| Involvement of Spinal Interleukin-1βin Inflammatory Pain and Electroacupuncture Analgesia in RatsInflammatory pain is one of the most common pathologic pains in clinic. Inflammatory pain arises as a debilitating consequence of injury to the peripheral tissue, which is characterized by combination of spontaneous burning pain, hyperalgesia and allodynia. The researches about its peripheral and central mechanism have been the important focus of the international pain research organization. Substantial evidences have indicated that a number of bioactive substances were involved in the formation and modulation of inflammatory pain. Unfortunately, the etiological mechanism of inflammatory pain has been poorly understood.Interleukin-1β(IL-1β) is a proinflammatory cytokine, which plays a major role in inflammation and immunity. Two types of IL-1 receptor proteins have been reported to be identified. IL-1 signalling activity appears to be mediated exclusively via the IL-1 receptor typeâ… (IL-1RI), whereas the IL-1 receptor typeâ…¡(IL-1RII) has no signalling property and acts as a "decoy" target for IL-1. Evidence shows that IL-1βis associated with the nociceptive modulation in the central nervous system and can be nociceptive or anti-nociceptive. Researchers have found that intracerebroventricular injection of IL-1βto the rats exerts biphasic responses on thermal and mechanical nociceptive thresholds depending upon the dosage, causing hyperalgesia at lower doses and analgesia at higher doses. Intrathecal (i. t.) administration of IL-1βalso shows varying nociceptive responses, and may promote the development of inflammatory and neuropathic pain or reduce inflammatory pain. However, the function of spinal endogenous IL-1βin inflammatory pain was still unclear, and therefore merits further investigation.Acupuncture, a traditional therapeutic modality from Traditional Chinese Medicine, has been used in China for thousands of years to treat a variety of diseases and symptoms with few side effects, which has been widely accepted by World Health Organization and other countries. Electrical stimulation of acupuncture points, which is called electroacupuncture (EA), is widely used both in clinic and in experimental studies. EA studies have also been performed on peripheral pathological pain animal models, such as carrageenan-induced inflammatory pain. It has been reported that peripheral IL-1βwas involved in EA analgesia during hyperalgesia. However, the mechanism of spinal IL-1βin EA analgesia has not been fully understood. Therefore, to investigate the role of IL-1βin inflammatory pain and EA analgesia is the main target of the present study.Protein kinase D (PKD), also known as protein kinase Cμ, is a newly described serine/threonine protein kinase with unique structural, enzymological, and regulatory properties that are different from those of the PKC family members. Previous study found that PKD functioned as a direct modulator of vanilloid receptor type 1, implied that it might be involved in the regulation of inflammatory responses. Moreover, protein kinase C (PKC) can be activated by IL-1β, while PKD can be activated via PKC-dependent pathways. However, whether PKD involved in IL-1βpathway is poorly known. Additionally, as the downstream molecules in the signal transduction pathway, p38 and nuclear factorκB (NF-κB) can also be activated by IL-1β, and control the gene expression of pain-related substances, such as interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α) and cyclooxygenase-2 (COX-2). However, it is also unclear whether there are intimate relationships between p38, NF-κB and PKD. Then, another part of our studies was to explore the role of PKD and its related downstream or upstream molecules in IL-1βpathway, in order to investigate the mechanism of spinal IL-1βin inflammatory pain.By using the molecular biological techniques, including RT-PCR, Western blot, ELISA, immunochemistry and neuron cultures, the present study was designed to explore: (1) effects of EA on carrageenan-induced inflammatory pain and spinal IL-1βand IL-1RI expression; (2) effects of antisense to IL-1RI on inflammatory pain and EA analgesia; (3) the role of PKD in IL-1βpathway in inflammatory pain and EA analgesia; (4) the role of PKC, p38 and NF-κB in IL-1β/PKD pathway.The results are following:1. Effects of EA on carrageenan-induced inflammatory pain and spinal IL-1βand IL-1RI expression 1.1 Inflammatory pain induced by carrageenanCarrageenan (2μg/100μl) was intraplantar (i. pl.) injected into unilateral hind paw of non-anesthetized rats. The contralateral paw was untreated. Paw withdrawal latency (PWL) of both hind paws were observed before carrageenan injection and 105 to 300 min post injection, respectively. The results indicated that carrageenan produced marked inflammation (edema and erythema) and thermal hyperalgesia in the injected paw.1.2 Effects of EA on carrageenan-induced inflammatory painAt 3 h following carrageenan injection, EA stimulation applied to the ipsilateral (carrageenan-injected) paw for 30 min. PWLs were observed before carrageenan injection and 180 to 300 min post injection, respectively. The results indicated that EA significantly reduced inflammation-induced thermal hyperalgesia, while sham EA demonstrated no effect on the PWLs.1.3 Effects of EA on carrageenan-induced spinal IL-1βexpressionAt 3 h following carrageenan injection, EA stimulation applied to the ipsilateral (carrageenan-injected) paw for 30 min. The expression of IL-1βin L4-L6 segments of the rats' spinal cord was examined by using ELISA. The results indicated that the expression of IL-1βin the spinal cord was raised post carrageenan injection, and EA reduced carrageenan-induced expression of IL-1β, while sham EA demonstrated no effect.1.4 Effects of EA on carrageenan-induced spinal IL-1RI expressionAt 3 h following carrageenan injection, EA stimulation applied to the ipsilateral (carrageenan-injected) paw for 30 min. The expression of IL-1βin L4-L6 segments of the rats' spinal cord was examined by RT-PCR and Western blot analysis, respectively. The results indicated that the mRNA and protein level of IL-1RI in the spinal cord was raised post carrageenan injection, and EA reduced carrageenan-induced mRNA and protein expression of IL-1RI, while sham EA demonstrated no effect.Summary: I. pl. carrageenan could induce inflammatory pain. EA could significantly reduce inflammation-induced thermal hyperalgesia. Spinal IL-1βand IL-1RI expression could be induced by inflammatory pain. EA could reduce carrageenan-induced expression of IL-1βand IL-1RI. These results suggested that IL-1βand IL-1RI in the spinal cord was involved in carrageenan-induced inflammatory pain, and EA might act its analgesic effect partly via reducing the expression of the IL-1β/IL-1RI system.2. Effects of antisense oligodeoxynueleotide (ODN) to IL-1RI on inflammatory pain and EA analgesia2.1 Intrathecal antisense ODN down-regulating spinal IL-1RI expressionTo verify the effect of antisense ODN, the expression of IL-1RI in L4-L6 segments of the spinal cord was detected by RT-PCR and Western blot analysis following i. t. delivery of antisense ODN (50μg/10μl), sense ODN (50μg/10μl) or normal saline (10μl) once daily for 3 days. The results indicated that antisense ODN reduced the mRNA and protein level of IL-1RI as compared with the control groups of normal saline and sense ODN treatment, suggesting that the expression of IL-1RI in the spinal cord could be significantly down-regulated by antisense ODN treatment.2.2 Effects of antisense ODN to IL-1RI and combination with EA on spinal IL-1βexpressionAntisense ODN and normal saline were i. t. injected respectively, once daily for 3 days. Carrageenan was i. pl. injected on the 3rd day following i. t. injection. EA was administered at 3 h post carrageenan injection, and lasting for 30 min. The expression of IL-1βin L4-L6 segments of the rats' spinal cord was examined by using ELISA. The results indicated that the expression of IL-1βin the spinal cord was not changed by antisense ODN to IL-1RI, and wasn't further inhibited by EA combination with antisense ODN. 2.3 Effects of antisense ODN to IL-1RI on PWLs of normal ratsAntisense ODN, sense ODN and normal saline were i. t. injected respectively, once daily for 3 days. PWLs were observed each day post injection. The results indicated that i. t. injection of ODNs or normal saline for 3 days to the normal rats led to no significant changes in the PWLs during the period.2.4 Effects of antisense ODN to IL-1RI on carrageenan-induced inflammatory painAntisense ODN, sense ODN and normal saline were i. t. injected respectively, once daily for 3 days. Carrageenan was i. pl. injected on the 3rd day following i. t. injection. PWLs were observed before carrageenan injection and 105 to 300 min post injection, respectively. The results indicated that down-regulating IL-1RI expression could significantly reduce inflammation-induced thermal hyperalgesia.2.5 Effects of antisense ODN to IL-1RI combined with EA on inflammatory painAntisense ODN, sense ODN and normal saline were i. t. injected respectively, once daily for 3 days. Carrageenan was i. pl. injected on the 3rd day following i. t. injection. EA was administered at 3 h post carrageenan injection, and lasting for 30 min. PWLs were observed before carrageenan injection and 180 to 300 min post injection, respectively. The results indicated that carrageenan-induced thermal hyperalgesia was significantly lower when EA was combined with antisense ODN than when EA or intrathecally injection of antisense ODN was used alone.Summary: Antisense ODN treatment could significantly down-regulate the expression of IL-1RI in the spinal cord, but had no effect on the expression of IL-1β. Down-regulating IL-1RI expression led to no significant changes in the normal rats, but could significantly reduce inflammation-induced thermal hyperalgesia. Carrageenan-induced thermal hyperalgesia was significantly lower when EA was combined with antisense ODN than when EA or intrathecally injection of antisense ODN was used alone. These results suggested that spinal endogenous IL-1βand IL-1RI contributed to the development of inflammatory pain, and played an antagonistic role in EA analgesia. 3. The role of PKD in IL-1βpathway in inflammatory pain and EA analgesia3.1 Effects of EA on carrageenan-induced PKD activationEA was administered at 3 h post carrageenan injection, and lasting for 30 min. PKD expression and activation were analyzed by Western blot. The results indicated that the activation of PKD in the spinal cord was raised post carrageenan injection, and EA reduced carrageenan-induced PKD phosphorylation, whereas the levels of PKD expression did not change.3.2 Effects of lipopolysaccharide (LPS) on the expression of IL-1βin primary spinal neuronsTo further investigate the role of PKD in IL-1βsignal pathway, primary cultures of spinal neurons were used. Following 10 days in culture, the cells were observed by immunostaining. It showed that the neurons grew well and was in proper density.On the 10th day of culture, spinal neurons were incubated with LPS (30μg/ml) for 0.5, 1, 3, 8 and 12 h, respectively. IL-1βexpression in cell iysates was analyzed by Western blot. The results indicated that the expression of IL-1βwas elevated from 0.5 h and reached a maximum at 8 h and declined at 12 h following LPS stimulation.3.3 Effects of LPS on the expression and activation of PKD in primary spinal neuronsOn the 10th day of culture, spinal neurons were incubated with LPS (30μg/ml) for 0.5, 1, 3, 8 and 12 h, respectively. PKD expression and activation in cell lysates was analyzed by Western blot. The results indicated that the phosphorylation of PKD was elevated from 0.5 h and reached a maximum at 8 h and declined at 12 h following LPS stimulation, whereas the levels of PKD expression did not change.3.4 Effects of IL-1 receptor antagonist (IL-1Ra) on LPS-induced PKD activation in primary spinal neurons On the 10th day of culture, spinal neurons were exposed to LPS and IL-IRa (50 to 1000 nM) for 8h. PKD expression and activation in cell lysates was analyzed by Western blot. IL-1Ra was observed to inhibit PKD activation in a concentration-dependent manner. lt inhibited PKD activation at a concentration of 50 nM and achieved maximum at 1000 nM, whereas the levels of PKD expression did not change.Summary: EA could reduce carrageenan-induced PKD phosphorylation. LPS could time-dependently induce IL-1βexpression and PKD phosphorylation in primary spinal neurons. IL-1Ra could concentration-dependently inhibit LPS-induced PKD activation. These results suggested that IL-1βmight participate in inflammatory pain modulation via activating PKD.4. The role of PKC, p38 and NF-KB in IL-1β/PKD pathway4.1 Effects of LPS on PKCs activation in primary spinal neuronsOn the 10th day of culture, spinal neurons were incubated with LPS for 0.5, 1, 3, 8 and 12 h, respectively. PKCs activation in cell lysates was analyzed by Western blot. p-PKC (pan), p-PKCα/βⅡ1, p-PKCγ, p-PKCδand p-PKCζ/λ, were determined by using specific antibodies. The results indicated that LPS induced PKCs phosphorylation within 0.5 h, and the activation reached a maximum at 3 or 8 h and declined at 12 h.4.2 Effects of IL-I Ra on LPS-induced PKCs activation in primary spinal neuronsOn the 10th day of culture, spinal neurons were exposed to LPS and IL-IRa (50 to 1000 nM) for 8h. PKCs activation in cell lysates were analyzed by Western blot. The results indicated that IL-1Ra significantly inhibited the phosphorylation of PKC (pan) and PKCα/βⅡat a concentration of 50 nM and achieved maximum at 1000 nM. Other isoforms of PKC phosphorylation weren't affected.4.3 Effects of PKC inhibitor on LPS-induced PKD activation in primary spinal neurons On the 10th day of culture, Ro-32-0432 (a selective ceil-permeable PKC inhibitor, principally for PKCαand PKCβ, 100 nM) was added to the culture medium at 7 h following LPS treatment and incubated for 1 h. The same amount of DMSO (Me2SO, the solvent for Ro-32-0432) was added in control cells and also incubated for 1 h. Compared to the DMSO control, Ro-32-0432 almost completely blocked PKD phosphorylation induced by LPS, suggesting that PKCs, probably PKCαor PKCβ, were involved in the LPS-induced PKD activation in spinal neurons.To further investigate which isoform of PKCs participated in PKD activation, the PKCβinhibitor (3-(1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino- lH-pyrrole-2, 5-dione, 100 nM) was added to the culture medium at 7 h following LPS treatment and incubated for 1 h. The result showed no effect on LPS-induced PKD activation. These data implied that PKCa might be the principal PKC isoform necessary for LPS-induced PKD activation in spinal neurons.4.4 Effects of LPS on the expression of p38 and NF-kB in primary spinal neuronsOn the 10th day of culture, spinal neurons were incubated with LPS for 0.5, 1, 3, 8 and 12 h, respectively. The expressions of p38 and NF-KB in cell lysates were analyzed by Western blot. The results indicated that LPS induced p38 activation and NF-kB expression within 0.5 h, and the activation reached a maximum at 8 h and declined at 12 h. The levels of p38 expression did not change.4.5 Effects of IL-1Ra on LPS-induced p38 activation and NF-kB expression in primary spinal neuronsOn the 10th day of culture, spinal neurons were exposed to LPS and IL-1Ra (50 to 1000 nM) for 8h. The expressions of p38 and NF-kB in cell lysates were analyzed by Western blot. The results indicated that IL-IRa significantly inhibited LPS-induced p38 activation and NF-kB expression at a concentration of 50 nM and achieved maximum at 1000 nM. The levels of p38 expression did not change. These data implied that 1L-1βwas involved in LPS-induced p38 activation and NF-kB expression. 4.6 Effects of PKC inhibitor on LPS-induced p38 activation and NF-kB expression in primary spinal neuronsOn the 10th day of culture, Ro-32-0432 and PKCβinhibitor was added to the culture medium at 7 h following LPS treatment and incubated for 1 h, respectively. The same amount of DMSO was added in control cells and also incubated for 1 h. Compared to the DMSO control, Ro-32-0432 inhibited p38 activation and NF-kB expression induced by LPS, while PKCβinhibitor showed no effect, suggesting that PKCαwas involved in the LPS-induced p38 activation and NF-kB expression in spinal neurons.4.7 Effects of down-regulating PKD expression on LPS-induced p38 activation and NF-kB expressionOn the 8th day of culture, spinal neurons were transfected with antisense ODN (0.2μM) to PKD and incubated for 48 h. The same amount of vehicle and sense ODN were added in control cells and also incubated for 48 h. Compared to the vehicle or sense ODN control, antisense ODN significantly reduced PKD expression.Following 48 h incubation of ODNs to PKD, spinal neurons were exposed to LPS for 8 h. Compared to the vehicle or sense ODN control, antisense ODN significantly reduced LPS-induced p38 activation and NF-KB expression. This result indicated that PKD was involved in LPS-induced p38 activation and NF-kB expression.Summary: In primary spinal neurons, LPS could time-dependently induce PKCs, p38 activation and NF-kB expression. IL-1Ra could concentration-dependently inhibit LPS-induced PKC (pan), PKCα/βⅡ, p38 activation and NF-kB expression. PKCαwas involved in the LPS-induced PKD, p38 activation and NF-KB expression. Down-regulating PKD expression could inhibit LPS-induced p38 activation and NF-KB expression. These results suggested that there might be an IL-1β/PKCα/PKD/p38 (NF-kB) pathway in pain modulation. 1. The expression of spinal IL-1βand IL-1RI changed in carrageenan-induced inflammatory pain and EA analgesia, suggested that spinal endogenous IL-1βwas involved in inflammatory pain, and EA might act its analgesic effect partly via reducing the expression of the IL-1β/IL-1RI system.2. Inflammation-induced thermal hyperalgesia was reduced by down-regulating IL-1RI expression. It was further reduced when EA was combined with antisense ODN. These results suggested that spinal endogenous IL-1βand IL-1RI contributed to the development of inflammatory pain, and played an antagonistic role in EA analgesia.3. In the spinal cord and primary spinal neurons, IL-1βmight participate in inflammatory pain modulation via activating PKD pathway.4. There might be an IL-1β/PKCα/PKD/p38 (NF-KB) pathway in pain modulation. |
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