Crosstalk Between Cdk5/p35 And ERK1/2 Signaling Pathway Mediates The Spinal PPARΓ Activity In A Rat Model Of Neuropathic Pain

Objective To investigate the crosstalk between Cdk5/p35 and ERK1/2 signaling mediates the spinal PPARy activity in a rat model of neuropathic pain.Part one The role of Cdk5/p35 in the neuropathic painMethod:Firstly,16 rats(male,weighing 220-250 g)were randomly divided into 2 groups,which received chronic sciatic nerve constricton injury(CCI group)and sham-operation(sham group)respectively.The right hind paw threshold mechanical value(PWMT)was measured by Von Frey filament and paw withdrawal thermal latency(PWTL)was measured by Hargreaves methods before Od and 3d,7d,14d,21d after surgery.40 rats were measured the time course and distribution of spinal p35 by immunofluorescence and western blot respectively according the time course.4 rats were established a model of CCI,the spinal cord lumbar enlargement was taken at 14d after surgery,we detected the co-expression of p35 with neuronal and glial cells and Cdk5 in the spinal cord by double-immunofluorescence.Secondly,32 rats were randomly divided into 4 groups(n=8):sham+DMSO group(dimethyl sulfoxide),sham+roscovitine group,CCI+DMSO group,CCI+roscovitine group.The corresponding drugs roscovitine 100μg/10μl or 5%DMSO 10μl were administered by intrathecal injection from the 3th day to 14th day once a day after CCI.The right hind paw threshold mechanical value(PWMT)was measured by Von Frey filament and Paw withdrawal thermal latency(PWTL)was measured by Hargreaves methods before Od and 3d,7d,14d,21d after surgery.Results:Compare with sham group,the PWMT and PWTL were both significantly decreased in CCI group(P<0.05).The protein level of p35 in CCI group increased significantly in a time-dependent manner(P<0.05)and distributed mainly in spinal cord,p35 was co-expressed with neurons(NeuN marker),astrocytes(GFAP marker)and Cdk5 but not with microglia(IBA1).The expression of Cdk5 have no significant change(P>0.05).Compared with CCI+DMSO group,roscovitine was administered by intrathecal injection alleviated mechanical allodynia and thermal hyperalgesia of CCI rats(P<0.05).Part two The role of ERK1/2 in the neuropathic painMethod:Firstly,40 rats were randomly divided into 5 groups according the time course:sham group,CCI-3d,CCI-7d,CCI-14d and CCI-21d.we measured the time course and distribution of spinal pERK1/2 by immunofluorescence and western blot respectively according the time course.4 rats were established a model of CCI,the spinal cord lumbar enlargement was taken at 14d after surgery,we detected the co-expression of pERK1/2 with p35 in the spinal cord by double-immunofluorescence.Secondly,32 rats were randomly divided into 4 groups(n=8):sham+DMSO group(dimethyl sulfoxide),sham+U0126 group,CCI+DMSO group,CCI+U0126 group.The corresponding drugs U01265μg/10μl or 5%DMSO 10μl were administered by intrathecal injection from the 3th day to 14th day once a day after CCI.The right hind paw threshold mechanical value(PWMT)was measured by Von Frey filament and paw withdrawal thermal latency(PWTL)was measured by Hargreaves methods before Od and 3d,7d,14d,21d after surgery.Results:Compared with sham group,the protein level of pERK1/2 in the spinal cord increased significantly in a time-dependent manner(P<0.05)and distributed mainly in spinal cord,and co-expressed with p35.The expression of total ERK1/2 have no significant change(P>0.05).Compared with CCI+DMSO group,U0126 was administered by intrathecal injection alleviated mechanical allodynia and thermal hyperalgesia of CCI rats(P<0.05).Part three The activation of PPARy mediated the maintenance of neuropathic pain,depending on Cdk5/p35 and ERK1/2.Method:Firstly,40 SD rats were randomly divided into 5 groups according the time course:sham group,CCI-3d,CCI-7d,CCI-14d and CCI-21d.we measured the time course and distribution of spinal pPPARy by immunofluorescence and western blot respectively according the time course.4 rats were established a model of CCI,the spinal cord lumbar enlargement was taken at 14d after surgery,we detected the co-expression of p35 with pPPARy in the spinal cord by double-immunofluorescence.Secondly,32 rats were randomly divided into 4 groups(n=8):sham+DMSO group(dimethyl sulfoxide),sham+pioglitazone group,CCI+DMSO group,CCI+pioglitazone group.The corresponding drug pioglitazone 100μg/10μl or 5%DMSO 10μl were administered by intrathecal injection from the 3th day to 14th day once a day after CCI.The right hind paw withdrawal mechanical threshold value(PWMT)was measured by Von Frey filament and paw withdrawal thermal latency(PWTL)was measured by Hargreaves methods before 0 d and 3d,7d,14d,21d after surgery.Thirdly,16 SD rats were randomly divided into 4 groups:sham+DMSO group(dimethyl sulfoxide),sham+roscovitine,CCI+DMSO group,CCI+roscovitine.The corresponding drug roscovitine 100μg/10μl or 5%DMSO 10μl were administered by intrathecal injection from the 3th day to 14th day once a day after CCI,we measured the expression of spinal pPPARy by western blot.Fourthly,12 SD rats were randomly divided into 4 groups:sham+DMSO group(dimethyl sulfoxide),sham+U0126 group,CCI+DMSO group,CCI+U0126 group.The corresponding drug U0126 5μg/lOμl or 5%DMSO 10 μl were administered by intrathecal injection from the 3th day to 14th day once a day after CCI.We measured the expression of spinal pPPARy by western blot.Results:Compared with sham group,the protein level of pPPARy in the spinal cord decreased significantly in a time-dependent manner(P<0.05)and distributed mainly in spinal cord,and co-expressed with p35.The expression of total PPARγ have no significant change(P>0.05).Compared with CCI+DMSO group,pioglitazone was administered by intrathecal injection alleviated mechanical allodynia and thermal hyperalgesia of CCI rats(P<0.05).Intrathecal injection of Cdk5/p35 inhibitor roscovitine and ERK1/2 upstream MEK inhibitor U0126 significantly increased expression of pPPARy in spinal dorsal horn(P<0.05).Part four Crosstalk between Cdk5/p35 and ERK1/2 mediated the development of NP and inflammation response.Method:Firstly,40 SD rats were randomly divided into 4 groups(n=10):sham+DMSO group(dimethyl sulfoxide),sham+roscovitine group,CCI+DMSO group,CCI+roscovitine group.The corresponding drug roscovitine 100μg/10μl or 5%DMSO 10μl were administered by intrathecal injection from the 3th day to 14th day once a day after CCI.We detected the effect of intrathecal injection of Cdk5/p35 inhibitor roscovitine on the expression of Cdk5,p35,pERK1/2 in CCI rats and on the enzyme activity assay of Cdk5/p35 Kinase by Cdk5 Kinase Activity kit.Secondly,40 SD rats were randomly divided into 4 groups(n=10):sham+DMSO group(dimethyl sulfoxide),sham+U0126 group,CCI+DMSO group,CCI+U0126 group.The corresponding drug U0126 5μg/10μl or 5%DMSO 10μl were administered by intrathecal injection from the 3th day to 14th day once a day after CCI.we detected the effect of intrathecal injection of MEK inhibitor U0126 on the expression of Cdk5,p35 and pERK1/2 in CCI rats,and on the enzyme activity assay of Cdk5/p35 Kinase by Cdk5 Kinase Activity kit.Thirdly,48 SD rats were randomly divided into 8 groups(n=6):sham+DMSO group,sham+roscovitine group,sham+U0126 group,sham+pioglitazone group,CCI+DMSO group,CCI+roscovitine group,CCI+U0126 group,CCI+ pioglitazone group.The corresponding drug or 5%DMSO were administered by intrathecal injection from the 3th day to 14th day once a day after CCI.The activity of inflammation response mediators(IL-1β、IL-6 and TNFa)were measured using ELISA kits.Results:Intrathecal injection of Cdk5/p35 inhibitor roscovitine or ERK1/2 upstream MEK inhibitor U0126 significantly inhibited expression of p35 and pERK1/2 in spinal dorsal horn by western blot(P<0.05).After roscovitine or U0126 treatment,Cdk5 kinase activity decreased significantly by Cdk5/p35 Kinase Activity Assay kits(P<0.05).Roscovitine,U0126,or pioglitazone exerted inhibitory effects on the production of proinflammatory cytokines(IL-1β、IL-6 and TNFa)in the spinal cord by ELISA(P<0.05).Conclusion:1、Cdk5/p35 and ERK1/2 mediated the induction and maintenance of NP.2、The activation of PPARy mediated the maintenance of NP depending on Cdk5/p35 and ERK1/2.3.Crosstalk between Cdk5/p35 and ERK1/2 mediated the induction and maintenance of NP and inflammation response.