RSK1 promotes mammalian axon regeneration by inducing the synthesis of regeneration-related proteins.

In contrast to the adult mammalian central nervous system (CNS), the neurons in the peripheral nervous system (PNS) can regenerate their axons. However, the underlying mechanism dictating the regeneration program after PNS injuries remains poorly understood. Combining chemical inhibitor screening with gain- and loss-of-function analyses, we identified p90 ribosomal S6 kinase 1 (RSK1) as a crucial regulator of axon regeneration in dorsal root ganglion (DRG) neurons after sciatic nerve injury (SNI). Mechanistically, RSK1 was found to preferentially regulate the synthesis of regeneration-related proteins using ribosomal profiling. Interestingly, RSK1 expression was up-regulated in injured DRG neurons, but not retinal ganglion cells (RGCs). Additionally, RSK1 overexpression enhanced phosphatase and tensin homolog (PTEN) deletion-induced axon regeneration in RGCs in the adult CNS. Our findings reveal a critical mechanism in inducing protein synthesis that promotes axon regeneration and further suggest RSK1 as a possible therapeutic target for neuronal injury repair.

The metabolomic profiling identifies N, N-dimethylglycine as a facilitator of dorsal root ganglia neuron axon regeneration after injury.

Identifying novel molecules involved in axon regeneration of neurons in the peripheral nervous system (PNS) will be of benefit in obtaining a therapeutic strategy for repairing axon damage both in the PNS and the central nervous system (CNS). Metabolism and axon regeneration are tightly connected. However, the overall metabolic processes and the landscape of the metabolites in axon regeneration of PNS neurons are uncovered. Here, we used an ultra high performance liquid tandem chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOFMS)-based untargeted metabolomics to analyze dorsal root ganglia (DRG) metabolic characteristics at different time points post sciatic nerve injury and acquired hundreds of differentially changed metabolites. In addition, the results reveal that several metabolic pathways were significantly altered, such as 'Histidine metabolism', 'Glycine serine and threonine metabolism', 'Arginine and proline metabolism', 'taurine and hypotaurine metabolism' and so on. Given metabolite could alter a cell's or an organism's phenotype, further investigation demonstrated that N, N-dimethylglycine (DMG) has a promoting effect on the regenerative ability post injury. Overall, our data may serve as a resource useful for further understanding how metabolites contribute to axon regeneration in DRG during sciatic nerve regeneration and suggest DMG may be a candidate drug to repair nerve injury.

Promoting AMPK/SR-A1-mediated clearance of HMGB1 attenuates chemotherapy-induced peripheral neuropathy.

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) is a serious side effect of chemotherapy with poorly understood mechanisms and few treatments. High-mobility group box 1 (HMGB1)-induced neuroinflammation is the main cause of CIPN. Here, we aimed to illustrate the role of the macrophage scavenger receptor A1 (SR-A1) in HMGB1 clearance and CIPN resolution. METHODS: Oxaliplatin (L-OHP) was used to establish a CIPN model. Recombinant HMGB1 (rHMGB1) (his tag) was used to evaluate the phagocytosis of HMGB1 by macrophages. RESULTS: In the clinic, HMGB1 expression and MMP-9 activity were increased in the plasma of patients with CIPN. Plasma HMGB1 expression was positively correlated with the cumulative dose of L-OHP and the visual analog scale. In vitro, engulfment and degradation of rHMGB1 increased and inflammatory factor expression decreased after AMP-activated protein kinase (AMPK) activation. Neutralizing antibodies, inhibitors, or knockout of SR-A1 abolished the effects of AMPK activation on rHMGB1 engulfment. In vivo, AMPK activation increased SR-A1 expression in the dorsal root ganglion, decreased plasma HMGB1 expression and MMP-9 activity, and attenuated CIPN, which was abolished by AMPK inhibition or SR-A1 knockout in the CIPN mice model. CONCLUSION: Activation of the AMPK/SR-A1 axis alleviated CIPN by increasing macrophage-mediated HMGB1 engulfment and degradation. Therefore, promoting HMGB1 clearance may be a potential treatment strategy for CIPN. Video abstract.

AMPK-autophagy-mediated inhibition of microRNA-30a-5p alleviates morphine tolerance via SOCS3-dependent neuroinflammation suppression.

BACKGROUND: The development of morphine tolerance is a clinical challenge for managing severe pain. Studies have shown that neuroinflammation is a critical aspect for the development of analgesic tolerance. We found that AMPK-autophagy activation could suppress neuroinflammation and improve morphine tolerance via the upregulation of suppressor of cytokine signaling 3 (SOCS3) by inhibiting the processing and maturation of microRNA-30a-5p. METHODS: CD-1 mice were utilized for the tail-flick test to evaluate morphine tolerance. The microglial cell line BV-2 was utilized to investigate the mechanism of AMPK-autophagy-mediated posttranscriptional regulation of SOCS3. Proinflammatory cytokines were measured by western blotting and real-time PCR. The levels of SOCS3 and miRNA-processing enzymes were evaluated by western blotting, real-time PCR and immunofluorescence staining. RESULTS: Based on experimental verification, miRNA-30a-5p could negatively regulate SOCS3. The AMPK activators AICAR, resveratrol and metformin downregulated miRNA-30a-5p. We found that AMPK activators specifically inhibited the processing and maturation of miRNA-30a-5p in microglia by degrading DICER and AGO2 via autophagy. Furthermore, a miRNA-30a-5p inhibitor significantly improved morphine tolerance via upregulation of SCOS3 in mice. It markedly increased the level of SOCS3 in the spinal cord of mice and subsequently inhibited morphine-induced phosphorylation of NF-κB p65. In addition, a miRNA-30a-5p inhibitor decreased the levels of IL-1ß and TNF-α caused by morphine in microglia. CONCLUSION: AMPK-autophagy activation suppresses neuroinflammation and improves morphine tolerance via the upregulation of SOCS3 by inhibiting miRNA-30a-5p.

High mobility group box-1-toll-like receptor 4-phosphatidylinositol 3-kinase/protein kinase B-mediated generation of matrix metalloproteinase-9 in the dorsal root ganglion promotes chemotherapy-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a significant side effect of chemotherapeutics. The mechanisms of CIPN remain substantially unidentified, although inflammation-induced peripheral sensitization has been indicated as an important factor. Here, we aimed to illustrate the role of the matrix metalloproteinase (MMP)-9-related signaling pathway in the process of CIPN. Oxaliplatin (L-OHP) was administered to mice to establish the CIPN model. Gelatin zymography was used to measure MMP-9/2 activities. Western blotting and immunohistochemistry were used to measure the expression of high-mobility group box-1 (HMGB-1), calcitonin gene-related peptide and ionized calcium-binding adapter molecule 1. Mechanical withdrawal was measured by von Frey hairs testing. Raw 264.7 cells and SH-SY5Y cells were cultured to investigate cell signaling in vitro. Here, we report that L-OHP-induced mechanical pain in mice with significant MMP-9/2 activation in dorsal root ganglion (DRG) neurons. MMP-9 inhibition or knockout alleviated the occurrence of CIPN directly. MMP-9/2 were released from macrophages and neurons in the DRG via the HMGB-1-toll-like receptor 4 (TLR4)-phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) axis, because MMP-9/2 activities could be reduced by macrophage scavengers or PI3Kγ knockout in CIPN mice. The in vitro data revealed that induced MMP-9 activity by recombinant HMGB-1 could be abolished by TLR4, PI3K or Akt inhibitors. Finally, it was shown that N-acetyl-cysteine (NAC) could reduce MMP-9/2 activities and attenuate CIPN effectively and safely. The HMGB-1-TLR4-PI3K/Akt-MMP-9 axis is involved in the crosstalk between macrophages and neurons in the pathological process of CIPN in mice. Direct inhibition of MMP-9 by NAC may be a potential therapeutic regimen for CIPN treatment.

Inhibition of apoptosis signal-regulating kinase by paeoniflorin attenuates neuroinflammation and ameliorates neuropathic pain.

BACKGROUND: Neuropathic pain is a serious clinical problem that needs to be solved urgently. ASK1 is an upstream protein of p38 and JNK which plays important roles in neuroinflammation during the induction and maintenance of chronic pain. Therefore, inhibition of ASK1 may be a novel therapeutic approach for neuropathic pain. Here, we aim to investigate the effects of paeoniflorin on ASK1 and neuropathic pain. METHODS: The mechanical and thermal thresholds of rats were measured using the Von Frey test. Cell signaling was assayed using western blotting and immunohistochemistry. RESULTS: Chronic constrictive injury (CCI) surgery successfully decreased the mechanical and thermal thresholds of rats and decreased the phosphorylation of ASK1 in the rat spinal cord. ASK1 inhibitor NQDI1 attenuated neuropathic pain and decreased the expression of p-p38 and p-JNK. Paeoniflorin mimicked ASK1 inhibitor NQDI1 and inhibited ASK1 phosphorylation. Paeoniflorin decreased the expression of p-p38 and p-JNK, delayed the progress of neuropathic pain, and attenuated neuropathic pain. Paeoniflorin reduced the response of astrocytes and microglia to injury, decreased the expression of IL-1ß and TNF-α, and downregulated the expression of CGRP induced by CCI. CONCLUSIONS: Paeoniflorin is an effective drug for the treatment of neuropathic pain in rats via inhibiting the phosphorylation of ASK1, suggesting it may be effective in patients with neuropathic pain.

Lidocaine alleviates morphine tolerance via AMPK-SOCS3-dependent neuroinflammation suppression in the spinal cord.

BACKGROUND: Morphine tolerance is a clinical challenge, and its pathogenesis is closely related to the neuroinflammation mediated by Toll-like receptor 4 (TLR4). In Chinese pain clinic, lidocaine is combined with morphine to treat chronic pain. We found that lidocaine sufficiently inhibited neuroinflammation induced by morphine and improved analgesic tolerance on the basis of non-affecting pain threshold. METHODS: CD-1 mice were utilized for tail-flick test to evaluate morphine tolerance. The microglial cell line BV-2 was utilized to investigate the mechanism of lidocaine. Neuroinflammation-related cytokines were measured by western blotting and real-time PCR. The level of suppressor of cytokine signaling 3 (SOCS3) and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-related signaling pathway was evaluated by western blotting, real-time PCR, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining. RESULTS: Lidocaine potentiated an anti-nociceptive effect of morphine and attenuated the chronic analgesic tolerance. Lidocaine suppressed morphine-induced activation of microglia and downregulated inflammatory cytokines, interleukin-1ß (IL-1ß), and tumor necrosis factor-alpha (TNF-α) via upregulating SOCS3 by activating AMPK. Lidocaine enhanced AMPK phosphorylation in a calcium-dependent protein kinase kinase ß (CaMKKß)-dependent manner. Furthermore, lidocaine decreased the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and inhibited the nuclear factor-κB (NF-κB) in accordance with the inhibitory effects to TLR4. CONCLUSIONS: Lidocaine as a prevalent local anesthetic suppresses morphine tolerance efficiently. AMPK-dependent upregulation of SOCS3 by lidocaine plays a crucial role in the improvement of analgesic tolerance.

Blocking ATP-sensitive potassium channel alleviates morphine tolerance by inhibiting HSP70-TLR4-NLRP3-mediated neuroinflammation.

BACKGROUND: Long-term use of morphine induces analgesic tolerance, which limits its clinical efficacy. Evidence indicated morphine-evoked neuroinflammation mediated by toll-like receptor 4 (TLR4) - NOD-like receptor protein 3 (NLRP3) inflammasome was important for morphine tolerance. In our study, we investigated whether other existing alternative pathways caused morphine-induced activation of TLR4 in microglia. We focused on heat shock protein 70 (HSP70), a damage-associated molecular pattern (DAMP), which was released from various cells upon stimulations under the control of KATP channel and bound with TLR4-inducing inflammation. Glibenclamide, a classic KATP channel blocker, can improve neuroinflammation by inhibiting the activation of NLRP3 inflammasome. Our present study investigated the effect and possible mechanism of glibenclamide in improving morphine tolerance via its specific inhibition on the release of HSP70 and activation of NLRP3 inflammasome induced by morphine. METHODS: CD-1 mice were used for tail-flick test to evaluate morphine tolerance. The microglial cell line BV-2 and neural cell line SH-SY5Y were used to investigate the pharmacological effects and the mechanism of glibenclamide on morphine-induced neuroinflammation. The activation of microglia was accessed by immunofluorescence staining. Neuroinflammation-related cytokines were measured by western blot and real-time PCR. The level of HSP70 and related signaling pathway were evaluated by western blot and immunofluorescence staining. RESULTS: Morphine induced the release of HSP70 from neurons. The released HSP70 activated microglia and triggered TLR4-mediated inflammatory response, leading to the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) p65 and the activation of NLRP3 inflammasome. Moreover, anti-HSP70 neutralizing antibody partly attenuated chronic morphine tolerance. The secretion of HSP70 was under the control of MOR/AKT/KATP/ERK signal pathway. Glibenclamide as a classic KATP channel blocker markedly inhibited the release of HSP70 induced by morphine and suppressed HSP70-TLR4-NLRP3 inflammasome-mediated neuroinflammation, which consequently attenuated morphine tolerance. CONCLUSIONS: Our study indicated that morphine-induced extracellular HSP70 was an alternative way for the activation of TLR4-NLRP3 in analgesic tolerance. The release of HSP70 was regulated by MOR/AKT/KATP/ERK pathway. Our study suggested a promising target, KATP channel and a new leading compound, glibenclamide, for treating morphine tolerance.

Grape seed-derived procyanidins alleviate gout pain via NLRP3 inflammasome suppression.

BACKGROUND: Gout is one of the common inflammatory arthritis which affects many people for inflicting unbearable pain. Macrophage-mediated inflammation plays an important role in gout. The uptake of monosodium urate (MSU) crystals by macrophages can lead to activation of NOD-like receptors containing a PYD 3 (NLRP3) inflammasome, thus accelerating interleukin (IL)-1ß production. Reactive oxygen species (ROS) promoted development of the inflammatory process through NLRP3 inflammasome. Our study aimed to find a food-derived compound to attenuate gout pain via the specific inhibition of the NLRP3 inflammasome in macrophages. METHODS: CD-1 mice were used to evaluate the degree of pain and the swelling dimension of joints after an intra-articular (IA) MSU injection in the ankle. The murine macrophage cell line Raw 264.7 was used to investigate the effects of procyanidins and the mechanism underlying such effects. Histological analysis was used to measure the infiltration of inflammatory cells. ROS produced from Raw 264.7 cells were evaluated by flow cytometry. Cell signaling was measured by Western blot assay and immunofluorescence. RESULTS: Procyanidins significantly attenuated gout pain and suppressed ankle swelling. Procyanidins also inhibited MSU-induced activation of the NLRP3 inflammasome and increase of IL-1ß. Furthermore, procyanidins decreased ROS levels in Raw 264.7 cells. CONCLUSIONS: Suppression of the NLRP3 inflammasome in macrophages contributes to the amelioration of gout pain by procyanidins.

Procyanidins alleviates morphine tolerance by inhibiting activation of NLRP3 inflammasome in microglia.

BACKGROUND: The development of antinociceptive tolerance following repetitive administration of opioid analgesics significantly hinders their clinical use. Evidence has accumulated indicating that microglia within the spinal cord plays a critical role in morphine tolerance. The inhibitor of microglia is effective to attenuate the tolerance; however, the mechanism is not fully understood. Our present study investigated the effects and possible mechanism of a natural product procyanidins in improving morphine tolerance via its specific inhibition on NOD-like receptor protein3 (NLRP3) inflammasome in microglia. METHODS: CD-1 mice were used for tail-flick test to evaluate the degree of pain. The microglial cell line BV-2 was used to investigate the effects and the mechanism of procyanidins. Reactive oxygen species (ROS) produced from BV-2 cells was evaluated by flow cytometry. Cell signaling was measured by western blot assay and immunofluorescence assay. RESULTS: Co-administration of procyanidins with morphine potentiated its antinociception effect and attenuated the development of acute and chronic morphine tolerance. Procyanidins also inhibited morphine-induced increase of interleukin-1ß and activation of NOD-like receptor protein3 (NLRP3) inflammasome. Furthermore, procyanidins decreased the phosphorylation of p38 mitogen-activated protein kinase, inhibited the translocation of nuclear factor-κB (NF-κB), and suppressed the level of reactive oxygen species in microglia. CONCLUSIONS: Procyanidins suppresses morphine-induced activation of NLRP3 inflammasome and inflammatory responses in microglia, and thus resulting in significant attenuation of morphine antinociceptive tolerance.

Resveratrol suppresses glial activation and alleviates trigeminal neuralgia via activation of AMPK.

BACKGROUND: Glial activation and neuroinflammation in the spinal trigeminal nucleus (STN) play a pivotal role in the genesis and maintenance of trigeminal neuralgia (TN). Resveratrol, a natural compound from grape and red wine, has a potential anti-inflammatory effect. We hypothesized that resveratrol could significantly suppress neuroinflammation in the STN mediated by glial activation and further relieve TN. In this study, we evaluated whether resveratrol could alleviate trigeminal allodynia and explore the mechanism underlying the antinociceptive effect of resveratrol. METHODS: Animals were orally injected with resveratrol after chronic constriction injury (CCI) of the infraorbital nerve. Mechanical thresholds of the affected whisker pad were measured to assess nociceptive behaviors. The STN was harvested to quantify the changing levels of p-NR1, p-PKC, TNF-α, and IL1-ß by western blotting and detect the expression of calcitonin gene-related peptide (CGRP) and c-Fos by immunofluorescence. Glial activation was observed by immunofluorescence and western blotting. Mitogen-activated protein kinase (MAPK) phosphorylation in vivo and in vitro was examined by western blotting. RESULTS: We found that resveratrol significantly attenuated trigeminal allodynia dose-dependently and decreased the increased expression of CGRP and c-Fos in the STN. Additionally, resveratrol showed an inhibitory effect on CCI-evoked astrocyte and microglia activation and reduced production of pro-inflammatory cytokines in the STN. Furthermore, the antinociceptive effect of resveratrol was partially mediated by reduced phosphorylation of MAP kinases via adenosine monophosphate-activated protein kinase (AMPK) activation. CONCLUSIONS: AMPK activation in the STN glia via resveratrol has utility in the treatment of CCI-induced neuroinflammation and further implicates AMPK as a novel target for the attenuation of trigeminal neuralgia.

Activation of Adenosine Monophosphate-activated Protein Kinase Suppresses Neuroinflammation and Ameliorates Bone Cancer Pain: Involvement of Inhibition on Mitogen-activated Protein Kinase.

BACKGROUND: Activation of adenosine monophosphate-activated kinase (AMPK) has been associated with the inhibition of inflammatory nociception and the attenuation of morphine antinociceptive tolerance. In this study, the authors investigated the impact of AMPK activation through resveratrol treatment on bone cancer pain. METHODS: The nociception was assessed by measuring the incidence of foot withdrawal in response to mechanical indentation in rats (n = 8). Cytokine expression was measured using quantitative polymerase chain reaction (n = 8). Cell signalings were assayed by western blot (n = 4) and immunohistochemistry (n = 5). The microglial cell line BV-2, primary astrocytes, and neuron-like SH-SY5Y cells were cultured to investigate the in vitro effects. RESULTS: Resveratrol and 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide, the AMPK activators, significantly attenuated bone cancer pain in rats with tumor cell implantation (TCI; threshold of mechanical withdrawal, resveratrol vs. vehicle: 10.1 ± 0.56 vs. 4.1 ± 0.37; 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide vs. vehicle: 8.2 ± 0.17 vs. 4.1 ± 0.37, mean ± SEM); these effects were reversed by the AMPK inhibitor compound C (compound C vs. resveratrol: 6.2 ± 1.35 vs. 10.1 ± 0.56, mean ± SEM). Resveratrol has an AMPK-dependent inhibitory effect on TCI-evoked astrocyte and microglial activation. The antinociceptive effects of resveratrol were partially mediated by the reduced phosphorylation of mitogen-activated protein kinases and decreased production of proinflammatory cytokines in an AMPK-dependent manner. Furthermore, resveratrol potently inhibited inflammatory factors-mediated protein kinase B/mammalian target of rapamycin signaling in neurons. Acute pain evoked by proinflammatory cytokines in the spinal cord was significantly attenuated by resveratrol. CONCLUSIONS: AMPK activation in the spinal glia by resveratrol may have utility in the treatment of TCI-induced neuroinflammation, and our results further implicate AMPK as a novel target for the attenuation of bone cancer pain.

Unleashing Axonal Regeneration Capacities: Neuronal and Non-neuronal Changes After Injuries to Dorsal Root Ganglion Neuron Central and Peripheral Axonal Branches.

Peripheral nerves obtain remarkable regenerative capacity while central nerves can hardly regenerate following nerve injury. Sensory neurons in the dorsal root ganglion (DRG) are widely used to decipher the dissimilarity between central and peripheral axonal regeneration as axons of DRG neurons bifurcate into the regeneration-incompetent central projections and the regeneration-competent peripheral projections. A conditioning peripheral branch injury facilitates central axonal regeneration and enables the growth and elongation of central axons. Peripheral axonal injury stimulates neuronal calcium influx, alters the start-point chromatin states, increases chromatin accessibility, upregulates the expressions of regeneration-promoting genes and the synthesis of proteins, and supports axonal regeneration. Following central axonal injury, the responses of DRG neurons are modest, resulting in poor intrinsic growth ability. Some non-neuronal cells in DRGs, for instance satellite glial cells, also exhibit diminished injury responses to central axon injury as compared with peripheral axon injury. Moreover, DRG central and peripheral axonal branches are respectively surrounded by inhibitory glial scars generated by central glial cells and a permissive microenvironment generated by Schwann cells and macrophages. The aim of this review is to look at changes of DRG neurons and non-neuronal cells after peripheral and central axon injuries and summarize the contributing roles of both neuronal intrinsic regenerative capacities and surrounding microenvironments in axonal regeneration.

Pyruvate dehydrogenase beta subunit (Pdhb) promotes peripheral axon regeneration by regulating energy supply and gene expression.

Key metabolic enzymes not only regulate Glucose, lipid, amino acid metabolism to serve the cellular energy needs, but also modulate noncanonical or nonmetabolic signaling pathway such as gene expression, cell-cycle progression, DNA repair, apoptosis and cell proliferation in regulating the pathologic progression of disease. However, the role of glycometabolism in peripheral nerve axon regeneration is little known. In this study, we investigated the expression of Pyruvate dehydrogenase E1(PDH), a key enzyme linking glycolysis and the tricarboxylic acid (TCA) cycle, with qRT-PCR and found that pyruvate dehydrogenase beta subunit (Pdhb) is up-regulated at the early stage during peripheral nerve injury. The knockdown of Pdhb inhibits neurite outgrowth of primary DRG neurons in vitro and restrains axon regeneration of sciatic nerve after crush injury. Pdhb overexpression promoting axonal regeneration is reversed by knockdown of Monocarboxylate transporter 2(Mct2), a transporter involved in the transport and metabolism of lactate, indicating Pdhb promoting axon regeneration depends on lactate for energy supply. Given the nucleus-localization of Pdhb, further analysis revealed that Pdhb enhances the acetylation of H3K9 and affecting the expression of genes involved in arachidonic acid metabolism and Ras signaling pathway, such as Rsa-14-44 and Pla2g4a, thereby promoting axon regeneration. Collectively, our data indicates that Pdhb is a positive dual modulator of energy generation and gene expression in regulating peripheral axon regeneration.

Neutrophil extracellular traps as a unique target in the treatment of chemotherapy-induced peripheral neuropathy.

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) is a severe dose-limiting side effect of chemotherapy and remains a huge clinical challenge. Here, we explore the role of microcirculation hypoxia induced by neutrophil extracellular traps (NETs) in the development of CIPN and look for potential treatment. METHODS: The expression of NETs in plasma and dorsal root ganglion (DRG) are examined by ELISA, IHC, IF and Western blotting. IVIS Spectrum imaging and Laser Doppler Flow Metry are applied to explore the microcirculation hypoxia induced by NETs in the development of CIPN. Stroke Homing peptide (SHp)-guided deoxyribonuclease 1 (DNase1) is used to degrade NETs. FINDINGS: The level of NETs in patients received chemotherapy increases significantly. And NETs accumulate in the DRG and limbs in CIPN mice. It leads to disturbed microcirculation and ischemic status in limbs and sciatic nerves treated with oxaliplatin (L-OHP). Furthermore, targeting NETs with DNase1 significantly reduces the chemotherapy-induced mechanical hyperalgesia. The pharmacological or genetic inhibition on myeloperoxidase (MPO) or peptidyl arginine deiminase-4 (PAD4) dramatically improves microcirculation disturbance caused by L-OHP and prevents the development of CIPN in mice. INTERPRETATION: In addition to uncovering the role of NETs as a key element in the development of CIPN, our finding provides a potential therapeutic strategy that targeted degradation of NETs by SHp-guided DNase1 could be an effective treatment for CIPN. FUNDING: This study was funded by the National Natural Science Foundation of China81870870, 81971047, 81773798, 82271252; Natural Science Foundation of Jiangsu ProvinceBK20191253; Major Project of "Science and Technology Innovation Fund" of Nanjing Medical University2017NJMUCX004; Key R&D Program (Social Development) Project of Jiangsu ProvinceBE2019732; Nanjing Special Fund for Health Science and Technology DevelopmentYKK19170.

Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine 5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway.

Opioids, such as morphine, are the most potent drugs used to treat pain. Long-term use results in high tolerance to morphine. High mobility group box-1 (HMGB1) has been shown to participate in neuropathic or inflammatory pain, but its role in morphine tolerance is unclear. In this study, we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days. We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1. HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1ß production by increasing Toll-like receptor 4 receptor expression in microglia, thereby inducing morphine tolerance. Glycyrrhizin, an HMGB1 inhibitor, markedly attenuated chronic morphine tolerance in the mouse model. Finally, compound C (adenosine 5'-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin (heme oxygenase-1 inhibitor) alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1ß production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tolerance, and alleviated morphine tolerance in the mouse model. These findings suggest that morphine induces HMGB1 release via the adenosine 5'-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway, and that inhibiting this signaling pathway can effectively reduce morphine tolerance.

Kinase DYRK2 acts as a regulator of autophagy and an indicator of favorable prognosis in gastric carcinoma.

Gastric cancer (GC) is the third leading cause of cancer-related death worldwide; therefore, new and more specific molecules for GC are needed. Here, we found that dual specificity tyrosine phosphorylation regulated kinase 2 (DYRK2) may be a specific marker for GC. Immunohistochemistry (IHC) and statistical and bioinformatics analyses were conducted to detect DYRK2 expression in stomach tissues. The role of DYRK2 in GC was analyzed with a nude mouse model and CCK-8, wound healing and Transwell assays. Western blotting and immunofluorescence experiments were also performed to elucidate the relationship between DYRK2 expression and both epithelial-mesenchymal transition (EMT) and autophagy progression. We found that DYRK2 expression in GC tissues was lower than that in benign or normal tissues, and patients with high DYRK2 expression had a good prognosis. The in vitro results showed that DYRK2 expression inhibited the tumorigenic activities of GC, including proliferation, migration, and invasion. By analyzing the expression of EMT markers after altering DYRK2 expression, we observed that DYRK2 inhibits the occurrence of EMT. The nude mouse model revealed that DYRK2 inhibits tumor growth. Finally, we used Western blotting and immunofluorescence assays and found that DYRK2 promotes autophagy. Based on these data, DYRK2 may be a good reference indicator for the clinical diagnosis of GC.

NET-Triggered NLRP3 Activation and IL18 Release Drive Oxaliplatin-Induced Peripheral Neuropathy.

Oxaliplatin is an antineoplastic agent frequently used in the treatment of gastrointestinal tumors. However, it causes dose-limiting sensorimotor neuropathy, referred to as oxaliplatin-induced peripheral neuropathy (OIPN), for which there is no effective treatment. Here, we report that the elevation of neutrophil extracellular traps (NET) is a pathologic change common to both cancer patients treated with oxaliplatin and a murine model of OIPN. Mechanistically, we found that NETs trigger NLR family pyrin domain containing 3 (NLRP3) inflammasome activation and the subsequent release of IL18 by macrophages, resulting in mechanical hyperalgesia. In NLRP3-deficient mice, the mechanical hyperalgesia characteristic of OIPN in our model was reduced. In addition, in the murine model, treatment with the IL18 decoy receptor IL18BP prevented the development of OIPN. We further showed that eicosapentaenoic acid (EPA) reduced NET formation by suppressing the LPS-TLR4-JNK pathway and thereby abolished NLRP3 inflammasome activation and the subsequent secretion of IL18, which markedly prevented oxaliplatin-induced mechanical hyperalgesia in mice. These results identify a role for NET-triggered NLRP3 activation and IL18 release in the development of OIPN and suggest that utilizing IL18BP and EPA could be effective treatments for OIPN.

An enzyme-linked immunosorbent assay to compare the affinity of chemical compounds for ß-amyloid peptide as a monomer.

Aß(1-42) is the proteolytic cleavage product of cleavage of the amyloid precursor protein by ß- and γ-secretases. The aggregation of Aß(1-42) plays a causative role in the development of Alzheimer's disease. To lock Aß(1-42) in a homogenous state, we embedded the Aß(1-42) sequence in an unstructured region of Bcl-x(L). Both the N-terminus and the C-terminus of Aß(1-42) were constrained in the disordered region, whereas the conjunction did not introduce any folding to Aß(1-42) but maintained the sequence as a monomer in solution. With Bcl-x(L)-Aß(42), we developed an enzyme-linked immunosorbent assay to compare the affinity of compounds for monomeric Aß(1-42). Bcl-x(L)-Aß(42) was coated on a microplate and this was followed by incubation with different concentrations of compounds. Compounds binding to Leu17-Val24 of Aß(1-42) inhibited the interaction between Bcl-x(L)-Aß(42) and antibody 4G8. The method can not only reproduce the activities of the reported Aß(1-42) inhibitors such as dopamine, tannin, and morin but can also differentiate decoy compounds that do not bind to Aß(1-42). Remarkably, using this method, we discovered a new inhibitor that binds to monomeric Aß(1-42) and inhibits Aß(1-42) fibril formation. As the structure of Bcl-x(L)-Aß(42) monomer is stable in solution, the assay could be adapted for high-throughput screening with a series of antibodies that bind the different epitopes of Aß(1-42). In addition, the monomeric form of the Aß(1-42) sequence in Bcl-x(L)-Aß(42) would also facilitate the identification of Aß(1-42) binding partners by coimmunoprecipitation, cocrystallization, surface plasmon resonance technology, or the assay as described here.

FIBCD1 overexpression predicts poor prognosis in patients with hepatocellular carcinoma.

Fibrinogen C domain-containing 1 (FIBCD1) is an acetyl-recognition receptor that affects the occurrence and development of certain tumors. However, the prognostic significance of FIBCD1 in hepatocellular carcinoma (HCC) remains unclear. This study aimed to explore FIBCD1 expression in HCC and to determine the prognostic value of FIBCD1 in patients with HCC. A total of 1,058 liver tissue samples with detailed and complete clinical information were collected, including 495 HCC samples. Tissue microarray immunohistochemistry analysis was used to evaluate FIBCD1 protein expression in the collected tissues. The Kaplan-Meier plotter online tool was used to investigate the association between FIBCD1 expression and prognosis of patients with HCC. Oncomine and the Gene Expression Profiling Interactive Analysis database were used for bioinformatics analysis of FIBCD1. Results showed that FIBCD1 expression was higher in HCC and was associated with tumor diameter (P=0.002), tumor number (P=0.001), tumor node metastasis stage (P<0.001), primary tumor (T; P<0.001), lymph node metastases (N; P=0.002), distant metastases (M; P=0.023), differentiation degree (P=0.003), vascular invasion (P<0.001) and liver cirrhosis (P=0.011). Patients with HCC and high FIBCD1 expression had worse overall survival than those with low FIBCD1 expression. High FIBCD1 expression (P<0.001), TNM stage (P=0.003), T (P<0.001), N (P=0.014), and vascular invasion (P<0.001) were independent prognostic factors in HCC. Hence, FIBCD1 may be a novel biomarker for prognosis evaluation of HCC.