The Involvement Of MCP-1/PI3K/Akt Signaling Pathway In A Rat Model Of Bone Cancer Pain

Part One:MCP-1 stimulates rat microglia in vitro via PI3K/Akt signaling pathwayObjective To observe the effect of MCP-1 on the activation of the PI3K/Akt signaling in rat microglia.Methods Cultured rat microglia in vitro were divided into 6 groups:blank control(Group I),MCP-1 1min(Group II),MCP-1 10min(Group III),MCP-1 20min(Group IV),MCP-1 30min(Group V) and MCP-1+ LY294002(Group VI).The cells of each groups were obtained to detect the expression of p-Akt and OX-42 by Immunohistochemistry(n=4)、Immunofluorescence(n=4) and Western blots(n=4).Results Immunohistochemistry showed that the MOD of p-Akt in Group II and Group III apparently increased compared with other groups(P<0.01).The expression of p-Akt in Group II and Group III had no remarkable difference(P>0.05),and there was no remarkable difference among Group I,IV,V and VI(P>0.05). Immunofluorescence and Western Blots showed that there was a significant increase in expression of both p-Akt and OX-42 in Group III compared with Group I(P<0.05),and an obvious decrease in Group VI compared with Group III(P<0.05).Conclusion MCP-1 may activate rat microglia via the PI3K/Akt signaling pathway in vitro.Part two: MCP-1 induces activation of PI3K/Akt signaling pathway in spinal dorsal horn in a rat model of bone cancer painObjective To observe the effect of intrathecal MCP-1 neutralizing antibody injection on pain behavior and the level of p-Akt expression in spinal cord in a rat model of bone cancer pain.Methods Forty SPF grade female Sprague-Dawley(SD) rats weighing 190~210g were randomly divided into five groups(n=8 each):Group I,sham operation group;Group II,sham+anti-MCP-1group;Group III,Bone Caner Pain(BCP) group;Group IV,BCP+control Ig G group;Group V,BCP+anti-MCP-1 group.BCP rat model was induced by inoculating Walker 256 breast carcinoma cells 10μl(2×107/ml) into the medullary cavity of the left tibia.Rats received i.t.Injections of either MCP-1 neutralizing antibody(1g/L) 10μl or control Ig G 10μl(1g/L) at the time of 7~9 days after the operation,once a day.Mechanical withdrawal threshold(MWT) test were performed one day before and at 1st,3rd,5th,7th,8th and 9th days after surgery.The rats were sacrificed on day 9 after inoculation after MWT test and the L4~6 segments of the spinal cord were removed for Immunofluorescence(n=4) and western blots(n=4) to determinate the expression changes of spinal p-Akt.Results Compared with Group I,the rats in BCP group(Group III,IV) appeared obvious mechanical hypealgesia(P<0.01), the expresstion of p-Akt in spinal dorsal horns also increased apparently(P<0.01),as Group II had no obvious statistical significance(P>0.05).Compared with Group III or Group IV,the i.t.Injections of anti-MCP-1 neutralizing antibody increased the mechanical withdrawal threshold(P<0.01) and obviously cut down the expression of p-Akt in the spinal dorsal horns(P<0.01).Conclusion MCP-1 may lead to bone cancer pain in rats through PI3K/Akt singaling pathway in spinal dorsal horns. Part three:Intrathecal injection of LY294002 attenuates bone cancer painObjective To investigate the effect of intrathecal injection of PI3 K inhibitor LY294002 on pain behavior and activation of microglia in spinal dorsal horns in a rat model of bone cancer pain.Methods Forty SPF grade female Sprague-Dawley(SD) rats weighing 190~210g were randomly divided into five groups(n=8 each):Group I,sham operation group;Group II,sham+LY294002 group;Group III,Bone cancer pain(BCP) group;Group IV,BCP+5%DMSO group;Group IV,BCP+LY294002 group.BCP rat model was induced by inoculating Walker 256 breast carcinoma cells 10μl(2×107/ml) into the medullary cavity of the left tibia.Rats received i.t.Injections of either LY294002 2.5ug/10μl or 5%DMSO 10μl at the time of 7~9 days after the operation. MWT test were performed one day before and at1 st,3rd,5th,7th,8th and 9th days after surgery.It was also measured before and each hour after injection until 8 hours on 7th day after inoculation.The rats were sacrificed after injection on day 9 after inoculation and the L4~6 segments of the spinal cord were removed for Immunofluorescence(n=4) and Western Blots(n=4) to determinate the expression changes of spinal OX-42.Results Compared with Group I,the rats in BCP group(III、IV) appeared obvious mechanical hyperalgesia(P<0.01), the expression of OX-42 in the spinal dorsal horns also increased apparently(P<0.01),as II group had no obvious statistical significance(P>0.05). Compared with Group III or Group IV,the MWT of V group increased apparently from the 2nd hour to the 4th hour on 7th day(P<0.05),and reached the peak in the 3rd hour(P<0.01).I.t.Injections of LY294002 for 3 days increased the MWT of Group V(P<0.05),obviously cut down the expression of OX-42 and the activation of microglia in the spinal dorsal horns(P<0.01).Conclusion PI3K/Akt signaling pathway may take part in the development of bone cancer pain by causing the activation of microglia in the spinal dorsal horns.