P300/CBP-based Histone Acetyltransferase Effects On Neuropathic Pain

Objectives:To investigate the variation regularity of histone acetyltransferase p300,CBP,acetylation level of histone H3 and pain relevant transcription factor c-Jun in a rat modal of chronic constriction injury (CCI). To detect the analgesic effects of intrathecal curcumin, a specific inhibitor of histone acetyltransferase (HAT) p300/CBP and use intrathecal p300 siRNA as a validation in order to search for the epigenetic modulation mechanism of histone acetyltransferase p300/CBP in neuropathic pain.Methods:1. Male SD rats were divided randomly into 2 groups. In group CCI, we established a rat modal of chronic constriction injury on the left sciatic nerve according to Bennett and Xie. In group sham rats only underwent surgery of exposing the left sciatic nerve without ligation. After detection of paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) after surgery, rats were sacrified on postoperative day 3,7,14,21 for immunohistochemisty,reverse transciptase polymerase chain reaction (RT-PCR),western-blot of p300,CBP and western-blot detection of acetylated histone H3 (Ac-H3),total histone H3,c-Jun.2. Male SD rats, fitted with intrathecal (i.t.) catheters, were divided randomly into 6 groups:group sham+NS,group sham+curcumin 500μg,group CCI+NS,group CCI+DMSO,group CCI+curcumin 100μg,group CCI+curcumin 500μg. High purity ((?)98.5%) curcumin (dissolved in 50% DMSO) or 50% DMSO 10μl was intrathecally injected beginning from day 3 to day 6 after surgery. On postoperative day 7,14,21, rats were sacrified after pain threshold measurement for morphology molecular biology detection. The lumbar spinal cord was stained with hematoxylin and eosin (HE) to check for toxic action.3. Male SD rats, fitted with intrathecal (i.t.) catheters, were divided randomly into 6 groups:group sham +NS,group CCI+NS,group CCI+vehie,group CCI+negative control siRN,group CCI+p300 siRNA,group CCI+positive control siRNA.2'Ome modified siRNAs were delivered 4μg/d (2μg, bid) intrathecally by a PEI (polyethyleneimine) system beginning from day 3 to day 6. The same scheme (10μl, bid) of intrathecal transfection agent was used for control. After detecting the pain threshold, rats were sacrified on day 7,14 for indexes detection.Results:1. Compared to group sham, the pain threshold in group CCI significantly decreased at all postoperative timepoints, beginning on day 1, reaching a nadir on day 7 and still existing on day 21. Immunonohistochemistry displayed that both p300 and CBP positive cells expressed mainly in the grey matter, abundantly in all layers, especially densely inⅠ-Ⅲlayers of the grey matter. The positive staining was mainly seen in cell nucleus, a small amounts in cytoplasm. In group CCI, p300 and CBP immuno-positive cells in superficial laminae obviously increased from day 3 after surgery, the most on day 7 and lasted to day 14. The mRNA expression of p300,CBP significantly increased and reached the apogee on operative day 3, lasted to day 14 and recovered to a level only slightly higher than group sham on day 21. The protein expression of p300,CBP,Ac-H3 and c-Jun also showed a similar change but with a peak on day 7.2. Intrathecal curcumin 100μg (0.33-0.4mg/kg),500μg (1.7-2mg/kg) can both obviously improve the pain-sensitive phenomenon in CCI rats from postoperative day 5(2 days after injection) and reached a greatest improvement on day 7 (4 days after injection) dose dependently. The analgesic effect of curcumin 100μg can last to day 11 (4 days after withdrawal) while curcumin 500μg can last to day 14 (7 days after withdrawal). Neither of the two curcumin groups had analgesic effect on day 21. Curcumin can dose dependently inhibit the mRNA,protein expression of p300,CBP and protein expression of Ac-H3,c-Jun on day 7. When on day 14, only curcumin 500μg can lower the p300 mRNA but not CBP, and only curcumin 500μg had inhibited all the proteins. Curcumin's inhition effect disappeared on postoperative day 21. In group sham, curcumin didn't present anly effect. HE staining showed that intrathecal curcumin didn't produce any overt toxic lesion to the spinal cord.3. There was an improvement of nociception in group p300 siRNA beginning on day 5(2 days after injection), a maximum relief on day 7 (4 days after injection) and a persistence to day 9 (2 days after withdrawal), but still couldn't recover to the normal level. mRNA and protein expression of p300 were obviously down regulated on day 7 but couldn't be lowered on day 14. The protein expressions of both Ac-H3 and c-Jun were significantly inhibited on day 7. Positive control siRNA affirmed the validity of this essay system. Meanwhile, negative control siRNA had no impaction on CBP, the homology of p300. There were no observed adverse reaction in all the rats.Conclusions:1. The expression of p300,CBP,Ac-H3 and c-Jun increased in the spinal cord of CCI rats of neuropathic pain and parallelled with the change of nociceptive behaviors.2. Intrathecal curcumin can dose dependently inhibit the development of neuropathic pain in CCI rats and depress the expression of p300,CBP,Ac-H3 and c-Jun in spinal cord. The analgesic effect of curcumin was implemented partly via inhibiting the histone acetyltransferase p300/CBP. 3. Intrathecal p300 siRNA can attenuate the neuropathic pain in CCI rats and lower the expression of p300,Ac-H3,c-Jun in spinal cord, further validating the effect of histone acetyltransferase p300 in neuropathic pain.4. p300/CBP could participate in the modulation of neuropathic pain by the role of histone acetyltransferase.