GLP-1R activation ameliorated novel-object recognition memory dysfunction via regulating hippocampal AMPK/NF-κB pathway in neuropathic pain mice.

Growing evidences indicate that neuropathic pain is frequently accompanied with cognitive impairments, which aggravate the decrease in the quality of life of chronic pain patients. Furthermore, it has been shown that the activation of Glucagon-like-peptide-1receptor (GLP-1R) improved memory deficit in multiple diseases, including Alzheimer's disease (AD), stroke. However, whether GLP-1R activation could improve memory impairment induced by neuropathic pain and the mechanisms underlying the effect of the activation of GLP-1R on memory protection have not yet been established. The spared nerve injury (SNI) model was established as a kind of neuropathic pain. And novel-object recognition memory (hippocampus-dependent memory) was tested by the novel object recognition test (NORT). The expression levels of GLP-1, GLP-1R, adenosine monophosphate-activated protein kinase (AMPK), p-AMPKThr172, nuclear factor κ B p65 (NF-κB p65), interleukin-1beta (IL-1ß), IL-1ß p17 (mature IL-1ß), tumor necrosis factor-alpha (TNF-α) and the synaptic proteins were tested in the murine hippocampus with memory deficits caused by neuropathic pain. Then, exenatide acetate (Ex-4, a GLP-1R agonist), exendin (9-39) (Ex(9-39), a GLP-1R antagonist) and Compound C dihydrochloride (CC, an AMPK inhibitor) were used to test the effects of the activation of GLP-1R in the mice with neuropathic pain. First, we uncovered that neuropathic pain could inhibit GLP-1/GLP-R axis, disturb inflammatory signaling pathway, increase the expression of IL-1ß, IL-1ß p17 and TNF-α, downregulate the synaptic proteins (postsynaptic density protein 95 (PSD95) and Arc). Subsequently, we reported that Ex-4 treatment could improve recognition memory impairment, increase the ratio of p-AMPKThr172/AMPK, inhibit the phosphorylation NF-κB p65 and decrease the expression of IL-1ß, IL-1ß p17 and TNF-α, upregulate the levels of PSD95 and Arc. Moreover, we found that Ex(9-39) and CC treatment could abrogate the memory protection of activation of GLP-1R in mice with neuropathic pain. The results indicated that the activation of GLP-1R could improve recognition memory impairment via regulating AMPK/NF-κB pathway, improving neuroinflammation, reversing the decreased level of synaptic proteins in neuropathic pain mice.

Targeting α7 nicotinic acetylcholine receptors for chronic pain.

Despite rapid advances in the field of chronic pain, it remains extremely challenging in the clinic. Pain treatment strategies have not improved for decades as opioids remain the main prescribed drugs for chronic pain management. However, long-term use of opioids often leads to detrimental side effects. Therefore, uncovering the mechanisms underlying the development and maintenance of chronic pain may aid the discovery of novel therapeutics to benefit patients with chronic pain. Substantial evidence indicates downregulation of α7 nicotinic acetylcholine receptors (α7 nAChR) in the sciatic nerve, dorsal root ganglia, and spinal cord dorsal horn in rodent models of chronic pain. Moreover, our recent study and results from other laboratories demonstrate that potentiation of α7 nAChR attenuates pain behaviors in various murine models of chronic pain. This review summarized and discussed the preclinical evidence demonstrating the therapeutic potential of α7 nAChR agonists and allosteric modulators in chronic pain. This evidence indicates that potentiation of α7 nAChR is beneficial in chronic pain, mostly by alleviating neuroinflammation. Overall, α7 nAChR-based therapy for chronic pain is an area with great promise, but more research regarding its detailed mechanisms is warranted.

Dysfunction of the Brain-derived Neurotrophic Factor-Tyrosine Kinase B Signaling Pathway Contributes to Learning and Memory Impairments Induced by Neuroinflammation in Mice.

Accumulating evidence suggests that neuroinflammation is the main mechanism in cognitive dysfunction and that brain-derived neurotrophic factor (BDNF) is involved in learning and memory by binding to tyrosine kinase B (TrkB) receptors. Herein, we tested the roles of the BDNF-TrkB signaling pathway and its downstream cascade in lipopolysaccharide (LPS) induced cognitive dysfunction in mice. Mice were treated with LPS (0.25 mg/kg) for 7 days, and learning and memory function was evaluated by the novel object recognition test (NORT). Western blotting was performed to elucidate roles of the BDNF-TrkB signaling pathway and its downstream cascades in LPS mice. The NORT showed that LPS induced learning and memory deficits in mice. The levels of IL-1ß, IL-6, and TNF-α in the serum and central nervous system decreased in LPS mice. In addition, LPS reduced the protein levels of BDNF, p-TrkB, Bcl-2, p-ERK1/2, p-CaMK2, p-CREB and p-GluR1 and increased the expression of Bax in the hippocampus and medial prefrontal cortex regions. In the entorhinal cortex, the protein levels of BDNF, p-TrkB, Bcl-2, p-CaMK2 and p-CREB were decreased, and the protein level of Bax was increased in LPS mice. Interestingly, 7,8-DHF alleviated these disorders in LPS mice and improved learning and memory function; however, the TrkB antagonist ANA12 effectively reversed effects of 7,8-DHF. Therefore, we conclude that the BDNF-TrkB signaling pathway and its downstream cascades disorders in different regions are main mechanisms of cognitive dysfunction, and 7,8-DHF maybe useful as a new treatment for preventing or treating cognitive dysfunction induced by neuroinflammation in neurodegenerative diseases.

Wnt signaling: A prospective therapeutic target for chronic pain.

Despite the rapid advance over the past decades to design effective therapeutic pharmacological interventions, chronic pain remains to be an unresolved healthcare concern. Long term use of opioids, the first line analgesics, often causes detrimental side effects. Therefore, a profound understanding of the mechanisms underlying the development and maintenance of chronic pain states is urgently needed for the management of chronic pain. Substantial evidence indicates aberrant activation of Wnt signaling pathways in sciatic nerve, dorsal root ganglia and spinal cord dorsal horn in rodent models of chronic pain. Moreover, growing evidence shows that pharmacological blockage of aberrant activation of Wnt signaling pathways attenuates pain behaviors in animal models of chronic pain. Importantly, both intrathecal injection of Wnt agonists and Wnt ligands to naïve rats lead to the development of mechanical allodynia, which was inhibited by Wnt inhibitors. In this review, we summarized and discussed the therapeutic potential of pharmacological inhibitors of Wnt signaling in chronic pain in preclinical studies. These evidence showed that aberrant activation of Wnt signaling pathways contributed to chronic pain via enhancing neuroinflammation, regulating synaptic plasticity and reducing intraepidermal nerve fiber density. However, these findings raise further questions. Overall, despite the future challenges, these pioneering studies suggest that Wnt signaling is a promising therapeutic target for chronic pain.

The Emerging Role of Quercetin in the Treatment of Chronic Pain.

Despite much research efforts being devoted to designing alternative pharmacological interventions, chronic pain remains to be an unresolved clinical problem. Quercetin, a compound that belongs to the flavonoids family, is abundantly found in fruits and vegetables. Emerging evidence indicates that quercetin possesses anti-nociceptive effects in different rodent models of chronic pain, including inflammatory pain, neuropathic pain and cancer pain. In this review, we summarize the mechanisms underlying the analgesic effect of quercetin in preclinical studies. These studies showed that quercetin exerts potent analgesic effects against chronic pain via suppressing neuroinflammation and oxidative stress as well as modulation of synaptic plasticity, GABAergic system, and opioidergic system. Considering that the safety of quercetin is well established, it has great potential for clinical use in pain treatment.

5-HT1F Receptor Agonist Ameliorates Mechanical Allodynia in Neuropathic Pain via Induction of Mitochondrial Biogenesis and Suppression of Neuroinflammation.

Neuropathic pain is a devastating disease that affects millions of people worldwide. Serotonin (5-hydroxytryptamine, 5-HT) is involved in pain modulation. Several lines of evidence have indicated that 5-HT1F receptor agonists are potent inducers of mitochondrial biogenesis. In this study, we tested the hypothesis that 5-HT1F receptor agonists ameliorate mechanical allodynia in neuropathic pain via the induction of mitochondrial biogenesis and suppression of neuroinflammation. Male Sprague-Dawley rats were used to establish a neuropathic pain model via spared nerve injury (SNI). The paw withdrawal threshold (PWT) was used to evaluate mechanical allodynia. Real-time polymerase chain reaction was used to examine the mitochondrial DNA (mtDNA) copy number. Western blotting and immunofluorescence were used to examine the expression of target proteins. Our results showed that mitochondrial biogenesis was impaired in the spinal cord of rats with SNI. Moreover, activation of PGC-1α, the master regulator of mitochondrial biogenesis, attenuates established mechanical allodynia in rats with neuropathic pain. In addition, the neuronal 5-HT1F receptor is significantly downregulated in the spinal cord of rats with neuropathic pain. Furthermore, the selective 5-HT1F receptor agonist lasmiditan attenuated established mechanical allodynia in rats with neuropathic pain. Finally, lasmiditan (Las) treatment restored mitochondrial biogenesis and suppressed neuroinflammation in the spinal cord of rats with SNI. These results provide the first evidence that lasmiditan ameliorates mechanical allodynia in neuropathic pain by inducing mitochondrial biogenesis and suppressing neuroinflammation in the spinal cord. Inducers of mitochondrial biogenesis may be an encouraging therapeutic option for the management of neuropathic pain.

The Role of the GABAergic System in Diseases of the Central Nervous System.

It is well known that the central nervous system (CNS) is a complex neuronal network and its function depends on the balance between excitatory and inhibitory neurons. Disruption of the excitatory/inhibitory (E/I) balance is the main cause for the majority of the CNS diseases. In this review, we will discuss roles of the inhibitory system in the CNS diseases. The GABAergic system as the main inhibitory system, is essential for the appropriate functioning of the CNS, especially as it is engaged in the formation of learning and memory. Many researchers have reported that the GABAergic system is involved in regulating synaptic plasticity, cognition and long-term potentiation. Some clinical manifestations (such as cognitive dysfunctions, attention deficits, etc.) have also been shown to emerge after abnormalities in the GABAergic system accompanied with concomitant diseases, that include Alzheimer's disease (AD), Parkinson's disease (PD), Autism spectrum disorder (ASD), Schizophrenia, etc. The GABAergic system consists of GABA, GABA transporters, GABAergic receptors and GABAergic neurons. Changes in any of these components may contribute to the dysfunctions of the CNS. In this review, we will synthesize studies which demonstrate how the GABAergic system participates in the pathogenesis of the CNS disorders, which may provide a new idea that might be used to treat the CNS diseases.

The therapeutic potential of Nrf2 inducers in chronic pain: Evidence from preclinical studies.

Chronic pain remains an enormous health problem affecting approximatively 30% of the world's population. Opioids as the first line analgesics often leads to undesirable side effects when used long term. Therefore, novel therapeutic targets are urgently needed to the development of more efficacious analgesics. Substantial evidence indicates that excessive reactive oxygen species (ROS) are extremely important to the development of chronic pain. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcription factor regulating endogenous antioxidant defense. Emerging evidence suggests that Nrf2 and its downstream effectors are implicated in chronic inflammatory and neuropathic pain. Notably, controversial results have been reported regarding the expression of Nrf2 and its downstream targets in peripheral and central regions involved in pain transmission. However, our recent studies and results from other laboratories demonstrate that Nrf2 inducers exert potent analgesic effects in various murine models of chronic pain. In this review, we summarized and discussed the preclinical evidence demonstrating the therapeutic potential of Nrf2 inducers in chronic pain. These evidence indicates that Nrf2 activation are beneficial in chronic pain mostly by alleviating ROS-associated pathological processes. Overall, Nrf2-based therapy for chronic pain is an area with great promise, but more research regarding its detailed mechanisms is warranted.

Src-family protein tyrosine kinases: A promising target for treating chronic pain.

Despite the growing knowledge of the mechanisms of chronic pain, the treatment of this disorder in the clinic remains a major challenge. Src-family protein tyrosine kinases (SFKs), a group of non-receptor protein tyrosine kinases, have been implicated in neuronal development and synaptic plasticity. SFKs are critical for the regulate of N-methyl-D-aspartic acid receptor (NMDAR) 2B subunit phosphorylation by various transmembrane receptors, e.g., G-protein coupled receptors (GPCRs), EphB receptors (EphBRs), increased intracellular calcium, epidermal growth factor (EGF) and other growth factors, and thus contribute to the development of chronic pain. SFKs have also been regarded as important points of convergence of intracellular signalling components for the regulation of microglial functions and the immune response. Additionally, the intrathecal administration of SFK inhibitors significantly alleviates mechanical allodynia in different chronic pain models. Here, we reviewed the current evidence for the role of SFKs in the development of chronic pain caused by complete Freund's adjuvant (CFA) injection, peripheral nerve injury (PNI), streptozotocin (STZ) injection and bone metastasis. Moreover, the role of SFKs in the development of morphine tolerance is also discussed. The regulation of SFKs therefore has emerged as a potential therapeutic target for the treatment of chronic pain in terms of safety and efficacy.

Disruption of the GABAergic system contributes to the development of perioperative neurocognitive disorders after anesthesia and surgery in aged mice.

AIMS: Perioperative neurocognitive disorders (PND) are associated with cognitive impairment in the preoperative or postoperative period, and neuroinflammation is thought to be the most important mechanisms especially during the postoperative period. The GABAergic system is easily disrupted by neuroinflammation. This study investigated the impact of the GABAergic system on PND after anesthesia and surgery. METHODS: An animal model of laparotomy with inhalation anesthesia in 16-month-old mice was addressed. Effects of the GABAergic system were assessed using biochemical analysis. Pharmacological blocking of α5GABAA Rs or P38 mitogen-activated protein kinase (MAPK) were applied to investigate the effects of the GABAergic system. RESULTS: After laparotomy, the hippocampus-dependent memory and long-term potentiation were impaired, the levels of IL-6, IL-1ß and TNF-α up-regulated in the hippocampus, the concentration of GABA decreased, and the protein levels of the surface α5GABAA Rs up-regulated. Pharmacological blocking of α5GABAA Rs with L655,708 alleviated laparotomy induced cognitive deficits. Further studies found that the P38 MAPK signaling pathway was involved and pharmacological blocking with SB203,580 alleviated memory dysfunctions. CONCLUSIONS: Anesthesia and surgery caused neuroinflammation in the hippocampus, which consequently disrupted the GABAergic system, increased the expressions of surface α5GABAA Rs especially through the P38 MAPK signaling pathway, and eventually led to hippocampus-dependent memory dysfunctions.

[Construction of rat constitutively active recombinant caspase-3 and investigation of its pro-apoptotic effect in vitro].

OBJECTIVE: To investigate the apoptosis induction effect by the rat recombinant caspase-3. METHODS: Reversed rat caspase-3 gene was gained by settling the small subunit prior to the large one through recombinant PCR, and cloned into the expression vector to transfect human 293T cells and rat immortalized neural progenitor cells. The expression and pro-apoptotic effect of recombinant caspase-3 was observed by the changes of morphology of the transfected cells through immunofluorescence and analyzed by Annexin V-FITC staining, Flowcytometry and MTT assay. RESULTS: The transfected cells presented obvious apoptosis as detected by immunofluorescence. MTT assay showed that the proliferation of recombinant caspase-3 transfected cells was significantly inhibited [(48.35 +/- 0.16)%, (44.61 +/- 0.15)%] (P < 0.05). Annexin V-FITC staining revealed that the percentage of apoptotic cells in the transfectants of recombinant caspase-3 gene was [(30.7 +/- 1.5)%, (16.0 +/- 1.0)%] (P < 0.05), which was much higher than that of control cells. CONCLUSIONS: Rat recombinant caspase-3 can be expressed and induce apoptosis effectively, and used safely both in vitro and in vivo. It can also cause neural cells to death.

The therapeutic potential of GABA in neuron-glia interactions of cancer-induced bone pain.

The development of effective therapeutics for cancer-induced bone pain (CIBP) remains a tremendous challenge owing to its unclear mechanisms. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). Emerging studies have shown that disinhibition in the spinal cord dorsal horn may account for the development of chronic pain. However, the role of GABA in the development of CIBP remains elusive. In addition, accumulating evidence has shown that neuroglial cells in the peripheral nervous system, especially astrocytes and microglial cells, played an important role in the maintenance of CIBP. In this study, we investigated the expression of GABA and Gamma-aminobutyric acid transporter-1 (GAT-1), a transporter of GABA. Our results demonstrate that GABA was decreased in CIBP rats as expected. However, the expression of glutamic acid decarboxylase (GAD) 65 was up-regulated on day 21 after surgery, while the expression of GAD 67 remained unchanged after surgery. We also found that the expression of GAT-1 was up-regulated mainly in the astrocytes of the spinal cord. Moreover, we evaluated the analgesic effect of exogenous GABA and the GAT-1 inhibitor. Intrathecal administration of exogenous GABA and NO-711 (a GAT-1 selective inhibitor) significantly reversed CIBP-induced mechanical allodynia in a dose-dependent manner. These results firstly show that neuron-glia interactions, especially on the GABAergic pathway, contribute to the development of CIBP. In conclusion, exogenous GABA and GAT-1 inhibitor might be alternative therapeutic strategies for the treatment of CIBP.

PGE2-EP3 signaling exacerbates hippocampus-dependent cognitive impairment after laparotomy by reducing expression levels of hippocampal synaptic plasticity-related proteins in aged mice.

AIM: Multifactors contribute to the development of postoperative cognitive dysfunction (POCD), of which the most important mechanism is neuroinflammation. Prostaglandin E2 (PGE2) is a key neuroinflammatory molecule and could modulate hippocampal synaptic transmission and plasticity. This study was designed to investigate whether PGE2 and its receptors signaling pathway were involved in the pathophysiology of POCD. METHODS: Sixteen-month old male C57BL/6J mice were exposed to laparotomy. Cognitive function was evaluated by fear conditioning test. The levels of PGE2 and its 4 distinct receptors (EP1-4) were assessed by biochemical analysis. Pharmacological or genetic methods were further applied to investigate the role of the specific PGE2 receptors. RESULTS: Here, we found that the transcription and translation level of the EP3 receptor in hippocampus increased remarkably, but not EP1, EP2, or EP4. Immunofluorescence results showed EP3 positive cells in the hippocampal CA1 region were mainly neurons. Furthermore, pharmacological blocking or genetic suppression of EP3 could alleviate surgery-induced hippocampus-dependent memory deficits and rescued the expression of plasticity-related proteins, including cAMP response element-binding protein (CREB), activity-regulated cytoskeletal-associated protein (Arc), and brain-derived neurotrophic factor (BDNF) in hippocampus. CONCLUSION: This study showed that PGE2-EP3 signaling pathway was involved in the progression of POCD and identified EP3 receptor as a promising treatment target.

Construction and identification of immortalized rat astrocyte cell line expressing enkephalin.

OBJECTIVE: To provide a sound cell source for further ex-vivo gene therapy for chronic pain, we attempt to develop an immortalized rat astrocyte cell line that expresses enkephalin regulated by doxycycline. METHODS: Retrovirus infection method was employed to develop an immortalized rat astrocyte cell line that could express enkephalin regulated by doxycycline. The hPPE gene expression level of immoralized astroyte cells (IAC)/ hPPE was detected by RT-PCR, indirect immunofluorescence staining and radioimmunoassay. RESULTS: IAC carrying Tet-on system transfected with preproenkephalin gene could secrete enkephalin that was regulated by doxycycline in a dose-dependent manner and hPPE gene activation could be repeated in on-off-on cycles through administration or removal of doxycycline. CONCLUSION: An immortalized rat astrocyte cell line that secrete enkephalin under the control of doxycycline is established successfully, which provides a research basis for transgenic cell transplantation for analgesia.

[The cellular location and significance of p38alpha/beta isoforms in the lumbar spinal cord of the bone cancer pain rats].

OBJECTIVE: To examine the cellular distribution and location of p38alpha/beta isoforms in the lumbar spinal cord of the bone cancer pain rats. METHODS: Twenty SD female rats weighing 180 - 220 g were randomly divided into 2 groups (n = 10 each): group A (control group): intra-tibial injection of 3 microl Hank's solution; group B (model group): intra-tibial injection of 3 microl MADB-106 mammary gland carcinoma cells of rats (4.8 x 10(3)/microl). Mechanical withdrawal threshold and radiant heat threshold of rats' hind paws were measured every other day from one day before operation until 14 days later. The lumbar 4 ~ 5 spinal cord was removed on the 14th day. The cellular distribution and location of the spinal p38alpha/beta immunoreactivity were detected by immunohistochemistry SABC and double immunofluorescence methods. RESULTS: No significant differences in mechanical withdrawal threshold and radiant heat threshold were found at all time points in group A. During the first 6 days of post-operation there was obvious difference in radiant heat stimulus between group A and B (P < 0.05); on the 14th day after operation, mechanical pain threshold and radiant heat threshold in group B were significantly different from that of group A (P < 0.05). The p38alpha/beta immunoreactivity of group B in laminae I approximately IV of dorsal horn showed stronger staining than group A (P < 0.05). Double immunofluorescence confocal micrographs showed that spinal p38alpha and the neuronal marker neuronal N (NeuN) were colocalized in the dorsal horn, indicating that p38alpha was expressed in neurons. Double immunofluorescence micrographs demonstrated that antibodies against p38beta and the microglia marker OX42 labeled the same cell, indicating that p38beta was expressed in microglia. CONCLUSION: Our studies indicate that p38alpha and p38beta are involved in the generation and maintenance of bone cancer pain states. p38alpha is predominantly expressed in neurons, while p38beta is expressed in microglia.