RNA-seq based transcriptomic map reveals multiple pathways of necroptosis in treating myocardial ischemia reperfusion injury.

Necroptosis has been shown to contribute to myocardial ischemia reperfusion injury (MIRI). This study aims to gain new insights into the signaling pathway of necroptosis in rat MIRI using RNA sequencing. MIRI was induced in male rats by ligating the left anterior descending coronary artery for 30 min, followed by reperfusion for 120 min. RNA sequencing was performed to obtain mRNA profiles of MIRI group and MIRI group treated with necrostatin-1 (Nec-1,an inhibitor of necroptosis). Differentially expressed genes (DEGs) were then identified. The DEGs were prominently enriched in the TNF-α signaling pathway, the MAPK signaling pathway and cytokine-cytokine receptor pathways. The majority of the results were associated with genes like Thumpd3,Egr2,Dot1l,Cyp1a1,Dbnl,which primarily regulate inflammatory response and apoptosis, particularly in myocardium. The above results suggested that Nec-1 might be involved in the regulation of necroptosis and the inflammatory response through the above-mentioned genes.

The cold-inducible RNA-binding protein-Thioredoxin 1 pathway ameliorates mitochondrial dysfunction and mitochondrial dynamin-related protein 1 level in the hippocampus of aged mice with perioperative neurocognitive dysfunction.

BACKGROUND: As a multi-disease model, neuroinflammation, mitochondrial dysfunction, and oxidative stress might be involved in the pathogenic process of perioperative neurocognitive dysfunction (PND). Dynamin-related protein 1 (Drp1) could mediate mitochondrial fission and play important roles in mitochondrial dynamic homeostasis and mitochondria function. The Drp1 may be involved in PND development. The cold-inducible RNA-binding protein (Cirbp) could bind to the 3'-UTR of the thioredoxin 1 (Trx1) mRNA, control oxidative stress, and improve mitochondrial function. In this study, we hypothesized that the Cirbp-Trx1 pathway could ameliorate mitochondrial dysfunction and Drp1 levels in PND mice. METHODS: Differentially expressed genes were screened using the Gene Expression Omnibus (GEO) database GSE95426 and validated using PCR. Eighteen-month-old C57BL/6 mice were subjected to tibial fracture surgery to generate a PND model. Cirbp was upregulated by hippocampal stereotaxic injections of over-Cirbp plasmid according to the manufacturer's instructions for the in vivo DNA transfection reagent. Cirbp expression was measured using western blot (WB) and immunofluorescence (IF). The Morris water maze (MWM) was used to assess cognitive function. After behavioral testing, the hippocampal tissue was extracted to examine changes in mitochondrial Drp1, mitochondrial function, neuroinflammation, and oxidative stress. RESULTS: Differential gene screening showed that Cirbp expression was significantly downregulated (fold change >1.5, p = 0.003272) in the PND model. In this study, we also found that Cirbp protein levels were downregulated, accompanied by an impairment of cognition, a decrease in superoxide dismutase (SOD) activity, and an increase in malondialdehyde (MDA) content, mitochondrial Drp1 levels, neuroinflammation, and apoptosis. Cirbp overexpression increased Trx1 protein levels and reversed the damage. However, this protective effect was abolished by PX-12 treatment with a Trx1 inhibitor. CONCLUSIONS: The Cirbp-Trx1 pathway may regulate mitochondrial dysfunction and mitochondrial Drp1 expression in the hippocampus of PND mice to ameliorate cognitive dysfunction.

CCL17 Blockade by CCL17mAb/GSK-J4 Ameliorates Hyperalgesia in a Rat Model of Postoperative Pain.

C-C motif chemokine ligand 17 (CCL17), an important chemokine, plays a vital role in regulating immune balance in the central nervous system. In this study, we explored the potential roles of CCL17 in a rat postoperative pain model and that of blocking CCL17 in the prevention of postoperative pain in rats. A right plantar incision in rat was used as a model of postoperative pain. A behavioral change was measured preoperatively and postoperatively using mechanical withdrawal thresholds and thermal withdrawal latency. CCL17 and its upstream Jmjd-3 mRNA levels in the spinal cord were detected using real-time PCR, CCL17 levels in the serum were measured using enzyme-linked immunosorbent assay (ELISA), and the expression of interferon regulatory factor 4 (IRF4), which interacts with Jmjd-3, was detected by immunohistochemistry staining. After that, rats were intraperitoneally injected with either anti-CCL17 monoclonal antibody (mAb) or GSK-J4 (the Jmjd3 inhibitor) to evaluate the protective effects of blocking CCL17 on postoperative pain. We found that CCL17 and Jmjd-3 were significantly increased in the spinal cords of the postoperative pain rat, consistent with changes in hyperalgesia. In addition, our results showed that the mechanical and thermal allodynia was significantly ameliorated using anti-CCL17mAb or GSK-J4. Moreover, we found that anti-CCL17mAb or GSK-J4 treatment decreased c-fos expression in response to peripheral stimulation. Finally, our preliminary exploration found that anti-CCL17mAb or GSK-J4 had a protective effect on tissue damage. These findings indicated that high expression of CCL17 played a critical role in postoperative pain induced by plantar incision and that CCL17 blockade may serve as an effective approach to managing postoperative pain.

Epitope alteration by small molecules and applications in drug discovery.

Small molecules and antibodies are normally considered separately in drug discovery, except in the case of covalent conjugates. We unexpectedly discovered several small molecules that could inhibit or enhance antibody-epitope interactions which opens new possibilities in drug discovery and therapeutic modulation of auto-antibodies. We first discovered a small molecule, CRANAD-17, that enhanced the binding of an antibody to amyloid beta (Aß), one of the major hallmarks of Alzheimer's disease, by stable triplex formation. Next, we found several small molecules that altered antibody-epitope interactions of tau and PD-L1 proteins, demonstrating the generality of this phenomenon. We report a new screening technology for ligand discovery, screening platform based on epitope alteration for drug discovery (SPEED), which is label-free for both the antibody and small molecule. SPEED, applied to an Aß antibody, led to the discovery of a small molecule, GNF5837, that inhibits Aß aggregation and another, obatoclax, that binds Aß plaques and can serve as a fluorescent reporter in brain slices of AD mice. We also found a small molecule that altered the binding between Aß and auto-antibodies from AD patient serum. SPEED reveals the sensitivity of antibody-epitope interactions to perturbation by small molecules and will have multiple applications in biotechnology and drug discovery.

Natural medicine HLXL targets multiple pathways of amyloid-mediated neuroinflammation and immune response in treating alzheimer's disease.

BACKGROUND: Based on the complex pathology of AD, a single chemical approach may not be sufficient to deal simultaneously with multiple pathways of amyloid-tau neuroinflammation. A polydrug approach which contains multiple bioactive components targeting multiple pathways in AD would be more appropriate. Here we focused on a Chinese medicine (HLXL), which contains 56 bioactive natural products identified in 11 medicinal plants and displays potent anti-inflammatory and immuno-modulatory activity. HYPOTHESIS/PURPOSE: We investigated the neuroimmune and neuroinflammation mechanisms by which HLXL may attenuate AD neuropathology. Specifically, we investigated the effects of HLXL on the neuropathology of AD using both transgenic mouse models as well as microglial cell-based models. STUDY DESIGN: The 5XFAD transgenic animals and microglial cell models were respectively treated with HLXL and Aß42, and/or lipopolysaccharide (LPS), and then analyzed focusing on microglia mediated Aß uptake and clearance, as well as pathway changes. METHODS: We showed that HLXL significantly reduced amyloid neuropathology by upregulation of microglia-mediated phagocytosis of Aß both in vivo and in vitro. HLXL displayed multi-modal mechanisms regulating pathways of phagocytosis and energy metabolism. RESULTS: Our results may not only open a new avenue to support pharmacologic modulation of neuroinflammation and the neuroimmune system for AD intervention, but also identify HLXL as a promising natural medicine for AD. CONCLUSION: It is conceivable that the traditional wisdom of natural medicine in combination with modern science and technology would be the best strategy in developing effective therapeutics for AD.

Degradation and inhibition of epigenetic regulatory protein BRD4 exacerbate Alzheimer's disease-related neuropathology in cell models.

Epigenetic regulation plays substantial roles in human pathophysiology, which provides opportunities for intervention in human disorders through the targeting of epigenetic pathways. Recently, emerging evidence from preclinical studies suggested the potential in developing therapeutics of Alzheimer's disease (AD) by targeting bromodomain containing protein 4 (BRD4), an epigenetic regulatory protein. However, further characterization of AD-related pathological events is urgently required. Here, we investigated the effects of pharmacological degradation or inhibition of BRD4 on AD cell models. Interestingly, we found that both degradation and inhibition of BRD4 by ARV-825 and JQ1, respectively, robustly increased the levels of amyloid-beta (Aß), which has been associated with the neuropathology of AD. Subsequently, we characterized the mechanisms by which downregulation of BRD4 increases Aß levels. We found that both degradation and inhibition of BRD4 increased the levels of BACE1, the enzyme responsible for cleavage of the amyloid-beta protein precursor (APP) to generate Aß. Consistent with Aß increase, we also found that downregulation of BRD4 increased AD-related phosphorylated Tau (pTau) protein in our 3D-AD human neural cell culture model. Therefore, our results suggest that downregulation of BRD4 would not be a viable strategy for AD intervention. Collectively, our study not only shows that BRD4 is a novel epigenetic component that regulates BACE1 and Aß levels, but also provides novel and translational insights into the targeting of BRD4 for potential clinical applications.

Gut microbiota-driven brain Aß amyloidosis in mice requires microglia.

We previously demonstrated that lifelong antibiotic (ABX) perturbations of the gut microbiome in male APPPS1-21 mice lead to reductions in amyloid ß (Aß) plaque pathology and altered phenotypes of plaque-associated microglia. Here, we show that a short, 7-d treatment of preweaned male mice with high-dose ABX is associated with reductions of Aß amyloidosis, plaque-localized microglia morphologies, and Aß-associated degenerative changes at 9 wk of age in male mice only. More importantly, fecal microbiota transplantation (FMT) from transgenic (Tg) or WT male donors into ABX-treated male mice completely restored Aß amyloidosis, plaque-localized microglia morphologies, and Aß-associated degenerative changes. Transcriptomic studies revealed significant differences between vehicle versus ABX-treated male mice and FMT from Tg mice into ABX-treated mice largely restored the transcriptome profiles to that of the Tg donor animals. Finally, colony-stimulating factor 1 receptor (CSF1R) inhibitor-mediated depletion of microglia in ABX-treated male mice failed to reduce cerebral Aß amyloidosis. Thus, microglia play a critical role in driving gut microbiome-mediated alterations of cerebral Aß deposition.

Molecular imaging of NAD+ -dependent deacetylase SIRT1 in the brain.

INTRODUCTION: Aging is an inevitable physiological process and the biggest risk factor of Alzheimer's disease (AD). Developing an imaging tracer to visualize aging-related changes in the brain may provide a useful biomarker in elucidating neuroanatomical mechanisms of AD. METHODS: We developed and characterized a new tracer that can be used to visualize SIRT1 in brains related to aging and AD by positron emission tomography imaging. RESULTS: The SIRT1 tracer displayed desirable brain uptake and selectivity, as well as stable metabolism and proper kinetics and distribution in rodent and nonhuman primate brains. This new tracer was further validated by visualizing SIRT1 in brains of AD transgenic mice, compared to nontransgenic animals. DISCUSSION: Our SIRT1 tracer not only enables, for the first time, the demonstration of SIRT1 in animal brains, but also allows visualization and recapitulation of AD-related SIRT1 neuropathological changes in animal brains.

An APP ectodomain mutation outside of the Aß domain promotes Aß production in vitro and deposition in vivo.

Familial Alzheimer's disease (FAD)-linked mutations in the APP gene occur either within the Aß-coding region or immediately proximal and are located in exons 16 and 17, which encode Aß peptides. We have identified an extremely rare, partially penetrant, single nucleotide variant (SNV), rs145081708, in APP that corresponds to a Ser198Pro substitution in exon 5. We now report that in stably transfected cells, expression of APP harboring the S198P mutation (APPS198P) leads to elevated production of Aß peptides by an unconventional mechanism in which the folding and exit of APPS198P from the endoplasmic reticulum is accelerated. More importantly, coexpression of APP S198P and the FAD-linked PS1ΔE9 variant in the brains of male and female transgenic mice leads to elevated steady-state Aß peptide levels and acceleration of Aß deposition compared with age- and gender-matched mice expressing APP and PS1ΔE9. This is the first AD-linked mutation in APP present outside of exons 16 and 17 that enhances Aß production and deposition.

TSPO Ligands PK11195 and Midazolam Reduce NLRP3 Inflammasome Activation and Proinflammatory Cytokine Release in BV-2 Cells.

Neuroinflammation related to microglial activation plays an important role in neurodegenerative diseases. Translocator protein 18 kDa (TSPO), a biomarker of reactive gliosis, its ligands can reduce neuroinflammation and can be used to treat neurodegenerative diseases. Therefore, we explored whether TSPO ligands exert an anti-inflammatory effect by affecting the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome, thereby inhibiting the release of inflammatory cytokines in microglial cells. In the present study, BV-2 cells were exposed to lipopolysaccharide (LPS) for 6 h to induce an inflammatory response. We found that the levels of reactive oxygen species (ROS), NLRP3 inflammasome, interleukin-1ß (IL-1ß), and interleukin-18 (IL-18) were significantly increased. However, pretreatment with TSPO ligands inhibited BV-2 microglial and NLRP3 inflammasome activation and significantly reduced the levels of ROS, IL-1ß, and IL-18. Furthermore, a combination of LPS and ATP was used to activate the NLRP3 inflammasome. Both pretreatment and post-treatment with TSPO ligand can downregulate the activation of NLRP3 inflammasome and IL-1ß expression. Finally, we found that TSPO was involved in the regulation of NLRP3 inflammasome with TSPO ligands treatment in TSPO knockdown BV2 cells. Collectively, these results indicate that TSPO ligands are promising targets to control microglial reactivity and neuroinflammatory diseases.

Molecular imaging of Alzheimer's disease-related gamma-secretase in mice and nonhuman primates.

The pathogenesis of Alzheimer's disease (AD) is primarily driven by brain accumulation of the amyloid-ß-42 (Aß42) peptide generated from the amyloid-ß precursor protein (APP) via cleavages by ß- and γ-secretase. γ-Secretase is a prime drug target for AD; however, its brain regional expression and distribution remain largely unknown. Here, we are aimed at developing molecular imaging tools for visualizing γ-secretase. We used our recently developed γ-secretase modulators (GSMs) and synthesized our GSM-based imaging agent, [11C]SGSM-15606. We subsequently performed molecular imaging in rodents, including AD transgenic animals, and macaques, which revealed that our probe displayed good brain uptake and selectivity, stable metabolism, and appropriate kinetics and distribution for imaging γ-secretase in the brain. Interestingly, rodents and macaques shared certain brain areas with high γ-secretase expression, suggesting a functional conservation of γ-secretase. Collectively, we have provided the first molecular brain imaging of γ-secretase, which may not only accelerate our drug discovery for AD but also advance our understanding of AD.

Upregulation of Alzheimer's Disease Amyloid-ß Protein Precursor in Astrocytes Both in vitro and in vivo.

BACKGROUND: The amyloid cascade hypothesis of Alzheimer's disease (AD) posits that amyloid-ß (Aß) protein accumulation underlies the pathogenesis of the disease by leading to the formation of amyloid plaques, a pathologic hallmark of AD. Aß is a proteolytic product of amyloid-ß protein precursor (AßPP; APP), which is expressed in both neurons and astrocytes. Although considerable evidence shows that astrocytes may play critical roles in the pathogenesis of AD, the longitudinal changes of amyloid plaques in relationship to AßPP expression in astrocytes and cellular consequences are largely unknown. OBJECTIVE: Here, we aimed to investigate astrocyte-related pathological changes of Aß and AßPP using immunohistochemistry and biochemical studies in both animal and cell models. METHODS/RESULTS: We utilized 5XFAD transgenic mice and found age-dependent upregulation of AßPP in astrocytes demonstrated with astrocytic reactive properties, which followed appearance of amyloid plaques in the brain. We also observed that AßPP proteins presented well-defined punctate immuno reactivity in young animals, whereas AßPP staining showed disrupted structures surrounding amyloid plaques in older mice. Moreover, we utilized astrocyte cell models and showed that pretreatment of Aß42 resulted in downstream astrocyte autonomous changes, including up regulation in AßPP and BACE1 levels, as well as prolonged amyloidogenesis that could be reduced by pharmacological inhibition of BACE1. CONCLUSION: Collectively, our results show that age-dependent AßPP up regulation in astrocytes is a key feature in AD, which will not only provide novel insights for understanding AD progression, but also may offer new therapeutic strategies for treating AD.

Negative evidence for a role of APH1B T27I variant in Alzheimer's disease.

γ-secretase is a macromolecular complex that catalyzes intramembranous hydrolysis of more than 100 membrane-bound substrates. The complex is composed of presenilin (PS1 or PS2), anterior pharynx defect-1 (APH-1), nicastrin (NCT) and PEN-2 and early-onset; autosomal dominant forms of Alzheimer's disease (AD) are caused by inheritance of mutations of PS. No mutations in genes encoding NCT, or PEN-2 have been identified to date that cause AD. In this regard, a large genetic meta-analysis of four cohorts consisting of more than 600 000 individuals identified a common missense variant, rs117618017 in the APH1B gene that results in a T27I mutation, as a novel genome-wide significant locus. In order to confirm the findings that rs117618017 is associated with risk of AD, we performed a genetic screen from deep whole genome sequencing of the large NIMH family-based Alzheimer's Disease (AD) dataset. In parallel, we sought to uncover potential molecular mechanism(s) by which APH-1B T27I might be associated with AD by generating stable HEK293 cell lines, wherein endogenous APH-1A and APH-1B expression was silenced and into which either the wild type APH-1B or the APH-1B T27I variant was stably expressed. We then tested the impact of expressing either the wild type APH-1B or the APH-1B T27I variant on γ-secretase processing of human APP, the murine Notch derivative mNΔE and human neuregulin-1. We now report that we fail to confirm the association of rs1047552 with AD in our cohort and that cells expressing the APH-1B T27I variant show no discernable impact on the γ-secretase processing of established substrates compared with cells expressing wild-type APH-1B.

A Curcumin Analog Reduces Levels of the Alzheimer's Disease-Associated Amyloid-ß Protein by Modulating AßPP Processing and Autophagy.

Alzheimer's disease (AD) is a devastating neurodegenerative disease with no cure currently available. A pathological hallmark of AD is accumulation and deposition of amyloid-ß protein (Aß), a ∼4 kDa peptide generated through serial cleavage of the amyloid-ß protein precursor (AßPP) by ß- and γ-secretases. Curcumin is a natural compound primarily found in the widely used culinary spice, turmeric, which displays therapeutic potential for AD. Recently, we reported the development of curcumin analogs and identified a lead compound, curcumin-like compound-R17 (CLC-R17), that significantly attenuates Aß deposition in an AD transgenic mouse model. Here, we elucidated the mechanisms of this analog on Aß levels and AßPP processing using cell models of AD. Using biochemical methods and our recently developed nanoplasmonic fiber tip probe technology, we showed that the lead compound potently lowers Aß levels in conditioned media and reduces oligomeric amyloid levels in the cells. Furthermore, like curcumin, the lead compound attenuates the maturation of AßPP in the secretory pathway. Interestingly, it upregulated α-secretase processing of AßPP and inhibited ß-secretase processing of AßPP by decreasing BACE1 protein levels. Collectively, our data reveal mechanisms of a promising curcumin analog in reducing Aß levels, which strongly support its development as a potential therapeutic for AD.

The high-affinity IgG receptor FcγRI modulates peripheral nerve injury-induced neuropathic pain in rats.

The Fc gamma receptor I (FcγRI; CD64) is the high-affinity receptor of the immunoglobulin G protein (IgG). It is usually expressed in immune cells and has recently been identified to distribute in the nervous system and play critical roles in various neurological disorders. Presently, the impacts of FcγRI in neuropathic pain was largely unknown. Here, we aimed to investigate the impacts of FcγRI in neuropathic pain through pain-related neurobehavioral studies and underlying mechanisms by biochemical methods in animal and cell models. Specifically, we first utilized the chronic constriction injury (CCI) rat model that displayed neuropathic pain related symptoms and signs, including thermal hyperalgesia and mechanical allodynia. These neurobehavioral defects were significantly attenuated by the anti-FcγRI antibody, which was associated with reduced levels of neuropeptide substance P, C3, and TNF-α. Furthermore, we validated our animal findings using the embryonically neural crest-originated PC12 cell model. We found that stimulation of the IgG immune complex led to increased levels of FcγRI and inflammatory mediators, which were attenuated by the anti-FcγRI antibody in these cells. Collectively, our results from animal and cell-based studies suggest that FcγRI is a critical player for peripheral nerve injury-induced neuropathic pain by mediating pain-related immunological events, which therefore may provide a new therapeutic target for protection against chronic pain.

Significant Upregulation of Alzheimer's ß-Amyloid Levels in a Living System Induced by Extracellular Elastin Polypeptides.

Alzheimer's disease (AD) is a neurodegenerative disorder and the primary cause of age-related dementia. The etiology of AD is complex and has not been completely elucidated. Herein, we report that treatment with elastin-like polypeptides (ELPs), a component of the brain extracellular matrix (ECM), significantly increased the levels of AD-related amyloid-ß peptides (Aß) both in vitro and in vivo. Regarding the molecular mechanism(s), the upregulation of Aß levels was related to increased proteolytic processing of the amyloid precursor protein. Furthermore, nesting tests demonstrated that the ELP-treated animals showed significant neurobehavioral deficits with cognitive impairment. These results suggest that the elastin is associated with AD-related pathological and behavioral changes. This finding presents a new aspect for Alzheimer's amyloidosis event and provides a great promise in developing ELP-based model systems to better understand the pathogenesis of AD.

Necrostatin-1 Ameliorates Peripheral Nerve Injury-Induced Neuropathic Pain by Inhibiting the RIP1/RIP3 Pathway.

Necrostatin-1 is an inhibitor of necroptosis, a form of programmed cell death that has been reported to be involved in various neurological diseases. Presently, the role of necroptosis in neuropathic pain induced by peripheral nerve injury is still unclear. This study was focused on investigating the potential effects of necroptosis in the development and progression of neuropathic pain in a rat model and the possible neuroprotective effects of necrostatin-1 in neuropathic pain. The results indicated that the necroptosis-related proteins RIP1 and RIP3 significantly increased postoperation in the spinal cord in a neuropathic pain model and peaked 7 days postoperation, which was consistent with the time-dependent changes of hyperalgesia. Additionally, we found that peripheral nerve injury-related behavioral and biochemical changes were significantly reduced by necrostatin-1. In particular, hyperalgesia was attenuated, and the levels of RIP1 and RIP3 were decreased. Furthermore, the ultrastructure of necrotic cell death and neuroinflammation were alleviated by necrostatin-1. Collectively, these results suggest that necroptosis is an important mechanism of cell death in neuropathic pain induced by peripheral nerve injury and that necrostatin-1 may be a promising neuroprotective treatment for neuropathic pain.

[FcγRIis involved in lipopolysaccharide-induced apoptosis of PC12 cells].

Objective To investigate the role of IgG Fc receptorI (FcγRI) in lipopolysaccharide (LPS)-induced apoptosis of the rat PC12 cells. Methods PC12 cells were treated with different concentrations of LPS (50, 125, 250, 500, 1000 µg/mL) for 24 hours and cell viability was analyzed by MTT assay. The appropriate concentration of LPS (500 µg/mL) was chosen for the following experiments. PC12 cells in the logarithmic growth phase were divided randomly into three groups: the control group without LPS, the 500 µg/mL LPS treated group and the 500 µg/mL LPS plus 0.2 µg/mL FcγRI neutralizer group. After24-hour different treatments, the mRNA and protein levels of FcγRIwere detected by quantitative real-time PCR and Western blotting, respectively. The apoptosis rate of PC12 cells was determined by flow cytometry combined with annexinV-FITC/PI double staining. The protein expression levels of caspase-3, Bcl-2 and BAX were measured by immunohistochemistry. Results PC12 cell viability decreased in a LPS dose-dependent manner. Compared to the control group, the protein and mRNA expression of FcγRI were upregulated, the expression levels of caspase-3, Bcl-2 and BAX proteins were elevated, and the apoptosis rate of PC12 cells was raised as well in the LPS treated group. Compared to the LPS treated group, the protein and mRNA levels of FcγRI were significantly lower along with significantly reduced expressions of Caspase-3 and BAX and inhibited cell apoptosis in the FcγRIneutralizer treated group, while Bcl-2 protein expression was upregulated. Conclusion FcγRIis involved in the LPS-induced apoptosis in PC12 cells.

Antinociceptive Effect of Najanalgesin from Naja Naja Atra in a Neuropathic Pain Model via Inhibition of c-Jun NH2-terminal Kinase.

BACKGROUND: Najanalgesin, a toxin isolated from the venom of Naja naja atra, has been shown to exert significant analgesic effects in a neuropathic pain model in rats. However, the molecular mechanism underlying this protective effect of najanalgesin is poorly understood. The present study sought to evaluate the intracellular signaling pathways that are involved in the antinociceptive effect of najanalgesin on neuropathic pain. METHODS: The antinociceptive properties of najanalgesin were tested in hind paw withdrawal thresholds in response to mechanical stimulation. We analyzed the participation of the mitogen-activated protein kinase p38, extracellular-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) by western blot analysis. This inhibition of JNK was confirmed by immunohistochemistry. RESULTS: The phosphorylation levels of JNK (as well as its downstream molecule c-Jun), p38, and ERK were significantly increased after injury. Najanalgesin only inhibited JNK and c-Jun phosphorylation but had no effect on either ERK or p38. This inhibition of JNK was confirmed by immunohistochemistry, which suggested that the antinociceptive effect of najanalgesin on spinal nerve ligation-induced neuropathic pain in rats is associated with JNK activation in the spinal cord. CONCLUSION: The antinociceptive effect of najanalgesin functions by inhibiting the JNK in a neuropathic pain model.

Behavioral and morphological evidence for the involvement of glial cells in the antinociceptive effect of najanalgesin in a rat neuropathic pain model.

Neuropathic pain is a devastating neurological disease that seriously affects patients' quality of life. Despite a high level of incidence, the underlying mechanisms of neuropathic pain are still poorly understood. However, recent evidence supports the prominent role of spinal glial cells in neuropathic pain states. In our laboratory, we observed that najanalgesin, a novel peptide isolated from the venom of Naja naja atra, exerts significant analgesic effects on acute pain in mice and neuropathic pain in rats. The objective of the present study was to determine whether spinal glia are associated with the antinociceptive effect of najanalgesin in an L5 spinal nerve ligation (SNL) rodent model of neuropathic pain. Mechanical allodynia developed after surgery, and hypersensitivity was significantly attenuated by the intrathecal administration of najanalgesin. The inhibitory effect of najanalgesin was significantly (p<0.05) enhanced after pretreatment with fluorocitrate (a glial cell antagonist). In addition, the astrocyte activation was attenuated following najanalgesin treatment in the dorsal horn of neuropathic rats, as assessed by immunohistology and Western blotting. The tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß) content of cerebral spinal fluid and cell culture supernatants changed significantly after najanalgesin administration. The results suggest that najanalgesin may exert its anti-allodynic effect by altering astrocyte cell function.