and compound 2 (isopaucifloral F) was further chosen to evaluate its anti-osteoporosis activity in vivo. Compared with the sham-operated and the positive control groups, isopaucifloral F (10 µg/kg) showed a notable anti-osteoporosis effect in the ovariectomized (OVX) female rats based on a micro-CT analysis and the following measurements: bone mineral density, bone volume/tissue volume, trabecular thickness, trabecular separation/spacing, and the serum biochemical parameters. LD50 of isopaucifloral F was found to be greater than 5 mg/kg and its effective dose (ED) was found to be about 10 µg/kg. Therefore, isopaucifloral F may be a promising lead compound for the treatment of postmenopausal osteoporosis.

Dose of incorporated immunodominant antigen in recombinant BCG impacts modestly on Th1 immune response and protective efficiency against Mycobacterium tuberculosis in mice.

One approach for improving BCG efficacy is to utilize BCG as vehicle to develop recombinant BCG (rBCG) strains overexpressing Mycobacterium tuberculosis (M. tb) antigens. Also expression level of a candidate antigen should impact the final T cell responses conferred by rBCG. In this study, based on our previously constructed differential expression system, we developed two rBCG strains overexpressing M. tb chimeric antigen Ag856A2 (coding a recombinant ag85a with 2 copies of esat-6 inserted at Acc I site of ag85a) at differential levels under the control of the subtly modified furA promoters. These two rBCG strains were used to vaccinate C57BL/6 mice and exploit dose of incorporated antigen in rBCG to optimize immune response and protective efficiency against M. tb challenge in mouse model. The results showed that rBCG strains overexpressing Ag856A2 at differential levels induced different antigen-specific IFN-γ production and comparable number of M. tb-specific CD4 T cells expressing IL-2. M. tb challenge experiment showed that rBCG strains afforded enhanced but comparable immune protection characterized by reduced bacillary load, lung pathology, and inflammation. These results suggested that the dose of antigens incorporated in rBCG can impact T cell immune responses but imposed no significantly differential protective efficacies.

Design, synthesis, and antibacterial activity of demethylvancomycin analogues against drug-resistant bacteria.

Five novel N-substituted demethylvancomycin derivatives were rationally designed and synthesized by using a structure-based approach. The in vitro antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA), gentamicin-resistant Enterococcus faecalis (GRE), methicillin-resistant Streptococcus pneumoniae (MRS), and vancomycin-resistant Enterococcus faecalis (VRE) were evaluated. One of the compounds, N-(6-phenylheptyl)demethylvancomycin (12 a), was found to exhibit more potent antibacterial activity than vancomycin and demethylvancomycin. Compound 12 a was also found to be ~18-fold more efficacious than vancomycin against MRSA; however, the two compounds were found to have similar efficacy against MRS. Furthermore, compound 12 a exhibited a favorable pharmacokinetic profile with a half-life of 5.11±0.52 h, which is longer than that of vancomycin (4.3±1.9 h). These results suggest that 12 a is a promising antibacterial drug candidate for further preclinical evaluation.

Neuroprotective effects of mercaptoethylleonurine and mercaptoethylguanidine analogs on hydrogen peroxide-induced apoptosis in human neuronal SH-SY5Y cells.

A series of mercaptoethylleonurine and mercaptoethylguanidine derivatives were designed and synthesized. Their neuroprotective effects toward H2O2-induced apoptosis were investigated in human SH-SY5Y cells. The results from these studies identified several potent compounds, with compound 8k emerging as the most effective. Further investigation demonstrated that 8k reduced H2O2-induced activation of mitochondrial apoptosis by inhibiting the expression of Bax and elevating the expression of Bcl-2. Moreover, the molecular mechanism underlying the observed neuroprotective effects of 8k was exerted via the Akt and JNK pathways. Compound 8k can be a lead compound for further discovery of neuroprotective medicine.

[Therapeutic effect of ovarian intra-arterial infusion of GE7-delivery system-mediated HSVl-tk/ganciclovir gene therapy in a rat model of malignant ovarian tumor].

OBJECTIVE: To observe the gene expression of herpes simplex virus type 1 thymidine kinase (HSVl-tk) in rat malignant ovarian tumor tissues and the therapeutic effect of ganciclovior (GCV) after intra-arterial infusion of HSVl-tk gene therapy mediated by GE7-delivery system. METHODS: A GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 4-element complex was constructed. Eighteen rats with DMBA-induced ovarian tumor were divided into 3 groups as Atk, ANS and Vtk groups. The 4-element complex GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 was injected via the ovarian artery into the rats of Atk group, saline buffer was injected in the ANS groups, and the 4-element complex was injected via the tail vein into the rats of Vtk group. All rats received intraperitoneal injection of GCV in a dose of 50 mg/kg daily for 10 days. The rats were sacrificed 3 days after the final dose of GCV, and the tumor weight was measured and tumor growth inhibition rate was calculated. Flow cytometry was used to assess the cell cycle and apoptosis. RESULTS: The tumor weight in the rats of Atk group was (4.77 ± 2.31) g, significantly lower than that of ANS group [(14.66 ± 6.26) g, P < 0.01] and Vtk group [(17.53 ± 7.19) g, P < 0.01]. The tumor growth inhibition rate of the Atk group was 67.5%, while that of Vtk group was -19.6%. The flow cytometry showed that S-phase tumor cells in the Atk group were (54.32 ± 9.65)%, significantly higher than that in the ANS (27.43 ± 9.22)% and (30.16 ± 11.57)% in the Vtk group (both P < 0.01). The tumor cell apoptosis rate in the Atk group was (39.15 ± 12.16)%, significantly higher than that in the ANS group [(11.86 ± 5.28)%, P < 0.01] and Vtk group [(14.32 ± 6.43)%, P < 0.01]. CONCLUSION: HSV1-tk/GCV gene therapy system mediated by GE7 non-viral delivery system via ovarian arterial infusion effectively causes cell cycle arrest at S phase and enhances cell apoptosis, therefore, exerts an inhibitory effect on tumor growth.

Incorporation of immunostimulatory motifs in the transcribed region of a plasmid DNA vaccine enhances Th1 immune responses and therapeutic effect against Mycobacterium tuberculosis in mice.

T-helper type 1 (Th1) immune response is involved in the development of protective immunity against Mycobacterium tuberculosis. Thus, an increase in Th1 and cellular immune responses should lead to enhanced anti-mycobacterial activity. In this study, we aimed to improve Th1 immune responses to a DNA vaccine by adding potentially immunostimulatory nucleotide sequences into the transcribed region downstream of the antigen. The Mycobacterium leprae gene for hsp65, codon-optimized for expression in mammalian cells, was inserted into pVAX1 with and without 3'-sequences containing CpG and dsRNA motifs. When the plasmid contained both motifs, transfected murine macrophage-like RAW264.7 cells showed markedly increased levels of mRNA for immune molecules of Th1 (IFN-α, IL-12) and Th17 (IL-17, IL-23 and IL-6) responses and for T cell co-stimulatory molecules (CD80 and CD86) but not for a Th2 response (IL-4 and IL-10). Immunized mice showed substantially increased serum anti-Hsp65 IgG2a antibody levels and IFN-γ production by spleen cells, confirming enhancement of the Th1 response in vivo. Furthermore, when non-vaccinated mice were infected with H37Rv by low-dose aerosol challenge, and then 4 weeks later were treated with plasmids by intramuscular injection, the mice that had been treated with plasmids containing immunostimulatory motifs showed an enhanced reduction in mycobacterial loads in lung and spleen. We conclude that DNA vaccines may be made more highly immunogenic and more effective for treatment by including transcribed stimulatory sequences.

[Psychological resilience features of urban migrant children and rural left-behind children in Sichuan province of China].

OBJECTIVE: To explore the characteristics of resilience of urban migrant children and rural left-behind children of Chinese farmer workers and to figure out the discrepancy between them. METHODS: The samples consisted of 1 391 primary students and middle school students from Chengdu, Guangyuan, Yibin, and Mianyang in Sichuan Province. The revised version of the Healthy Kids Resilience Assessment was used for the measurement of resilience. And ANOVA was performed for data analysis. RESULTS: The results of the present study indicated that among all the junior high students, urban migrant children got a significantly lower score of resilience (128.11±21.70) than rural left-behind children (135.61±22.77) and the control group (132.87±23.22), F(0.05 (2, 884))=8.076, P<0.001. And migrant children got lower scores on three external protective factors of resilience-family, school, community as well as on resilience traits than left-behind children and the control group (Family: F(0.05(2, 884))=7.820, P<0.001; School: F(0.05(2, 884))=5.041, P=0.007; Community: F(0.05(2, 884))=9.261, P<0.001; Resilience traits: F(0.05(2, 884))=3.510, P=0.030). No significant difference was noted between left-behind children and control group. Gender difference and grade difference were noted for each group. CONCLUSION: The resilience of migrant children was not so good as non-migrant children. Migrant children were enjoying less intimate interpersonal relationship in their schools, their families as well as their community, and they could get less psychological support from the external environment, so that migrant children could not develop some resilience traits to promote the sound development of themselves. Suggestions for intervention were also discussed.

[Evaluation of GE7-transferring system-mediated HSV(1)-tk gene transfer in a rat model of ovarian tumor via intra-arterial route].

OBJECTIVE: To observe the gene and protein expression of herpes simplex virus type I-thymidine kinase (HSV(1)-tk) in the ovarian tumor tissues and other organs after arterial infusion of HSV(1)-tk gene mediated by GE7 delivery system. METHODS: GE7-polylysine/pCMV-HSV(1)-tk/polylysine-HA20 complexes were constructed. Nine rats with induced ovarian tumor were divided into 3 groups, injecting the 4-element complexes or saline buffer through the ovarian artery and complexes through the tail vein, respectively. The ovarian tumors, hearts, livers, spleens, lungs and kidneys were obtained at 72 hours after injection. RT-PCR and Western Blot were preceeded to determine the expression of HSV(1)-tk gene and protein in the tumor tissues and other organs. RESULTS: In the group of arterial injection with 4-element complexes, the HSV(1)-tk gene and protein were expressed strongly in the tumor tissues, while little or none was detected in other organs. In the group of arterial injection with saline buffer, no HSV(1)-tk gene and protein was detected in both tumor tissues and other organs. In the group of tail vein injection, none was detected in tumor tissues and only little was found in the livers, spleens, lungs and kidneys. CONCLUSION: High target and gene transfer rates can be obtained when HSV(1)-tk gene is transferred via the artery route mediated by GE7 delivery system. HSV(1)-tk protein can be expressed after the gene transfer. The results may provide a new strategy for target killing of HSV(1)-tk/GCV system in ovarian tumors.

Medicated rat serum containing Gengnianchun decoction reduces apoptosis of pheochromocytoma cells insulted by amyloid beta protein.

OBJECTIVE: To investigate the effects of medicated rat serum containing Gengnianchun (GNC) decoction and its protection to pheochromocytoma cells (PC12 cells) from amyloid beta (Abeta)(25-35)-insulted apoptosis and to find the possible mechanism. METHODS: Medicated rat serum was prepared by administering ovariectomized Sprague-Dawley (SD) rats with GNC decoction. The effects of medicated rat serum on viability of PC12 cells were evaluated by cell counting kit-8 (CCK-8) assay. The PC12 cells were cultured with different doses of Abeta(25-35) to induce a model of Alzheimer's disease in vitro. Then, the protective effects of medicated rat serum on Abeta(25-35)-insulted PC12 cells were evaluated by using CCK-8 assay to detect the cell viability, using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry to detect cell apoptosis rate and using Western blotting assay to analyze the expressions of Bcl-2, Bax and active caspase-3 proteins. RESULTS: PC12 cells cultured with 20% medicated rat serum containing GNC decoction for 24 h or 48 h had higher viability than those cultured with normal culture medium (P<0.05). After 24- or 48-hour treatment of different concentrations of Abeta(25-35), cell viabilities were all decreased as compared with normal medium (P<0.05). Cells underwent apoptosis, which showed the neurotoxicity of Abeta(25-35). The cell apoptosis induced by Abeta 25-35 was significantly decreased in PC12 cells which were pretreated with 20% medicated rat serum or nerve growth factor (NGF) according to CCK-8 assay and Annexin V-FITC/PI flow cytometry (P<0.05). The ratio of Bax expression to Bcl-2 expression and the expression of active caspase-3 were decreased in the cells treated with medicated serum or NGF as compared with the cells cultured with Abeta(25-35) only. CONCLUSION: The GNC-medicated rat serum at concentration of 20% can promote viability of Abeta(25-35)-insulted PC12 cells and decrease the cell apoptosis by regulating the expressions of Bcl-2, Bax and active caspase 3.

An in vitro study on neuroprotective effects of serum containing Gengnianchun decoction and its main monomers against amyloid beta protein-induced cellular toxicity.

OBJECTIVE: To observe the effects of serum containing Gengnianchun (GNC) decoction, a compound traditional Chinese herbal medicine, and its monomers (paeoniflorin, berberine, timosaponin A-III and icariin) on neurotoxicity in PC12 cells induced by amyloid beta-protein (Abeta). METHODS: Injury of PC12 cells was induced by incubating with Abeta(25-35) in vitro. Ovariectomized rats were intragastrically administered with GNC decoction twice daily for 5 days and then sera were obtained. PC12 cells were cultured with different concentrations of serum containing GNC decoction and its main monomers including paeoniflorin, berberine, timosaponin and icariin and the monomer mixtures to determine the best concentration of the drugs by methyl thiazolyl tetrazolium (MTT) method. The effective concentration of Abeta(25-35) was determined by culturing PC12 cells with different concentrations of Abeta(25-35). Then, the activity of PC12 cells with Abeta(25-35)-induced injury was observed with MTT method. Cellular morphological change was observed by phase contrast microscopy. Flow cytometry and fluorescence microscopy were employed to observe the Abeta(25-35)-induced early apoptosis of PC12 cells. RESULTS: After Abeta(25-35) induction, the PC12 cells were fewer in number, less viable with shrinked cel1 body and nuclei, and many fragments, and the cells were less adhered. The cell proliferation was inhibited by Abeta(25-35) concentration- and time-dependently. Abeta(25-35) at concentration of 20 micromol/ L was selected to construct the Alzheimer's disease model in vitro. The sera containing GNC decoction could reinforce PC12 cell activity, and concentration at 20% was better than other concentrations after 24-, 48- and 72-hour culture. The 20% serum containing GNC decoction, 0.1 micromol/L berberin and monomer mixture 3 including 1 mg/mL paeoniflorin, 1 micromol/L berberine, 1 micromol/L timosaponin and 1 ng/mL icariine could antagonize neurotoxicity induced by Abeta(25-35). Moreover, they could inhibit Abeta(25-35)-induced early apoptosis of PC12 cells, with the effect of 20% serum containing GNC decoction better than 0.1 micromol/L berberine and monomer mixture 3. CONCLUSION: Serum containing GNC decoction at 20% concentration has a potential neuroprotective effect on Abeta-induced cellular impairment. The serum containing GNC decoction was found to be stronger in action than the main monomers.

[Proteomic analysis of plasma membrane of HBV transgene mice livers].

OBJECTIVE: To study liver plasma membrane (PM) proteins affected by hepatitis B virus (HBV), and to study the molecular mechanism of HBV infection through plasma membrane proteome analysis of transgene mice livers. METHODS: Plasma membrane was purified using second antibody superparamagnetic beads that combine subcellular fractionation and immunoisolation strategies. Western blot was used to verify the purification of plasma membrane and the expression of HBV envelope protein. The proteins from plasma membrane were extracted, quantified and separated by two-dimensional electrophoresis. The gels were stained by silver nitrate or Coomassie Blue and analyzed by Imagemaster software. The different expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF-TOF MS). RESULTS: The plasma membrane of mice livers was enriched 3-fold and the contamination from mitochondria was reduced 2-fold in comparison with the density gradient centrifugation method. HBV envelope protein was found only in HBV transgene mice livers. More than 500 protein spots were separated in Coomassie Blue stained gels. Twenty-six proteins with two-fold differences were found through Imagemaster software analysis. Seven proteins were identified. CONCLUSION: An optimized plasma membrane purification method was developed; protein profile of liver plasma membrane changed in the transgene mice livers; some proteins related to HBV infection were found, and this work may be helpful in the research of the molecular mechanism of HBV infection.

[Effect of Gengnianchun Recipe on learning memory function and hippocampal cholinergic system in ovariectomized rats].

OBJECTIVE: To evaluate the effect of Gengnianchun Recipe (GNC) on learning memory function and its regulatory effect on hippocampal cholinergic system in ovariectomized rats. METHODS: Female rats 10-12 months old were randomized into 5 groups, the sham-operation group, the model group treated with normal saline, the positive control group treated with Nilestriol, and the two GNC groups treated with high and low dose GNC respectively. A little fat around ovary was cut in the sham-operation group. The treatment lasted for 12 weeks after ovariectomy. Changes of learning memory function were tested by Morris water maze; serum level of estradiol (E2) was measured by chemical fluorescent method; hippocampal acetylcholinesterase (AChE) mRNA was determined with Real-time PCR; and the activities of acetylcholine (ACh), AChE and choline acetyltransferase (ChAT) in hippocampus were detected by immunohistochemistry respectively. RESULTS: Twelve weeks after ovariectomy, serum E2 and learning memory function markedly decreased in the ovariectomized rats (P < 0.01, P < 0.05). Nilestriol and high dose GNC showed an effect in improving the symptoms of learning memory functional deprivation and elevating the activities of hippocampal ACh, AChE and ChAT (all P < 0.05). CONCLUSION: GNC can improve learning memory function of ovariectomized rats, and its mechanism might be realized by regulating the cholinergic system in hippocampus.

Effect of Gengnianchun Recipe on bone mineral density, bone biomechanical parameters and serum lipid level in ovariectomized rats.

OBJECTIVE: To observe the effect of Gengnianchun Recipe (GNC) on bone mineral density (BMD), bone biomechanical parameters and serum lipid level in the bilaterally ovariectomized (OVX) rats and to explore the prophylactic and therapeutic action of GNC on ovariectomy induced osteoporosis and hyperlipidemia. METHODS: OVX SD rats, 10 - 12 months old, were divided into different groups and fed with GNC 2 g/d, GNC 1 g/d and Nilestriol 0.125 mg/week, respectively for 4 months to observe the change of BMD and bone biomechanical parameters of the lumbar vertebrae, and the serum levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), and to compare the effect of the two drugs on the morphology of the uterus. RESULTS: There was marked reduction in BMD and biomechanical parameters in lumbar vertebrae (P < 0.01) and increase of serum TC and LDL-C levels (P < 0.01) in rats after OVX. GNC or Nilestriol significantly improved the decreased BMD and biomechanical parameters of the lumbar vertebrae (P < 0.05 or P < 0.01), and reduced the serum TC and LDL-C levels (P < 0.01). In the Nilestriol group, the wet weight of uterus got increased obviously (P < 0.01), the number of uterine glands increased, uterine columnar epithelium thickened, and the mitotic figures in the epithelial stroma and myointimal cells augmented. But no such effect in wet weight and morphology of uterus was found in the GNC group. CONCLUSION: GNC could increase the BMD and biomechanical parameters of the lumbar vertebrae, reduce the serum TC and LDL-C levels, yet produce no adverse reaction in stimulating proliferation and hypertrophy of uterus.

Enhanced efficiency and specificity of ovarian cancer gene therapy in rats with a novel nonviral gene delivery system (GE7) via intraovarian artery perfusion approach.

Transfer of the herpes simplex virus type I-thymidine kinase gene, followed by the administration of ganciclovir (HSV1-tk/GCV) into ovarian cancer-derived cell line either in vitro or transplanted into nude mice has been shown to provide a potential strategy for the gene therapy of ovarian cancer. We investigated the antitumor effects of HSV1-tk/GCV strategy with a chemically induced rat ovarian cancer model and a tumor-selective gene delivery by a novel nonviral gene delivery system (GE7) through the ovarian artery and tail vein. We demonstrated the expression of a reporter gene, beta-gal gene, as well as HSV1-tk gene in tumors and other organs, evaluated the overall antitumor effects after the GCV treatment and analyzed the tumor cell cycle phase distribution. Via the ovarian artery route, the expressions of beta-gal and HSV1-tk in tumors were significantly stronger than those expressed in such organs as the hearts, livers, spleens, lungs and kidneys. However, no beta-gal and HSV1-tk were detected in the tumor tissues when administrated via the tail vein, and little was found in other organs. The cell cycle analysis showed that the total S-phase of tumor cells in the test intra-arterial treatment group was considerably higher than that of the controls. The weight of the tumor tissues in the group treated by the intra-arterial route (4.06+/-2.12 g) was much less than the group treated intravenously (18.25+/-8.34 g) (P<.01). These findings indicated that the administration of GE7/HSV1-tk complex via the ovarian artery route could be a promising avenue of future human ovarian cancer treatment.

[Study of GE7-transferring system mediated beta-galactosidase gene transfer in a rat model of ovarian tumor by intraarterial route].

OBJECTIVE: To investigate the efficiency and targeting of the GE7 system mediated gene transfer in the chemically induced ovarian tumor by intraarterial route. METHODS: Animal models were chemically induced by 7,12-dimethylbenz[a]anthracene (DMBA). GE7-polylysine/beta-galactosidase (beta-gal)/polylysine-HA20 complexes were constructed. Fifteen rats with induced ovarian tumor were divided into three groups, the complexes were injected through ovarian artery and tail vein, respectively. The tumor, heart, liver, spleen, lung and kidney were obtained at 72 h. X-gal staining was used to check expression of beta-gal. RESULTS: beta-gal was expressed strongly and well-distributed in tumor in the first group, and stained blue in the liver, spleen, lung and kidney, but much weaker than that of tumor. In the second group, beta-gal was expressed in the tumor, and weakly expressed in the liver, none was detected in the other organs. In the third group beta-gal was detected in the lung slightly, none was detected in the tumor and other organs. CONCLUSIONS: When GE7 complexes were administrated through ovarian artery, the introduced gene expressed preferentially in ovary. This result was the basis of intraarterial administration of therapeutic gene to treat ovarian cancer.