| Cancer-induced pain is one of the most severe types of chronic pain and serious impact on daily work, study and life. Skeletal involvement is a frequent and troublesome complication affecting many patients with neoplastic disease. It is the third most common metastatic site after the lung and liver. So to define the mechanisms that give rise to cancer-induced bone pain (CIBP) is very important.There are many works have been done to explore the mechanism of CIBP. Up to now, it is reported that the neurochemical changes observed in the spinal cord and sensory neurons in mice with CIBP are distinct from those induced by inflammatory or neuropathic conditions thus suggesting that CIBP is a unique type of persistent pain. The work from our laboratory has demonstrated that in the spinal cord of CIBP model, the astrocyte and microglia are proliferated obviously with the expression of pro-inflammatory cytokines, such as IL-1β, IL-6, TNF-α, Preprodynorphin et al. We called it as neuroinflammation, which is related to the CIBP.Acute inflammation is now widely appreciated in the pathogenesis of many human diseases. The acute inflammatory response is mainly a protective mechanism: it destroys and/or separates the injurious agent, removes damaged tissue and repairs the area as best possible. While inflammation is a fundamental component of normal host defense and wound repair. However, failure to completely resolve an acute inflammatory response can lead to chronic inflammation, scarring, and eventual loss of tissue function.Lipoxins (LXs) are generated from arachidonic acid (AA) via sequential actions of lipoxygenases and subsequent reactions to give specific trihydroxytetraene-containing eicosanoids. LXs were the first mediators recognized to carry out both endogenous anti-inflammatory and pro-resolving actions in vivo that can function as "Braking signals" in inflammation. Lipoxin A4(LXA4) and lipoxin B4(LXB4) are positional isomers that each possesses potent cellular and in vivo actions. Aspirin has a direct impact in the LX circuit by triggering the biosynthesis of endogenous epimers of LX, termed as aspirin-triggered15-epi-LX (ATL), that share the potent anti-inflammatory actions of LX. It has been reported that LXA4inhibits LPS-induced pro-inflammatory mediators, including COX-2, PGE2and TNF-a, possibly by mediation through HO-1pathway in RAW264.7macrophages. Svensson et al report that intrathecal or intravenous injection of LXA4, as well as stable analogues, attenuates inflammation-induced pain. The analgesic effect of LXA4was also demonstrated in chronic compression of DRG (CCD) model. However, there is no report about the effect of LXs in CIBP. So the effect of LXA4and its analogues in CIBP and the mechanism of analgesic effect are the first part of our research.Two major routes of LX biosynthesis in human cell types have been clarified. One pathway involves peripheral blood platelet-leukocyte interaction. The leukocytes5-LO converts AA to the epoxide product LTA4, which is then released and further transformed by adherent platelets to LXA4via the LX syntheses activity of human12-LO. The second biosynthetic route is initiated at the mucosal surfaces by15-LO that inserts molecular oxygen into AA at the carbon15position to produce15S-hydroxyleicosatetraenoic acid (15S-HETE); this latter metabolite is rapidly taken up by polymorphonuclear cells (PMNs) and is converted subsequently via5-LO to LXs. Another source of LXs that is from aspirin-acetylated cyclooxygenase-2(COX-2) converts AA to15R-HETE (vide infra) to generate ATL. The lipoxygenase of5-LO,12-LO and15-LO play important role in the biosynthesis of LX. It is reported that Baicalein (BE) is a potent inhibitor against both12-LO and15-LO in vitro. BE is one of the major constituents of Scutellaria baicalensis. Scutellaria baicalensis is one of the most popular and multi-purpose herb used in China traditionally for treatment of inflammation, hypertension, cardiovascular diseases, and bacterial and viral infections. It has been demonstrated that BE has the potent anti-inflammatory, anti-tumor cell invasion and antioxidant are largely due to its abilities to scavenge oxidative radicals, to block MEK-ERK signals, to decrease the expression of inflammatory cytokines, to attenuate NF-κB activity, to inhibit several genes important for regulation of the cell cycle, to suppress COX-2gene expression and to prevent viral infections. So whether BE could alleviate the CIBP and the related molecular mechanisms are the other part of our research.According to the above reports, based on the Walker256rat mammary gland carcinoma cells induced bone cancer pain model, using behavioral test, Real-Time PCR, immunohistochemistry, Western Blot, the presented study was aimed to:1) Observe the effect of LX and its analogues in CIBP model and the related molecular mechanism;2) The effect of BE on CIBP model and the related mechanism of it.The results are as follows:1. LXA4and its analogues alleviate the mechanical allodynia of CIBP1.1The development of CIBPAfter the baseline behavioral test, the rats in the cancer group received carcinoma cells (Walker256carcinoma cells,4×105) that were injected into the right tibia cavity, as previously described (day0). The sham group received an equal volume of PBS (day0). The naive group received no treatment. In order to assess the development of mechanical allodynia in CIBP, PWT was tested on day3,7,9and11after surgery. On day3after surgery, the rats in the cancer group displayed a profound decrease in PWT to von Frey hair stimulation in the ipsilateral right limb compared with the naive group and displayed significant body weight loss. This decrease lasted until day11when the observation ended. In contrast, there was no significant difference in PWT between the naive group and the sham group at the different time points. Moreover, X-ray and HE staining showed that the rats inoculated with tumor cells showed significant tibia bone destruction as compared with naive rats.1.2Intrathecal injection of LXA4, ATL or LXB4reduces the mechanical allodynia in CIBPIn order to examine the analgesic effects of LXs on the mechanical allodynia in CIBP, on day7after surgery, cancer rats were randomly divided into four groups:one group was treated i.t. with NS (20μl) as vehicle, and the others were treated i.t. with equal doses of LXA4, LXB4or ATL (0.1μg/20μl). The ipsilateral PWT was tested at2,4and6hours after drug administration. Compared with vehicle-treated animals, the PWT was profoundly increased from2to6hours in the ATL-injected animals, while the PWT was significantly increased after2hours in the LXA4-injected animals and in the LXB4-injected animals. However, i.t. treatment with equimolar doses of LXA4, LXB4or ATL did not alter the nociceptive thresholds of the naive animals.Summary:Intrathecal injection of LXA4, LXB4or ATL reduces the mechanical allodynia in CIBP and the effect of ATL is better than the others. However, i.t. treatment with equimolar doses of LXA4, LXB4or ATL did not alter the nociceptive thresholds of the naive rats.2. ATL could alleviate the mechanical allodynia and inhibit the expression of proinflammatory cytokines in spinal cord2.1Expression of ALX in the spinal cordThe possible distribution of ALX in the spinal cord was detected by immunohistochemistry. Lumbar spinal cords from the naive, sham and cancer groups were sectioned and incubated with ALX antibody. Similar and moderate ALX-like immunoreactivity was observed in the spinal cord of naive, sham and cancer rats. No significant difference in the protein level of the expression of ALX was found among naive, sham and cancer groups. Double labeling of spinal sections revealed that the ALX-like immunoreactivity was mainly co-localized with the astrocyte marker glial fibrillary acidic protein (GFAP), partly co-localized with the neuronal marker NeuN but not with the microglia marker CDllb.2.2BOC-2could inhibit the analgesic effect of ATL in CIBPOn day11after surgery, cancer rats were randomly divided into four groups: Cancer with DMSO, Cancer with BOC-2100μg, Cancer with ATL0.1μg, Cancer with BOC-2and ATL. As compared with Cancer with DMSO, single i.t. with BOC-2has no influence in CIBP. However, i.t. with BOC-2before ATL could inhibit the analgesic effect of ATL in CIBP.2.3Intrathecal injection with different doses of ATL alleviate mechanical allodynia in CIBPWe assessed the effects of i.t. with different doses of ATL on the ipsilateral PWT of animals on day11after surgery. Cancer rats were randomly divided into four groups:one group was treated with i.t. NS (20μl) as vehicle, and the others were treated i.t. with different doses of ATL (0.01,0.1or0.15μg/20μl). PWT was tested at2,4,6,8and12hours after drug administration. Compared with the vehicle-treated animals, the animals treated i.t. with ATL (0.01,0.1or0.15μg) showed significant increased PWT from2to6hours. The dose of0.15μg showed longer effect (for8hours) than the others. These results indicate that a single intrathecal injection of ATL could reduce the mechanical allodynia in CIBP. 2.4To compare the analgesic effect of ATL with morphineIn order to evaluate the analgesic effect of ATL on the mechanical allodynia in CIBP, on day11after surgery, cancer rats were randomly divided into four groups:one group was treated i.t. with NS (20μl) as vehicle, and the others were treated i.t. with equal doses of ATL or morphine (0.1μg/20μl), the fourth group was i.t. with5μg morphine. The ipsilateral PWT was tested at0.5,1,1.5,2,2.5,3,4,5,6hours after drug administration. Compared with vehicle-treated animals, the PWT was profoundly increased after injected with drug, while the group with5μg morphine showed the highest PWT than the others. However, i.t. with equimolar doses of ATL or morphine, ATL showed a longer effect.2.5The effect of intravenous injection with different dose of ATL in CIBPTo assess the possible analgesic effect of systemically administered with ATL, the effects of i.v. ATL on the mechanical allodynia in cancer rats were observed on day11after surgery. From1to4hours after the injection of10or40μg/kg ATL, the PWT increased significantly compared with vehicle indicating an anti-allodynic effect of i.v. ATL in CIBP rats.Summary:Intrathecal or intravenous injection with different dose of ATL could attenuate the mechanical allodynia in CIBP. The analgesic effect of ATL could be inhibited by BOC-2. No significant difference in the level of the expression of the ALX protein was found among naive, sham or cancer groups. The distribution of ALX in the spinal cord is mainly co-localized with GFAP, partly co-localized with NeuN, but none with CDllb.3. The analgesic effect of ATL is mainly through inhibit the expression of proinflammatory cytokines and the activation of phosphorylated MAPK signals in CIBP3.1Effects of ATL on the expression of the mRNA of pro-inflammatory cytokines and PPD in CIBPTo investigate the molecular mechanisms associated with the anti-allodynic effect of ATL in rats with CIBP, we evaluated the expression of the mRNA of several pro-inflammatory mediators, including IL-1β, IL-6and TNF-α in the spinal cord by Real-Time PCR. Rats were randomly divided into three groups: naive, cancer with NS and cancer with ATL0.1μg. As compared with the naive group, cancer with saline groups showed significant increased pro-inflammatory mediators and PPD. Intrathecal injection of ATL significantly decreased the expression of the mRNA of IL-1β, TNF-α and PPD.3.2Intrathecal injection with ATL could inhibit the activation of phosphorylated MAPK signals and lipoxygenases in spinal cord of CIBPThe Western Blot results suggested that i.t. injection of ATL0.1μg after2hours decreased the p-ERK, p-p38and p-JNK level in spinal cord of CIBP on day11after surgery. Moreover, the expression of5-LO and15-LO are also inhibited. But there is no effect on p-STAT3and12-LO levels.Summary:Intrathecal injection of ATL in CIBP rats significantly decreased the expression of the mRNA of IL-1βTNF-α and PPD and decrease the level of p-ERK, p-p38, p-JNK,5-LO and15-LO in spinal cord.4. The effect of BE in CIBP and related molecular mechanism4.1Intraperitoneal or intrathecal injection with BE alleviate the mechanical allodynia in CIBPThe behavior results suggested that an intraperitoneal injection of BE (300mg/kg) attenuated mechanical allodynia on the2hour on day11after surgery. And intrathecal injection of100μg BE attenuated mechanical allodynia on the0.5hour and lasted until4hour on day11after surgery. Moreover, Intraperitoneal or intrathecal injection of BE has no influence for naive rats.4.2Intrathecal injection with BE decrease the expression of pro-inflammatory cytokines and the activation of MAPK signals in the spinal cord of CIBP4.2.1Intrathecal injection with BE decrease the expression of pro-inflammatory cytokines in the spinal cord of CIBPTo investigate the molecular mechanisms associated with the anti-allodynic effect of BE in rats with CIBP, we evaluated the expression of the mRNA of several pro-inflammatory mediators, including IL-1β, IL-6and TNF-a in the spinal cord by Real-Time PCR. Rats were randomly divided into three groups:naive, cancer with NS and cancer with BE100μg. As compared with the naive group, cancer with saline groups showed significant increases in pro-inflammatory mediators. I.t. injection of ATL markedly decreased the expression of the mRNA of IL-6and TNF-a in CIBP rats.4.2.2The effect of intrathecal injection of BE on MAPK and STAT3activity in the spinal cord of CIBPThe Western Blot results suggested that i.t. injection of BE100μg after2hours decreased the p-p38and p-JNK level in spinal cord of CIBP on day11after surgery. Moreover, the expression of5-LO was also inhibited. But there are no effect on the expression of p-ERK, p-STAT3and12-LO level in CIBP rats.Summary:Both intraperitoneal and intrathecal injection of BE increase PWT in CIBP. Intrathecal injection of BE100μg inhibited the expression of the mRNA of IL-6and TNF-a and decreased the level of p-p38, p-JNK and5-LO in spinal cord of CIBP rats on day11after surgery.Conclusion:1. Intrathecal injection of LXA4, LXB4or ATL attenuated mechanical allodynia in CIBP and ATL showed a better analgesic effect than others. Both intrathecal and intravenous injection of ATL increase the PWT in CIBP and the analgesic effect can be inhibited by BOC-2.2. The distribution of ALX in the spinal cord is mainly co-localized with GFAP, partly co-localized with NeuN, but none with CD11b. Intrathecal injection of0.1μg ATL after2hours inhibited the expression of the mRNA of IL-1β, TNF-α and decreased the level of p-ERK, p-p38, p-JNK,5-LO and15-LO in spinal cord of CIBP on day11after surgery.3. Both intraperitoneal and intrathecal injection of BE increase PWT in CIBP. Intrathecal injection of BE100μg inhibited the expression of the mRNA of IL-6, TNF-α and decreased the level of p-p38, p-JNK and5-LO in spinal cord of CIBP on day11after surgery. |
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