Activation Of JNK And P38 Affected By P2X3 Up-regulation In Neuropathic Pain

OBJECTIVESExperiments in this paper applicated of P2X3 agonist β,γ-meATP to activate P2X3 receptor in Chronic Compression of Dorsal Root Ganglion (CCD) neuropathic pain (NP) rats’ model, to explore whether activation of P2X3 receptor in NP can cause changement of JNK and P38 expression and activation in the DRG, further improving the understanding of the mechanisms of P2X3 receptor in NP.MEASUREMENTS1.12 Sprague-Dawley (SD) male rats were built NP model (CCD) group (n=6) or the control (SHAM) group (n=6) randomly:detection of 50% Mechanical Paw Withdrawal Threshold (50%MPWT) the day before surgery,postoperative 1,4,7,10,14, 21,28 and 35 day on the ipsilateral and contralateral hinder paws.2. Testing the expressional trendency of P2X3 receptor within postoperative 28 d after CCD operation:15 SD male rats randomly divided into untreated rats (NAIVE) (n= 3) and CCD building rats (n=12);CCD rats were randomly executed at 7 d after operation (POD07) (n=3),14 d after operation (POD14) (n=3),21 d after operation (POD21) (n=3) or 28 d (POD28) after surgery (n=3) and L4, L5 DRG were rapidly removed, applicating quantitative Western Blotting to test P2X3 levels.3. Detection duration of paw lift-one nociceptive behavior of the CCD rats and SHAM rats after P2X3 agonists applied intraplantely:36 SD male rats were randomly divided into CCD group (18 rats) and SHAM group (18 rats); respectively,18 rats were randomly given a intraplant injection 14 d after surgery:H2O (n=6),β,γ-meATP 100nmol (n=6) and β,γ-meATP 500nmol (n=6), the volume were 50μl all; all followed by testing Paw Lift Time per Minute(PLTPM).4. Testing changes of JNK, pJNK (phosphorylation activational typeof JNK), P38 and pP38 (phosphorylation activational type of P38) expression in the DRG of CCD rats and SHAM rats after surgery:9 SD male rats were randomly divided into NAIVE group (n=3), SHAM group (n=3) and CCD group (n=3); L4, L5 DRG were rapidly removed and applicating quantitative Western Blotting to test JNK, pJNK, P38 and pP38 levels.5. Testing changes of P2X3, JNK, pJNK, P38 and pP38 expression in the DRG of CCD rats and SHAM rats after surgery:18 SD male rats were randomly to build CCD (9 rats) or SHAM group (9 rats); respectively,9 rats were randomly given a intraplant injection 14 d after surgery:H2O (n=6),β,γ-meATP 100nmol (n=6) and β,γ-meATP 500nmol (n=6), the volume were 50μl all; followed by L4 and L5 DRG rapid removement and Western Blotting administration to detect P2X3, JNK,pJNK,P38 and pP38 quantitatively.RESULTS1.Compared with CCD contralateral hinder paws or Sham ipsilateral hinder paws, 50%MPWTs of ipsilateral hinder paws of CCD rats at postoperative 1,4,7,10,14,21, 28 and 35 day were decreased significantly (P<0.001);2.P2X3 which expresses in DRG of CCD rats on postoperative day 14 enhanced (P <0.05) compared with NAIVE rats;3. Among the 18 SHAM rats,1 min after appling β,γ-meATP 100nmol to SHAM rats the PLTPM increased obviously compared with SHAM rats applied H2O (P<0.01), 1 min after appling β,γ-meATP 500nmol to SHAM rats the PLTPM increased dramatically compared with SHAM rats applied H2O(P<0.001); Among the 18 CCD rats,1 min after appling β,γ-meATP 100nmol to CCD rats the PLTPM increased dramatically compared with CCD rats applied H2O (P<0.001),1,2,3,5 min after appling β,γ-meATP 500nmol to CCD rats the PLTPM increased dramatically compared with CCD rats applied H2O (P<0.001),6 min after the treatment PLTPM increases (P <0.05);In addition,2 min after appling β,γ-meATP 500nmol to CCD rats PLTPM increased dramatically compared with β,γ-meATP 500nmol application to SHAM rats (P <0.001).4. Comparing JNK, pJNK, P38 and pP38 in DRG among The NAIVE group, SHAM group and CCD group, all of them had no statistically significance.5. Testing changes of P2X3, JNK, pJNK, P38 and pP38 expression in the DRG of CCD rats and SHAM rats after P2X3 agonists applied intraplantely:(1) P2X3:CCD rats applied H2O increased than SHAM rats applied H2O (P<0.05), CCD rats applied β,γ-meATP 100nmol increased significantly than SHAM rats applied β,γ-meATP 100nmol (P<0.01);In addition, CCD rats applied β,γ-meATP 100nmol increased than CCD rats applied H2O (P<0.05). (2) JNK:among the 9 CCD rats, applied β,γ-meATP 100nmol or β,γ-meATP 500nmol increased than rats applied H2O (P<0.05); In addition, CCD rats applied β,γ-meATP 100nmol significantly increased than SHAM rats applied β,γ-meATP 100nmol (P<0.01); CCD rats applied β,γ-meATP 500nmol increased than SHAM rats appliedβ,γ-meATP 500nmol (P<0.05). (3) pJNK:CCD rats applied β,γ-meATP 500nmol decreased than SHAM rats appliedβ,γ-meATP 500nmol (P<0.05). (4) P38:there was no statistical significance among groups. (5) pP38:CCD rats applied β,γ-meATP 100nmol decreased than SHAM rats applied β,γ-meATP 100nmol (P<0.05).CONCLUSIONSP2X3 activation in CCD rats followed by enhanced JNK expression but decreased phosphorylation-activated JNK on the DRG; no P38 viriation but phosphorylation-activated P38 lessening on the DRQ which reminded us a speculation of "P2X3 activation may be followed by activational lessening of JNK and P38 in NP".