Epiberberine inhibits bone metastatic breast cancer-induced osteolysis.

ETHNOPHARMACOLOGICAL RELEVANCE: The anti-tumor related diseases of Coptidis Rhizoma (Huanglian) were correlated with its traditional use of removing damp-heat, clearing internal fire, and counteracting toxicity. In the recent years, Coptidis Rhizoma and its components have drawn extensive attention toward their anti-tumor related diseases. Besides, Coptidis Rhizoma is traditionally used as an anti-inflammatory herb. Epiberberine (EPI) is a significant alkaloid isolated from Coptidis Rhizoma, and exhibits multiple pharmacological activities including anti-inflammatory. However, the effect of epiberberine on breast cancer and the inflammatory factors of metastatic breast cancer-induced osteolysis has not been demonstrated clearly. AIM OF THE STUDY: Bone metastatic breast cancer can lead to osteolysis via inflammatory factors-induced osteoclast differentiation and function. In this study, we try to analyze the effect of epiberberine on breast cancer and the inflammatory factors of metastatic breast cancer-induced osteolysis. METHODS: To evaluate whether epiberberine could suppress bone metastatic breast cancer-induced osteolytic damage, healthy female Balb/c mice were intratibially injected with murine triple-negative breast cancer 4T1 cells. Then, we examined the inhibitory effect and underlying mechanism of epiberberine on breast cancer-induced osteoclastogenesis in vitro. Xenograft assay was used to study the effect of epiberberine on breast cancer cells in vivo. Moreover, we also studied the inhibitory effects and underlying mechanisms of epiberberine on RANKL-induced osteoclast differentiation and function in vitro. RESULTS: The results show that epiberberine displayed potential therapeutic effects on breast cancer-induced osteolytic damage. Besides, our results show that epiberberine inhibited breast cancer cells-induced osteoclast differentiation and function by inhibiting secreted inflammatory cytokines such as IL-8. Importantly, we found that epiberberine directly inhibited RANKL-induced differentiation and function of osteoclast without cytotoxicity. Mechanistically, epiberberine inhibited RANKL-induced osteoclastogensis via Akt/c-Fos signaling pathway. Furthermore, epiberberine combined with docetaxel effectively protected against bone loss induced by metastatic breast cancer cells. CONCLUSIONS: Our findings suggested that epiberberine may be a promising natural compound for treating bone metastatic breast cancer-induced osteolytic damage by inhibiting IL-8 and is worthy of further exploration in preclinical and clinical trials.

Salvianolic acid A provides neuroprotective effects on cerebral ischemia-reperfusion injury in rats via PKA/CREB/c-Fos signaling pathway.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) is a phenomenon that pathological injury of ischemic brain tissue is further aggravated after the restoration of blood supply. The complex pathological mechanism of CIRI has led to the failure of multiple neuroprotective agents in clinical studies. Salvianolic acid A (SAA) is a neuroprotective extract from Salvia miltiorrhiza Bge., with significant pharmacological activities in the treatment of brain injury. However, the neuroprotective mechanisms of SAA remain unclear. PURPOSE: To explore the potential protective effect of SAA on CIRI and its mechanism, and to provide experimental basis for the research of new drugs for CIRI. STUDY DESIGN: A model of transient middle cerebral artery occlusion (tMCAO) in rats was used to simulate clinical CIRI, and the neuroprotective effect of SAA on tMCAO rats was investigated within 14 days after reperfusion. The improvement effects of SAA on cognitive impairment of tMCAO rats were investigated by behavioral tests from days 7-14. Finally, the neuroprotective mechanism of SAA was investigated on day 14. METHODS: The neuroprotective effects and mechanism of SAA were investigated by behavioral tests, HE and TUNEL staining, RNA sequence (RNA-seq) analysis and Western blot in tMCAO rats. RESULTS: The brain protective effects of SAA were achieved by alleviating cerebral infarction, cerebral edema, cerebral atrophy and nerve injury in tMCAO rats. Meanwhile, SAA could effectively improve the cognitive impairment and pathological damage of hippocampal tissue, and inhibit cell apoptosis in tMCAO rats. Besides, SAA could provide neuroprotective effects by up-regulating the expression of Bcl-2, inhibiting the activation of Caspase 3, and regulating PKA/CREB/c-Fos signaling pathway. CONCLUSION: SAA can significantly improve brain injury and cognitive impairment in CIRI rats, and this neuroprotective effect may be achieved through the anti-apoptotic effect and the regulation of PKA/CREB/c-Fos signaling pathway.

Activation of neurons in the insular cortex and lateral hypothalamus during food anticipatory period caused by food restriction in mice.

Mice fed a single meal daily at a fixed time display food anticipatory activity (FAA). It has been reported that the insular cortex (IC) plays an essential role in food anticipation, and lateral hypothalamus (LH) regulates the expression of FAA. However, how these areas contribute to FAA production is still unclear. Thus, we examined the temporal and spatial activation pattern of neurons in the IC and LH during the food anticipation period to determine their role in FAA establishment. We observed an increase of c-Fos-positive neurons in the IC and LH, including orexin neurons of male adult C57BL/6 mice. These neurons were gradually activated from the 1st day to 15th day of restricted feeding. The activation of these brain regions, however, peaked at a distinct point in the food restriction procedure. These results suggest that the IC and LH are differently involved in the neural network for FAA production.

Antinociceptive effects of vitamin B-complex: A behavioral and histochemical study in rats.

B-vitamins have been evaluated as a useful adjuvant therapy to treat pain. In spite of clinical and experimental evidence indicating the analgesic effect of B-vitamins, few studies have investigated their effect on aspects of the inflammatory pain response. In the present study, we investigated the analgesic effect of chronic application of B-complex vitamins (Neurobion) using an inflammatory experimental pain model in rats. Nociceptive behavioral responses were evaluated in male Wistar rats after plantar injection of formalin, comparing the treatment group (TG) with Neurobion pretreatment to the control group (CG) without the pretreatment. In addition, neuronal activity in the central pain pathway was evaluated using c-Fos immunohistochemical reactivity and NADPH-d histochemistry. A highly significant reduction of painful behaviors such as licking and flinching were observed in TG, especially during the secondary phase of the formalin test compared to CG. Results suggest that long-term pre-treatment using Neurobion can have a beneficial effect in reducing the chronic phase of pain. In addition, we observed a downregulation of c-Fos and NADPH-d in dorsal spinal neurons, suggesting that the antinociceptive effect induced by Neurobion could be due to a suppression of nociceptive transmission at the spinal level, particularly in the afferent regions of the dorsal spinal horn, which these neurons utilizing nitric oxide at least as one of their pain neurotransmitters.

Gleditsiae fructus regulates osteoclastogenesis by inhibiting the c­Fos/NFATc1 pathway and alleviating bone loss in an ovariectomy model.

Medical and economic developments have allowed the human lifespan to extend and, as a result, the elderly population has increased worldwide. Osteoporosis is a common geriatric disease that has no symptoms and even a small impact can cause fractures in patients, leading to a serious deterioration in the quality of life. Osteoporosis treatment typically involves bisphosphonates and selective estrogen receptor modulators. However, these treatments are known to cause severe side effects, such as mandibular osteonecrosis and breast cancer, if used for an extended period of time. Therefore, it is essential to develop therapeutic agents from natural products that have fewer side effects. Gleditsiae fructus (GF) is a dried or immature fruit of Gleditsia sinensis Lam. and is composed of various triterpenoid saponins. The anti­inflammatory effect of GF has been confirmed in various diseases, and since the anti­inflammatory effect plays a major role in inhibiting osteoclast differentiation, GF was expected to be effective in osteoclast differentiation and menopausal osteoporosis; however, to the best of our knowledge, it has not yet been studied. Therefore, the present study was designed to examine the effect of GF on osteoclastogenesis and to investigate the mechanism underlying inhibition of osteoclast differentiation. The effects of GF on osteoclastogenesis were determined in vitro by tartrate­resistant acid phosphatase (TRAP) staining, pit formation assays, filamentous actin (F­actin) ring formation assays, western blotting and reverse transcription­quantitative PCR analyses. Furthermore, the administration of GF to an animal model exhibiting menopausal osteoporosis allowed for the analysis of alterations in the bone microstructure of the femur using micro­CT. Additionally, assessments of femoral tissue and serum were conducted. The present study revealed that the administration of GF resulted in a reduction in osteoclast levels, F­actin rings, TRAP activity and pit area. Furthermore, GF showed a dose­dependent suppression of nuclear factor of activated T­cells cytoplasmic, c­Fos and other osteoclastogenesis­related markers.

Rhubarb Peony Decoction Downregulates the Expression of c-Fos after Intestinal Epithelial Cells Hypoxia-reoxygenation Injury.

BACKGROUND: Intestinal ischemia-reperfusion (I/R) injury occurs in several clinical situations and after intestinal transplantation. This study aimed to examine the role of rhubarb peony decoction (RPD) in intestinal I/R injury. METHODS: Different concentrations of RPD were set to treat IEC-6 and Caco-2 cells. Cell proliferation and apoptosis were measured by CCK-8 and flow cytometry assays. High-throughput transcriptome sequencing was performed on IEC-6 cells treated with hypoxia-reoxygenation (HR) or HR and RPD. RESULTS: RPD treatment significantly promoted the proliferation of IEC-6 and Caco-2 cells and inhibited apoptosis. Sequencing results identified 109 significantly up-regulated genes and 36 significantly down-regulated genes in the RPD group. In addition, the results of western blot suggested that HR induced the expression of c-Fos, and the treatment of RPD prevented the HR-induced c- Fos expression. Importantly, knockdown of c-Fos rescued the HR-inhibited cell proliferation and HR-induced apoptosis. CONCLUSIONS: In conclusion, RPD was beneficial in protecting the survival of intestinal epithelial cells under HR stress. Furthermore, the increase in c-Fos expression after HR stress was closely related to the proliferation and apoptosis of intestinal epithelial cells.

Effect of an herbal formulation containing Peganum harmala L. and Fraxinus excelsior L. on oxidative stress, memory impairment and withdrawal syndrome induced by morphine.

Background: Traditional Persian medicine has introduced effective remedies in opioid dependence care. One of the most widely used remedies is an herbal formulation containing Peganum harmala L. and Fraxinus excelsior L. (HF). This study investigated the effects of HF to attenuate the withdrawal signs and rewarding effects in morphine-dependent rats.Methods: Forty-nine male Wistar rats were randomly divided into seven groups. The control and vehicle groups received normal saline and sodium carboxymethyl cellulose, respectively. The morphine group received morphine for one week. The single and daily dose of HF groups received morphine similar to the morphine group, and HF (1.4 and 2.8 g/kg) once a day in the daily dose group and only on the last day of the experiment in the single dose of HF group. Finally, the withdrawal signs as well biochemical tests were evaluated. The behavioral parameters were assessed by conditioned place preference (CPP), elevated plus-maze and Y-maze tests. The antioxidant activity of HF was evaluated by measurement of serum contents of malondialdehyde, stable nitric oxide metabolites and total antioxidant capacity (TAC). Moreover, the protein expression of c-fos was assessed by western blotting.Results: Daily treatment with HF significantly reduced the score of CPP behavioral test, all of the withdrawal signs, TAC and the c-fos protein level.Conclusions: The results indicated that HF might be a promising complementary treatment in reducing morphine-induced physical and psychological dependence probably through modulation of c-fos protein expression.

Pre-Synaptic GABAA in NaV1.8+ Primary Afferents Is Required for the Development of Punctate but Not Dynamic Mechanical Allodynia following CFA Inflammation.

Hypersensitivity to mechanical stimuli is a cardinal symptom of neuropathic and inflammatory pain. A reduction in spinal inhibition is generally considered a causal factor in the development of mechanical hypersensitivity after injury. However, the extent to which presynaptic inhibition contributes to altered spinal inhibition is less well established. Here, we used conditional deletion of GABAA in NaV1.8-positive sensory neurons (Scn10aCre;Gabrb3fl/fl) to manipulate selectively presynaptic GABAergic inhibition. Behavioral testing showed that the development of inflammatory punctate allodynia was mitigated in mice lacking pre-synaptic GABAA. Dorsal horn cellular circuits were visualized in single slices using stimulus-tractable dual-labelling of c-fos mRNA for punctate and the cognate c-Fos protein for dynamic mechanical stimulation. This revealed a substantial reduction in the number of cells activated by punctate stimulation in mice lacking presynaptic GABAA and an approximate 50% overlap of the punctate with the dynamic circuit, the relative percentage of which did not change following inflammation. The reduction in dorsal horn cells activated by punctate stimuli was equally prevalent in parvalbumin- and calretinin-positive cells and across all laminae I-V, indicating a generalized reduction in spinal input. In peripheral DRG neurons, inflammation following complete Freund's adjuvant (CFA) led to an increase in axonal excitability responses to GABA, suggesting that presynaptic GABA effects in NaV1.8+ afferents switch from inhibition to excitation after CFA. In the days after inflammation, presynaptic GABAA in NaV1.8+ nociceptors constitutes an "open gate" pathway allowing mechanoreceptors responding to punctate mechanical stimulation access to nociceptive dorsal horn circuits.

Mussaendoside O, a N-triterpene cycloartane saponin, attenuates RANKL-induced osteoclastogenesis and inhibits lipopolysaccharide-induced bone loss.

BACKGROUND: Elevated activity of osteoclasts (OCs) is linked to osteolytic bone diseases, such as osteoporosis and rheumatoid arthritis. Developing natural anti-osteoclastogenic compounds with greater efficacy and fewer adverse effects is crucial for preventing or treating osteolytic bone diseases. N-triterpene cycloartane saponins (NTCSs) are rarely found in nature, and their inhibitory effects on OC differentiation in vitro and in vivo have not yet been explored. PURPOSE: This study was aimed to investigate the effect of mussaendoside O, an NTCS isolated from Mussaenda pubescens, on RANKL-induced OC differentiation and its underlying mechanism in vitro, and lipopolysaccharide (LPS)-induced bone resorption in a mouse model. METHODS: The content of mussaendoside O in methanol extract of M. pubescens was determined by HPLC. The inhibitory effects of mussaendoside O on RANKL-induced OC formation were assessed using TRAP staining, western blotting, immunofluorescence staining, and real-time qPCR. Meanwhile, the effects of mussaendoside O on LPS-induced inflammatory responses were assessed using a Griess reagent and qPCR. The effects of mussaendoside O on LPS-induced bone resorption in a mouse model were evaluated using micro-CT and immunohistochemical staining. RESULTS: Mussaendoside O inhibited RANKL-induced TRAP-positive multinucleated OC formation in a concentration-dependent manner without affecting cell viability. However, mussaendoside O did not inhibit LPS-induced mRNA expression of COX-2, iNOS, and TNF-α. Mice orally administrated with mussaendoside O exhibited significant protection from LPS-induced bone resorption and OC formation. At the molecular level, mussaendoside O suppressed RANKL-activated phosphorylation of p38 MAPK and JNK, as well as c-Fos expression. In addition, mussaendoside O suppressed RANKL-induced NFATc1 activation and the expression of its target genes, including OSCAR, DC-STAMP, CtsK, and TRAP. CONCLUSION: Mussaendoside O attenuates OC differentiation in vitro and LPS-induced bone resorption in a mouse model by inhibiting the RANKL-activated c-Fos/NFATc1 signaling pathways. Therefore, mussaendoside O may be a valuable lead compound for preventing or treating of osteolytic bone diseases.

Behavioral and Immunohistochemical Evidence for Suppressive Effects of Goshajinkigan on Salicylate-Induced Tinnitus in Rats.

Many people are affected by tinnitus, a sensation of ringing in the ear despite the absence of external sound. Goshajinkigan (GJG) is one of the formulations of Japanese traditional herbal medicine and is prescribed for the palliative treatment of patients with tinnitus. Although GJG is clinically effective in these patients, its behavioral effects and the underlying neuroanatomical substrate have not been modeled in animals. We modeled tinnitus using salicylate-treated rats, demonstrated the effectiveness of GJG on tinnitus, and examined the underlying neuronal substrate with c-Fos expression. Intraperitoneal injection of sodium salicylate (400 mg/kg) into rats for three consecutive days significantly increased false positive scores, which were used to assess tinnitus behavior. When GJG was orally administered one hour after each salicylate injection, the increase in tinnitus behavior was suppressed. The analysis of c-Fos expression in auditory-related brain areas revealed that GJG significantly reduced the salicylate-induced increase in the number of c-Fos-expressing cells in the auditory cortices, inferior colliculus, and dorsal cochlear nucleus. These results suggest a suppressive effect of GJG on salicylate-induced tinnitus in animal models.

Hypothalamic response with PKA/CREB signaling is associated with direct cerebroventricular administration of bombesin-induced scratching.

Bombesin (BN) is an itch-specific mediator that causes intense itch-scratching activity in mammals. Although most examinations of BN-induced itch processing have focused on the spinal cord, the involvement of central nervous system mechanisms remains unclear. Here, we investigated how relationships among hypothalamic regions regulate BN-mediated itch-scratch processes. We found that intracerebroventricular (i.c.v.) administration of BN (0.04-4 µg) elicited intense itch scratching in mice, whereas BN (0.4-400 µg) administered via intravenous tail injection failed to evoke a scratching response. Additionally, nalfurafine had no significant effects on BN-induced scratching behavior, indicating that central modulation of BN is distinct from histamine-mediated histaminergic itch and chloroquine-mediated non-histaminergic itch signaling pathways. We labeled BN with a fluorescent tag, 7-nitrobenz-2-oxa-1 (NBD), and traced its fluorescence in the hypothalamus for 30 min following i.c.v. NBD-BN administration. Accordingly, we confirmed that i.c.v. administration of BN enhanced c-Fos expression in the dorsal medial nucleus of the hypothalamus, where neuromedin B receptors and gastrin-releasing peptide receptors are highly expressed. Interestingly, in situ injection of BN into the hypothalamus immediately and robustly induced itch-scratching behavior. Moreover, gene transcripts and western blot assay revealed that BN receptor-dependent PKA/CREB signaling was upregulated in the hypothalamus after i.c.v. administration of BN. Consistently, pretreatment with a PKA inhibitor, Rp-cAMP, significantly reduced BN-induced scratching behavior. Our results indicate that the dorsal medial nucleus of the hypothalamus may be a key nucleus in mediating BN-mediated itch and hypothalamic PKA/CREB signaling is involved in regulating BN-mediated itch.

Auricular Transcutaneous Vagus Nerve Stimulation Acutely Modulates Brain Connectivity in Mice.

Brain electrical stimulation techniques take advantage of the intrinsic plasticity of the nervous system, opening a wide range of therapeutic applications. Vagus nerve stimulation (VNS) is an approved adjuvant for drug-resistant epilepsy and depression. Its non-invasive form, auricular transcutaneous VNS (atVNS), is under investigation for applications, including cognitive improvement. We aimed to study the effects of atVNS on brain connectivity, under conditions that improved memory persistence in CD-1 male mice. Acute atVNS in the cymba conchae of the left ear was performed using a standard stimulation protocol under light isoflurane anesthesia, immediately or 3 h after the training/familiarization phase of the novel object-recognition memory test (NORT). Another cohort of mice was used for bilateral c-Fos analysis after atVNS administration. Spearman correlation of c-Fos density between each pair of the thirty brain regions analyzed allowed obtaining the network of significant functional connections in stimulated and non-stimulated control brains. NORT performance was enhanced when atVNS was delivered just after, but not 3 h after, the familiarization phase of the task. No alterations in c-Fos density were associated with electrostimulation, but a significant effect of atVNS was observed on c-Fos-based functional connectivity. atVNS induced a clear reorganization of the network, increasing the inter-hemisphere connections and the connectivity of locus coeruleus. Our results provide new insights into the effects of atVNS on memory performance and brain connectivity extending our knowledge of the biological mechanisms of bioelectronics in medicine.

Effect of Hibiscus syriacus Linnaeus extract and its active constituent, saponarin, in animal models of stress-induced sleep disturbances and pentobarbital-induced sleep.

Treatment of sleep disorders promotes the long-term use of commercially available sleep inducers that have several adverse effects, including addiction, systemic fatigue, weakness, loss of concentration, headache, and digestive problems. Therefore, we aimed to limit these adverse effects by investigating a natural product, the extract of the Hibiscus syriacus Linnaeus flower (HSF), as an alternative treatment. In the electric footshock model, we measured anxiety and assessed the degree of sleep improvement after administering HSF extract. In the restraint model, we studied the sleep rate using PiezoSleep, a noninvasive assessment system. In the pentobarbital model, we measured sleep improvement and changes in sleep-related factors. Our first model confirmed the desirable effects of HSF extract and its active constituent, saponarin, on anxiolysis and Wake times. HSF extract also increased REM sleep time. Furthermore, HSF extract and saponarin increased the expression of cortical GABAA receptor α1 (GABAAR α1) and c-Fos in the ventrolateral preoptic nucleus (VLPO). In the second model, HSF extract and saponarin restored the sleep rate and the sleep bout duration. In the third model, HSF extract and saponarin increased sleep maintenance time. Moreover, HSF extract and saponarin increased cortical cholecystokinin (CCK) mRNA levels and the expression of VLPO c-Fos. HSF extract also increased GABAAR α1 mRNA level. Our results suggest that HSF extract and saponarin are effective in maintaining sleep and may be used as a novel treatment for sleep disorder. Eventually, we hope to introduce HSF and saponarin as a clinical treatment for sleep disorders in humans.

Diterbutyl phthalate attenuates osteoarthritis in ACLT mice via suppressing ERK/c-fos/NFATc1 pathway, and subsequently inhibiting subchondral osteoclast fusion.

Osteoarthritis (OA) is the most common arthritis with a rapidly increasing prevalence. Disease progression is irreversible, and there is no curative therapy available. During OA onset, abnormal mechanical loading leads to excessive osteoclastogenesis and bone resorption in subchondral bone, causing a rapid subchondral bone turnover, cyst formation, sclerosis, and finally, articular cartilage degeneration. Moreover, osteoclast-mediated angiogenesis and sensory innervation in subchondral bone result in abnormal vascularization and OA pain. The traditional Chinese medicine Panax notoginseng (PN; Sanqi) has long been used in treatment of bone diseases including osteoporosis, bone fracture, and OA. In this study we established two-dimensional/bone marrow mononuclear cell/cell membrane chromatography/time of flight mass spectrometry (2D/BMMC/CMC/TOFMS) technique and discovered that diterbutyl phthalate (DP) was the active constituent in PN inhibiting osteoclastogenesis. Then we explored the therapeutic effect of DP in an OA mouse model with anterior cruciate ligament transaction (ACLT). After ACLT was conducted, the mice received DP (5 mg·kg-1·d-1, ip) for 8 weeks. Whole knee joint tissues of the right limb were harvested at weeks 2, 4, and 8 for analysis. We showed that DP administration impeded overactivated osteoclastogenesis in subchondral bone and ameliorated articular cartilage deterioration. DP administration blunted aberrant H-type vessel formation in subchondral bone marrow and alleviated OA pain assessed in Von Frey test and thermal plantar test. In RANKL-induced RAW264.7 cells in vitro, DP (20 µM) retarded osteoclastogenesis by suppressing osteoclast fusion through inhibition of the ERK/c-fos/NFATc1 pathway. DP treatment also downregulated the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and d2 isoform of the vacuolar (H+) ATPase V0 domain (Atp6v0d2) in the cells. In conclusion, we demonstrate that DP prevents OA progression by inhibiting abnormal osteoclastogenesis and associated angiogenesis and neurogenesis in subchondral bone.

Anti-rheumatic, and analgesic effects by the parent tuberous roots of Aconitum jaluense in adjuvant induced arthritis rats.

AIM OF THE STUDY: The aim of this study was to test the anti-rheumatic effects of A. jaluense tubers in acute and chronic arthritis rats, and to assign its ingredients through UHPLC-TOF/MS. MATERIALS AND METHODS: Subcutaneous injection of carrageenan for acute arthritis and complete Freund's adjuvant (CFA) for chronic arthritis was carried out in the hind paw of SD rats. The paw volume was measured by a plethysmometer thermal hyperalgesia was tested using a thermal plantar tester, and mechanical hyperalgesia was evaluated by ankle flexion evoked vocalizations. The expression of c-Fos in the brain hippocampus was measured with the avidin-biotin-peroxidase technique. The ingredients were assigned by UHPLC-TOF/MS, chromatography was performed by UHPLC system with DAD detector and BEH C18 column, and spectroscopy was conducted by ESI-MS system. RESULTS AND DISCUSSION: The 80% ethanoic extract of A. jaluense tubers showed an acute anti-inflammatory effect by suppressing the edema volume in the hind paw of carrageenan-stimulated rats. In addition, A. jaluense tubers exerted an anti-rheumatic activity by reducing the secondary swelling volume from an immunological reaction in the left hind paw of CFA-induced chronic arthritis rats. Additionally, oral treatment with the 80% ethanoic extract -showed potent analgesic effects in the arthritis rats by recovering the paw withdrawal latency stimulated by the thermal hyperalgesia and by reducing the vocalization scores evoked by ankle flexion on both hind paws. Moreover, its treatment also indicated an anti-psychiatric effect by controlling the c-Fos protein expression of the brain hippocampus in CFA-stimulated arthritis rats. These results suggested that these therapeutic effects were exhibited by less toxic mono-esterified diterpenoid alkaloids (MDAs), and nontoxic non-esterified diterpenoid alkaloids (NDAs). CONCLUSION: A. jaluense tubers may act as viable therapeutic or preventive candidates for acute and chronic arthritis, particularly, for immune-inflammatory rheumatoid arthritis to suppress the pain and psychiatric condition.

Anti-nociceptive effects of ECa 233 a standardized extract of Centella asiatica (L.) Urban on chronic neuropathic orofacial pain in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: ECa 233 is a standardized extract of Centella asiatica (L.) Urban, a herb traditionally used to treat a number of diseases including neurological disorders. Accordingly, ECa 233 showed benefits on animal models of cognitive deficits, chronic stress and Parkinson's disease. Analgesic activity of ECa 233 was shown in Tail's flick test in rodent and relieving aphthous ulcer pain in man. Moreover, acute and sub-chronic toxicity testing in rodents and pharmacokinetic study in healthy volunteers, clinical trial phase I demonstrated good safety profiles of ECa 233. AIM OF THE STUDY: This study aims to evaluate the anti-nociceptive effects of ECa 233 and its synergistic effect with gabapentin on chronic neuropathic orofacial pain after 3 weeks infraorbital nerve chronic constriction injury in mice. The peripheral and central nociceptive activities are also examined. MATERIALS AND METHODS: Chronic neuropathic orofacial pain was induced by 3 weeks infraorbital nerve chronic constriction injury. Mice were treated with ECa 233 (30, 100 and 300 mg/kg) and gabapentin (10 mg/kg) by oral gavage starting on day 21 and going on for 14 consecutive days. Mechanical hyperalgesia and allodynia were measured on day 7, 14, 21, 28 and 35 after infraorbital nerve chronic constriction injury. At the end of the experiment, mice were observed for the sedative effect using the locomotor activity, the calcitonin gen-related peptide in trigeminal ganglion and c-fos expression in trigeminal nucleus caudalis were investigated after euthanasia. RESULTS: Infraorbital nerve chronic constriction injury gradually induced marked ipsilateral mechanical hyperalgesia and allodynia. The maximum hyperalgesia and allodynia response presented on day 21 and the response was remained constant until day 35. Treatment with either 300 mg/kg ECa 233 or 10 mg/kg gabapentin were able to attenuate mechanical hyperalgesia and allodynia. The downregulation of calcitonin gen-related peptide on ipsilateral trigeminal ganglion were observed in ECa 233 at 100 and 300 mg/kg and 10 mg/kg gabapentin-treated groups. The c-fos expression on ipsilateral trigeminal nucleus caudalis was also decreased in 300 mg/kg ECa 233 and 10 mg/kg gabapentin-treated groups. CONCLUSION: ECa 233 reduced hyperalgesia and allodynia by modulating the peripheral calcitonin gen-related peptide expression consequently alleviate the nociceptive activity in trigeminal nucleus caudalis. Further clinical trial to proof ECa 233's efficacy in neuropathic pain in man as well as possible attributable mechanism of action should be further investigated.

Transgenic approaches to opening up new fields of vasopressin and oxytocin research.

Transgenic approaches have been applied to generate transgenic rats that express exogenous genes in arginine vasopressin (AVP)- and oxytocin (OXT)-producing magnocellular neurosecretory cells (MNCs) of the hypothalamic-neurohypophyseal system (HNS). First, the fusion gene that expresses AVP-enhanced green fluorescent protein (eGFP) and OXT-monomeric red fluorescent protein 1 (mRFP1) was used to visualize AVP- and OXT-producing MNCs and their axon terminals in the HNS under fluorescence microscopy. Second, the fusion gene that expresses c-fos-eGFP and c-fos-mRFP1 was used to identify activated neurons physiologically in the central nervous system, including MNCs, circumventricular organs and spinal cord. In addition, AVP-eGFP x c-fos-mRFP1 and OXT-mRFP1 × c-fos-eGFP double transgenic rats were generated to identify activated AVP- and OXT-producing MNCs using appropriate physiological stimuli. Third, the fusion gene that expresses AVP-chanelrhodopsin 2 (ChR2)-eGFP and AVP-hM3Dq-mCherry was used to activate AVP- and OXT-producing MNCs by optogenetic and chemogenetic approaches. In each step, these transgenic approaches in rats have provided new insights on the physiological roles of AVP and OXT not only in the HNS, but also in the whole body. In this review, we summarize the transgenic rats that we generated, as well as related physiological findings.

Selected Molecular Targets for Antiepileptogenesis.

The term epileptogenesis defines the usually durable process of converting normal brain into an epileptic one. The resistance of a significant proportion of patients with epilepsy to the available pharmacotherapy prompted the concept of a causative treatment option consisting in stopping or modifying the progress of epileptogenesis. Most antiepileptic drugs possess only a weak or no antiepileptogenic potential at all, but a few of them appear promising in this regard; these include, for example, eslicarbazepine (a sodium and T-type channel blocker), lamotrigine (a sodium channel blocker and glutamate antagonist) or levetiracetam (a ligand of synaptic vehicle protein SV2A). Among the approved non-antiepileptic drugs, antiepileptogenic potential seems to reside in losartan (a blocker of angiotensin II type 1 receptors), biperiden (an antiparkinsonian drug), nonsteroidal anti-inflammatory drugs, antioxidative drugs and minocycline (a second-generation tetracycline with anti-inflammatory and antioxidant properties). Among other possible antiepileptogenic compounds, antisense nucleotides have been considered, among these an antagomir targeting microRNA-134. The drugs and agents mentioned above have been evaluated in post-status epilepticus models of epileptogenesis, so their preventive efficacy must be verified. Limited clinical data indicate that biperiden in patients with brain injuries is well-tolerated and seems to reduce the incidence of post-traumatic epilepsy. Exceptionally, in this regard, our own original data presented here point to c-Fos as an early seizure duration, but not seizure intensity-related, marker of early epileptogenesis. Further research of reliable markers of early epileptogenesis is definitely needed to improve the process of designing adequate antiepileptogenic therapies.

Solanum nigrum Line inhibits osteoclast differentiation and suppresses bone mineral density reduction in the ovariectomy­induced osteoporosis model.

Bone homeostasis is maintained by osteoclasts that absorb bone and osteoblasts that form bone tissue. Menopausal osteoporosis is a disease associated with aging and hormonal changes due to menopause causing abnormal activation of osteoclasts, resulting in a decrease in bone density. Existing treatments for osteoporosis have been reported to have serious side effects, such as jawbone necrosis and breast and uterine cancer; therefore, their use by patients is decreasing, whilst studies focusing on alternative treatments are increasingly popular. Solanum nigrum Line (SL) has been used as a medicinal plant that possesses several pharmacological effects, such as anti­inflammatory and hepatotoxic protective effects. To the best of our knowledge, however, its effects on osteoporosis and osteoclasts have not been demonstrated previously. In the present study, the anti­osteoporotic effect of SL was investigated using a postmenopausal model of osteoporosis in which Sprague­Dawley rat ovaries were extracted. In addition, the inhibitory effects on osteoclast differentiation and function of SL was confirmed using an osteoclast model treated with receptor activator of NF­κB ligand (RANKL) on murine RAW 264.7 macrophages. In vivo experiments showed that SL reduced the decrease in bone mineral density and improved changes in the morphological index of bone microstructure, such as trabecular number and separation. In addition, the number of tartrate resistant acid phosphatase­positive cells in the femur and the expression levels of nuclear factor of activated T­cells cytoplasmic 1 (NFATc1) and cathepsin K protein were inhibited. In vitro, SL suppressed RANKL­induced osteoclast differentiation and bone resorption ability; this was mediated by NFATc1/c­Fos, a key transcription factor involved in osteoclast differentiation, ultimately inhibiting expression of various osteoclast­associated genes. These experimental results show that SL may be an alternative treatment for osteoporosis caused by abnormal activation of osteoclasts in the future.

Effects of Melandrium firmum Rohrbach on RANKL­induced osteoclast differentiation and OVX rats.

Osteoporosis is a systemic skeletal disease characterized by reduced bone mineral density (BMD), which results in an increased risk of fracture. Melandrium firmum (Siebold & Zucc.) Rohrbach (MFR), 'Wangbulryuhaeng' in Korean, is the dried aerial portion of Melandrii Herba Rohrbach, which is a member of the Caryophyllaceae family and has been used to treat several gynecological conditions as a traditional medicine. However, to the best of our knowledge, the effect of MFR on osteoclast differentiation and osteoporosis has not been assessed. To evaluate the effects of MFR on osteoclast differentiation, tartrate­resistant acid phosphatase staining, actin ring formation and bone resorption assays were used. Additionally, receptor activator of nuclear factor­κB ligand­induced expression of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1) and c­Fos were measured using western blotting and reverse transcription­PCR. The expression levels of osteoclast­related genes were also examined. To further investigate the anti­osteoporotic effects of MFR in vivo, an ovariectomized (OVX) rat model of menopausal osteoporosis was established. Subsequently, the femoral head was scanned using micro­computed tomography. The results revealed that MFR suppressed osteoclast differentiation, formation and function. Specifically, MFR reduced the expression levels of osteoclast­related genes by downregulating transcription factors, such as NFATc1 and c­Fos. Consistent with the in vitro results, administration of MFR water extract to OVX rats reduced BMD loss, and reduced the expression levels of NFATc1 and cathepsin K in the femoral head. In conclusion, MFR may contribute to alleviate osteoporosis­like symptoms. These results suggested that MFR may exhibit potential for the prevention and treatment of postmenopausal osteoporosis.