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Inhibition of CaMKIV relieves streptozotocin-induced diabetic neuropathic pain through regulation of HMGB1.

ABSTRACT: The pathogenesis of diabetic neuropathic pain is complicated and its underlying mechanisms remain unclear. Calmodulin-dependent protein kinases (CaMKs) IV (CaMKIV), one of CaMKs, regulates several transcription factors in pain mechanisms. High-mobility group box 1 (HMGB1) is a key mediator in diabetic neuropathic pain. This study aims to find the roles and mechanisms of CaMIV in diabetic neuropathic pain.Diabetic animal models were constructed by injecting with streptozotocin (STZ) intraperitoneally. They were randomly divided into seven groups (n = 6 per group): Naive, Normal Saline, STZ, STZ + Sham, STZ + DMSO and STZ + KN93 (an inhibitor of CaMKIV) (50 μg), STZ + KN93 (100 μg), which received KN93 (50 or 100 μg) intrathecally after the administration of STZ. Phospho-CaMKIV (pCaMKIV) and HMGB1 expression in rat dorsal root ganglion (DRG) and RAW264.7 cell line were measured by western blot. Distribution of pCaMKIV immune reactivity in different subpopulations of DRG neurons was measured by double-immunofluorescence staining.The pCaMKIV and HMGB1 in DRG significantly increased after STZ administration, and pCaMKIV can regulate the expression of HMGB1 based on both cellular and animal models. Pretreatment with CaMKIV inhibitor attenuated STZ-induced mechanical allodynia and thermal hyperalgesia, as well as reduced HMGB1 expression in the DRG.This study demonstrates that CaMKIV can relieve STZ-induced diabetic neuropathic pain. The mechanism of this function depended on the process: pCaMKIV localized in the nuclei of DRG neurons and regulated HMGB1 which was an important mediator of neuropathic pain. These findings reported CaMKIV may be a potential target or important node in relieving diabetic neuropathic pain.

SUBMITTER: Zhao X

PROVIDER: S-EPMC4877728 | biostudies-other | 2016

REPOSITORIES: biostudies-other

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Project description:Diabetic neuropathy (DN) is a serious microvascular complication of diabetes mellitus (DM) that impacts the nervous system. Several risk factors are involved in the progression and maintenance of DN-associated pain, such as higher expression of various inflammatory mediators, e.g., tumor necrotic factor-alpha (TNF-α), nuclear factor-kappa B (NF-κB), and cyclo-oxygenase-2 (COX-2). The present research explores the neuroprotective potential of natural isolates, including berbamine, bergapten, and carveol, on the DM-induced neuroinflammation and neurodegeneration that cause neuropathic pain. The study utilized computerized techniques, including computational analysis (a docking assay and a molecular dynamic simulation) before moving to in vivo protocols. Diabetic neuropathy was induced by intraperitonial injection (IP) of streptozotocin (65 mg/kg), and the animal subjects (rats) were kept for 4 weeks for the development of DN. Once diabetic neuropathy was confirmed, the subjects were treated with berbamine, bergapten, and carveol until the sixth week (i.e., 2 weeks of treatment). At the sixth week, the rats were sacrificed, and the sciatic nerve and spinal cord of each was collected for further molecular investigation. Docking and a molecular dynamic simulation (MDS) delivered the information that the natural compounds (berbamine, bergapten, and carveol) were interacting with the selected target protein (i.e., mitogen-activated protein kinase). After IP, it was found that berbamine, bergapten, and carveol had ameliorated mechanical allodynia and thermal hyperalgesia by the 28th day of the study (2 weeks after treatment) without affecting blood glucose levels. Berbamine, bergapten, and carveol markedly elevated the levels of glutathione (GSH) and glutathione s-transferase (GST), in both the sciatic nerve and spinal cord, and also reduced lipid peroxidase (LPO) and nitric oxide (NO). The abovementioned natural isolates reduced pathologic alterations provoked through DN, a finding confirmed through histopathological assays (hematoxylin and eosin staining and immuno-histochemical analysis). Treatment down regulated higher expressions of the inflammatory mediatorcyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and nuclear factor kappa B (NF-κB), as confirmed by ELISA and polymerase chain reaction (PCR). The outcomes of berbamine, bergapten, and carveol are compared with those of pregabalin as a positive control group. Compared to pregabalin, treatment with the aforementioned three natural compounds improved nociception and reduced hyperalgesic effects, and consequently reduced pain perception and inflammation. Our results suggest the mechanism for the neuro-protective impact of berbamine, bergapten, and carveol might possibly be arbitrated via COX-2, TNF-α, and NF-κB, and regulated by mitogen-activated protein kinase, ultimately ameliorating STZ-provoked, DM-induced neuroinflammation and neurodegeneration, and associated neuropathic pain.

Project description:BackgroundWhether neuroinflammation causes comorbid mood disorders in neuropathic pain remains elusive. Here we investigated the role of high mobility group box 1 protein (HMGB1), a proinflammatory cytokine, in the medial prefrontal cortex (mPFC) in anxiety comorbidity of neuropathic pain.MethodsNeuropathic pain was induced by partial transection of the infraorbital nerve (p-IONX) or partial sciatic nerve ligation (PSL) in mice and evaluated by measuring nociceptive thresholds to mechanical and heat stimulation. Anxiety-like behaviors were assessed by elevated plus maze, light dark box and open field tests. Aversive or anti-aversive effect was detected by conditioned place preference test. Neuronal activity was evaluated by single-unit and patch clamp recordings. The contribution of mPFC pyramidal neurons to anxiety was further examined by selectively inhibiting them by optogenetics. HMGB1 expression was measured by immunohistochemistry and western blotting. Antagonism of HMGB1 was achieved by injecting anti-HMGB1 monoclonal antibody (mAb) intracerebrally or intraperitoneally.ResultsAnxiety-like behaviors were presented earlier after p-IONX than after PSL. HMGB1 expression was upregulated in the mPFC temporally in parallel to anxiety onset, rather than in other regions associated with anxiety. The upregulation of HMGB1 expression and its translocation from the nucleus to cytoplasm in the mPFC occurred predominantly in neurons and were accompanied with activation of microglia and astrocytes. Infusion of anti-HMGB1 mAb into the mPFC during the early and late phases after either p-IONX or PSL alleviated anxiety-like behaviors and aversion without changing pain sensitization, while local infusion of exogenous ds-HMGB1, the proinflammatory form of HMGB1, into the mPFC induced anxiety and aversion but not pain sensitization in naïve mice. In addition to reversing established pain sensitization and anxiety simultaneously, intraperitoneal injection of anti-HMGB1 mAb reduced HMGB1 upregulation and suppressed the hyperexcitability of layer 2/3 pyramidal neurons in the mPFC after p-IONX. Moreover, optogenetic inhibition of mPFC pyramidal neurons alleviated anxiety in p-IONX mice.ConclusionThese results demonstrate that HMGB1 in the mPFC drives and maintains anxiety comorbidity in neuropathic pain by increasing the excitability of layer 2/3 pyramidal neurons, and justify antagonism of HMGB1, e.g., neutralization by mAb, as a promising therapeutic strategy for neuropathic pain with anxiety comorbidity.

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Inhibition of CaMKIV relieves streptozotocin-induced diabetic neuropathic pain through regulation of HMGB1.

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