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.

Bulleyaconitine A attenuates hyperexcitability of dorsal root ganglion neurons induced by spared nerve injury: The role of preferably blocking Nav1.7 and Nav1.3 channels.

Background Oral administration of Bulleyaconitine A, an extracted diterpenoid alkaloid from Aconitum bulleyanum plants, is effective for treating chronic pain in rats and in human patients, but the underlying mechanisms are poorly understood. Results As the hyperexcitability of dorsal root ganglion neurons resulting from the upregulation of voltage-gated sodium (Nav) channels has been proved critical for development of chronic pain, we tested the effects of Bulleyaconitine A on Nav channels in rat spared nerve injury model of neuropathic pain. We found that Bulleyaconitine A at 5 nM increased the threshold of action potentials and reduced the firing rate of dorsal root ganglion neurons in spared nerve injury rats but not in sham rats. Bulleyaconitine A preferably blocked tetrodotoxin-sensitive Nav channels over tetrodotoxin-resistant ones in dorsal root ganglion neurons of spared nerve injury rats. Bulleyaconitine A was more potent for blocking Nav1.3 and Nav1.7 than Nav1.8 in cell lines. The half maximal inhibitory concentration (IC50) values for resting Nav1.3, Nav1.7, and Nav1.8 were 995.6 ± 139.1 nM, 125.7 ± 18.6 nM, and 151.2 ± 15.4 µM, respectively, which were much higher than those for inactivated Nav1.3 (20.3 ± 3.4 pM), Nav1.7 (132.9 ± 25.5 pM), and Nav1.8 (18.0 ± 2.5 µM). The most profound use-dependent blocking effect of Bulleyaconitine A was observed on Nav1.7, less on Nav1.3, and least on Nav1.8 at IC50 concentrations. Bulleyaconitine A facilitated the inactivation of Nav channels in each subtype. Conclusions Preferably blocking tetrodotoxin-sensitive Nav1.7 and Nav1.3 in dorsal root ganglion neurons may contribute to Bulleyaconitine A's antineuropathic pain effect.

Upregulation of N-type calcium channels in the soma of uninjured dorsal root ganglion neurons contributes to neuropathic pain by increasing neuronal excitability following peripheral nerve injury.

N-type voltage-gated calcium (Cav2.2) channels are expressed in the central terminals of dorsal root ganglion (DRG) neurons, and are critical for neurotransmitter release. Cav2.2 channels are also expressed in the soma of DRG neurons, where their function remains largely unknown. Here, we showed that Cav2.2 was upregulated in the soma of uninjured L4 DRG neurons, but downregulated in those of injured L5 DRG neurons following L5 spinal nerve ligation (L5-SNL). Local application of specific Cav2.2 blockers (ω-conotoxin GVIA, 1-100 µM or ZC88, 10-1000 µM) onto L4 and 6 DRGs on the operated side, but not the contralateral side, dose-dependently reversed mechanical allodynia induced by L5-SNL. Patch clamp recordings revealed that both ω-conotoxin GVIA (1 µM) and ZC88 (10 µM) depressed hyperexcitability in L4 but not in L5 DRG neurons of L5-SNL rats. Consistent with this, knockdown of Cav2.2 in L4 DRG neurons with AAV-Cav2.2 shRNA substantially prevented L5-SNL-induced mechanical allodynia and hyperexcitability of L4 DRG neurons. Furthermore, in L5-SNL rats, interleukin-1 beta (IL-1ß) and IL-10 were upregulated in L4 DRGs and L5 DRGs, respectively. Intrathecal injection of IL-1ß induced mechanical allodynia and Cav2.2 upregulation in bilateral L4-6 DRGs of naïve rats, whereas injection of IL-10 substantially prevented mechanical allodynia and Cav2.2 upregulation in L4 DRGs in L5-SNL rats. Finally, in cultured DRG neurons, Cav2.2 was dose-dependently upregulated by IL-1ß and downregulated by IL-10. These data indicate that the upregulation of Cav2.2 in uninjured DRG neurons via IL-1ß over-production contributes to neuropathic pain by increasing neuronal excitability following peripheral nerve injury.

ATF4 inhibits TRPV4 function and controls itch perception in rodents and nonhuman primates.

ABSTRACT: Acute and chronic itch are prevalent and incapacitating, yet the neural mechanisms underlying both acute and chronic itch are just starting to be unraveled. Activated transcription factor 4 (ATF4) belongs to the ATF/CREB transcription factor family and primarily participates in the regulation of gene transcription. Our previous study has demonstrated that ATF4 is expressed in sensory neurons. Nevertheless, the role of ATF4 in itch sensation remains poorly understood. Here, we demonstrate that ATF4 plays a significant role in regulating itch sensation. The absence of ATF4 in dorsal root ganglion (DRG) neurons enhances the itch sensitivity of mice. Overexpression of ATF4 in sensory neurons significantly alleviates the acute and chronic pruritus in mice. Furthermore, ATF4 interacts with the transient receptor potential cation channel subfamily V member 4 (TRPV4) and inhibits its function without altering the expression or membrane trafficking of TRPV4 in sensory neurons. In addition, interference with ATF4 increases the itch sensitivity in nonhuman primates and enhances TRPV4 currents in nonhuman primates DRG neurons; ATF4 and TRPV4 also co-expresses in human sensory neurons. Our data demonstrate that ATF4 controls pruritus by regulating TRPV4 signaling through a nontranscriptional mechanism and identifies a potential new strategy for the treatment of pathological pruritus.

Endophilin A2 controls touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking.

BACKGROUND: Tactile and mechanical pain are crucial to our interaction with the environment, yet the underpinning molecular mechanism is still elusive. Endophilin A2 (EndoA2) is an evolutionarily conserved protein that is documented in the endocytosis pathway. However, the role of EndoA2 in the regulation of mechanical sensitivity and its underlying mechanisms are currently unclear. METHODS: Male and female C57BL/6 mice (8-12 weeks) and male cynomolgus monkeys (7-10 years old) were used in our experiments. Nerve injury-, inflammatory-, and chemotherapy-induced pathological pain models were established for this study. Behavioral tests of touch, mechanical pain, heat pain, and cold pain were performed in mice and nonhuman primates. Western blotting, immunostaining, co-immunoprecipitation, proximity ligation and patch-clamp recordings were performed to gain insight into the mechanisms. RESULTS: The results showed that EndoA2 was primarily distributed in neurofilament-200-positive (NF200+) medium-to-large diameter dorsal root ganglion (DRG) neurons of mice and humans. Loss of EndoA2 in mouse NF200+ DRG neurons selectively impaired the tactile and mechanical allodynia. Furthermore, EndoA2 interacted with the mechanically sensitive ion channel Piezo2 and promoted the membrane trafficking of Piezo2 in DRG neurons. Moreover, as an adaptor protein, EndoA2 also bound to kinesin family member 5B (KIF5B), which was involved in the EndoA2-mediated membrane trafficking process of Piezo2. Loss of EndoA2 in mouse DRG neurons damaged Piezo2-mediated rapidly adapting mechanically activated currents, and re-expression of EndoA2 rescued the MA currents. In addition, interference with EndoA2 also suppressed touch sensitivity and mechanical hypersensitivity in nonhuman primates. CONCLUSIONS: Our data reveal that the KIF5B/EndoA2/Piezo2 complex is essential for Piezo2 trafficking and for sustaining transmission of touch and mechanical hypersensitivity signals. EndoA2 regulates touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking in sensory neurons. Our findings identify a potential new target for the treatment of mechanical pain.

Hen Egg Lysozyme Alleviates Static Mechanical Pain Via NRF1-Parkin-TACAN Signaling Axis in Sensory Neurons.

Mechanical allodynia impinges on the life quality of patients. Hen Egg Lysozyme (HEL) is a substance extracted from eggs that is commonly used to inhibit bacterial activity. The role of HEL in regulating and treating pain is unclear. Here, we find that HEL selectively attenuates static mechanical allodynia of mice induced by complete Freund's adjuvant (CFA), spinal nerve ligation (SNL) and chemotherapeutic agent. RNA-seq screening reveals that CFA significantly reduces the expression of Parkin in dorsal root ganglion (DRG) neurons of mice, while pre-administration of HEL increases the expression of Parkin and remits the static mechanical allodynia induced by Parkin-siRNA. Moreover, HEL increases the interaction between nuclear respiratory factor 1 (NRF1) and histone acetyltransferase P300 and then enhances the NRF1 mediated histone acetylation in prkn promoter region in DRGs of mice. Further, Parkin interacts with mechanotransducing ion channel TACAN (Tmem120a) and knockdown of Parkin significantly increases the membrane trafficking of TACAN in sensory neurons of mice. While pre-administration of HEL inhibits the increased membrane trafficking of TACAN in sensory neurons of mice induced by Parkin-siRNA. In addition, pre-given of HEL also significantly attenuates the static mechanical allodynia induced by overexpression of TACAN in mice, and the effect of HEL can be blocked by Parkin-siRNA. This indicates that HEL increases the expression of Parkin through epigenetic mechanisms and then decreases TACAN membrane trafficking in sensory neurons to relieve static mechanical hypersensitivity. Therefore, we reveal a novel function of HEL, which is a potential substance for the treatment of static mechanical pain.

The Causal Role of Magnesium Deficiency in the Neuroinflammation, Pain Hypersensitivity and Memory/Emotional Deficits in Ovariectomized and Aged Female Mice.

PURPOSE: Postmenopausal women often suffer from chronic pain, memory decline and mood depression. The mechanisms underlying the neuronal disorders are not fully understood, and effective treatment is still lacking. METHODS: Oral administration of magnesium-L-threonate was tested to treat the neuronal disorders in ovariectomized and aged female mice. The pain hypersensitivity, memory function and depression-like behaviors were measured with a set of behavioral tests. Western blots, immunochemistry and in situ hybridization were used to assess molecular changes. RESULTS: Chronic oral administration of magnesium-L-threonate substantially prevented or reversed the chronic pain and memory/emotional deficits in both ovariectomized and aged female mice. We found that phospho-p65, an active form of nuclear factor-kappaB, tumor necrosis factor-alpha and interleukin-1 beta were significantly upregulated in the neurons of dorsal root ganglion, spinal dorsal horn and hippocampus in ovariectomized and aged mice. The microglia and astrocytes were activated in spinal dorsal horn and hippocampus. Calcitonin gene-related peptide, a marker for peptidergic C-fibers, was upregulated in dorsal horn, which is associated with potentiation of C-fiber-mediated synaptic transmission in the model mice. In parallel with neuroinflammation and synaptic potentiation, free Mg2+ levels in plasma, cerebrospinal fluid and in dorsal root ganglion neurons were significantly reduced. Oral magnesium-L-threonate normalized the neuroinflammation, synaptic potentiation and Mg2+ deficiency, but did not affect the estrogen decline in ovariectomized and aged mice. Furthermore, in cultured dorsal root ganglion neurons, estrogen at physiological concentration elevated intracellular Mg2+, and downregulated phospho-p65, tumor necrosis factor-alpha and interleukin-1 beta exclusively in the presence of extracellular Mg2+. CONCLUSION: Estrogen decline in menopause may cause neuroinflammation by reducing intracellular Mg2+ in neurons, leading to chronic pain, memory/emotional deficits. Supplement Mg2+ by oral magnesium-L-threonate may be a novel approach for treating menopause-related neuronal disorders.

ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking.

Effective treatments for patients suffering from heat hypersensitivity are lacking, mostly due to our limited understanding of the pathogenic mechanisms underlying this disorder. In the nervous system, activating transcription factor 4 (ATF4) is involved in the regulation of synaptic plasticity and memory formation. Here, we show that ATF4 plays an important role in heat nociception. Indeed, loss of ATF4 in mouse dorsal root ganglion (DRG) neurons selectively impairs heat sensitivity. Mechanistically, we show that ATF4 interacts with transient receptor potential cation channel subfamily M member-3 (TRPM3) and mediates the membrane trafficking of TRPM3 in DRG neurons in response to heat. Loss of ATF4 also significantly decreases the current and KIF17-mediated trafficking of TRPM3, suggesting that the KIF17/ATF4/TRPM3 complex is required for the neuronal response to heat stimuli. Our findings unveil the non-transcriptional role of ATF4 in the response to heat stimuli in DRG neurons.

Difference of pain vulnerability in adult and juvenile rodents: the role of SIRT1-mediated ClC-3 trafficking in sensory neurons.

ABSTRACT: Adults are more likely to suffer from chronic pain than minors, and its underlying mechanism remains unclear. SIRT1 an important age-related protein with function of lifespan extension; whether SIRT1 plays a role in the different pain vulnerability of adult and juvenile remains unclear. Here, we found that the expression level of SIRT1 in dorsal root ganglia (DRG) was related to the pain vulnerability. After nerve injury, the expression of SIRT1 in DRG was decreased in adult rodents whereas increased in juvenile rodents. Differential manipulation of SIRT1 abolished the different pain vulnerability between adult and juvenile rodents. Furthermore, SIRT1 interacted with ClC-3 channel and mediated ClC-3 membrane trafficking and Cl- current in DRG neurons. Differential manipulation of ClC-3 also abolished the difference in pain vulnerability between adult and juvenile rodents. The different anti-inflammatory ability determined the different change trends of SIRT1 and ClC-3 trafficking contributed to the different pain vulnerability in adult and juvenile rodents. In addition, the serum SIRT1 level was negatively correlated with the pain score in patients with chronic pain. These findings revealed the mechanism of the difference in pain vulnerability between adult and juvenile rodents and provided evidence for age-specific treatment of chronic pain.

Foxo1 selectively regulates static mechanical pain by interacting with Nav1.7.

ABSTRACT: Mechanical allodynia is a debilitating condition for millions of patients with chronic pain. Mechanical allodynia can manifest in distinct forms, including brush-evoked dynamic and filament-evoked static allodynia. In the nervous system, the forkhead protein Foxo1 plays a critical role in neuronal structures and functions. However, the role of Foxo1 in the somatosensory signal remains unclear. Here, we found that Foxo1 selectively regulated static mechanical pain. Foxo1 knockdown decreased sensitivity to static mechanical stimuli in normal rats and attenuated static mechanical allodynia in rat models for neuropathic, inflammatory, and chemotherapy pain. Conversely, Foxo1 overexpression selectively enhanced sensitivity to static mechanical stimuli and provoked static mechanical allodynia. Furthermore, Foxo1 interacted with voltage-gated sodium Nav1.7 channels and increased the Nav1.7 current density by accelerating activation rather than by changing the expression of Nav1.7 in dorsal root ganglia neurons. In addition, the serum level of Foxo1 was found to be increased in chronic pain patients and to be positively correlated with the severity of chronic pain. Altogether, our findings suggest that serum Foxo1 level could be used as a biological marker for prediction and diagnosis of chronic pain. Moreover, selective blockade of Foxo1/Nav1.7 interaction may offer a new therapeutic approach in patients with mechanical pain.

Nuclear Factor-kappaB Gates Nav1.7 Channels in DRG Neurons via Protein-Protein Interaction.

It is well known that nuclear factor-kappaB (NF-κB) regulates neuronal structures and functions by nuclear transcription. Here, we showed that phospho-p65 (p-p65), an active form of NF-κB subunit, reversibly interacted with Nav1.7 channels in the membrane of dorsal root ganglion (DRG) neurons of rats. The interaction increased Nav1.7 currents by slowing inactivation of Nav1.7 channels and facilitating their recovery from inactivation, which may increase the resting state of the channels ready for activation. In cultured DRG neurons TNF-α upregulated the membrane p-p65 and enhanced Nav1.7 currents within 5 min but did not affect nuclear NF-κB within 40 min. This non-transcriptional effect on Nav1.7 may underlie a rapid regulation of the sensibility of the somatosensory system. Both NF-κB and Nav1.7 channels are critically implicated in many physiological functions and diseases. Our finding may shed new light on the investigation into the underlying mechanisms.

Puerarin Relieves Paclitaxel-Induced Neuropathic Pain: The Role of Nav1.8 ß1 Subunit of Sensory Neurons.

Currently there is no effective treatment available for clinical patients suffering from neuropathic pain induced by chemotherapy paclitaxel. Puerarin is a major isoflavonoid extracted from the Chinese medical herb kudzu root, which has been used for treatment of cardiovascular disorders and brain injury. Here, we found that puerarin dose-dependently alleviated paclitaxel-induced neuropathic pain. At the same time, puerarin preferentially reduced the excitability and blocked the voltage-gated sodium (Nav) channels of dorsal root ganglion (DRG) neurons from paclitaxel-induced neuropathic pain rats. Furthermore, puerarin was a more potent blocker of tetrodotoxin-resistant (TTX-R) Nav channels than of tetrodotoxin-sensitive (TTX-S) Nav channels in chronic pain rats' DRG neurons. In addition, puerarin had a stronger blocking effect on Nav1.8 channels in DRG neurons of neuropathic pain rats and ß1 subunit siRNA can abolish this selective blocking effect on Nav1.8. Together, these results suggested that puerarin may preferentially block ß1 subunit of Nav1.8 in sensory neurons contributed to its anti-paclitaxel induced neuropathic pain effect.

Activation of RAGE/STAT3 pathway by methylglyoxal contributes to spinal central sensitization and persistent pain induced by bortezomib.

Bortezomib is a first-line chemotherapeutic drug widely used for multiple myeloma and other nonsolid malignancies. Although bortezomib-induced persistent pain is easily diagnosed in clinic, the pathogenic mechanism remains unclear. Here, we studied this issue with use of a rat model of systemic intraperitoneal administration of bortezomib for consecutive 5days. Consisted with our previous study, we found that bortezomib treatment markedly induced mechanical allodynia in rats. Furthermore, we first found that bortezomib treatment significantly induced the upregulation of methylglyoxal in spinal dorsal horn of rats. Spinal local application of methylglyoxal also induced mechanical allodynia and central sensitization in normal rats. Moreover, administration of bortezomib upregulated the expression of receptors for advanced glycation end products (RAGE) and phosphorylated STAT3 (p-STAT3) in dorsal horn. Importantly, intrathecal injection of metformin, a known scavenger of methylglyoxal, significantly attenuated the upregulation of methylglyoxal and RAGE in dorsal horn, central sensitization and mechanical allodynia induced by bortezomib treatment, and blockage of RAGE also prevented the upregulation of p-STAT3, central sensitization and mechanical allodynia induced by bortezomib treatment. In addition, inhibition of STAT3 activity by S3I-201 attenuated bortezomib-induced mechanical allodynia and central sensitization. Local knockdown of STAT3 also ameliorated the mechanical allodynia induced by bortezomib administration. Our results suggest that accumulation of methylglyoxal may activate the RAGE/STAT3 signaling pathway in dorsal horn, and contributes to the spinal central sensitization and persistent pain induced by bortezomib treatment.

Bulleyaconitine A preferably reduces tetrodotoxin-sensitive sodium current in uninjured dorsal root ganglion neurons of neuropathic rats probably via inhibition of protein kinase C.

Oral Bulleyaconitine A (BLA) is effective for treating neuropathic pain in human patients, but the underlying mechanism is poorly understood. Here, we tested whether BLA blocked voltage-gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons. Compelling evidence shows that voltage-gated sodium channels are upregulated in uninjured DRG neurons but downregulated in injured ones following peripheral nerve injury. We found that BLA preferably inhibited Na currents in uninjured DRG neurons in neuropathic rats. Compared to sham rats, IC50 values for resting and inactivated Na currents were 113 and 74 times lower in injured and uninjured neurons of L4-6 DRGs in spared nerve injury (SNI) rats (4.55 and 0.56 nM) and were 688 and 518 times lower in the uninjured L4 and L6 DRG neurons of L5 spinal nerve ligation (L5-SNL) rats. The use-dependent blockage of BLA on Na currents was more potent in neuropathic rats compared to sham rats. Bulleyaconitine A facilitated the inactivation of Na channels in each group. IC50 values for resting and inactivated tetrodotoxin-sensitive (TTX-S) channels were 1855 and 1843 times lower than those for TTX-resistant channels in the uninjured neurons of L5 spinal nerve ligation rats. The upregulation of protein kinase C was associated with the preferable effect of BLA on TTX-S Na channels in the uninjured DRG neurons. Local application of BLA onto L4-6 DRGs at 0.1 to 10 nM dose-dependently alleviated the mechanical allodynia and thermal hyperalgesia in L5 spinal nerve ligation model. Thus, preferable blockage of TTX-S Na channels in uninjured DRG neurons may contribute to BLA's antineuropathic pain effect.

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.