The Role Of HMGB1 In The Development Of Morphine Analgesic Tolerance In Rats

Bachground Morphine is an opioid receptor agonist, which is the gold standard for clinical analgesia.However,long-term use of these drugs is often limited by the development of analgesic tolerance and hyperalgesia that can be overcome by escalating doses to achieve equivalent pain relief. Although a lot of studies have been carried out on morphine tolerance, the mechanism of morphine tolerance is still not clear. It is well documented that repeated and long-term use of morphine causes opioid receptor-mediated adaptive changes within the nervous system, including desensitization, internalization,downregulation and phosphorylation of opioid receptors or heterodimerization with other receptors.Moreover,chronic opioid exposure leads to adaptive changes in different neuronal circuits, including activation of anti-opioid systems including excitatory neurotransmitter release from primary afferents and spinal dorsal horn glutamate receptor activation;descending spinal facilitation; glial cell activation and cytokine release.And these cytokine factors maybe the primary cause of Morphine tolerance and hyperalgesia.High mobility group box-1protein is a pro-inflammatory cytokine belong to a family of three nuclear proteins present in mammals.It can be released into the extracellular milieu from immune and non-immune cells in response to various stimuli.Extracellular HMGB1 contributes to the pathogenesis of numerous chronic inflammatory and autoimmune diseases.Recently research find that t peripheral nerve injury can induce HMGB1 in the dorsal root ganglion(dorsal root ganglia DRG and spinal cord dorsal horn)expression changes and blocking the role of HMGB1 can alleviate the neuropathic pain in rats.At present, most scholars believe that neuropathic pain and morphine tolerance have the similar mechanism.Although the role of HMGB1 in pathological pain has been achieved clear results, but the role of HMGB1 in morphine tolerance and molecular mechanisms have not been reported in the literature.The main purpose of this study was to demonstrate the role of HMGB1 in the formation of morphine tolerance through animal experiments.Firstly,repeatedly(2times a day, for 6 consecutive days) intrathecal microinjection of morphine for established morphine tolerance model,through the change in the tail flick latency(Tail-flick latency) in due to observation of morphine analgesic effect,through paw withdrawal threshold(PWT) and paw withdrawal latency elevate of pain behavior in Rats after morphine withdrawal.During different periods of morphine tolerance formation(before the formation of morphine tolerance or after) by intrathecal injection of glycyrrhizin(GL)(HMGB1 small molecule inhibitors) or HMGB1 si RNA through combination of above behavior tests to observe the effect of blocking HMGB1 or inhibiting the synthesis of HMGB1 on the formation of morphine tolerance.By using of Western Blot we observed HMGB1 expression changes in DRG and Spinal Cord and the effect of intrathecal injection of HMGB1 si RNA on the expression of HMGB1 during the formation of morphine tolerance.The expression of cell type of HMGB1 in DRG and Spinal Cord by Immunofluorescence.The experimental results will provide theoretical support for further elucidation of the mechanism of morphine tolerance, and have a certain practical reference value for the prevention and treatment of morphine tolerance.Results1.The formation of morphine tolerance model and the expression of HMGB1 in spinal cord and DRG and distribution in different cell types.The results showed that intrathecal injection of morphine 10 μg/10 μl twice a daily for 6 consecutive days.In the fifth day of drug administration %MPAE in about 20%, in the seventh day %MPAE almost 0%.After stopping drug administration can cause bilateral mechanical and thermal hyperalgesia of rats compared with Saline group and its baseline at eighth day.Western blot was used to detect morphine-induced expression of HMGB1 increased in DRG and spinal cord, according to immunofluorescence found that HMGB1 in the spinal dorsal co-location with neurons and glia and microglia cells. In dorsal root ganglion HMGB1 and type A and type C neurons and satellite glial cells co-located with each other.2.Intrathecal injection of HMGB1 small molecule inhibitors Glycyrrhizin can partially inhibit the symptoms of morphine tolerance.Compared with intrathecal injection of GL group and Saline group the %MPAE has been significantly improved in the fifth day and seventh day.After withdrawal of drugs the PWMT and PWTl were improved compared with saline group in eighth day.In this way, the hyperalgesia had been alleviated that were dose- and time-dependent.3.After intrathecal injection of GL can partially reverse the symptoms of morphine tolerance.Compared with saline group, intrathecal injection of Gl group has a remarkable increased in %MPAE during 7d to 11 d.At the twelveth day,withdrawal drugs compared with saline group of PWT and PWL were significantly elevated to relieve morphine withdrawal induced hyperalgesia.4. Intrathecal injection of HMGB1 si RNA can partially inhibit the symptoms of morphine tolerance.Compared with intrathecal injection of HMGB1 Scramble si RNA,Transfection Regant and Saline group the %MPAE has been significantly improved in the fifth day and seventh day.After withdrawal of drugs the PWT and PWL were improved compared with HMGB1 Scramble si RNA,Transfection Regant and saline group in eighth day.In this way,hyperalgesia had been alleviated.There is no significant difference,the results of HMGB1 Scramble si RNA group and Transfection Regant group behavioral text comparing with saline group.Western blot shows the expression of HMGB1 in spinal cord of HMGB1 si RNA group was the lowest.5.After the paw withdrawal mechanical threshold and paw withdrawal thermal latency test, the motor function test was carried out.The rats did not have motor malfunction when tested by placing reflex,grasping reflex and righting reflex after administration of drug.Conclusion By inhibiting HMGB1 expression or function, can effectively alleviate morphine induced analgesic tolerance,and mechanical allodynia and thermal hyperalgesia after morphine withdrawal.