| Objective To investigate the role of spinal HSP27 in morphine tolerance and its relationship with PDGFRβ,and further explore the possible signaling pathways associated with HSP27 activation.Methods 1.Adult male SD rats were anesthetized and Intrathecal catheters were implanted into subarachnoid cavity between L4 and L5 vertebrae of rats.Rats were intrathecally administered with morphine for 9 days to induce chronic tolerance to morphine.Western blot and q RT-PCR were used to explore the expression of HSP27 and PDGFR β,and immunofluorescence was performed to observe the localization of HSP27 and PDGFR β.2.Small interfering RNA(si RNA)lentiviral vectors against HSP27(HSP27 RNAi-LV)was prepared and injected into lumbar spinal cord of rats 7 days before intrathecal injection.Then,rats were intrathecally administered with morphine for9 days.The effect of HSP27 inhibition on the development of morphine tolerance was observed by behavioral test.The transfection effect of lentivirus was verified by fluorescence microscopy,q RT-PCR and western blot.3.Rats were intrathecally injected with PDGFRβ inhibitor imatinib 30 min before morphine administration from day 1 to 9,or from day 5 to 9,respectively.And the effect of PDGFR β on the formation,development and maintenance of morphine tolerance was verified by behavior test.In addition,the effect of PDGFR β on morphine tolerance was further verified by PDGF β agonist PDGF-BB.Then q RT-PCR and Western blot were employed to investigate the effect of PDGFR β on the total protein and phosphorylated protein of HSP27.4.Rats were intrathecally administrated with PDGFRβ inhibitor imatinib 30 min before morphine administration from day 5 to 9.Western blot was used to explore some of MAPK and PI3 K / Akt signaling pathways which were influenced by PDGFRβ.Then,rats were intrathecally injected with the inhibitor of signaling pathways which were involved in PDGFRβ-mediated morphine tolerance 30 min before morphine administration from day 5 to 9,respectively.Western blot was performed to investigate the effect of associated signaling pathways on HSP27.Results 1.The results of behavioral test showed that the pain threshold of rats on the first day(P < 0.001)and the third day(P < 0.01)after morphine administration was significantly higher than that of normal rats.From day 5 to day 9,there was no significant difference between long-term morphine administration and normal rats(P > 0.05),indicating that the construction of morphine tolerance model was successful.q RT PCR showed that the chronic morphine injection increased the expression of HSP27 m RNA(P < 0.05),Western blot showed that the total protein and phosphorylated protein of HSP27 were increased after chronic morphine administration(P < 0.05),while PDGFR β only phosphorylated protein increased(P < 0.05).According to results of double immunofluorescence,the total HSP27 protein in the spinal dorsal horn of rats was mainly co-located with astrocytes in the saline-treated rats,and astrocytes and neurons in the morphine-tolerance rats,while the phosphorylation of HSP27 was co-located with the nucleus of neurons.In addition,the result of immunofluorescence showed that phosphorylated PDGFR β was mainly co-located with microglia in the saline-treated rats,and microglia and neurons in morphine-tolerance rats.2.Inhibition of HSP27 Expression after HSP27 RNAi-LV injection partially attenuated the development of morphine tolerance(P < 0.05).Fluorescence microscopy showed that the lentivirus was successfully transfected.The results of q RT-PCR and Western blot showed that HSP27 RNAi-LV inhibited HSP27 expression successfully(P < 0.05).3.Treatment with imatinib from day 5 to 9 partially reversed developed morphine tolerance(P < 0.05).And treatment with imatinib from day 1 to 9 partially attenuated development of morphine tolerance(P < 0.05).In addition,chronic PDGF-BB injection reduced analgesia of morphine(P < 0.05).The increased expression and phosphorylation of HSP27 induced by morphine treatment were inhibited by imatinib measured by western blot(P < 0.05).And chronic PDGF-BB administration increased the total protein and phosphorylation of HSP27(P < 0.05).4.The phosphorylation of Akt and p38 induced by chronic morphine injection were reduced by treatment with imatinib from day 5 to 9(P < 0.05),while this treatment with imatinib had no influence in the change of JNK and ERK(1/2)induced by morphine administration(P > 0.05).Treatment with PI3K/Akt and p38 inhibitor from day 5 to 9 partially reversed developed morphine tolerance,respectively,measured by behavioral test(P < 0.05).And phosphorylations of HSP27 induced morphine treatment were reduced by these inhibitors,respectively,measured by western blot(P < 0.05).Conclusion 1.Inhibition of HSP27 expression and phosphorylation could partially alleviate the development of morphine tolerance.2.PDGFRβ could regulate the expression and activity of HSP27 in morphine tolerance.3.PI3 K / Akt and p38 signaling pathways might be involved in the regulation of PDGFRβ on HSP27 during morphine tolerance.4.After long-term injection of morphine,except for phosphorylation of HSP27,the location of HSP27 total protein and phosphorylated PDGFRβ could change,and the cells involved in these three proteins may participate in the development of morphine tolerance... |
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