Mechanisms for therapeutic effect of bulleyaconitine A on chronic pain.

Bulleyaconitine A, a diterpenoid alkaloid isolated from Aconitum bulleyanum plants, has been used for the treatment of chronic pain in China since 1985. Clinical studies show that the oral administration of bulleyaconitine A is effective for treating different kinds of chronic pain, including back pain, joint pain, and neuropathic pain with minimal side effect in human patients. The experimental studies have revealed that bulleyaconitine A at therapeutic doses potently inhibits the peripheral sensitization and central sensitization that underlie chronic pain and has no effect on acute pain. Bulleyaconitine A preferably blocks tetrodotoxin-sensitive voltage-gated sodium channels in dorsal root ganglion neurons by inhibition of protein kinase C, and the effect is around 600 times more potent in neuropathic animals than in naïve ones. Bulleyaconitine A at 5 nM inhibits the hypersensitivity of dorsal root ganglion neurons in neuropathic rats but has no effect on excitability of dorsal root ganglion neurons in sham group. Bulleyaconitine A inhibits long-term potentiation at C-fiber synapses in spinal dorsal horn, a synaptic model of pathological pain, preferably in neuropathic pain rats over naïve rats. The following mechanisms may underlie the selective effect of bulleyaconitine A on chronic pain. (1) In neuropathic conditions, protein kinase C and voltage-gated sodium channels in dorsal root ganglion neurons are upregulated, which enhances bulleyaconitine A's effect. (2) Bulleyaconitine A use-dependently blocks voltage-gated sodium channels and therefore inhibits the ectopic discharges that are important for neuropathic pain. (3) Bulleyaconitine A is shown to inhibit neuropathic pain by the modulation of spinal microglia, which are involved in the chronic pain but not in acute (nociceptive) pain. Moreover, bulleyaconitine A facilitates the anesthetic effect of morphine and inhibits morphine tolerance in rats. Together, bulleyaconitine A is able to inhibit chronic pain by targeting at multiple molecules. Further clinical and experimental studies are needed for evaluating the efficacy of bulleyaconitine A in different forms of chronic pain in patients and for exploring the underlying mechanisms.

Calpain-2 contributes to neuropathic pain following motor nerve injury via up-regulating interleukin-6 in DRG neurons.

Motor nerve injury by L5 ventral root transection (L5-VRT) initiates interleukin-6 (IL-6) up-regulation in primary afferent system contributing to neuropathic pain. However, the early upstream regulatory mechanisms of IL-6 after L5-VRT are still unknown. Here, we monitored both the activity of calpain, a calcium-dependent protease suggested as one of the earliest mediators for cytokine regulation, and the expression of IL-6 in bilateral L4-L6 dorsal root ganglias (DRGs) soon after L5-VRT. We found that the protein level of calpain-2 in DRGs, but not calpain-1 was increased transiently in the first 10 min(-1)h ipsilaterally and 20 min(-1)h contralaterally after L5-VRT, long before mechanical allodynia was initiated (5-15 h ipsilaterally and 15 h(-1)d contralaterally). The early activation of calpain evaluated by the generation of spectrin breakdown products (SBDP) correlated well with IL-6 up-regulation in bilateral DRGs. Double immunofluorescence staining revealed that almost all the calpain-2 positive neurons expressed IL-6, indicating an association between calpain-2 and IL-6. Inhibition of calpain by pre-treatment with MDL28170 (25mg/kg, i.p.) attenuated the rat mechanical allodynia and prevented the early up-regulation of IL-6 following L5-VRT. Addition of exogenous calpain-2 onto the surface of left L5 DRG triggered a temporal allodynia and increased IL-6 in bilateral DRGs simultaneously. Taken together, the early increase of calpain-2 in L5-VRT rats might be responsible for the induction of allodynia via up-regulating IL-6 in DRG neurons.

Up-regulation of CX3CL1 via Nuclear Factor-κB-dependent Histone Acetylation Is Involved in Paclitaxel-induced Peripheral Neuropathy.

BACKGROUND: Up-regulation of CX3CL1 has been revealed to be involved in the neuropathic pain induced by nerve injury. However, whether CX3CL1 participates in the paclitaxel-induced painful peripheral neuropathy remains unknown. The aim of the current study was to elucidate the involvement of transcriptional factors nuclear factor-κB (NF-κB) and its causal interaction with CX3CL1 signaling in the paclitaxel-induced painful peripheral neuropathy. METHODS: Painful peripheral neuropathy induced by paclitaxel treatment was established in adult male Sprague-Dawley rats. The von Frey test were performed to evaluate neuropathic pain behavior, and real-time quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, Western blot, immunohistochemistry, and small interfering RNA were performed to understand the molecular mechanisms. RESULTS: The application of paclitaxel induced an up-regulation of CX3CL1 expression in the spinal neurons, which is reduced significantly by NF-κB inhibitor ammonium pyrrolidinedithiocarbamate or p65 small interfering RNA. Blockade of either CX3CL1 (n = 12 each) or NF-κB (n = 12 each) signaling pathway attenuated mechanical allodynia induced by paclitaxel. Chromatin immunoprecipitation further found that paclitaxel induced an increased recruitment of nuclear factor-κB (NF-κB)p65 to the Cx3cl1 promoter region. Furthermore, an increased acetylation level of H4, but not H3, in Cx3cl1 promoter region in spinal neurons was detected after paclitaxel treatment, which was reversed by inhibition of NF-κB with ammonium pyrrolidinedithiocarbamate or p65 small interfering RNA. CONCLUSIONS: These findings suggest that up-regulation of CX3CL1 via NF-κB-dependent H4 acetylation might be critical for paclitaxel-induced mechanical allodynia.

CX3CL1-mediated macrophage activation contributed to paclitaxel-induced DRG neuronal apoptosis and painful peripheral neuropathy.

Painful peripheral neuropathy is a dose-limiting side effect of paclitaxel therapy, which hampers the optimal clinical management of chemotherapy in cancer patients. Currently the underlying mechanisms remain largely unknown. Here we showed that the clinically relevant dose of paclitaxel (3×8mg/kg, cumulative dose 24mg/kg) induced significant upregulation of the chemokine CX3CL1 in the A-fiber primary sensory neurons in vivo and in vitro and infiltration of macrophages into the dorsal root ganglion (DRG) in rats. Paclitaxel treatment also increased cleaved caspase-3 expression, induced the loss of primary afferent terminal fibers and decreased sciatic-evoked A-fiber responses in the spinal dorsal horn, indicating DRG neuronal apoptosis induced by paclitaxel. In addition, the paclitaxel-induced DRG neuronal apoptosis occurred exclusively in the presence of macrophage in vitro study. Intrathecal or systemic injection of CX3CL1 neutralizing antibody blocked paclitaxel-induced macrophage recruitment and neuronal apoptosis in the DRG, and also attenuated paclitaxel-induced allodynia. Furthermore, depletion of macrophage by systemic administration of clodronate inhibited paclitaxel-induced allodynia. Blocking CX3CL1 decreased activation of p38 MAPK in the macrophage, and inhibition of p38 MAPK activity blocked the neuronal apoptosis and development of mechanical allodynia induced by paclitaxel. These findings provide novel evidence that CX3CL1-recruited macrophage contributed to paclitaxel-induced DRG neuronal apoptosis and painful peripheral neuropathy.

Downregulation of ClC-3 in dorsal root ganglia neurons contributes to mechanical hypersensitivity following peripheral nerve injury.

ClC-3 chloride channel/antiporter has been demonstrated to play an important role in synaptic transmission in central nervous system. However, its expression and function in sensory neurons is poorly understood. In present work, we found that ClC-3 is expressed at high levels in dorsal root ganglia (DRG). Co-immunofluorescent data showed that ClC-3 is mainly distributed in A- and C-type nociceptive neurons. ClC-3 expression in DRG is decreased in the spared nerve injury (SNI) model of neuropathic pain. Knockdown of local ClC-3 in DRG neurons with siRNA increased mechanical sensitivity in naïve rats, while overexpression of ClC-3 reversed the hypersensitivity to mechanical stimuli after peripheral nerve injury. In addition, genetic deletion of ClC-3 enhances mouse mechanical sensitivity but did not affect thermal and cold threshold. Restoration of ClC-3 expression in ClC-3 deficient mice reversed the mechanical sensitivity. Mechanistically, loss of ClC-3 enhanced mechanical sensitivity through increasing the excitability of DRG neurons. These data indicate that ClC-3 is an endogenous inhibitor of neuropathic pain development. Downregulation of ClC-3 by peripheral nerve injury is critical for mechanical hypersensitivity. Our findings suggest that ClC-3 is a novel therapeutic target for treating neuropathic pain.

Bulleyaconitine A depresses neuropathic pain and potentiation at C-fiber synapses in spinal dorsal horn induced by paclitaxel in rats.

Paclitaxel, a widely used chemotherapeutic agent, often induces painful peripheral neuropathy and at present no effective drug is available for treatment of the serious side effect. Here, we tested if intragastrical application of bulleyaconitine A (BLA), which has been approved for clinical treatment of chronic pain in China since 1985, could relieve the paclitaxel-induced neuropathic pain. A single dose of BLA attenuated the mechanical allodynia, thermal hyperalgesia induced by paclitaxel dose-dependently. Repetitive administration of the drug (0.4 and 0.8 mg/kg, t.i.d. for 7 d) during or after paclitaxel treatment produced a long-lasting inhibitory effect on thermal hyperalgesia, but not on mechanical allodynia. In consistency with the behavioral results, in vivo electrophysiological experiments revealed that spinal synaptic transmission mediated by C-fiber but not A fiber was potentiated, and the magnitude of long-term potentiation (LTP) at C-fiber synapses induced by the same high frequency stimulation was ~50% higher in paclitaxel-treated rats, compared to the naïve rats. Spinal or intravenous application of BLA depressed the spinal LTP, dose-dependently. Furthermore, patch clamp recordings in spinal cord slices revealed that the frequency but not amplitude of both spontaneous excitatory postsynaptic current (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs) in lamina II neurons was increased in paclitaxel-treated rats, and the superfusion of BLA reduced the frequency of sEPSCs and mEPSCs in paclitaxel-treated rats but not in naïve ones. Taken together, we provide novel evidence that BLA attenuates paclitaxel-induced neuropathic pain and that depression of spinal LTP at C-fiber synapses via inhibiting presynaptic transmitter release may contribute to the effect.

Interleukin-10 down-regulates voltage gated sodium channels in rat dorsal root ganglion neurons.

The over-expression of voltage-gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons following peripheral nerve injury contributes to neuropathic pain by generation of the ectopic discharges of action potentials. However, mechanisms underlying the change in VGSCs' expression are poorly understood. Our previous work has demonstrated that the pro-inflammatory cytokine TNF-α up-regulates VGSCs. In the present work we tested if anti-inflammatory cytokine IL-10, which had been proven to be effective for treating neuropathic pain, had the opposite effect. Western blot and immunofluorescence results showed that IL-10 receptor was localized in DRG neurons. Recombinant rat IL-10 (200 pg/ml) not only reduced the densities of TTX-sensitive and Nav1.8 currents in control DRG neurons, but also reversed the increase of the sodium currents induced by rat recombinant TNF-α (100 pg/ml), as revealed by patch-clamp recordings. Consistent with the electrophysiological results, real-time PCR and western blot revealed that IL-10 (200 pg/ml) down-regulated VGSCs in both mRNA and protein levels and reversed the up-regulation of VGSCs by TNF-α. Moreover, repetitive intrathecal administration of rrIL-10 for 3 days (4 times per day) attenuated mechanical allodynia in L5 spinal nerve ligation model and profoundly inhibited the excitability of DRG neurons. These results suggested that the down-regulation of the sodium channels in DRG neurons might contribute to the therapeutic effect of IL-10 on neuropathic pain.

Peripheral nerve injury leads to working memory deficits and dysfunction of the hippocampus by upregulation of TNF-α in rodents.

Patients with chronic pain usually suffer from working memory deficits, which may decrease their intellectual ability significantly. Despite intensive clinical studies, the mechanism underlying this form of memory impairment remains elusive. In this study, we investigated this issue in the spared nerve injury (SNI) model of neuropathic pain, a most common form of chronic pain. We found that SNI impaired working memory and short-term memory in rats and mice. To explore the potential mechanisms, we studied synaptic transmission/plasticity in hippocampus, a brain region critically involved in memory function. We found that frequency facilitation, a presynaptic form of short-term plasticity, and long-term potentiation at CA3-CA1 synapses were impaired after SNI. Structurally, density of presynaptic boutons in hippocampal CA1 synapses was reduced significantly. At the molecular level, we found that tumor necrosis factor-α (TNF-α) increased in cerebrospinal fluid, in hippocampal tissue and in plasma after SNI. Intracerebroventricular or intrahippocampal injection of recombinant rat TNF mimicked the effects of SNI in naive rats, whereas inhibition of TNF-α or genetic deletion of TNF receptor 1 prevented both memory deficits and synaptic dysfunction induced by SNI. As TNF-α is critical for development of neuropathic pain, we suggested that the over-production of TNF-α following peripheral nerve injury might lead to neuropathic pain and memory deficits, simultaneously.