Aldosterone-producing adenoma (APA) is a major cause of primary aldosteronism, the most frequent form of secondary hypertension. Although somatic mutations in ion channels within APA have been shown to activate Ca2+ signaling and drive aldosterone production, the pathophysiology of primary aldosteronism remains partially understood. SP (Substance P), encoded by the TAC1 gene, is a neuropeptide of the tachykinin family, known for its role in stimulating aldosterone production through activation of the neurokinin 1 receptor (NK1R) in the human adrenal cortex. The aim of our work was to investigate the presence of SP nerve fibers and the NK1R in a large series of APA to assess the potential role of tachykinins in the pathophysiology of primary aldosteronism. Using molecular, immunohistochemical, and functional techniques, 56 APA tissues were analyzed to assess the expression of SP and NK1R and their impact on aldosterone secretion. SP-positive nerve fibers were detected in 90% of the APA tissues, localized both within and around the adenomas, which also showed strong NK1R expression. Functional studies revealed that SP stimulated aldosterone secretion in 6 of 10 APA cultures. The NK1R antagonist aprepitant inhibited SP-induced aldosterone secretion in 3 of the 4 SP-responsive APA cultures on which the antagonist was tested. Additionally, in perifused APA explants, SP influenced aldosterone pulsatility, resulting in enhanced mineralocorticoid secretion. These findings suggest that the SP-NK1R signaling pathway may contribute to APA pathophysiology and represent a novel potential target for the pharmacological treatment of PA in a subset of patients.

Involvement of substance P/NK1 receptor system in central sensitization in chronic pain.

Batatasin III alleviates slow transit constipation by regulating gut microbiota and inhibiting the NLRP3-IL-1β pathway.

Arthritis represents a group of chronic joint diseases characterised by persistent inflammation, pain, and progressive tissue damage. Despite advances in therapeutic management, many patients experience incomplete symptom relief, highlighting the need to better understand the underlying mechanisms that sustain inflammation and pain. Emerging evidence indicates that neuroimmune interactions within the joint microenvironment play a central role in the pathogenesis of arthritis. The synovium, a thin membrane that lines the joint cavity, is the primary site of pathology in arthritis and serves as a dynamic interface integrating immune, vascular, and neural components. Under physiological conditions, tissue-resident macrophages, fibroblasts, and sensory nerve fibres maintain joint homeostasis. However, during arthritis, the synovium undergoes extensive remodelling, including hyperplasia, angiogenesis, and nerve fibre sprouting, which together amplify inflammatory and nociceptive signalling. Distinct macrophage subsets within the synovium exhibit specialised roles in mediating inflammation and communicating with neurons. Macrophage-derived cytokines such as IL-1β, IL-6, and TNF-α can directly sensitise nociceptors, whilst chemokines like CCL2 engage neuronal receptors to enhance excitability. Conversely, activated sensory neurons release neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P (SP), which can modulate immune cell behaviour. Sympathetic signalling further contributes to immune modulation and correlates with disease severity. Together, these studies reveal that arthritis progression and chronic pain are shaped by reciprocal signalling between the nervous and immune systems. Understanding these complex pathways offers new perspectives for therapeutic intervention, suggesting that targeting neuroimmune crosstalk could provide dual benefits-reducing inflammation whilst alleviating chronic pain in arthritic disease.

Temporomandibular disorder (TMD) pain originates from muscular or intracapsular disorders, with the latter being represented by arthralgia. The aim of this study was to investigate the effects of photobiomodulation therapy (PBMT) on nociceptor activity, proinflammatory cytokine expression, neuropeptide expression, and tissue alterations in temporomandibular joint (TMJ) discs following unilateral injury in rats. Disc injury was induced via surgical access to the TMJ under general anesthesia. Forty rats were divided into four groups (n = 10 each). Group 1: surgical injury to the articular disc with PBMT; Group 2: sham surgery with PBMT; Group 3: surgical injury to the articular disc without PBMT; and Group 4: naïve (control). Ten PBMT sessions were conducted with a GaAs laser at a wavelength of 904nm and an energy density of 6J/cm². TMJs were analyzed for histological and histomorphometric parameters to evaluate tissue changes and protein levels of substance P (SP), transient receptor potential vanilloid 1 (TRPV-1), calcitonin gene-related peptide (CGRP), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α). Statistical analysis was performed by ANOVA with Tukey's post-hoc test (p < 0.050 indicating a significant difference). The results revealed increased expression of SP, TRPV-1, CGRP, IL-6, IL-1β, and TNF-α following TMJ injury, with significantly lower levels after PBMT than in the other groups, leading to an improvement in the initial phases of tissue repair. These findings suggest that PBMT effectively modulates nociceptive activity and reduces proinflammatory cytokine expression, optimizing tissue regeneration and improving the treatment of TMD.

Osteoarthritis (OA) is a debilitating joint disease affecting over 500 million people globally, characterized by cartilage degradation, chronic pain, and failed tissue repair. Neurogenic inflammation, driven by neuropeptides including Substance P (SP) and calcitonin gene-related peptide (CGRP), plays a key role in the pathogenesis of OA. This study explores the therapeutic potential of extracellular vesicles (EVs) derived from infrapatellar fat pad mesenchymal stem/stromal cells (IFP-MSCs) transduced with CGRP antagonist CGRP8-37 (aCGRP IFP-MSC EVs). These EVs are enriched in anti-inflammatory miRNAs and proteins, and they express neprilysin (CD10), enabling SP degradation. Herein, several LncRNAs were identified, which have been known to interact with miRNAs that affect the knee joint homeostasis. Specifically, 11 LncRNAs (ZFAS1, EMX2OS, HOTAIRM1, RPS6KA2-AS1, DANCR, LINC-ROR, GACAT1, GNAS-AS1, HAR1A, OIP5-AS1, TERC) interact with miRNAs that promote cell proliferation, prevent apoptosis, and preserve homeostasis. In vitro, aCGRP IFP-MSC EVs downregulated pro-inflammatory markers (TNF, TLR4, MAPK8) in dorsal root ganglia and promoted chondrocyte gene expression consistent with anabolism and matrix remodeling. In vivo, intra-articular EV delivery attenuated pain behaviors, preserved the cartilage structure, restored PRG4+ stem/progenitor cell localization, and trended toward reduced SP levels. Histological analysis confirmed improved collagen organization and reduced matrix degradation. These findings suggest that aCGRP IFP-MSC EVs exert multimodal effects on neuroinflammation, cartilage regeneration, and joint homeostasis. This cell-free, gene-enhanced EV therapy offers a promising disease-modifying strategy for the treatment of OA, with the potential to address both structural changes and chronic pain associated with this disease.

Neurokinin 1 receptor (NK1R) is a class A G protein-coupled receptor (GPCR) that interacts with the endogenous undecapeptide agonist Substance P (SP) to activate its role in key physiological processes such as nausea, inflammation, and neurological disorders. Structure determinations by cryogenic electron microscopy (cryo-EM) for NK1R with SP bound provided high-quality views of the bulk of these complexes but failed to observe the N-terminal peptide segments of both components. Here, 19F-NMR spectroscopy was combined with function assays to explore the impact of these chain ends on structure and function. The 19F-NMR probe 3-trifluoromethyl-l-phenylalanine (mtfF) was introduced into the SP sequence positions 0, 3, 5, 7, and 8 and observed in aqueous solution, bound to lauryl-maltose-neopentyl-glycol (LMNG)/cholesteryl hemisuccinate (CHS) micelles, and bound to micelle-solubilized NK1R. The NMR data showed for NK1R-bound SP that the N-terminal pentapeptide segment is flexible and solvent-exposed, while the C-terminal hexapeptide segment is rigidly anchored within the orthosteric pocket. Cyclic adenosine monophosphate function tests further showed that transient interactions of the side chains of Lys3 and Gln5 in SP with sites on the NK1R impacted the functional properties of SP. Combined with the rates measured for exchange of SP between all of its states in solutions with micelle-solubilized NK1R, these observations identify specific dynamic intermolecular interactions between the N-terminal peptide segments of SP and NK1R that impact the functional potency of SP.

BackgroundAbdominal massage is a therapeutic intervention in traditional Chinese medicine for managing insomnia; however, its underlying mechanisms remain incompletely understood. This study aimed to investigate the effects of abdominal massage on the gut microbiota and brain-gut peptides in a rat model of insomnia from the perspective of the microbiota–gut–brain axis.MethodsForty-eight male Wistar rats were randomized into control, model, abdominal massage (Abd massage), and zolpidem groups (n = 12). An insomnia model was induced by intraperitoneal injection of 4-chloro-DL-phenylalanine (PCPA). The Abd massage group and the zolpidem group, respectively, received 14 days of abdominal massage and zolpidem treatment. Hippocampal histopathology was evaluated with hematoxylin and eosin (HE) staining. Serum levels of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), vasoactive intestinal peptide (VIP), growth hormone (GH), substance P (SP), and cholecystokinin-8 (CCK8) were measured by enzyme-linked immunosorbent assay (ELISA). The gut microbiota composition was examined using 16S rRNA sequencing.ResultsBehavioral experiments in a rat model of insomnia demonstrated that abdominal massage significantly extended sleep duration. The treatment alleviated histopathological damage in the hippocampus and regulated brain-gut peptide levels in both colon and brain tissues. Additionally, abdominal massage modulated gut microbiota structure, reducing the relative abundance of Bacteroidetes and Proteobacteria and increasing that of Firmicutes, Lachnospiraceae_NK4A136_group, Clostridia, and Clostridiales. Spearman correlation analysis revealed significant associations between microbial abundance and biochemical indicators. PICRUSt2 analysis further implicated carbohydrate metabolism, amino acid metabolism, and transcriptional regulation in the pathogenesis of insomnia.ConclusionThe results of this study demonstrate that abdominal massage ameliorates insomnia and increases sleep duration. This effect is associated with the regulation of brain-gut peptide levels and the restoration of gut microbiota diversity and structure. These findings suggest that the microbiota-gut-brain axis may be involved in the therapeutic mechanism of abdominal massage for insomnia.

BackgroundAllergic conjunctivitis (AC) is a prevalent ocular surface disorder, with chronic or severe manifestations of AC frequently resulting in substantial ocular surface damage. The neuropeptide substance P (SP) plays a critical role in inflammation and immune regulation; however, its involvement in AC remains unclear. The objective of this study was to establish a murine model of persistent AC through repeated allergen exposure to investigate how SP and its downstream STAT3/STAT5 pathway modulate Th17/Treg imbalance and exacerbate ocular surface damage.MethodsA persistent AC mouse model was established through repeated ovalbumin (OVA) challenges. The ocular surface damage in the mice was evaluated using slit lamp examination, H&E staining, fluorescein staining, PAS staining, and TUNEL staining. The percentages of Th17/Treg cells and the expression of associated proteins (IL-17 and FOXP3) in cervical lymph nodes and conjunctival-corneal tissues were assesed using flow cytometry, immunofluorescence and Western blot. RNA sequencing was subsequently conducted to identify differentially expressed genes. The expression of substance P (SP) and its receptor (NK1R) was validated using a reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blotting, immunofluorescence and immunohistochemistry. SP and the NK1R antagonist Spantide II were administered to examine their effects on Th17/Treg balance and STAT3/STAT5 signaling (pSTAT3/pSTAT5).ResultsExposure to continuous OVA induced severe allergic symptoms, such as eyelid swelling and conjunctival hyperaemia, as well as ocular surface damage. This damage included inflammatory infiltration, corneal barrier dysfunction, loss of goblet cells and apoptosis. The number of Th17 cells increased while the number of Tregs decreased, resulting in an elevated Th17/Treg ratio. RNA-seq revealed that SP expression was upregulated in the conjunctiva of AC, correlating with sensitization frequency; a similar trend was observed for NK1R. Both in vitro and in vivo experiments confirmed that SP exacerbated the Th17/Treg imbalance by promoting Th17 differentiation and inhibiting Treg differentiation while also activating the STAT3/STAT5 signaling pathway, as evidenced by increased pSTAT3 and decreased pSTAT5 levels. Intervention with Spantide II markedly alleviated ocular inflammation, restored corneal integrity, reduced apoptosis, rebalanced Th17/Treg ratios, and suppressed STAT3/STAT5 activation.ConclusionsSubstance P contributes to persistent AC by disrupting the Th17/Treg equilibrium via STAT3/STAT5 signaling, thereby exacerbating ocular surface damage. Targeting the SP/NK1R pathway may offer a novel therapeutic approach for chronic AC.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-07460-9.

Background: Substance P (SP), a neuropeptide identified in 1931, is ubiquitously present in both the central (CNS) and peripheral (PNS) nervous systems, playing a pivotal role in nociceptive signal transmission and immune system responses. Aims and Objectives: This research aims to explore the correlation of SP levels with chronic pain in various syndromes, specifically focusing on osteoarthritis (OA), rheumatoid arthritis (RA), widespread pain (WSP), and fibromyalgia (FM). Materials and Methods: This cross-sectional study involved 60 participants aged 18-65 years with chronic pain persisting for at least 180 days. Patient selection criteria included evaluation of pain severity using the Visual Analogue Scale (VAS). Substance P concentration was measured in both serum and cerebrospinal fluid (CSF). Statistical analysis methodologies employed included the t-test, ANOVA, and Pearson's correlation coefficient. All participants provided informed consent prior to their inclusion in the study. Results: The study population's features included a mean age of 42.08±10.57 years, with a gender distribution of 38 females and 22 males. Mean VAS scores were 73.5±13.0, while mean serumSP concentrations were 477.52±288.39 pg/mL and mean CSF SP concentrations were 653.95±262.23 pg/mL. Among the 60 patients classified with severe pain, 32 were diagnosed with osteoarthritis, 26 with rheumatoid arthritis, and 2 with widespread pain syndrome. Correlation analysis revealed a weak positive correlation between cerebrospinal fluid SP levels and chronic pain syndromes, and a moderate positive correlation with VAS scores. Weaker associations were observed between serum SP levels and both pain syndromes and VAS scores. These findings suggest potential influences of biomarkers on pain states and diagnostic categories. Conclusion: The study concludes that Substance P levels positively correlate with chronic pain perception across various pain syndromes. While SP levels do not appear to vary significantly with the specific underlying cause of chronic pain, these findings support the need for a larger, multi-centric trial to comprehensively assess SP's role in a more diverse patient population.

To explore the inhibitory effects of Nardostachys Jatamansi DC. volatile oil (NJVO) on psychological factors substance P (SP)/cortisol (CORT)-induced hyperpigmentation. The model of psychologically-induced hyperpigmentation of B16F10 cells was created using SP (10 nmol/L) + CORT (10 µmol/L) for 72 h. The levels of melanin content, tyrosinase (TYR) activity using NaOH lysis and L-dihydroxyphenylalanine (L-DOPA) oxidation methods were assessed, respectively. The effect of NJVO on SP/CORT-induced normal human skin tissue pigmentation was detected by Masson staining. Protein expressions of tyrosinase-related protein 1 (TRP-1), tyrosinase-relative protein 2 (DCT), and microphthalmia-associated transcription factor were determined using Western blot. The melanosome number, maturation, and melanosomal structure changes were detected through transmission electron microscopy and immunofluorescence experiments. In vivo, zebrafish pigment content was evaluated in SP/CORT-induced zebrafish hyperpigmentation model. NJVO significantly reduced the melanin content (P<0.01) and inhibited tyrosinase activity (P<0.01), the pigmentation of the normal skin tissue in the NJVO group was significantly lower than that in the SP/CORT group (P<0.05). And NJVO considerably downregulated expressions of melanogenesis-related proteins (TYR, TRP-1, DCT) in cells (P<0.01). In addition, the number of melanosomes was decreased and the dentrites formation of B16F10 cells was inhibited after NJVO treatment (P<0.01). In vivo, NJVO significantly reduced the pigment content in the zebrafish body (P<0.01). NJVO effectively reversed SP/CORT-induced hyperpigmentation by suppressing the activity and expression of TYR and TRPs and inhibiting melanosome maturation in mouse B16F10 melanoma cells.

The neuro-cutaneous axis: the role of nerve cells in wound healing.

To investigate the analgesic effect of sufentanil plus dexmedetomidine following cesarean section and to determine its influence on placental hypoxia-inducible factors. A cohort of 150 puerperae who underwent prenatal examinations and cesarean section in our hospital were randomized into a control group (n=75) and a study group (n=75). Anesthesia and analgesia were carried out using sufentanil alone in the control group and sufentanil plus dexmedetomidine in the study group. Measurements were made before anesthesia (T0), 5 minutes (T1) and 10 minutes (T2) after anesthesia, and immediately after delivery (T3) and after the end of surgery (T4). Mean arterial pressure (MAP) and heart rate (HR) decreased in both groups at T1 - T4 compared with T0, but were higher in the study group compared to the control group (p < 0.05). The visual analogue scale (VAS) score, and levels of substance P (SP), neuropeptide Y (NPY) and malondialdehyde (MDA) in the study group were lower than those in the control group (p<0.05). The beginning and duration of labor and the dose of analgesics within the 48-hour observation period were all lower in the study group compared to the control group (p<0.05). Sufentanil plus dexmedetomidine can effectively maintain hemodynamic stability during cesarean section without marked changes in placental hypoxia-inducible factors and oxidative stress responses and has a limiting effect on the secretion of pain mediators.

ABSTRACTClinical studies have established that repeated blast traumatic brain injury (rbTBI) can result in chronic pain conditions, with outcomes exhibiting notable sex‐dependent differences. However, limited preclinical rbTBI models have systematically investigated the behavioral and neuropathological outcomes of female subjects. In the present study, adult female rats were subjected to repeated blast exposures, and the subsequent development of chronic pain–related behaviors and neuropathological changes was assessed. Repeated blast events induced robust mechanical and thermal hypersensitivity beginning 48 h post‐injury and persisted through 12 weeks, accompanied by anxiety and depressive‐like behaviors at the chronic time point. These behavioral alterations were associated with increased glial activity, as evidenced by Glial Fibrillary Acidic Protein (GFAP) and Ionized Calcium‐Binding Adaptor Molecule 1 (IBA‐1) in the frontal cortex and posterior nucleus regions at 12 weeks following injury. Notably, expression levels of neuropeptide markers, Calcitonin Gene‐Related Peptide (CGRP) and Substance P (SP), remained unchanged. Collectively, these findings suggest that chronic pain behaviors following rbTBI in females are mediated primarily by sustained glial activation rather than neuropeptide dysregulation.

This study compared the outcomes of laparoscopic ovariectomy (LOVE) and open ovariectomy (OVE) in dogs, assessing pain, quality of recovery, and systemic inflammatory biomarkers. A prospective clinical study. Twenty anestrous female dogs, ten for each group. The parameters compared between the two groups were: incision length, surgical time, pain, along with concentrations of serum amyloid A (SAA), C-reactive protein (CRP), and substance P (SP), measuring one hour before surgery (T0), two hours (T1), 24h (T2), and seven days post-surgery (T3); quality of recovery (QoR-15) at T0, T2 and T3. LOVE results in a longer surgery time, while a shorter incision length. CRP was higher (P < 0.05) at T1 but lower (P < 0.001) at T2 and T3 in LOVE than in OVE group. At T1 and T2 lower SAA concentrations were found in OVE groups compared to LOVE group (P < 0.001); and at T3, SP was higher (P < 0.05) in the OVE group compared to the LOVE group. Pain at T1 and T2, and QoR-15 at T2 and T3, showed better scores (P < 0.01) in LOVE compared to OVE group. This study showed that laparoscopic ovariectomy resulted in moderate surgical trauma and postoperative pain, reducing the time for recovery compared to a laparotomic procedure. Serum concentrations of CRP and SP, predictive biomarkers of postoperative systemic inflammation, confirm a greater inflammatory response in the LOVE group, which may explain the prolonged recovery times observed in these group. Our results indicate that SAA is not a reliable marker of mild surgical stress, whereas CRP remains a valid indicator of postoperative inflammation in canine ovariectomy. SP showed promising consistency with the Glasgow Composite Pain Scale, supporting its potential as a biomarker for perioperative pain. Preliminary findings also suggest the feasibility of using the adapted QoR-15 scale in dogs. Overall, laparoscopic ovariectomy confirmed clear advantages over open laparotomy, including reduced inflammation, lower pain levels, and faster recovery. Further studies with larger cohorts and long-term follow-up are needed to validate these tools and assess the broader welfare implications of different surgical approaches. This study supports the advantages of laparoscopic techniques for ovariectomy, particularly in terms of the patient's recovery quality.

This study aimed to identify hematological markers that can objectively assess postpartum recovery in dairy cows, which is essential for determining an individualized voluntary waiting period (VWP). Twenty Holstein cows were divided into early recovery and late recovery groups based on their clinical uterine recovery. Blood samples were collected from coccygeal vessels and analyzed for inflammatory and metabolic markers, including serum amyloid A (SAA), haptoglobin (HPT), cortisol (COR), substance P (SP), interleukin-6 (IL-6), total cholesterol (T-Chol), beta-hydroxybutyrate, and non-esterified fatty acids (NEFA). Both SAA and HPT significantly decreased after recovery compared to immediately after parturition (p < 0.001), whereas T-Chol and NEFA significantly increased and decreased over time, respectively, indicating recovery from a negative energy balance status. No significant changes were observed in COR, SP, or IL-6 levels. Thus, SAA and HPT are sensitive indicators of postpartum inflammation, whereas T-Chol and NEFA levels reflect metabolic recovery. Using these hematological markers enables a more objective and customized setting of VWP for each cow, potentially improving reproductive efficiency and farm profitability. Further studies are warranted to establish cutoff values for each parity and validate the applicability of individualized VWP models.

Background:Acupuncture effectively reduces chronic neck pain and plasma Substance P (SP) levels, but upstream molecular mechanisms remain unknown.Objectives:We aimed to identify circulating microRNAs (miRNAs) associated with acupuncture-induced SP reduction and explore potential neuroplasticity mechanisms.Methods:We performed longitudinal plasma miRNA profiling (Affymetrix miRNA 4.0 Array, ~4600 miRNAs) in chronic neck pain patients: Acupuncture group (n = 3; 0, 4, 8 weeks) and Control group (n = 3; 0, 4 weeks). Linear Mixed-Effects Models (LMMs) tested associations between each miRNA and SP dynamics (miRNA × Time × Group interaction). Statistical significance was validated using permutation testing (2000 iterations).Results:Screening identified 53 miRNAs significantly associated with SP, validated by permutation testing (p < 0.001). Fourteen high-confidence miRNAs showed significant three-way interactions, indicating treatment-specific SP relationships. The most significant was miR-1302-6 (p = 7.65 × 10−6), followed by miR-181b-2. These miRNAs displayed diverse temporal patterns: some (miR-196b, miR-6788) increased during treatment, while others (let-7d, miR-1302-6) decreased parallel to SP. Functional enrichment revealed striking convergence on neuroplasticity pathways: axon guidance (p = 2.61 × 10−6), MAPK signaling (p = 4.18 × 10−5), neuron projection development (p = 7.52 × 10−10), and synaptic structures (p = 9.52 × 10−12).Conclusions:This exploratory study provides first molecular evidence for an acupuncture-miRNA-SP axis in chronic pain. The enrichment of neuroplasticity pathways suggests acupuncture may induce structural remodeling of nociceptive circuits rather than simply suppressing inflammation, offering novel mechanistic insights and potential biomarkers for personalized acupuncture therapy. The trial was registered with the Korean Clinical Trial Registry (KCT0005363).

Substance P (SP) is a key neuropeptide that has an important role in the neurotransmission, inflammation and immune system regulation. Its high degree of conservation and wide range of biological functions, including interaction with NK1 receptors, emphasize its importance in physiological and pathological processes. SP significantly influences the development of dermatological diseases such as psoriasis, atopic dermatitis and rosacea, and is also involved in processes of wound healing and immune response modulation. Modern researches focus on its role in activating both the innate and adaptive immunity making it an important regulator of the immune activity. Multiple functions of SP indicate its potential as a therapeutic target especially in the treatment of inflammatory skin diseases. In addition, substance P is involved in pain sensations regulation and neuroimmune interactions that expands its role in the pathophysiology of pain syndromes. The development of NK1 receptors antagonists such as aprepitant offer prospects for the treatment of inflammatory skin diseases and immune system disorders. These data emphasize the importance of targeting SP and its signal pathways as a new approach to the treatment of dermatological and immunological diseases.

We aimed to assess the effects of intravesical cocktail instillation on the outcomes and serum pain factors of patients with bladder pain syndrome (BPS). The clinical data of 86 female BPS patients hospitalized between March 2017 and March 2024 were collected for retrospective analysis. All patients were treated with oral medication (amitriptyline) + local intravesical instillation of drugs, and then assigned to a control group (sodium hyaluronate intravesical instillation) and a research group [intravesical instillation of dimethyl sulfoxide (DMSO) + chondroitin sulfate (CS) + low-molecular-weight heparin (LMWH) cocktail]. The research group (n=43) had lower urination frequency in 24 h, Pelvic Pain and Urgency/Frequency Patient Symptom Scale score, and O'Leary-Sant interstitial cystitis symptom index and interstitial cystitis problem index, as well as larger single urination volume than those of the control group (n=43) after 3 and 6 months of treatment (P<0.05). In the serum, the levels of substance P (SP), 5-hydroxytryptamine (5-HT), prostaglandin E2 (PGE2), neuropeptide Y (NPY), β-endorphin (β-EP), and dopamine (DA) declined in the two groups after 1 month of treatment in comparison to the pre-treatment levels. The levels of SP, 5-HT, PGE2, and NPY were lower, while the levels of β-EP and DA were higher in the research group than those in the control group (P<0.05). The intravesical instillation of DMSO + CS + LMWH cocktail is superior in long-term outcomes. It is more conducive to improving the levels of serum pain factors, with a good safety profile and without increasing adverse reactions.

The bionic drug-containing cerebrospinal fluid(CSF) of the herb pair Ephedrae Herba-Armeniacae Semen Amarum was prepared and analyzed by experiments on the chemical composition, effects, and correlations, on the basis of which the central active ingredients of the pair and their mechanisms of relieving dyspnea were clarified and the feasibility of the bionic drug-containing CSF method for the neuropharmacological study of traditional Chinese medicine(TCM) in vitro was explored. UPLC-Q-TOF-MS was used to detect and identify the ingredients in the mass spectra of the cerebrospinal fluid containing the herb pair mixed in three ratios. A total of 40 compounds were identified, and 13 differential components were obtained by OPLS-DA. The comparison with the data from the database of the active ingredients of the herb pair for dyspnea alleviation preliminarily identified ephedrine, pseudoephedrine, and methylephedrine as the main differentially active ingredients of the herb pair in the brain. The blood-brain barrier(BBB) model was established by contact co-culture of rat brain microvascular endothelial cells(RBE4) and astrocytes(AC), and the drug-containing CSF was prepared with the BBB model after obtaining drug-containing serum with the herb pair mixed in different ratios. Then, the mass spectra of RBE4 and AC were compared with that of the drug-containing CSF. It was found that the compositional similarity between the bionic drug-containing CSF and the drug-containing CSF was as high as 91.43%, while the content of differential active ingredients in the bionic systems with different mixture ratios was higher than that in the drug-containing CSF. A Transwell chamber was used to construct a PC12 and AC co-culture system, and 6.4×10~(-3) mol·L~(-1) histamine phosphate was used to establish an in vitro cortical neuronal injury model. After the herb pair in a 2∶1 ratio was used to treat different final concentrations of the bionic drug-containing CSF and the drug-containing CSF, biochemical kits were used to measure the content of neurotransmitters, such as nerve growth factor(NGF), substance P(SP), acetylcholine(Ach), 5-hydroxytryptamine(5-HT), and norepinephrine(NA), and their relative standard deviation(RSD) values were calculated. RT-PCR and Western blot were employed to determine the mRNA and protein levels, respectively, of neurokinin-1 receptor(NK-1R), mitogen-activated protein kinase(MAPK), and proto-oncogene C-Fos(C-FOS). The results showed that compared with the normal group, the model group showed increased content of neurotransmitters and up-regulated mRNA and protein levels of NK-1R, MAPK, and C-FOS. Compared with the model group, the drug intervention groups showed reduced content of neurotransmitters and down-regulated mRNA and protein levels of NK-1R, MAPK, and C-FOS. Moreover, the RSD values of neurotransmitter changes in the bionic drug-containing CSF group were generally smaller than those in the drug-containing CSF group. Regression analyses were performed for the content of the active ingredients of the above two drug-containing systems and each effect indicator. The results showed that each effect indicator had higher correlation with the content of the ingredients in the bionic drug-containing CSF than with that in the drug-containing CSF. In conclusion, ephedrine, pseudoephedrine, and methylephedrine may be the main central active ingredients of the herb pair for relieving dyspnea. They exert their dyspnea-relieving effects by inhibiting the expression of NK-1R, MAPK, and C-FOS in the central nervous system and reducing the content of neurotransmitters such as NGF, SP, Ach, 5-HT, and NA. The preliminary results indicate that the bionic drug-containing CSF method has comparative advantages such as good reproducibility and high correlation between ingredients and effects in the in vitro neuropharmacological study of TCM.