Tanshinone ⅡA And Melatonin Regulate Inflammatory Response Of Spinal Cord In Rats Of Chronic Pain

Chronic pain is a common clinical pain,which exists more than three months.Chronic pain can be persistent after the healing of primary disease or tissue injury,and seriously affect the life quality of patients.Chronic pain includes neuropathic pain,inflammatory pain,and cancer-related pain.Because the mechanism is not clear,the treatment of chronic pain is lack of effective measures.Recent studies have suggested that spinal inflammation could play an important role on the development and maintenance of chronic pain.Previous studies have demonstrated that TSN IIA inhibits HMGB1 release in vitro and in vivo.But the role of TSN IIA in inflammatory pain is not clear.Therefore,we established a chronic pancreatitis(CP)rat model to observe the role of TSN IIA in inflammatory pain by behavioral,pharmacological,morphological,and molecular methods.Melatonin has been shown to have anti-allodynia effects in both preclinical and clinical studies.But its role in neuropathic pain has not been reported.In the present study,we investigated the role of systemic administration of melatonin in the rat model of oxaliplatin-induced pain.In conclusion,this study observed the analgesic effect and associated mechanisms of TSN IIA and melatonin in rat models of inflammatory pain and neuropathic pain.Our study will provide a new theoretical basis for analgesic therapy of chronic pain targeting the neuro-inflammation of spinal cord.Part One Tanshinone IIA regulates the inflammatory response of spinal cord in rats of chronic pancreatitis-induced painBackgroundChronic pancreatitis(CP)is a long-standing inflammation of the exocrine pancreas which typically results in severe and constant abdominal pain.Most previous studies on the mechanisms underlying CP-induced pain have focused on the peripheral nociceptive system,whereas a central mechanism in the mediation or modulation of abdominal pain is largely unknown.Previous studies have shown that high-mobility group protein B1(HMGB1),a late proinflammatory cytokine,binds and activates Toll-like receptor 4(TLR4)to induce spinal astrocyte activation and proinflammatory cytokine release in neuropathic pain.Tanshinone IIA(TSN IIA),an active component of the traditional Chinese medicine Danshen,exhibits its anti-inflammatory property via downregulation of the expression of HMGB1.ObjectiveIn the present study,we investigated the effect of TSN IIA on pain responses in rats with trinitrobenzene sulfonic acid(TNBS)-induced CP.The role of central mechanismin mediating or modulating CP was also investigated.MethodsCP was induced in rats by intrapancreatic infusion of trinitrobenzene sulfonic acid(TNBS).Pancreatic histopathological changes were measured with semi-quantitative scores.The abdomen nociceptive behaviors were assessed with von Frey filaments.The effect of intraperitoneally administered TSN IIA on CP-induced mechanical allodynia was tested.The spinal protein expression of HMGB1 was determined by western blot.The spinal mRNA and protein expression of proinflam-matory cytokines IL-10,TNF-α,and IL-6 were determined by RT-PCR and western blot,respectively.The spinal expression of the HMGB1 receptor TLR4 and the astrocyte activation marker glial fibrillary acidic protein(GFAP)were determined by western blot or immunohistological staining after intraperitoneal injection of TSN IIA or intrathecal administration of a neutralizing anti-HMGB1 antibody.ResultsTNBS infusion resulted in pancreatic histopathological changes of chronic pancreatitis and mechanical allodynia in rats.TSN IIA significantly attenuated TNBS-induced mechanical allodynia in a dose-dependent manner.TNBS significantly increased the spinal expression of HMGB1 and proinflammatory cytokines IL-1β,TNF-α,and IL-6.These TNBS-induced changes were significantly inhibited by TSN IIA in a dose-dependent manner.Furthermore,TSN ⅡA,but not the neutralizing anti-HMGB1 antibody,significantly inhibited TNBS-induced spinal TLR4 and GFAP expression.ConclusionsOur results suggest that spinal HMGB1 contribute to the development of CP-induced pain and could potentially be a therapeutic target.TSN IIA attenuates CP-induced pain via the downregulation of spinal HMGB1 and TLR4 expressions.Therefore,TSN IIA might be a potential anti-nociceptive drug for the treatment of CP-induced pain.Part Two Melatonin regulates the inflammatory response of spinal cord in rats of oxaliplatin-induced pain.BackgroundNeuroinflammatory response in spinal dorsal horn has been demonstrated to be a critical factor in oxaliplatin-induced pain.Melatonin has been shown to have anti-inflammatory and anti-allodynia effects in both preclinical and clinical studies.ObjectiveIn the present study,we investigated the role of systemic administration of melatonin in oxaliplatin-induced pain.MethodsRats were intraperitoneally(i.p.)injected with oxaliplatin to induce pain.Rats in the melatonin groups were treated with a single i.p.injection of melatonin.The nociceptive behaviors were assessed with von Frey filaments.The spinal mRNA and protein expression of proinflammatory cytokines IL-1β,TNF-α,IL-6,MCP-1 and MIP-la were determined by RT-PCR and western blot,respectively.The astrocyte activation marker GFAP was determined by western blot or immunohistological staining.Primary astrocytes cultures were prepared from cortex of PO or P1 rats for experiments of lipopolysaccharide(LPS,1 μg/ml)stimulation and melatonn treatment.ResultsIntraperitoneal(i.p.)injection with oxaliplatin induced significantly mechanical allodynia and thermal hyperalgesia.Melatonin(i.p.)significantly.alleviated oxaliplatin-induced mechanical allodynia and thermal hyperalgesia.The attenuation of nociceptive response persisted at least for 3 days after melatonin injection,throughout the entire observing window.Immunohistochemistry showed that oxaliplatin induced a significant increase of glial fibrillary acidic protein(GFAP),which could be suppressed by melatonin.Western blotting showed that GFAP protein levels were significantly elevated in the oxaliplatin-vehicle group,whereas melatonin significantly decreased the oxaliplatin induced up-regulation of GFAP expressions.Oxaliplatin injection also enhanced the mRNA expressions of cytokines including interleukin-1β(IL-1α)and tumor necrosis factor-α(TNF-α)and chemokines including monocyte chemoattractant protein-1(MCP-1)and monocyte inflammatory protein-1(MIP-la)in the spinal dorsal horn,which could be significantly repressed by melatonin.In vitro study showed that mRNA levels of TNF-α,IL-1β,MCP-1 and MIP-la in primarily astrocytes were significantly increased after lipopolysaccharide(LPS,1 μg/ml)stimulation,whereas melatonin(10μM and 100 μM)greatly inhibited the synthesis of these inflammatory mediators,in a dose-related manner.ConclusionsOur study demonstrated that systemic treatment with melatonin significantly attenuated oxaliplatin-induced nociceptive response.The anti-nociceptive effect of melatonin might be mediated through inhibition of astrocytic activation and the consequent production of astrocyte-derived inflammatory mediators in the spinal cord.