Long-term Stimulation of α7 Nicotinic Acetylcholine Receptor Rescues Hemorrhagic Neuron Loss via Apoptosis of M1 Microglia.

We previously revealed that long-term treatment with nicotine suppresses microglial activation, resulting in a protective effect against thrombin-induced shrinkage of the striatal tissue in organotypic slice cultures. Here, the effect of nicotine on impaired M1 and protective M2 microglial polarization was investigated using the BV-2 microglial cell line in the presence or absence of thrombin. Following nicotine treatment, α7 nicotinic acetylcholine receptor expression transiently increased and then gradually decreased until 14 days. Treatment with nicotine for 14 days slightly polarized M0 microglia to M2b and d subtypes. Co-exposure of thrombin and low concentration of interferon-γ recruited inducible NO synthase (iNOS)- and interleukin-1ß-double-positive M1 microglia in a thrombin-concentration-dependent manner. Treatment with nicotine for 14 days significantly decreased the thrombin-induced increase of iNOS mRNA levels and conversely showed a tendency to increase arginase1 mRNA levels. Moreover, treatment with nicotine for 14 days suppressed thrombin-induced phosphorylation of p38 MAPK through the α7 receptor. Repeated intraperitoneal administration of α7 agonist PNU-282987 for 14 days selectively evoked the apoptosis of iNOS-positive M1 microglia at the perihematomal area and showed a neuroprotective effect in an in vivo intracerebral hemorrhage model. These findings revealed that long-term stimulation of α7 receptor causes suppression of thrombin-induced activation of p38 MAPK followed by apoptosis in neuropathic M1 microglia.

Indoleamine 2, 3-dioxygenase is responsible for low stress tolerance after intracerebral hemorrhage.

In the chronic phase after intracerebral hemorrhage (ICH), the aftereffect-associated lowering of motivation burdens many patients; however, the pathogenic mechanism is unclear. Here, we revealed for the first time that indoleamine 2, 3-dioxygenase (IDO) expression and enzyme activity are increased in the collagenase-induced murine ICH model. IDO is a rate-limiting enzyme situated at the beginning of the kynurenine pathway and converts tryptophan, a source of serotonin (5-hydroxytryptamine; 5-HT), to kynurenine. In this study, we showed that IDO is localized in 5-HTergic neurons. After ICH, the synaptosomal 5-HT level decreased, but this effect was neutralized by subcutaneous injections of 1-methyl tryptophan (MT), a specific IDO inhibitor. These results suggest that ICH-induced IDO weakens the activity of 5-HTergic neurons. Accordingly, we next investigated whether the IDO increase contributes to the depression-like behaviors of ICH mice. The immobility times of tail suspension and forced swimming tests were significantly prolonged after ICH but shortened by the administration of 1-MT. In conclusion, the increased IDO after ICH was found to decrease 5-HT levels and subsequently reduce stress tolerance. These findings indicate that IDO is a novel therapeutic target for the ICH aftereffect-associated lowering of motivation.

A Novel Amphotericin B Hydrogel Composed of Poly(Vinyl Alcohol)/Borate Complex for Ophthalmic Formulation.

In this study, we developed a water-soluble complex-hydrogel viscosity-controlled formulation of amphotericin B (AmB). AmB is insoluble in water, but borax makes it soluble by forming a complex with AmB. Borax also forms complexes with poly(vinyl alcohol) (PVA) to produce viscous hydrogels. Furthermore, boric acid interacts with mucin expressed in corneal epithelial cells. Accordingly, by utilizing these properties of borax simultaneously, we prepared a water-soluble AmB complex-hydrogel with poly(vinyl alcohol)/borate (PVA-B-AmB), which is suitable for eye drops. PVA-B-AmB was easily prepared by simply mixing aqueous AmB solution dissolved in borax, PVA solution, and water. The 11B-NMR results suggested that PVA-B-AmB existed by bonding PVA and AmB via boronic acid. PVA-B-AmB (gel ratio = 0.55) has a viscosity of 18.3 ± 0.5 mPa·s and is suitable for ophthalmic formulations. This formulation exhibited sustained release of AmB of approximately 45% at 24 h. It was also shown that this formulation interacts with mucin. These results suggest that PVA-B-AmB can be used as a water-soluble AmB preparation suitable for ophthalmic use.

Amphotericin B nanohydrogel ocular formulation using alkyl glyceryl hyaluronic acid: Formulation, characterization, and in vitro evaluation.

The present study focused on the development of an amphotericin B (AmB) nanoformulation for ophthalmic applications. Accordingly, AmB nanohydrogels (AHA/AmB) using alkyl glyceryl hyaluronic acid (Hyalorepair®, AHA), a hydrophobized hyaluronic acid, were prepared by employing the dialysis method, followed by assessments of physical properties, drug efficacy, and toxicity. In the AHA/AmB formulation, AmB existed in a self-aggregated and amorphous state in the hydrophobic environment of the AHA moiety. AHA/AmB was shown in vitro to interact with mucin, which is known to be expressed in the corneal epithelium and was expected to improve its corneal retention. Compared with the conventional AmB formulation, amphotericin B sodium deoxycholate, AHA/AmB had the same in vitro antifungal activity but significantly lower in vitro toxicity. These findings indicate that nanohydrogels prepared with AHA possess high fungal selectivity and serve as a promising system for ophthalmic AmB delivery.

Posterior Reversible Encephalopathy Syndrome Due to Acute Water Intoxication in a Patient with Schizophrenia.

Posterior reversible encephalopathy syndrome (PRES) is a clinical syndrome that presents as transient cerebral edema (vasogenic edema), usually on a background of hypertensive encephalopathy, puerperal eclampsia, or immunosuppressant drug use. We describe a case of PRES that arose in the context of a psychiatric disorder. The patient was a 26-year-old woman with schizophrenia who was hospitalized upon falling into a catatonic stupor and then suffered acute anxiety leading to impulsive polydipsia and subsequent water intoxication. She lost consciousness, and brain magnetic resonance imaging revealed a high density area, primarily affecting the cortex and subcortical white matter in areas in the occipital and parietal lobes, leading to the diagnosis. We did not treat the hyponatremia by means of aggressive sodium supplementation but rather balanced the extracellular fluid by continuous infusion of isotonic electrolyte replacement fluid. The patient's level of consciousness improved gradually, but a total 141 days passed before hospital discharge was appropriate. The prognosis for PRES is generally favorable, but irreversible neurological damage can occur. We believe, therefore, that brain magnetic resonance imaging should be performed promptly whenever PRES is suspected and that timely, appropriate treatment is of utmost importance. If PRES is observed in a psychiatric patient, it is important to investigate whether the condition might have been caused by water intoxication and to treat the condition accordingly.

Gadolinium causes M1 and M2 microglial apoptosis after intracerebral haemorrhage and exerts acute neuroprotective effects.

OBJECTIVES: Gadolinium (Gd) affects microglial polarization during remyelination. We previously reported that the suppression of proinflammatory microglia was neuroprotective in intracerebral haemorrhage (ICH). The objective of the present study was to investigate the effects of Gd on microglial polarization and neuronal injury after ICH. METHODS: Gadolinium was intraperitoneally administered to ICH mice prepared by an intrastriatal microinjection of collagenase type VII. The polarization of M1, 2a, b and c microglia was evaluated by real-time PCR using the respective markers. Changes in representative mRNAs were also confirmed by immunological methods. Neuroprotective effects were evaluated by counting NeuN-positive cells and a behavioural analysis. KEY FINDINGS: One day after ICH, the mRNA levels of proinflammatory M1 microglial markers, such as inducible nitric oxide synthase (iNOS), and anti-inflammatory M2 microglial markers, such as arginase1 (M2a, c), Ym1 (M2a), and transforming growth factor-ß (M2c), increased, while those of chemokine CCL1 (M2b) only increased after 3 days. Gd decreased the levels of all M1 and M2 markers. Arginase1 and iNOS protein levels also increased, and Gd reduced them due to apoptotic cell death. Gadolinium attenuated oedema, neuron loss, neurological deficits and the mortality rate without affecting haematoma sizes. CONCLUSIONS: Gadolinium induced M1 and M2 microglial apoptosis and exerted acute neuroprotective effects after ICH.

Bradykinin and interleukin-1ß synergistically increase the expression of cyclooxygenase-2 through the RNA-binding protein HuR in rat dorsal root ganglion cells.

Synergistic expression of cyclooxygenase-2 (COX-2) by interleukin-1ß (IL-1ß) and bradykinin (BK) in peri-sensory neurons results in the production of prostanoids, which affects sensory neuronal activity and responsiveness and causes hyperalgesia. To evaluate the effects of pro-inflammatory mediators on COX-2 expression, cultured rat dorsal root ganglion (DRG) cells were treated with IL-1ß and BK, which caused persistent increased COX-2 expression. Co-treatment increased COX-2 transcriptional activities in an additive manner by a COX-2 promoter luciferase assay. Immunoprecipitated HuR, an RNA-binding protein, in co-treated DRG cells contained more COX-2 mRNA than that of the control. The synergistic effects of IL-1ß and BK on COX-2 expression may be a result of RNA stabilization mediated by HuR in peri-sensory neurons. Multiple pro-inflammatory cytokines and mediators are produced during neurogenic inflammation and aberrant control of COX-2 mRNA turnover may be implicated in diseases including chronic inflammation, which results in inflammation-derived hyperalgesia around primary sensory neurons.

Shogaol but not gingerol has a neuroprotective effect on hemorrhagic brain injury: Contribution of the α, ß-unsaturated carbonyl to heme oxygenase-1 expression.

We investigated the effects of shogaol, which has an α, ß-unsaturated carbonyl group, and gingerol, which does not, on primary-cultured microglia to understand how the α, ß-unsaturated carbonyl interacts with Kelch-like ECH-associated protein (Keap)1. Shogaol (1 µM) but not the same concentration of gingerol significantly increased heme oxygenase (HO)-1 protein levels in cultured microglia without cytotoxicity. In addition, shogaol suppressed the release of the inflammation marker nitric oxide induced by 30 U/ml thrombin treatment. A docking simulation suggested that the α, ß-unsaturated carbonyl of shogaol but not gingerol interacts with Keap1. Nuclear import of nuclear factor E2-related factor 2 and increased binding of the HO-1 E2 enhancer support the docking-simulation prediction. The transcription inhibitor actinomycin D (0.1 µg/ml) markedly blocked the increase of HO-1 mRNA levels by shogaol. To evaluate whether the α, ß-unsaturated carbonyl can be used for intracerebral hemorrhage (ICH) therapy, we investigated the effect of shogaol on an in vivo mouse ICH model. Intracerebroventricular injection of 0.2 nmol shogaol increased striatal HO-1 protein levels and rescued ICH-induced neuron loss. Thus, the α, ß-unsaturated carbonyl is necessary for the interaction of compounds, such as shogaol, with Keap1, and these findings may be useful for screening novel ICH therapeutic agents that increase HO-1 expression.

Curcuma sp.-derived dehydrocurdione induces heme oxygenase-1 through a Michael reaction between its α, ß-unsaturated carbonyl and Keap1.

To elucidate the anti-inflammatory mechanism of Curcuma sp., we investigated whether dehydrocurdione, a sesquiterpene contained in Curcuma sp., induces heme oxygenase (HO)-1, an antioxidative enzyme, in RAW 264.7 macrophages. Dehydrocurdione was extracted from the rhizome of Curcuma sp., and its purity was verified by high performance liquid chromatography. Treatment with 10-100 µM dehydrocurdione transiently and concentration-dependently increased HO-1 mRNA and protein levels. Docking simulation suggested the presence of the Michael reaction between dehydrocurdione and Kelch-like ECH-associated protein (Keap)1 keeping nuclear factor-erythroid2-related-factor (Nrf)2, a transcription factor, in the cytoplasm. Nrf2 that was definitely free from Keap1 was detected in the nuclei after dehydrocurdione treatment. Subsequently, the HO-1 E2 enhancer, a target of Nrf2, was activated, resulting in HO-1 expression. Also, an investigation using 6-shogaol and 6-gingerol supported the concept that the α, ß-unsaturated carbonyl structure plays an important role in the interaction with Keap1. Dehydrocurdione suppressed lipopolysaccharide-induced NO release, a marker of inflammation. Clarification of the HO-1 synthesis increase mechanism revealed in this study will help contribute to the development of novel phytotherapeutic strategies against inflammation-associated diseases.

Fabrication and characterization of water-dispersed chitosan nanofiber/poly(ethylene glycol) diacrylate/calcium phosphate-based porous composites.

We evaluated the capacity of chitosan nanofiber (CNF)- and poly(ethylene glycol) (PEG)-based hydrogel/calcium phosphate hybrid (CNF-PEG/CaP) composites to act as scaffolding materials. CNF-PEG/CaP composites were fabricated by mineralization of CNF-PEG hydrogels using an alternate soaking method. The amount of CaP mineralized on CNF-PEG hydrogels increased as the ratio of CNF in the CNF-PEG hydrogel increased. Young's modulus of CNF-PEG/CaP hydrogels was enhanced by increase in CNF ratio. It was further confirmed that osteoblasts embedded on the CNF-PEG/CaP composites were viable after incubation for 5days and firmly attached to the CaP porous layer, forming an extensive cell-scaffold leading to cell-cell interactions. These results indicated that the micro-porous structure of CNF-PEG hydrogels is suitable for CaP to be utilized as a scaffold for bone regeneration.

Claudin domain containing 1 contributing to endothelial cell adhesion decreases in presence of cerebellar hemorrhage.

The claudin family comprises four-pass transmembrane proteins involved in the formation of tight junctions (TJs). Relatively recently, claudin domain containing (CLDND) 1, also known as claudin-25, was identified as a novel member of the claudin family. In the present study, we revealed that in the adult murine brain, CLDND1 is abundant in the cerebellum among common sites of intracerebral hemorrhage. Thus, the dynamics of CLDND1 after cerebellar hemorrhage were examined. Both CLDND1 mRNA and protein levels transiently decreased at 24 hr after hemorrhagic insult. For immunostaining, an anti-CLDND1 antibody that recognizes the specific epitope in the extracellular first loop was prepared. Dual immunohistochemical staining with CD31 using coronal cryosections of intact murine cerebellum tissue revealed that CLDND1 is expressed on endothelial cells. We therefore performed an in vitro permeability test using a human brain endothelial cell (HBEC) line to reveal whether CLDND1 contributes to cell adhesion like other claudins. CLDND1 was expressed on HBECs as well as in murine cerebellum tissue, and a strong signal was observed at TJs. RNA interference against CLDND1 decreased both the mRNA and protein levels without cytotoxicity. The permeability to small molecules, but not to large ones, across confluent HBECs increased on CLDND1 knockdown compared with mock-treated cells. These results suggest that the transient decrease of CLDND1 after cerebellar hemorrhage is responsible for low-molecular-weight selective vascular hyperpermeability. © 2017 Wiley Periodicals, Inc.

Selective enhancement of wnt4 expression by cyclic AMP-associated cooperation between rat central astrocytes and microglia.

The wnt protein family has important members involved in cell differentiation, proliferation and plasticity expression; however, little is known about its biosynthesis processes. On the other hand, an increase in the intracerebral cyclic adenosine 3', 5'-monophosphate (cAMP) level leads to synaptic plasticity via the de novo synthesis of any protein. Here, the effect of dibutyryl cAMP (dbcAMP), a membrane permeability cAMP analog, on the wnt family was investigated in rat primary-cultured glial cells containing astrocytes and microglia. Among wnt3a, 4, 5a, 7a and 11 mRNA, only wnt4 expression was increased by longer treatment (24 h), compared with short treatment (2 h), with dbcAMP in a concentration-dependent manner, and its effect reached statistical significance at 1 mM. In cultures of isolated astrocytes or microglia, wnt4 expression was not affected by 1 mM dbcAMP for 24 h, and microglial wnt4 protein was undetectable even when cells were treated with the drug. Mixed glial cells treated for 24 h with 1 mM dbcAMP showed significantly increased wnt4 protein, as well as mRNA. Immunofluorescence manifested that cells that expressed wnt4 protein were astrocytes, but not microglia. Intraperitoneal injection of 1.25 mg/kg rolipram, a phosphodiesterase (PDE) IV inhibitor that can pass through the blood brain barrier and inhibits cAMP degradation specifically, showed a tendency to increase wnt4 expression in the adult rat brain after 24 h, and the increases in wnt4 mRNA and protein levels reached statistical significance in the hippocampus and striatum, respectively. This is the first finding to help elucidate the selective biosynthesis of central wnt4 through cAMP-stimulated microglia and astrocytes interaction.

Sesamin increases heme oxygenase-1 protein in RAW 264.7 macrophages through inhibiting its ubiquitination process.

Sesamin is a major component in lignans of sesame seed oil, known to possess potent anti-oxidative capacity. In this study, the variation of heme oxygenase (HO)-1, a kind of anti-oxidative enzyme, by sesamin in murine macrophage cell line RAW 264.7 cells was investigated. Lipopolysaccharide (LPS; 10µg/ml) exposure tended to increase HO-1 protein expression. Co-treatment with 100µM sesamin for 12h up-regulated the HO-1 protein level increased by LPS; however, HO-1 mRNA was unaffected. Sesamin delayed the reversal, by the protein synthesis inhibitor cycloheximide (1µM), of the LPS-induced increase of HO-1 protein level. Meanwhile, sesamin suppressed LPS-induced expression of inducible nitric oxide (NO) synthase (iNOS) protein and associated NO release. LPS-induced increase of iNOS protein expression was also reversed by cycloheximide, which was not affected by sesamin, unlike HO-1. To clarify the mechanisms that underlie the up-regulation of HO-1 protein level by sesamin, the human embryonic kidney (HEK) 293T cell line transfected with Flag-tagged HO-1 was used. A proteasome inhibitor, MG-132 (10µM), stabilized HO-1 protein in HEK 293T cells. Co-treatment with sesamin decreased ubiquitinated HO-1 protein accumulation by MG-132. However, sesamin did not affect the proteasome activity. These findings suggest that sesamin disturbs the degradation of HO-1 protein through inhibiting its ubiquitination, resulting in HO-1 protein up-regulation.

High-mobility group box 1 up-regulates aquaporin 4 expression via microglia-astrocyte interaction.

To clarify the mechanism of high-mobility group box (HMGB) 1-induced brain edema formation, this study focused on the effect of HMGB1 on aquaporin (AQP) 4, a water channel, in rat brain. Treatments for 6h with 100-1000ng/ml HMGB1, not showing self-toxicity, of primary-cultured rat astrocytes didnot increase AQP4 mRNA, unexpectedly. In contrast, intracerebroventricular (i.c.v.) injection of 300ng of HMGB1 significantly increased AQP4 protein after 8h and formed edema after 24h in vivo. Thus, we investigated the roles of microglia as well as astrocytes. HMGB1 (1000ng/ml) drastically increased interleukin (IL)-1ß in the primary-cultured rat microglia after 2h. The exposure of microglia to conditioned medium with HMGB1 and 3mM adenosine 5'-triphosphate for 6h significantly increased AQP4 mRNA in astrocytes after 6h. Although 1000ng/ml HMGB1 didnot induce transfer of nuclear factor (NF)-κB into the nucleus in astrocytes after 1h, the conditioned medium containing IL-1ß led to its nuclear import. As factors likely to be involved in the nuclear import of NF-κB besides IL-1ß, nitric oxide and tumor necrosis factor-α didnot contribute under these conditions. Finally, i.c.v. injection of 30nmol parthenolide, an NF-κB inhibitor, reversed 300ng of HMGB1 injection-induced AQP4 protein increase after 8h in vivo. The effect of parthenolide and the outcomes obtained so far suggest that HMGB1 indirectly up-regulates AQP4 expression through diffusible factor(s) such as IL-1ß from microglia since HMGB1 by itself didnot affect NF-κB intracellular localization in astrocytes.

Protein kinase A-dependent substance P expression by pituitary adenylate cyclase-activating polypeptide in rat sensory neuronal cell line ND7/23 cells.

The neurotrophic effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on rat sensory neuronal cell line ND7/23 cells were investigated. PACAP caused a concentration-dependent increase in the number of neurite-bearing cells and the expression of the substance P precursor (PPT) mRNA in 24 h. The effects of PACAP were mimicked by vasoactive intestinal polypeptide with lower potency and dibutyryl-cyclic AMP, and inhibited by inhibitors of protein kinase A, ERK kinase or p38 kinase, KT5720, U0126, or SB203580, respectively. In a PPT promoter luciferase reporter assay, the increase of PPT mRNA was the result of an increase in PPT gene transcriptional activity by PACAP. The increasing effects of PACAP on PPT mRNA were similarly observed in primary cultured rat dorsal root ganglion cells. Thus, PACAP could induce differentiation-like phenomena in sensory neurons in a cAMP-, protein kinase A-, ERK kinase-, and p38 kinase-dependent manner. These results provide evidence of the neurotrophic action of PACAP, which may function to rescue damaged neurons or to switch the neuronal phenotype in injured or inflamed sensory neurons.

HMGB1 inhibitor glycyrrhizin attenuates intracerebral hemorrhage-induced injury in rats.

Thrombin activates immunocompetent microglia and increases release of inflammatory cytokines under intracerebral hemorrhage (ICH) insults. Also, thrombin injection into the striatum evokes acute necrosis and delayed apoptosis of neurons. A nucleoprotein high-mobility group box 1 (HMGB1) that is released from necrotic cells has been suggested to behave like a cytokine and cause over-facilitation of immune functions. Here we examined the effect of glycyrrhizin, known as an inhibitor of HMGB1, on thrombin-induced injury in rat cortico-striatal slice cultures and in vivo rat ICH model. In slice cultures, thrombin-induced a drastic increase in propidium iodide fluorescence indicating necrotic cell death in the cortical region, and robust shrinkage of the striatal tissue. Glycyrrhizin (10-100 µM) attenuated thrombin-induced cortical injury in a concentration-dependent manner. The protective effect of glycyrrhizin was not mediated by glucocorticoid receptors or modulation of nitric oxide production, but was reversed by exogenous HMGB1 application. The injury induced by a high concentration of HMGB1 was suppressed by glycyrrhizin. In vivo, unilateral injection of type IV collagenase into rat striatum induced ICH associated with brain edema formation, contralateral paralysis and neuron death. Once daily intraperitoneal administration of glycyrrhizin attenuated ICH-induced edema in both the cortex and the basal ganglia, and improved behavioral performance of rats in forelimb placing. Moreover, glycyrrhizin partially but significantly ameliorated ICH-induced neuron loss inside hematoma. These findings suggest that an HMGB1 inhibitor glycyrrhizin is a potential candidate for a remedy for ICH.

Four novel furanocoumarin glucosides, candinosides A, B, C and D, from Heracleum candicans Wall.

Four novel furanocoumarin glucosides, candinosides A, B, C and D (1-4), were isolated from the roots of Heracleum candicans Wall. Their structures were established using chemical and spectral methods.

Five condensed furanocoumarins from the root of Heracleum candicans Wall.

Three new spirotrifuranocoumarins, canditririns C-E (1-3), a new spirotetrafuranocoumarin, canditetrarin A (4), and a new tetrafuranocoumarin, canditetrarin B (5), were isolated from the roots of Heracleum candicans Wall. Their structures were established using spectral methods.

Acting performance and flow state enhanced with sensory-motor rhythm neurofeedback comparing ecologically valid immersive VR and training screen scenarios.

Actors were trained in sensory-motor rhythm (SMR) neurofeedback interfaced with a computer rendition of a theatre auditorium. Enhancement of SMR led to changes in the lighting while inhibition of theta and high beta led to a reduction in intrusive audience noise. Participants were randomised to a virtual reality (VR) representation in a ReaCTor, with surrounding image projection seen through glasses, or to a 2D computer screen, which is the conventional neurofeedback medium. In addition there was a no-training comparison group. Acting performance was evaluated by three experts from both filmed, studio monologues and Hamlet excerpts on the stage of Shakespeare's Globe Theatre. Neurofeedback learning reached an asymptote earlier as did identification of the required mental state following training in the ReaCTor training compared with the computer screen, though groups reached the same asymptote. These advantages were paralleled by higher ratings of acting performance overall, well-rounded performance, and especially the creativity subscale including imaginative expression, conviction and characterisation. On the Flow State scales both neurofeedback groups scored higher than the no-training controls on self-ratings of sense of control, confidence and feeling at-one. This is the first demonstration of enhancement of artistic performance with eyes-open neurofeedback training, previously demonstrated only with eyes-closed slow-wave training. Efficacy is attributed to psychological engagement through the ecologically relevant learning context of the acting-space, putatively allowing transfer to the real world otherwise achieved with slow-wave training through imaginative visualisation. The immersive VR technology was more successful than a 2D rendition.

Chemical studies on the root of Heracleum candicans Wall. (Part 3).

Two new ester coumarins, candinols B and C (1 and 2), and three new coumarin dimers, candibirins F-H (3-5), were isolated from the roots of Heracleum candicans Wall. Their structures were established by using chemical and spectral means.