Tadashi Hisamitsu

Cutaneous hyperalgesia induced by peripheral injection of interleukin-1β in the rat

The contribution of the activity of afferent fiber filaments to pain and hyperalgesia after administration of a plantar injection of interleukin-1 beta (IL-1 beta) to the hind-paw skin was investigated by recording action potentials of the rat dorsal root in response to mechanical and thermal stimuli. Touch stimuli were delivered by stroking with a cotton-tipped applicator and thermal stimulation was applied by cooling or heating of the skin. After the administration of IL-1 beta (100 pg-1 microgram), responses to touch, cold, and heat stimulation increased to 143%, 200%, and 392%, respectively, of control values on average. IL-1 beta induced transient spontaneous discharge in 50% of experiments. The effects of IL-1 beta were apparent within 1 min. To examine responses to pressure stimulation, an area of 1 mm2 of the hind-paw skin was pressed by a mechanical stimulator. IL-1 beta (0.1 pg-200 ng) decreased the threshold value to 58% of the control pressure required for firing. IL-1 beta also increased responses to various levels of pressure (range: 1-20 g/mm2). These data suggest that IL-1 beta may play an important role in cutaneous hyperalgesia by activating polymodal receptors to mechanical and thermal stimulation.

Descending pain inhibitory system involved in acupuncture analgesia

The descending pain inhibitory system (DPIS) associated with acupuncture analgesia (AA), caused by low frequency stimulation of an acupuncture point, was identified by the results of lesion and stimulation procedures previously determined to differentiate the afferent and efferent paths in rats. The DPIS starts in the posterior arcuate nucleus and descends to the hypothalamic ventromedian nucleus (HVM) from whence it divides into two pathways: one path, the serotonin mediated path, descends through the ventral periaqueductal central gray (V-PAG) and then to the raphe magnus (RM). The other, the noradrenaline mediated path, descends through the reticuloparagigantocellular nucleus (NRPG) and part of the reticulogigantocellular nucleus (NRGC). The afferent and efferent paths are both present in the RM and NRGC, and were separately identified by means of the analgesia (SPA) produced by stimulation of the separate regions in AA responders and nonresponders, because SPA of these regions in nonresponders produced only efferent pathway mediated analgesia.

The acupuncture point and its connecting central pathway for producing acupuncture analgesia

Characteristics of the acupuncture point in producing acupuncture analgesia (AA) were examined by the inhibition of noxious responses in the brain stem reticular formation, potentials, and neuronal activity in the dorsal periaqueductal central gray (D-PAG), and analgesia caused by low frequency stimulation of the acupuncture point. As a result, stimulation of the muscle beneath the acupuncture point was found to be effective in producing AA. AA measured by tail flick, vocalization, and writhing tests was abolished by hypophysectomy, and by antiserum of beta-endorphin administered into the 3rd ventricle. The pathway from the D-PAG to the anterior hypothalamus (AA-AH) in the AA afferent pathway from the acupuncture point to the pituitary gland was determined. The lateral hypothalamus, lateral septum, cingulate bundle, dorsal-hippocampus, and habenulo-interpeduncular tract were found, in addition to regions previously found, to belong to the AA afferent pathway. A network of divergence and convergence in their rostral and caudal relations was observed. The AA afferent pathway diverges from the D-PAG, converges to the HP, and then projects to the AA-AH.

Role of glutamate and NMDA receptors in the descending limb of the spinobulbospinal micturition reflex pathway of the rat

MK-801, an NMDA receptor antagonist administered intravenously or intrathecally to the L6-S1 spinal cord inhibited in a dose dependent manner the amplitude of isovolumetric bladder contractions evoked by electrical stimulation in the pontine micturition center (PMC) in urethane anesthetized rats. The mean threshold dose of MK-801 was 10 +/- 6 micrograms/kg i.v. and 10 +/- 1 micrograms i.t. Bladder contractions were completely inhibited at doses ranging from 300 to 3000 micrograms/kg i.v. and from 18 to 48 micrograms i.t. These data indicate that NMDA glutamatergic receptors play an important role in excitatory transmission in the descending pathway from the PMC to the spinal segmental circuitry involved in the control of the urinary bladder.

Role of endogenous interferon-γ on the enhancement of splenic NK cell activity by electroacupuncture stimulation in mice

Successive electro-acupuncture (EA) stimulation applied to bilateral anterior tibial muscles, where Zusanli (ST36) acupoints are located, once a day (30 min) for 3 successive days significantly enhanced splenic natural killer (NK) cell activity in BALB/c mice. The percentage of splenic NK cells, as measured by flow cytometry, was not affected in these mice. Interferon (IFN)-gamma level in splenic aqueous extract, prepared from the ST36 acupoint-stimulated mice, was significantly higher than that of the controls. In vivo treatment with neutralizing monoclonal antibody against mouse IFN-gamma completely abrogated the increase in splenic NK cell activity induced by ST36 acupoint stimulation. The same stimulation also significantly increased the concentration of splenic beta-endorphin, which coincided with the significant increase in splenic IFN-gamma production. Pre-administration of 10 mg/kg naloxone before initiation of EA stimulation every day reduced the enhancements of NK cell activity and IFN-gamma level. These observations strongly suggest that endogenous IFN-gamma mediates the up-regulation of NK cell activity by EA stimulation at the ST36 acupoints. Furthermore, endogenous beta-endorphin secreted by EA stimulation also plays an important role in the up-regulation of NK cell function, which may be realized through regulating IFN-gamma production.

Suppressive activity of a macrolide antibiotic, roxithromycin, on pro-inflammatory cytokine production in vitro and in vivo

This study was designed to examine the influence of a macrolide antibiotic, roxithromycin (RXM), on the production of pro-inflammatory cytokines, interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. In the first experiments, we examined the effect of RXM on in vitro cytokine production from lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes. The monocytes were cultured in the presence of various doses of the agent. After 24 h, the culture supernatants were obtained and assayed for IL-1beta and TNF-alpha contents by enzyme-linked immunosorbent assay. RXM suppressed the in vitro production of IL-1beta and TNF-alpha in response to LPS stimulation. This was dose dependent and first noted at a concentration of as little as 0.05 microg/ml, which is much lower than therapeutic blood levels. In the second part of the experiments, we examined the influence of RXM on the appearance of IL-1beta and TNF-alpha in mouse lung extract induced by LPS inhalation. RXM was administered orally into BALB/c mice at a single dose of 2.5 mg/kg once a day for 5-12 weeks. These mice were then instilled with LPS into the trachea and examined for the presence of cytokines in aqueous lung extracts. Pretreatment of mice with RXM for 5 weeks did not influence of the appearance of both IL-1beta and TNF-alpha in aqueous lung extracts. However, pretreatment for more than 7 weeks dramatically suppressed the cytokine appearance in the extracts.

Interleukin-17A promotes rheumatoid arthritis synoviocytes migration and invasion under hypoxia by increasing MMP2 and MMP9 expression through NF-κB/HIF-1α pathway.

Both hypoxia and interleukin-17A (IL-17A) promote the migration and invasion of fibroblast-like synoviocytes (FLSs), which are critical for the pathogenesis of rheumatoid arthritis (RA). However, the biochemical pathways regulating IL-17A combined with hypoxia are not well defined. In this study, we found that co-stimulating RA-FLSs with IL-17A and hypoxia did not appear to promote the epithelial-mesenchymal transition (EMT), but did increase cell motility. We further showed that a proinvasive effect of IL-17A on FLSs under hypoxia might be through upregulation of matrix metalloproteinase 2 (MMP2) and MMP9. Moreover, IL-17A-induced expression of MMP2 and MMP9 under hypoxia was accompanied by increased activation of nuclear factor-κB (NF-κB)/hypoxia-inducible factor-1α (HIF-1α). Knockdown or inhibition of HIF-1α and NF-κB by small interfering RNA or specific small molecule inhibitors blocked IL-17A-mediated and hypoxia-mediated MMP2 and MMP9 expression, cell migration, and invasion. In addition, the inhibition of NF-κB led to a marked decrease in the expression of HIF-1α, which indicated that IL-17A activated HIF-1α via the NF-κB pathway in hypoxia. Taken together, our observations suggest a synergetic effect of IL-17A and hypoxia that might contribute to the migration and invasion of RA-FLSs by upregulating the expression of MMP2 and MMP9 by activation of the NF-κB/HIF-1α pathway.

Celastrol Inhibits Lipopolysaccharide-Stimulated Rheumatoid Fibroblast-Like Synoviocyte Invasion through Suppression of TLR4/NF-κB-Mediated Matrix Metalloproteinase-9 Expression

Invasion of fibroblast-like synoviocytes (FLSs) is critical in the pathogenesis of rheumatoid arthritis (RA). The metalloproteinases (MMPs) and activator of Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway play a critical role in RA-FLS invasion induced by lipopolysaccharide (LPS). The present study aimed to explore the anti-invasive activity of celastrol on LPS-stimulated human RA-FLSs, and to elucidate the mechanism involved. We investigated the effect of celastrol on LPS-induced FLS migration and invasion as well as MMP expression and explored the upstream signal transduction. Results showed that celastrol suppressed LPS-stimulated FLS migration and invasion by inhibiting MMP-9 expression and activity. Furthermore, our results revealed that celastrol inhibited the transcriptional activity of MMP-9 by suppressing the binding activity of NF-κB in the MMP-9 promoter, and suppressed the TLR4/MyD88/NF-κB pathway. Administration of celastrol (0.5 mg/kg and 1 mg/kg, intraperitoneally) daily for 3 weeks in a collagen-induced arthritis rat model markedly alleviated the clinical signs, synovial hyperplasia and inflammatory cell infiltration of joints. In conclusion, celastrol might inhibit FLS migration and invasion induced by LPS by suppressing TLR4/NF-κB-mediated MMP-9 expression, providing a theoretical foundation for the clinical treatment of RA with celastrol.

Suppressive effects of Tripterygium wilfordii Hook f., a traditional Chinese medicine, on collagen arthritis in mice.

The effect of chloroform extract of Tripterygium wilfordii Hook f. (TWH extract), a traditional immunosuppressive Chinese herb, on type II collagen (C II)-induced arthritis (CIA) in DBA/1J mice was studied. In the first set of experiments, we examined the effect of TWH extract on cellular immune responses to C II. As compared with mice treated with saline, TWH extract administered orally at doses of more than 400 microg kg(-1) once a day for 14 days inhibited the ability of inguinal lymph node cells to produce T cell cytokines interleukin-2 and interferon-gamma when the cells were obtained from mice 21 days after immunization and cultured in vitro with C II. Treatment with TWH extract also inhibited production of macrophage cytokines interleukin-1beta and tumor necrosis factor-alpha in response to in vitro stimulation of lymph node cells with C II. In the second part of the experiment, we evaluated the influence of TWH extract on the incidence and development of arthritis in murine CIA. Mice were immunized twice at a 3-week interval with bovine C II, with TWH extract being given orally once a day for 14 days with four different regimens. A 14-day course of TWH extract treatment at a daily dose of 400 microg kg(-1), which began on the day of the first C II immunization, suppressed the development of arthritis, as well as antibody production and delayed-type hypersensitivity to C II. Treatment with TWH extract, which started on the same day as the booster immunization, also resulted in inhibition of development of arthritis and of immune responses to C II. On the other hand, therapeutic administration with TWH extract did not affect the clinical course of the disease and the immune response to C II.

Non-NMDA glutamatergic excitatory transmission in the descending limb of the spinobulbospinal micturition reflex pathway of the rat.

I.v. administration of GYKI-52466, a non-competitive AMPA/kainate glutamatergic receptor antagonist, inhibited bladder contractions elicited by electrical stimulation in the pontine micturition center (PMC) in urethane-anesthetized rats. The mean threshold dose of GYKI-52466 was 2 mg/kg i.v. (range = 1-4 mg/kg). Maximum inhibition (mean = 57.7 +/- 8.2%, range = 24-83.3% of control) occurred at a dose of 8 mg/kg. CNQX, a competitive AMPA/kainate glutamatergic receptor antagonist, did not significantly alter the evoked contractions. These results indicate that AMPA/kainate receptors are involved in bulbospinal excitatory pathway from the PMC to the parasympathetic nucleus in the lumbosacral spinal cord in the rat.