Neurokinin-1 Receptor Expression Research Articles

Elevated Neurokinin-1 Receptor Expression in Uterine Products of Conception Is Associated With First Trimester Miscarriages

BackgroundMiscarriage is a common complication of early pregnancy, mostly occurring in the first trimester. However, the etiological factors and prognostic and diagnostic biomarkers are not well known. Neurokinin-1 receptor (NK-1R) is a receptor of tachykinin peptide substance P (SP) and has a role in various pathological conditions, cancers, but its association with miscarriages and significance as a clinicopathological parameter are not studied. Accordingly, the present study aimed to clarify the localization and expression for NK-1R in human retained products of conception (POC). The role of NK-1R is not known in miscarriages.Materials and MethodsNK-1R expression was assessed in POC and normal placental tissues by immunohistochemistry. Three- to four-micrometer-thin sections of formalin-fixed paraffin-embedded tissues were used for this purpose. Tissues were processed and then immunohistochemically stained with NK-1R antibody. Brain tissue was used as control for antibody. Protein expression was evaluated using the nuclear labeling index (%). Tissues were counterstained with 3,3′-diaminobenzidine (DAB), and microscopy was performed at 10×, 20×, and 40× magnifications.ResultsTen human POC tissues and 10 normal placental tissues were studied by immunohistochemistry to demonstrate the localization of NK-1R. The expression of NK-1R protein was high in all the cases of both groups. NK-1R expression showed no notable differences among different cases of miscarriages as well as normal deliveries at full term regardless of the mother’s age and gestational age at which the event occurred. Statistically, no difference was found in both groups, which is in agreement with our hypothesis and previous findings.ConclusionThe expression of NK-1R was similar in all the cases, and it was intense. It shows that dysregulation of NK-1R along with its ligand SP might be involved in miscarriages and also involved in normal delivery. Our results provide fundamental data regarding this anti-NK-1R strategy. Thus, the present study recommends that SP/NK-1R system might, therefore, be considered as an emerging and promising diagnostic and therapeutic strategy against miscarriages. Hence, we report for the first time the expression and localization of NK-1R in POC. We suggest NK-1R antagonist in addition to the immunoglobulins and human chorionic gonadotropin to diagnose and treat spontaneous miscarriages.

Evaluation of Serum Substance P Level and Tissue Distribution of NK-1 Receptor in Papillary Thyroid Cancer

Background: Papillary thyroid carcinoma (PTC) is the most prevalent malignancy of the endocrine system. This study was aimed at evaluating the serum substance P (SP) levels, the tissue distribution of Neurokinin-1 receptors (NK1-R), and their possible diagnostic value in PTC. Method: The present case-control study included 31 healthy volunteers and 31 cases (age range: 25-64, 40.26 ±12.77) who were primarily diagnosed with PTC and were candidates for total thyroidectomy. Pre-operative serum level of SP was measured using a commercial ELISA kit. The tissue distribution of NK1-R was assessed immunohistochemically. Results: The serum level of SP in the patient group was higher than the healthy volunteers (P-value =.005). Besides, the expression of NK1-R was higher in tumoral tissues compared with their normal surroundings (P-value =.005). However, we observed no significant correlation between either SP level or NK1-R expression and the disease stage or lymph node involvement. Conclusion: SP level and NK1-R expression were upregulated in PTC patients, showing the involvement of SP/NK1R complex in PTC pathophysiology. Nonetheless, proposing SP/NK1R as a diagnostic factor requires further studies because we found no correlation between SP/NK1R and clinical stage or lymph node involvement.

Substance P Mediates Estrogen Modulation Proinflammatory Cytokines Release in Intervertebral Disc.

Intervertebral disc degeneration (IDD) is a main contributor to low back pain. A close relationship exists between inflammation and pain. Estrogen can affect inflammation and may play a crucial role in IDD and pain. Substance P (SP) can also regulate the expression of pro-inflammatory cytokines in intervertebral disc (IVD). This study aimed to investigate the potential role of SP in estrogen regulation of IDD. Nine-week-old C57BL/6 female mice were divided into four groups as follows: sham surgery (sham), ovariectomy (OVX), ovariectomy plus estrogen replacement therapy (ERT) group (OVX+E2), and ovariectomy, ERT plus neurokinin 1 receptor (NK1R) agonist (OVX+E2+G). Serum E2, body, and uterus weight were recorded. Immunohistochemistry study and quantitative real-time PCR were used for SP, NK1R, IL-1β, IL-6, and TNF-α examination and comparison in IVD at protein and gene levels. After OVX, the gene and protein expression of TNF-α, IL-1β, IL-6, SP, and NK1R in NP cells significantly increased compared with the sham group. ERT can reverse these impacts. ERT plays anti-inflammatory and anti-hyperalgesic roles in IDD of OVX mice. The estrogen-induced changes of the pro-inflammatory cytokines, TNF-α, IL-1β, and IL-6, are significantly inhibited by NK1R agonists. SP may be a mediator of estrogen regulating pro-inflammatory factors in IDD. Estrogen may affect IVD inflammation through two ways: one is to directly affect the level of pro-inflammatory cytokines and the other is by means of modulation of SP.

Detection of neurokinin-1 receptor by immunohistochemistry in canine mammary gland tumours.

Canine mammary gland tumour (cMGT) is the most common tumour in intact female dogs. Surgery is the only effective treatment for cMGT, and dogs with metastasis at the time of diagnosis or those diagnosed at an advanced stage have poorer prognosis. Thus, novel diagnostic biomarkers and therapeutic targets are needed. Neurokinin‐1 receptor (NK‐1 receptor) is involved in cancer progression and has been detected in various malignant tumours including breast cancer in humans. Furthermore, NK‐1 receptor antagonists inhibit cancer progression. We evaluated NK‐1 receptor expression in malignant and benign cMGT compared with that in normal mammary gland tissues and analysed the relationship between the expression of NK‐1 receptor and histopathological type or malignancy grade. Specimens from 34 malignant MGT and 35 benign MGT cases were used for immunohistochemistry and scored according to intensity and percentage. Healthy margins from each tumour were used as internal controls. The scores for NK‐1 receptor intensity, percentage of positive cells and overall immunohistochemistry were higher in malignant MGT than in benign MGT and normal tissue (p < .000). NK‐1 receptor expression was not correlated with either malignancy grade or histopathological type. Expression of the NK‐1 receptor in malignant MGT was higher than that in benign MGT and normal tissues. Thus, NK‐1 receptor could be considered a novel therapeutic target for cMGT. Further studies using other quantitative tests such as western blotting or PCR and the evaluation of substance P in patient tumour tissue or serum are needed.

The NK-1R Antagonist Aprepitant Prevents LPS-Induced Oxidative Stress and Inflammation in RAW264.7 Macrophages.

BackgroundThe macrophage is one of the most important types of immune cells that protect against harmful stimuli. Macrophage activation plays a pivotal role in the progression and development of various inflammatory diseases. The neurokinin 1 receptor (NK-1R) is a G protein-coupled receptor that plays an important role in inflammatory diseases. Aprepitant is a kind of NK-1R antagonist. The purpose of this study is to determine the protective effect of aprepitant in lipopolysaccharide (LPS)-induced inflammatory responses in macrophages.MethodsWe examined the anti-inflammatory and anti-oxidant effects of aprepitant in LPS-treated RAW264.7 macrophages by using real-time PCR, ELISA, and Western blot analysis. We also assessed cellular oxidative stress signaling by measuring the levels of cellular MDA, total ROS, and NADPH oxidase expression. Cellular NO production was measured by DAF-FM DA staining. The inhibitory effect of aprepitant against NF-κB signaling was evaluated by luciferase assay and Western blot analysis.ResultsThe expression of NK-1R is increased in LPS-induced macrophages, suggesting a potential role of the receptor in the inflammatory response. We show that aprepitant protects macrophages against oxidative stress by reducing the generation of ROS and the expression of NOX-4. Furthermore, aprepitant inhibits the secretion of pro-inflammatory cytokines and chemotactic factors by mediating the NF-κB signaling pathway.ConclusionThe NK-1R receptor antagonist aprepitant acts as an anti-inflammatory agent, indicating that the blockage of the NK-1R pathway in macrophages has the potential to suppress inflammation.

The substance P and neurokinin-1 receptor system in human thyroid cancer: an immunohistochemical study.

To develop a new therapeutic strategy against thyroid cancer (TC), the expression of both substance P (SP) and neurokinin-1 receptor (NK-1R) must be demonstrated in TC cells. This study aims to examine by immunohistochemistry the localization of SP and the NK-1R in human TC samples (papillary, follicular, medullary, anaplastic) in metastasis and in healthy thyroid samples. SP and the NK-1R were expressed in all normal and TC samples. In healthy glands, SP was located in follicular cells (nucleus) and colloid, and NK-1R in follicular cells (cytoplasm) and stroma. In TC samples, SP was visualized in follicular cells (nucleus and cytoplasm), stroma and colloid, and NK-1R in follicular cells (cytoplasm), stroma and colloid. A semiquantitative scoring system (Allred Unit Scoring System) was applied. The expression (Allred total score) of SP and NK-1R was weaker in normal thyroid glands than in TC. In comparison with TC samples, a lower intensity/proportion of SP (nucleus and cytoplasm of follicular cells; stroma) was observed in normal samples. By contrast, in the colloid of TC samples the presence of SP was lower than in normal samples. In comparison with TC samples, the presence of the NK-1R in the cytoplasm of follicular cells and colloid was lower in normal thyroid samples, whereas the expression of this receptor in the stroma was higher. The results reported in this study suggest that the NK-1R could be a new target for the treatment of TC and use of the NK-1R antagonists could serve as a new anti-TC therapeutic strategy.

A HEATED SITUATION: THERMAL INJURY INDUCING AN IDIOPATHIC PULMONARY FIBROSIS EXACERBATION

A HEATED SITUATION: THERMAL INJURY INDUCING AN IDIOPATHIC PULMONARY FIBROSIS EXACERBATION

Neurokinin 1 receptor antagonists exhibit peripheral effects in prurigo nodularis including reduced ERK1/2 activation.

Aprepitant is a neurokinin 1 receptor (NK1R) antagonist used for its antipruritic properties in dermatoses and systemic diseases. The mode of action is still unclear. A peripheral effect is assumed as aprepitant shows efficacy in inflammatory skin diseases including prurigo nodularis (PN). To investigate the peripheral effects of NK1R antagonism in PN and cell culture models. Subjects with PN received an aprepitant treatment. Clinical, morphological and immunohistochemical changes were investigated in skin biopsies before and after treatment. Expression of NK1R was analysed by immunohistochemistry and for downstream pathways ((p)ERK1/2) by Western blotting in PN patients and matched healthy volunteers. Effects of NK1R blocking were analysed in cell cultures of primary keratinocytes by Western blotting for (p)ERK1/2 and by qPCR for NK1R, interleukin (IL)-1beta, IL-6, IL-8 and TNFalpha. Aprepitant treatment showed significant reduction in pruritus intensity (P<0.05) in PN and relevant immunohistochemical changes (down: CD5, CD25, up: CD79a, IL4). NK1R expression was higher in keratinocytes of PN patients compared to healthy controls. After treatment, epidermal NK1R expression increased while expression and activation of ERK1/2 decreased. In vitro, receptor up-regulation and reduced expression and activation of ERK1/2 were confirmed and reduced IL-expression shown when blocking NK1R. Our data confirm that NK1R antagonists such as aprepitant exhibit effects in the skin. Epidermal receptor expression, epidermal inflammatory ILs, ERK1/2 MAPK signalling and cutaneous inflammatory infiltrate were targets of NK1R antagonism. This may explain partly the antipruritic effect of NK1R antagonists next to its role in the central nervous system.

Neurokinin-1 receptor is an effective target for treating leukemia by inducing oxidative stress through mitochondrial calcium overload

Substance P (SP) regulates multiple biological processes through its high-affinity neurokinin-1 receptor (NK-1R). While the SP/NK-1R signaling axis is involved in the pathogenesis of solid cancer, the role of this signaling pathway in hematological malignancy remains unknown. Here, we demonstrate that NK-1R expression is markedly elevated in the white blood cells from acute myeloid leukemia patients and a panel of human leukemia cell lines. Blocking NK-1R induces apoptosis in vitro and in vivo via increase of mitochondrial reactive oxygen species. This oxidative stress was triggered by rapid calcium flux from the endoplasmic reticulum into mitochondria and, consequently, impairment of mitochondrial function, a mechanism underlying the cytotoxicity of NK-1R antagonists. Besides anticancer activity, blocking NK-1R produces a potent antinociceptive effect in myeloid leukemia-induced bone pain by alleviating inflammation and inducing apoptosis. These findings thus raise the exciting possibility that the NK-1R antagonists, drugs currently used in the clinic for preventing chemotherapy-induced nausea and vomiting, may provide a therapeutic option for treating human myeloid leukemia.

264 Neurokinin 1 receptor antagonists exhibit peripheral effects in prurigo nodularis including reduced ERK1/2 activation

Blocking the neurokinin 1 receptor (NK1R) in chronic pruritus has recently been shown to reduce pruritus intensity. Both, central effects involving spinal dorsal horn neurons and peripheral effects involving cutaneous components are discussed as underlying mechanisms. A peripheral effect is assumed as aprepitant shows efficacy in inflammatory skin diseases including prurigo nodularis (PN). Within this study we therefore investigated in vivo and in vitro peripheral effects mediated by NK1R antagonism. For the in vivo study 12 patients suffering from PN received an oral four weeks treatment with aprepitant (80 mg/d). Clinical and immunohistochemical parameter were assessed before and after treatment. Expression of NK1R was analyzed by immunohistochemistry and for downstream pathways ((p)ERK1/2) by western blotting in PN patients and matched healthy volunteers. Studies were continued in vitro using cell cultures of primary keratinocytes. Effects of NK1R blocking were analyzed by western blotting for (p)ERK1/2 and by qPCR for NK1R, interleukin (IL)-1beta, -6, -8, and TNFalpha. Treatment with aprepitant reduced significantly pruritus intensity in PN patients and showed relevant immunohistochemical changes (down: CD5, CD25, up: CD79a, IL4). NK1R expression was higher in keratinocytes of PN patients compared to healthy controls. After treatment, epidermal NK1R expression increased while expression and activation of ERK1/2 decreased. In vitro, receptor upregulation and reduced expression and activation of ERK1/2 were confirmed. Furthermore, a reduced IL-expression could be shown when blocking NK1R. Our data confirm that NK1R antagonists such as aprepitant exhibit effects in the skin. Epidermal receptor expression, inflammatory interleukins and ERK1/2 signaling were targets of NK1R antagonism. This may explain partly the antipruritic effect of NK1R antagonists next to its role in the central nervous system.

Structural and functional defects of the respiratory neural system in the medulla and spinal cord of Pax6 mutant rats

Pax6 is an important transcription factor expressed in several discrete domains of the developing central nervous system. It has been reported that Pax6 is involved in the specification of subtypes of hindbrain motor neurons. Pax6 homozygous mutant (rSey2/rSey2) rats die soon after birth, probably due to impaired respiratory movement. To determine whether the respiratory center in the medulla functions normally, we analyzed the histological and neurophysiological properties of the medulla and spinal cord in fetal rats with this mutation. First, the medulla of rSey2/rSey2 at embryonic (E) 21.5–E22.5 tended to be smaller than those from heterozygous mutant (rSey2/+) and wild-type (+/+) littermates. Through immunohistochemical analysis, we confirmed normal distribution of Phox2b-expressing cells in the parafacial respiratory group (pFRG) of rSey2/rSey2 rats. Expression of neurokinin-1 receptor (NK-1R) was weak and dispersed in rSey2/rSey2 rats. In addition, rSey2/rSey2 rats have a defect of the hypoglossal nerve root. Electrophysiological analysis using brainstem-spinal cord preparations (E21.5–E22.5) revealed that rSey2/rSey2 rats showed larger fluctuation of the amplitude of inspiratory activity monitored from the fourth cervical root although there was no significant difference in the respiratory rate among rSey2/rSey2, rSey2/+, and +/+ littermates. The response of respiratory rhythm to high CO2 was similar among all genotypes. Optical recordings of neuronal activity revealed that the activity of the pFRG tended to be weaker and inspiratory activity appeared in more scattered areas in the caudal ventral medulla in the rSey2/rSey2 rats. These results suggest that the basal activity of the respiratory system was preserved with mild impairment of the inspiratory activity in the rSey2/rSey2 rats and that the Pax6 gene is involved in the functional development of the neuronal system producing effective inspiratory motor outputs for survival.

SP promotes cell proliferation in esophageal squamous cell carcinoma through the NK1R/Hes1 axis

BackgroundSubstance P (SP) plays an important role in several types of cancer promotion and progression by binding to its preferential neurokinin 1 receptor (NK1R). However, the clinical significance and downstream mechanism of NK1R in esophageal squamous cell carcinoma (ESCC) have not been elucidated. The aim of this study was to investigate the role of SP/NK1R in the proliferation of ESCC and to screen related downstream molecules. MethodsIn the current investigation, the expression of NK1R was detected via immunohistochemistry (IHC), western blot (WB) analysis and real-time reverse transcription-polymerase chain reaction (RT-qPCR) in ESCC tissues and cell lines. Thereafter, the optimal concentration of SP was determined in vitro. The proliferation ability of SP/NK1R was assessed by cell counting kit-8 (CCK-8) and colony formation assays and subcutaneous tumour formation in nude mice with EC109 cells. Moreover, the related downstream molecules were screened by performing isobaric tags for relative and absolute quantitation (iTRAQ) protein spectrum analysis. ResultsNK1R was upregulated in ESCC, and its overexpression correlated with larger tumour size, deeper tumour invasion, more perineural invasion and eventually caused poorer overall survival (OS). Both intrinsic and SP-activated NK1R upregulation could promote the proliferation and clonogenic capacity of ESCC cells. In nude mice, tumour growth was suppressed by EC109 cells of NK1R downregulation. Further experiments demonstrated that Hairy and Enhancer of Split 1 (Hes1) was markedly reduced upon NK1R downregulation in EC109 cell lines and could regulate cell proliferation in the downstream of SP/NK1R. ConclusionsThe significant role of NK1R in mediating ESCC cell proliferation depended on the activation of SP and might be related to the downstream regulation of Hes1.

Mispositioned Neurokinin-1 Receptor-Expressing Neurons Underlie Heat Hyperalgesia in Disabled-1 Mutant Mice.

Reelin (Reln) and Disabled-1 (Dab1) participate in the Reln-signaling pathway and when either is deleted, mutant mice have the same spinally mediated behavioral abnormalities, increased sensitivity to noxious heat and a profound loss in mechanical sensitivity. Both Reln and Dab1 are highly expressed in dorsal horn areas that receive and convey nociceptive information, Laminae I-II, lateral Lamina V, and the lateral spinal nucleus (LSN). Lamina I contains both projection neurons and interneurons that express Neurokinin-1 receptors (NK1Rs) and they transmit information about noxious heat both within the dorsal horn and to the brain. Here, we ask whether the increased heat nociception in Reln and dab1 mutants is due to incorrectly positioned dorsal horn neurons that express NK1Rs. We found more NK1R-expressing neurons in Reln-/- and dab1-/- Laminae I-II than in their respective wild-type mice, and some NK1R neurons co-expressed Dab1 and the transcription factor Lmx1b, confirming their excitatory phenotype. Importantly, heat stimulation in dab1-/- mice induced Fos in incorrectly positioned NK1R neurons in Laminae I-II. Next, we asked whether these ectopically placed and noxious-heat responsive NK1R neurons participated in pain behavior. Ablation of the superficial NK1Rs with an intrathecal injection of a substance P analog conjugated to the toxin saporin (SSP-SAP) eliminated the thermal hypersensitivity of dab1-/- mice, without altering their mechanical insensitivity. These results suggest that ectopically positioned NK1R-expressing neurons underlie the heat hyperalgesia of Reelin-signaling pathway mutants, but do not contribute to their profound mechanical insensitivity.

Antagonism of NK-1R using aprepitant suppresses inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes

Chronic inflammation in fibroblast-like synoviocytes (FLSs) induced by pro-inflammatory cytokines such as TNF-α plays a key role in the pathogenesis of rheumatoid arthritis (RA). The neurokinin-1 receptor (NK-1R) is one of the G protein–coupled receptors (GPCRs) mediating the intracellular signalling of substance P (SP). However, the possible implications of NK-1R in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) and the pathogenesis of RA have not yet been reported. In the current study, we report that NK-1R is expressed in FLSs. Importantly, NK-1R expression was found to be significantly increased in RA-FLSs compared to normal FLSs. Interestingly, we found that treatment with tumour necrosis factor (TNF)-α increased the expression of NK-1R at both the gene and protein levels. Treatment with the NK-1R antagonist aprepitant reduced TNF-α-induced expression of NADPH oxidase 4 (NOX-4) and generation of reactive oxygen species (ROS) in FLSs. Our results also display that blockage of NF-1R using aprepitant inhibited TNF-α-induced expression and secretion of proinflammatory cytokines, including interleukin-1β (IL-1β), IL-6, and IL-8. Consistently, aprepitant prevented TNF-α-induced expression of matrix metalloproteinases (MMPs), including matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-13 (MMP-13). Mechanistically, our data demonstrate that treatment with aprepitant inhibited TNF-α-induced phosphorylation and degradation of inhibitor of NF-κB (IκBα). Notably, aprepitant attenuated TNF-α-induced nuclear translocation of nuclear factor κB (NF-κB) p65 and reduced luciferase activity of NF-κB in FLSs. The findings implicated a novel function of NK-1R in RA-FLSs. Blockage of NK-1R using its specific antagonist aprepitant might provide a new therapeutic strategy for RA.

Menadione (vitamin K3) inhibits hydrogen sulfide and substance P via NF-кB pathway in caerulein-induced acute pancreatitis and associated lung injury in mice

ObjectiveWe aim to study the protective effect of menadione on caerulein-induced acute pancreatitis (AP) and associated lung injury and to explore the possible mechanism. MethodsMale Swiss mice randomized into control and different experimental groups. AP was induced in mice by six hourly intraperitoneal (i.p) injections of caerulein (50 μg/kg at 1 h interval). Menadione (10 mg/kg) was administered one hour (i.p, 10 mg/kg) after the first caerulein injection and control animals were given hourly intraperitoneal (i.p) injection of isotonic sodium chloride solution for 6 hours. ResultsAdministration of menadione attenuated the severity of AP and associated lung injury as shown by the histopathology, reduced MPO and serum amylase activity. Further, the anti-inflammatory effect of menadione was associated with a reduction of pancreatic and pulmonary proinflammatory cytokine interleukin 1β (IL-1β) and hydrogen sulfide (H2S). Moreover, menadione inhibited caerulein-induced cystathionine-γ-lyase, preprotachykinin-A (PPTA) and neurokinin-1 receptor (NK-1R) expression in pancreas and lungs. Also menadione further enhances the beneficial effect by reducing caerulein-induced nuclear factor (NF) -κB activation in both pancreas and lung. ConclusionThe present findings show for the first time that in AP, menadione may exhibit an anti-inflammatory effect by down-regulating substance-P and H2S signaling via the NF-кB pathway.

Tachykinin NK1 receptor antagonist L-733,060 and substance P deletion exert neuroprotection through inhibiting oxidative stress and cell death after traumatic brain injury in mice.

Substance P (SP) is believed to play a role in traumatic brain injury (TBI), and the inhibition of binding of SP to the tachykinin neurokinin-1 receptor (NK1R) using NK1R antagonists had made favorable effects on TBI. Our current study addresses the functional roles and underlying mechanisms of SP and NK1R antagonist L-733,060 following TBI. Adult male wild type C57BL/6 J and SP knock out (SPKO) mice received a controlled cortical impact and outcome parameters were assessed. The results showed that TBI-induced motor and spatial memory deficits, lesion volume, brain water content and blood-brain barrier disruption were alleviated both in L-733,060-treated C57BL/6 J mice and vehicle-treated SPKO mice. L-733,060 treatment and SP deletion inhibited TBI-induced the release of cytochrome c from mitochondria to cytoplasm, activation of caspase-3, oxidative stress and neuroinflammation. Higher SP levels in serum and cortex were observed in wild type mice undergoing TBI relative to wild type sham group, but very little expression of cortical SP was detected in the SP-/- mice either TBI or not. Upregulation of NK1R expression after TBI was observed, and there was no significant difference between wild type and SPKO groups. in vitro, L-733,060 and SP deletion inhibited scratch injury-induced cell death, loss of mitochondrial membrane potential and reactive oxygen species (ROS) production following TBI. Together, the results of this study implicate a functional role for NK1-R antagonist L-733,060 and deletion of SP in TBI-induced neurological outcome, oxidative damage, neuroinflammation and cell death. Upregulation of NK1R maybe a consequence of TBI, independent of the levels of substance P. This study raises the possibility that targeting SP through its receptor NK1R or genetic deletion may have therapeutic efficacy in TBI.

MiR-34b/c-5p and the neurokinin-1 receptor regulate breast cancer cell proliferation and apoptosis.

ObjectivesMiR‐34 is a tumour suppressor in breast cancer. Neurokinin‐1 receptor (NK1R), which is the predicted target of the miR‐34 family, is overexpressed in many cancers. This study investigated the correlation and clinical significance of miR‐34 and NK1R in breast cancer.Materials and MethodsWestern blotting, quantitative reverse transcription‐PCR (qRT‐PCR) and luciferase assays were conducted to analyse the regulation of NK1R by miR‐34 in MDA‐MB‐231, MCF‐7, T47D, SK‐BR‐3 and HEK‐293 T cells. MiR‐34b/c‐5p, full‐length NK1R (NK1R‐FL) and truncated NK1R (NK1R‐Tr) expression in fifty patients were quantified by qRT‐PCR and correlated with their clinicopathological parameters. CCK‐8 assays, colony formation assays and flow cytometry were used to measure cell proliferation and apoptosis in MDA‐MB‐231 and MCF‐7 cells transfected with miR‐34b/c‐5p or NK1R‐siRNA and before treatment with or without Substance P (SP), an endogenous peptide agonists of NK1R. The effect of NK1R antagonist aprepitant was also investigated. In vivo xenograft models were used to further verify the regulation of NK1R by miR‐34b/c‐5p.ResultsExpression levels of miR‐34b/c‐5p and NK1R‐Tr, but not NK1R‐FL, were associated with enhanced malignant potential, such as tumour stage and Ki67 expression. The overexpression of miR‐34b/c‐5p or NK1R silencing potently suppressed cell proliferation and induced G2/M phase arrest and the apoptosis of MDA‐MB‐231 and MCF‐7 cells. The NK1R antagonist aprepitant had similar effects. In vivo studies confirmed that miR‐34b/c‐5p overexpression or NK1R silencing reduced the tumorigenicity of breast cancer. In addition, SP rescued the effects of miR‐34b/c‐5p overexpression or NK1R silencing on cell proliferation and apoptosis in vitro and in vivo assays.ConclusionsMiR‐34b/c‐5p and NK1R contribute to breast cancer cell proliferation and apoptosis and are potential targets for breast cancer therapeutics.

Neurokinin-1 receptor antagonism improves postoperative neurocognitive disorder in mice

Postoperative neurocognitive disorder (PND) is a major complication in surgical patients, especially the elderly, leading to mild memory impairment after surgery. The underlying pathophysiology remains unknown, although neuroinflammation and blood-brain barrier (BBB) disruption have been increasingly implicated in PND. Emerging evidence suggests that neurokinin-1 receptor (NK-1R), the principal target of proinflammatory neuropeptide substance P (SP), plays a pivotal role in modulating neuroinflammation and BBB integrity. In this study, we used an established mouse model for PND to investigate the effects of a selective NK-1R antagonist L-733,060 on PND-like features after peripheral surgery. Hippocampal SP started to increase at 6 h, peaked at 1 day, and returned to baseline at 3 days after surgery. At 1 day after surgery, NK-1R expression was increased in the hippocampus. At this time point, NK-1R antagonist pretreatment attenuated microgliosis and prevented neutrophil infiltration after surgery. Similarly, proinflammatory cytokines interleukin-1 beta and interleukin-6 were reduced in the hippocampus in NK-1R antagonist-treated mice at 6 h after surgery. Furthermore, surgery-induced BBB disruption, assessed by albumin deposition and expression of tight junction protein claudin-5, was attenuated by NK-1R antagonism at postoperative day 1. Finally, trace fear conditioning test revealed NK-1R antagonism reversed surgery-induced cognitive impairment at 3 days after surgery. Our findings suggest that inhibition of NK-1R signaling protects hippocampus-dependent memory from surgical insult, probably through modulations of neuroinflammation and BBB integrity.

Evaluation of serum level of substance P and tissue distribution of NK-1 receptor in endometrial cancer.

Substance P (SP), a neuropeptide belonging to the tachykinin family, exerts different biological activities mainly through neurokinin-1 receptor (NK1R). The role of SP/NK1R system in tumoral growth and spread is reported in several cancers. We aimed to evaluate the serum SP concentration and NK1R tissue distribution in endometrial cancer, and to study the relationship between these factors with tumor size, lymph node involvement, disease stage and cancer grade. Recruiting 22 patients with endometrial cancer and 21 patients with leiomyoma as the control group, serum SP concentration was measured using an ELISA method, and NK1R tissue distributions were immunohistochemically analyzed. Serum SP concentration in patients was significantly higher than the control group (p-value = 0.005). The expression level of NK1R in tumoral tissue was more than normal tissue (p-value < 0.001). The NK1R expression had a significant relationship with lymph node involvement (p-value = 0.005) and disease stage (p-value = 0.017). The NK1R expression was higher in more advanced and less-differentiated tumors. SP/NK1R system seems to play a role in tumor growth and development in endometrial cancer. As well, the NK1R expression increased in endometrial cancer, and may be considered as a prognostic factor; but further studies are needed in this field.

Neuropeptide Initiated Mast Cell Activation by Transcutaneous Electrical Acupoint Stimulation of Acupoint LI4 in Rats

Transcutaneous electrical acupoint stimulation (TEAS) has been consistently used clinically for its ease of operation, non-invasiveness and painlessness, in contrast to the characteristics of inserted needles. However, the mechanism remains unknown. The aim of this study was to investigate the local response of TEAS at Hegu acupoint (LI4). Immunohistochemistry was used to measure the expression of tryptase-positive mast cells, neuropeptides of the calcitonin gene-related peptide (CGRP) and substance P (SP) in LI4. Mast cells were also labelled with serotonin (5-HT), neurokinin-1 receptor (NK-1R) and toluidine blue. The results showed that cutaneous CGRP and SP immune-positive (CGRP-IP or SP-IP) nerve fibres in LI4 were more highly expressed. There were high degrees of mast cell aggregation and degranulation with release of 5-HT near the CGRP-IP or SP-IP nerve fibres and blood vessels after TEAS. The degranulation of mast cells (MCs) was accompanied by expression of NK-1R after TEAS. Either mast cell membrane stabilizer (Disodium cromoglycate) or NK-1R antagonist (RP 67580) diminished the accumulation and degranulation of MCs induced by TEAS. Taken together, the findings demonstrated that TEAS induced sensory nerve fibres to express CGRP and SP, which then bound to the NK-1R on MCs, after which MCs degranulated and released 5-HT, resulting in TEAS-initiated acupuncture-like signals.