| Cancer, one of the most difficult diseases to treat in the medical profession, induces chronic pathological pain, which decreased the patients’quality of life greatly. Approximately50-70%of all cancer patients will experience moderate to severe pain during the progressing process. According to the guidelines of the World Health Organization’s’analgesic ladder’, treatment with non-steroidal anti-inflammatory drugs and/or opioids, great progress has been made in cancer pain relieving. However, pain management still remains inadequate in approximately40%of cancer patients because of the side effects of current treatments. This highlights the need to find molecular mechanisms and effective new approaches for cancer pain.Although bone cancer pain is one of the most severe and common chronic pain, relatively little is known about the mechanisms that generate and maintain this pain. In order to define the mechanisms that give rise to cancer pain, animal models of localized but progressive bone destruction are published. Schwei et al. developed the first mice model of bone cancer pain by inoculating of NCTC2472tumor cells into the intramedullary cavity of femur of C3H/HeJ mice, which produced a series of behavioral, cellular, and neurochemical changes correlated with cancer growth and bone destruction. In this article, we described a modified model of bone cancer pain with4T1mammary carcinoma cells injected into the femur cavity of mice. Bone destruction and the time course of pain-related behavioral changes were evaluated, including the changes of limb use, the development of mechanical allodynia and thermal hyperalgesia.Interleukin (IL)-33is a recently described member of the IL-1family, which mediates its biological effects as a ligand for ST2. IL-33is thought to combine with ST2L on the cell membrane in the peripheral system, and this leads to the activation of transcription factors such as NF-κB and MAP kinases and the production of inflammatory mediators and leads to severe pathological changes in immune diseases. However, the effects of IL-33are either pro-or anti-inflammatory depending on the diseases and the models. Though the IL-33/ST2signaling pathway has been shown to regulate a broad range of diseases, the investigation on its role in the transmission and modulation of pain is rare. It is reported that IL-33, like other IL-1cytokines, could induce inflammatory pain in the peripheral nervous system and mediate antigen-induced cutaneous and articular hyper-nociception in mice, suggesting IL-33play a pivotal role in the modulation of arthritic pain in peripheral nervous system.However, roles of IL-33in the pain modulation in the central nervous system (CNS) are under-appreciated. Previous study of our group suggested that spinal IL-33and its ST2receptor mediate formalin-induced inflammatory pain. Meanwhile, lots of reports showed that it was expressed at a high level in CNS and localized in astrocytes, which were the main mediators for central sensitization, suggesting that IL-33may have a close relationship with pain modulation in CNS. Bone cancer pain has components of both inflammatory and neuropathic pain. Therefore, we hypothesize that the spinal IL-33/ST2act a role in bone cancer induced pain.According to the reported animal model of cancer pain, the presented study was aimed to:1) Establishing mice models of cancer pain, and evaluation of the animals by pain behaviors, radiological and histological methods.2) Examining expression of IL-33, ST2and astrocytes released cytokines(TNF-a, IL-6, IL-1β) via RT-PCR, Western Blot and Bio-Plex.3) Examining distribution of IL-33and ST2in the spinal cord using immuno-histochemistry.4) Examining the effects of intrathecal administrations of ST2neutralizing antibody(100ng,200ng,400ng) or koncking out of the ST2gene on the pain behaviors in bone cancer mice.5) Examining the effects of koncking out of the ST2gene in the concentrations of TNF-a, IL-6and IL-1β.The results are as follows:1. Establishment of murine models of cancer painAccording to the mice model of femur bone cancer pain first reported by Schwei, the modified mice model of femur bone cancer pain was established by inoculating the4T1mammary carcinoma cells into the intramedullary space of the mouse femur. The tumor bearing mice displayed significant ambulatory pain, mechanical allodynia and thermal hyperalgesia. X-ray and HE staining showed that the mice inoculated with tumor cells showed significant bone destruction as time extension.2. Up-regulation of spinal IL-33and relative cytokines in bone cancer pain miceThe expression of IL-33, ST2and other relative cytokines in the cancer-induced pain model was investigated using RT-PCR, Western Blot and Bio-Plex. Both the mRNA level and the protein level of IL-33, TNF-a, IL-6, IL-1β were significantly increased in the spinal cord on day7,10and14after injection of carcinoma cells. However, no significant change in the levels of spinal ST2was found among the five groups. Koncking out of the ST2gene could reduce the protein level of TNF-a, IL-6and IL-1β.3. Both IL-33and ST2were mainly expressed in astrocytes in the spinal cord.We used double immunofluorescence to identify the cell types that expressed IL-33and ST2. Both IL-33and ST2were co-expressed with astrocytes, while co-expression of ST2with neurons was also found in few numbers.4. Administration of ST2antibody or ST2gene Knockout suppresses pain behaviors in bone cancer pain miceTo further confirm the role of IL-33/ST2in the bone cancer pain, ST2neutralizing antibody (100ng,200ng,400ng, i.t.) was given on the10th day after cancer cells injection. PWL as well as PWT of tumor-bearing mice was ameliorated dose-dependently. ST2gene knockout (ST2KO) mice showed the same results.Taken together, the present study indicates that activation of IL-33/ST2in spinal dorsal horn contributes to the development and maintenance of bone cancer pain. And the modulation of IL-33to bone cancer pain may be involved in the activation of spinal glial cells. Thus, IL-33might become a novel target for treating bone cancer pam. |
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