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Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-β superfamily. It is widely distributed in the central and peripheral nervous systems. Whether and how GDF-15 modulates nociceptive signaling remains unclear. Behaviorally, we found that peripheral GDF-15 significantly elevated nociceptive response thresholds to mechanical and thermal stimuli in naïve and arthritic rats. Electrophysiologically, we demonstrated that GDF-15 decreased the excitability of small-diameter dorsal root ganglia (DRG) neurons. Furthermore, GDF-15 concentration-dependently suppressed tetrodotoxin-resistant sodium channel Nav1.8 currents, and shifted the steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction. GDF-15 also reduced window currents and slowed down the recovery rate of Nav1.8 channels, suggesting that GDF-15 accelerated inactivation and slowed recovery of the channel. Immunohistochemistry results showed that activin receptor-like kinase-2 (ALK2) was widely expressed in DRG medium- and small-diameter neurons, and some of them were Nav1.8-positive. Blockade of ALK2 prevented the GDF-15-induced inhibition of Nav1.8 currents and nociceptive behaviors. Inhibition of PKA and ERK, but not PKC, blocked the inhibitory effect of GDF-15 on Nav1.8 currents. These results suggest a functional link between GDF-15 and Nav1.8 in DRG neurons via ALK2 receptors and PKA associated with MEK/ERK, which mediate the peripheral analgesia of GDF-15.
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Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-β superfamily. It is widely distributed in the central and peripheral nervous systems. Whether and how GDF-15 modulates nociceptive signaling remains unclear. Behaviorally, we found that peripheral GDF-15 significantly elevated nociceptive response thresholds to mechanical and thermal stimuli in naïve and arthritic rats. Electrophysiologically, we demonstrated that GDF-15 decreased the excitability of small-diameter dorsal root ganglia (DRG) neurons. Furthermore, GDF-15 concentration-dependently suppressed tetrodotoxin-resistant sodium channel Nav1.8 currents, and shifted the steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction. GDF-15 also reduced window currents and slowed down the recovery rate of Nav1.8 channels, suggesting that GDF-15 accelerated inactivation and slowed recovery of the channel. Immunohistochemistry results showed that activin receptor-like kinase-2 (ALK2) was widely expressed in DRG medium- and small-diameter neurons, and some of them were Nav1.8-positive. Blockade of ALK2 prevented the GDF-15-induced inhibition of Nav1.8 currents and nociceptive behaviors. Inhibition of PKA and ERK, but not PKC, blocked the inhibitory effect of GDF-15 on Nav1.8 currents. These results suggest a functional link between GDF-15 and Nav1.8 in DRG neurons via ALK2 receptors and PKA associated with MEK/ERK, which mediate the peripheral analgesia of GDF-15.
Assuntos
Animais , Ratos , Analgesia , Gânglios Espinais , Fator 15 de Diferenciação de Crescimento , Células Receptoras Sensoriais , Canais de Sódio , Tetrodotoxina/farmacologiaRESUMO
Objective To evaluate the relationship betweenα2 adrenergic receptors and expression of Nav1. 8 in dorsal root ganglion (DRG) neurons, and to illuminate the mechanism of dexmedetomidine-induced reduction of acute visceral pain in rats. Methods Thirty-two clean-grade healthy adult male Spra-gue-Dawley rats, aged 6-8 weeks, weighing 180-220 g, were divided into 4 groups ( n=8 each) using a random number table method: control group ( group C ) , acute visceral pain group ( group VP ) , dexmedetomidine group (group D), and dexmedetomidine plus atipamezole group (group DA). In VP, D and DA groups, 10-3 mmol∕L capsaicin 1. 3 ml was injected into the rectum at a dose of 10-3 mmol∕L to establish the acute visceral pain model, while the equal volume of normal saline was given instead in group C. Atipamezole 1 mg∕kg was subcutaneously injected through the back of the neck at 20 min before establishing the model in group DA. Dexmedetomidine 10μg∕kg was injected through the tail vein at 15 min before establishing the model in D and DA groups, while the equal volume of normal saline was given instead at the correspording time points in C and VP groups. The visceral pain behavior score was recorded at 1 h after establishing the model. The animals were then sacrificed, and DRGs of the lumbar segment (L3-6) were removed for determination of the number of Nav1. 8 positive DRG neurons (by immunohisto-chemistry) and expression of Nav1. 8 mRNA (by quantitative real-time polymerase chain reaction). Results Compared with group C, the visceral behavior scores and the number of Nav 1. 8 positive DRG neurons were significantly increased, and the expression of Nav 1. 8 mRNA was up-regulated in VP, D and DA groups (P<0. 05). Compared with group VP, the visceral behavior score and the number of Nav1. 8 positive DRG neurons were significantly decreased, and the expression of Nav 1. 8 mRNA was down-regulated in D and DA groups (P<0. 05). Compared with group D, the visceral behavior scores and the number of Nav1. 8 positive DRG neurons were significantly increased, and the expression of Nav1. 8 mRNA was up-regulated in group DA ( P<0. 05) . Conclusion The mechanism by which dexmedetomidine reduces acute visceral pain is related to activatingα2 adrenergic receptors and to down-regulating the expression of Nav1. 8 in DRG neu-rons of rats.
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Objective To observe the effect of miR-145 on pain threshold and explore the pos-sible underlying positive role of miR-145 in rats with diabetic neuropathic pain.Methods The total of 36 rats with diabetic neuropathic pain were randomly divided into three groups respectively with nor-mal control group (group N)(n=12 for each group):diabetic neuropathic pain(DNP)group (group D),DNP-NC group (group DN)and DNP-agomiR-145 group (group agomiR-145).The rats received agomiR-145 intrathecal injection in group agomiR-145 (10 μl,1×106TU/ml),or the negative control virus in group DN (10 μl,1×106TU/ml),or equal volume of normal saline in other two groups. Paw mechanical withdrawal threshold(MWT)and paw withdrawal latency(TWL)were measured on the day before intrathecal injection and day 1,days 3,7 and 14 after intrathecal injection.On the days 14 after pain-related behavioral test,the RNA expression of miR-145 in the dorsal root ganglion (DRG)was detected using reverse transcription-quantitative polymerase chain reaction(RT-PCR)as-say and the expression of Nav 1.8 in DRG were detected by fluorescent immunofluorescence.In addi-tion,a dual luciferase activity assay was used to testify the target genes of miR-145.Results MWT and TWL were decreased at 1 d before intrathecal injectionin groups D,DN and agomiR-145 than that in group N (P<0.05).The significant increase of MWT was observed in group agomiR-145 on day 3,7,14 than those in group D and group DN (P<0.05).TWL in group agomiR-145 was increased significantly on day 7 and day 14 compared with those in groups D and DN (P<0.05).Compared with group N,miR-145 expression level in DRG in groups D and DN were significantly lower (P<0.05).In addition,the protein expression of Nav1.8 was significantly increased in group D and DN compared with that in group N (P<0.05).Compared with groups D and DN,miR-145 expression was increased significantly and the expression of Nav1.8 in DRG was decreased significantly in group agomiR-145 (P<0.05).In addition,a dual luciferase reporter assay demonstrated that miR-145 can bind with the 3'-UTR region of Nav1.8 and regulate its expression.Conclusion Intrathecal agomiR-145 can effectively attenuate neuropathic pain of DNP rats,which may be related with down-regulation of Nav1.8 in DRG..
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Objective:To evaluate the analgesic effect of botulinum toxin type A (BTX-A) in osteoarthritis model and the changes of Nav1.8 protein expression in spinal ganglia changes.Method:Animal model of knee osteoarthritis (KOA) was established by intra-articular injection of 4％ papain solution 0.3ml into SD rat right knee.After the formation of arthritis,they were randomly divided into two groups at the 2nd day:BTX-A group (n=10):intra-articular injection of 5μl BTX-A 0.1IU;WFI group (n=10):intra-articular injection of 5μl water.No papain or BXT-A was given to the sham group (n=10).At the 1st,3rd,5th day after injection,we tested the pain behavior,thermal pain threshold,and sodium channel 1.8 (Nav1.8) protein expression in spinal ganglion by using immunohistochemistry.Result:Analysis of spontaneous pain behavior showed abnormal gait caused by rat osteoarthritis.Comparing with the WFI group,abnormal gait caused by osteoarthritis improved significantly in BTX-A group.At day 5 it improved more significantly than day 1 and day 3.Thermal pain threshold of BTX-A group increased more than that of WFI group (P＜0.05) at any time point.Abnormal high Nav1.8 protein in model rats decreased in spinal ganglia for BTX-A group.Conclusion:The intra-articular injection of BTX-A may play the analgesic effect in the model of KOA by down-regulating of the expression of Nav1.8 protein in spinal ganglia and reduction in the central sensitization to pain stimulation.
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@#To study the effects of quercetin(Que)on the electric current of Nav1. 8(INav1. 8)in rat dorsal root ganglion(DRG)neurons, dose-effect relationship of Que on INav1. 8 and the activation and inactivation properties of voltage-dependent Nav1. 8 influenced by Que were studied by using the whole-cell patch clamp technique in fresh isolated rat DRG neurons. It was found that diffeent concerntrations of Que(10, 30, 100 μmol/L)could inhibit INav1. 8 peak value of DRG neurons in concentration-dependent manner. The inhibition of peak currents were(15. 32±3. 43)%, (22. 92±8. 24)% and(47. 29±11. 42)% respectively, the IC50 was 121. 38 μmol/L and the Hill coefficient was 0. 76. In the existence of Que(100 μmol/L), the activation curve of Nav1. 8 channel in DRG was slightly shifted to depolarizing direction for 0. 83 mV, and the inactivation curve was shifted to hyperpolarizing direction for 1. 86 mV. Compared with the stage before intervention, the half-activation voltage(V1/2=-40. 23±0. 25 mV)was significantly different(P< 0. 01). In conclusion, Que inhibits the Nav1. 8 channel activity in dose and voltage-dependent manner, which may count for the reduction of algesthesia transmission and the alleviation of chronic visceral pain.
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Neuropathic pain has been hypothesized to be the result of aberrant expression and function of sodium channels at the site of injury. To investigate the effects of NaV1.8 antisense oligonucleotide on the expression of sodium channel mRNA in dorsal root ganglion (DRG) neurons in chronic neuropathic pain. 24 Sprague-Dawley rats weighing 200-260 g were anesthetized with the in of sciatic nerve trunk by 4-0 chromic gut. The mechanical and thermal pain threshold were measured before operation and 1, 3, 5, 7, 9, 11, 13 days after operation. A PE-10 catheter was implanted in subarachnoid space at lumbar region. On the 7th postoperative day the animals were randomly divided into 4 groups. The drugs were injected intrathecally twice a day for 5 consecutive days in group 2-4. The animals were decapitated 14 days after the surgery. The L4-L6 DRG of the operated side was removed and crushed, and total RNA was extracted with Trizol reagent. The contralateral side was used as control. The change of NaV1.8 sodium channel transcripts was determined by RT-PCR. Pain threshold was significantly lowered after CCI as compared with that in control group and was elevated 3 days after antisense oligonucleotide injection. Sensory neuron specific TTX-R sodium channel NaV1.8 transcript was down-regulated after antisense oligonucleotide injection at the dosage of 45 μg as compared with that in CCI group (P＜0.01), and it was even greater at the dosage of 90 μg. The intrathecally injected NaV1.8 antisense oligonucleotide can reduce the mechanical allodynia and thermal hyperalgesia partially by downregulating the SNS transcript expression.
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Objective To investigate the effect of inhibiting the expression of Navl. 8 sodium channels by an antisense oligodeoxynudeotide(ODN) on visceral hypersensitivity. Methods The visceral hypersensitivity animal models induced by giving neonatal Sprague-Dawley rats colorectal distention(CRD) after postnatal days 8, 10 and 12. Animals were injected intrathecally either Navl. 8 antisense ODN (research group) or mismatch ODN (control group) twice a day for 3 days at the 8th week. After that, the expression of Navl. 8 in each group was examined by RT-PCR, and abdomiral withdrawal reflex(AWR) score and spinal c-Fos expression were evaluated to assess whether the intervention can relieve the visceral hypersensitivity. Results After intrathecal injection with the antisense ODN, the expression of Navl. 8 in each group decreased.AWR score and the number of c-Fos positive neurons in the spinal cord, especially in the area 1 and 3, decreased in the model group, while they did not change in the control group. Conclusions Interventing the expression of Nav1.8 may relieve the visceral hypersensitivity in this model of irritable bowel syndrome.
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AIM To invesigate the effect of subarachnoid transplantation of APA microcapsulized bovine chromaffin cells (BCCs) on mRNA expression for Nav1.8 in the dorsal root ganglia neurons(DRG) of rats with neuropathic pain by means of in situ hybridization. METHODS SD rats were randomly divided into four groups of five. Normal rats were used as control group (group C). Rats with right sciatic nerve been ligated were used as CCI group. Five to six hundred empty APA microcapsules(group APA) or 5?10 6 APA microcapsulized BCCs (group APA-BCCs) were grated into subarachnoid space of CCI rats 7 days after operation. Allodynia and hyperalgesia were measured by Von-Frey filaments and CO 2 laser 7 days after transplantation. DRG in lumbar four and five was taken out and 15 ?m freezing sections were made 7 days after tansplantation. Sections was used to detect mRNA expression for TTX-resistent Na + Nav1.8 by in situ hybridization with Dig-labeled RNA probe. RESULTS The mRNA hybridization signal for Nav1.8 in DRG of group CCI and group APA was lower than that of group C. The expression of mRNA for Nav1.8 in DRG was higher in group APA-BCCs than that in group CCI and group APA with abatement of allodynia and hyperalgesia. There was no difference in the mRNA hybridization signal for Nav1.8 in DRG between group APA-BCCs and group C. CONCLUSION mRNA expression for Nav1.8 in DRG of CCI ratswas down-regulated. APA microcapsulized BCCs grafting can reverse the down-regulation of mRNA expression for Nav1.8 in DRG of CCI rats. Restoration of mRNA expression for Nav1.8 in DRG contributes to the analgesic effect of subarachnoid transplantation of APA microcapsulized BCCs.
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Objective To establish a model of bone cancer pain in rats and to evaluate the role of voltage gated sodium channel Nav1.8 in the course of bone cancer pain by observing the expression of Nav1.8 in dorsal root ganglion in the model with bone cancer pain.Method Female SD rats received intra-tibial injection of syngenetic Walker256 mammary gland carcinoma cells in different concertration(103/?l,104/?l or 105/?l).Pain threshold of mechanical hyperalgesia and thermal hyperalgesia were tested at 1d,3d,5d,7d,10d,and 14d after cell injection.The development of the bone tumor was verified by pathological examination 14d after cell injection.The L5-6 DRG was obtained from normal rats and rats with bone cancer pain.Expression of voltage gated sodium channel Nav1.8 was investigated by RT-PCR.Result Intra-tibial injection of Walker256 cells produced a rapidly expanding tumor within the boundaries of the tibia,causing marked remodeling of the bone.Rats receiving intra-tibial injection of Walker256 cells displayed gradual development of both mechanical and thermal hyperalgesia 7-14 days after the injection.The expression of Nav1.8 in DRG was up-regulated in the model of bone cancer pain in rats(P