Lycium Barbarum Polysaccharides Improves Cognitive Functions in ICV-STZ-Induced Alzheimer's Disease Mice Model by Improving the Synaptic Structural Plasticity and Regulating IRS1/PI3K/AKT Signaling Pathway.

Lycium barbarum polysaccharide (LBP) have a certain curative effect on hypoglycemic and neuroprotective effects, but the specific mechanism is unclear and needs to be further explored. This study aimed to clarify the mechanisms of LBP in the treatment of ICV-STZ mice model of AD from the perspectives of insulin resistance, IRS1/PI3K/AKT signaling pathway, and synaptic protein expression. We used male C57BL/6J mice injected with STZ (3 mg/kg) in the lateral ventricle as an AD model. After treatment with LBP, the learning and memory abilities of ICV-STZ mice were enhanced, and the pathological changes in brain tissue were alleviated. LBP can regulate the expression of proteins related to the IRS1/PI3K/AKT signaling pathway and thereby reducing Aß deposition and tau protein phosphorylation in the brain of ICV-STZ mice. In addition, LBP also can up-regulate the expression of synaptic proteins. The results indicated that LBP played a neuroprotective role by regulating the IRS1/PI3K/AKT pathway, inhibiting tau protein hyperphosphorylation and improving the expression levels of synapse-related proteins.

Upregulation of Nav1.7 by endogenous hydrogen sulfide contributes to maintenance of neuropathic pain.

Nav1.7 is closely associated with neuropathic pain. Hydrogen sulfide (H2S) has recently been reported to be involved in numerous biological functions, and it has been shown that H2S can enhance the sodium current density, and inhibiting the endogenous production of H2S mediated by cystathionine ß­synthetase (CBS) using O­(carboxymethyl)hydroxylamine hemihydrochloride (AOAA) can significantly reduce the expression of Nav1.7 and thus the sodium current density in rat dorsal root ganglion (DRG) neurons. In the present study, it was shown that the fluorescence intensity of H2S was increased in a spared nerve injury (SNI) model and AOAA inhibited this increase. Nav1.7 is expressed in DRG neurons, and the expression of CBS and Nav1.7 were increased in DRG neurons 7, 14 and 21 days post­operation. AOAA inhibited the increase in the expression of CBS, phosphorylated (p)­MEK1/2, p­ERK1/2 and Nav1.7 induced by SNI, and U0126 (a MEK blocker) was able to inhibit the increase in p­MEK1/2, p­ERK1/2 and Nav1.7 expression. However, PF­04856264 did not inhibit the increase in CBS, p­MEK1/2, p­ERK1/2 or Nav1.7 expression induced by SNI surgery. The current density of Nav1.7 was significantly increased in the SNI model and administration of AOAA and U0126 both significantly decreased the density. In addition, AOAA, U0126 and PF­04856264 inhibited the decrease in rheobase, and the increase in action potential induced by SNI in DRG neurons. There was no significant difference in thermal withdrawal latency among each group. However, the time the animals spent with their paw lifted increased significantly following SNI, and the time the animals spent with their paw lifted decreased significantly following the administration of AOAA, U0126 and PF­04856264. In conclusion, these data show that Nav1.7 expression in DRG neurons is upregulated by CBS­derived endogenous H2S in an SNI model, contributing to the maintenance of neuropathic pain.

Safflower Yellow Improves the Synaptic Structural Plasticity by Ameliorating the Disorder of Glutamate Circulation in Aß1-42-induced AD Model Rats.

Safflower yellow (SY) is the main effective component of Carthamus tinctorius L., and Hydroxysafflor yellow A (HSYA) is the single active component with the highest content in SY. SY and HSYA have been shown to have neuroprotective effects in several AD models. In this study, we aimed to clarify whether the effects of SY and HSYA on the learning and memory abilities of Aß1-42-induced AD model rats are related to the enhancement of synaptic structural plasticity in brain tissues and the amelioration of disorder of glutamate circulation. We used rats injected with Aß1-42 into the bilateral hippocampus as a model of AD. After treatment with SY and HSYA, the learning and memory abilities of the Aß1-42-induced AD model rats were enhanced, Aß deposition in the AD model rats was decreased, structural damage to dendritic spines and the loss of synaptic-associated proteins were alleviated, and the disorder of glutamate circulation was ameliorated. The results indicated that SY and HSYA improve synaptic structural plasticity by ameliorating the disorder of glutamate circulation in Aß1-42-induced AD model rats.

Safflower Yellow Improves Synaptic Plasticity in APP/PS1 Mice by Regulating Microglia Activation Phenotypes and BDNF/TrkB/ERK Signaling Pathway.

Alzheimer's disease (AD) is a common neurodegenerative disease that is always accompanied by synaptic loss in the brain. Safflower yellow (SY) is the extract of safflower, a traditional Chinese medicine, which has shown neuroprotective effects in recent studies. However, the mechanism of SY in protecting synapses remains unclear. In this study, we are going to study the mechanism of how SY treats AD in terms of synaptic plasticity. We found, via behavioral experiments, that SY treatment could improve the abilities of learning and memory in APP/PS1 mice. In addition, using Golgi staining and HE staining, we found that SY treatment could reduce the loss of dendritic spines in the pathological condition and could maintain the normal physiological state of the cells in cortex and in hippocampus. In addition, the results of immunofluorescence staining and western blotting showed that SY treatment could significantly increase the expression of synapse-related proteins. Moreover, after being treated with SY, the expression of iNOS (marker of M1 microglia) declined remarkably, and the level of Arginase-1 (marker of M2 microglia) increased significantly. Finally, we found BDNF/TrkB/ERK signaling cascade was activated. These results indicate that SY enhances synaptic plasticity in APP/PS1 mice by regulating microglia activation phenotypes and BDNF/TrkB/ERK signaling pathway.

Geniposidic acid ameliorates spatial learning and memory deficits and alleviates neuroinflammation via inhibiting HMGB-1 and downregulating TLR4/2 signaling pathway in APP/PS1 mice.

Geniposidic acid (GPA) is an extract from Eucommia ulmoides Oliv. Bark (Eucommiaceae). Accumulating evidences have reported GPA has anti-aging, anti-oxidative stress, anti-inflammatory and neurotrophic effects on neurons. However, whether GPA could alleviate memory deficits in Alzheimer's disease animal models is not clear. We aimed to investigate the effect of GPA treatment on cognitive performance, Aß deposition and glial cells activation in the transgenic mouse model of AD. 6-7 months APP/PS1 mice were given GPA for 90 days; behavioral experiments were executed to estimate the memory and spatial learning abilities of mice, and the mechanism of neuroprotective effect of GPA was investigated with a focus on amyloid-ß deposition, astrocytes and microglia activation and neuroinflammation. GPA treatment significantly improved the spatial learning and memory abilities and also decreased cerebral amyloid-ß deposition in APP/PS1 mice. Via HE staining, we found that GPA could ameliorate histopathological changes in cerebrum. We also found that GPA treatment inhibited the activation of astrocytes and microglia, down-regulated the expression of pro-inflammatory cytokines and iNOS, and up-regulated the expression of anti-inflammatory cytokines and Arg-1. In addition, GPA down-regulated the gene expression of HMGB-1 receptors (TLR2, TLR4 and RAGE) then mediated MyD88, TRAF6 and phospho-ERK1/2, subsequently modulated the expression of key AP-1 and NF-κB family members (c-Fos, c-Jun and p65). The reversal of the pro-inflammatory state suggested GPA can serves as a multi-target candidate by alleviating Aß deposition and neuroinflammation for the auxiliary therapy of Alzheimer's disease.

Safflower yellow attenuates learning and memory deficits in amyloid ß-induced Alzheimer's disease rats by inhibiting neuroglia cell activation and inflammatory signaling pathways.

Safflower yellow (SY) is an aqueous extract of natural safflower. Our laboratory has reported protective effects of alleviating memory impairment with SY in a transgentic mouse model of Alzheimer's disease. The possible beneficial effects of SY on amyloid-ß-induced neuroinflammation in dementia remain unclarified. This study we hypothesize that astrocytes and microglia may cause amyloid-ß deposition and produce a neuroinflammatory response, aims to explain the role and mechanism of SY in regulating glial activation and reducing Aß deposition in Aß1-42 induced rat model. Wistar rats were treated with SY for one month after bilateral hippocampal injection of aggregated Aß1-42; behavioral tests were performed to demonstrate the amelioration of cognitive function. After that, the contents of iNOS, IL-1ß, IL-6, and TNF-α in AD brain was detected. Western blot and real-time PCR were used to detect the M1 and M2-associated markers to demonstrate the activation of microglia. The conducted experiments have revealed that SY could strengthen spatial learning and memory ability of dementia rats, decrease the contents of iNOS, IL-1ß, IL-6, and TNF-α and depress the activation of glial cells. Moreover, the SY treatment inhibited the M1 release of pro-inflammatory cytokines (iNOS and CD86), increased the expression of arginase-1, CD206, and YM-1 thereby reduced inflammation in model rats. Thus our results indicated that SY has very important theoretical and clinical value for the research and development of Chinese medicine for the treatment of AD.

Prolactin potentiates the activity of acid-sensing ion channels in female rat primary sensory neurons.

Prolactin (PRL) is a polypeptide hormone produced and released from the pituitary and extrapituitary tissues. It regulates activity of nociceptors and causes hyperalgesia in pain conditions, but little is known the molecular mechanism. We report here that PRL can exert a potentiating effect on the functional activity of acid-sensing ion channels (ASICs), key sensors for extracellular protons. First, PRL dose-dependently increased the amplitude of ASIC currents with an EC50 of (5.89 ± 0.28) × 10(-8) M. PRL potentiation of ASIC currents was also pH dependent. Second, PRL potentiation of ASIC currents was blocked by Δ1-9-G129R-hPRL, a PRL receptor antagonist, and removed by intracellular dialysis of either protein kinase C inhibitor GF109203X, protein interacting with C-kinase 1(PICK1) inhibitor FSC-231, or PI3K inhibitor AS605240. Third, PRL altered acidosis-evoked membrane excitability of DRG neurons and caused a significant increase in the amplitude of the depolarization and the number of spikes induced by acid stimuli. Four, PRL exacerbated nociceptive responses to injection of acetic acid in female rats. Finally, PRL displayed a stronger effect on ASIC mediated-currents and nociceptive behavior in intact female rats than OVX female and male rats and thus modulation of PRL may be gender-dependent. These results suggest that PRL up-regulates the activity of ASICs and enhances ASIC mediated nociceptive responses in female rats, which reveal a novel peripheral mechanism underlying PRL involvement in hyperalgesia.

Prokineticin 2 facilitates mechanical allodynia induced by α,ß-methylene ATP in rats.

Prokineticin 2 (PK2), a new chemokine, causes mechanical hypersensitivity in the rat hind paw, but little is known about the molecular mechanism. Here, we have found that ionotropic P2X receptor is essential to mechanical allodynia induced by PK2. First, intraplantar injection of high dose (3 or 10 pmol) of PK2 significantly increased paw withdrawal response frequency (%) to innocuous mechanical stimuli (mechanical allodynia). And the mechanical allodynia induced by PK2 was prevented by co-administration of TNP-ATP, a selective P2X receptor antagonist. Second, although low dose (0.3 or 1 pmol) of PK2 itself did not produce an allodynic response, it significantly facilitated the mechanical allodynia evoked by intraplantar injection of α,ß-methylene ATP (α,ß-meATP). Third, PK2 concentration-dependently potentiated α,ß-meATP-activated currents in rat dorsal root ganglion (DRG) neurons. Finally, PK2 receptors and intracellular signal transduction were involved in PK2 potentiation of α,ß-meATP-induced mechanical allodynia and α,ß-meATP-activated currents, since the potentiation were blocked by PK2 receptor antagonist PKRA and selective PKC inhibitor GF 109203X. These results suggested that PK2 facilitated mechanical allodynia induced by α,ß-meATP through a mechanism involved in sensitization of cutaneous P2X receptors expressed by nociceptive nerve endings.

17ß-Estradiol Enhances ASIC Activity in Primary Sensory Neurons to Produce Sex Difference in Acidosis-Induced Nociception.

Sex differences have been reported in a number of pain conditions. Women are more sensitive to most types of painful stimuli than men, and estrogen plays a key role in the sex differences in pain perception. However, it is unclear whether there is a sex difference in acidosis-evoked pain. We report here that both male and female rats exhibit nociceptive behaviors in response to acetic acid, with females being more sensitive than males. Local application of exogenous 17ß-estradiol (E2) exacerbated acidosis-evoked nociceptive response in male rats. E2 and estrogen receptor (ER)-α agonist 1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, but not ERß agonist 2,3-bis(4-hydroxyphenyl)-propionitrile, replacement also reversed attenuation of the acetic acid-induced nociceptive response in ovariectomized females. Moreover, E2 can exert a rapid potentiating effect on the functional activity of acid-sensing ion channels (ASICs), which mediated the acidosis-induced events. E2 dose dependently increased the amplitude of ASIC currents with a 42.8 ± 1.6 nM of EC50. E2 shifted the concentration-response curve for proton upward with a 50.1% ± 6.2% increase of the maximal current response to proton. E2 potentiated ASIC currents via an ERα and ERK1/2 signaling pathway. E2 also altered acidosis-evoked membrane excitability of dorsal root ganglia neurons and caused a significant increase in the amplitude of the depolarization and the number of spikes induced by acidic stimuli. E2 potentiation of the functional activity of ASICs revealed a peripheral mechanism underlying this sex difference in acetic acid-induced nociception.

Spinal vasopressin alleviates formalin-induced nociception by enhancing GABAA receptor function in mice.

Arginine vasopressin (AVP) plays a regulatory role in nociception. Intrathecal administration of AVP displays an antinociceptive effect. However, little is understood about the mechanism underlying spinal AVP analgesia. Here, we have found that spinal AVP dose dependently reduced the second, but not first, phase of formalin-induced spontaneous nociception in mice. The AVP analgesia was completely blocked by intrathecal injected SR 49059, a vasopressin-1A (V1A) receptor antagonist. However, spinal AVP failed to exert its antinociceptive effect on the second phase formalin-induced spontaneous nociception in V1A receptor knock-out (V1A-/-) mice. The AVP analgesia was also reversed by bicuculline, a GABAA receptor antagonist. Moreover, AVP potentiated GABA-activated currents in dorsal root ganglion neurons from wild-type littermates, but not from V1A-/- mice. Our results may reveal a novel spinal mechanism of AVP analgesia by enhancing the GABAA receptor function in the spinal cord through V1A receptors.

Inhibition of acid-sensing ion channels by levo-tetrahydropalmatine in rat dorsal root ganglion neurons.

Levo-tetrahydropalmatine (l-THP), a main bioactive Chinese herbal constituent from the genera Stephania and Corydalis, has been in use in clinical practice for years in China as a traditional analgesic agent. However, the mechanism underlying the analgesic action of l-THP is poorly understood. This study shows that l-THP can exert an inhibitory effect on the functional activity of native acid-sensing ion channels (ASICs), which are believed to mediate pain caused by extracellular acidification. l-THP dose dependently decreased the amplitude of proton-gated currents mediated by ASICs in rat dorsal root ganglion (DRG) neurons. l-THP shifted the proton concentration-response curve downward, with a decrease of 40.93% ± 8.45% in the maximum current response to protons, with no significant change in the pH0.5 value. Moreover, l-THP can alter the membrane excitability of rat DRG neurons to acid stimuli. It significantly decreased the number of action potentials and the amplitude of the depolarization induced by an extracellular pH drop. Finally, peripherally administered l-THP inhibited the nociceptive response to intraplantar injection of acetic acid in rats. These results indicate that l-THP can inhibit the functional activity of ASICs in dissociated primary sensory neurons and relieve acidosis-evoked pain in vivo, which for the first time provides a novel peripheral mechanism underlying the analgesic action of l-THP.

Inhibition of acid-sensing ion channels by chlorogenic acid in rat dorsal root ganglion neurons.

Chlorogenic acid (CGA) is one of the most abundant polyphenol compounds in the human diet. Recently, it is demonstrated to have potent antinociceptive effect. However, little is understood about the mechanism underlying CGA analgesia. Here, we have found that CGA can exert an inhibitory effect on the functional activity of native acid-sensing ion channels (ASICs) in rat dorsal root ganglion (DRG) neurons. First, CGA decreased the peak amplitude of proton-gated currents mediated by ASICs in a concentration-dependent manner. Second, CGA shifted the proton concentration-response curve downward, with a decrease of 41.76 ± 8.65% in the maximum current response to protons but with no significant change in the pH0.5 value. Third, CGA altered acidosis-evoked membrane excitability of rat DRG neurons and caused a significant decrease in the amplitude of the depolarization and the number of action potentials induced by acid stimuli. Finally, peripheral administered CGA attenuated nociceptive response to intraplantar injection of acetic acid in rats. ASICs are distributed in peripheral sensory neurons and participate in nociception. Our findings CGA inhibition of native ASICs indicated that CGA may exert analgesic action by modulating ASICs in the primary afferent neurons, which revealed a novel cellular and molecular mechanism underlying CGA analgesia.

Oxytocin inhibits the activity of acid-sensing ion channels through the vasopressin, V1A receptor in primary sensory neurons.

BACKGROUND AND PURPOSE: A growing number of studies have demonstrated that oxytocin (OT) plays an analgesic role in modulation of nociception and pain. Most work to date has focused on the central mechanisms of OT analgesia, but little is known about whether peripheral mechanisms are also involved. Acid-sensing ion channels (ASICs) are distributed in peripheral sensory neurons and participate in nociception. Here, we investigated the effects of OT on the activity of ASICs in dorsal root ganglion (DRG) neurons. EXPERIMENTAL APPROACH: Electrophysiological experiments were performed on neurons from rat DRG. Nociceptive behaviour was induced by acetic acid in rats and mice lacking vasopressin, V1A receptors. KEY RESULTS: OT inhibited the functional activity of native ASICs. Firstly, OT dose-dependently decreased the amplitude of ASIC currents in DRG neurons. Secondly, OT inhibition of ASIC currents was mimicked by arginine vasopressin (AVP) and completely blocked by the V1A receptor antagonist SR49059, but not by the OT receptor antagonist L-368899. Thirdly, OT altered acidosis-evoked membrane excitability of DRG neurons and significantly decreased the amplitude of the depolarization and number of action potentials induced by acid stimuli. Finally, peripherally administered OT or AVP inhibited nociceptive responses to intraplantar injection of acetic acid in rats. Both OT and AVP also induced an analgesic effect on acidosis-evoked pain in wild-type mice, but not in V1A receptor knockout mice. CONCLUSIONS AND IMPLICATIONS: These results reveal a novel peripheral mechanism for the analgesic effect of OT involving the modulation of native ASICs in primary sensory neurons mediated by V1A receptors.

Gastrodin inhibits the activity of acid-sensing ion channels in rat primary sensory neurons.

Acid-sensing ion channels (ASICs), a family of proton-gated cation channels, are believed to mediate pain caused by extracellular acidification. Gastrodin is a main bioactive constituent of the traditional herbal Gastrodia elata Blume, which has been widely used in Oriental countries for centuries. As an analgesic, gastrodin has been used clinically to treat pain such as migraine and headache. However, the mechanisms underlying analgesic action of gastrodin are still poorly understood. Here, we have found that gastrodin inhibited the activity of native ASICs in rat dorsal root ganglion (DRG) neurons. Gastrodin dose-dependently inhibited proton-gated currents mediated by ASICs. Gastrodin shifted the proton concentration-response curve downwards, with a decrease of 36.92 ± 6.23% in the maximum current response but with no significant change in the pH0.5 value. Moreover, gastrodin altered acid-evoked membrane excitability of rat DRG neurons and caused a significant decrease in the amplitude of the depolarization and the number of action potentials induced by acid stimuli. Finally, peripheral applied gastrodin relieved pain evoked by intraplantar injection of acetic acid in rats. Our results indicate that gastrodin can inhibit the activity of ASICs in the primary sensory neurons, which provided a novel mechanism underlying analgesic action of gastrodin.

Morphine inhibits acid-sensing ion channel currents in rat dorsal root ganglion neurons.

Extracellular acidosis is a common feature in pain-generating pathological conditions. Acid-sensing ion channels (ASICs), pH sensors, are distributed in peripheral sensory neurons and participate in nociception. Morphine exerts potent analgesic effects through the activation of opioid receptors for various pain conditions. A cross-talk between ASICs and opioid receptors in peripheral sensory neurons has not been shown so far. Here, we have found that morphine inhibits the activity of native ASICs in rat dorsal root ganglion (DRG) neurons. Morphine dose-dependently inhibited proton-gated currents mediated by ASICs in the presence of the TRPV1 inhibitor capsazepine. Morphine shifted the proton concentration-response curve downwards, with a decrease of 51.4±3.8% in the maximum current response but with no significant change in the pH0.5 value. Another µ-opioid receptor agonist DAMGO induced a similar decrease in ASIC currents compared with morphine. The morphine inhibition of ASIC currents was blocked by naloxone, a specific opioid receptor antagonist. Pretreatment of forskolin, an adenylyl cyclase activator, or the addition of cAMP reversed the inhibitory effect of morphine. Moreover, morphine altered acid-evoked excitability of rat DRG neurons and decreased the number of action potentials induced by acid stimuli. Finally, peripheral applied morphine relieved pain evoked by intraplantar of acetic acid in rats. Our results indicate that morphine can inhibit the activity of ASICs via µ-opioid receptor and cAMP dependent signal pathway. These observations demonstrate a cross-talk between ASICs and opioid receptors in peripheral sensory neurons, which was a novel analgesic mechanism of morphine.