Astroglial morphological changes in periaqueductal grey in different pain and itch mice models.

BACKGROUND: The periaqueductal gray (PAG) plays a well-established pivotal role in the descending pain modulatory circuit. The objective of this study was to investigate morphological changes in the astroglia in models that are commonly used in pain and itch studies. METHODS: Five different mouse models of pain, as well as two models of chronic itch, were established using complete Freund's adjuvant (CFA), spared nerve injury (SNI), bone cancer pain (BCP), cisplatin (CIS), and paclitaxel (PTX) for pain, and diphenylcyclopropenone (DCP) and acetone and diethyl ether followed by water (AEW) for chronic itch. von Frey tests and video recordings were employed to assess pain and itching behaviors. The immunofluorescence of S100ß, pSTAT3, and glial fibrillary acidic protein (GFAP) was examined. Two- and three-dimensional studies were used to evaluate changes in astrocyte morphology. RESULTS: Significant scratching was caused by DCP and AEW, whereas the administration of CFA, SNI, BCP, CIS, and PTX produced clear mechanical allodynia. The expression of GFAP in the lPAG/vlPAG was upregulated in CFA, SNI, BCP, CIS, PTX, and DCP mice but decreased in AEW mice. According to Sholl analysis, CFA, SNI, PTX, and BCP mice showed substantially higher astrocyte intersections in the vlPAG, whereas CFA, SNI, BCP, CIS, and DCP mice presented longer peak lengths. In three-dimensional analysis, CFA, SNI, PTX, and DCP mice showed increased astrocyte surface areas, while CIS and AEW mice showed both reduced surface areas and/or volumes of astrocytes. CONCLUSION: The findings showed that different pain and itching conditions have different astrocyte morphologies, and these variations in morphological changes help to explain the pathophysiology of these conditions.

A regulatory role of the medial septum in the chloroquine-induced acute itch through local GABAergic system and GABAergic pathway to the anterior cingulate cortex.

Itch, a common somatic sensation, serves as a crucial protective system. Recent studies have unraveled the neural mechanisms of itch at peripheral, spinal cord as well as cerebral levels. However, a comprehensive understanding of the central mechanism governing itch transmission and regulation remains elusive. Here, we report the role of the medial septum (MS), an integral component of the basal forebrain, in modulating the acute itch processing. The increases in c-Fos+ neurons and calcium signals within the MS during acute itch processing were observed. Pharmacogenetic activation manipulation of global MS neurons suppressed the scratching behaviors induced by chloroquine or compound 48/80. Microinjection of GABA into the MS or pharmacogenetic inhibition of non-GABAergic neurons markedly suppressed chloroquine-induced scratching behaviors. Pharmacogenetic activation of the MS-ACC GABAergic pathway attenuated chloroquine-induced acute itch. Hence, our findings reveal that MS has a regulatory role in the chloroquine-induced acute itch through local increased GABA to inhibit non-GABAergic neurons and the activation of MS-ACC GABAergic pathway.

Analgesic Effect of Exercise on Neuropathic Pain via Regulating the Complement Component 3 of Reactive Astrocytes.

BACKGROUND: Exercise has been proven to be an efficient intervention in attenuating neuropathic pain. However, the underlying mechanisms that drive exercise analgesia remain unknown. In this study, we aimed to examine the role of complement component 3 (C3) in neuropathic pain and whether antinociceptive effects are produced by exercise via regulating C3 in mice. METHODS: In this study, using a spared nerve injury (SNI)-induced neuropathic pain mice model, C57BL/6J mice were divided into 3 groups: Sham mice, SNI mice, and SNI + Exercise (Ex) mice with 30-minute low-intensity aerobic treadmill running (10 m/min, no inclination). Paw withdrawal threshold; thermal withdrawal latency; and glial fibrillary acidic protein, C3, tumor necrosis factor-α, and interlukin-1ß expression in the spinal cord were monitored. C3 knockout (KO) mice were further used to verify the role of C3 in neuropathic pain. RESULTS: von Frey test, acetone test, and CatWalk gait analysis revealed that treadmill exercise for 4 weeks reversed pain behaviors. In addition, exercise reduced astrocyte reactivity (SNI mean = 14.5, 95% confidence interval [CI], 12.7-16.3; SNI + Ex mean = 10.3, 95% CI, 8.77-11.9, P = .0003 SNI + Ex versus SNI) and inflammatory responses in the spinal cord after SNI. Moreover, it suppressed the SNI-induced upregulation of C3 expression in the spinal cord (SNI mean = 5.46, 95% CI, 3.39-7.53; SNI + Ex mean = 2.41, 95% CI, 1.42-3.41, P = .0054 SNI + Ex versus SNI in Western blot). C3 deficiency reduced SNI-induced pain and spinal astrocyte reactivity (wild type mean = 7.96, 95% CI, 6.80-9.13; C3 KO mean = 5.98, 95% CI, 5.14-6.82, P = .0052 C3 KO versus wild type). Intrathecal injection of recombinant C3 (rC3) was sufficient to produce mechanical (rC3-Ex mean = 0.77, 95% CI, 0.15-1.39; rC3 mean = 0.18, 95% CI, -0.04 to 0.41, P = .0168 rC3-Ex versus rC3) and cold (rC3-Ex mean = 1.08, 95% CI, 0.40-1.77; rC3 mean = 3.46, 95% CI, 1.45-5.47, P = .0025 rC3-Ex versus rC3) allodynia in mice. Importantly, exercise training relieved C3-induced mechanical and cold allodynia, and the analgesic effect of exercise was attenuated by a subeffective dose of intrathecal injection of C3. CONCLUSIONS: Overall, these results suggest that exercise suppresses neuropathic pain by regulating astroglial C3 expression and function, thereby providing a rationale for the analgesic effect of exercise as an acceptable alternative approach for treating neuropathic pain.

Spinal Neuronal miR-124 Inhibits Microglial Activation and Contributes to Preventive Effect of Electroacupuncture on Chemotherapy-Induced Peripheral Neuropathy in Mice.

Chemotherapy-induced peripheral neuropathy (CIPN) is a persistent and irreversible side effect of antineoplastic agents. Patients with CIPN usually show chronic pain and sensory deficits with glove-and-stocking distribution. However, whether spinal neuronal microRNA (miR)-124 is involved in cisplatin-induced peripheral neuropathy remains to be studied. In this study, miR-124 was significantly reduced in the spinal dorsal horn in CIPN mice. Overexpression of neuronal miR-124 induced by injecting adeno-associated virus with neuron-specific promoter into the spinal cord of mice prevented the development of mechanical allodynia, sensory deficits, and the loss of intraepidermal nerve fibers induced by cisplatin. Meanwhile, cisplatin-induced M1 microglia activation and the release of proinflammatory cytokines were significantly inhibited by overexpression of neuronal miR-124. Furthermore, electroacupuncture (EA) treatment upregulated miR-124 expression in the spinal dorsal horn of CIPN mice. Interestingly, downregulation of spinal neuronal miR-124 significantly inhibited the regulatory effect of EA on CIPN and microglia activity as well as spinal neuroinflammation induced by cisplatin. These results demonstrate that spinal neuronal miR-124 is involved in the prevention and treatment of EA on cisplatin-induced peripheral neuropathy in mice. Our findings suggest that spinal neuronal miR-124 might be a potential target for EA effect, and we provide, to our knowledge, a new experimental basis for EA prevention of CIPN.

Foxg1 Modulation of the Prkcd Gene in the Lateral Habenula Mediates Trigeminal Neuralgia-Associated Anxiety-Like Behaviors in Mice.

Trigeminal Neuralgia (TN) is a debilitating disorder frequently accompanied by mood complications such as depression and anxiety. The current study sought to elucidate the molecular underpinnings that contribute to the pathogenesis of TN and its associated anxiety. Employing a partial transection of the infraorbital nerve (pT-ION) in a murine model, we successfully induced sustained primary and secondary orofacial allodynia alongside anxiety-like behavioral manifestations. Transcriptome-wide gene microarray analyses revealed a marked upregulation of Foxg1 subsequent to pT-ION. Targeted knockdown of Foxg1, achieved through bilateral microinjection of adeno-associated virus harboring Foxg1-specific shRNA into the lateral habenula (LHb), resulted in a significant attenuation of both orofacial pain and anxiety-like behaviors. Subsequent RNA sequencing implicated Prkcd as a downstream effector gene modulated by Foxg1. Pharmacological inhibition of protein kinase C delta, encoded by Prkcd, within the LHb markedly ameliorated pT-ION-induced symptomatology. The dual luciferase assay revealed that Foxg1 substantially enhances the transcriptional activity of the Prkcd gene. Collectively, these findings indicate that trigeminal nerve injury leads to Foxg1 upregulation in the LHb, which in turn elevates the expression of Prkcd, culminating in the manifestation of orofacial pain and anxiety-like behaviors. This work offers promising therapeutic targets and a conceptual framework for the clinical management of TN and its psychological comorbidities.

Xanthotoxol relieves itch in mice via suppressing spinal GRP/GRPR signaling.

Although pruritus, commonly known as itch, is a common and debilitating symptom associated with various skin conditions, there is a lack of effective therapies available. Xanthotoxol (XAN), a biologically active linear furocoumarin, shows potential in the treatment of various neurological disorders. In this study, we discovered that administering XAN either through intraperitoneal or intrathecal injections effectively reduced scratching behavior induced by compound 48/80 or chloroquine. Importantly, XAN also substantially alleviates chronic itch in dry skin and allergic contact dermatitis mice. Substantial progress has highlighted the crucial role of gastrin-releasing peptide (GRP)-gastrin-releasing peptide receptor (GRPR) signaling in the dorsal spinal cord in transmitting various types of itch. Our behavior tests revealed that XAN significantly alleviated scratching behaviors induced by intrathecal administration of GRP or GRPR agonist bombesin. Furthermore, XAN reduced the activation of neurons in the spinal cord caused by intrathecal administration of GRP in mice. Moreover, XAN attenuates the activation of spinal GRPR-positive neurons in itchy mice. These findings suggest that XAN mitigates itch in mice by suppressing spinal GRP/GRPR signaling, thereby establishing XAN as a promising therapeutic option for treating pruritus.

IL-33/ST2 Signaling in the Pathogenesis of Chronic Pain and Itch.

Interleukin-33 (IL-33) is an inflammatory factor with an extensive range of biological effects and pleiotropic roles in diseases. Evidence suggests that IL-33 and its receptor ST2 play a pivotal role in chronic pain and itch at the level of primary sensory neurons, the spinal cord, and the brain. In this review, we outline an evolving understanding of the roles and mechanisms of IL-33 in chronic pathological pain, including inflammatory, neuropathic, and cancer, and chronic pruritus, such as allergic contact dermatitis, atopic dermatitis, and dry skin. Understanding the key roles of IL-33/ST2 signaling may provide exciting insights into the mechanisms of chronic pain and itch and lead to new clues for therapeutic approaches to the resolution of chronic pain and itch.

Antipruritic effects of geraniol on acute and chronic itch via modulating spinal GABA/GRPR signaling.

BACKGROUND AND PURPOSE: Itch (pruritus) is a common unpleasant feeling, often accompanied by the urge of scratching the skin. It is the main symptom of many systemic and skin diseases, which can seriously affect the patient's quality of life. Geraniol (GE; trans-3,7-dimethyl-2,6-octadien-1-ol) is a natural monoterpene with diverse effects, including anti-inflammatory, antioxidant, neuroprotective, anti-nociceptive, and anticancer properties. The study aims to examine the effects of GE on acute and chronic itch, and explore the underlying mechanisms. METHODS: Acute itch was investigated by using Chloroquine and compound 48/80 induced model, followed by manifestation of diphenylcyclopropenone (DCP)-induced allergic contact dermatitis and the acetone-ether-water (AEW)-induced dry skin model in mice. The scratching behavior, skin thickness, c-Fos expression, and GRPR protein expression in the spinal cord were subsequently monitored and evaluated by behavioral tests as well as pharmacological and pharmacogenetic technologies. RESULTS: Dose-dependent intraperitoneal injection of GE alleviated the acute itch, induced by chloroquine and compound 48/80, as well as increased the spinal c-Fos expression. Intrathecal administration of GE suppressed the GABAA receptor inhibitor bicuculline-induced itch, GRP-induced itch, and the GABAergic neuron inhibition-induced itch. Furthermore, the subeffective dose of bicuculline blocked the anti-pruritic effect of GE on the chloroquine and compound 48/80 induced acute itch. GE also attenuated DCP and AEW-induced chronic itch, as well as the increase of spinal GRPR expression in DCP mice. CONCLUSION AND IMPLICATIONS: GE alleviates both acute and chronic itch via modulating the spinal GABA/GRPR signaling in mice. Findings of this study reveal that GE may provide promising therapeutic options for itch management. Also, considering the pivotal role of essential oils in aromatherapy, GE has great application potential in aromatherapy for treating skin diseases, and especially the skin with severe pruritus.

Interleukin-33 Potentiates TGF-ß Signaling to Regulate Intestinal Stem Cell Regeneration After Radiation Injury.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal radiation injury. Accumulating evidence indicates that the interleukin family members play critical roles in intestinal stem-cell-mediated epithelial regeneration. However, little is known about the relationship between interleukin 33 (IL-33)/ST2 axis and intestinal regeneration after radiation injury. We demonstrate here that IL-33 expression significantly increased after radiation treatment. Deficiency of IL-33/ST2 promotes intestinal epithelial regeneration, resulting in a reduction of mortality during radiation-induced intestine injury. Using ex vivo organoid cultures, we show that recombinant IL-33 promotes intestinal stem cell differentiation. Mechanistically, the effects of IL-33 were mediated by activation of transforming growth factor-ß signaling. Our findings reveal a fundamental mechanism by which IL-33 is able to regulate the intestinal crypt regeneration after tissue damage.

Crosstalk between Interleukin-1 Receptor-Like 1 and Transforming Growth Factor-ß Receptor Signaling Promotes Renal Fibrosis

IL-33, a member of the IL-1 family, acts as an alarmin in immune response. Epithelial-mesenchymal transition and transforming growth factor-ß (TGF-ß)­induced fibroblast activation are key events in the development of renal interstitial fibrosis. The current study found increased expression of IL-33 and interleukin-1 receptor-like 1 (IL1RL1, alias ST2), the receptor for IL-33, in human fibrotic renal tissues. In addition, IL-33­ or ST2-deficient mice showed significantly reduced levels of fibronectin, α-smooth muscle actin, and vimentin, and increased E-cadherin levels. In HK-2 cells, IL-33 promotes the phosphorylation of the TGF-ß receptor (TGF-ßR), Smad2, and Smad3, and the production of extracellular matrix (ECM), with reduced expression of E-cadherin. Blocking TGF-ßR signaling or suppressing ST2 expression impeded Smad2 and Smad3 phosphorylation, thereby reducing ECM production, suggesting that IL-33­induced ECM synthesis requires cooperation between the two pathways. Mechanistically, IL-33 treatment induced a proximate interaction between ST2 and TGF-ßRs, activating downstream Smad2 and Smad3 for ECM production in renal epithelial cells. Collectively, this study identified a novel and essential role for IL-33 in promoting TGF-ß signaling and ECM production in the development of renal fibrosis. Therefore, targeting IL-33/ST2 signaling may be an effective therapeutic strategy for renal fibrosis.

Tachykinin receptor 3 in the lateral habenula alleviates pain and anxiety comorbidity in mice.

The coexistence of chronic pain and anxiety is a common clinical phenomenon. Here, the role of tachykinin receptor 3 (NK3R) in the lateral habenula (LHb) in trigeminal neuralgia and in pain-associated anxiety was systematically investigated. First, electrophysiological recording showed that bilateral LHb neurons are hyperactive in a mouse model of trigeminal neuralgia made by partial transection of the infraorbital nerve (pT-ION). Chemicogenetic activation of bilateral LHb glutamatergic neurons in naive mice induced orofacial allodynia and anxiety-like behaviors, and pharmacological activation of NK3R in the LHb attenuated allodynia and anxiety-like behaviors induced by pT-ION. Electrophysiological recording showed that pharmacological activation of NK3R suppressed the abnormal excitation of LHb neurons. In parallel, pharmacological inhibition of NK3R induced orofacial allodynia and anxiety-like behavior in naive mice. The electrophysiological recording showed that pharmacological inhibition of NK3R activates LHb neurons. Neurokinin B (NKB) is an endogenous high-affinity ligand of NK3R, which binds NK3R and activates it to perform physiological functions, and further neuron projection tracing showed that the front section of the periaqueductal gray (fPAG) projects NKB-positive nerve fibers to the LHb. Optogenetics combined with electrophysiology recordings characterize the functional connections in this fPAG NKB → LHb pathway. In addition, electrophysiological recording showed that NKB-positive neurons in the fPAG were more active than NKB-negative neurons in pT-ION mice. Finally, inhibition of NKB release from the fPAG reversed the analgesic and anxiolytic effects of LHb Tacr3 overexpression in pT-ION mice, indicating that fPAG NKB → LHb regulates orofacial allodynia and pain-induced anxious behaviors. These findings for NK3R suggest the cellular mechanism behind pT-ION in the LHb and suggest that the fPAG NKB → LHb circuit is involved in pain and anxiety comorbidity. This previously unrecognized pathway might provide a potential approach for relieving the pain and anxiety associated with trigeminal neuralgia by targeting NK3R.

Neuronal toll like receptor 9 contributes to complete Freund's adjuvant-induced inflammatory pain in mice.

Toll like receptor 9 (TLR9) is a critical sensor for danger-associated molecular patterns (DAMPs) and a crucial marker of non-sterile/sterile inflammation among all TLRs. However, the significance of TLR9 in inflammatory pain remains unclear. Here, we subcutaneously injected Complete Freund's adjuvant (CFA) into the plantar surface of the hind paw, to established a mouse model of inflammatory pain, and we examined expression and distribution of TLR9 in this model. There was a significant increase of TLR9 mRNA and reduction of mechanical paw withdrawal threshold in mice intraplantar injected with CFA. By contrast, mechanical paw withdrawal threshold significantly increased in mice treated with TLR9 antagonist ODN2088. Furthermore, TLR9 is found predominantly distributed in the neurons by immunofluorescence experiment. Accordingly, neuronal TLR9 downregulation in the spinal cord prevented CFA-induced persistent hyperalgesia. Overall, these findings indicate that neuronal TLR9 in the spinal cord is closely related to CFA-induced inflammatory pain. It provides a potential treatment option for CFA-induced inflammatory pain by applying TLR9 antagonist.

A dysregulated sebum-microbial metabolite-IL-33 axis initiates skin inflammation in atopic dermatitis.

Microbial dysbiosis in the skin has been implicated in the pathogenesis of atopic dermatitis (AD); however, whether and how changes in the skin microbiome initiate skin inflammation, or vice versa, remains poorly understood. Here, we report that the levels of sebum and its microbial metabolite, propionate, were lower on the skin surface of AD patients compared with those of healthy individuals. Topical propionate application attenuated skin inflammation in mice with MC903-induced AD-like dermatitis by inhibiting IL-33 production in keratinocytes, an effect that was mediated through inhibition of HDAC and regulation of the AhR signaling pathway. Mice lacking sebum spontaneously developed AD-like dermatitis, which was improved by topical propionate application. A proof-of-concept clinical study further demonstrated the beneficial therapeutic effects of topical propionate application in AD patients. In summary, we have uncovered that the dysregulated sebum-microbial metabolite-IL-33 axis might play an initiating role in AD-related skin inflammation, thereby highlighting novel therapeutic strategies for the treatment of AD.

A Simplified Murine Model to Imitate Flexor Tendon Adhesion Formation without Suture.

Peritendinous adhesion (PA) around tendons are daunting challenges for hand surgeons. Tenotomy with various sutures are considered classical tendon repair models (TRM) of tendon adhesion as well as tendon healing. However, potential biomimetic therapies such as anti-adhesion barriers and artificial tendon sheaths to avoid recurrence of PA are sometimes tested in these models without considering tendon healing. Thus, our aim is to create a simplified model without sutures in this study by using three 6 mm longitudinal and parallel incisions called the longitudinal incision model (LCM) in the murine flexor tendon. We found that the adhesion score of LCM has no significant difference to that in TRM. The range of motion (ROM) reveals similar adhesion formation in both TRM and LCM groups. Moreover, mRNA expression levels of collagen I and III in LCM shows no significant difference to that in TRM. The breaking force and stiffness of LCM were significantly higher than that of TRM. Therefore, LCM can imitate flexor tendon adhesion formation without sutures compared to TRM, without significant side effects on biomechanics with an easy operation.

Cytokines and Chemokines Modulation of Itch.

Itch (pruritus) is a common cutaneous symptom widely associated with many skin complaints, and chronic itch can be a severe clinical problem. The onset and perpetuation of itch are linked to cytokines, such as interleukin (IL)-31, IL-4, IL-13, IL-33, thymic stromal lymphopoietin, and tumor necrosis factor-alpha, and chemokines, such as chemokine (C-C motif) ligand 2 and C-X-C motif chemokine ligand 10. This review highlights research that has attempted to determine the attributes of various cytokines and chemokines concerning the development and modulation of itch. Through such research, clinical approaches targeting cytokines and/or chemokines may arise, which may further the development of itch therapeutics.

Very-low-density lipoprotein receptor-enhanced lipid metabolism in pancreatic stellate cells promotes pancreatic fibrosis.

Lipoprotein disorder is a common feature of chronic pancreatitis (CP); however, the relationship between lipoprotein disorder and pancreatic fibrotic environment is unclear. Here, we investigated the occurrence and mechanism of pancreatic stellate cell (PSC) activation by lipoprotein metabolites and the subsequent regulation of type 2 immune responses, as well as the driving force of fibrotic aggressiveness in CP. Single-cell RNA sequencing revealed the heterogeneity of PSCs and identified very-low-density lipoprotein receptor (VLDLR)+ PSCs that were characterized by a higher lipid metabolism. VLDLR promoted intracellular lipid accumulation, followed by interleukin-33 (IL-33) expression and release in PSCs. PSC-derived IL-33 strongly induced pancreatic group 2 innate lymphoid cells (ILC2s) to trigger a type 2 immune response accompanied by the activation of PSCs, eventually leading to fibrosis during pancreatitis. Our findings indicate that VLDLR-enhanced lipoprotein metabolism in PSCs promotes pancreatic fibrosis and highlight a dominant role of IL-33 in this pro-fibrotic cascade.

Geraniol enhances inhibitory inputs to the paraventricular thalamic nucleus and induces sedation in mice.

BACKGROUND: Plant extracts with sedative effects have a long history of clinical use for treating insomnia and epilepsy. Geraniol (GE), a plant-derived acyclic monoterpene, reduces locomotion and prolongs barbiturate-induced anesthesia in rats. However, the mechanisms of GE in sedation remain elusive. PURPOSE: This study aimed to investigate the mechanisms of GE in sedation in mice. METHODS: GE was administered systemically by nebulization and intraperitoneal injection. Open field tests, acute seizure tests, and electroencephalogram (EEG) recordings were performed to examine the sedative effects of GE in mice. The time of loss of the righting reflex and return of the righting reflex were recorded in anesthesia experiments to examine the effect of GE on anesthesia. In vitro c-Fos staining and in vivo fiber photometry recordings were performed to detect the activity change of the paraventricular thalamic nucleus (PVT). Microinjection of GE into PVT and related behavioral tests were performed to confirm that PVT was a critical target for GE. Whole-cell recordings were performed to dissect the effects of GE on PVT neurons via GABAA receptors. Molecular docking was performed to examine the interaction between GE and GABAA receptor subunits. RESULTS: We found that GE reduced locomotion, relieved acute seizures, altered the EEG, and facilitated general anesthesia in mice. Next, we found that GE decreased c-Fos expression and suppressed the calcium activity in PVT. Microinjection of GE into PVT reduced locomotion and facilitated anesthesia. Furthermore, electrophysiology results showed that GE induced dramatic membrane hyperpolarization and suppressed the activity of PVT neurons, mainly by prolonging spontaneous inhibitory postsynaptic currents and inducing tonic inhibitory currents. Molecular docking results indicated that the ß3 subunit might be a potential target for GE. CONCLUSION: By combined using behavioral tests, immunohistochemistry, calcium recording, and electrophysiology, we systematically revealed that GE inhibits PVT and induces sedation in mice. Essential oils have long been considered part of traditional medicine, and they are playing a critical role in aromatherapy. Since GE has a comparatively ideal safety property and multiple delivery methods, GE has great application potential in aromatherapy. Our study also provides a potential candidate for further development of sedatives and anaesthetics.

Neuronal GRK2 regulates microglial activation and contributes to electroacupuncture analgesia on inflammatory pain in mice.

BACKGROUND: G protein coupled receptor kinase 2 (GRK2) has been demonstrated to play a crucial role in the development of chronic pain. Acupuncture is an alternative therapy widely used for pain management. In this study, we investigated the role of spinal neuronal GRK2 in electroacupuncture (EA) analgesia. METHODS: The mice model of inflammatory pain was built by subcutaneous injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the hind paws. The mechanical allodynia of mice was examined by von Frey test. The mice were subjected to EA treatment (BL60 and ST36 acupuncture points) for 1 week. Overexpression and downregulation of spinal neuronal GRK2 were achieved by intraspinal injection of adeno associated virus (AAV) containing neuron-specific promoters, and microglial activation and neuroinflammation were evaluated by real-time PCR. RESULTS: Intraplantar injection with CFA in mice induced the decrease of GRK2 and microglial activation along with neuroinflammation in spinal cord. EA treatment increased the spinal GRK2, reduced neuroinflammation, and significantly decreased CFA-induced mechanical allodynia. The effects of EA were markedly weakened by non-cell-specific downregulation of spinal GRK2. Further, intraspinal injection of AAV containing neuron-specific promoters specifically downregulated neuronal GRK2, and weakened the regulatory effect of EA on CFA-induced mechanical allodynia and microglial activation. Meanwhile, overexpression of spinal neuronal GRK2 decreased mechanical allodynia. All these indicated that the neuronal GRK2 mediated microglial activation and neuroinflammation, and subsequently contributed to CFA-induced inflammatory pain. CONCLUSION: The restoration of the spinal GRK2 and subsequent suppression of microglial activation and neuroinflammation might be an important mechanism for EA analgesia. Our findings further suggested that the spinal GRK2, especially neuronal GRK2, might be the potential target for EA analgesia and pain management, and we provided a new experimental basis for the EA treatment of pain.

Neuropilin-1 mediates lung tissue-specific control of ILC2 function in type 2 immunity.

Group 2 innate lymphoid cells (ILC2s) are highly heterogeneous tissue-resident lymphocytes that regulate inflammation and tissue homeostasis in health and disease. However, how these cells integrate into the tissue microenvironment to perform tissue-specific functions is unclear. Here, we show neuropilin-1 (Nrp1), which is induced postnatally and sustained by lung-derived transforming growth factor beta-1 (TGFß1), is a tissue-specific marker of lung ILC2s. Genetic ablation or pharmacological inhibition of Nrp1 suppresses IL-5 and IL-13 production by ILC2s and protects mice from the development of pulmonary fibrosis. Mechanistically, TGFß1-Nrp1 signaling enhances ILC2 function and type 2 immunity by upregulating IL-33 receptor ST2 expression. These findings identify Nrp1 as a tissue-specific regulator of lung-resident ILC2s and highlight Nrp1 as a potential therapeutic target for pulmonary fibrosis.

Spinal Neuronal GRK2 Contributes to Preventive Effect by Electroacupuncture on Cisplatin-Induced Peripheral Neuropathy in Mice.

BACKGROUND: The main symptoms of chemotherapy-induced peripheral neuropathy (CIPN) include pain and numbness. Neuronal G protein-coupled receptor kinase 2 (GRK2) plays an important role in various pain models. Cisplatin treatment can induce the activation of proinflammatory microglia in spinal cord. The purpose of this study was to investigate the role of spinal neuronal GRK2 in cisplatin-induced CIPN and in the prevention of CIPN by electroacupuncture (EA). METHODS: The pain and sensory deficit behaviors of mice were examined by von Frey test and adhesive removal test. The expression of neuronal GRK2 in the spinal cord is regulated by intraspinal injection of adeno-associated virus (AAV) containing neuron-specific promoters. The protein levels of GRK2, triggering receptor expressed on myeloid cells 2 (TREM2), and DNAX-activating protein of 12 kDa (DAP12) in spinal dorsal horn were detected by Western blot, the density of intraepidermal nerve fibers (IENFs) was detected by immunofluorescence, and microglia activation were evaluated by real-time polymerase chain reaction (PCR). RESULTS: In this study, cisplatin treatment led to the decrease of GRK2 expression in the dorsal horn of spinal cord. Overexpression of neuronal GRK2 in spinal cord by intraspinal injection of an AAV vector expressing GRK2 with human synapsin (hSyn) promotor significantly inhibited the loss of IENFs and alleviated the mechanical pain and sensory deficits induced by cisplatin. Real-time PCR analysis showed that the overexpression of neuronal GRK2 significantly inhibited the messenger RNA (mRNA) upregulation of proinflammatory cytokine interleukin (IL)-1ß, IL-6, inducible nitric oxide synthase (iNOS), and M1 microglia marker cluster of differentiation (CD)16 induced by cisplatin. Furthermore, the TREM2 and DAP12, which has been demonstrated to play a role in microglia activation and in the development of CIPN, were also downregulated by overexpression of neuronal GRK2 in this study. Interestingly, preventive treatment with EA completely mimics the effect of overexpression of neuronal GRK2 in the spinal cord in this mouse model of cisplatin-induced CIPN. EA increased GRK2 level in spinal dorsal horn after cisplatin treatment. Intraspinal injection of AAV vector specifically downregulated neuronal GRK2, completely reversed the regulatory effect of EA on CIPN and microglia activation. All these indicated that the neuronal GRK2 mediated microglial activation contributed to the process of CIPN. CONCLUSIONS: Neuronal GRK2 in the spinal cord contributed to the preventive effect of EA on CIPN. The neuronal GRK2 may be a potential target for CIPN intervention.