Excitatory toxicity(ET) is an important factor of neuropathic pain(NPP) induced by central sensitization(CS), and the association of pannexin-1(Panx1)-Src-N-methyl-D-aspartate receptor subunit 2 B(NMDAR-2 B) is an important new pathway for ET to initiate CS. The present study confirmed whether the central analgesic effect of Chuanxiong Rhizoma extract(CRE) was achieved through the synchronous regulation of the brain and spinal pathways of Panx1-Src-NMDAR-2 B. In this study, dynamic and simulta-neo-us microdialysis of the brain and spinal cord in vivo combined with behavioristics, high performance liquid chromatography(HPLC)-fluorescence detection, microdialysis analysis(ISCUS~(flex)), ultrasensitive multifactorial electrochemiluminescence immunoassay, ELISA, and Western blot was employed to investigate the protein expression of NMDAR-2 B, Src, and Panx1, extracellular excitatory amino acids, cytokines, energy metabolites, and substance P in spinal dorsal horn(SDH) and anterior cingulate cortex(ACC) after CRE intervention with the rat model of spared sciatic nerve injury(SNI) as the experimental tool. Compared with the sham group, the SNI group exhibited diminished mechanical withdrawal threshold(MWT)(P<0.01), increased cold spray scores(P<0.01), glutamate(Glu), D-serine(D-Ser), and glycine(Gly) in extracellular fluids of ACC, and Glu, D-Ser, interleukin-1β(IL-1β), and lactic acid(Lac) in extracellular fluids of SDH(P<0.05), dwindled tumor necrosis factor(TNF-α)(P<0.05), and elevated protein levels of NMDAR-2 B, Src, and Panx1 in ACC(P<0.05). Compared with the SNI model rats, high-and medium-dose CRE(CRE-H/M) could potentiate the analgesic activity as revealed by the MWT test(P<0.05) and CRE-M enabled the decrease in cold spray scores(P<0.05). CRE-H/M could inhibit the levels of Glu, D-Ser and Gly in the extracellular fluids of ACC(P<0.05), and the levels of Glu in the extracellular fluids of SDH(P<0.05) in SNI rats. CRE-M significantly increased the levels of glucose(Gluc), Lac, interferon-gamma(IFN-γ), keratinocyte chemoattractant/human growth-regulated oncogenes(KC/GRO), and IL-4 in extracellular fluids of SDH in SNI rats(P<0.05). CRE-H/M/L could also inhibit the levels of NMDAR-2 B, Src and Panx1 in ACC and SDH in SNI rats(P<0.05). The central analgesic effect of CRE is presumedly related to the inhibited release of excitatory amino acid transmitters(Glu, D-Ser and Gly) in ACC and SDH of SNI rats, decreased protein expression of NMDAR-2 B, Src and Panx1 in the two regions, and the regulation of the Panx1-Src-NMDAR-2 B pathway in the spinal cord and brain. The above findings partially clarified the scientific basis of clinical analgesic effect of Chuanxiong Rhizoma.
Animals ; Central Nervous System Sensitization ; Neuralgia/drug therapy* ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate/metabolism* ; Signal Transduction ; Spinal Cord/metabolism*

Objective To investigate the effects of simvastatin on diabetic neuropathic pain and systematic inflammation in diabetic rats and explore their molecular mechanisms.Methods Totally 24 rats were equally randomized into the normal+vehicle(NV)group,diabetic+vehicle(DV)group,and diabetic+simvastatin(DS)group using the random number table.Streptozotocin(STZ)was used to establish the rat models of diabetes.Blood glucose,body mass,paw withdrawal mechanical threshold(PWMT),and paw withdrawal thermal latency(PWTL)in each group were observed on days 7,14,21,and 28 after STZ injection.On day 28 after STZ injection,rats were sacrificed,and the lumbar spinal dorsal horn and serum were collected.Western blotting was used to detect the expression of receptor for advanced glycation end products(RAGE)and the phosphorylation levels of protein kinase B(AKT),extracellular signal-regulated kinase(ERK),p38,and c-Jun N-terminal kinase(JNK)in the spinal dorsal horn of rats in each group.Enzyme-linked immunosorbent assay was performed to determine the serum concentrations of oxidized low density lipoprotein(ox-LDL)and interleukin-1β(IL-1β).Results On days 14,21 and 28 after STZ injection,the PWMT in DV group were(8.6 ± 0.8),(7.1 ± 1.6),and(7.8 ± 0.8)g respectively,which were significantly lower than (12.0 ± 0.9)(=8.482, =0.000),(11.6 ± 1.5)(=11.309, =0.000),and(11.7 ± 1.5)g(=9.801, =0.000)in NV group.The PWMT in DS group on days 21 and 28 were(9.4 ± 1.4)(=5.780, =0.000)and(9.7 ± 0.9)g(=4.775, =0.003),respectively,which were significantly improved comparing with those of DV group.On days 7,14,21,and 28,there were no significant differences in PWTL among these three groups (all <0.05).The expression of RAGE in the spinal dorsal horn of DV group was significantly higher than those of NV group(=6.299, =0.000)and DS group(=2.891, =0.025).The phosphorylation level of AKT in the spinal dorsal horn of DV group was significantly higher than those of NV group(=8.915,=0.000)and DS group(=4.103,=0.003).The phosphorylation levels of ERK( =8.313,=0.000),p38( =2.965, =0.022),and JNK(=7.459, =0.000)in the spinal dorsal horn of DV group were significantly higher than those of NV group;the phosphorylation level of JNK in the spinal dorsal horn of DS group was significant lower than that of DV group(=3.866, =0.004);however,there were no significant differences in the phosphorylation levels of ERK(=1.987,=0.122)and p38(=1.260,=0.375)in the spinal dorsal horn between DS group and DV group.The serum concentrations of ox-LDL and IL-1β in DV group were(41.86 ± 13.40)ng/ml and(108.16 ± 25.88)pg/ml,respectively,which were significantly higher than those in NV group [(24.66 ± 7.87)ng/ml(=3.606,=0.003)and(49.32 ± 28.35)pg/ml(=5.079,=0.000)] and DS group [(18.81 ± 5.62)ng/ml (=4.833, =0.000)and(32.73 ± 11.73)pg/ml(=6.510, =0.000)].Conclusions Simvastatin can relieve the mechanical allodynia of diabetic rats possibly by inhibiting the activation of RAGE/AKT and the phosphorylation of JNK in the spinal dorsal horn.Simvastatin can also decrease the serum concentrations of ox-LDL and IL-1β in diabetic rats,which may contribute to the relief of systematic inflammation.
Animals ; Diabetes Mellitus, Experimental ; complications ; Hyperalgesia ; Inflammation ; drug therapy ; Interleukin-1beta ; blood ; Lipoproteins, LDL ; blood ; Neuralgia ; drug therapy ; Proto-Oncogene Proteins c-akt ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor for Advanced Glycation End Products ; metabolism ; Simvastatin ; pharmacology

The voltage-gated Na channel subtype Nav1.7 is important for pain and itch in rodents and humans. We previously showed that a Nav1.7-targeting monoclonal antibody (SVmab) reduces Na currents and pain and itch responses in mice. Here, we investigated whether recombinant SVmab (rSVmab) binds to and blocks Nav1.7 similar to SVmab. ELISA tests revealed that SVmab was capable of binding to Nav1.7-expressing HEK293 cells, mouse DRG neurons, human nerve tissue, and the voltage-sensor domain II of Nav1.7. In contrast, rSVmab showed no or weak binding to Nav1.7 in these tests. Patch-clamp recordings showed that SVmab, but not rSVmab, markedly inhibited Na currents in Nav1.7-expressing HEK293 cells. Notably, electrical field stimulation increased the blocking activity of SVmab and rSVmab in Nav1.7-expressing HEK293 cells. SVmab was more effective than rSVmab in inhibiting paclitaxel-induced mechanical allodynia. SVmab also bound to human DRG neurons and inhibited their Na currents. Finally, potential reasons for the differential efficacy of SVmab and rSVmab and future directions are discussed.
Animals ; Antibodies, Monoclonal ; therapeutic use ; Biotin ; metabolism ; Cells, Cultured ; Disease Models, Animal ; Female ; Ganglia, Spinal ; cytology ; HEK293 Cells ; Humans ; Hybridomas ; chemistry ; Hyperalgesia ; drug therapy ; Male ; Mice ; Mice, Inbred C57BL ; NAV1.5 Voltage-Gated Sodium Channel ; metabolism ; NAV1.7 Voltage-Gated Sodium Channel ; chemistry ; immunology ; metabolism ; Neuralgia ; drug therapy ; metabolism ; Protein Binding ; drug effects ; Recombinant Proteins ; biosynthesis ; therapeutic use ; Sensory Receptor Cells ; drug effects ; physiology

Increasing evidence suggests that spinal microglia regulate pathological pain in males. In this study, we investigated the effects of several microglial and astroglial modulators on inflammatory and neuropathic pain following intrathecal injection in male and female mice. These modulators were the microglial inhibitors minocycline and ZVEID (a caspase-6 inhibitor) and the astroglial inhibitors L-α-aminoadipate (L-AA, an astroglial toxin) and carbenoxolone (a connexin 43 inhibitor), as well as U0126 (an ERK kinase inhibitor) and D-JNKI-1 (a c-Jun N-terminal kinase inhibitor). We found that spinal administration of minocycline or ZVEID, or Caspase6 deletion, reduced formalin-induced inflammatory and nerve injury-induced neuropathic pain primarily in male mice. In contrast, intrathecal L-AA reduced neuropathic pain but not inflammatory pain in both sexes. Intrathecal U0126 and D-JNKI-1 reduced neuropathic pain in both sexes. Nerve injury caused spinal upregulation of the astroglial markers GFAP and Connexin 43 in both sexes. Collectively, our data confirmed male-dominant microglial signaling but also revealed sex-independent astroglial signaling in the spinal cord in inflammatory and neuropathic pain.
2-Aminoadipic Acid ; toxicity ; Animals ; Anti-Inflammatory Agents ; therapeutic use ; Astrocytes ; pathology ; Carbenoxolone ; pharmacology ; Caspase 6 ; deficiency ; metabolism ; Connexin 43 ; metabolism ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Female ; Glial Fibrillary Acidic Protein ; metabolism ; Male ; Mice ; Mice, Transgenic ; Microglia ; pathology ; Minocycline ; therapeutic use ; Neuralgia ; chemically induced ; drug therapy ; pathology ; Pain Measurement ; Phenylurea Compounds ; pharmacology ; Sex Characteristics ; Spinal Cord ; pathology ; Time Factors

Along with the development of economy and society, type 2 diabetic mellitus (T2DM) has become one of the most common diseases at the global level. As one of the complications of T2DM, diabetic neuropathic pain (DNP) stubbornly and chronically affects the health and life of human beings. In the pain field, dorsal root ganglion (DRG) is generally considered as the first stage of the sensory pathway where the hyperexcitability of injured neurons is associated with different kinds of peripheral neuropathic pains. The abnormal electrophysiology is mainly due to the changed properties of voltage-gated sodium channels (VGSCs) and the increased sodium currents (I(Na)). Curcumin is an active ingredient extracted from turmeric and has been demonstrated to ameliorate T2DM and its various complications including DNP effectively. The present study demonstrates that the I(Na) of small-sized DRG neurons are significantly increased with the abnormal electrophysiological characteristics of VGSCs in type 2 diabetic neuropathic pain rats. And these abnormalities can be ameliorated efficaciously by a period of treatment with curcumin.
Animals ; Curcumin ; pharmacology ; Diabetes Mellitus, Experimental ; complications ; Diabetes Mellitus, Type 2 ; complications ; Diabetic Neuropathies ; drug therapy ; Ganglia, Spinal ; cytology ; drug effects ; metabolism ; Neuralgia ; drug therapy ; Neurons ; drug effects ; metabolism ; Rats ; Sodium ; Voltage-Gated Sodium Channels ; physiology

BACKGROUNDNajanalgesin, 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.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONThe antinociceptive effect of najanalgesin functions by inhibiting the JNK in a neuropathic pain model.

Animals ; Elapid Venoms ; therapeutic use ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Immunohistochemistry ; JNK Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Male ; Neuralgia ; drug therapy ; enzymology ; Proto-Oncogene Proteins c-jun ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

OBJECTIVETo explore the analgesia of oxymatrine (OMT) affecting high voltage-dependent calcium channels (HVDCCs) and GABA release under neuropathic pain condition.

METHODSTotally 66 C57BL/6 mice were randomly divided into the sham-operation group, the model group, and the OMT group, 22 in each group. Neuropathic pain models were established by partial sciatic nerve ligation (PSNL). Hind paw plantar mechanical response threshold (MWT) was measured by up-and-down method with Von-Frey filament. mRNA expression of HVDCCs in brains and spinal cords was detected with Real-time PCR and concentration of GABA was determined using ELISA kit.

RESULTSCompared with day 0, the left hind paw MWTwas decreased on day 7, 10, and 14 in the model group (P < 0.05). Compared with the sham-operation group, the left hind paw MWT was significantly reduced in the model group on day 7 (P < 0.05). The MWT of PSNL ipsilateral hind paw was decreased on day 7 before OMT administration, when compared with day 0 (P < 0.05), and increased after OMT administration (P < 0.05). Compared with the sham-operation group, mRNA levels of Cav1.2, Cav1.3, Cav2.1, and Cav2.3 in brain tissues were increased and those of Cav2.2 were decreased significantly in the model group (P < 0.05). In spinal cord tissues, mRNA levels of Cav1.2 and Cav1.3 were increased, but those of Cav2.1, Cav2.2, and Cav2. 3 were decreased significantly in the model group, when compared with those of the sham-operation group (P < 0.05). Compared with the model group, mRNA levels of Cavl.2, Cavl.3, Cav2.1, and Cav2. 3 in brain tissues were decreased, and those of Cav2.2 were increased significantly in the OMT group (P < 0.05). In spinal cord tissues of the OMT group, mRNA levels of Cav1.3 decreased and those of Cav2.1, Cav2.2, and Cav2.3 increased significantly with statistical difference, when compared with those of the model group (P < 0.05). Compared with the sham-operation group, GABA levels in brain tissues decreased in the model group (P < 0.05). Compared with the model group, GABA levels in brain tissues increased in the OMT group (P < 0.05). There was no statistical difference in GABA levels of spinal cord tissues among these groups (P > 0.05).

CONCLUSIONSOMT had analgesic effect on neuropathic pain, which might be probably related to HVDDCs. Cav2.2 might directly affect GABA release.

Alkaloids ; pharmacology ; therapeutic use ; Analgesia ; methods ; Animals ; Calcium ; Calcium Channels ; drug effects ; metabolism ; Disease Models, Animal ; Mice ; Mice, Inbred C57BL ; Neuralgia ; drug therapy ; Pain Management ; Quinolizines ; pharmacology ; therapeutic use ; Spinal Cord ; metabolism ; gamma-Aminobutyric Acid

Autophagy acts as an important homoeostatic mechanism by degradation of cytosolic constituents and plays roles in many physiological processes. Recent studies demonstrated that autophagy can also regulate the production and secretion of the proinflammatory cytokine interleukin-1β (IL-1β), which plays a critical role in the development and maintenance of neuropathic pain. In the present study, the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were significantly decreased after spinal nerve ligation (SNL), and the changes were accompanied by inhibited autophagy in the spinal microglia and increased mRNA and protein levels of IL-1β in the ipsilateral spinal cord. We then investigated the antinociceptive effect of rapamycin, a widely used autopahgy inducer, on SNL-induced neuropathic pain in rats and found that treatment with intrathecal rapamycin significantly attenuated the mechanical allodynia and thermal hyperalgesia. Moreover, rapamycin significantly enhanced autophagy in the spinal microglia, whereas it reduced the mRNA and protein levels of IL-1β in the ipsilateral spinal cord. Our results showed that rapamycin could ameliorate neuropathic pain by activating autophagy and inhibiting IL-1β in the spinal cord.
Animals ; Autophagy ; drug effects ; Immunosuppressive Agents ; Interleukin-1beta ; antagonists & inhibitors ; metabolism ; Male ; Neuralgia ; drug therapy ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sirolimus ; pharmacology ; Spine ; metabolism ; pathology

BACKGROUNDActivation of glial cells and the extracellular signal-regulated kinase (ERK) signaling pathway play an important role in the development and maintenance of neuropathic pain. Curcumin can alleviate the symptom of inflammatory pain by inhibiting the production and release of interleukin and tumor necrosis factor. However, whether curcumin affects neuropathic pain induced by nerve injury and the possible mechanism involved are still unknown. This study investigated the effects of tolerable doses of curcumin on the activation of astrocytes and ERK signaling in the spinal dorsal horn in rat model of neuropathic pain.

METHODSAdult male Sprague-Dawley rats were randomly divided into three groups: a control (sham operated) group, and chronic constriction injury groups (to induce neuropathic pain) that were either untreated or treated with curcumin. Thermal and mechanical hyperalgesia thresholds were measured. The distribution and morphological changes of astrocytes were observed by immunofluorescence. Western blotting was used to detect changes in the expression of glial fibrillary acid protein (GFAP) and phosphorylated ERK.

RESULTSInjured rats showed obvious mechanical allodynia and thermal hyperalgesia. The number of GFAP-positive astrocytes, and the fluorescence intensity of GFAP were significantly increased in the spinal dorsal horn of injured compared with control rats. The soma of astrocytes also appeared hypertrophied in injured animals. Expression of GFAP and phosphorylated ERK was also significantly increased in the spinal dorsal horn of injured compared with control rats. Curcumin reduced the injury-induced thermal and mechanical hyperalgesia, the increase in the fluorescence intensity of GFAP and the hypertrophy of astrocytic soma, activation of GFAP and phosphorylation of ERK in the spinal dorsal horn.

CONCLUSIONSCurcumin can markedly alleviate nerve injury-induced neuropathic pain in rats. The analgesic effect of curcumin may be attributed to its inhibition of astrocyte hypertrophy in the spinal dorsal horn and phosphorylation of the ERK signaling pathway.

Analgesics ; therapeutic use ; Animals ; Astrocytes ; drug effects ; Blotting, Western ; Constriction, Pathologic ; metabolism ; Curcumin ; therapeutic use ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Glial Fibrillary Acidic Protein ; metabolism ; Hyperalgesia ; metabolism ; Male ; Neuralgia ; drug therapy ; Phosphorylation ; drug effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects

We examined the possible anti-inflammatory mechanisms of gabapentin in the attenuation of neuropathic pain and the interaction between the anti-allodynic effects of gabapentin and interleukin-10 (IL-10) expression in a rat model of neuropathic pain. The anti-allodynic effect of intrathecal gabapentin was examined over a 7-day period. The anti-allodynic effects of IL-10 was measured, and the effects of anti-IL-10 antibody on the gabapentin were assessed. On day 7, the concentrations of pro-inflammatory cytokines and IL-10 were measured. Gabapentin produced an anti-allodynic effect over the 7-day period, reducing the expression of pro-inflammatory cytokines but increasing the expression of IL-10 (TNF-alpha, 316.0 +/- 69.7 pg/mL vs 88.8 +/- 24.4 pg/mL; IL-1beta, 1,212.9 +/- 104.5 vs 577.4 +/- 97.1 pg/mL; IL-6, 254.0 +/- 64.8 pg/mL vs 125.5 +/- 44.1 pg/mL; IL-10, 532.1 +/- 78.7 pg/mL vs 918.9 +/- 63.1 pg/mL). The suppressive effect of gabapentin on pro-inflammatory cytokine expression was partially blocked by the anti-IL-10 antibody. Expression of pro-inflammatory cytokines was significantly attenuated by daily injections of IL-10. The anti-allodynic effects of gabapentin may be caused by upregulation of IL-10 expression in the spinal cord, which leads to inhibition of the expression of pro-inflammatory cytokines in the spinal cords.
Amines/pharmacology/*therapeutic use ; Analgesics/pharmacology/*therapeutic use ; Animals ; Antibodies/immunology/pharmacology ; Behavior, Animal/drug effects ; Cyclohexanecarboxylic Acids/pharmacology/*therapeutic use ; Cytokines/*metabolism ; Disease Models, Animal ; Injections, Spinal ; Interleukin-10/genetics/immunology/*metabolism ; Male ; Neuralgia/*drug therapy/metabolism/pathology ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins/biosynthesis/genetics/pharmacology ; Spinal Cord/metabolism ; Up-Regulation ; gamma-Aminobutyric Acid/pharmacology/*therapeutic use