Preventative treatment with Fluorothiazinon suppressed Acinetobacter baumannii-associated septicemia in mice.

The high prevalence of multidrug-resistant Acinetobacter baumannii has emerged as a serious problem in the treatment of nosocomial infections in the past three decades. Recently, we developed a new small-molecule inhibitor belonging to a class of 2,4-disubstituted-4H-[1,3,4]-thiadiazine-5-ones, Fluorothiazinon (FT, previously called CL-55). FT effectively suppressed the T3SS of Chlamydia spp., Pseudomonas aeruginosa, and Salmonella sp. without affecting bacterial growth in vitro. In this study, we describe that prophylactic use of FT for 4 days prior to challenge with resistant clinical isolates of A. baumannii (ABT-897-17 and 52TS19) suppressed septic infection in mice, resulting in improved survival, limited bacteraemia and decreased bacterial load in the organs of the mice. We show that FT had an inhibitory effect on A. baumannii biofilm formation in vitro and, to a greater extent, on biofilm maturation. In addition, FT inhibited Acinetobacter isolate-induced death of HeLa cells, which morphologically manifested as apoptosis. The mechanism of FT action on A. baumannii is currently being studied. FT may be a promising candidate for the development of a broad-spectrum anti-virulence drug to use in the prevention of nosocomial infections.

Antitumor effects of the multi-target tyrosine kinase inhibitor cabozantinib: a comprehensive review of the preclinical evidence.

INTRODUCTION: Altered receptor tyrosine kinase (RTK) signaling contributes to tumorigenesis and suppression of immune-mediated destruction of cancer cells. Cabozantinib is an oral tyrosine kinase inhibitor that inhibits several RTKs involved in tumorigenesis, and is approved for the treatment of patients with progressive metastatic medullary thyroid cancer, advanced renal cell carcinoma, and hepatocellular carcinoma that has been previously treated with sorafenib. AREAS COVERED: We present an up-to-date evaluation of preclinical evidence for RTK inhibition with cabozantinib, specifically VEGFR, MET, KIT, RET, AXL, FLT3, and associated antitumor effects. Preclinical investigations of cabozantinib in combination with other anticancer drugs are also reviewed. EXPERT OPINION: Preclinical evidence shows that cabozantinib has antitumor activity against various cancer cells and exhibits synergy with other anticancer agents, including immune checkpoint inhibitors and hormone receptor or metabolic pathway inhibitors. Further optimization of cabozantinib treatment requires the identification of biomarkers of response and resistance, and exploration of complementary drug targets. Investigation of mechanisms of adaptive resistance, such as epithelial to mesenchymal transition (cancer intrinsic) and immunomodulation by the tumor microenvironment (cancer extrinsic), as well as identification of novel drug targets based on characterization of cancer stem cell metabolomic phenotypes, appear to be promising approaches.

Rice Bran Extract Protected against LPS-Induced Neuroinflammation in Mice through Targeting PPAR-γ Nuclear Receptor.

PPAR-γ anti-inflammatory functions have received significant attention since its agonists have been shown to exert a wide range of protective effects in many experimental models of neurologic diseases. Rice bran is very rich in polyunsaturated fatty acids, which are reported to act as PPAR-γ partial agonists. Herein, the anti-inflammatory effect of rice bran extract (RBE) through PPAR-γ activation was evaluated in LPS-induced neuroinflammatory mouse model in comparison to pioglitazone (PG) using 80 Swiss albino mice. RBE (100 mg/kg) and PG (30 mg/kg) were given orally for 21 days and LPS (0.25 mg/kg) was injected intraperitoneally for the last 7 days. TNF-α and COX-2 brain contents were evaluated by real-time PCR and immunohistochemical analysis. In addition, NFκB binding to its response element was evaluated alongside with the effect of treatments on IκB gene expression. Furthermore, PPAR-γ sumoylation was also studied. Finally, histopathological examination was performed for different brain areas. RBE administration was found to protect against the LPS-induced inflammatory effects by decreasing the inflammatory mediator expression in mice brains. It also decreased PPAR-γ sumoylation without significant effect on IκB expression or NFκB binding to its response element. The majority of the effects were attenuated in presence of PPAR-γ antagonist (GW9662). Level of significance was set to P < 0.05. Such findings highlight the agonistic effect of RBE component(s) on PPAR-γ and support the hypothesis of involvement of PPAR-γ activation in its neuroprotective effect.

Iron Induces Cell Death and Strengthens the Efficacy of Antiandrogen Therapy in Prostate Cancer Models.

PURPOSE: In search of novel strategies to improve the outcome of advanced prostate cancer, we considered that prostate cancer cells rearrange iron homeostasis, favoring iron uptake and proliferation. We exploited this adaptation by exposing prostate cancer preclinical models to high-dose iron to induce toxicity and disrupt adaptation to androgen starvation. EXPERIMENTAL DESIGN: We analyzed markers of cell viability and mechanisms underlying iron toxicity in androgen receptor-positive VCaP and LNCaP, castration-resistant DU-145 and PC-3, and murine TRAMP-C2 cells treated with iron and/or the antiandrogen bicalutamide. We validated the results in vivo in VCaP and PC-3 xenografts and in TRAMP-C2 injected mice treated with iron and/or bicalutamide. RESULTS: Iron was toxic for all prostate cancer cells. In particular, VCaP, LNCaP, and TRAMP-C2 were highly iron sensitive. Toxicity was mediated by oxidative stress, which primarily affected lipids, promoting ferroptosis. In highly sensitive cells, iron additionally caused protein damage. High-basal iron content and oxidative status defined high iron sensitivity. Bicalutamide-iron combination exacerbated oxidative damage and cell death, triggering protein oxidation also in poorly iron-sensitive DU-145 and PC-3 cells.In vivo, iron reduced tumor growth in TRAMP-C2 and VCaP mice. In PC-3 xenografts, bicalutamide-iron combination caused protein oxidation and successfully impaired tumor expansion while single compounds were ineffective. Macrophages influenced body iron distribution but did not limit the iron effect on tumor expansion. CONCLUSIONS: Our models allow us to dissect the direct iron effect on cancer cells. We demonstrate the proof of principle that iron toxicity inhibits prostate cancer cell proliferation, proposing a novel tool to strengthen antiandrogen treatment efficacy.

Peroxisome proliferator-activated receptor-γ mediates the antihypertensive effects of acupuncture in spontaneously hypertensive rats.

We investigated a central antihypertensive effect of acupuncture in rostral ventrolateral medulla (RVLM) in spontaneously hypertensive rats (SHRs). In total, 56 rats were randomly divided into seven groups as follows: the SHR group, SHR+acupuncture (SHR+Acu) group, SHR+nonacupuncture (SHR+Non-acu) group, GW9662+acupuncture (GW9662+Acu) group, GW9662+GW1929 group, GW9662 group, and 2% DMSO group (n = 8 per group). The whole eight Wistar-Kyoto rats were assigned to the WKY group. The acupuncture treatment lasting for 14 days was performed at the Taichong acupoint (LR3) or at a nonacupoint (non-acu) once daily. The peroxisome proliferator-activated receptor (PPAR)-γ agonist GW1929 and the PPAR-γ inhibitor GW9662 were microinjected by the brain stereotactic technique. Blood pressure was measured by the tail-cuff method. Sympathetic vasomotor activity was determined by implanting in a telemetry electrocardiogram radio transmitter. The expression of PPARs in the RVLM of the rats was detected using Western blot. We demonstrated that acupuncture attenuated blood pressure, heart rate, and sympathetic vasomotor activity in SHRs. The protein expression of PPAR-γ was significantly increased in SHRs treated with acupuncture. The antihypertensive effects of acupuncture in SHRs were abrogated by microinjection bilaterally into RVLM of GW9662. Microinjection of GW1929 mimicked the antihypertensive effect of acupuncture. PPAR-γ expression was negatively correlated with blood pressure and sympathetic vasomotor activity in SHRs treated with acupuncture. These results suggested that acupuncture promoted a central antihypertensive effect by increasing the expression of PPAR-γ in RVLM of SHRs.

Optimizing treatment of renal cell carcinoma with VEGFR-TKIs: a comparison of clinical pharmacology and drug-drug interactions of anti-angiogenic drugs.

Anti-angiogenic treatment is an important option that has changed the therapeutic landscape in various tumors, particularly in patients affected by renal cell carcinoma (RCC). Agents that block signaling pathways governing tumor angiogenesis have raised high expectations among clinicians. Vascular endothelial growth factor receptor-tyrosine kinase inhibitors (VEGFR-TKIs) comprise a heterogeneous class of drugs with distinct pharmacological profiles, including potency, selectivity, pharmacokinetics and drug-drug interactions. Among them, tivozanib is one of the last TKIs introduced in the clinical practice; this drug selectively targets VEGFRs, it is characterized by a favorable pharmacokinetics and safety profile and has been approved as first-line treatment for patients with metastatic RCC (mRCC). In this article, we describe the clinical pharmacology of selected VEGFR-TKIs used for the treatment of mRCC, highlighting the relevant differences; moreover we aim to define the main pharmacologic characteristics of these drug.

CDK12 inhibition mediates DNA damage and is synergistic with sorafenib treatment in hepatocellular carcinoma.

OBJECTIVES: Hepatocellular carcinoma (HCC) is one of the most frequent malignancies and a major leading cause of cancer-related deaths worldwide. Several therapeutic options like sorafenib and regorafenib provide only modest survival benefit to patients with HCC. This study aims to identify novel druggable candidate genes for patients with HCC. DESIGN: A non-biased CRISPR (clustered regularly interspaced short palindromic repeats) loss-of-function genetic screen targeting all known human kinases was performed to identify vulnerabilities of HCC cells. Whole-transcriptome sequencing (RNA-Seq) and bioinformatics analyses were performed to explore the mechanisms of the action of a cyclin-dependent kinase 12 (CDK12) inhibitor in HCC cells. Multiple in vitro and in vivo assays were used to study the synergistic effects of the combination of CDK12 inhibition and sorafenib. RESULTS: We identify CDK12 as critically required for most HCC cell lines. Suppression of CDK12 using short hairpin RNAs (shRNAs) or its inhibition by the covalent small molecule inhibitor THZ531 leads to robust proliferation inhibition. THZ531 preferentially suppresses the expression of DNA repair-related genes and induces strong DNA damage response in HCC cell lines. The combination of THZ531 and sorafenib shows striking synergy by inducing apoptosis or senescence in HCC cells. The synergy between THZ531 and sorafenib may derive from the notion that THZ531 impairs the adaptive responses of HCC cells induced by sorafenib treatment. CONCLUSION: Our data highlight the potential of CDK12 as a drug target for patients with HCC. The striking synergy of THZ531 and sorafenib suggests a potential combination therapy for this difficult to treat cancer.

Cabozantinib in patients with hepatocellular carcinoma failing previous treatment with sorafenib.

Over the past 10 years, sorafenib has been the only systemic agent approved for the treatment of patients with unresectable hepatocellular carcinoma. Recently, lenvatinib was demonstrated noninferior to sorafenib, and regorafenib and ramucirumab were demonstrated superior to placebo in patients progressing on sorafenib and in patients with elevated α-fetoprotein-failing sorafenib, respectively. Phase I-II trials of immune checkpoint inhibitors reported promising efficacy signals. Recently, the randomized, placebo-controlled, Phase III CELESTIAL trial demonstrated statistically and clinically significant increase in overall survival from 8 months with placebo to 10.2 months with cabozantinib in patients failing sorafenib. Furthermore, the study showed a significant improvement in all the efficacy end points. Main adverse events were palmar-plantar erythrodysesthesia, hypertension, increased aspartate aminotransferase, fatigue and diarrhea.

Ellagic acid dose and time-dependently abrogates d-galactose-induced animal model of aging: Investigating the role of PPAR-γ.

AIMS: The world's population is becoming aged and the proportion of older persons is growing in almost every country in the world. Ellagic acid (EA) shows abundant pharmacological properties. Therefore, we aimed to determine the mechanism of anti-aging effects of low and high doses of EA. MAIN METHODS: Aging model was induced by d-galactose (DG), and the anti-aging effect of EA alone or in the presence of PPAR-γ antagonist GW9662, and in combination with metformin were evaluated. The activities of ALT, AST, and AChE, the levels of FBS, HbA1c, testosterone and DHEA-SO4, MDA, GSH, TNF-α, IL-6, advanced glycation end products (AGEs), and BDNF were measured in serum, liver or brain. KEY FINDINGS: DG led to increasing in the levels of IL-6, TNF-α, MDA, AChE, AGEs, ALT, AST, FBS, and HbA1c, in which decrease in the levels of body weight, GSH, BDNF, DHEA-SO4 and testosterone. Metformin (300 mg/kg) abrogated the effects of DG-induced aging model. We also found that the low dose of EA (30 mg/kg) decreases the deteriorative effects of DG-induced aging at 10 weeks of treatment only, however, high dose of EA (100 mg/kg) was effective at both 6 and 10 weeks of treatment. The addition of GW9662 completely reversed the effects of the low dose of EA, but not for the high dose, on DG-induced aging model. SIGNIFICANCE: We revealed that daily and oral administration of EA provides anti-aging effects at low dose in a PPAR-γ receptor-dependent fashion, but not at the high dose.

Structural optimization on a virtual screening hit of smoothened receptor.

The Hedgehog (Hh) pathway plays a critical role during embryonic development by controlling cell patterning, growth and migration. In adults, the function of Hh pathway is curtailed to tissue repair and maintenance. Aberrant reactivation of Hh signaling has been linked to tumorigenesis in various cancers, such as basal cell carcinoma (BCC) and medulloblastoma. The Smoothened (Smo) receptor, a key component of the Hh pathway which is central to the signaling transduction, has emerged as an attractive therapeutic target for the treatment of human cancers. Taking advantage of the availability of several crystal structures of Smo in complex with different antagonists, we have previously conducted a molecular docking-based virtual screening to identify several compounds which exhibited significant inhibitory activity against the Hh pathway activation (IC50 < 10 µM) in a Gli-responsive element (GRE) reporter gene assay. The most potent compound (ChemDiv ID C794-1677: 47 nM) showed comparable Hh signaling inhibition to the marketed drug vismodegib (46 nM). Herein, we report our structural optimization based on the virtual screening hit C794-1677. Our efforts are aimed to improve potency, decrease cLogP, and remove potentially metabolic labile/toxic pyrrole and aniline functionalities presented in C794-1677. The optimization led to the identification of numerous potent compounds exemplified by 25 (7.1 nM), which was 7 folds more potent compared with vismodegib. In addition, 25 was much less lipophilic compared with C794-1677 and devoid of the potentially metabolic labile/toxic pyrrole and aniline functional groups. Furthermore, 25 exhibited promising efficacy in inhibiting Gli1 mRNA expression in NIH3T3 cells with either wildtype Smo or D473H Smo mutant. These results represented significant improvement over the virtual screening hit C794-1677 and suggested that compound 25 can be used as a good starting point to support lead optimization.

Evidence of a PPARγ-mediated mechanism in the ability of Withania somnifera to attenuate tolerance to the antinociceptive effects of morphine.

Notwithstanding the experimental evidence indicating Withania somnifera Dunal roots extract (WSE) ability to prolong morphine-elicited analgesia, the mechanisms underlying this effect are largely unknown. With the aim of evaluating a PPARγ-mediated mechanism in such WSE effects, we verified the ability of the PPARγ antagonist GW-9662 to modulate WSE actions. Further, we evaluated the influence of GW-9662 upon WSE / morphine interaction in SH-SY5Y cells since we previously reported that WSE hampers the morphine-induced µ-opioid receptor (MOP) receptor down-regulation. Nociceptive thresholds / tolerance development were assessed in different groups of rats receiving vehicles (control), morphine (10 mg/kg; i.p.), WSE (100 mg/kg, i.p.) and PPARγ antagonist GW-9662 (1 mg/kg; s.c.) in acute and chronic schedules of administration. Moreover, the effects of GW-9662 (5 and 10 µM) applied alone and in combination with morphine (10 µM) and/or WSE (0.25 and 1.00 mg/mL) on the MOP gene expression were investigated in cell cultures. Data analysis revealed a functional effect of the PPARγ antagonist in attenuating the ability of WSE to prolong morphine analgesic effect and to reduce tolerance development after repeated administration. In addition, molecular experiments demonstrated that the blockade of PPARγ by GW-9662 promotes MOP mRNA down-regulation and counteracts the ability of 1.00 mg/mL of WSE to keep an adequate MOP receptor availability. In conclusion, our results support the involvement of a PPARγ-mediated mechanism in the WSE effects on morphine-mediated nociception and the likely usefulness of WSE in lengthening the analgesic efficacy of opioids in chronic therapy.

Phytohormone Abscisic Acid Protects Human Neuroblastoma SH-SY5Y Cells Against 6-Hydroxydopamine-Induced Neurotoxicity Through Its Antioxidant and Antiapoptotic Properties.

Parkinson's disease (PD) is a destroying and prevalent neurodegenerative disease that is characterized by a progressive death of midbrain dopaminergic neurons. It is important to understand the possible neuroprotective effects of reagents that rescue the neurons from death and apoptosis. In this study, we investigated the effects of abscisic acid (ABA) on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in human dopaminergic neuroblastoma SH-SY5Y cell line as an in vitro model of PD. Cell damage was induced by 150 µM 6-OHDA and the cell viability was examined by 2-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) assay. Reactive oxygen species (ROS) and mitochondrial membrane potential were assessed by fluorescence probe methods. Biochemical markers of apoptosis were also determined by immunoblotting. The data showed that 6-OHDA caused a significant loss of cell viability and mitochondrial membrane potential. In addition, intracellular ROS, cleaved caspase-3, Bax:Bcl-2 ratio, and cytochrome c release were significantly increased in 6-OHDA-incubated cells. ABA (100 µM) elicited a significant protective effect and reduced biochemical markers of cell damage and death. Blockage of peroxisome proliferator-activated receptor (PPAR)γ completely prevented the effect of ABA on 6-OHDA-induced cell toxicity. The results suggest that ABA has neuroprotective property against 6-OHDA-induced neurotoxicity, which is performed through PPARγ signaling. However, ABA antioxidant and antiapoptotic properties are involved, at least in part, in such protection.

Evaluation of ostarine as a selective androgen receptor modulator in a rat model of postmenopausal osteoporosis.

Selective androgen receptor modulators (SARMs) have shown beneficial effects on muscle wasting, general physical function and bone properties in male mammals. However, data on the effects of SARMs in postmenopausal osteoporotic bone are scarce. We evaluated the effects of the SARM drug ostarine on postmenopausal osteoporotic bone in a rat osteoporosis model. Ovariectomy was performed on 46 of 56 3-month-old female Sprague-Dawley rats. Eight weeks after ovariectomy, ostarine was orally administered daily for 5 weeks in dosages of 0.04 (low, OVX + Ost. 0.04), 0.4 (intermediate, OVX + Ost. 0.4), and 4 mg/kg (high, OVX + Ost. 4) body weight. Another ovariectomized group received no ostarine. Lumbar vertebrae and femora were removed for biomechanical, gene expression, ashing, and computer tomography analyses. Low dose showed no effects. The effects of intermediate and high doses were comparable overall. Improvements were mainly seen in structural properties such as bone mineral density and bone volume density. However, the effects in femora were superior to effects in vertebrae. Ostarine treatment for 5 weeks did not improve significantly biomechanical properties. mRNA expression of the receptor activator of NF-κB ligand decreased after treatment, and uterine weight increased. Serum levels of phosphorus increased following ostarine treatment in intermediate and high-dose groups. Short-term treatment of osteoporotic bone with ostarine leads to improvement of several microstructural bone indices. While we did not observe changes in biomechanics, it is conceivable that longer treatment may also improve biomechanical properties. Further studies are needed to characterize longer time effects and side effects of ostarine in osteoporosis.

NeuroEPO Preserves Neurons from Glutamate-Induced Excitotoxicity.

Many experimental studies show that erythropoietin (EPO) has a neuroprotective action in the brain. EPO in acute and chronic neurological disorders, particularly in stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, has neuroprotective effects. We previously reported the neuroprotective effect of NeuroEPO, a low sialic form of EPO, against oxidative stress induced by glutamate excitotoxicity. In this paper, we analyze the effect of NeuroEPO against apoptosis induced by glutamate excitotoxicity in primary neuronal cultures obtained from the forebrains of Wistar rat embryos after 17 days of gestation. Excitotoxicity was induced after nine days of in vitro culture by treatment with a culture medium containing 100µM glutamate for 15 min. To withdraw glutamate, a new medium containing 100 ng NeuroEPO/mL was added. Apoptosis was analyzed after 24 h. Images obtained by phase contrast microscopy show that neurons treated with glutamate exhibit cell body shrinkage, loss of dendrites that do not make contact with neighboring cells, and that NeuroEPO was able to preserve the morphological characteristics of the control. Immunocytochemistry images show that the culture is essentially pure in neurons; that glutamate causes cell mortality, and that this is partially avoided when the culture medium is supplemented with NeuroEPO. Activation of intrinsic apoptotic pathways was analyzed. The decreases in Bcl-2/Bax ratio, increase in the release of cytochrome c, and in the expression and activity of caspase-3 observed in cells treated with glutamate, were restored by NeuroEPO. The results from this study show that NeuroEPO protects cortical neurons from glutamate-induced apoptosis via upregulation of Bcl-2 and inhibit glutamate-induced activation of caspase-3.

The effects of urolithins on the response of prostate cancer cells to non-steroidal antiandrogen bicalutamide.

BACKGROUND: Urolithins are bioavailable products of gut microbiota metabolism of ellagitannins. Their biological activity includes anti-cancer effects. PURPOSE: The aim of this study was to explore the effects of urolithins on prostate cancer cells and activity of clinically used anti-androgen, bicalutamide. METHODS: Prostate cancer cells were treated with urolithin A, urolithin B, urolithin C or their combinations with bicalutamide. Cell proliferation was determined by DNA fluorescence with Hoechst 33258. The combination index method was used to examine interactions. Apoptosis and androgen receptor (AR) localization were analysed by flow cytometry. Prostate specific antigen (PSA) secretion was measured by ELISA. RESULTS: Urolithins inhibited proliferation of LNCaP prostate cancer cells. The mixtures of bicalutamide with uroA and uroB had additive anti-proliferative effect. All tested urolithins induced apoptosis of LNCaP cells. However, the combinations of bicalutamide with urolithin A and urolithin B had attenuated pro-apoptotic activity. UroA and uroC decreased DHT-induced PSA secretion. In contrast, uroB impaired PSA lowering effect of bicalutamide. UroA, individually and in combination with bicalutamide, promoted cytoplasmic localization of AR. CONCLUSION: Urolithins might contribute to chemopreventive activity of ellagitannin rich preparations. Our results support use of ellagitannin rich preparations in prostate cancer chemoprevention, but advise caution in their potential use in complementary therapy of prostate cancer. The differences in activity profiles of urolithins indicate that possible health benefits and interactions will depend on the type of produced ellagitannins metabolite.

Unveiling some FDA-approved drugs as inhibitors of the store-operated Ca2+ entry pathway.

The store-operated calcium entry (SOCE) pathway is an important route for generating cytosolic Ca2+ signals that regulate a diverse array of biological processes. Abnormal SOCE seem to underlie several diseases that notably include allergy, inflammation and cancer. Therefore, any modulator of this pathway is likely to have significant impact in cell biology under both normal and abnormal conditions. In this study, we screened the FDA-approved drug library for agents that share significant similarity in 3D shape and surface electrostatics with few, hitherto best known inhibitors of SOCE. This has led to the identification of five drugs that showed dose-dependent inhibition of SOCE in cell-based assay, probably through interacting with the Orai1 protein which effectively mediates SOCE. Of these drugs, leflunomide and teriflunomide could suppress SOCE significantly at clinically-relevant doses and this provides for an additional mechanism towards the therapeutic utility of these drugs as immunosuppressants. The other three drugs namely lansoprazole, tolvaptan and roflumilast, were less potent in suppressing SOCE but were more selective and thus they may serve as novel scaffolds for future development of new, more efficacious SOCE inhibitors.

Asiatic acid inhibits LPS-induced inflammatory response in human gingival fibroblasts.

Asiatic acid, a triterpenoid component isolated from Centella asiatica (L.) Urban, possesses antioxidative and anti-inflammatory activities. In this study, we aimed to investigate the anti-inflammatory effects of asiatic acid both in vivo and in vitro. HGFs or RAW264.7 cells were treated with asiatic acid 1h before LPS treatment. Cell viability was measured by MTT assay. The levels of PGE2, NO, IL-6, and IL-8 were detected by ELISA. Protein expression levels were detected by western blot analysis. In vivo, asiatic acid significantly inhibited LPS-induced IL-6 and IL-8 expression levels in gingival tissues. In vitro, LPS-induced PGE2, NO, IL-6, and IL-8 production was significantly attenuated by asiatic acid. Asiatic acid also inhibited p65 NF-κB phosphorylation induced by LPS in HGFs. The expression of PPAR-γ was up-regulated by asiatic acid. Furthermore, GW9662, a PPAR-γ inhibitor, attenuated the inhibitory effect of asiatic acid on PGE2, NO, IL-6, and IL-8 production. Our results suggest that asiatic acid activates PPAR-γ, which subsequently inhibits LPS-induced NF-κB activation and inflammatory mediators production. Asiatic acid may offer therapeutic potential for the treatment of periodontitis.

N-(4-methoxyphenyl) caffeamide-induced melanogenesis inhibition mechanisms.

BACKGROUND: The derivative of caffeamide exhibits antioxidant and antityrosinase activity. The activity and mechanism of N-(4-methoxyphenyl) caffeamide (K36E) on melanogenesis was investigated. METHODS: B16F0 cells were treated with various concentrations of K36E; the melanin contents and related signal transduction were studied. Western blotting assay was applied to determine the protein expression, and spectrophotometry was performed to identify the tyrosinase activity and melanin content. RESULTS: Our results indicated that K36E reduced α-melanocyte-stimulating hormone (α-MSH)-induced melanin content and tyrosinase activity in B16F0 cells. In addition, K36E inhibited the expression of phospho-cyclic adenosine monophosphate (cAMP)-response element-binding protein, microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1 (TRP-1). K36E activated the phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3 beta (GSK3ß), leading to the inhibition of MITF transcription activity. K36E attenuated α-MSH induced cAMP pathways, contributing to hypopigmentation. CONCLUSIONS: K36E regulated melanin synthesis through reducing the expression of downstream proteins including p-CREB, p-AKT, p-GSK3ß, tyrosinase, and TRP-1, and activated the transcription factor, MITF. K36E may have the potential to be developed as a skin whitening agent.

Regional osteoporosis due to osteoclast activation as a trigger for the pain-like behaviors in tail-suspended mice.

Pathological conditions with refractory skeletal pain are often characterized by regional osteoporotic changes such as transient osteoporosis of the hip, regional migratory osteoporosis, or complex regional pain syndrome (CRPS). Our previous study demonstrated that the acidic microenvironment created by osteoclast activation under high bone turnover conditions induced pain-like behaviors in ovariectomized mice through the stimulation of acid-sensing nociceptors. The aim of the present study was to examine whether regional transient osteoporotic changes are related to pain-like behaviors in the hind limb using tail-suspended model mice. The hind limbs of tail-suspended mice were unloaded for 2 weeks, during which time the mice revealed significant regional osteoporotic changes in their hind limbs accompanied by osteoclast activation. In addition, these changes were significantly recovered by the resumption of weight bearing on the hind limbs for 4 weeks. Consistent with the pathological changes in the hind limbs, pain-like behaviors in the mice were induced by tail suspension and recovered by the resumption of weight bearing. Moreover, treatment with bisphosphonate significantly prevented the triggering of the regional osteoporosis and pain-like behaviors, and antagonists of the acid-sensing nociceptors, such as transient receptor potential channel vanilloid subfamily member 1 and acid-sensing ion channels, significantly improved the pain-like behaviors in the tail-suspended mice. We, therefore, believe that regional transient osteoporosis due to osteoclast activation might be a trigger for the pain-like behaviors in tail-suspended model mice. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1226-1236, 2017.

Positive allosteric modulators of the α7 nicotinic acetylcholine receptor potentiate glutamate release in the prefrontal cortex of freely-moving rats.

Positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (α7nAChRs) exhibit pro-cognitive effects in animal models of schizophrenia and are targets for the discovery of cognition-enhancing drugs. However, little is known about their in vivo mechanism of action because such studies have been performed in vitro. Here we test the hypothesis that PAMs' potentiation of glutamate release in prefrontal cortex depends upon the level of endogenous cholinergic activity. NMDA stimulation of the nucleus accumbens shell (0.05-0.30 µg in 0.5 µL) increased extracellular choline (0.87 ± 0.15 - 1.73 ± 0.31 µM) and glutamate (0.15 µg, 3.79 ± 0.87 µM) in medial prefrontal cortex, and the glutamate release was prevented by local infusions of MLA (6.75 µg, 0.19 ± 0.06 µM). The lower dose (1 mg/kg) of AVL3288 (type I) potentiated the glutamate release to a greater degree after the high dose of NMDA (0.30 µg; 84.7% increase vs AVL vehicle) versus the low dose of NMDA (0.05 µg; 24.2% increase), whereas glutamate release was inhibited when the high dose of NMDA was combined with the high dose of AVL3288 (64.2% decrease). In contrast, PNU120596 (type II) only potentiated glutamate release when the high dose (9 mg/kg) was combined with the low dose of NMDA (0.05 µg; 211% increase from PNU vehicle). Collectively, the results suggest a potential in vivo mechanism for the pro-cognitive effects of PAMs and provide the proof-of-concept for the continued focus on allosteric modulation of cortical α7nAChRs for cognition-enhancing drug development.