Subtle structural modifications at the 3-position of opioid peptides have been reported to modulate their receptor binding affinity and functional activity. Our previous work identified a novel cyclic hexapeptide, Tyr-c[D-Lys-Gly-Phe-Asp]-D-Pro-NH2, as a multitarget full agonist at μ-, κ-, and δ-opioid receptors (MOR, KOR, and DOR). In the present study, 16 novel analogs of this hexapeptide were synthesized by replacing the Gly3 residue with phenylalanine and its derivatives to investigate their structure-activity relationships. Functional characterization demonstrated that these modifications generated analogs exhibiting varying selectivity and efficacy profiles at opioid receptors. Notably, analog 9 containing p-F-Phe3 exhibited MOR and KOR agonism/DOR antagonism and produced potent antinociception following peripheral administration, with reduced antinociceptive tolerance, physical dependence, and respiratory depression. Surprisingly, analog 11 containing p-Br-Phe3 acted as a potent and selective MOR antagonist. Pharmacological blockade of the peripheral MOR by analog 11 significantly attenuated fentanyl-induced antinociceptive tolerance, highlighting its therapeutic potential.

Opioid receptors, as members of the G-protein-coupled receptor (GPCR) superfamily, represent promising therapeutic targets for managing nociception. Herein, we designed, synthesized, and pharmacologically characterized the novel bifunctional decapeptide, MKOP002. In vitro, MKOP002 activated both µ- and κ-opioid receptors (MOR and KOR), exhibiting G protein-biased agonism at MOR. Subcutaneous (s.c.) injection of MKOP002 produced significant antinociceptive effects through the peripheral MOR and KOR in the tail-flick test. Notably, MKOP002 demonstrated potent and dose-dependent antinociceptive efficacy across various models of acute, inflammatory, and neuropathic pain. Additionally, MKOP002 exhibited antipruritic effects comparable to CR845, mediated by both the peripheral and central KOR. Similar to CR845, at the high doses, MKOP002 induced sedation via central KOR at least partially, and also resulted in depressive-like behaviors. Importantly, MKOP002 did not elicit MOR-associated adverse effects such as tolerance, constipation, respiratory depression, or addiction. These findings underscore the therapeutic potential of MKOP002 as a lead compound for antinociceptive and antipruritic therapy, supporting the development of bifunctional MOR/KOR agonists as a safer opioid strategy.

Recently, κ-opioid receptor (KOR) agonists have been attractive therapeutic candidates for the treatment of pain and pruritus. To advance the development of effective and safer peripherally restricted KOR agonists, we designed, synthesized, and evaluated 21 novel chimeric peptides incorporating the N-terminal tetrapeptide sequence of CR845 and the C-terminal “address” domain derived from dynorphin A. In vitro calcium mobilization assays confirmed the selective and full KOR agonism for all the compounds. Among these, analogues 4, 9, and 17 emerged as optimized analogues, exhibiting potent analgesic and antipruritic effects comparable to those of CR845 after subcutaneous administration, through the activation of the peripheral KOR. Additionally, subcutaneous administration of these 3 optimized analogues induced sedation without eliciting aversion or depressive-like side effects. Notably, analogue 4 was selected as the final candidate due to its high potency in KOR agonism and minimal sedative effects, making it the most promising drug candidate for the development of efficient and safe antinociceptive and antipruritic therapies.

Opioid analgesics are critical for managing moderate-to-severe pain，yet are limited by adverse gastrointestinal (GI) effects, notably constipation. This necessitates developing novel opioid agonists with robust analgesia and reduced GI side effects. The cyclic hexapeptide Tyr-c[D-Lys-Gly-p-F-Phe-Asp]-D-Pro-NH2 (analog 15), a recently characterized multifunctional agonist of μ-opioid receptor (MOR), κ-opioid receptor (KOR), and δ-opioid receptor (DOR), exhibits potent antinociception following subcutaneous (s.c.) administration with constipation observed only at high doses. To further evaluate its GI impact, we assessed the effects of analog 15 on intestinal motility using in vivo upper GI transit and colonic bead expulsion assays. Our results indicated that fentanyl, analog 15, and its parent peptide analog 0 dose-dependently slowed upper GI transit and colonic expulsion after s.c. administration, with the upper GI tract exhibiting greater sensitivity. Mechanistically, fentanyl inhibited the GI motility via both central and peripheral opioid receptors, whereas analog 15 inhibited upper GI transit exclusively through the peripheral MOR, KOR, and DOR, and suppressed colonic transit via the peripheral MOR and KOR, both effects were independent of the central opioid receptor pathway. In conclusion, we demonstrated that the high doses of analog 15 inhibited GI motility through peripherally restricted activation of multiple opioid receptors. This finding aligns with analog 15's limited blood-brain barrier (BBB) permeability, which explains its reduced constipating effects while preserving potent analgesia, thereby supporting the therapeutic potential of multi-target peripheral opioid agonists.

The aim of this study was to investigate the combination effects of α-glycerol monolaurate (GML) and glyceryl tributyrate (TB) on growth performance, nutrient digestibility, gut microbiota, and immune function in weaned piglets. A total of 120 weaned piglets with an average body weight (BW) of 6.88 kg were randomly allocated to one of the three dietary treatments: (1) CON: a basal diet; (2) 0.1%: a basal diet with 0.1% MSCFA (GML/TB = 1:1); (3) 0.2%: a basal diet with 0.2% MSCFA (GML/TB = 1:1). The experiment lasted 28 days. There were no differences on average daily growth (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR). Supplementation with 0.1% MSCFA increased apparent total tract digestibility (ATTD) of crude protein (CP) and gross energy (GE, p < 0.05) on d 14 and increased GE (p < 0.05) on d 28 compared with the CON group. The ATTD of dry matter (DM), organic matter (OM) and crude protein (CP) of piglets supplemented with 0.1% MSCFA was higher (p < 0.05). Compared with the CON group, supplementation with 0.1% MSCFA increased immunoglobulin M (IgM) concentration, decreased interleukin-6 (IL-6) content (p < 0.05) on d 14 and decreased malonaldehyde (MDA), interleukin-1beta (IL-1β), IL-6 concentrations (p < 0.05) on d 28. Supplementation with 0.1% MSCFA increased total antioxidant capacity (T-AOC) concentration (p < 0.05), decreased GSH-Px, MDA content (p < 0.05) in jejunum compared with the CON group. Moreover, supplementation with MSCFA increased the activity of duodenal lipase (p < 0.05) and the abundance of firmicutes and decreased the abundance of proteobacteria compared with the CON group. Overall, supplementation with MSCFA can improve nutrient digestibility, enhance immunity and antioxidant capacity, and improve the intestinal health of piglets. The combined use of MSCFA is a nutrition regulation strategy worthy of further exploration in modern animal husbandry.

BACKGROUND AND PURPOSE:The opioid crisis caused by the proliferation of fentanyl-related drugs has intensified concerns about the utilisation of opioid analgesics. Herein, a novel peptide-drug conjugate FENPFF01, incorporating the neuropeptide FF (NPFF) and fentanyl pharmacophores, was synthesised and pharmacologically characterised. EXPERIMENTAL APPROACH:The agonist activities of FENPFF01 at opioid and NPFF receptors were characterised in in vitro functional assays. Antinociceptive effects and underlying pharmacological mechanisms of FENPFF01 were assessed in multiple mouse pain models. The side effects of FENPFF01 were further investigated through antinociceptive tolerance, respiratory function, addiction potential and gastrointestinal transit tests. KEY RESULTS:FENPFF01 functioned as a mixed partial agonist at the μ-opioid and NPFF2 receptors, and a full agonist at NPFF1 receptor. Subcutaneous FENPFF01 produced potent antinociception in acute, inflammatory and post-operative pain, which was mediated by the μ receptor. The in vivo pharmacological and pharmacokinetic data demonstrated that FENPFF01 could penetrate the blood-brain barrier (BBB) and activate the μ receptor in the brain to elicit antinociception. Importantly, chronic administration of FENPFF01 did not induce antinociceptive tolerance or observable effects on spinal microglial activation, which may be partly attributable to the activation of the NPFF2 receptor. FENPFF01 also did not affect arterial gas parameters, respiratory rate, locomotor activity, conditioned place preference response, physical dependence and gastrointestinal transit. CONCLUSIONS AND IMPLICATIONS:The newly developed peptide-drug conjugate FENPFF01 exhibited strong antinociception with reduced opioid-like side effects. It represents an interesting candidate for developing novel analgesics with multi-targeted agonist properties, aiming to address the widespread issues associated with opioid misuse.

Objective(s): This study aims to investigate the effects of fucoidan-alginate combined dressings on wound healing in rats with full-thickness skin defects and to explore the underlying mechanisms. Materials and Methods: Male SD rats were divided into three groups (n=15): Control, 2% fucoidan, and 5% fucoidan. Full-thickness skin wounds were created on each rat. Fucoidan-alginate dressings were prepared by applying 20 mg/ml and 50 mg/ml fucoidan solutions to alginate dressings (2×2 cm), resulting in 2% and 5% (w/v) fucoidan-alginate combined dressings, respectively. The control group utilized alginate dressings. Wound healing was assessed through various methods, including wound area measurement, histopathological analysis, white blood cell counts, ELISA for TNF-α and IL-1β, Masson’s trichrome staining for collagen, immunohistochemistry for TGF-β1, and western blotting for TGF-β1 and Smad-related proteins.Results: The results revealed that wound healing was significantly more effective in rats treated with 5% fucoidan-alginate combined dressings. Compared to the control group (P<0.01) and the 2% FUC group (P<0.05), the 5% FUC group exhibited reduced inflammatory cell infiltration and lower levels of TNF-α and IL-1β. Moreover, in comparison to the control group, the 5% FUC group demonstrated a significant up-regulation in the mean density of TGF-β1 (P<0.01) and significantly elevated protein expression levels of Col I, α-SMA, and p-Smad2/3 (P<0.01). Additionally, a notable amount of collagen production was observed.Conclusion: The findings suggested that fucoidan-alginate dressings promote wound healing, reduce inflammation, and enhance collagen synthesis in rats, likely via the TGF-β1/Smad signaling pathway.

BACKGROUND:Accumulating evidence suggests that glial mechanisms are pivotal in regulating chronic pain. Our previous findings revealed that the interactions between spinal microglia and astrocytes are crucial for burn-induced pain hypersensitivity. However, the mechanisms underlying burn-induced peripheral sensitisation remain incompletely understood. METHODS:Sensory neurone-satellite glial cell (SGC) interactions within peripheral dorsal root ganglia were investigated using in vitro and in vivo experiments. Behavioural tests were conducted to evaluate the therapeutic potential of targeting peripheral sensitisation mechanisms for burn pain management. RESULTS:Burn injury upregulated calcitonin gene-related peptide (CGRP) expression in sensory neurones (1.5-fold; P=0.013) through transient receptor potential vanilloid 1 (TRPV1) channels. Pharmacological blockade of the TRPV1/CGRP signalling pathway effectively attenuated burn-induced mechanical allodynia and thermal hyperalgesia. Additionally, neurone-derived CGRP triggered SGC activation (from 6.8% pre-injury to 41.6% at day 5 post-injury), concomitant with enhanced gap junction-mediated SGC coupling (from 16.7% pre-injury to 40.5% at day 5 post-injury). Furthermore, chemokine expression (particularly CXCL1) in SGCs was elevated after burn injury, which potentiated sensory neurone excitability and exacerbated pain hypersensitivity. Blocking SGC coupling exerted potent analgesic effects in this burn pain model. CONCLUSIONS:A novel neurone-SGC interaction mechanism drives burn-induced peripheral sensitisation, providing translational implications for burn pain therapeutics.

The neuropeptide FF (NPFF) system regulates various physiological and pharmacological functions, particularly pain modulation. However, the modulatory effect of NPFF system on itch remains unclear. To investigate the modulatory effect and functional mechanism induced by NPFF system on acute itch, we examined the effects of supraspinal administration of NPFF and related peptides on acute itch induced by intradermal (i.d.) injection of histamine or chloroquine in male mice. Our results indicated that intracerebroventricular (i.c.v.) administration of NPFF dose-dependently prevented histamine- or chloroquine-induced acute itch behaviors. In addition, the modulatory effect of NPFF was not affected by the selective NPFF receptor antagonist RF9. Furthermore, we investigated the effects of NPVF and dNPA, the selective agonists of NPFF1 and NPFF2 receptors respectively, on the acute itch. The results demonstrated that both NPFF agonists effectively prevented acute itch induced by histamine or chloroquine in a manner similar to NPFF, and their effects were also not modified by RF9. To further investigate the possible mechanism of the NPFF receptors agonists, the NPFF-derived analogues [Phg8]-NPFF and NPFF(1-7)-NH2 that could not activate NPFF receptors in cAMP assays were subsequently tested in the acute itch model. Interestingly, [Phg8]-NPFF, but not NPFF(1-7)-NH2, prevented acute itch behavior after i.c.v. administration. In conclusion, our findings reveal that NPFF and related peptides prevent histamine- and chloroquine-induced acute itch through a NPFF receptor-independent mechanism. And it was revealed that the C-terminal phenyl structure of NPFF may play a crucial role in these modulatory effects on acute itch.

With the current unmet demand for effective pain relief, analgesics without major central adverse effects are highly appealing, such as peripherally restricted kappa-opioid receptor (KOR) agonists. In this study, Conorphin-66, an analog of the selective KOR peptide agonist Conorphin T, was pharmacologically characterized in a series of experiments, with CR845 serving as the reference compound. Firstly, in vitro functional assay indicated that Conorphin-66 selectively activates KOR and exhibits weak β-arrestin2 signaling bias (-1.54 versus -4.35 for CR845). Additionally, subcutaneous Conorphin-66 produced potent antinociception in mouse pain models with ED50 values ranged from 0.02 to 3.28 μmol/kg, including tail-flick test, post-operative pain, formalin pain, and acetic acid-induced visceral pain. Similarly, CR845 exert potent antinociception in mouse pain models ranged from 0.15 to 1.47 μmol/kg. Notably, antagonism studies revealed that the analgesic effects of Conorphin-66 were mainly mediated by the peripheral KOR. Furthermore, Conorphin-66 produced non-tolerance-forming antinociception over 8 days. Unlike CR845, subcutaneous Conorphin-66 did not promote the sedation, anxiogenic effects, depressive-like effects, but did exhibit diuretic activity. Further study showed that Conorphin-66 does not have apparent antipruritic effects in an acute itch model. Overall, Conorphin-66 emerges as a novel peripherally restricted KOR agonist that produced potent antinociception with reduced side effects.

Venom-derived NaV1.7 channel blockers have promising prospects in pain management. The 34-residue tarantula peptide GpTx-1 is a potent NaV1.7 channel blocker. Its powerful analog [Ala5, Phe6, Leu26, Arg28]GpTx-1 (GpTx-1-71) displayed excellent NaV1.7 selectivity and analgesic properties in mice. The current study aimed to elucidate the anti-hyperalgesic activities of GpTx-1-71 in inflammatory pain and reveal the underlying mechanisms. Our results demonstrated that intrathecal and intraplantar injections of GpTx-1-71 dose-dependently attenuated CFA-induced inflammatory hypersensitivity in rats. Moreover, GpTx-1-71-induced anti-hyperalgesia was significantly reduced by opioid receptor antagonists and the enkephalin antibody and diminished in proenkephalin (Penk) gene knockout animals. Consistently, GpTx-1-71 treatment increased the enkephalin level in the spinal dorsal horn and promoted the Penk transcription and enkephalin release in primary dorsal root ganglion (DRG) neurons, wherein sodium played a crucial role in these processes. Mass spectrometry analysis revealed that GpTx-1-71 mainly promoted the secretion of Met-enkephalin but not Leu-enkephalin from DRG neurons. In addition, the combination of subtherapeutic Met-enkephalin and GpTx-1-71 produced synergistic anti-hyperalgesia in CFA-induced inflammatory hypersensitivity. These findings suggest that the endogenous enkephalin pathway is essential for GpTx-1-71-induced spinal and peripheral analgesia in inflammatory pain. PERSPECTIVE: This article presents a possible pharmacological mechanism underlying NaV1.7 blocker-induced analgesia in inflammatory pain, which helps us to better understand and develop venom-based painkillers for incurable pain.

The most commonly used opioid analgesics are limited by their severe side-effects in the clinical treatment of pain. Preliminary reports indicate that the combination of classical opioids and N/OFQ receptor (NOP) ligands may be an effective strategy to reduce unwanted side-effects and improve antinociception. But the interaction of these two receptor ligands in pain regulation at the peripheral level remains unclear. In this study, the antinociception of a designed amide analogue of the mu opioid receptor (MOP) peptide agonist DAMGO, DAMGO-NH2, and its antinociceptive interaction with the peripherally limited NOP peptide agonist NOP01 was investigated in two mouse models of formalin pain. Our results showed that DAMGO-NH2 acted as a MOP agonist in in vitro functional assays. Moreover, local subcutaneous or intraplantar injection of DAMGO-NH2 exerted dose-related antinociception in both phases of the formalin orofacial and intraplantar pain, which could be mediated by the classical opioid receptor. Peripheral but not central pretreatment with the peripherally restricted opioid antagonist naloxone methiodide inhibited local DAMGO-NH2-induced antinociception, supporting the involvement of the peripheral opioid receptor in local DAMGO-NH2-induced antinociception. Furthermore, co-administration of the inactive doses of DAMGO-NH2 and NOP01 produced effective antinociception. More importantly, isobolographic analysis indicates that the combination of DAMGO-NH2 and NOP01 elicited supra-additive antinociception in these two models of formalin pain. In addition, the combination of DAMGO-NH2 and NOP01 did not change motor function of mice in rotarod test. In conclusion, these data suggest that peripheral DAMGO-NH2 and particularly its combination therapy with NOP01 may be effective for pain management.

Burn injury-induced pain (BIP) is an extremely complicated condition usually resistant to analgesic drugs, while its pathogenesis remains unknown. Considerable attention has been attracted to elucidate the glial mechanisms in chronic pain. In this study, we initiatively used a mouse model of second-degree BIP to investigate the underlying non-neuronal mechanisms at the spinal cord level. Our behavioral results showed that hind-paw burn injury caused persistent allodynia and hyperalgesia for 2 weeks in mice. Further studies revealed that both microglia and astrocytes activated in a spatially-and temporally-dependent manner in spinal cord after burn injury. In addition, the phosphorylated p38 mitogen-activated protein kinase (MAPK)-mediated tumor necrosis factor (TNF) release in spinal microglia is essentially attributed to the early stage of BIP, while the c-Jun N terminal kinase (JNK) MAPK-dependent chemokine CXCL1 expression is mainly involved in the maintenance of pain hypersensitivity. Most strikingly, burn injury-induced pain symptoms and the activation of astrocytes were significantly suppressed by TNF inhibitor Thalidomide. On the contrary, intrathecal injection of TNF caused apparent pain hypersensitivity, accompanied by the activation of astrocytes and the upregulation of CXCL1 via the JNK MAPK signaling pathway, indicating that TNF is the key cytokine in the interaction between microglia and astrocytes at the spinal level. Moreover, treatment with the CXCR2 receptor antagonist SB225002 to block the biological activities of CXCL1 significantly attenuated the mechanical allodynia and thermal hyperalgesia in this BIP model. Taken together, this study indicates that intervention of glial pathways provides a new perspective in the management of BIP.

Grazing is the main utilization of native grassland, and forage fungal disease is one of the limiting factors of grassland productivity. The present research in the Hulunber meadow steppe grassland was conducted to investigate the responses of the dominant plant Leymus chinensis (Trin.) to beef cattle grazing, rust, and their interaction influence. Six grazing intensity treatments with three replicates were established. The response of L. chinensis to grazing and rust was systematically studied for two consecutive years. The main findings were that grazing and rust had significant effects (p < 0.05) on the growth and nutrient elements content of L. chinensis. Compared with the 0 cattle ha−1 treatment, the dry matter of L. chinensis in the 0.42, 0.63, and 1.67 cattle ha−1 treatments decreased by 42.2%, 90.5%, and 339.5%, respectively. Compared with non-infected plants, dry matter of rust-infected L. chinensis plants decreased by 45.6%. The N:C and P:C ratios of rust-infected plants were lower than in non-infected plants, and positively correlated with their relative growth rates. Therefore, we concluded that the growth rate hypothesis still applied in L. chinensis under the interactive effects of grazing and disease. Additionally, grazing can alleviate the loss of dry matter caused by disease.

Orofacial pain is one of the most common medical challenges. A preliminary report indicates that the NOP receptor may act as a therapeutic target in orofacial pain. Previous studies have shown that [(pF)Phe4, Aib7, Aib11, Arg14, Lys15]N/OFQ-NH2 (NOP01) functions as a potent NOP receptor peptide agonist. This work aims to investigate the antinociception of NOP01 and its possible action mechanisms in a formalin-induced mouse orofacial pain model at different levels. Our results demonstrated that local, intraperitoneal (i.p.) or intrathecal (i.t.) injection of NOP01 produced dose-related antinociception in both phases of the formalin pain, which could be inhibited by the NOP receptor antagonist but not the classical opioid receptor antagonist. Furthermore, the antinociception induced by systemic NOP01 was blocked by local but not spinal pretreatment with the NOP receptor antagonist, suggesting the involvement of the peripheral NOP receptor in NOP01-induced systemic antinociception. Moreover, local injection of NOP01 markedly suppressed the expression of c-Fos protein induced by formalin in ipsilateral trigeminal ganglion (TG) neurons. In conclusion, this work suggests that NOP01 exerts significant antinociception on orofacial pain at both peripheral and spinal levels via the NOP receptor. Notably, NOP01 cannot readily penetrate the blood-brain barrier. Thus, NOP01 may behave as a potential compound for developing peripherally restricted analgesics.

Burn injury is one of the main causes of mortality worldwide and frequently associated with severe and longlasting pain that compromises the quality of patient life. Several studies have shown that the mu-opioid system plays an important role in burn pain relief. In this study, we investigated the spinal antinociception induced by the endogenous mu-opioid receptor (MOR) agonists endomorphins and explored their mechanisms of actions in burn injury-induced pain model. Our results showed that intrathecal injection of endomorphin-1 and -2 dosedependently attenuated mechanical allodynia and thermal hyperalgesia via the mu-opioid receptor in mice on day 3 after burn injury, which was consistent with the data obtained from the mu-opioid receptor knockout mice. Western blot showed that the phosphorylation levels of extracellular signal-regulated kinase1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) in ipsilateral spinal cord tissues were significantly upregulated after burn injury. Intrathecal injection of endomorphins selectively inhibited the activation of p38 MAPK on day 3 after burn injury via the mu-opioid receptor. Further studies found that repeated application of the specific p38 MAPK inhibitor SB203580 dose-dependently inhibited burn-injury pain, as well as the activation of spinal p38 MAPK. Taken together, our present study demonstrates that intrathecal injection of endomorphins attenuates burn-injury pain in male mice by affecting the spinal activation of p38 MAPK via the mu-opioid receptor.

2D Sb2MoO6 was designed as a Z-scheme heterojunction in the construction of the 2D hybridized g-C3N4/Sb2MoO6/Bi2O3 dual Z-scheme structure.

不孕不育症患者之间的个体差异性较大,中医以辩证的角度对患者实施治疗,患者的中药治疗方法也有所不同,以辨证施治的方法对不孕不育症患者进行治疗,均能够获得较为可观的疗效.本文从不孕不育症的发病原因、中医临床辨证治愈不孕不育症的治疗方法和治疗效果等多方面进行阐述,目的是为了给有关中医临床辨证治愈不孕不育症实践及治疗研究提供一定的参考.

目的:比较子宫内膜息肉不孕症患者采用不同宫腔镜手术治疗的效果.方法:抽取80例患者的进行观察,对患者分别实施不同的宫腔镜手术治疗方法,对比分析临床相关指标.结果:在相关指标方面,电切术组优越性较强,(P＜0.05).结论:子宫内膜息肉不孕症患者采用不同宫腔镜手术的治疗效果相当,都能够获得可观的疗效,但是电切术在减少月经量和减少疾病复发方面有一定的优势,临床可以采纳该治疗方法.