| BachgroundAs we all know,now opioid morphine is still the gold standard for the clinical treatment of refractory pain.However,long-term use of these drugs is often produce a decline in analgesic effect,we called it analgesic tolerance.Although the phenomenon can be overcome by escalating doses of morphine,its side effects often make the patient unbearable.Therefore,its clinical application is limited by the development of tolerance.It is thought that the change of opioid receptor function and the activation of central nervous system caused by repeated application of morphine is the main cause of morphine tolerance.A good deal of experiments have proved that the activation of spinal glial cells caused by application of morphine,and then promote the expression of cytokines,chemokines and other pain-causing substances played an important role in the morphine tolerance formation and maintenance process.HMGB1(High-Mobility Group Box-1)belong to a family of three nuclear proteins present in mammals,is a pro-inflammatory cytokine.It is well documented that HMGB1 plays a vital role in bone cancer pain and neuropathic pain,but the molecular mechanism of HMGB1 in morphine tolerance and effect has not been reported in the literature.According to HMGB1 can promote the release of cytokines and its cytokine-like effect,we speculate that like those cytokines HMGB1 may also play an important role in the formation of morphine tolerance.Accordingly,our laboratory conducted a series of studies,the results proved that spinal cord HMGB1 upregulation involved in the formation and maintenance of morphine tolerance.However,the mechanism of HMGB1 expression secreted by application of morphine and the molecular principles of HMGB1-mediated morphine tolerance still need further study.ObjectiveThis experiment is to determine the expression changes of HMGB1 in spinal cord horn(SC)and dorsal root ganglion(DRG)in rats,and observe the expression changes of HMGB1 signaling molecules in rats of morphine induced analgesis tolerance and subsequent hyperalgesia.According to those results we can determine the expression of HMGB1 in morphine tolerance and the pathways of HMGB1-mediated morphine tolerance.Thus we may know the molecular mechanism of HMGB1 involved in morphine tolerance formation.Results 1.The effects of intrathecal injection of morphine on the expression of HMGB1 and its receptors,phosphorylated NF-?B p65,TNF-? and IL-1? in spinal cordWe intrathecal injection of morphine 10 ?g/10?l twice a daily for 6 consecutive days,and inductive dose was given on day 7.The results showed that in the first and third day of morphine administration the %MAPE in about 90%-100%,the fifth day the %MAPE in about 20%,and in the seventh day the %MAPE almost 0%.The spinal cord of the rat L4-L5 was drawn on the first day,third day,fifth day and seventh day of the administration of morphine and the third day after the withdrawal of morphine followed by western blot.The results of western blot showed that the expression of HMGB1 and its receptors RAGE and TLR4 were maintained at a high level(* P <0.05 ** P <0.01)on the first day,third day,fifth day and seventh day of morphine injection and the level to reach the peak on the third day,and its expression to return to normal levels on the third day after the withdrawal.The results showed that intrathecal microinjection of morphine could induce HMGB1 and its receptor,and the expression of phosphorylated NF-?B p65,TNF-? and IL-1? increased.2.The effects of morphine on the expression of HMGB1,phosphorylated NF-?B p65 and its downstream proinflammatory cytokines TNF-? and IL-1? in rat spinal cord neuronsIn vitro spinal cord neurons were cultured for 10 days and the cell growth was stable,then 1 mg / ml of morphine at 30?l was added to the mediun and the final concentration of morphine was 20 ?M / L,and for 0 h?3 h?6 h?12 h?24 h and 48 hours later the protein was extracted.The results showed that compared with the 0h group,the expression of HMGB1 ?phosphorylated NF-?B p65 and its downstream proinflammatory cytokines TNF-? and IL-1?was significantly increased(*P <0.05 **P <0.01 ***P <0.001),and the level of HMGB1 ?phosphorylated NF-?B p65 and its downstream proinflammatory cytokines TNF-? and IL-1?reach the peak on the 6 h.As described above,added drugs to the medium in the same manner,saline and 1 ?l / ml of morphine at 15 ?l,30 ?l,60 ?l,120 ?l and 240 ?l were added to the culture medium at a final concentration of 0,10,20,40,80 and 160?M / L,respectively.The results showed that the expression of HMGB1 in morphine group was significantly higher than that in saline group(***P <0.001),and the level reach the peak on morphine concentration of 20 ?M / L.From those above results,we conclude that morphine can play an inhibitory role in the expression of HMGB1 and TNF-? and IL-1? in neurons of isolated rat spinal cord neurons,and it may be time-and dose-dependent for morphine.3.Effects of TLR4 and ? receptor blockers on the expression of HMGB1 and TNF-? in spinal cord neuronsTLR4 blocker TAK-242(10 ?M / L)and ?-receptor blocker CTOP(10 ?M / L)were preliminarily added to the culture medium after incubation of the rat spinal cord neurons for 10 days,after 30 min,morphine was added to a final concentration of 20 ?M / L,and the cells were harvested for 6 h later.The western blot results showed that compared with morphine plussolvent group,the expression of HMGB1 and TNF-? in morphine plus TAK-242 group decreased(*P <0.05).The expression level of TAK-242 group was not statistically significant compared with the solvent group,morphine plus TAK-242 group.Compared with morphine plus solvent group,the expression of HMGB1 was not changed in morphine plus CTOP group,and the expression of HMGB1 in these two groups was significantly higher than that in solvent group and CTOP group.We concluded that block the activition of TLR4 can inhibit the expression of HMGB1 and TNF-?,but block the activition of ? receptor can not inhibit the expression of HMGB1 and TNF-?,thus we determined that TLR4 was involved in the formation of HMGB1-mediated morphine tolerance,while the ? receptor did not participate in the process.4.The effects of intrathecal TLR4 blocker on expression of HMGB1,phosphorylated NF-?B p65 and morphine tolerance in spinal cord and dorsal root ganglionTAK-242(5,10,20 ?g/10?l)was injected 30 min before morphine injection,TAK-242 once a day for six consecutive days,morphine twice a day for six consecutive days.In the control group,the rats were treated with morphine twice a day for six consecutive days.The results showed that intrathecal pre-injection of TAK-242 group compared with intrathecal pre-injection solvent group on the fifth day(***P <0.001)and seventh day(***P <0.001 *P <0.05)morphine maximum analgesic efficiency was significantly increased.On the eighth day after morphine injection(the first day after stopping to intrathecal injection of morphine injection),the PWMT and PWTL of rats were improved,but compared with own baseline was no significant difference.In this way,the hyperalgesia had been alleviated that were dose-and time-dependent.The western blot was taken on the second day after discontinuation,the western blot results showed that compared with morphine group the expression of HMGB1 and p-p65 reduced in SC and DRG.These results suggest that blocking TLR4 can relieve the formation of morphine tolerance and inhibit the release of HMGB1 and p-p65 in the SC and DRG.5.Effects of intrathecal injection of HMGB1 siRNA on intracellular signal pathway of morphine toleranceIn the experimental group,HMGB1 siRNA,scramble siRNA and transfection regent were injected 30 min before morphine injection.HMGB1 siRNA,scramble siRNA and transfection regent were once a day and morphine was twice a day for six consecutive days;In the control group,the rats were treated with morphine twice a day for six consecutive days.The western blot was taken on the second day after discontinuation,the western blot results showed that compared with the other three groups,the expression of HMGB1?p-p65?p-JNK in SC and DRG reduced in HMGB1 siRNA group(*P < 0.05 **P < 0.01 ***P < 0.001).This suggest that intrathecal injection HMGB1 siRNA can relieve morphine tolerance and inhibit the release of HMGB1,p-p65 and p-JNK expression in the SC and DRG.The results suggest that intrathecal morphine-induced HMGB1 upregulation may be mediated by activation of NF-?B and JNK signaling pathways leading to morphine tolerance.ConclusionMorphine mediates the expression of HMGB1 in the spinal cord through the TLR4-NF-?B signaling pathway.HMGB1 mediates the formation of morphine tolerance by activating NF-?B and p-JNK signaling pathway. |
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