Two new chemical constituents from Lonicera japonica.

Two new chemical constituents, japopenoid D (1), and japopenoid E (2), were isolated and identified from the flower buds of Lonicera japonica Thunb. The structures of these compounds were elucidated based on spectroscopic analysis (HR-ESI-MS, NMR), and the absolute configurations of 1 and 2 were determined by comparison of their electronic circular dichroism (ECD) spectra with literature and theoretical calculation. The anti-inflammatory activities of the isolates were evaluated by measuring their inhibitory effects on PGE2 and IL-6 production in LPS stimulated RAW 264.7 macrophages. As a result, compound 1 could reduce PGE2 and IL-6 levels in LPS-activated RAW 264.7 macrophages with IC50 values of 6.78 and 42.07 µM, respectively.[Formula: see text].

Phosphatidylserine inhibits inflammatory responses in interleukin-1ß-stimulated fibroblast-like synoviocytes and alleviates carrageenan-induced arthritis in rat.

Recently, phosphatidylserine (PS) has received attention for its anti-inflammatory effect; however, the molecular mechanisms of its action have not been fully understood. Thus, we hypothesized that PS might have antiarthritic and anti-inflammatory effects. To test this hypothesis, the in vitro anti-inflammatory effect of soybean-derived PS was tested on interleukin (IL)-1ß-stimulated fibroblast-like synoviocytes from rheumatoid arthritis patients (RA-FLS) by measuring the levels of IL-6, IL-8, prostaglandin E(2), and vascular endothelial growth factor by enzyme-linked immunosorbent assay. The analgesic and antiarthritic activities of PS were investigated in rat models of carrageenan-induced acute paw pain and arthritis. The former was evaluated with a paw pressure test; the latter, by measuring paw volume and weight distribution ratio. In addition, the participation of mitogen-activated protein kinase signaling in the anti-inflammatory and antiarthritic effects of PS was investigated in RA-FLS. Phosphatidylserine inhibited the production of inflammatory mediators IL-6; IL-8; vascular endothelial growth factor; and, in particular, prostaglandin E(2) in IL-1ß-stimulated RA-FLS. These effects were associated with abrogation of inhibitor of nuclear factor-κBα phosphorylation and suppression of p38 and c-jun amino terminal kinase but not extracellular signal-regulated kinase 1/2 phosphorylation. In rats, PS also showed a significant inhibitory effect on arthritic and nociceptive symptoms induced by carrageenan. These findings suggest that PS has anti-inflammatory and antiarthritic effects in vitro and in in vivo animal models; thus, PS should be further studied to determine its potential use as either a pharmaceutical or dietary supplement for alleviating arthritic symptoms.

Molecular mechanisms of a novel selenium-based complementary medicine which confers protection against hyperandrogenism-induced polycystic ovary.

The objective was to evaluate ovarian functionality and oxidative response in hyperandrogenism-induced polycystic ovary (PCO) and the protective effects of immunomodulator drug (IMOD), an electromagnetically-treated, selenium-based, herbal medicine. Daily oral administration of letrozole (1 mg/kg) for 21 consecutive days induced ovarian cysts in female rats. An effective dose of IMOD (30 mg/kg per day) was given intraperitoneally for 21 days. Biomarkers of ovarian function, serum concentrations of estradiol, progesterone, testosterone, and ovarian prostaglandin-E (PGE), were analyzed. To determine the role of oxidative stress (OS) in hyperandrogenism-induced PCO, concentrations of cellular lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), peroxynitrite (ONOO), and tumor necrosis factor (TNF)-α as a marker of inflammation and apoptosis were measured in serum and ovaries. Letrozole-induced PCO resulted in significant increases in concentrations of lipid peroxidation and peroxynitrite in serum and ovary, but significantly decreased superoxide dismutase, catalase, and glutathione peroxidase. Serum concentrations of testosterone and TNF-α, and ovarian prostaglandin-E were increased (P < 0.001) in animals with cysts versus control, whereas estradiol and progesterone were decreased (P < 0.01 and P < 0.001, respectively). When compared with controls, letrozole induced irregular cycles and PCO characterized by a high incidence of subcapsular ovarian cysts with a diminished granulosa cell layer, luteinized granulosa cells in the cyst wall, significantly more atretic preantral and antral follicles, and absence of CL. There were almost no intact primary, secondary, and tertiary follicles in PCO rats. All end points assessed were significantly improved by IMOD and reached close to normal levels. In conclusion, the present study provided evidence that toxic free radicals and TNF-α were involved in the pathogenesis of PCO; furthermore, IMOD prevented ovarian histopathologic, endocrine, and biochemical alterations induced by hyperandrogenism.

Endometrial gene expression of acute phase extracellular matrix components following estrogen disruption of pregnancy in pigs.

In pigs, administration of estrogen to gilts on Days 9 and 10 of pregnancy causes conceptus fragmentation and death between Days 15 and 18 of gestation. Conceptus degeneration is associated with breakdown of the microvilli surface glycocalyx on the lumenal epithelium (LE). We previously identified endometrial expression of inter-α-trypsin inhibitor (ITI) and hyaluronic acid (HA), which are key components of extracellular matrix (ECM), during the period of conceptus attachment to the uterine surface in the pig. Tumor necrosis factor-α-inducible protein-6 (TNFAIP6) serves as a linker for ECM expansion and is stimulated by prostaglandin E (PGE). We hypothesized that early estrogen administration alters the normal ECM components forming glycocalyx on the LE. Bred gilts (4 gilts/trt/day) were treated with either 5mg estradiol cypionate (E) or corn oil (CO) on Days 9 and 10 of gestation. The uterus was surgically removed on either Days 10, 12, 13, 15 and 17 of gestation and endometrial tissue snap frozen in liquid nitrogen. Endometrial tumor necrosis factor-α (TNF), TNFAIP6, interleukin 6 (IL6), and inter-α-trypsin inhibitor heavy chains (ITIH) were detected during early pregnancy thereby indicating all components for maintenance of the extracellular glycocalyx are present in the endometrium of pigs. However, only gene expression of ITIH2 was suppressed by E-treatment. TNFAIP6 protein was detected across all days of gestation but was not affected by E-treatment. The present study demonstrates that while the pig endometrium expresses key components of ECM only ITIH2 gene expression was altered by E-treatment. A decrease in ITIH2 could lead to the possible loss of the uterine glycocalyx leading to conceptus degeneration; however, other factors may be involved with the loss of glycocalyx during implantation in the pig following E-treatment.

Effect of epidermal growth factor and prostaglandin on the expression of aromatase (CYP19) in human adrenocortical carcinoma cell line NCI-H295R cells.

We investigated the effects of epidermal growth factor (EGF) and prostaglandins (PG) on the expression of aromatase (CYP19) in human adrenocortical carcinoma cell line NCI-H295R cells. EGF significantly increased aromatase activity and CYP19 gene transcript in NCI-H295R cells. Exon PII was selected from among several tissue-specific exon I regions. Promoter II that abuts on exon PII was activated by EGF. PGE(2) also significantly increased aromatase activity, CYP19 gene transcript, and promoter II activity. The results of experiments using protein kinase (PK) inhibitors suggest that the cAMP-PKA signaling pathway is involved in the up-regulation of aromatase expression by EGF. PGE(2) activated promoter II activity in 4 h, while 12 h was required for its activation by EGF. In addition, PGE(2) was secreted from NCI-H295R cells in response to EGF. Selective agonists for prostaglandin receptors EP(1) and EP(2) significantly increased aromatase activity, which was decreased by the corresponding antagonists. Finally, antagonists for EP(1) and EP(2) inhibited the up-regulation of aromatase expression following EGF. These results suggest that PGE(2) secondarily acts as an autocrine signal in the up-regulation of aromatase expression by EGF in NCI-H295R cells.

Effects of dehydroepiandrosterone on ovarian cystogenesis and immune function.

The purpose of the present report was to study the possible relationship between ovarian functionality and the immune response during cystogenesis induced by androgenization with dehydroepiandrosterone (DHEA). Daily injection of DHEA (6 mg/kg body weight) for 20 consecutive days induced ovarian cysts in BALB/c mice. As markers of ovarian function, serum estradiol (E) and progesterone (P) and the ovarian inmunomodulator prostaglandin E (PGE) were analyzed. In order to know how the integrity of the tissue was altered after induction of cystogenesis, the oxidative status was also evaluated. Serum E and P levels, and ovarian PGE concentration, were increased in animals with cysts compared with healthy controls. The oxidant status (quantified by malondialdehyde (MDA) formed after the breakdown of the cellular membrane by free radical mechanisms) was augmented, meanwhile the antioxidant (evaluated by the glutathione (GSH) content) diminished during the induction of cystogenesis. Both immunohistochemical and flow cytometry assays demonstrated that DHEA treatment increased the number of T lymphocytes infiltrating ovarian tissue. Therefore, while ovarian controls showed equivalent expression of CD4+ and CD8+ T cell subsets, injection of DHEA yielded a selective ovarian T cell infiltration as demonstrated by enhanced CD8+ and diminished CD4+ T lymphocyte expression. These results show that the development of cysts involves changes in ovarian function and an imbalance in the oxidant-antioxidant equilibrium. We observed also both an increased and selective T lymphocyte infiltration.

Localization of isoketal adducts in vivo using a single-chain antibody.

Isoketals are highly reactive gamma-ketoaldehydes formed by the oxidation of arachidonic acid that rapidly adduct to proteins. To investigate the formation of isoketal adducts in vivo, we isolated and characterized a single-chain antibody from a phage displayed recombinant ScFv library that bound a model peptide adducted with synthetic 15-E2-isoketal. Recognition of isoketal adduct by this anti-isoketal adduct single-chain antibody was essentially independent of the amino acid sequence of adducted peptides or proteins. The antibody did not cross-react with 4-hydroxynonenal or 4-oxononanal adducts or with 15-F2t-isoprostane (8-iso-prostaglandin F2alpha). We investigated the formation of isoketal adducts in a well-established model of oxidative injury, hyperoxia. Exposure to >98% oxygen for 7 h dramatically increased both the number of immunoreactive airway epithelial cells and the intensity of immunoreactivity compared with animals exposed to normal room air (21% oxygen). We conclude that isoketal adducts form in epithelial cells as a result of high oxygen exposure and that this single-chain antibody provides a valuable tool to localize the formation of isoketal adducts in tissues in vivo.

The impact of induced stress during days 13 and 14 of pregnancy on the composition of allantoic fluid and conceptus development in sows.

Stress due to regrouping of breeding females is difficult to avoid completely in loose-housing systems. The effects of stress during the maternal recognition of pregnancy on fetal development and survival at Day 30 of pregnancy was, therefore, studied in 17 sows allocated into one control (C-) group, one group deprived of food during Days 13 and 14 (FD-), and one group (A-), which was treated with ACTH (0.01 mg/kg body weight of Synacthen Depot) every sixth hour during the same period. Total number of fetuses, fetal survival rate, volume of allantoic fluid, and the weight and length of total fetal unit, placentas, allantochorion and fetuses were determined. The concentrations of progesterone (P4), PGFM, PGF2, PGE, estrone-sulfate, and estradiol-17beta in the allantoic fluid were analyzed. No significant differences between groups were found for any parameter measured except for P4. Food deprivation increased P4 concentration in the allantoic fluid, and there was a positive correlation between the P4 concentration and the weight of the placenta. It is, therefore, suggested that P4 influences the placenta size among food-deprived sows.

Liver resistance to CCl(4)-induced injury after stimulation of macrophages with various preparations.

Acute toxic hepatitis in male Wistar rats was produced by single injection of 40% CCl(4) (0.2 ml per 100 g body weight in oil). Pretreatment with various immunostimulators (bacterial polysaccharides prodigiozan and salmozan; yeast polysaccharides zymosan, peptidoglycan, and mannan; and hydrolytic enzyme egg lysozyme) produced a hepatoprotective effect correlating which the stimulatory influence on macrophages and increasing in the following order: mannan

Bone marrow cells inhibit the generation of autologous EBV-specific CTL.

Recently, we reported that human bone marrow cells (BMC) inhibited the proliferative (recall) response of lymphocytes to Epstein-Barr virus (EBV) and cytomegalovirus (CMV) protein antigens [12]. To clarify further the effect of BMC on the immune response to viral antigens, we obtained PBL from EBV IgG antibody positive kidney transplant recipients (R) and their living-related donors (LRD) 1 year after renal transplantation and generated EBV-specific CTL in vitro in the presence or absence of autologous BMC. The addition of freshly aspirated autologous iliac crest BMC from either R or LRD caused a significant inhibitory effect on the generation of EBV-specific CTL from CTL precursors, in contrast to the addition of autologous PBL used as controls (62.29 +/- 10.85% inhibition using BMC from the kidney transplant recipients; 74.47 +/- 15.21% inhibition using BMC from the living-related donors). This inhibitory effect was only exerted during the CTL generation phase; but not in the effector CTL killing phase. The expression of CD94, a component of the killer inhibitory receptor (KIR) on CD3(+) cells was elevated in the cultures with BMC, in contrast to the cultures without BMC. The BMC inhibitory effect was partially abrogated by pre-incubation of the CTL effectors with anti-CD94 monoclonal antibody, in contrast with its isotype control. In addition, supernatants obtained from the CTL generating cultures with BMC contained high levels of prostaglandin E(2) (PGE(2)), and EBV-specific CTL activity was inhibited by the addition of exogenous PGE(2) in the absence of BMC. The induction of CD40L cell surface expression by anti-CD3 was also decreased on the effector T cell population when BMC were added. There was a concomitant reduction in protein kinase C (PKC) activity. These studies demonstrate that BMC exert an inhibitory effect on T cell-mediated immunity to viral antigens in humans by regulating autologous effector T cell generation and early T cell activation signaling pathways.

Evaluation of the effects of omega-3 fatty acid-containing diets on the inflammatory stage of wound healing in dogs.

OBJECTIVES: To ascertain the effects of dietary omega-3 (n-3) fatty acids on biochemical and histopathologic components of the inflammatory stage of wound healing. ANIMALS: 30 purpose-bred Beagles. PROCEDURE: Dogs were allotted to 5 groups of 6. Each group was fed a unique dietary fatty acid ratio of omega-6 to n-3--diet A, 5.3:1; diet B, 10.4:1; diet C, 24.1:1; diet D, 51.6:1; and diet E, 95.8:1. Dogs were fed once daily for 12 weeks, then biopsy specimens were taken from 4-day-old wounds of each dog and analyzed by gas chromatography-mass spectrometry for: prostaglandin E2 (PGE2) metabolites, and ratios of omega-6 to n-3 fatty acids, arachidonic acid (AA) to eicosapentaenoic acid (EPA), adrenic acid to docosahexaenoic acid, and PGE2 to prostaglandin E3 (PGE3) metabolites. RESULTS: Qualitative analysis was carried out on AA, EPA, adrenic acid, docosahexaenoic acid, and the major metabolite from the PGE2 and PGE3 pathway. These molecules were further quantified with respect to diet to determine significant differences. By analysis of the AA-to-EPA ratio, diet A was different from diets D and E and diets B and C were different from diet E (P < 0.05). By analysis of the PGE2-to-PGE3 metabolite ratio, diet A was different from diet E (P < 0.05). Though biochemical analysis indicated dietary dependence, histopathologic data indicated no significant difference with respect to diet groups. CONCLUSION: The biochemical component of the inflammatory stage of wound healing can be manipulated by diet. CLINICAL RELEVANCE: Omega-3 fatty acid-enriched diets can be used to control inflammation associated with dermatologic conditions.

Calcium and protein kinase C play a significant role in response to Shigella toxin in rabbit ileum both in vivo and in vitro.

The role of second messengers in Shigella toxin (STx) induced fluid secretion in rabbit ileum was evaluated. In vivo and in vitro studies were carried out in presence or absence of following modulators: Ca2+ ionophore A23187 (15 microM), l-verapamil (200 microM), phorbol-12-myristate-13-acetate (PMA, 200 ng), 1-(5-isoquinolinyl-sulphonyl)-2-methyl-piperazine (H-7, 15 microg) and indomethacin (20 microM). In in vivo studies, the fluid accumulation into rabbit ileal loops in response to STx was measured in presence or absence of these modulators. In in vitro studies, unidirectional fluxes of Na+ and Cl- were carried out in presence or absence of these modulators. The addition of Ca2+ ionophore A23187 along with STx further increases the amount of fluid already induced by STx. Whereas the presence of l-verapamil along with STx did not decrease the amount of fluid induced by STx. In vitro findings were in consonance with the in vivo studies. A significant increase in inositol triphosphate (IP3) levels was observed in enterocytes isolated from STx treated rabbit ileum. The addition of PMA into rabbit ileal loops in presence of STx mimicked the effect of STx while the presence of H-7 reversed the secretion caused by STx to absorption. Similar results were obtained while determining unidirectional fluxes of Na+ and Cl- in presence of PMA and also with H-7. A significant increase in PKC levels was observed in the membrane fraction of enterocytes isolated from STx treated rabbit ileum as compared to control. Further a marked decrease in PKC levels was observed in the presence of H-7 in membrane fraction of enterocytes isolated from STx treated rabbit ileum. The addition of indomethacin into rabbit ileal loops reversed the secretion (caused by STx) to absorption. In vitro findings were in consonance with in vivo studies. Besides, there was a significant increase in PG-E levels in enterocytes isolated from STx treated rabbit ileum as compared to control. These findings suggested that STx induced enteritis involves the role of PKC, intracellular calcium stores and prostaglandins. The extracellular calcium pool probably does not play a significant role in this process.

Prostaglandins and ovum implantation in mice.

The possible involvement of prostaglandins (PGs) in events of ovum implantation is investigated. The levels of PGF and PGE-A were measured by radioimmunoassay in the implantation and interimplantation sites on days 4, 5, 6, and 7 of pregnancy. The concentration of prostaglandins was determined in morulae and blastocysts also. The levels of PGE-A were higher in implantation sites in comparison to PGF. PGE-A level showed a peak on d5 and remained significantly higher on d6 and d7, in comparison to d4. The concentration of PGF was found to be very low in both interimplantation sites and implantation sites before implantation. The concentration of PGF showed a significant increase on d6 in the interimplantation sites which peaked up to 32.61 +/- 2.01 ng on d7. The embryos showed an increase in PGE-A concentration along with development. However, PGF could not be found in the embryos. The present result shows that in mice PGE is the main prostaglandin involved in ovum implantation and PGF is associated with maintaining the embryos in the uterus. It is also predicted that in mice, blastocysts differentially stimulate PG synthesis in the uterus between implantation sites and interimplantation sites.

In vitro effects of prostaglandin E2 or indomethacin on the proliferation of lymphokine-activated killer cells and their cytotoxicity against bladder tumor cells in patients with bladder cancer.

PURPOSE: To investigate the combined effects of interleukin-2 (IL-2) with either prostaglandin E2 (PGE2) or indomethacin (IM) on the proliferation and cytolysis of bladder tumor cells by lymphokine-activated killer (LAK) cells in patients with bladder cancer. METHODS: LAK cell proliferation was assayed in the presence of various concentrations of either PGE2 or IM by cell counting. Bladder cancer cell lines BIU-87, EJ and bladder tumor cells (BTC) from the patients were cultured as target cells, and the cytotoxicity of LAK cells was determined by 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, PGE2 in samples of conditioned medium from bladder cancer cells or peripheral blood mononuclear cells (PBMC) as well as plasma from 21 patients with bladder cancer and 20 healthy donors were determined by radioimmunoassay (RIA). RESULTS: The proliferation of LAK cells induced by IL-2 was inhibited by PGE2 (0.05 to 5 ng/mL) in concentration-dependent manner. The enhanced growth of LAK cells was observed at certain concentrations of IM (100-400 ng/mL) from 48 to 96 h. Pretreatment of LAK cells with IM (200 ng/mL) significantly enhanced cytotoxicity against BIU-87, EJ cells, or BTC. More PGE2 was present in conditioned medium from BIU-87 cells than in the conditioned medium from PBMC. CONCLUSIONS: These studies indicate that LAK cell proliferation induced by IL-2 in patients with bladder cancer is inhibited by PGE2 produced by PBMC and bladder cancer cells. This inhibition can be overcome by IM, which may be of use in immunotherapy of bladder cancer.

[Prostaglandins of series E in morphologic variants of osteogenic sarcoma and evaluation of the degree of therapeutic pathomorphism of the tumor]. / Prostaglandiny serii E v razlichnykh morfologicheskikh variantakh osteogennoi sarkomy i otsenke stepeni lechebnogo patomorfoza opukholi.

The content of prostaglandins E (PGE) is studied in osteogenic sarcomas from 191 patients. The level of PGE after a neo-adjuvant therapy of osteogenic sarcoma depends upon the individual susceptibility to the drug. Inverse correlation is found between the PGE content in the tumor and the degree of therapeutic pathomorphosis when adriamycin and methotrexate are used before the operation. Single transfusion of the allogenic bone-marrow suspension results in a considerable decrease of the PGE level in the osteogenic sarcoma. Possible mechanisms of action of different therapeutical methods on the PGE level as well as use of the drugs influencing arachidonic acid metabolism in the tumor are discussed.

Ammonium urate nephrolithiasis in a variant of Bartter's syndrome with intact renal tubular function.

In two patients with Bartter's syndrome proximal tubular function and distal chloride reabsorption were intact on admission; however, chloride reabsorption and distal tubular acidifying capacity decreased in one patient over a period of 10 years. Renal prostaglandin E excretion and urinary and plasma uric acid were in the normal range, but urinary ammonium was significantly elevated during controlled diet. One patient developed ammonium urate nephrolithiasis. In both patients renal biopsy demonstrated lymphocytic infiltration of the interstitial tissue and hypercellularity of the macula densa. Indomethacin treatment improved serum potassium concentration and decreased plasma renin activity, plasma aldosterone concentration, and urinary prostaglandin E but had to be discontinued because of side effects. It is likely that our patients represent a variant form of the syndrome originally described by Bartter.

[The characteristics of prostaglandin E synthesis in epithelial ovarian tumors]. / Osobennosti sinteza prostaglandinov serii E v épitelial'nykh opukholiakh iaichnikov.

Comparative investigation of prostaglandin E (PGE) levels in primary malignant and benign epithelial tumors, metastases, and normal tissue of the ovaries has revealed more significant variations in PGE levels in malignant tumors than in benign ones. PGE content was reliably higher in adenocarcinomas as against benign tumors and normal ovarian tissue. No noticeable differences in PGE levels of benign tumors and normal tissue were revealed. The possibility of using arachidonic acid cyclooxygenase metabolism blockers in therapy of ovarian carcinoma, besides the common methods of treatment, is discussed.

[De-Nol in the treatment of patients with duodenal peptic ulcer]. / De-Nol v likuvanni khvorykh na vyrazkovu khvorobu dvanadtsiatypaloï kyshky.

A clinico-morphological study was made of processes in the gastro-duodenal mucosa during treatment of duodenal ulcer by de-nol. Results indicate that the dyspeptic and pain syndrome disappeared after de-nol treatment within 4-7 days in 78.3% of patients, the ulcer scarred in the course of 21.4 days. Advantages of de-nol treatment are shown electron-microscopically, by studying biopsy specimens of the mucosa before and after treatment.