Mechanism Of Resveratrol In The Treatment Of Bone Cancer Pain And Experimental Study Of Baicalin Inhibiting Morphine Tolerance

BackroundBone cancer pain is one of the most common symptoms presents by patients in late stage cancer.It has been reported that 70-90%of patients with advanced cancer experience intractable pain.Cancer-induced pain is most disruptive to cancer patient's quality of life.Among the different types of cancer pains,bone cancer pain is the most common,severe and refractory pain.Despite decades of study,the cellular and molecular mechanisms underlying bone cancer pain remain elusive and the clinical approaches for treating bone cancer pain are limited.Mechanisms of bone cancer pain are complex and may involve a combination of inflammatory and neuropathic pain.with unique characteristics.Increasing evidences have shown that microglia and astrocytes play important roles in the de-velopment of bone cancer pain.Upound activation,microglia and astrocytes release the proinflammatory cytokines interleukin-1?(IL-1?)and tumor necrosis factor-?(TNF-?),which cause central sensitization.In the present study,rat model of tibia bone cancer pain was used to investigate the role of AMPK activation in regulation of bone cancer pain.Methods(1)Tibia bone cancer pain model was established in rats.Effects of AMPK activation on the tumor cell implantation(TCI)-induced mechanical hyperalgesia were tested with vonfrey test.(2)To investigate the role of resveratrol on bone cancer pain.The expressions of GFAP and IBA-1 in spinal cord were detected by western blot.And immunofluorescence was used to identify the distribution and location of GFAP and IBA-1.Meanwhile,the proinflammatory cytokines interleukin-1?(IL-1[)and tumor necrosis factor-a(TNF-a)in the spinal cord after bone cancer pain treatment was determined with western blot.(3)To investigate the mechanisms of resveratrol on TCI-induced glia activation.Effects of AMPK agonist,resveratrol,on the tumor cell implantation(TCI)-induced the actvation of MAP Kinase family were tested with western blot(4)In order to clarify the role of resveratrol on inhibition of glial cell activation in bone cancer pain.Western blot was used to determine effects of resveratrol on proinflammatory cytokines TNF-a plus IL-1?-induced astrocyte and microglia activation.Immunofluorescence was used to determine effects of resveratrol on proinflammatory cytokines TNF-a plus IL-1?-induced astrocyte proliferation.Meanwhile,the role of resveratrol on mechanical hyperalgesia evoked by TNF-a plus IL-1? stimulation was also be test with vonfrey test.And,western blot was used to detect effects of resveratrol on TNF-a plus IL-1?-induced glia cell activation.Results(1)Resveratrol suppressed TCI-induced mechanical hyperalgesia in a dose dependment manner.Repetitive administration of resveratrol(each 300mg/kg X 5,p.o.)almost reversed bone cancer pain.Spinal administration of AMPK antagonists,Compound C,abolished the role of resveratrol inhibiting pain related-behaviors after TCI.Meanwhile,Spinal administration of AMPK agonist AICAR and metformin mimicked the role of resveratrol and significantly attenuted TCI-induced mechanical hyperalgesia.(2)TCI-induced activation of the astrocytes and microglial cells were significantly inhibited by resveratrol;Resveratrol inhibited TCI-induced increased IL-1? and TNF-a in the spinal cord.(3)Resveratrol inhibited TCI-induced upregulation of p-JNK,p-p38 and p-ERK levels in the spinal cord.And,the inhibition of resveratrol inhibiting TCI-induced upgulation of p-JNK,p-p38 and p-ERK levels in the spinal cord was cancelled by Compound C.(4)Resveratrol inhibited TNF-a and IL-1?-induced astrocyte activation and proliferation.Meanwhile,resveratrol inhibited TNF-a plus IL-1?-induced microglial cell activation.Additionally,Compound C cancelled the role of resveratrol inhibiting TNF-a plus IL-1?-induced astrocytes and microglial cells activation.And AICAR mimicked this role of resveratrol.CompoundC also cancelled the role of resveratrol inhibiting TNF-a plus IL-1?-induced astrocytes proliferation,and AICAR mimicked the role of resveratrol.In addition,resveratrol significantly suppressed TNF-a and IL-1?-induced activation of astrocyte(GFAP)and microglial cells in SD rats spinal cord and attenuated mechanical hyperalgesia evoked by TNF-a and IL-1?stimulation.Conclusion(1)Resveratrol can reverse bone cancer pain and suppress tumor cell implantation-induced astrocytes and microglial activation.(2)The role of resveratrol reversing bone cancer pain may depend on AMPK activation and is associated with inhibition on MAPK signaling in the spinal cord.The present study was aim to investigate the effects and possible mechanisms of baicalin on morphine induced microglia activation and morphine tolerance.Cytokine IL-1? and TNF-? expression was analyzed by reverse transcription polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA),the level of p38 MAP Kinase phosphorylation and IBA-1 expression was determined by Western blot assay,The antinociception and morphine tolerance were assessed in mice using hot-plate test.Result showed baicalin(1?mol/L,10?mol/L and 100?mol/L)inhibited morphine induced up-regulated levels of IL-1?,TNF-? in BV-2 microglia cells in a dose dependent manner;100?mol/L baicain significantly inhibited morphine induced up-regulated level of p38MAP Kinase phosphorylation expression in BV2 microglia cells.Intrathecal(it.)injection of baicain(20?g/10?L,40?g/10?L and 60?g/10?L)attenuated the development of chronic morphine tolerance in a dose-dependent manner;Baicalin(60?g/10?L)mimicked the role of minocycline and significantly suppressed chronic morphine treatment-induced up-regulated level of p38MAP Kinase phosphorylation and IBA-1 expression in mice spinal cord.In conclusion,baicalin suppresses morphine-induced microglia activation through suppressing p38MAPK signaling,resulting in significant attenuation of morphine antinociceptive tolerance.