Vicarious defeat stress induces increased alcohol consumption in female mice: Role of neurokinin-1 receptor and interleukin-6.

There is a high frequency of comorbidity of alcohol use disorder (AUD) and depression in human populations. We have studied this relationship in our lab using the social defeat stress (SDS) model, which results in both depression-like behaviours and increased alcohol consumption in male mice. However, standard SDS procedures are difficult to use in female mice due to a lack of territorial aggression. In the experiments presented here, we used vicarious defeat stress (VDS) to assess social withdrawal and alcohol consumption in female C57BL6/J mice. We also assessed the expression of interleukin-6 (IL6), which is a proinflammatory cytokine that is associated with depression in humans and sensitivity to SDS in mice. In these experiments, C57BL/6 female mice underwent 10 days of VDS where they witnessed the physical defeat of a male conspecific by an aggressive CD1 mouse. After the end of VDS, mice were either given access to alcohol or sacrificed for the measurement of IL6 expression. We found that VDS increased alcohol consumption and IL6 expression in the frontal cortex and hippocampus. Given that the neurokinin-1 receptor (NK1R) can mediate both stress-induced alcohol consumption and IL6 expression, we tested the ability of NK1R antagonism to reduce VDS-induced alcohol consumption and found that this treatment reduced alcohol intake in both VDS-exposed mice and in unstressed controls. The observed increase in alcohol consumption suggests that VDS is a model that can be utilized to study stress-induced alcohol consumption in female mice, and that this is sensitive to NK1R antagonism.

The Repurposing of Non-Peptide Neurokinin-1 Receptor Antagonists as Antitumor Drugs: An Urgent Challenge for Aprepitant.

The substance P (SP)/neurokinin-1 receptor (NK-1R) system is involved in cancer progression. NK-1R, activated by SP, promotes tumor cell proliferation and migration, angiogenesis, the Warburg effect, and the prevention of apoptosis. Tumor cells overexpress NK-1R, which influences their viability. A typical specific anticancer strategy using NK-1R antagonists, irrespective of the tumor type, is possible because these antagonists block all the effects mentioned above mediated by SP on cancer cells. This review will update the information regarding using NK-1R antagonists, particularly Aprepitant, as an anticancer drug. Aprepitant shows a broad-spectrum anticancer effect against many tumor types. Aprepitant alone or in combination therapy with radiotherapy or chemotherapy could reduce the sequelae and increase the cure rate and quality of life of patients with cancer. Current data open the door to new cancer research aimed at antitumor therapeutic strategies using Aprepitant. To achieve this goal, reprofiling the antiemetic Aprepitant as an anticancer drug is urgently needed.

Illuminating the structural basis of human neurokinin 1 receptor (NK1R) antagonism through classical all-atoms molecular dynamics simulations.

Advances in structural biology have bestowed insights into the pleiotropic effects of neurokinin 1 receptors (NK1R) in diverse patho-physiological processes, thereby highlighting the potential therapeutic value of antagonists directed against NK1R. Herein, we investigate the mode of antagonist recognition to discern the obscure atomic facets germane for the function and molecular determinants of NK1R. To commence discernment of potent antagonists and the conformational changes in NK1R, induced upon antagonist binding, state-of-the-art classical all-atoms molecular dynamics (MD) simulations in lipid mimetic bilayers have been utilized. MD simulations of structural ensembles reveals the involvement of TM5 and TM6 in tight anchoring of antagonists through a network of interhelical hydrogen-bonds, while, the extracellular loop 2 (ECL2) governs the overall size and nature of the pocket, thereby modulating NK1R. Consistent comparison between experiments and MD simulation results discerns the predominant role of TM3, TM4, and TM6 in lipid-NK1R interaction. Correlation between hydrophobic index and helicity of TM domains elucidates their importance in maintaining the structural stability in addition to regulating NK1R antagonism. Taken together, we anticipate that our computational study marks a comprehensive structural basis of NK1R antagonism in lipid bilayers, which may facilitate designing of new therapeutics against associated diseases targeting human neurokinin receptors.

Deciphering specificity and cross-reactivity in tachykinin NK1 and NK2 receptors.

The tachykinin receptors neurokinin 1 (NK1R) and neurokinin 2 (NK2R) are G protein-coupled receptors that bind preferentially to the natural peptide ligands substance P and neurokinin A, respectively, and have been targets for drug development. Despite sharing a common C-terminal sequence of Phe-X-Gly-Leu-Met-NH2 that helps direct biological function, the peptide ligands exhibit some degree of cross-reactivity toward each other's non-natural receptor. Here, we investigate the detailed structure-activity relationships of the ligand-bound receptor complexes that underlie both potent activation by the natural ligand and cross-reactivity. We find that the specificity and cross-reactivity of the peptide ligands can be explained by the interactions between the amino acids preceding the FxGLM consensus motif of the bound peptide ligand and two regions of the receptor: the ß-hairpin of the extracellular loop 2 (ECL2) and a N-terminal segment leading into transmembrane helix 1. Positively charged sidechains of the ECL2 (R177 of NK1R and K180 of NK2R) are seen to play a vital role in the interaction. The N-terminal positions 1 to 3 of the peptide ligand are entirely dispensable. Mutated and chimeric receptor and ligand constructs neatly swap around ligand specificity as expected, validating the structure-activity hypotheses presented. These findings will help in developing improved agonists or antagonists for NK1R and NK2R.

Peptide TK-HR from the Skin of Chinese Folk Medicine Frog Hoplobatrachus Rugulosus Accelerates Wound Healing via the Activation of the Neurokinin-1 Receptor.

Wound healing is a complex process and remains a considerable challenge in clinical trials due to the lack of ideal therapeutic drugs. Here, a new peptide TK-HR identified from the skin of the frog Hoplobatrachus rugulosus was tested for its ability to heal cutaneous wounds in mice. Topical application of TK-HR at doses of 50-200 µg/mL significantly accelerated wound closure without causing any adverse effects in the animals. In vitro and in vivo investigations proved the regulatory role of the peptide on neutrophils, macrophages, keratinocytes, and vein endothelial cells involved in the inflammatory, proliferative, and remodeling phases of wound healing. Notably, TK-HR activated the MAPK and TGF-ß-Smad signaling pathways by acting on NK1R in RAW264.7 cells and mice. The current work has identified that TK-HR is a potent wound healing regulator that can be applied for the treatment of wounds, including diabetic foot ulcers and infected wounds, in the future.

Gabapentin alleviated the cough hypersensitivity and neurogenic inflammation in a guinea pig model with repeated intra-esophageal acid perfusion.

OBJECTIVE: The anti-tussive effect of gabapentin and its underlying neuromodulatory mechanism were investigated via a modified guinea pig model of gastroesophageal reflux-related cough (GERC). METHODS: Intra-esophageal perfusion with hydrochloric acid (HCl) was performed every other day 12 times to establish the GERC model. High-dose gabapentin (48 mg/kg), low-dose gabapentin (8 mg/kg), or saline was orally administered for 2 weeks after modeling. Cough sensitivity, airway inflammation, lung and esophagus histology, levels of substance P (SP), and neurokinin-1 (NK1)-receptors were monitored. RESULTS: Repeated intra-esophageal acid perfusion aggravated the cough sensitivity in guinea pigs in a time-dependent manner. The number of cough events was significantly increased after 12 times HCl perfusion, and the hypersensitivity period was maintained for 2 weeks. The SP levels in BALF, trachea, lung, distal esophagus, and vagal ganglia were increased in guinea pigs receiving HCl perfusion. The intensity of cough hypersensitivity in the GERC model was significantly correlated with increased SP expression in the airways. Both high and low doses of gabapentin administration could reduce cough hypersensitivity exposed to HCl perfusion, attenuate airway inflammatory damage, and inhibit neurogenic inflammation by reducing SP expression from the airway and vagal ganglia. CONCLUSIONS: Gabapentin can desensitize the cough sensitivity in the GERC model of guinea pig. The anti-tussive effect is associated with the alleviated peripheral neurogenic inflammation as reflected in the decreased level of SP.

Moving out of CF3 -Land: Synthesis, Receptor Affinity, and in silico Studies of NK1 Receptor Ligands Containing a Pentafluorosulfanyl (SF5 ) Group.

The NK1 receptor (NK1R) is a molecular target for both approved and experimental drugs intended for a variety of conditions, including emesis, pain, and cancers. While contemplating modifications to the typical NK1R pharmacophore, we wondered whether the CF3 groups common for many NK1R ligands, could be replaced with some other moiety. Our attention was drawn by the SF5 group, and so we designed, synthesized, and tested ten novel SF5 -containing compounds for NK1R affinity. All analogues exhibit detectable NK1R binding, with the best of them, compound 5 a, (3-bromo-5-(pentafluoro-λ6 -sulfanyl)benzyl acetyl-L-tryptophanate) binding only slightly worse (IC50 =34.3 nM) than the approved NK1R-targeting drug, aprepitant (IC50 =27.7 nM). Molecular docking provided structural explanation of SAR. According to our analysis, the SF5 group in our compounds occupies a position similar to that of one of the CF3 groups of aprepitant as found in the crystal structure. Additionally, we checked whether the docking scoring function or energies derived from Fragment Molecular Orbital quantum chemical calculations may be helpful in explaining and predicting the experimental receptor affinities for our analogues. Both these methods produce moderately good results. Overall, this is the first demonstration of the utility of the SF5 group in the design of NK1R ligands.

The SP/NK1R system promotes the proliferation of breast cancer cells through NF-κB-mediated inflammatory responses.

BACKGROUND: Numerous molecules have been introduced to participate in the formation of breast cancer, the most common malignancy in women. Among them, neuropeptide substance P (SP) and its related receptor neurokinin-1 receptor (NK1R) have attracted unprecedented attention in tumorigenesis processes. In this study, we investigated the effect of the SP/NK1R pathway on the induction of oxidative stress in breast cancer and examine the therapeutic potential of NK1R inhibition in this malignancy. METHODS: MCF-7 cells were treated with varying concentrations of SP and aprepitant, an FDA-approved NK1R antagonist, either as a single drug or in a combined modality. Resazurin assay was used to evaluate the anti-cancer ability of aprepitant. The alteration in the intracellular levels of reactive oxygen species (ROS) and gene expression were determined using ROS assay and the qRT-PCR analysis, respectively. RESULTS: The stimulation of the SP/NK1R axis in the MCF-7 cells was coupled with the accumulation of ROS as well as upregulation of NF-κB and its related pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and IL-6. In contrast, the suppression of NK1R by aprepitant halted the viability of MCF-7 cells, at least partly due to p53-mediated upregulation of p21. Moreover, aprepitant attenuated the oncogenic properties of SP by preventing the oxidative property of this neuropeptide. CONCLUSION: Overall, our results suggest that the SP/NK1R pathway might play a critical role in breast cancer pathogenesis, probably through inducing ROS/NF-κB-mediated inflammatory responses. Moreover, it seems that blockage of the axis has promising therapeutic value against breast cancer cells. Schematic representation proposed for the plausible mechanism by which the stimulation of the SP/NK1R might induce oxidative stress in breast cancer-derived MCF-7 cells. Once SP interacts with NK1R, this signaling axis could disturb the balance between the expression of p53 and NF-κB, an event that leads to the accumulation of ROS within MCF-7 cells. The produced ROS, in turn, elevates the expression of pro-inflammatory cytokines (TNF-α and IL-6) and downregulates the expression of p21. On the other hand, aprepitant, an antagonist of NK1R, could reduce the survival of proliferative capacity of MCF-7 cells by decreasing the intracellular levels of ROS and p53-mediated up-regulation of p21. Along with the effect on p53, aprepitant could also reduce the expression of NF-κB and its related pro-inflammatory cytokines.

C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

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.

Involvement of Substance P (SP) and Its Related NK1 Receptor in Primary Sjögren's Syndrome (pSS) Pathogenesis.

Primary Sjögren's Syndrome (pSS) is a systemic autoimmune disease that primarily attacks the lacrimal and salivary glands, resulting in impaired secretory function characterized by xerostomia and xerophthalmia. Patients with pSS have been shown to have impaired salivary gland innervation and altered circulating levels of neuropeptides thought to be a cause of decreased salivation, including substance P (SP). Using Western blot analysis and immunofluorescence studies, we examined the expression levels of SP and its preferred G protein-coupled TK Receptor 1 (NK1R) and apoptosis markers in biopsies of the minor salivary gland (MSG) from pSS patients compared with patients with idiopathic sicca syndrome. We confirmed a quantitative decrease in the amount of SP in the MSG of pSS patients and demonstrated a significant increase in NK1R levels compared with sicca subjects, indicating the involvement of SP fibers and NK1R in the impaired salivary secretion observed in pSS patients. Moreover, the increase in apoptosis (PARP-1 cleavage) in pSS patients was shown to be related to JNK phosphorylation. Since there is no satisfactory therapy for the treatment of secretory hypofunction in pSS patients, the SP pathway may be a new potential diagnostic tool or therapeutic target.

Targeting the Nerve-Cancer Circuit.

The tumor microenvironment is innervated by sensory, sympathetic, and parasympathetic nerves that actively stimulate cancer growth and dissemination. The cross-talk among the peripheral nerves, cancer cells, and stromal cells is mediated by a diverse set of bioactive ligands and their corresponding receptors. Dissecting the specific neuronal subtypes and molecular signals that drive cancer-nerve interaction holds the hope of developing targeted therapies for cancer. A recent study by Restaino and colleagues demonstrated that regardless of tumor type, origin, or anatomic location, tumors are densely innervated, predominantly by transient receptor potential cation channel subfamily V member 1 positive (TRPV1+) sensory fibers. The intratumoral fibers likely have functional connectivity and contribute to increased electrical activity in the tumor bed. Importantly, the neuropeptide substance P produced by intratumoral fibers stimulates its neurokinin 1 receptor (NK1R) expressed on tumor cells to drive tumor proliferation and migration. The findings raised the intriguing possibility of a generalizable molecular pathway that mediates cancer-nerve interaction that can be targeted to inhibit tumor growth and metastasis across different tumor types.

The Role of Substance P Within Traumatic Brain Injury and Implications for Therapy.

This review examines the role of the neuropeptide substance P within the neuroinflammation that follows traumatic brain injury. It examines it in reference to its preferential receptor, the neurokinin-1 receptor, and explores the evidence for antagonism of this receptor in traumatic brain injury with therapeutic intent. Expression of substance P increases following traumatic brain injury. Subsequent binding to the neurokinin-1 receptor results in neurogenic inflammation, a cause of deleterious secondary effects that include an increased intracranial pressure and poor clinical outcome. In several animal models of TBI, neurokinin-1 receptor antagonism has been shown to reduce brain edema and the resultant rise in intracranial pressure. A brief overview of the history of substance P is presented, alongside an exploration into the chemistry of the neuropeptide with a relevance to its functions within the central nervous system. This review summarizes the scientific and clinical rationale for substance P antagonism as a promising therapy for human TBI.

Novel trajectories of the NK1R antagonist aprepitant in rotenone-induced Parkinsonism-like symptoms in rats: Involvement of ERK5/KLF4/p62/Nrf2 signaling axis.

Regulation of the interplay between autophagy and oxidative stress is vital in maintaining neuronal homeostasis during neurotoxicity. The interesting involvement of NK1 receptor (NK1R) in neurodegeneration has highlighted the value of investigating the neuroprotective effect of aprepitant (Aprep), an NK1R antagonist in Parkinson's disease (PD). This study was conducted to disclose Aprep's ability to modulate extracellular signal-regulated kinase 5/Krüppel-like factor 4 (ERK5/KLF4) cue as molecular signaling implicated in regulating autophagy and redox signaling in response to rotenone neurotoxicity. Rotenone (1.5 mg/kg) was administered on alternate days, and rats were given Aprep simultaneously with or without PD98059, an ERK inhibitor, for 21 days. Aprep ameliorated motor deficits as verified by restored histological features, and intact neurons count in SN and striata along with tyrosine hydroxylase immunoreactivity in SN. The molecular signaling of Aprep was illustrated by the expression of KLF4 following the phosphorylation of its upstream target, ERK5. Nuclear factor erythroid 2-related factor 2 (Nrf2) was up-regulated, shifting the oxidant/antioxidant balance towards the antioxidant side, as evidenced by elevated GSH and suppressed MDA levels. In parallel, Aprep noticeably reduced phosphorylated α-synuclein aggregates due to autophagy induction as emphasized by marked LC3II/LC3I elevation and p62 level reduction. These effects were diminished upon PD98059 pre-administration. In conclusion, Aprep showed neuroprotective effects against rotenone-induced PD, which may be partially attributed to the activation of the ERK5/KLF4 signaling pathway. It modulated p62-mediated autophagy and Nrf2 axis which act cooperatively to counter rotenone-associated neurotoxicity pointing to Aprep's prospect as a curious candidate in PD research.

Aprepitant inhibits the progression of esophageal squamous cancer by blocking the truncated neurokinin­1 receptor.

Increasing evidence showed that the substance P (SP)/neurokinin­1 receptor (NK1R) complex is involved in the development of several cancers. However, little is known about the mechanisms by which SP/NK1R complex plays a role in esophageal squamous cell carcinoma (ESCC) progression. RT­qPCR, CCK­8, Transwell, western blotting, immunohistochemical, immunofluorescence, ELISA and analysis of apoptosis were employed in the present study. It was aimed to investigate the function and therapeutic potential of the SP/tr­NK1R system in human ESCC progression. The results revealed that both SP and tr­NK1R were highly expressed in ESCC cell lines and specimens. In ESCC tissues, SP was mainly derived from ESCC cells and M2 macrophages. The NK1R antagonist aprepitant inhibited the SP­induced proliferation of human ESCC cell lines. Aprepitant inhibited cell migration and invasion and induced apoptosis of ESCC cells by downregulating the PI3K/AKT/mTOR signaling pathways. Animal experiments revealed that aprepitant inhibited tumor progression of ESCC in xenograft mice. In conclusion, high expression of SP plus tr­NK1R indicated poor prognosis in ESCC, suggesting that aprepitant has a potential application in ESCC. To the best of our knowledge, high SP and tr­NK1R expression in ESCC cell lines was reported for the first time in the present study. These findings provided evidence for a novel therapeutic strategy for patients with ESCC.

Targeting NK-1R attenuates renal fibrosis via modulating inflammatory responses and cell fate in chronic kidney disease.

Background: Renal fibrosis is the final common pathway of chronic kidney disease (CKD), which is clinically irreversible and without effective therapy. Renal tubules are vulnerable to various insults, and tubular injury is involving in the initiation and evolution of renal inflammation and fibrosis. Neurokinin-1 receptor (NK-1R) functions by interacting with proinflammatory neuropeptide substance P (SP), exerting crucial roles in various neurological and non-neurological diseases. However, its roles in renal inflammation and fibrosis are still unknown. Methods: We collected renal biopsy specimens and serum samples of individuals with or without CKD. Additionally, knockout mice lacking NK-1R expression, SP addition and NK-1R pharmacological antagonist treatment in the unilateral ureteral obstruction (UUO) model, and NK-1R-overexpressed HK-2 cells were employed. Results: Renal SP/NK-1R and serum SP were increased in patients with CKD and mice experiencing UUO and correlated with renal fibrosis and function. SP addition enhanced UUO-induced progressive inflammatory responses and renal fibrosis, whereas genetically or pharmacologically targeting NK-1R attenuated these effects. Mechanistically, TFAP4 promoted NK-1R transcription by binding to its promoter, which was abolished by mutation of the binding site between TFAP4 and NK-1R promoter. Furthermore, SP acted through the NK-1R to activate the JNK/p38 pathways to modulate cell fate of tubular epithelial cells including growth arrest, apoptosis, and expression of profibrogenic genes. Conclusion: Our data reveals that SP/NK-1R signaling promotes renal inflammatory responses and fibrosis, suggesting NK-1R could be a potential therapeutic target for the patients with CKD.

Inflammation and Organ Injury the Role of Substance P and Its Receptors.

Tightly controlled inflammation is an indispensable mechanism in the maintenance of cellular and organismal homeostasis in living organisms. However, aberrant inflammation is detrimental and has been suggested as a key contributor to organ injury with different etiologies. Substance P (SP) is a neuropeptide with a robust effect on inflammation. The proinflammatory effects of SP are achieved by activating its functional receptors, namely the neurokinin 1 receptor (NK1R) receptor and mas-related G protein-coupled receptors X member 2 (MRGPRX2) and its murine homolog MRGPRB2. Upon activation, the receptors further signal to several cellular signaling pathways involved in the onset, development, and progression of inflammation. Therefore, excessive SP-NK1R or SP-MRGPRX2/B2 signals have been implicated in the pathogenesis of inflammation-associated organ injury. In this review, we summarize our current knowledge of SP and its receptors and the emerging roles of the SP-NK1R system and the SP-MRGPRX2/B2 system in inflammation and injury in multiple organs resulting from different pathologies. We also briefly discuss the prospect of developing a therapeutic strategy for inflammatory organ injury by disrupting the proinflammatory actions of SP via pharmacological intervention.

The effects of lipopolysaccharide exposure on social interaction, cytokine expression, and alcohol consumption in male and female mice.

Much recent research has demonstrated a role of inflammatory pathways in depressive-like behavior and excess alcohol consumption. Lipopolysaccharide (LPS) is a cell wall component of gram-negative bacteria that can be used to trigger a strong inflammatory response in rodents in a preclinical research setting to study the mechanisms behind this relationship. In our study, we exposed male and female mice to LPS and assessed depressive-like behavior using the social interaction (SI) test, alcohol consumption in the two-bottle choice procedure, and expression of inflammatory mediators using quantitative PCR. We found that LPS administration decreased SI in female mice but had no significant impact on male mice when assessed 24 h after injection. LPS resulted in increased proinflammatory cytokine expression in both male and female mice; however, some aspects of the cytokine upregulation observed was greater in female mice as compared to males. A separate cohort of male and female mice underwent drinking for 12 days before receiving a saline or LPS injection, which we found to increase alcohol intake in both males and females. We have previously observed a role of the neurokinin-1 receptor (NK1R) in escalated alcohol intake, and in the inflammatory and behavioral response to LPS. The NK1R is the endogenous target of the neuropeptide SP, and this system has wide ranging roles in depression, anxiety, drug/alcohol seeking, pain, and inflammation. Thus, we administered a NK1R antagonist prior to alcohol access. This treatment reduced escalated alcohol consumption in female mice treated with LPS but did not affect drinking in males. Taken together, these results indicate that females are more sensitive to some physiological and behavioral effects of LPS administration, but that LPS escalates alcohol consumption in both sexes. Furthermore, NK1R antagonism can reduce alcohol consumption that is escalated by LPS treatment, in line with our previous findings.

Expression of substance P, neurokinin 1 receptor, Ki-67 and pyruvate kinase M2 in hormone receptor negative breast cancer and evaluation of impact on overall survival.

BACKGROUND: Chronic inflammation is a hallmark of cancer, and it can be stimulated by many factors. Substance P (SP), through binding to neurokinin 1 receptor (NK1R), and pyruvate kinase M2 (PKM2) play critical roles in cancer development and progression via modulating the tumor microenvironment. This study aimed to investigate the prognostic significance of SP and PKM2 in combination with NK1R and Ki-67 in hormone receptor negative (HR-ve) breast cancer. METHODS: Immunohistochemical expression levels of SP, NK1R, PKM2, and Ki-67 were measured in 144 paraffin-embedded breast cancer tissues (77 h -ve and 67 h + ve). SP, NK1R, and PKM2 were scored semiquantitatively, while Ki-67 was obtained by the percentage of total number of tumor cells with nuclear staining. The optimal cutoff value for SP, NK1R, PKM2, and Ki-67 were assessed by Cutoff Finder. RESULTS: High SP expression in HR -ve breast cancer was associated with TNM stage (p = 0.020), pT stage (p = 0.035), pN stage (p = 0.002), axillary lymph node metastasis (p = 0.003), and NK1R expression level (p = 0.010). In HR + ve breast cancer, SP expression was associated with HER2 status (p = 0.001) and PKM2 expression level (p = 0.012). Regarding PKM2 expression level, it significantly associated with HER2 status (p = 0.001) and history of DCIS (p = 0.046) in HR-ve tumors, and with HER2 status (p < 0.001) and SP expression level (p = 0.012) in HR + ve tumors. Survival analysis revealed that high SP level negatively impacted overall survival in HR-ve tumors that had low NK1R level (p = 0.021). Moreover, high SP negatively impacted overall survival in HR-ve tumors that had low Ki-67 level (p = 0.005). High PKM2 negatively impacted overall survival in HR-ve cases with low SP (p = 0.047). CONCLUSION: Combined expression levels of SP with NK1R or Ki-67, and PKM2 with SP could be used to predict survival in breast cancer patients with HR-ve tumors. Our findings suggest a role of SP/NK1R pathway and PKM2 in HR-ve breast cancer pathogenesis which should be further investigated to unveil the underlying molecular mechanisms.

Stigmasterol alleviates allergic airway inflammation and airway hyperresponsiveness in asthma mice through inhibiting substance-P receptor.

CONTEXT: Stigmasterol has significant anti-arthritis and anti-inflammatory effects, but its role in immune and inflammatory diseases is still unclear. OBJECTIVE: The potential advantages of stigmasterol in asthma were explored in IL-13-induced BEAS-2B cells and asthmatic mice. MATERIALS AND METHODS: The optimal target of stigmasterol was confirmed in asthma. After detecting the cytotoxicity of stigmasterol in BEAS-2B cells, 10 µg/mL and 20 µg/mL stigmasterol were incubated with the BEAS-2B cell model for 48 h, and anti-inflammation and antioxidative stress were verified. Asthmatic mice were induced by OVA and received 100 mg/kg stigmasterol for 7 consecutive days. After 28 days, lung tissues and BAL fluid were collected for the following study. To further verify the role of NK1-R, 0.1 µM WIN62577 (NK1-R specific antagonist), and 1 µM recombinant human NK1-R protein were applied. RESULTS: NK1-R was the potential target of stigmasterol. When the concentration of stigmasterol is 20 µg/mL, the survival rate of BEAS-2B cells is about 98.4%, which is non-toxic. Stigmasterol exerted anti-inflammation and antioxidant stress in a dose-dependent manner and decreased NK1-R expression in IL-13-induced BEAS-2B. Meanwhile, in vivo assay also indicated the anti-inflammation and antioxidant stress of stigmasterol after OVA challenge. Stigmasterol inhibited inflammation infiltration and mucus hypersecretion, and NK1-R expression. DISCUSSION AND CONCLUSIONS: The protective effect of stigmaterol on asthma and its underlying mechanism have been discussed in depth, providing a theoretical basis and more possibilities for its treatment of asthma.