Neurokinin-1 Receptor Protein Expression Research Articles

TNF-α evokes blood-brain barrier dysfunction through activation of Rho-kinase and neurokinin 1 receptor

Ischaemic stroke, accompanied by neuroinflammation, impairs blood–brain barrier (BBB) integrity through a complex mechanism involving activation of both RhoA/Rho kinase/myosin light chain-2 and neurokinin 1 receptor (NK1R). Using an in vitro model of human BBB composed of brain microvascular endothelial cells (BMEC), astrocytes and pericytes, this study examined the potential contributions of these elements to BBB damage induced by elevated availability of pro-inflammatory cytokine, TNF-α. Treatment of human BMECs with TNF-α significantly enhanced RhoA activity and the protein expressions of Rho kinase and phosphorylated Ser19MLC-2 while decreasing that of NK1R. Pharmacological inhibition of Rho kinase by Y-27632 and NK1R by CP96345 neutralised the disruptive effects of TNF-α on BBB integrity and function as ascertained by reversal of decreases in transendothelial electrical resistance and increases in paracellular flux of low molecular weight permeability marker, sodium fluorescein, respectively. Suppression of RhoA activation, mitigation of actin stress fibre formation and restoration of plasma membrane localisation of tight junction protein zonula occludens-1 appeared to contribute to the barrier-protective effects of both Y-27632 and CP96345. Attenuation of TNF-α-mediated increases in NK1R protein expression in BMEC by Y-27632 suggests that RhoA/Rho kinase pathway acts upstream to NK1R. In conclusion, specific inhibition of Rho kinase in cerebrovascular conditions, accompanied by excessive release of pro-inflammatory cytokine TNF-α, helps preserve endothelial cell morphology and inter-endothelial cell barrier formation and may serve as an important therapeutic target.

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.

Alcohol extract from Vernonia anthelmintica willd (L.) seed counteracts stress-induced murine hair follicle growth inhibition

BackgroundVernonia anthelmintica (L.) willd is a traditional urgur herb in China for a long history. Its alcohol extract (AVE) has been proved to promote hair follicle growth in C57BL/6 mice. We conducted this study to investigate the hair-growth effects of AVE in stressed mice and its possible mechanism of action.MethodsThe hair-follicle growth effects of AVE were examined by in vivo and in vitro study. We exposed C57BL/6 male mice to chronic restraint stress to induce murine hair follicle growth inhibition. The effects of AVE were examined by histological analysis, immunofluorescence for Ki67 and cytokeratin 19 immunoreactivity, western blot assay in tyrosinase and related proteins expressions and immunofluorescence for nerve fibers. In organ culture of mouse vibrissae follicles, we used substance P as a catagen-inducing factor of hair follicle growth, and measured the elongation of hair shafts and expression of neurokinin-1 receptor protein by application of AVE.ResultsOur results showed that AVE counteract murine hair follicle growth inhibition caused by chronic restraint stress via inducing the conversion of telogen to anagen and inhibiting catagen premature, increasing bulb keratinocytes and bulge stem cells proliferation, promoting melanogenesis, and reducing the numbers of substance P and calcitonin gene-related peptide nerve fibers. Furthermore, AVE also counteracted murine hair follicle growth inhibition caused by substance P in organ culture.ConclusionThese results suggest that AVE counteract stress-induced hair follicle growth inhibition in C57BL/6 mice in vivo and in vitro, and may be an effective new candidate for treatment of stress-induced hair loss.

Effects of Mycoplasma pneumoniae infection on airway neurokinin-1 receptor expression in BALB/c mice.

The aim of this study was to establish a BALB/c mouse model of Mycoplasma pneumoniae (MP) infection and to explore the expression of neurokinin-1 receptor (NK1-R) in the trachea and lung tissue and changes in its relative content at different time points (on the 3rd, 7th, 14th, 21st, and 30th days after infection) in MP-infected BALB/c mice. Immunohistochemistry and Western blot analysis were performed to determine NK1-R expression in the trachea and lung tissue and changes in relative content in MP-infected BALB/c mice. After MP infection, the expression of NK1-R on the surfaces of upper tracheal and bronchial epithelial cells, submucosa, and alveolar epithelial cells, as well as around the smooth muscle, was upregulated more significantly in the infection group than in the control group (P < 0.05); NK1-R protein expression was enhanced on the 3rd, 7th, 14th, 21st, and 30th days after infection compared with that of the control group (P < 0.05). NK1-R expression in the trachea, bronchus, and lung tissue increased in MP-infected BALB/c mice, which may explain why wheezing occurs after MP infection.

Pregnancy-induced changes in substance P and neurokinin 1 receptor (NK1-R) expression in the rat uterus

Adrenergic nerve fibres of the mammalian uterus degenerate during pregnancy. The behaviour of peptidergic fibres, such as substance P-positive fibres and of its preferred neurokinin 1 receptor (NK1-R), is poorly studied in the pregnant rat uterus. The present study analysed the changes in substance P immunoreactivity and in the expression of NK1-R protein in the uterus of non-pregnant, pregnant (days 7, 14 and 21) and postpartum rats (days 1, 8 and 22) by immunohistology, dot blot analysis and western blot analysis. In non-pregnant rats, substance P-positive fibres were localized to the myometrium; these fibres progressively disappeared during gestation and were almost absent at term (day 21). At day 22 post partum, substance P-positive fibres had recovered to numbers comparable with those in the non-pregnant uterus. Dot blot analysis revealed a significant decrease in the immunoreactivity of substance P in the uterus at mid-pregnancy (day 14) and especially at term. Expression of the NK1-R protein showed a progressive increase throughout pregnancy reaching a peak on day 1 post partum; downregulation of NK1-R protein occurred on day 8 post partum. The low and high expressions of NK1-R protein were coincident with a large number of eosinophils and almost no eosinophils in the uterus at oestrus and at term, respectively. It was concluded that substance P immunoreactivity is inversely correlated with NK1-R protein expression in the pregnant and postpartum uterus. The marked upregulation of NK1-R protein at term and after birth indicates that the NK1-R may be involved in the complex regulation of labour and postpartum physiology. However, it is likely that the NK1-protein is not involved in the recruitment of eosinophils into the uterus at oestrus.

Pregnancy-induced changes in substance P and neurokinin 1 receptor (NK1-R) expression in the rat uterus.

Adrenergic nerve fibres of the mammalian uterus degenerate during pregnancy. The behaviour of peptidergic fibres, such as substance P-positive fibres and of its preferred neurokinin 1 receptor (NK1-R), is poorly studied in the pregnant rat uterus. The present study analysed the changes in substance P immunoreactivity and in the expression of NK1-R protein in the uterus of non-pregnant, pregnant (days 7, 14 and 21) and postpartum rats (days 1, 8 and 22) by immunohistology, dot blot analysis and western blot analysis. In non-pregnant rats, substance P-positive fibres were localized to the myometrium; these fibres progressively disappeared during gestation and were almost absent at term (day 21). At day 22 post partum, substance P-positive fibres had recovered to numbers comparable with those in the non-pregnant uterus. Dot blot analysis revealed a significant decrease in the immunoreactivity of substance P in the uterus at mid-pregnancy (day 14) and especially at term. Expression of the NK1-R protein showed a progressive increase throughout pregnancy reaching a peak on day 1 post partum; downregulation of NK1-R protein occurred on day 8 post partum. The low and high expressions of NK1-R protein were coincident with a large number of eosinophils and almost no eosinophils in the uterus at oestrus and at term, respectively. It was concluded that substance P immunoreactivity is inversely correlated with NK1-R protein expression in the pregnant and postpartum uterus. The marked upregulation of NK1-R protein at term and after birth indicates that the NK1-R may be involved in the complex regulation of labour and postpartum physiology. However, it is likely that the NK1-protein is not involved in the recruitment of eosinophils into the uterus at oestrus.

A neurokinin 1 receptor antagonist reduces an ongoing ileal pouch inflammation and the response to a subsequent inflammatory stimulus.

Ileal pouch-anal anastomosis (IPAA) is an excellent surgical option for patients with chronic ulcerative colitis (CUC) requiring colectomy; however, persistent episodes of ileal pouch inflammation, or pouchitis, may result in debilitating postoperative complications. Because considerable evidence implicates substance P (SP) as an inflammatory mediator of CUC, we investigated whether SP participates in the pathophysiology of pouchitis. With the use of a rat model of IPAA that we developed, we showed that ileal pouch MPO levels and neurokinin 1 receptor (NK-1R) protein expression by Western blot analysis were significantly elevated 28 days after IPAA surgery. In situ hybridization and immunohistochemistry showed that the increase in NK-1R protein expression was localized to the lamina propria and epithelia of pouch ileum. The intraperitoneal administration of the NK-1R antagonist (NK-1RA) CJ-12,255 for 4 days, starting on day 28, was effective in reducing MPO levels. Starting on day 28, animals with IPAA were given 5% dextran sulfate sodium (DSS) in their drinking water for 4 days, which caused histological and physical signs of clinical pouchitis concomitant with significant increases in ileal pouch MPO concentrations as well as NK-1R protein expression by Western blot analysis. In situ hybridization and immunohistochemistry showed that the increase in NK-1R protein expression was especially evident in crypt epithelia of pouch ileum. When the NK-1RA was administered 1 day before starting DSS and continued for the duration of DSS administration, the physical signs of clinical pouchitis and the rise in MPO were prevented. These data implicate SP in the pathophysiology of pouchitis and suggest that NK-1RA may be of therapeutic value in the management of clinical pouchitis.

Impaired renal sensory responses after unilateral ureteral obstruction in the rat.

Renal responses to the activation of renal sensory receptors were examined in rats after release of 24-h unilateral ureteral obstruction of the left kidney. The integrity of the renorenal reflex was examined in both 24-h unilateral ureteral obstruction-treated (UUO) and sham-operated (Sham) rats. Increased ipsilateral afferent renal nerve activity (ARNA) and reflexly decreased efferent renal nerve activity (ERNA) and increased contralateral diuresis and natriuresis produced by increasing the left intrapelvic pressure were observed in Sham rats but not in UUO rats. The lack of responsiveness of the renorenal reflex in UUO rats was associated with lower release of substance P (SP) and increased neutral endopeptidase (NEP) activity in the renal pelvis in the postobstructive kidney. Compared with Sham rats, urine and sodium excretion after acute saline loading was significantly reduced in the postobstructive kidney. The blunted excretory responses were accompanied by lower activation of ARNA and less reflex inhibition of ERNA. Renal sensory dysfunction in the postobstructive kidney was further examined by stimulation of renal mechanoreceptors and chemoreceptors. Graded increases in intrapelvic pressure or renal pelvic perfusion with hypertonic saline solution elicited, respectively, a pressure- or concentration-dependent increase in ARNA in the control kidney of Sham rats, this response being greatly attenuated in the postobstructive kidney. Western blots showed no quantitative difference in the expression of renal pelvic neurokinin 1 (NK-1) receptors between the two groups. It was concluded that renal sensory function is impaired in the postobstructive kidney of UUO rats and that this defective activation of renal sensory receptors results in an impaired renorenal reflex, which is associated with enhanced NEP activity and catabolism of SP released in the renal pelvis and is not related to the expression of NK-1 receptor protein.