Lipopeptide surfactin ameliorates the cell uptake of platensimycin and enhances its therapeutic effect on treatment of MRSA skin infection.

OBJECTIVES: The rapid development of drug-resistant bacteria, especially MRSA, poses severe threats to global public health. Adoption of antibiotic adjuvants has proved to be one of the efficient ways to solve such a crisis. Platensimycin and surfactin were comprehensively studied to combat prevalent MRSA skin infection. METHODS: MICs of platensimycin, surfactin or their combinations were determined by resazurin assay, while the corresponding MBCs were determined by chequerboard assay. Growth inhibition curves and biofilm inhibition were determined by OD measurements. Membrane permeability analysis was conducted by propidium iodide staining, and morphological characterizations were performed by scanning electron microscopy. Finally, the therapeutic effects on MRSA skin infections were evaluated in scald-model mice. RESULTS: The in vitro assays indicated that surfactin could significantly improve the antibacterial performance of platensimycin against MRSA, especially the bactericidal activity. Subsequent mechanistic studies revealed that surfactin not only interfered with the biofilm formation of MRSA, but also disturbed their cell membranes to enhance membrane permeability, and therefore synergistically ameliorated MRSA cellular uptake of platensimycin. Further in vivo assessment validated the synergistic effect of surfactin on platensimycin and the resultant enhancement of therapeutical efficacy in MRSA skin-infected mice. CONCLUSIONS: The combination of effective and biosafe surfactin and platensimycin could be a promising and efficient treatment for MRSA skin infection, which could provide a feasible solution to combat the major global health threats caused by MRSA.

Screening for Active Compounds Targeting Human Natural Killer Cell Activation Identifying Daphnetin as an Enhancer for IFN-γ Production and Direct Cytotoxicity.

Natural killer (NK) cells are a potent weapon against tumor and viral infection. Finding active compounds with the capacity of enhancing NK cell effector functions will be effective to develop new anti-cancer drugs. In this study, we initially screened 287 commercially available active compounds by co-culturing with peripheral blood mononuclear cells (PBMCs). We found that five compounds, namely, Daphnetin, MK-8617, LW6, JIB-04, and IOX1, increased the IFN-γ+ NK cell ratio in the presence of IL-12. Further studies using purified human primary NK cells revealed that Daphnetin directly promoted NK cell IFN-γ production in the presence of IL-12 but not IL-15, while the other four compounds acted on NK cells indirectly. Daphnetin also improved the direct cytotoxicity of NK cells against tumor cells in the presence of IL-12. Through RNA-sequencing, we found that PI3K-Akt-mTOR signaling acted as a central pathway in Daphnetin-mediated NK cell activation in the presence of IL-12. This was further confirmed by the finding that both inhibitors of PI3K-Akt and its main downstream signaling mTOR, LY294002, and rapamycin, respectively, can reverse the increase of IFN-γ production and cytotoxicity in NK cells promoted by Daphnetin. Collectively, we identify a natural product, Daphnetin, with the capacity of promoting human NK cell activation via PI3K-Akt-mTOR signaling in the presence of IL-12. Our current study opens up a new potential application for Daphnetin as a complementary immunomodulator for cancer treatments.

Homoadamantane and Adamantane Acylphloroglucinols from Hypericum hirsutum.

1H NMR-guided fractionation of the petroleum ether extract of the aerial parts from Hypericum hirsutum yielded to the isolation of 19 polyprenylated polycyclic acylphloroglucinols. Structure elucidation based on 1D and 2D NMR spectroscopy together with high-resolution electrospray ionization mass spectroscopy revealed 14 acylphloroglucinols with a homoadamantane scaffold (1: -14: ), while 5 further compounds showed an adamantane skeleton (15: -19: ). Except for hookerione C (15: ), all isolated metabolites are hitherto unknown. While structurally-related metabolites have been isolated from other Hypericum species, it is the first report of admantan and homoadamantan type acylphloroglucinols in section Taeniocarpium Jaub. & Spach (Hypericaceae). The isolated compounds have been tested in a crystal violet-based in vitro assay on their properties to reduce the proliferation of human microvascular endothelial cells compared to hyperforin as the positive control. They showed a moderate reduction of proliferation with IC50 values in the range ~ 3 - 22 µM, with the homoadamantane-based compounds 2: and 4: being the most active. In addition, inhibition of the TNF-α-induced ICAM-1 expression was determined for 1:  - 5, 7,: and 10:  - 12: . Substances 3: and 12: reduced the ICAM-1 expression significantly (to 46.7% of control for 3: , 62.3% for 12,: at 50 µM).

Nanoparticle-Hydrogel Systems Containing Platensimycin for Local Treatment of Methicillin-Resistant Staphylococcus aureus Infection.

Skin and soft tissue infections require effective and sustained topical administration. Platensimycin (PTM) is a natural drug lead that targets bacterial fatty acid synthases and has a great potential to treat infections caused by methicillin-resistant Staphylococcus aureus (MRSA). To facilitate the use of PTM against local MRSA infections, we prepared polyacrylamide hydrogels containing polyamidoamine (PAMAM)/PTM nanoparticles (NP-gel(PTM)) for the controlled release of PTM. NP-gel(PTM) can continuously inhibit the growth of MRSA and its biofilm formation in simulated drug flow models in vitro. In situ implantation of NP-gel(PTM) could treat MRSA-infected subcutaneous soft tissues without toxicity. For MRSA-infected skin wounds, NP-gel(PTM) not only showed strong anti-MRSA activity but also accelerated more wound healing than the widely used antibiotic mupirocin. Collectively, PTM is expected to be used in this safe and effective NP-gel delivery platform for the treatment of local infections, which might help to alleviate the current antibiotic resistance crisis.

An overview of therapeutic potential of N-alkylated 1-deoxynojirimycin congeners.

Polyhydroxylated alkaloids display a wide range of biological activities, suggesting their use in the treatment of various diseases. Their most famous representative, 1-deoxynojirimycin (DNJ), is a natural product that shows α- and ß-glucosidase inhibition. This molecule has been since converted into two clinically approved drugs i.e., Zavesca® and Glyset®, targeting type I Gaucher's disease and type II diabetes mellitus, respectively. This review examines the therapeutic potential of important DNJ congeners reported in last decade and presents concise mechanism of glycosidase inhibition. A brief overview of substituents conjugation's impact on DNJ scaffold (including N-alkylated DNJ derivatives, mono-valent, di-valent and multivalent DNJ congeners, N-[5-(adamantan-1-yl-methoxy)-pentyl]-1-deoxynojirimycin (AMP-DNM) look alike DNJ based lipophilic derivatives, AMP-DNM based neoglycoconjugates, DNJ click derivatives with varying carboxylic acids and aromatic moieties, conjugates of DNJ and glucose, and N-bridged DNJ analogues) towards various enzymes such as α/ß glucosidase, porcine trehalase, as F508del-CFTR correctors, α-mannosidase, human placental ß-glucocerebrosidase, N370S ß-GCase, α-amylase and insect trehalase as potent and selective inhibitors have been discussed with potential bioactivities, which can provide inspiration for future studies.

1,3,5-Triaza-7-Phosphaadamantane (PTA) as a 31P NMR Probe for Organometallic Transition Metal Complexes in Solution.

Due to the rigid structure of 1,3,5-triaza-7-phosphaadamantane (PTA), its 31P chemical shift solely depends on non-covalent interactions in which the molecule is involved. The maximum range of change caused by the most common of these, hydrogen bonding, is only 6 ppm, because the active site is one of the PTA nitrogen atoms. In contrast, when the PTA phosphorus atom is coordinated to a metal, the range of change exceeds 100 ppm. This feature can be used to support or reject specific structural models of organometallic transition metal complexes in solution by comparing the experimental and Density Functional Theory (DFT) calculated values of this 31P chemical shift. This approach has been tested on a variety of the metals of groups 8-12 and molecular structures. General recommendations for appropriate basis sets are reported.

A comparative study of acarbose, vildagliptin and saxagliptin intended for better efficacy and safety on type 2 diabetes mellitus treatment.

As a complicated metabolic disorder, type 2 diabetes mellitus (T2DM) is becoming a major health concern worldwide. Drugs including acarbose, saxagliptin and vildagliptin are applied, but their efficacy is still required to be compared. Therefore, the study aimed to evaluate the efficacy and safety of acarbose, saxagliptin and vildagliptin in the treatment of T2DM. Ninety patients diagnosed with T2DM were treated with acarbose, saxagliptin and vildagliptin, respectively (30 patients for each drug). All patients were examined at 0, 4 and 12 weeks after treatment with vital signs recorded. Fasting blood glucose and blood biochemical indices were analyzed. In addition, fecal samples were taken for microbial macrogenome sequencing and safety evaluation within 12 weeks after treatment. Blood glucose level decreased at 4 and 12 weeks after treatment, and the total cholesterol (TC) and high-density lipoprotein (HDL) levels at 12 weeks were different. Genus abundance of intestinal flora was altered at different time points. Acarbose increased Butyricimonas level first and then decreased it during drug treatment. Saxagliptin increased Megamonas and decreased Turicibacter genus level gradually. Pseudomonas, Klebsiella, Blautia, Faecalibacterium and Roseburia levels fluctuated after Vildagliptin treatment, which increased fasting C-peptide level greater than the other two drugs. Saxagliptin showed higher adverse reactions than acarbose and vildagliptin. Collectively, acarbose, vildagliptin, and saxagliptin can effectively reduce the HbA1c level and affect the intestinal flora distribution in T2DM patients, and the adverse reactions of acarbose and vildagliptin are less than saxagliptin, providing alternative strategies for the treatment of T2DM.

Neurotensin receptor 1 agonist provides neuroprotection in pre-diabetic rats.

Exogenous treatment of a neurotensin receptor 1 (NTR1) agonist exerted the neuroprotection in an obese and Alzheimer's model. However, the effects of NTR1 modulation on peripheral/hippocampal impairment and cognitive deficit following sustained HFD consumption are poorly understood. Forty rats received a normal diet (ND) or HFD for 16 weeks. At week 13, the ND group received a vehicle (n = 8). Thirty-two HFD-fed group were randomized into four subgroups (n = 8/subgroup) with a vehicle, 1 mg/kg of NTR1 agonist, 1 mg/kg of NTR antagonist, and combined treatment (NTR1 agonist-NTR antagonist) for 2 weeks, s.c. injection. Then, the cognitive tests and peripheral/hippocampal parameters were determined. Our findings demonstrated that NTR1 activator reversed obesity and attenuated metabolic impairment in pre-diabetic rats. It also alleviated hippocampal pathologies and synaptic dysplasticity, leading to deceleration or prevention of cognitive impairment progression. Therefore, NTR1 activation would be a possible novel therapy to decelerate or prevent progression of neuropathology and cognitive impairment in the pre-diabetes.

Effect of stevia aqueous extract on the antidiabetic activity of saxagliptin in diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Stevia rebaudiana Bertoni is a perennial herb that belongs to the Asteraceae family. It is a natural sweetener plant known as "Sweet Leaf", "Sweet Herbs" and "Honey Leaf", which is estimated to be 300 times more sweetening than sugar cane. Stevia has been used as a traditional treatment for diabetes in many countries for hundreds of years. Several animal studies referred to the antihyperglycemic activity of stevia. However, the combined use of stevia with saxagliptin has not been studied so far, so this study has been done. The aim of the present study was to evaluate the antihyperglycemic effect of stevia alone and in combination with saxagliptin. MATERIALS AND METHODS: Diabetes was induced in rats by i.p. injection of streptozotocin and nicotinamide. Animals were divided into five groups, each contains eight rats. Group I: included negative controland group II: included diabetic control that received saline. Group III: included diabetic rats that received 400 mg/kg/day stevia aqueous extract. Group IV: included diabetic rats that received saxagliptin 10 mg/kg/day. Group V: included diabetic rats that received stevia 400 mg/kg + saxagliptin 10 mg/kg. Food and water intake were measured daily while body weight was measured weekly. After 3 weeks animals were sacrificed and blood and tissue samples were collected. Fasting blood glucose (FBG), serum insulin, serum dipeptidylepeptidase-4 (DPP-4), TC, TGs, LDL, HDL, GSH and MDA were measured in treated and control rats by colorimetric and ELISA methods. RESULTS: Both stevia and saxagliptin significantly reduced food, water intake, body weight and FBG. Stevia with saxagliptin produced more significant decrease in FBG. While serum insulin increased significantly in stevia, saxagliptin treated groups and their combination. Serum DPP-4 decreased significantly in all treated groups, concerning lipid profile, stevia and saxagliptin notably lowered TC, TGs, and LDL and increased HDL. Both stevia and saxagliptin remarkably decreased MDA and increased GSH compared to diabetic rats. In addition, stevia significantly improved the antidiabetic effects of saxagliptin. CONCLUSION: Stevia has an antihyperglycemic effect and could enhance the antidiabetic activity of saxagliptin. DPP-4 attenuation, antihyperlipidemic and antioxidant activity as well as improvement of insulin sensitivity may be involved in the antidiabetic action of stevia.

Anti-adipogenicadamantane type polycyclic polyprenylated acylphloroglucinols from Hypericum subsessile.

Hypersubones D-H (1-5), five new polycyclic polyprenylated acylphloroglucinols (PPAPs) type metabolites with intriguing adamantane and homo-adamantane skeletons, were characterized from aerial parts of Hypericum subsessile. Compounds 1-2 were elucidated to share an adamantane core with 28,29-expoxide moiety, while 3-5 were homo-adamantane type PPAPs sharing a1,2-dioxepane ring system. Their structures were determined on the basis of comprehensive NMR and MS spectroscopic data.The anti-adipogenesis activities of these isolates were evaluated through employing 3T3-L1 cells as an in vitro system using oil red O staining, and compounds 1, 2 and 5 were able to significant inhibit the adipocyte differentiation, which implied that these compounds possessed anti-adipogenic activity.

[Delivery of Anticancer Drugs Using a Supramolecular Complex to Cancer Tissues Having High Interstitial Fluid Pressure].

Pancreatic cancer is the fourth-leading cause of death from cancer in Japan, after lung, colorectal, and stomach cancers and has the lowest survival among these tumors, because of not only no symptoms, no screening tool and no biomarkers but also high rates of recurrence and metastasis. In addition, pancreatic cancer has excessive stroma which serves as a severe biological barrier for anticancer drug delivery and successful treatment. Therefore, there are many challenges for drug delivery systems for the treatment of pancreatic cancer. Recently, we developed self-assembly PEGylation retaining activity (SPRA) technology, which comprises a reversible pegylated protein complex without loss of bioactivity. SPRA technology is based on a host-guest interaction between PEGylated ß-cyclodextrin and adamantane-appended protein. In this review, first pancreatic cancer is introduced, second, principle drug delivery systems for the treatment of pancreatic cancer are described, and third the concept of SPRA technology as well as examples of SPRA proteins, especially focusing on the potential of SPRA-bromelain for treatment of pancreatic cancer, are introduced.

Hyperacmosins E-G, three new homoadamantane-type polyprenylated acylphloroglucinols from Hypericum acmosepalum.

Three new homoadamantane-type polyprenylated acylphloroglucinols, hyperacmosins E-G (1-3), with seven known compounds were isolated from the air-dried aerial parts of Hypericum asmosepalum. Their structures were determined by NMR, HRESIMS and experimental electronic circular dichroism (ECD) spectra. The hepatoprotective activity of these compounds were evaluated. Compounds 4 and 8 exhibited hepatoprotective activity against paracetamol-induced HepG2 cell damage.

Actin Cytoskeleton-Disrupting and Magnetic Field-Responsive Multivalent Supramolecular Assemblies for Efficient Cancer Therapy.

Actin cytoskeleton disruption is a promising and intriguing anticancer strategy, but their efficiency is frequently compromised by severe side effects of the actin cytoskeleton-disrupting agents. In this study, we constructed the biocompatible actin cytoskeleton-targeting multivalent supramolecular assemblies that specifically target and disrupt the tumor actin cytoskeleton for cancer therapy. The assemblies were composed of ß-cyclodextrin-grafted hyaluronic acid (HACD) and iron oxide magnetic nanoparticles (MNPs) grafted by an actin-binding peptide (ABP) and adamantane (Ada)-modified polylysine. Owing to the multivalent binding between cyclodextrin and Ada, HACD, and peptide-grafted MNPs (MNP-ABP-Ada) could self-assemble to form MNP-ABP-Ada⊂HACD nanofibers in a geomagnetism-dependent manner. Furthermore, the presence of ABP rendered the assemblies to efficiently target the actin cytoskeleton. Interestingly, with the acid of a low-frequency alternating magnetic field (200 Hz), the actin cytoskeleton-targeting nanofibers could induce severe actin disruption, leading to a remarkable cell cycle arrest and drastic cell death of tumor cells both in vitro and in vivo, but showed no obvious toxicity to normal cells. The actin cytoskeleton-targeting/disrupting supramolecular assembly implies an excellent strategy for realizing efficient cancer therapy.

The extracellular calcium-sensing receptor promotes porcine egg activation via calcium/calmodulin-dependent protein kinase II.

Extracellular calcium is required for intracellular Ca2+ oscillations needed for egg activation, but the regulatory mechanism is still poorly understood. The present study was designed to demonstrate the function of calcium-sensing receptor (CASR), which could recognize extracellular calcium as first messenger, during porcine egg activation. CASR expression was markedly upregulated following egg activation. Functionally, the addition of CASR agonist NPS R-568 significantly enhanced pronuclear formation rate, while supplementation of CASR antagonist NPS2390 compromised egg activation. There was no change in NPS R-568 group compared with control group when the egg activation was performed without extracellular calcium addition. The addition of NPS2390 precluded the activation-dependent [Ca2+ ]i rise. When egg activation was conducted in intracellular Ca2+ chelator BAPTA-AM and NPS R-568 containing medium, CASR function was abolished. Meanwhile, CASR activation increased the level of the [Ca2+ ]i effector p-CAMKII, and the presence of KN-93, an inhibitor of CAMKII, significantly reduced the CASR-mediated increasement of pronuclear formation rate. Furthermore, the increase of CASR expression following activation was reversed by inhibiting CAMKII activity, supporting a positive feedback loop between CAMKII and CASR. Altogether, these findings provide a new pathway of egg activation about CASR, as the extracellular Ca2+ effector, promotes egg activation via its downstream effector and upstream regulator CAMKII.

Impact of baseline characteristics on glycemic effects of add-on saxagliptin or acarbose to metformin therapy: Subgroup analysis of the SMART study in Chinese patients with type 2 diabetes mellitus.

AIMS/INTRODUCTION: This secondary analysis of the 24-week SMART study examined the efficacy of add-on saxagliptin or acarbose to metformin across different patient subgroups with type 2 diabetes mellitus, based on baseline characteristics. MATERIALS AND METHODS: Randomized patients (n = 481) were classified into subgroups based on their baseline age (<65, ≥65 years), body mass index (BMI; <24, 24-<28, ≥28 kg/m2 ), glycated hemoglobin (HbA1c; <8%, 8-<9%, 9-<10%, ≥10%) and renal function (creatinine clearance 50-<80, ≥80 mL/min). Treatment effects on primary outcome (HbA1c) and key secondary outcomes of fasting plasma glucose (FPG), 2-h postprandial glucose and homeostatic model assessment of ß-cell function were assessed across patient subgroups. RESULTS: For saxagliptin, reductions in HbA1c from baseline to week 24 were consistent across different subgroups regardless of baseline age, body mass index, HbA1c and renal function (range -0.66 to -1.16%). Saxagliptin was associated with consistent reductions in FPG (-0.60 to -1.33 mmol/L) and 2-h postprandial glucose (-0.48 to -1.95 mmol/L) across the majority of subgroups studied. The efficacy of acarbose on FPG attenuated progressively with increasing baseline HbA1c (+0.86 to -1.43 mmol/L); an increase from baseline FPG was observed in patients with HbA1c >9%. The effect of acarbose on postprandial glucose was also variable (+0.23 to -3.38 mmol/L). CONCLUSIONS: As add-on to metformin, both saxagliptin and acarbose reduced HbA1c regardless of baseline HbA1c, age, body mass index and renal function; however, only saxagliptin was effective at a stable glycemic control (FPG and PPG). The efficacy of acarbose on FPG and PPG was significantly attenuated in patients with higher baseline HbA1c (≥8%).

Kisspeptin and RF9 prevent paroxetine-induced changes in some parameters of seminal vesicle fluid in the male rats.

The aim of the study was to examine possible impacts of paroxetine and agomelatine on the levels of some components that constitute the seminal vesicle fluid. As a second purpose, it was also aimed to examine how possible negative effects induced by paroxetine on seminal vesicle fluid components were affected by kisspeptin and RF9 (an RFamide-related peptide antagonist, RFRP). Forty-two male rats, aged 21 days, divided into six groups; control, sham, paroxetine, agomelatine, paroxetine + kisspeptin and paroxetine + RF9. Paroxetine (3.6 mg/kg) and agomelatine (10 mg/kg) were administrated by oral gavage. Kisspeptin (1 nmol) and RF9 (20 nmol) were administered intracerebroventricular (i.c.v). The experiments were ended on post-natal 120 days; fructose, vitamin E, sodium, potassium and magnesium levels were measured in seminal vesicle fluid. Fructose, vitamin E, magnesium and potassium levels were significantly decreased in seminal vesicle fluid from the rats treated with paroxetine but did not show significant differences following agomelatine administration. The co-administration of kisspeptin or RF9 with paroxetine prevented the paroxetine-induced negative effects on seminal vesicle fluid components. These results suggest that reduction in sperm fertilising ability caused by changes in seminal vesicle fluid can be seen in long-term antidepressant use. RF-9 and kisspeptin might have positive effects on long-term antidepressant use-induced infertility.

Potential of the zebrafish model for the forensic toxicology screening of NPS: A comparative study of the effects of APINAC and methiopropamine on the behavior of zebrafish larvae and mice.

The increased diffusion of the so-called novel psychoactive substances (NPS) and their continuous change in structure andconceivably activity has led to the need of a rapid screening method to detect their biological effects as early as possible after their appearance in the market. This problem is very felt in forensic pathology and toxicology, so the preclinical study is fundamental in the approach to clinical and autopsy cases of difficult interpretation intoxication. Zebrafish is a high-throughput suitable model to rapidly hypothesize potential aversive or beneficial effects of novel molecules. In the present study, we measured and compared the behavioral responses to two novel neuroactive drugs, namely APINAC, a new cannabimimetic drug, and methiopropamine (MPA), a methamphetamine-like compound, on zebrafish larvae (ZL) and adult mice. By using an innovative statistical approach (general additive models), it was found that the spontaneous locomotor activity was impaired by the two drugs in both species: the disruption extent varied in a dose-dependent and time-dependent manner. Sensorimotor function was also altered: i) the visual object response was reduced in mice treated with APINAC, whereas it was not after exposure to MPA; ii) the visual placing responses were reduced after treatment with both NPS in mice. Furthermore, the visual motor response detected in ZL showed a reduction after treatment with APINAC during light-dark and dark-light transition. The same pattern was found in the MPA exposed groups only at the dark-light transition, while at the transition from light to dark, the individuals showed an increased response. In conclusion, the present study highlighted the impairment of spontaneous motor and sensorimotor behavior induced by MPA and APINAC administration in both species, thus confirming the usefulness of ZL as a model for a rapid behavioural-based drug screening.

RF9: May it be a new therapeutic option for hypogonadotropic hypogonadism?

Hypogonadotropic hypogonadism (secondary hypogonadism), congenital or acquired, is a form of hypogonadism that is due to problems with either the hypothalamus or pituitary gland affecting gonadotropin levels. Pulsatile secretion of gonadotropin-releasing hormone (GnRH) by hypothalamus is a primer step to initiate the release of pituitary gonadotropins. Kisspeptin and gonadotropin-inhibitory hormone (GnIH) are accepted as two major players in the activation and inhibition of GnRH regarding the neuroendocrine functioning of the hypothalamic pituitary gonadal axis. Kisspeptin is known as the most potent activator of GnRH. Regarding the inhibition of GnRH, RF-amide-related peptide-3 (RFRP-3) is accepted as the mammalian orthologue of GnIH in avian species. RF9 (1-adamantane carbonyl-Arg-Phe-NH2) is an antagonist of RFRP-3/GnIH receptor (neuropeptide FF receptor 1 (NPFFR1; also termed as GPR147). In recent years, several studies have indicated that RF9 activates GnRH neurons and gonadotropins in a kisspeptin receptor (Kiss1r, formerly known as GPR54) dependent manner. These results suggest that RF9 may have a bimodal function as both an RFRP-3 antagonist and a kisspeptin agonist or it may be a kiss1r agonist rather than an RFRP-3/GnIH receptor antagonist. These interactions are possible because Kisspeptin and GnIH are members of the RF-amide family, and both possibilities are not far from explaining the potent gonadotropin stimulating effects of RF9. Therefore, we hypothesize that RF9 may be a new therapeutic option for the hypogonadotropic hypogonadism due to its potent GnRH stimulating effects. A constant or repeated administration of RF9 provides a sustained increase in plasma gonadotrophin levels. However, applications in the same way with GnRH analogues and kisspeptin may result in desensitization of the gonadotropic axis. The reasons reported above contribute to our hypothesis that RF9 may be a good option in the GnRH stimulating as a kisspeptin agonist. We suggest that further studies are needed to elucidate the potential effects of RF9 in the treatment of the hypogonadotropic hypogonadism.

Opioid receptors inhibit the spinal AMPA receptor Ca2+ permeability that mediates latent pain sensitization.

Acute inflammation induces sensitization of nociceptive neurons and triggers the accumulation of calcium permeable (CP) α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) in the dorsal horn of the spinal cord. This coincides with behavioral signs of acute inflammatory pain, but whether CP-AMPARs contribute to chronic pain remains unclear. To evaluate this question, we first constructed current-voltage (IV) curves of C-fiber stimulus-evoked, AMPAR-mediated EPSCs in lamina II to test for inward rectification, a key characteristic of CP-AMPARs. We found that the intraplantar injection of complete Freund's adjuvant (CFA) induced an inward rectification at 3 d that persisted to 21 d after injury. Furthermore, the CP- AMPAR antagonist IEM-1460 (50 µM) inhibited AMPAR-evoked Ca2+ transients 21d after injury but had no effect in uninflamed mice. We then used a model of long-lasting vulnerability for chronic pain that is determined by the balance between latent central sensitization (LCS) and mu opioid receptor constitutive activity (MORCA). When administered 21 d after the intraplantar injection of CFA, intrathecal administration of the MORCA inverse agonist naltrexone (NTX, 1 µg, i.t.) reinstated mechanical hypersensitivity, and superfusion of spinal cord slices with NTX (10 µM) increased the peak amplitude of AMPAR-evoked Ca2+ transients in lamina II neurons. The CP-AMPAR antagonist naspm (0-10 nmol, i.t.) inhibited these NTX-induced increases in mechanical hypersensitivity. NTX had no effect in uninflamed mice. Subsequent western blot analysis of the postsynaptic density membrane fraction from lumbar dorsal horn revealed that CFA increased GluA1 expression at 2 d and GluA4 expression at both 2 and 21 d post-injury, indicating that not just the GluA1 subunit, but also the GluA4 subunit, contributes to the expression of CP-AMPARs and synaptic strength during hyperalgesia. GluA2 expression increased at 21 d, an unexpected result that requires further study. We conclude that after tissue injury, dorsal horn AMPARs retain a Ca2+ permeability that underlies LCS. Because of their effectiveness in reducing naltrexone-induced reinstatement of hyperalgesia and potentiation of AMPAR-evoked Ca2+ signals, CP-AMPAR inhibitors are a promising class of agents for the treatment of chronic inflammatory pain.

homo-Adamantane type polycyclic polyprenylated acylphloroglucinols from Hypericum hookerianum.

Hookeriones I-Q (1-9), nine new homo-adamantane type polycyclic polyprenylated acylphloroglucinols (PPAPs), were isolated from Hypericum hookerianum, along with twenty known analogues. These structures were determined on the basis of comprehensive NMR and MS spectroscopic data. Comprehensive analysis of the NMR data revealed the correlations between the configuration of H-18 and H-28 and the chemical shifts of related signals. The cytotoxicity and anti-allergic activities of the new isolates were evaluated, and several ones exhibited moderate cytotoxicity against ECA-109 cell lines.