Myocardial infarction (MI) is characterized by a paucity of cardiomyocyte regeneration, leading to significant morbidity and mortality. Contemporary therapeutic modalities, while mitigating ischemic effects, fail to reconstitute the impaired electromechanical coupling within the infracted myocardium. Emerging evidence supports the utility of electroconductive hydrogels (ECHs) in facilitating post-MI cardiac function recovery by restoring the conductive microenvironment of the infarcted tissue. This comprehensive review delineates the taxonomy of ECHs predicated on their constituent conductive materials. It also encapsulates prevailing research trends in ECH-mediated MI repair, encompassing innovative design paradigms and microenvironment-sensitive strategies. The review also provides a critical appraisal of various implantation techniques, underscored by a thorough examination of the attendant considerations. It elucidates the mechanistic underpinnings by which hydrogels exert salutary effects on myocardial repair, namely by augmenting mechanical and electrical integrity, exerting anti-inflammatory actions, fostering angiogenesis, and curtailing adverse remodeling processes. Furthermore, the review engages with the pressing challenge of optimizing ECH functionality to achieve superior reparative outcomes post-MI. The discourse concludes with an anticipatory perspective on the evolution of ECH scaffolds, advocating for a tailored approach that integrates multifaceted physicochemical properties to cater to the nuances of personalized medicine.

G protein-coupled receptor 75 (GPR75), a novel member of the rhodopsin-like G protein-coupled receptor (GPCR) family, has been identified across various tissues and organs, where it contributes to biological regulation and disease progression. Recent studies suggest potential interactions between GPR75 and ligands such as 20-hydroxyeicosatetraenoic acid (20-HETE) and C-C motif chemokine ligand 5 (CCL5/RANTES); however, its definitive endogenous ligand remains unidentified, and GPR75 is currently classified as an orphan receptor by International Union of Basic and Clinical Pharmacology (IUPHAR). Research on GPR75 deorphanization has underscored its critical roles in disease models, particularly in metabolic health, glucose regulation, and stability of the nervous and cardiovascular systems. However, the signaling pathways of GPR75 across different pathological conditions require further investigation. Importantly, ongoing studies are targeting GPR75 for drug development, exploring small molecule inhibitors, antibodies, and gene silencing techniques, positioning GPR75 as a promising GPCR target for treating related diseases. This review summarizes the recent advancements in GPR75 deorphanization research, examines its functions across tissues and systems, and highlights its links to metabolic, cardiovascular, and neurological disorders, thereby providing a resource for researchers to better understand the biological functions of this receptor.

Cerebrovascular diseases (CVDs) have become a global public health problem and ischemia‑reperfusion injury, the major cause of neurological impairment exacerbation, is closely related to excitotoxicity. The present study aimed to investigate the effects of changes in heat shock protein (HSP)90β expression and verify whether HSP90β regulates EAAT2 expression in a cerebral ischemia‑reperfusion injury model. Healthy adult Sprague‑Dawley (SD) male rats were used to establish a control group, sham‑operated group, middle cerebral artery occlusion (MCAO) group, empty virus group and lentivirus group. A model of cerebral ischemia‑reperfusion was established using the MCAO method. Lentivirus construction and injection were used to interfere with the expression of HSP90β. The modified neurological severity score was used to assess neurological deficits. Triphenyltetrazolium chloride staining was used to detect infarct areas. Immunofluorescence was used to detect HSP90β expression localization and the expression levels of HSP90β and EAAT2 were determined using western blotting and reverse transcription‑quantitative PCR. An MCAO model was successfully established and it was found that HSP90β, but not HSP90α, was upregulated after MCAO. HSP90β expression coincided with astrocyte markers in the ischemic penumbra area, while no expression was observed in microglia. Inhibition of HSP90β expression improved neurological deficits and alleviated brain injury by increasing EAAT2 expression. These results suggested that HSP90β is involved in the process of cerebral ischemia‑reperfusion injury in rats and that inhibition of HSP90β expression increases EAAT2 levels, conferring a neuroprotective effect in MCAO model rats.

The periaqueductal gray (PAG) is an important relay center for the descending pathways that regulate nociceptive information transduction. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play critical roles in the nerve injury-induced pain hypersensitivity. Previous studies have identified that HCN1 and HCN2 channel protein located in the ventral-lateral periaqueductal gray (vlPAG), a region important for pain regulation. However, it is not clear whether the HCN channel in vlPAG is involved in bone cancer pain (BCP). In this study, we assessed the role of HCN channels in BCP by measuring changes of HCN channel expression and activity in vlPAG neurons in bone cancer rats. In the present study, the BCP model was established by injecting SHZ-88 breast cancer cells into the right tibia bone marrow in rats. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured to evaluate pain behavior in rats. HCN1 and HCN2 channels expression in vlPAG were detected by using Western Blot and immunohistochemistry. In addition, the cAMP level in vlPAG neurons was detected by ELISA, and HCN channel current (I h ) of vlPAG neurons was recorded by whole cell patch-clamp to evaluate HCN channel activity. As a result, decreased MWT and TWL were observed in rats on 7d after SHZ-88 cell inoculation, and the allodynia was sustained until 21d after inoculation. At the same time, HCN1 and HCN2 channels expression and neuronal I h in vlPAG were significantly increased in BCP rats. In addition, the level of cAMP in vlPAG also increased after SHZ-88 cell inoculation. Furthermore, intravlPAG injection of ZD7288 (HCN channels antagonist) could significantly reduce hyperalgesia and the elevation of cAMP in vlPAG in BCP rats. Our observations suggest that the elevation of cAMP may promote the activation of HCN channels in vlPAG in bone cancer rats, thereby promoting the development of bone cancer pain.

Hyperpolarization-activated cyclic nucleotide-gated channels and purinergic P2X receptors play critical roles in the nerve injury-induced pain hypersensitivity. Both HCN channels and P2XR are expressed in dorsal root ganglia sensory neurons. However, it is not clear whether the expression and function of P2X2 and P2X3 receptors can be modulated by HCN channel activity. For this reason, in rats with chronic constriction injury of sciatic nerve, we evaluated the effect of intrathecal administration of HCN channel blocker ZD7288 on nociceptive behavior and the expression of P2X2 and P2X3 in rat DRG. The mechanical withdrawal threshold was measured to evaluate pain behavior in rats. The protein expression of P2X2 and P2X3 receptor in rat DRG was observed by using Western Blot. The level of cAMP in rat DRG was measured by ELISA. As a result, decreased MWT was observed in CCI rats on 1 d after surgery, and the allodynia was sustained throughout the experimental period. In addition, CCI rats presented increased expression of P2X2 and P2X3 receptor in the ipsilateral DRG at 7 d and 14 d after CCI operation. Intrathecal injection of ZD7288 significantly reversed CCI-induced mechanical hyperalgesia, and attenuated the increased expression of P2X2 and P2X3 receptor in rat DRG, which open up the possibility that the expression of P2X2 and P2X3 receptor in DRG is down-regulated by HCN channel blocker ZD7288 in CCI rats. Furthermore, the level of cAMP in rat DRG significantly increased after nerve injury. Intrathecal administration of ZD7288 attenuated the increase of cAMP in DRG caused by nerve injury. Subsequently, effects of HCN channel activity on ATP-induced current (IATP) in rat DRG neurons were explored by using whole-cell patch-clamp techniques. ATP (100 μM) elicited three types of currents (fast, slow and mixed IATP) in cultured DRG neurons. Pretreatment with ZD7288 concentration-dependently inhibited three types of ATP-activated currents. On the other hand, pretreatment with 8-Br-cAMP (a cell-permeable cAMP analog, also known as an activator of PKA) significantly increased the amplitude of fast, slow and mixed IATP in DRG neurons. The enhanced effect of 8-Br-cAMP on ATP-activated currents could be reversed by ZD7288. In a summary, our observations suggest that the opening of HCN channels could enhance the expression and function of P2X2 and P2X3 receptor via the cAMP-PKA signaling pathway. This may be important for pathophysiological events occurring within the DRG, for where it is implicated in nerve injury-induced pain hypersensitivity.

TLR4 and Cx43 signaling in dorsal spinal cord has been shown to be involved in the development of neuropathic pain. However, it is not clear whether TLR4 signaling is associated with the expression of MCP-1, CXCL1, and Cx43 in LPS (lipopolysaccharide)-treated rat dorsal spinal cord astrocytes under in vitro condition. In the present study, we found that TLR4 antagonist TAK-242 significantly inhibited LPS-induced MCP-1, CXCL1, and Cx43 expression, suggesting the role of TLR4 in response to LPS in cultured dorsal spinal cord astrocytes. Application of TAK-242 significantly blocked LPS-induced NF-κB and AP-1 activity and the expression of MCP-1, CXCL1 and Cx43. Furthermore, NF-κB inhibitor PDTC and AP-1 inhibitor SR11302 significantly blocked LPS-induced MCP-1, CXCL1, and Cx43 expression. DNA-binding activity of NF-κB, its effect on MCP-1 expression was suppressed by PDTC and SR11302. On the other hand, DNA-binding activity of AP-1, its effect on CXCL1 or Cx43 expression was also suppressed by PDTC and SR11302. In addition, PDTC was found to inhibit the nuclear translocation of AP-1 and the expression of c-Jun induced by LPS, which suggested that NF-κBp65 is essential for the AP-1 activity. Similarly, SR11302 significantly blocked LPS-induced the nuclear translocation of NF-κBp65 and the expression of NF-κBp65 induced by LPS. Pretreatment with CBX, Gap26, or Gap19 (Cx43 blockers) significantly inhibited abnormal astrocytic hemichannel opening and chemokines (MCP-1 and CXCL1) release in LPS-stimulated astrocytes. In summary, cell culture experiments revealed that LPS stimulation could evoke TLR4 signaling with the subsequent activation of NF-κB and AP-1, resulting in the expression of MCP-1, CXCL1, and Cx43. TLR4 activation increased Cx43 hemichannel, but not gap-junction activities and induced the release of the MCP-1 and CXCL1 from astrocytes via Cx43 hemichannel. These findings may help us to understand the role of astrocytic signaling in inflammatory response within dorsal spinal cord tissue.

Recent reports have suggested that abnormal miR-29c expression in hippocampus have been implicated in the pathophysiology of some neurodegenerative and neuropsychiatric diseases. However, the underlying effect of miR-29c in regulating hippocampal neuronal function is not clear. In this study, HT22 cells were infected with lentivirus containing miR-29c or miR-29c sponge. Cell counting kit-8 (CCK8) and lactate dehydrogenase (LDH) assay kit were applied to evaluate cell viability and toxicity before and after TNF-α administration. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Hoechst 33258 staining and TUNEL assay were used to evaluate cell apoptosis. The expression of key mRNA/proteins (TNFR1, Bcl-2, Bax, TRADD, FADD, caspase-3, -8 and -9) in the apoptosis pathway was detected by PCR or WB. In addition, the protein expression of microtubule-associated protein-2 (MAP-2), nerve growth-associated protein 43 (GAP-43) and synapsin-1 (SYN-1) was detected by WB. As a result, we found that miR-29c overexpression could improve cell viability, attenuate LDH release, reduce ROS production and inhibit MMP depolarization in TNF-α-treated HT22 cells. Furthermore, miR-29c overexpression was found to decrease apoptotic rate, along with decreased expression of Bax, cleaved caspase-3, cleaved caspase-9, and increased expression of Bcl-2 in TNF-α-treated HT22 cells. However, miR-29c sponge exhibited an opposite effects. In addition, in TNF-α-treated HT22 cells, miR-29c overexpression could decrease the expressions of TNFR1, TRADD, FADD and cleaved caspase-8. However, in HT22 cells transfected with miR-29c sponge, TNF-α-induced the expressions of TNFR1, TRADD, FADD and cleaved caspase-8 was significantly exacerbated. At last, TNF-α-induced the decreased expression of MAP-2, GAP-43 and SYN-1 was reversed by miR-29c but exacerbated by miR-29c sponge. Overall, our study demonstrated that miR-29c protects against TNF-α-induced HT22 cells injury through alleviating ROS production and reduce neuronal apoptosis. Therefore, miR-29c might be a potential therapeutic agent for TNF-α accumulation and toxicity-related brain diseases.

BACKGROUND Despite previous reports on cerebral structures and functional connectivity in patients with myofascial pain (MFP), it is not clear whether alterations in neurovascular coupling occur in these patients. OBJECTIVES We analyzed the coupling between resting-state cerebral blood flow (CBF) and functional connectivity strength (FCS) for observation of neurovascular coupling in patients with chronic MFP. STUDY DESIGN Observational study. SETTING University hospital. METHODS Resting-state functional magnetic resonance imaging and arterial spin labeling were performed in 23 patients with chronic MFP and 23 healthy controls (HC) for the calculation of FCS and CBF. The whole-brain gray matter CBF-FCS correlations and CBF/FCS ratios of the various voxels of the 2 groups were subsequently compared. RESULTS Compared with the HC, the patients with MFP experienced a decrease in whole-brain gray matter CBF-FCS coupling. In patients with MFP, a decrease in CBF/FCS was found in the bilateral superior temporal gyri, right parahippocampal gyrus, right hippocampus, caudate nucleus, right medial prefrontal cortex, and the periaqueductal gray matter (PAG), whereas an increase in CBF/FCS was found in the bilateral lingual gyri, posterior cingulate cortex, and bilateral inferior parietal lobules. In addition, the CBF/FCS of the PAG in patients with MFP was significantly negatively correlated with the pain visual analog scale score and pain duration. LIMITATIONS Alterations in neurovascular coupling in patients with MFP were observed only before treatment. Therefore, there is a lack of data on the alterations that occurred after treatment. CONCLUSION This study demonstrated for the first time that impairment of neurovascular coupling in the brain may be a potential neuropathological mechanism of chronic MFP.

The cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signaling acts a pivotal part in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels-mediated neuropathic and inflammatory pain. However, there has been no evidence of cAMP-PKA signaling is involved in regulation of spinal HCN channels function in the occurrence of diabetic neuropathic pain (DNP). The study aimed to elucidate the impact of HCN channels on neuropathic pain in a rat model of diabetes induced by streptozotocin, and whether cAMP-PKA signaling is involved in regulation of HCN channels function. In this report, we evaluated the effect of intrathecal administration of HCN channel blockers ZD7288, cAMP inhibitor SQ22536 and PKA inhibitor H-89 on nociceptive behavior in DNP rats. The mechanical withdrawal threshold (MWT) was measured to evaluate pain behavior in rats. Protein expression levels of HCN2, HCN4 channels and PKA in the spinal dorsal horn of rats were assessed. Furthermore, the levels of cAMP in rat spinal dorsal horn was analyzed. We discovered that DNP rats showed significant mechanical allodynia and are related to the increased HCN2 and HCN4 channels expression, enhanced cAMP production and elevated the expression of PKA protein in the spinal dorsal horn, which were attenuated by intrathecal ZD7288. Furthermore, intrathecal injection of SQ22536 and H-89 significantly reduced the HCN2 and HCN4 channels expression in the spinal dorsal horn of DNP rats. Our findings indicate that HCN channels of the spinal dorsal horn participate in the pathogenesis of allodynia in rats with DNP, which could be regulated by cAMP-PKA signaling. Therefore, HCN channels and cAMP-PKA signaling are potential targets for hyperalgesia treatment in DNP patients.

Epilepsy is a chronic nervous system disease. Excessive increase of the excitatory neurotransmitter glutamate in the body results in an imbalance of neurotransmitters and excessive excitation of neurons, leading to epileptic seizures. Long-term recurrent seizures lead to behavior and cognitive changes, and even increase the risk of death by 2- to 3-fold relative to the general population. Adenosine A1 receptor (A1R), a member of the adenosine system, has notable anticonvulsant effects, and adenosine levels are controlled by the type 1 equilibrative nucleoside transporter (ENT1); in addition the p38 MAPK signaling pathway is involved in the regulation of ENT1, although the effect of its inhibitors on the expression levels of A1R and ENT1 is unclear. Therefore, in the present study, SB203580 was used to inhibit the p38 MAPK signaling pathway in rats, and the expression levels of A1R and ENT1 in the brain tissue of rats with acute LiCl-pilocarpine-induced status epilepticus was detected. SB203580 decreased pathological damage of hippocampal neurons, prolonged seizure latency, reduced the frequency of seizures, and decreased levels of A1R and ENT1 protein in rats.

Background: OLFM3 (olfactomedin-3) is a member of the olfactomedin domain family, which has been found to stimulate the formation and adhesion of tight cell connections and to regulate cytoskeleton formation and cell migration. Differences in the gene coding for OLFM3 have been found between patients with epilepsy and controls. However, the exact role of OLFM3 in epilepsy has not been thoroughly investigated. Methods: Biochemical methods were used to assess OLFM3 expression and localization in the cortex of patients with temporal lobe epilepsy and in the hippocampus and cortex of epileptic mice. Electrophysiological recordings were used to measure the role of OLFM3 in regulating hippocampal excitability in a model of magnesium-free-induced seizure in vitro. Behavioral experiments were performed in a pentylenetetrazol (PTZ)-induced seizure model, and electroencephalograms (EEGs) were recorded in the chronic phase of the kainic acid (KA)-induced epilepsy model in vivo. OLFM3 and its interaction with AMPAR (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor) subunits were analyzed by co-immunoprecipitation. Results: The expression of OLFM3 was increased in the cortex of patients with temporal lobe epilepsy and in the hippocampus and cortex of epileptic mice compared with controls. Interestingly, lentivirus-mediated overexpression of OLFM3 in the hippocampus increased the susceptibility of mice to PTZ-induced seizures, and OLFM3 knockdown had the opposite effect. OLFM3 affected AMPAR currents in a brain-slice model of epileptiform activity induced by Mg2+-free medium. We found that OLFM3 co-immunoprecipitation with GluA1 and GluA2. Furthermore, downregulation or overexpression of OLFM3 in the hippocampus affected the membrane expression of GluA1 and GluA2 in epileptic mice. Conclusion: These findings reveal that OLFM3 may enhance seizure activity by interacting with GluA1 and GluA2, potentially indicating a molecular mechanism for new therapeutic strategies.

Background: The importance of P2X purinoceptors, CB2 receptor and microRNA-124(miR-124) in spinal cord microglia to the development of neuropathic pain was demonstrated in numerous previous studies. The upregulation of P2X 4 and P2X 7 receptors in spinal dorsal horn microglia is involved in the development of pain behavior caused by peripheral nerve injury. However, it is not clear whether the expression of P2X 4 and P2X 7 receptors at dorsal spinal cord will be influenced by CB2 receptor or miR-124 in rats after chronic sciatic nerve injury. Methods: Chronic constriction injury (CCI) of the sciatic nerve was performed in rats to induce neuropathic pain. Tests of the mechanical withdrawal threshold (MWT) were carried out to assess the response of the paw to mechanical stimulus. The expression of miR-124, P2X 4 , P2X 7 and CB2 receptor were detected with RT-PCR. The protein expression of P2X 4 , P2X 7 and CB2 receptor, RhoA, ROCK1, ROCK2, p-p38MAPK and p-NF-kappaBp65 was detected with Western blotting analysis. Results: Intrathecal administration of CB2 receptor agonist AM1241 significantly attenuated CCI-induced mechanical allodynia and significantly inhibited the increased expression of P2X 4 and P2X 7 receptors at the mRNA and protein levels, which imply that P2X 4 and P2X 7 receptors expression are down-regulated by AM1241 in CCI rats. Western blot analysis showed that AM1241 suppressed the elevated expression of RhoA, ROCK1, ROCK2, p-p38MAPK and NF-κBp65 in the dorsal spinal cord induced by CCI. After administration with Y-27632 (ROCK inhibitor), SB203580 (P38MAPK inhibitor) or PDTC (NF-κB inhibitor), the levels of P2X 4 and P2X 7 receptors expression in the dorsal spinal cord were lower than those in CCI rats, which imply that the ROCK/P38MAPK pathway and NF-κB activation may contribute to the increased expression of P2X 4 and P2X 7 receptor. On the other hand, in CCI rats, AM1241 treatment evoked the increased expression of CB2 receptor and miRNA-124, which can be inhibited by intrathecal injection of CB2 receptor antagonist AM630, which indicate that the increased expression of miRNA-124 may be medicated by CB2 receptor activation. In addition, the increased expression of P2X 4 and P2X 7 receptors in the dorsal spinal cord of CCI rats were inhibited by miRNA-124 agomir. Furthermore, intrathecal injection of miRNA-124 agomir could efficiently inhibit the ROCK/P38MAPK pathway and NF-κB activation in CCI rats. Moreover, AM1241 treatment significantly inhibited the expression of P2X 4 and P2X 7 receptors, and this suppression is enhanced by pretreatment with miRNA-124 agomir. On the contrast, the inhibitory effect of AM1241 on the expression of P2X 4 and P2X 7 receptor can be reversed by pretreatment with miRNA-124 antagomir. Conclusions: In CCI rats, intrathecal injection of AM1241 could efficiently induce the increased expression of miRNA-124, while inhibiting the ROCK/P38MAPK pathway and NF-κB activation in dorsal spinal cord. CB2 receptor/miRNA-124 signaling induced the decreased P2X 4 and P2X 7 receptors expression via inhibit the ROCK/P38MAPK pathway and NF-κB activation.

DATA REPORT article Front. Mol. Neurosci., 24 October 2019Sec. Pain Mechanisms and Modulators Volume 12 - 2019 | https://doi.org/10.3389/fnmol.2019.00248

Transforming growth factor-β (TGF-β) is a key factor that promotes fibrosis or scar formation, which could become an obstacle in the repair of impaired axons in the central nervous system (CNS) of the human body resulting from diseases or injuries. Considering that major pathological reactions occur during this process, we focused on TGF-secreting M2 macrophages to identify the interactions between M2 macrophages and astrocytes (AS) and verify the specific mechanism of fibrosis or glial scar formation. In the present study, we used the Transwell coculturing technique and found an increase in glial fibrillary acidic protein (GFAP), neurocan, IL-13, and TGF-β expression after incubation for 48 h; the expression of these proteins decreased when additional inhibitors of the TGF-β receptor were added. We concluded that fibrosis or glial scar formation would be enhanced by the secretion of neurocan from AS, resulting from the release of TGF-β from M2 macrophages. We also used M2 macrophage–conditioned medium to further confirm this finding in a subsequent experiment. We hope that the findings in this research could provide a foundation for locating new targets for treating CNS diseases or injuries.

OBJECTIVE:The dorsal horn P2Y13 receptor is involved in the development of pain behavior induced by peripheral nerve injury. It is unclear whether the expression of proinflammatory cytokines interleukin (IL)-1β and IL-6 at the spinal dorsal horn are influenced after the activation of P2Y13 receptor in rats with diabetic neuropathic pain (DNP).METHODS:A rat model of type 1 DNP was induced by intraperitoneal injection of streptozotocin (STZ). We examined the expression of P2Y13 receptor, Iba-1, IL-1β, IL-6, JAK2, STAT3, pTyr1336, and pTyr1472 NR2B in rat spinal dorsal horn.RESULTS:Compared with normal rats, STZ-diabetic rats displayed obvious mechanical allodynia and the increased expression of P2Y13 receptor, Iba-1, IL-1β, and IL-6 in the dorsal spinal cord that was continued for 6 weeks in DNP rats. The data obtained indicated that, in DNP rats, administration of MRS2211 significantly attenuated mechanical allodynia. Compared with DNP rats, after MRS2211 treatment, expression of the P2Y13 receptor, Iba-1, IL-1β, and IL-6 were reduced 4 weeks after the STZ injection. However, MRS2211 treatment did not attenuate the expression of the P2Y13 receptor, Iba-1, IL-1β, and IL-6 at 6 weeks after the STZ injection. MRS2211 suppressed JAK2 and STAT3 expression in the early stage, but not in the later stage. Moreover, pTyr1336 NR2B was significantly decreased, whereas pTyr1472 NR2B was unaffected in the dorsal spinal cord of MRS2211-treated DNP rats.CONCLUSION:Intrathecal MRS2211 produces an anti-nociceptive effect in early-stage DNP. A possible mechanism involved in MRS2211-induced analgesia is that blocking the P2Y13 receptor downregulates levels of IL-1β and IL-6, which subsequently inhibit the activation of the JAK2/STAT3 signaling pathway. Furthermore, blocking the activation of the P2Y13 receptor can decrease NR2B-containing NMDAR phosphorylation in dorsal spinal cord neurons, thereby attenuating central sensitization in STZ-induced DNP rats.

Seizures induce the release of excitatory amino acids (EAAs) from the intracellular fluid to the extracellular fluid, and the released EAAs primarily comprise glutamic acid (Glu) and asparaginic acid (Asp). Glu neurotransmission functions via EAA transporters (EAATs) to maintain low concentrations of Glu in the extracellular space and avoid excitotoxicity. EAAT2, the most abundant Glu transporter subtype in the central nervous system (CNS), plays a key role in the regulation of glutamate transmission. Previous studies have shown that SB203580 promotes EAAT2 expression by inhibiting the p38 mitogen-activated protein kinase (MAPK) signaling pathway, but whether SB203580 upregulates EAAT2 expression in epileptic rats is unknown. This study demonstrated that EAAT2 expression was increased in the brain tissue of epileptic rats. Intraperitoneal injection of a specific inhibitor of p38 MAPK, SB203580, reduced the time to the first epileptic seizure and attenuated the seizure severity. In addition, SB203580 treatment increased the EAAT2 expression levels in the brain tissue of epileptic rats. These results suggest that SB203580 could regulate epileptic seizures via EAAT2.

Purinergic signaling in spinal cord microglia plays an important role in the pathogenesis of neuropathic pain. Among all P2 receptors, P2Y(6) receptor is expressed in rat dorsal spinal cord. However, it's not clear that the role of P2Y(6) receptor in the chronic constriction injury (CCI) model of neuropathic pain rats. We evaluated the effect of repeated intrathecal administration of MRS2578 (selective P2Y(6) receptor antagonist) on CCI-induced nociceptive behaviors in rats. After CCI, MRS2578 (10(-11)-10(-4) M) was administration. The thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were assessed. The expression of P2Y(6) receptor and lba-1 at rat dorsal spinal cord was observed by using RT-PCR. We found that intrathecal injection of MRS2578 suppressed CCI-induced mechanical allodynia and thermal hyperalgesia with a dose-dependent manner. The CCI rats presented increased expression of P2Y(6) receptor and lba-1 at the mRNA level in the ipsilateral dorsal spinal cord than that in sham group. Treatment with either minocycline or SB203580 effectively inhibited P2Y(6) receptor expression compared to CCI rats. Intrathecal injection of UDP enhanced mechanical and thermal allodynia than that in CCI group. To the further study, intrathecal injection of UDP causes mechanical allodynia and thermal hyperalgesia in naive rats. The increased expression of P2Y(6) receptor and lba-1 were observed in UDP-treated rats. Intrathecal injection of MRS2578 alleviates pain response in UDP-treated rats. These observations suggested that P2Y(6) receptor in dorsal spinal cord contribute to mechanical allodynia and thermal hyperalgesia in CCI-induced neuropathic pain. (C) 2018 Elsevier Ltd. All rights reserved.

Spinal cannabinoid receptor 1 (CB1R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB1R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB1 receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca2+ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca2+]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca2+]i increase in cultured DH neurons. ATP-evoked [Ca2+]i increase in DH neurons was blocked by chelating extracellular Ca2+ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca2+]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca2+]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca2+ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca2+ response was mimicked by ACEA (CB1R agonist), but was not influenced by AM1241 (CB2R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca2+ mobilization was blocked by AM251 (CB1 receptor antagonist), but was not influenced by AM630 (CB2 receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB1R rather than CB2R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB1R on P2X-channel-induced Ca2+ mobilization.

BACKGROUND:More evidence suggests that dorsal spinal cord microglia is an important site contributing to CB2 receptor-mediated analgesia. The upregulation of P2Y12 and P2Y13 purinoceptors in spinal dorsal horn microglia is involved in the development of pain behavior caused by peripheral nerve injury. However, it is not known whether the expression of P2Y12 and P2Y13 receptors at spinal dorsal horn will be influenced after CB2 receptor activation in neuropathic pain rats.METHODS:Chronic constriction injury (CCI) and intrathecal ADPbetaS injection were performed in rats to induce neuropathic pain. The paw withdrawal latency (PWL) was used to evaluate thermal hyperalgesia in neuropathic rats. The expression of P2Y12 and P2Y13 receptors, p-p38MAPK, and NF-kappaBp65 was detected with RT-PCR and western blotting analysis.RESULTS:Treatment with AM1241 produces a pronounced inhibition of CCI-induced thermal hyperalgesia and significantly inhibited the increased expression of P2Y12 and P2Y13 receptors at the mRNA and protein levels, which open up the possibility that P2Y12 and P2Y13 receptor expression are downregulated by CB2 receptor agonist AM1241 in CCI rats. Western blot analysis demonstrated that AM1241 reduced the elevated expression of p-p38MAPK and NF-κBp65 in the dorsal spinal cord induced by CCI. After administration with either SB203580 (p38MAPK inhibitor) or PDTC (NF-kappaB inhibitor), the levels of P2Y13 receptor expression in the dorsal spinal cord were lower than those in the CCI group. However, in CCI rats, the increased expression of P2Y12 receptor was prevented by intrathecal administration of PDTC but not by SB203580. In addition, minocycline significantly decreased the increased expression of P2Y12 and P2Y13 receptors. The similar results can be observed in ADPbetaS-treated rats. Intrathecal injection of ADPbataS causes thermal hyperalgesia and increased expression of P2Y12 and P2Y13 receptors in the dorsal spinal cord of naive rats. Moreover, intrathecal injection of AM1241 alleviates pain response and reduces the elevated expression of P2Y12 and P2Y13 receptors, p-p38MAPK, and NF-κBp65 in the dorsal spinal cord of ADPbetaS-treated rats. Intrathecal injection of SB203580 significantly inhibited the ADPbetaS-induced P2Y13 receptor expression, without affecting P2Y12 receptor expression. However, treatment with either SB203580 or PDTC effectively inhibited P2Y13 receptor expression compared to ADPbetaS-treated rats.CONCLUSIONS:In CCI- and ADPbetaS-treated rats, AM1241 pretreatment could efficiently activate CB2 receptor, while inhibiting p38MAPK and NF-kappaB activation in the dorsal spinal cord. CB2 receptor stimulation decreased P2Y13 receptor expression via p38MAPK/NF-kappaB signaling. On the other hand, CB2 receptor activation decreased P2Y12 receptor expression via p38MAPK-independent NF-kappaB signaling pathway.

Objective: P2 receptors have been implicated in the release of neurotransmitter and pro-inflammatory cytokines due to their response to neuroexcitatory substances in the microglia. Dorsal horn P2Y(12) and P2Y(13) receptors are involved in the development of pain behavior induced by peripheral nerve injury. However, it is not known whether P2Y(12) and P2Y(13) receptors activation is associated with the expression and the release of interleukin-1B (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-alpha) in cultured dorsal spinal cord microglia. For this reason, we examined the effects of ADP beta s (ADP analog) on the expression and the release of IL-1 beta, IL-6, and TNF-alpha.Methods and results: In this study, we observed the effect of P2Y receptor agonist ADP beta s on the expression and release of IL-1 beta, IL-6 and TNF-alpha by using real-time fluorescence quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). ADP beta s induced the increased expression of Iba-1, IL-1 beta, IL-6 and TNF-alpha at the level of messenger RNA (mRNA). ADPs-evoked increase in I beta a-1, IL-1 beta, IL-6 and TNF-a mRNA expression was inhibited only partially by P2Y(12) receptor antagonist MRS2395 or P2Y(13) receptor antagonist MRS2211, respectively. Similarly, ADP beta s-evoked release of IL-1 beta, IL-6 and TNF-alpha was inhibited only partially by MRS2395 or MRS2211. Furthermore, ADP beta s-evoked increased expression of Iba-1, IL-1 beta, IL-6 and TNF-alpha mRNA, and release of IL-1 beta, IL-6 and TNF-alpha were nearly all blocked after co-administration of MRS2395 plus MRS2179. Further evidence indicated that P2Y(12) and P2Y(13) receptor-evoked increased gene expression of IL-1 beta, IL-6 and TNF-alpha were inhibited by Y-27632 (ROCK inhibitor), SB203580 (P38MAPK inhibitor) and PDTC (NF-kappa b inhibitor), respectively. Subsequently, P2Y(12) and P2Y(13) receptor-evoked release of IL-1 beta, IL-6 and TNF-alpha, were also inhibited by Y-27632, SB203580 and PDTC, respectively.Conclusion: These observations suggest that P2Y(12) and P2Y(13) receptor-evoked gene expression and release of IL-1 beta, IL-6 and TNF-alpha are associated with ROCK/P38MAPK/NF-kappa b signaling pathway.