Abeta(31-35)-induced neuronal apoptosis is mediated by JNK-dependent extrinsic apoptosis pathway.

OBJECTIVE: To investigate whether JNK-caspase-dependent apoptotic pathway is involved in Abeta(31-35)-induced apoptosis of cultured cortical neurons. METHODS: Cultured cortical neurons were treated with Abeta(31-35) (25 micromol/L) for 4 h, 8 h, 16 h and 24 h, respectively. Caspase activities were measured using a spectrophotometer. Levels of c-Jun phosphorylation (p-c-Jun) and Fas ligand (FasL) expression were assessed by immunocytochemistry method and quantified using Image-pro plus11.0 image processing and analysis software. RESULTS: Treatment with Abeta(31-35) (25 micromol/L) for 24 h induced significant increases in the activities of caspase-3 and caspase-8 in the cortical neurons. Besides, Abeta(31-35) could time-dependently enhance the expression of p-c-Jun protein. Moreover, SP600125 application (100 nmol/L) could completely abolish Abeta(31-35) neurotoxicity. The increase in FasL expression was detected at 8 h, 16 h and 24 h after Abeta(31-35) treatment, and SP600125 (100 nmol/L) significantly inhibited FasL expression. CONCLUSION: JNK-c-Jun-FasL-caspase-dependent extrinsic apoptotic pathway plays a critical role in mediating Abeta(31-35)-induced apoptosis of cultured neurons.

Electroacupuncture attenuates bone-cancer-induced hyperalgesia and inhibits spinal preprodynorphin expression in a rat model.

Cancer pain impairs the quality of life of cancer patients, but opioid intervention can cause significant side effects that further decrease quality of life. Although electroacupuncture (EA) has been used to treat cancer pain, its mechanisms are largely unknown. To examine its effects and underlying mechanisms on cancer pain, we injected AT-3.1 prostate cancer cells into the tibia to induce bone cancer in the male Copenhagen rat. The resulting pain was treated with 10Hz/2mA/0.4ms pulse EA for 30min daily at the point equivalent to the human acupoint GB30 (Huantiao) between days 14 and 18 after the injection. For sham control, EA needles were inserted into GB30 without stimulation. Thermal hyperalgesia, a decrease in paw withdrawal latency (PWL) to a noxious thermal stimulus, and mechanical hyperalgesia, a decrease in paw withdrawal pressure threshold (PWPT), was measured at baseline and 20min after the EA treatment. Preprodynorphin mRNA and dynorphin were determined by RT-PCR and immunohistochemistry, respectively. Thermal and mechanical hyperalgesia developed ipsilaterally between days 12 and 18 after cancer cell inoculation. EA significantly (P<0.05) attenuated this hyperalgesia, as shown by increased PWL and PWPT, and inhibited up-regulation of preprodynorphin mRNA and dynorphin compared to sham control. Intrathecal injection of antiserum against dynorphin A (1-17) also significantly inhibited the cancer-induced hyperalgesia. These results suggest that EA alleviates bone cancer pain at least in part by suppressing dynorphin expression, and they support the clinical use of EA in the treatment of cancer pain.

Electroacupuncture attenuates bone cancer pain and inhibits spinal interleukin-1 beta expression in a rat model.

BACKGROUND: Although pain affects the quality of life of cancer patients, current medical treatments are either ineffective or have side effects. In the present study we investigated the effect of electroacupuncture (EA) on cancer-induced hyperalgesia and expression of interleukin-1beta (IL-1beta), upregulation of which is related to the maintenance of persistent pain, in a rat model of bone cancer pain. METHODS: Cancer was induced by injecting AT-3.1 prostate cancer cells into the tibia of male Copenhagen rats. The resulting pain was treated with 10 Hz/2 mA/0.4 ms pulse EA for 30 min daily at the equivalent of the human acupoint GB30 (Huantiao) between Days 14 and 18 after cancer cell inoculation. For sham control, EA needles were inserted into GB30 without stimulation. Thermal hyperalgesia, a decrease in paw withdrawal latency to a noxious thermal stimulus, was measured at baseline and 20 min after EA treatment. IL-1beta and its mRNA were respectively determined by immunohistochemistry and reverse transcription-polymerase chain reaction analysis. RESULTS: Thermal hyperalgesia developed between Days 12 and 18 after cancer cell inoculation. EA significantly (P < 0.05) attenuated this hyperalgesia, increasing paw withdrawal latency from 7.0 +/- 0.3 s to 9.2 +/- 0.4 s, and inhibited the upregulation of IL-1beta and its mRNA compared to the sham control. Intrathecal injection of IL-1 receptor antagonist (IL-1ra, 0.1 mg/rat) also significantly inhibited cancer-induced thermal hyperalgesia. CONCLUSION: The data suggest that EA alleviates bone cancer pain, at least in part by suppressing IL-1beta expression. The results support the clinical use of EA in the treatment of cancer pain.

Is protein kinase C (PKC) involved in nociception?

The study was designed to determine whether the protein kinase C (PKC) is involved in nociceptive c-Fos expression and the concomitant signaling processes of endogenous opioid-like substances (OLS) that modulate c-Fos expression in the spinal dorsal horn following formalin injection into the unilateral hindpaw in rats by using immunocytochemical techniques. In the first part of experiments in which rats were pretreated with intrathecal (i.t.) chelerythrine (Chel), an inhibitor of PKC, the nociceptive c-Fos-like immunoreactive (Fos-LI) neurons in the lumbar dorsal horn ipsilateral to the formalin injection were significantly suppressed with a reduction rate of 60.3% (p < .001) as compared to that in the control group with i.t. saline. In the second part of experiments in which rats were pretreated with i.t. naloxone (Nal), the nociceptive Fos-LI neurons were significantly increased by 53.2% (p < .01) as compared to that in the control group; however, when rats were pretreated with combined i.t. Nal + Chel, the nociceptive Fos-LI neurons exhibited a percentage reduction similar to that in group with i.t. Chel alone, although the real number of Fos-LI neurons in group with i.t. Nal + Chel still significantly surpassed that in group with i.t. Chen only. These results suggest that: (1) PKC may play an important role in the induction of nociceptive c-Fos expression; (2) nociceptive c-Fos expression is subject to the modulation of endogenous OLS that suppress the nociceptive responses of the dorsal horn neurons; and (3) PKC may not be involved in the signaling processes by which the endogenous OLS modulate the nociceptive c-Fos expression in the spinal level.

[Recent progress in neuroprotection of humanin against Alzheimer's disease-relevant neurotoxicity].

Alzheimer's disease (AD) is the leading cause of dementia for aging people, and far from control due to its obscure mechanism. Humanin, a 24-aa peptide encoded by a newly identified gene cloned from an apparently normal region of AD brain, can specifically attenuate AD-related neurotoxicity. It protects neurons from insults of various AD genes, anti-APP antibodies and Abeta by forming a homodimer outside and interfering directly or indirectly with the activity of Abeta. Humanin seems, however, not to inhibit other toxic insults to neurons, such as Fas or etoposide, an agent against carcinomatous cells in clinical therapy. So Humanin rescues neurons from various AD-related toxicity specifically with efficiency.

Spinal glial activation in a new rat model of bone cancer pain produced by prostate cancer cell inoculation of the tibia.

Studies suggest that astrocytes and microglia in the spinal cord are involved in the development of persistent pain induced by tissue inflammation and nerve injury. However, the role of glial cells in bone cancer pain is not well understood. The present study evaluated the spinal glial activation in a novel rat model of bone cancer pain produced by injecting AT-3.1 prostate cancer cells into the unilateral tibia of male Copenhagen rats. The structural damage to the tibia was monitored by radiological analysis. The thermal hyperalgesia, mechanical hyperalgesia and allodynia, and spontaneous flinch were measured. The results showed that: (1) inoculation of prostate cancer cells, but not the vehicle Hank's solution, induced progressive bone destruction at the proximal epiphysis of the tibia from day 7-20 post inoculation; (2) the inoculation also induced progressive thermal hyperalgesia, mechanical hyperalgesia, mechanical allodynia, and spontaneous flinches; (3) astrocytes and microglia were significantly activated in the spinal cord ipsilateral to the cancer leg, characterized by enhanced immunostaining of both glial fibrillary acidic protein (GFAP, astrocyte marker) and OX-42 (microglial marker); (4) IL-1beta was up-regulated in the ipsilateral spinal cord, evidenced by an increase of IL-1beta immunostained astrocytes. These results demonstrate that injection of AT-3.1 prostate cancer cells into the tibia produces progressive hyperalgesia and allodynia associated with the progression of tibia destruction, indicating the successful establishment of a novel male rat model of bone cancer pain. Further, bone cancer activates spinal glial cells, which may release IL-1beta and other cytokines and contribute to hyperalgesia.

c-Fos antisense oligodeoxynucleotide offsets behavioral nociceptive responses and both up-regulations of c-Fos protein and dynorphin a (1-8) in dorsal horn: a study using the formalin test in rats.

The formalin test was used to elicit acute and chronic pain in rats, and antisense oligodeoxynucleotide (AS-ODN) was used as a tool to modulate the expression of nociceptive behavioral and neurochemical responses. AS-ODN complementary to c-Fos mRNA was administered intrathecally (i.t.) 4 h before formalin injection in the experimental group. Normal saline or reverse AS-ODN was pre-administered i.t. at the same time in two control groups (saline and reverse AS-ODN). The results showed that the acute phase of nociceptive behavior showed no change by AS-ODN administration, whereas the tonic phase of nociceptive licking and biting behavior was significantly suppressed by AS-ODN as compared with the saline or the reverse AS-ODN group, respectively (p < .05 and p < .01). At the same time, both Fos-like immunoreactive (FLI) neurons and density of dynorphin-like immunoreactivities (DLI) were decreased significantly (p < .05 and p < .01) in the AS-ODN group as compared with that in two control groups. The results indicate that the long-lasting nociceptive responses elicited by sustained noxious inputs are based on the up-regulation of c-Fos gene expression, which in turn induces the upregulation of Dyn A production. It is proposed that intensified Dyn A production in the dorsal horn may be pivotal for the appearance of chronic pain.

ATP-sensitive potassium channels and endogenous adenosine are involved in spinal antinociception produced by locus coeruleus stimulation.

The effects of locus coeruleus stimulation on nociceptive evoked discharges of thalamic parafascicular (PF) neurons were investigated in lightly urethane-anesthetized rats, aiming to study the mechanisms underlying these effects. Intrathecal (i.t.) administration of aminophylline (an adenosine antagonist), glibenclamide (an ATP-sensitive potassium [K+(ATP)] channels blocker), nicrorandil (Nico; an agonist of K+(ATP) channel and a K+(ATP) channel opener), and 5'-N-ethylcarboxamido-adenosine (NECA; an adenosine agonist) were used. The results showed that (1) locus coeruleus stimulation significantly inhibited the nociceptive evoked discharges of parafascicular neurons, (2) locus coeruleus stimulation-produced antinociception in PF neurons was blocked by both it. glibenclamide and i.t. aminophylline, (3) nociceptive discharges of PF neurons were also suppressed by both i.t. NECA and i.t. nicorandil, and (4) i.t. glibenclamide showed no effect on the suppression of nociceptive discharges induced by NECA, whereas aminophylline blocked the suppression of nociceptive discharges induced by nicorandil. These results suggest that (a) K+(ATP) channels and endogenous adenosine may be involved in the mediation of antinociception induced by norepinephrine, which is released in the dorsal horn by descending fibers originating from the locus coeruleus and (b) the opening of K+(ATP) channels may precede the release of endogenous adenosine in the process of suppressing nociceptive transmission at the spinal level.

Protein kinase C is partly involved in c-fos protein expression of nocuously-activated neurons but may not in concomitant modulatory action through opioid receptors at the spinal level in rats.

The present study was aimed to examine if protein kinase C (PKC) activation is necessarily involved in both the c-fos protein expression in the nocuously-activated c-fos protein-like immunoreactive (Fos-LI) neurons and the concomitant opioid receptor-mediated modulation in the dorsal horn circuitry of the spinal cord. Formalin was injected into a hindpaw of rats 5 min after the rats were pretreated with intrathecal (i.t.) administration of chelerythrine (Chel), an inhibitor of PKC, naloxone (Nal), combined administration of these two (Chel + Nal), or vehicle (n=5 in each group),respectively. By using immunocytochemical techniques, the formalin-induced Fos-LI neurons in the lumbar dorsal horn were calculated 1 h after formalin injection. The results showed that: (1) i.t. Chel significantly reduced the number of Fos-LI neurons in the dorsal horn of the spinal cord on the side ipsilateral to the formalin injection, showing a decrease by 60.3% (P<0.001) as compared to that observed in the i.t.vehicle group; (2) i.t. Nal significantly increased the number of Fos-LI neurons in the ipsilateral dorsal horn, with an increase of 46.0% (P<0.01) as compared to that in the i.t.vehicle group, the highest percentage increase being found in the deeper laminae of the dorsal horn; and (3) i.t. Chel + Nal also exhibited a significant decrease in Fos-LI neurons in the ipsilateral dorsal horn as compared to i.t. Nal group, showing a reduction of 53.2%, a value similar to that in the i.t. Chel group. These results suggest that: (1) PKC plays a role in the c-fos protein expression only in nearly one half of the Fos-LI neurons in the dorsal horn; and (2) PKC is possibly not involved in the concomitant modulation on the nociception mediated by micro- (and also partly delta-) opioid receptors in the spinal cord.

Effects of testosterone and 17-beta-estradiol on TNF-alpha-induced E-selectin and VCAM-1 expression in endothelial cells. Analysis of the underlying receptor pathways.

This study investigated the effects of testosterone and 17-beta-estradiol on tumor necrosis factor-alpha (TNF-alpha)-induced endothelial expression of E-selectin and vascular cell adhesion molecule-1 (VCAM-1) and the potential roles of hormone receptors involved in these actions. Human umbilical vein endothelial cells (HUVEC) were stimulated with TNF-alpha in the presence or absence of testosterone or 17-beta-estradiol, and the expression of E-selectin and VCAM-1 was investigated. As shown by Western blot analysis, co-administration with testosterone or 17-beta-estradiol increased the expression of E-selectin and VCAM-1 induced by TNF-alpha at 6 h and 3 h, respectively. Similarly, RT-PCR analysis revealed a significant increase in the amount of mRNA for E-selectin and VCAM-1 after co-administration with testosterone or 17-beta-estradiol in TNF-alpha-stimulated HUVEC. The presence of mRNA and proteins for androgen receptor and estrogen receptor alpha in HUVEC was verified by RT-PCR and Western blot. Flow cytometric analysis showed that preincubation with androgen receptor antagonist cyproterone and estrogen receptor antagonist tamoxifen completely abrogated the upregulating effects of testosterone and 17-beta-estradiol on TNF-alpha-induced E-selectin and VCAM-1 expression, respectively. Expression of TNF receptors in TNF-alpha-stimulated HUVEC was not influenced by testosterone and 17-beta-estradiol. The data indicate that both testosterone and 17-beta-estradiol increase TNF-alpha-induced E-selectin and VCAM-1 expression in endothelial cells via a receptor-mediated system, and expression of TNF receptors are not changed in these actions. The implications of these results for the facilitory effects of both sex hormones on immune reactions are discussed.

ATP-sensitive potassium channels and endogenous adenosine are involved in spinal antinociception produced by locus coeruleus stimulation.

It has been known that locus coeruleus (LC) stimulation suppresses nociceptive discharges of the thalamic parafascicular (PF) neurons through the spinally descending adrenergic terminals which inhibit the transmission of nociceptive signals in the spinal dorsal horn. This experimental model was used in the present study to analyze the detailed processes that happened in the dorsal horn following norepinephrine release by preemptive intrathecal (i.t.) administration of related drugs in lightly urethane-anesthetized rats. The results showed that: (1) LC stimulation significantly inhibited the noxiously-evoked discharges of PF neurons; (2) the LC stimulation-produced antinociception in PF neurons could be blocked either by i.t. glibenclamide, an ATP-sensitive potassium (K(+)(ATP)) channel blocker, or by i.t. aminophylline, an adenosine receptor antagonist; (3) nociceptive discharges of PF neurons were also suppressed both by i.t. 5 -N-ethylcarboxamido-adenosine (NECA, an adenosine receptor agonist) and by i.t. nicorandil (a K(+)(ATP) channel opener); and (4) i.t. aminophylline blocked the suppression of PF nociceptive discharges induced by i.t. nicorandil, while i.t. glibenclamide showed no effect on the suppression of nociceptive discharges induced by i.t. NECA. These results suggest that: (1) K(+)(ATP) channels and endogenous adenosine may be involved in the mediation of spinal antinociception induced by descending adrenergic fibers originating from the LC; and (2) the opening of K(+)(ATP) channels precedes the release of adenosine in the cascade of mediation.