Effects of Korean ginseng berry extract (GB0710) on penile erection: evidence from in vitro and in vivo studies

Several reports have promoted the root-derived Korean red ginseng (KRG; Panax ginseng) as alternative treatment for erectile dysfunction (ED), and ginsenosides are known to be the principal active ingredients of ginseng. Recent studies showed that ginseng berries produce more ginsenosides than KRG; thus, we investigated the ability of the Korean ginseng berry extract GB0710 to relax the penile corpus cavernosum smooth muscle (CCSM) in this study. As a comparative control, the results were compared to those obtained using KRG. In addition, possible mechanisms of action for GB0710 were investigated. While KRG and GB0710 both displayed dose-dependent relaxation effects on precontracted rabbit CCSM in vitro, GB0710 was shown to be more potent than KRG. The GB0710-induced relaxation could be partially reduced by removing the endothelium. In addition, pre-treatment with several nitric oxide (NO) inhibitors significantly inhibited the relaxation of muscle strips. Furthermore, administration of GB0710 increased intracavernosal pressure (ICP) in a rat in vivo model in both a dose- and duration-dependent manner. Intracellular NO production in human microvascular endothelial cells could be induced by GB0710 and inhibited by N^G-monomethyl-L-arginine. In conclusion, GB0710 had a greater relaxation effect on rabbit CCSM than did KRG extract, and increased ICP in a rat model in both a dose- and a duration-dependent manner. This relaxing effect might be mediated by NO production.     

Gal alpha(1-3)Gal expression of the cornea in vitro, in vivo and in xenotransplantation

BACKGROUND: To investigate alpha Gal (Gal alpha1-3Gal beta 1-4GlcNAc-R) expression of the porcine cornea in vitro, in vivo and after xenotransplantation. METHODS: Using the GS-IB4 lectin (Griffonia simplifolia I isolectin B4), the expression of alpha Gal was evaluated in the normal porcine cornea and in cultured corneal stromal and endothelial cells of the second passages. The distribution of alpha Gal epitopes was also evaluated in corneal grafts at 4, 7 and 10 days after pig-to-rat orthotopic corneal transplantation. RESULTS: The expression of alpha Gal was mostly confined to the anterior stromal keratocytes of the normal porcine cornea. However, reactivity for GS-IB4 was markedly increased during cell culture passage (P)-by 18.0% (P1) and 39.0% (P2) in cultivated keratocytes, and by 62.1% (P1) and 87.1% (P2) in cultured endothelial cells. The expression of alpha Gal epitopes was gradually enhanced in all corneal layers of the graft after transplantation. CONCLUSION: Alpha Gal expression in the cornea was gradually induced during in vitro culture and after xenotransplantation, suggesting a role of putative Gal-related acute humoral rejection in porcine corneal xenotransplantation.     

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.     

Medroxyprogesterone acetate,enoxaparin and pentoxyfylline cause alterations in lipid peroxidation,paraoxonase (PON1) activities and homocysteine levels in the acute oxidative stress in an experimental model of spinal cord injury

Summary.  Background: Effects of medroxyprogesterone acetate, enoxaparin and pentoxyfylline on lipid peroxidation, antioxidant defence system, paraoxonase activities, and homocysteine levels in an experimental model of spinal cord injury were investigated.  Method: Sixty-three male albino Wistar rats were anaesthetized by 400 mg/kg chloral hydrate and divided into 5 groups. G1 (n 7) = control group provided the baseline levels. G2–G5 underwent T3–6 total laminectomies and spinal cord injuries by clip compression at T4–5 levels. Medications were applied to G3–G5 right after the injury. Hence, G2 constituted laminectomy + injury (lam+I); G3 = lam + I + medroxyprogesterone acetate (MPA), G4 = lam + I + enoxaparin (E), and G5 = lam+I+pentoxyfylline (P) groups. Animals were decapitated either at the 1st or 4th hour after injury. Tissue and blood malonyldialdehyde (MDA) and plasma homocysteine and erythrocyte superoxide dismutase (SOD) levels, and erythrocyte glutathione peroxidase (GSH-Px) and plasma paraoxonase (PON1) activities were assayed. SPSS 9.0 program was used for statistical analysis and graphics. Intergroup comparisons were made by Bonferroni corrected Mann Whitney U test (P<0.025), and intragroups comparisons by Wilcoxon Rank test (P<0.03).  Findings: In intergroup comparison, G1–G2, G1–G3, G1–G5, G2–G3, G2–G4, and G4–5 groups differed from each other for all parameters (P<0.025, MWU) except for G4–G5 4th hour MDA levels. G1–G4 was similar for all 1st hour parameters (P>0.025, MWU), but different for 4th hour (P<0.025, MWU) except for GSH-Px and SOD levels. For G2–G5, all parameters for 1st and 4th hour were similar except for 4th PON1, Hcy and SOD levels. For G3–G4, all 1st hour parameters were different from each other (P<0.025, MWU); whereas all 4th hour parameters were similar except for SOD level. For G3–G5, all parameters at 1st and 4th hour were similar except for 4th hour GSH-Px, PON1, and Hcy. In intragroup comparison, all parameters differed from each other at all times (P<0.03, WRT) except for 1st hour G4 MDA, Hcy and SOD levels compared to basal levels.  Interpretation: In injury groups, plasma Hcy levels decreased and PON1 activities increased as erythrocyte SOD level and GSH-Px activities decreased in parallel to increases of tissue and blood MDA levels. These changes were relatively suppressed by MPA, enoxaparin and pentoxyfylline administrations at varying degrees. Enoxaparin was the most powerful agent, particularly at 1st hour. MPA was also effective, particularly at 4th hour. Pentoxyfylline despite having slight effect at 4th hour, was not effective according to both control and injury groups. Enoxaparin and MPA can be used in the treatment of spinal cord injuries. PON1 and Hcy are helpful in monitoring the antioxidant defence system as well as SOD and GSH-Px, both in injury and medically treated groups. Published online October 10, 2002 Acknowledgments  The experiments comply with the current laws of the country on the protection of animals. We wish to thank “Erciyes University Medical Faculty, Hakan Cetinsaya Experimental and Clinical Research Centre, Kayseri, Turkey” for providing us the animals for this study. Correspondence: Dr. Cahide Topsakal, Firat Universitesi Tip Fakultesi, Norosirurji Departmani, Elazig/Turkey.     

Anti-androgenic,anti-oxidant and anti-apoptotic effects of the aqueous and methanol extracts of Pterorhachis zenkeri (Meliaceae): Evidence from in vivo and in vitro studies

The aim of this study was to evaluate the effects of Pterorhachis zenkeri (Meliaceae) on sex organ growth in immature male rats and, oxidative stress and apoptosis markers in CCL-97 (R2C) Leydig cells. For the in vivo studies, 70 immature male Wistar rats (n = 10/group) were treated for 2 or 4 weeks with: distilled water (10 ml/kg, per os) plus soya oil (1 ml/kg, sc), bicalutamide (10 mg/kg, per os), aqueous or methanol extract of P. zenkeri (10 mg/kg or 62 mg/kg, per os) or testosterone propionate (3 mg/kg, sc). After each treatment period, body and sexual organ weights, plasmatic testosterone, total proteins and total cholesterol levels were measured. In the in vitro test, the effects of the methanol extract of P. zenkeri on cell viability, apoptosis, reactive oxygen species (ROS) production, intracellular calcium release and caspases 3/9 were assessed using CCL-97 Leydig cells. Pterorhachis zenkeri extracts decreased sex organ weights, plasmatic testosterone and protein levels in rats. In the in vitro studies, P. zenkeri inhibited apoptosis, ROS production, calcium release and caspase 3/9 activities. These results suggest that P. zenkeri has anti-androgenic, anti-oxidant and anti-apoptotic activities with methanol extract being the most active and could be an effective alternative for the management of androgen-related diseases.     

In vitro and in vivo investigation of a novel monoclonal antibody to plasma cells (W5 mAb)

Abstract:  Natural antibodies (Abs), predominately anti-Gal α 1–3Gal (Gal) Abs, in non-human primates and human beings present a major hurdle to successful pig-to-primate xenotransplantation. Attempts to inhibit anti-Gal Ab production in naïve baboons using non-specific immunosuppressive or B cell-specific reagents have failed. A new rat monoclonal antibody (W5 mAb) has been generated, which binds to all B cells, including memory cells, and to the majority of plasma cells, but not to T cells. It has been tested in vitro and in vivo. By immunoprecipitation, W5 mAb bound a human leukocyte antigen class II (HLA-DR) determinant. Sorting splenic or bone marrow W5+ cells resulted in a highly enriched anti-Gal Ab and total immunoglobulin (Ig)-secretory population. In vivo studies in baboons demonstrated that W5 mAb was safe but, despite the concomitant administration of an anti-CD154 mAb to inhibit sensitization, anti-rat Abs were detected within 10 days and inhibited the effect of the W5 mAb. High levels of W5 mAb were able to completely deplete B cells in the blood, but not in lymphoid tissues. Enzyme-linked spot-forming assay (ELISPOT) demonstrated that only 50 to 60% of secreting cells (SC) were depleted in the bone marrow. No reduction in the serum levels of anti-Gal Ab was observed. W5 mAb did not cause complete inhibition of anti-Gal Ab production, probably as a result of its inability to completely deplete B and plasma cells from all lymphoid compartments.     

Differential expression of adenosine A1 and A2A receptors after upper cervical (C2) spinal cord hemisection in adult rats

BACKGROUND: In an animal model of spinal cord injury, a latent respiratory motor pathway can be pharmacologically activated via adenosine receptors to restore respiratory function after cervical (C2) spinal cord hemisection that paralyzes the hemidiaphragm ipsilateral to injury. Although spinal phrenic motoneurons immunopositive for adenosine receptors have been demonstrated (C3-C5), it is unclear if adenosine receptor protein levels are altered after C2 hemisection and theophylline administration. OBJECTIVE: To assess the effects of C2 spinal cord hemisection and theophylline administration on the expression of adenosine receptor proteins. METHODS: Adenosine A1 and A2A receptor protein levels were assessed in adult rats classified as (a) noninjured and theophylline treated, (b) C2 hemisected, (c) C2 hemisected and administered theophylline orally (3x daily) for 3 days only, and (d) C2 hemisected and administered theophylline (3x daily for 3 days) and assessed 12 days after drug administration. Assessment of A1 protein levels was carried out via immunohistochemistry and A2A protein levels by densitometry. RESULTS: Adenosine A1 protein levels decreased significantly (both ipsilateral and contralateral to injury) after C2 hemisection; however, the decrease was attenuated in hemisected and theophylline-treated animals. Attenuation in adenosine A1 receptor protein levels persisted when theophylline administration was stopped for 12 days prior to assessment. Adenosine A2A protein levels were unchanged by C2 hemisection; however, theophylline reduced the levels within the phrenic motoneurons. Furthermore, the decrease in A2A levels persisted 12 days after theophylline was withdrawn. CONCLUSION: Our findings suggest that theophylline mitigates the effects of C2 hemisection by attenuating the C2 hemisection-induced decrease in A1 protein levels. Furthermore, A2A protein levels are unaltered by C2 hemisection but decrease after continuous or interrupted theophylline administration. The effects on protein levels may underlie the stimulant actions of theophylline.     

Effect of platelet-activating factor (PAF) receptor blockers on smooth muscle cell replication in vitro and allograft arteriosclerosis in vivo

Platelet-activating factor (PAF) stimulates smooth muscle cell (SMC) replication both in vivo and in vitro. In this study we have investigated whether PAF receptor-blocking molecules modulate SMC replication in vitro and the generation of allograft arteriosclerosis in vivo. SMC cultures were established from baby rat aorta media and fibroblast control cultures from the adventitia. Identification of the cultured cell types was determined both by immunohistochemistry and electron microscopy. Both cell types replicated in culture with 10% fetal calf serum (FCS). The addition of PAF-C₁₈ enhanced, and the addition of three PAF receptor inhibitors — WEB 2086, WEB 2170, and BN 50739-reduced, SMC replication and protein synthesis in a dose-dependent fashion in vitro until toxic concentrations were reached. The most potent of these drugs, WEB 2170, was then delivered at the rate of 12 mg/kg per day to recipients of rat aortic allografts. The responses were quantitated by autoradiography after short-term labeling of the recipients with tritium-labeled thymidine (³H-TdR) and by quantitative morphology. Administration of the PAF receptor blocker had no impact on the replication of the inflammatory cells in the allograft adventitia nor on the replication of SMCs in the media and intima. Administration of the PAF receptor blocker delayed the generation of allograft arteriosclerosis slightly, but not significantly. These results suggest that PAF is not an essential component in the inflammatory cascade leading to allograft arteriosclerosis.     

Contragestazol （DL111-IT） inhibits proliferation of human androgen-independent prostate cancer cell line PC3 in vitro and in vivo

Aim： To evaluate the antiproliferative activity of contragestazol （DL111-IT） on the human prostate cancer cell line PC3 in vitro and in vivo and to elucidate its potential molecular mechanisms. Methods： The cell killing ability of DL111-IT was measured by the 3-（4,5-dimethylthia-zol,2-yl）-2,5-diphenyltetrazolium bromide （MTT） reagent assay method and the tumor xenograft model. The cell cycle was analyzed by flow cytometry and protein expression, including retinoblastoma （pRb）, cyclin-dependent kinase 4 （CDK4） and cyclin D 1, was detected by Western blotting. Results： DL111-IT exhibited high efficiency on cell growth inhibition of the human androgen-independent prostate cancer cell line PC3. The drug concentration that yielded 50 % cell inhibition （IC50 value） was 9.9 mg/mL. In the PC3 tumor xenograft study, DL111-IT （1.25 mg/kg-20.0 mg/kg） given once a day for 10 days significantly inhibited tumor growth, with the inhibition rate ranging from 21% to 50 %. Flow cytometric analysis indicated that DL111-IT could cause GI arrest in the PC3 cell line, but not apoptosis. DL111-IT enhanced pRb expression and down-regulated CDK4 and cyclin D 1 expression, suggesting that cell cycle regulation might contribute to the anticancer property of DL 111- IT. Conclusion： DL111-1T inhibits the proliferation of human androgen-independent prostate cancer cell line PC3 in vitro and in vivo by a cell cycle regulation pathway.     

脂氧合酶抑制剂NDGA对乳腺癌MCF-7细胞VEGF和CD44V6表达影响的研究

目的探讨NDGA对乳腺癌MCF-7细胞VEGF和CD44V6表达的影响。方法将乳腺癌MCF-7细胞系培养至对数生长期,分别以0、1、10、100μmol/L的NDGA处理48h后,RT-PCR法检测细胞VEGFmRNA的表达水平,流式细胞仪检测细胞CD44V6的表达情况。结果在1、10、100μmol/L的NDGA作用下,3组VEGFmRNA和CD44V6的表达显著低于对照组(F=2996.13,P<0.001;F=3435.08,P<0.001)。结论NDGA明显下调MCF-7细胞VEGFmRNA和CD44V6的表达,抑制肿瘤细胞的侵袭转移能力。     

Effect in vitro and in vivo of a rat anti-CD2 monoclonal antibody (LO-CD2b) on pig-to-baboon xenogeneic cellular (T and natural killer cells) immune response

Abstract: Although hyperacute rejection of discordant xenogeneic grafts can be prevented, baboon or human anti-pig cellular response may lead to acute xenograft rejection. Among the immune cellular actors participating in such a xenograft rejection are both T and natural killer (NK) cells. In the pre-clinical model of pig-to-baboon discordant xenograft, there is however, a lack of specific immunological therapeutic agent, in particular antibaboon T-cell monoclonal antibodies do not exist. We therefore developed a rat anti-CD2 monoclonal antibody (LO-CD2b) that recognizes both baboon and human CD2 + cells. In this study, we show that in vitro LO-CD2b inhibits a pig-to-baboon mixed lymphocyte reaction, the direct cytotoxicity of baboon peripheral blood lymphocytes to pig aortic endothelial cells, as well as the baboon NK activity against K562 cell line. In vivo, LO-CD2b produces a strong depletion of all peripheral CD2+ cells including NK CD2+ cells. In summary, LO-CD2b represents an important immunological tool that can be used in the preclinical model of discordant pig-to-baboon vascularized xenograft.     

NBQX (2, 3–dihydroxy–6–nitro–7–sulfamoyl–benzo(F)quinoxaline) did not affect recovery of high energy phosphates and pH in early reperfusion in a rat model of transient forebrain ischemia, or:An in vivo 31PNMR spectroscopy study

The new non–NMDA (N–methyl–D–aspartate) receptor antagonist NBQX (2, 3–dihydroxy–6–nitro–7–sulfamoyl–benzo(F)quinoxaline) has previously been shown to exert a neuroprotective effect in animal models of cerebral ischemia when administered in the post–ischemic phase. In this investigation the effect of NBQX on acidosis and energy recovery in early reperfusion after 10 min of transient forebrain ischemia with the 2–vessel occlusion model in the rat was studied with ³¹P NMR spectroscopy. In the intervention group the animals received a bolus dose of NBQX 30 mg kg^-1 iv at the start of reperfusion. ³¹P NMR spectroscopy was used to measure intracellular pH, ATP and phosphocreatine continuously in–vivo during, and after, the ischemic event. The recovery of high energy phosphates and pH was followed during 30 min of reperfusion. Pre–ischemic levels of phosphocreatine were reached after approximately 9–10 min in both groups. Although a slight improvement could be seen in the intervention group there was no significant difference in the rate of recovery between the two groups. ATP reached 90% of preischemic levels after about 8 min without significant difference between the two groups. With respect to the recovery of intracellular pH, no difference could be shown. Our results do not contradict previously published results, but suggest that the potential protective effect of NBQX is not mediated through improved recovery of energy metabolism in early reperfusion.