Substance P-Derived Extracellular-Matrix-Mimicking Peptide Hydrogel as a Cytocompatible Biomaterial Platform.

Self-assembled short peptide-based hydrogel platforms have become widely applicable biomedical therapeutic maneuvers for their soft, tunable architecture, which can influence cellular behavior and morphology to an inordinate extent. In this work, a short supramolecular hydrogelator peptide, substance P, has been designed and synthesized from the C terminus conserved "FFGLM" section of a biologically abundant neuropeptide by using a fusion approach. In addition, to incorporate a good hydrophobic-hydrophilic balance, the truncated pentapeptide segment was further C-terminally modified by the incorporation of an integrin-binding "RGD" motif. Thanks to its N-terminal Fmoc group, this octapeptide ensemble "FFGLMRGD" undergoes rapid self-assembly to give rise to an injectable, pH-responsive, hydrogel-based self-supporting platform that exhibited good cytocompatibility with the cultured mammalian cells under both 2D and 3D culture conditions without exerting any potent cytotoxic effect in a Live/Dead experiment. A rheological experiment demonstrated its hydrogel-like mechanical properties, including thixotropicity. The atomic force microscopy and field emission scanning electron microscopy images of the fabricated hydrogel show a tangled fibrous surface topography owing to the presence of the N-terminal Fmoc-FF residue. Furthermore, an in-vitro scratch assay performed on fibroblast cell lines confirmed the wound-ameliorating potency of this designed hydrogel; this substantiates its future therapeutic prospects.

In Situ Blood Vessel Regeneration Using SP (Substance P) and SDF (Stromal Cell-Derived Factor)-1α Peptide Eluting Vascular Grafts.

OBJECTIVE: The objective of this study was to develop small-diameter vascular grafts capable of eluting SDF (stromal cell-derived factor)-1α-derived peptide and SP (substance P) for in situ vascular regeneration. APPROACH AND RESULTS: Polycaprolactone (PCL)/collagen grafts containing SP or SDF-1α-derived peptide were fabricated by electrospinning. SP and SDF-1α peptide-loaded grafts recruited significantly higher numbers of mesenchymal stem cells than that of the control group. The in vivo potential of PCL/collagen, SDF-1, and SP grafts was assessed by implanting them in a rat abdominal aorta for up to 4 weeks. All grafts remained patent as observed using color Doppler and stereomicroscope. Host cells infiltrated into the graft wall and the neointima was formed in peptides-eluting grafts. The lumen of the SP grafts was covered by the endothelial cells with cobblestone-like morphology, which were elongated in the direction of the blood flow, as discerned using scanning electron microscopy. Moreover, SDF-1α and SP grafts led to the formation of a confluent endothelium as evaluated using immunofluorescence staining with von Willebrand factor antibody. SP and SDF-1α grafts also promoted smooth muscle cell regeneration, endogenous stem cell recruitment, and blood vessel formation, which was the most prominent in the SP grafts. Evaluation of inflammatory response showed that 3 groups did not significantly differ in terms of the numbers of proinflammatory macrophages, whereas SP grafts showed significantly higher numbers of proremodeling macrophages than that of the control and SDF-1α grafts. CONCLUSIONS: SDF-1α and SP grafts can be potential candidates for in situ vascular regeneration and are worthy for future investigations.

Effect of systemic delivery of Substance P on experimental tooth movement in rats.

INTRODUCTION: The purpose of this study was to investigate the effect of systemic delivery of Substance P (SP) on experimental tooth movement. METHODS: Forty-eight adult Sprague-Dawley rats were randomly divided into 2 groups and their maxillary first molars were mesially moved with the use of closed-coil springs. The experiment group received systemic injection of SP and the control group received phosphate-buffered saline solution. Transportation distances of first molars were measured. Hematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, and immunohistochemistry staining were performed to evaluate alveolar bone remodeling. Then the interferon (IFN) γ and tumor necrosis factor (TNF) α concentrations in peripheral blood and local periodontal tissue were measured. Finally, the effects of SP on bone marrow-derived stem cell (BMSC) proliferation and migration were tested in vitro. RESULTS: Systemic delivery of SP significantly increased the distance of tooth movement and stimulated both osteoclast and osteoblast activities. The concentrations of IFN-γ and TNF-α increased in peripheral blood at early phases of the experiment and decreased in periodontal tissue at late phases. In vitro, the proliferation and migration of BMSCs were promoted by SP. CONCLUSIONS: Systemic delivery of SP can accelerate orthodontic tooth movement and promote alveolar bone remodeling potentially through immunomodulation and mobilizing endogenous mesenchymal stem cells.

A Novel Substance P-Based Hydrogel for Increased Wound Healing Efficiency.

The neuropeptide substance P (SP) is known to stimulate wound healing by regulating the production of relevant cytokines as well as cell proliferation and migration. However, the therapeutic application of SP is limited by its low stability under biological conditions and oxidation during purification, formulation, and storage. To address this problem, we developed a novel formulation of SP as an SP gel, and investigated its wound healing activity both in vitro and in vivo. SP in SP gel was stable at various temperatures for up to 4 weeks. In vitro, SP gel exhibited more potential as a candidate wound-healing agent than SP alone, as evidenced by the observed increases in the proliferation and migration of human epidermal keratinocytes and human dermal fibroblasts. In vivo experiments showed that SP gel treatment enhanced the healing of full-thickness wounds in mice as compared to SP alone. These results demonstrate the benefits of SP gel as a promising topical agent for wound treatment.

The effects of P. gingivalis and E. coli LPS on the expression of proinflammatory mediators in human mast cells and their relevance to periodontal disease.

Mast cells (MCs) are tissue-resident immune cells that participate in a variety of allergic and inflammatory conditions, including periodontal disease, through the release of cytokines, chemokines and proteolytic enzymes. Porhyromonas gingivalis (P. g) is widely recognized as a major pathogen in the development and progression of periodontitis. Here we compared the differential effects of lipopolysaccharides (LPS) from P. g and E. coli on the expression and production of tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein (MCP-1) by human MCs. Human LAD2 MCs were stimulated with LPS from either P. g or E. coli (1-1000 ng/ml). MCs were also stimulated with SP (2µM) serving as the positive control or media alone as the negative control. After 24 h, the cells and supernatant fluids were collected and analyzed for ß-Hexosaminidase (ß-hex) spectrophotometrically, TNF, VEGF and MCP-1 release by ELISA and real-time polymerase chain reaction (PCR) for mediator gene expression, respectively. To assess the functional role of tolllike receptors (TRL) in mediator release, MCs were pre-incubated with either anti-TLR2 or anti- TLR4 (2 µg/ml) polyclonal antibody for 1 h before stimulation with LPS. When MCs were stimulated with SP (2 µM), there was a statistically significant ß-hex release as well as release of TNF, VEGF and MCP-1. Stimulation of MCs with either type of LPS did not induce degranulation based on the lack of ß-hex release. However, both types of LPS stimulated expression and release of TNF, VEGF and MCP-1. Although, P. g LPS induced significant release of TNF, VEGF and MCP-1, the effect was not concentration-dependent. There was no statistically significant difference between the effects of P. g and E. coli LPS. P. g LPS stimulated TNF through TLR-2 while E. coli utilized TRL-4 instead. In contrast, VEGF release by P. g LPS required both TRL-2 and TRL-4 while E. coli LPS required TLR-4. Release of MCP-1 was independent of TLR-2 or TLR-4. P. g LPS activates human MCs to generate and release TNF, VEGF and MCP-1 through different TLRs than E. coli LPS. MCs may, therefore, be involved in the inflammatory processes responsible for periodontal disease.

In situ vascular regeneration using substance P-immobilised poly(L-lactide-co-ε-caprolactone) scaffolds: stem cell recruitment, angiogenesis, and tissue regeneration.

In situ tissue regeneration holds great promise for regenerative medicine and tissue engineering applications. However, to achieve control over long-term and localised presence of biomolecules, certain barriers must be overcome. The aim of this study was to develop electrospun scaffolds for the fabrication of artificial vascular grafts that can be remodelled within a host by endogenous cell recruitment. We fabricated scaffolds by mixing appropriate proportions of linear poly (l-lactide-co-ε-caprolactone) (PLCL) and substance P (SP)-immobilised PLCL, using electrospinning to develop vascular grafts. Substance P was released in a sustained fashion from electrospun membranes for up to 30 d, as revealed by enzyme-linked immunosorbent assay. Immobilised SP remained bioactive and recruited human bone marrow-derived mesenchymal stem cells (hMSCs) in an in vitro Trans-well migration assay. The biocompatibility and biological performance of the scaffolds were evaluated by in vivo experiments involving subcutaneous scaffold implantations in Sprague-Dawley rats for up to 28 d followed by histological and immunohistochemical studies. Histological analysis revealed a greater extent of accumulative host cell infiltration and collagen deposition in scaffolds containing higher contents of SP than observed in the control group at both time points. We also observed the presence of a large number of laminin-positive blood vessels and Von Willebrand factor (vWF+) cells in the explants containing SP. Additionally, scaffolds containing SP showed the existence of CD90+ and CD105+ MSCs. Collectively, these findings suggest that the methodology presented here may have broad applications in regenerative medicine, and the novel scaffolding materials can be used for in situ tissue regeneration of soft tissues.

Effect of the neuropeptides vasoactive intestinal peptide, peptide histidine methionine and substance P on human major salivary gland secretion.

OBJECTIVE: The parasympathetic transmitters vasoactive intestinal peptide (VIP) and substance P (SP) are secretagogues in salivary glands of animals. Currently, we hypothesise that in human salivary glands, these neuropeptides and the VIP-related peptide histidine methionine (PHM) also exert secretory actions, reflected morphologically by exocytosis of acinar protein/glycoprotein-storing granules. MATERIALS AND METHODS: Submandibular and parotid gland tissues, exposed in vitro to VIP and PHM, and SP, respectively, were examined by light and transmission electron microscopy. For comparison, the response to in vitro stimulation of isoproterenol, phenylephrine and carbachol was examined. Moreover, the peptidergic innervation of the glands was examined by immunohistochemistry. RESULTS: Vasoactive intestinal peptide- and PHM-immunoreactive nerves were in close proximity to acini and ducts in the two glands, while these elements lacked a SP-positive innervation. While no morphological changes occurred in response to SP (parotid glands), VIP and PHM administration (submandibular glands) caused conspicuous acinar degranulation accompanied by luminal space broadening. In the two glands, both α1 - and ß-adrenergic receptor stimulation and muscarinic receptor stimulation caused similar changes as to VIP/PHM, although to varying extent. CONCLUSIONS: Vasoactive intestinal peptide and PHM, but not SP, are likely transmitters in the parasympathetic control of salivary (protein) secretion in humans.

Temporospatial Expression of Neuropeptide Substance P in Dental Pulp Stem Cells During Odontoblastic Differentiation in Vitro and Reparative Dentinogenesis in Vivo.

INTRODUCTION: Substance P (SP) is a neuropeptide released from the nervous fibers in response to injury. In addition to its association with pain and reactions to anxiety and stress, SP exerts various physiological functions by binding to the neurokinin-1 receptor (NK1R). However, the expression and role of SP in reparative dentinogenesis remain elusive. Here, we explored whether SP is involved in odontoblastic differentiation during reparative dentinogenesis. METHODS: Dental pulp stem cells (DPSCs) were isolated from healthy human dental pulp tissues and subjected to odontoblastic differentiation. The expression of SP and NK1R during odontoblastic differentiation was investigated in vitro. The effects of SP on odontoblastic differentiation of DPSCs were evaluated using alizarin red staining, alkaline phosphatase staining, and real-time polymerase chain reaction. After direct pulp capping with mineral trioxide aggregate, the expression of SP and NK1R during reparative dentin formation in rats were identified using histological and immunohistochemical staining. RESULTS: SP and NK1R expression increased during the odontoblastic differentiation of DPSCs. SP translocated to the nucleus when DPSCs were exposed to differentiation medium. NK1R was always present in the nuclei of DPSCs and odontoblast-like cells. Additionally, we discovered that 10-8 M SP marginally enhanced the odontoblastic differentiation of DPSCs, and that these effects could be impaired by the NK1R antagonist. Furthermore, SP and NK1R were expressed in odontoblast-like and dental pulp cells during reparative dentin formation in vivo. CONCLUSIONS: SP contributes to odontoblastic differentiation during reparative dentin formation by binding to the NK1R.

Atypical antipsychotics--effects of amisulpride on salivary secretion and on clozapine-induced sialorrhea.

OBJECTIVE: Amisulpride is suggested for treatment of clozapine-induced sialorrhea. However, objective measurements of its effectiveness are lacking and, preclinically, amisulpride has no effect. We currently hypothesise that amisulpride acts by reducing the nervous- rather than the clozapine-driven salivary secretion. MATERIAL AND METHODS: Effects of intravenous amisulpride (as well as of clozapine and raclopride, a dopamine D2/D3 antagonist) were investigated in rats, including those subjected to chronic preganglionic parasympathetic denervation (submandibular glands) or combined postganglionic parasympathetic and sympathetic denervation (parotid glands). In duct-cannulated glands, secretion was evoked reflexly, at low and maximum flow rates, and by electrical stimulation of the parasympathetic and sympathetic innervations, and administration of autonomimetics (including substance P). RESULTS: Unlike clozapine, amisulpride had no effect on the reflexly evoked secretion at maximum rate. With respect to reflex secretion at low rate and to the secretion evoked by muscarinic, α-adrenergic, ß-adrenergic and substance P receptors, amisulpride (in contrast to raclopride) dose dependently potentiated the responses. Amisulpride had no effect on gland blood flow. CONCLUSIONS: No support for any inhibitory influence of amisulpride was found. Conversely, amisulpride universally enhanced secretion, suggesting that amisulpride is a potential drug for dry-mouth treatment. The mechanism behind the potentiation is currently unknown.

Effect of bioactive glasses used as dentin desensitizers on the dentin-pulp complex in rats.

Bioactive glasses have been recommended for the occlusion of dentinal tubules in treating cervical dentin hypersensitivity. This study evaluates an in vivo model of dentin exposure, and tests the efficacy of bioglass treatments. Thirty male Wistar rats received gingival recession surgery on the upper left first molar. The treatments were applied over the surface of the exposed dentin every 4 days for 28 days. The groups were as follows: Naive; Gingival recession; Cavity varnish; Biosilicate®; Strontium bioglass; and Potassium bioglass. Changes in the dentin-pulp complex, and the presence of substance P, were evaluated through hematoxylin-eosin and immunohistochemical staining. The groups had similar results. Teeth with exposed dentinal tubules in rats showed a typical pattern in the dentin-pulp complex and immunotracing for substance P. The materials did not cause pulp damage. The effects of gingival recession and open dentinal tubules on pulp tissue require further clarification.

Nacre-mimetic hydroxyapatite/chitosan/gelatin layered scaffolds modifying substance P for subchondral bone regeneration.

Nacre, exhibiting excellent mechanical strengths due to its hierarchical structures, becomes a potential model to design bone implants. Herein, we firstly fabricated nacre-mimetic hydroxyapatite/chitosan/gelatin (HA/CS/Gel) layered scaffolds and incorporated substance P (SP) peptides. The CS scaffolds with a layered architecture could regulate the deposition of HA nanoplates on the interlamellar CS sheets by using CaCO3 as precursors. The HA/CS/Gel layered scaffolds exhibited a great flexure strength of 11.42 MPa due to a brick-and-mortar structure. The biocompatible components, layered macropores and SP peptides in the HA/CS/Gel therapeutic scaffolds facilitated the spreading and proliferation mesenchymal stem cells (MSCs). Notably, the incorporation of SP peptides induced MSC osteogenic differentiation and extracellular matrix mineralization. Rabbit knee subchondral defect models further proved that the HA/CS/Gel-SP layered scaffolds promoted in vivo subchondral bone regeneration. Hence, the combination of nacre-mimetic bone implants and therapeutic drugs may become an attractive strategy for subchondral bone regeneration.

Dental pulp fibroblasts express neuropeptide Y Y1 receptor but not neuropeptide Y.

AIM: To investigate whether dental pulp fibroblasts express neuropeptide Y (NPY) and NPY-Y1 in vitro and to determine the effects of the cytokines including interleukin-1ß (IL-1ß), TGF- ß(1) , substance P and NPY on the expression of NPY Y1. METHODOLOGY: Three primary fibroblast cell strains were obtained from freshly extracted human third molar teeth. RT-PCR was utilized to detect expression of NPY and mRNA expression. Membrane protein samples were isolated, and protein expression was determined by Western blotting. Radioimmunoassay was used to quantify NPY expression in healthy (n = 35) and carious (n = 39) whole pulp samples, and the student's t-test was used to test for statistical significance. In addition, the 3-(4,5-Dimethylthiazol,2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assay fibroblast cell growth. RESULTS: mRNA transcripts were found in all three fibroblast cell populations with the cytokines having a stimulatory effect on its expression (P < 0.05). NPY mRNA was not detected in the cell strains. NPY-Y1 receptor protein expression was visualized by Western blotting, and there was no effect of IL-1ß or TGF- ß(1) on its expression. The mean concentration of NPY-Ir determined by radioimmunoassay in non-carious teeth was 19.40 ng x g(-1) (±17.03 SD) compared to 29.95 ng x g(-1) (±20.99 SD) in carious teeth (P < 0.05). CONCLUSION: Human dental pulp fibroblasts express, but do not synthesize, NPY, demonstrating that the fibroblast is a target cell for NPY. The effect of proinflammatory cytokines suggests that fibroblasts play a neuroimmunomodulatory role in the pulpal response to dental caries and injury.

[Anti-inflammatory and repair activity of two toothpastes in an ex-vivo human gingival mucosa model]. / Comparaison de l'effet anti-inflammatoire et réparateur de deux dentifrices sur un modèle expérimental de muqueuse gingivale humaine.

INTRODUCTION: Our aim was to compare the anti-inflammatory activity of two toothpastes, one including enoxolone 1%, the other including plant extracts and sodium bicarbonate. MATERIAL AND METHODS: Gingival fragments were kept alive ex-vivo. Inflammation, inflammatory mediators (SP and LPS) were applied to culture medium on contact with corium to induce inflammation. The effect of both toothpastes was assessed with histological and biochemical parameters (inflammatory cytokine IL8) of inflammation on the synthesis of collagen and cellular viability. RESULTS: Both toothpaste "A" including enoxolone at 1% and "P" including plant extracts and sodium bicarbonate were effective on edema and vasodilatation. "A" acted on IL8 synthesis, unlike "P". Both toothpastes boosted collagen synthesis by fibroblasts. The percentage of cellular viability for "A" was superior to the currently admitted standard (80%), unlike to "P". DISCUSSION: The mechanisms of action of each toothpaste seem to be different. "A" modulates pro-inflammatory cytokine IL8 expression, unlike "P". The toothpaste "A" seems to be better tolerated.

Substance P-loaded electrospun small intestinal submucosa/poly(ε-caprolactone)-ran-poly(l-lactide) sheet to facilitate wound healing through MSC recruitment.

In this work, we prepared an electrospun small intestinal submucosa/poly(ε-caprolactone)-ran-poly(l-lactide) (SIS/PCLA) sheet onto which substance P (SP) was loaded, and this was employed as a cell-free scaffold for wound healing through the mobilization of human mesenchymal stem cells (hMSCs). SP release from the SP-loaded scaffold was 42% at 12 h and 51% at 24 h due to an initial burst of SP, but after 1 day, it exhibited a linear release profile and was released at a sustained rate for 21 days. The SP-loaded SIS/PCLA sheet exhibited higher in vitro and in vivo hMSC migration than did the PCLA and SIS/PCLA sheets. Large hMSCs injected into the tail vein of mice models migrated towards the wound to a greater extent in the presence of the SP-loaded SIS/PCLA sheet than with the PCLA and SIS/PCLA sheets, as confirmed by the CD44 and CD29 markers of recruited hMSCs. In animal wound models, significantly higher wound contraction (∼97%) in the group treated with the SP-loaded SIS/PCLA sheet was observed compared with the PCLA (∼74%) and SIS/PCLA (∼84%) groups at 3 weeks. In addition, SP-loaded SIS/PCLA-treated animals showed significant epidermal regeneration and collagen density (56%) in the mature granulation tissue at 3 weeks compared to the PCLA and SIS/PCLA groups. The wound area after SP-loaded SIS/PCLA sheet treatment also showed high blood vessel formation at the early stage, resulting in enhanced wound healing. Furthermore, the SP-loaded SIS/PCLA group exhibited a lower macrophage count (2.9%) than did the PCLA (7.7%) and SIS/PCLA (3.4%) groups. It was thus confirmed that the use of SP-loaded SIS/PCLA sheet as a cell-free scaffold could effectively enhance wound healing through MSC recruitment.

Desensitization of the inhibition of the M-current in sympathetic neurons: effects of ATP analogs, polyanions, and multiple agonist applications.

Desensitization occurs when the response to a neurotransmitter receptor agonist wanes in the continued presence of agonist. In amphibian sympathetic neurons, both muscarinic and peptidergic receptor agonists inhibit a K+ current, the M-current (IM), and this inhibition desensitizes. We have studied the desensitization to substance P (SP) by whole-cell recordings from dissociated sympathetic neurons from bullfrogs. When ATP in the recording pipette was replaced with AMP-PNP, SP still inhibited IM, but no desensitization was observed, indicating that ATP hydrolysis is required for desensitization. Desensitization inhibitors of beta-adrenergic receptors did not block desensitization to SP. When a low dose of muscarine sufficient to inhibit IM, but not to elicit desensitization, was applied simultaneously with a desensitizing dose of SP, IM remained depressed and did not desensitize. Thus, there may be separate systems controlling desensitization for different agonists, or the enzyme(s) involved is "compartmentalized."

Substance P regulates the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in cultured human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Substance P may play a role in the pathogenesis of periodontal disease; however, its mechanisms of modulation are not clear. This study evaluated the effect of two concentrations of Substance P on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in cultured human gingival fibroblasts. MATERIAL AND METHODS: Fibroblasts were stimulated for 48 h with 10(-4) or 10(-9) m Substance P; untreated fibroblasts served as controls. The expression of MMP-1, -2, -3, -7 and -11 and of TIMP-1 and -2 was evaluated using real-time polymerase chain reaction and western blotting. RESULTS: There was a significant, concentration-dependent stimulatory effect of Substance P on MMP-1, -2, -3 and -7 and TIMP-2 gene expression (p < 0.05), and a probable effect on MMP-11 (p = 0.06). At the higher concentration (10(-4) m Substance P), MMP-1, -2, -3, -7 and -11 and TIMP-2 showed the greatest up-regulation; at the lower concentration (10(-9) m Substance P), MMP-1, -3 and -7 and TIMP-2 exhibited diminished up-regulation, with MMP-2 and -11 showing down-regulation (p < 0.05). Expression of TIMP-1 was not affected by Substance P (p > 0.05). Western blotting confirmed that Substance P up-regulated MMP-1, -2, -3 and -11 and TIMP-2. MMP-1, -3 and -11 and TIMP-2 showed greater up-regulation at the higher Substance P concentration and diminished up-regulation at the lower concentration. MMP-2 was up-regulated to a similar degree at both Substance P concentrations. CONCLUSION: In gingival fibroblast cells, Substance P at the higher concentration (10(-4) m) induced greater up-regulation of MMP-1, -3 and -11 and TIMP-2 expression, but at the lower concentration (10(-9) m) induced diminished up-regulation, which may represent a mechanism for modulating periodontal breakdown.

Substance P influenced gelatinolytic activity via reactive oxygen species in human pulp cells.

AIM: To investigate the effects of substance P (SP) on gelatinolytic activity of matrix metalloproteinases (MMPs) in human pulp cells. METHODOLOGY: Human dental pulp cells were isolated and cultured. Subconfluent cells, between the third and sixth passages, were maintained under serum deprivation for 18 h followed by the treatment of varying doses of SP (1 pmol L(-1), 100 pmol L(-1), 10 nmol L(-1), 1 micromol L(-1) and 100 micromol L(-1)). Conditioned media were then subjected to gelatin zymography using 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis minigels containing 1.5 g L(-1) gelatin. The effect of SP on intracellular reactive oxygen species (ROS) was also examined by confocal microscopy. ROS scavenger N-Acetyl-L-cysteine (NAC, 5 mmol L(-1)) was utilized to evaluate the roles of ROS pathway in mediating the impact of SP on cellular gelatinolytic activity. Data were analysed using analysis of variance with Bonferroni correction for multiple comparisons or an unpaired Student's t-test. RESULTS: Substance P, at levels above 1 micromol L(-1), remarkably enhanced MMP-2 activity reflected by the band migrating at 66 kDa (P < 0.05). A gelatinolytic band at approximately 44 kDa appeared to be intensified in a SP dose-dependent manner. In addition, it was demonstrated that SP could induce ROS production in pulp cells and ROS scavenger NAC was further found to significantly reduce MMP-2 activity (P < 0.05), as well as other bands of gelatinolytic proteinases. CONCLUSION: Substance P can influence gelatinolytic activity in human pulp cells via ROS pathway.

Substance P increases production of proinflammatory cytokines and formation of osteoclasts in dental pulp fibroblasts in patients with severe orthodontic root resorption.

INTRODUCTION: The objective of this study was to determine the extent to which substance P (SP) increases proinflammatory cytokine production and osteoclast formation of human dental pulp fibroblasts (HDPF) in patients with severe orthodontically induced inflammatory root resorption (OIIRR). METHODS: HDPF were obtained from 5 patients with severe apical root resorption after orthodontic treatment. The levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha were determined after 24 hours by using ELISA kits. Furthermore, culture supernatants were added to cultured human osteoclasts, and osteoclast formation was observed after tartrate-resistant acid phosphatase (TRAP) staining and the formation of resorption cavities. RESULTS: Stimulation with SP increased the levels of IL-1beta, IL-6, and TNF-alpha, in a time- and concentration-dependent manner, although the increase was greater in the severe root resorption (SRR) group than in the nonresorption (NR) group (P < 0.001, 3-way repeated measures ANOVA). As for osteoclast formation, the numbers of TRAP-positive multinucleate cells and resorptive pits were significantly increased in the SRR group compared with the NR group (P < 0.001, 2-way repeated measures ANOVA). CONCLUSIONS: These results suggest that HDPF stimulated with SP might be deeply involved in the progress of inflammation in pulp tissue and the incidence of SRR during orthodontic treatment.

The protease-activated receptor-2 agonist induces gastric mucus secretion and mucosal cytoprotection.

Protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, modulates smooth muscle tone and exocrine secretion in the salivary glands and pancreas. Given that PAR-2 is expressed throughout the gastrointestinal tract, we investigated effects of PAR-2 agonists on mucus secretion and gastric mucosal injury in the rat. PAR-2-activating peptides triggered secretion of mucus in the stomach, but not in the duodenum. This mucus secretion was abolished by pretreatment with capsaicin, which stimulates and ablates specific sensory neurons, but it was resistant to cyclo-oxygenase inhibition. In contrast, capsaicin treatment failed to block PAR-2-mediated secretion from the salivary glands. Intravenous calcitonin gene-related peptide (CGRP) and neurokinin A markedly elicited gastric mucus secretion, as did substance P to a lesser extent. Specific antagonists of the CGRP1 and NK2, but not the NK1, receptors inhibited PAR-2-mediated mucus secretion. Pretreatment with the PAR-2 agonist strongly prevented gastric injury caused by HCl-ethanol or indomethacin. Thus, PAR-2 activation triggers the cytoprotective secretion of gastric mucus by stimulating the release of CGRP and tachykinins from sensory neurons. In contrast, the PAR-2-mediated salivary exocrine secretion appears to be independent of capsaicin-sensitive sensory neurons.

Substance P regulates macrophage inflammatory protein 3alpha/chemokine C-C ligand 20 (CCL20) with heme oxygenase-1 in human periodontal ligament cells.

Although substance P (SP), a potent proinflammatory peptide, is involved in inflammation and immune responses, the effect of SP on the expression of macrophage inflammatory protein 3alpha[MIP-3alpha, chemokine C-C ligand 20 (CCL20)] in periodontal ligament (PDL) cells is unknown. Equally enigmatic is the link between SP, the stress protein heme oxygenase-1 (HO-1), and CCL20 production. We investigated whether SP induces the release of chemokine CCL20 from immortalized PDL (IPDL) cells, and further clarify SP-mediated pathways. We also examined the relationship between HO-1 and CCL20 by treating PDL cells with SP. Incubating IPDL cells with SP increased expression of CCL20 mRNA and CCL20 protein in a dose-time-dependent manner. Highly selective p38 and extracellular-regulated kinase 1/2 (ERK1/2) inhibitors abrogated SP-induced expression of CCL20 in IPDL cells. SP is also responsible for initiating phosphorylation of IkappaB, degradation of IkappaB and activation of nuclear factor (NF)-kappaB. SP induced expression of HO-1 in both a concentration- and time-dependent manner, and CCL20 reflected similar patterns. The inductive effects of SP on HO-1 and CCL20 were enhanced by HO-1 inducer hemin and the membrane-permeable guanosine 3',5'-monophosphate (cGMP) analogue 8-bromo-cGMP. Conversely, this pathway was inhibited by the HO-1 inhibitor zinc protoporphyrin IX (ZnPP IX) and the selective inhibitor of guanylate cyclase, 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ). We report herein the pathway that connects SP along with other modulators of neuroimmunoregulation to the induction of HO-1 and the inflammatory mediator macrophage inflammatory protein (MIP)-3alpha/CCL20 in IPDL cells, which play an important role in the development of periodontitis or inflammation during orthodontic tooth movement.