Intrathecal application of shRNA has become a suitable approach for studies on small molecules such as drugs, receptors, or other proteins [25, 27, 28]. Small interference RNA (siRNA)-induced knockdown can be generated by the expression of the vector-mediated siRNA in the genome . In this study, we adopted the system of lentiviral vector-mediated hairpin RNAs as it has a more efficient transfection rate in tissues in vivo compared with the system based on polyethylenimine/DNA complexes. Our results showed that LV-shp300 was successful transfected into the cells in the spinal cord (Figure 1), and it significantly silenced the p300 gene in LV-shp300-treated rats (Figure 3).
CCI induces neuropathy, hyperalgesia and allodynia normally develop on the 3rd day and stabilize on the 7th day after surgery . In the present study, to ensure a steady-state of the pain, we began our treatments from the 7th day following CCI. In the LV-shp300 and C646 treated groups, behavioral testing revealed that both the mechanical allodynia and thermal hyperexcitability gradually recovered to the baseline level, and reached their peaks on the 14th day, indicating that these two p300 inhibition interferences had exerted their effects. Therefore, at the maximum time point of the functional changes, our study was mainly focused on the potential mechanism.
Functioning as an integrator for many signal transduction pathways through interaction with numerous critical transcription factors and other functional proteins, p300 is found to be highly sensitive to promoters and potential enhancers. The binding for p300 analyzed by ChIP, followed by sequencing, is positively correlated with gene expression and acetylation levels, suggesting that p300 is involved in transcriptional initiation . As epigenetic processes such as histone acetylation and RNA interference have profound effects on inflammatory cytokine metabolism, steroid responsiveness, and opioid sensitivity, these processes are likely essential in the development of chronic pain [32, 33]. DNA is wrapped around histone proteins, composing the chromatin; chromatin remodeling occurs through chemical modifications of histone proteins. These modifications decide how tightly or loosely the chromatin is packaged, thus controlling gene accessibility and expression . p300, as a HAT, via acetylating the N-terminal tail lysines of core histones correlates strongly with active transcription of genes [35, 36].
To identify the cellular and epigenetic functions relevant to p300 activities, we looked into the possibility of a molecular interaction between p300 and COX-2. COX-2, an inducible enzyme, strongly expresses in the spinal cord under proinflammatory stimulation. It is crucial in the generation of neuropathic pain  and in the maintenance of pain states . Inflammation and nerve degeneration, seen as direct effects of nerve injury, are critical in the development of neuropathic pain states [9, 38, 39]. COX-2 regulates the synthesis of multiple inflammatory mediators by producing prostaglandins that are potent algetic factors in sensitizing neuronal responses. The resultant prostaglandin production augments neuronal excitability in the spinal cord (central sensitization) and in somatosensory pathways . On the contrary, administering COX-2 inhibitor or inhibiting COX-2 activity decreases neuropathy-induced prostaglandin levels and reduces generated pain hypersensitivity .
The effect of p300 on the expression of COX-2 gene was monitored in the present study. The results revealed that the COX-2 gene expression was regulated at the chromosomal level, and the p300 recruitment to COX-2 promoter region determined the level of COX-2 gene expression in the CCI rats. These results were consistent with the previous studies indicating that p300 is essential for the regulation of COX-2 promoter activity [42–45]. Moreover, with the aid of acetylating the lysine residues in the DNA binding regions, p300 was found to regulate the expression of other pain-associated genes, such as c-Jun, in another report .
We also found that the binding of p300 to COX-2 was enhanced in vehicle treated CCI rats and further augmented by a p300 overexpression. The enhanced p300 binding to COX-2 was correlated with an up-regulation of COX-2 promoter activities. This enhancement in p300 activities was abrogated by p300shRNA. These results indicated p300’s importance in regulating COX-2 transcription in the neuropathic pain model. Our results also showed that the p300-mediated COX-2 transcriptional activation was dependent on its own HAT. The inhibition of the HAT by C646 resulted in an approximate reduction of 52.24% in COX-2 promoter activity. p300 HAT contributes to the accessibility of COX-2 promoter regulatory elements for transactivators to bind. Therefore, it is reasonable to conclude that the overexpression of p300 in CCI rats actually enhances the downstream transcription of pain-associated gene (such as COX-2), and the upregulation of p300 expression in the spinal cord may be a critical process in neuropathic pain induced by CCI. In summary, the importance of epigenetic mechanism for the development of chronic neuropathic pain is clearly evidenced by the changes of p300 expression during the course of neuropathy after CCI and through inhibition of p300 expression. This is additionally highlighted by the effect of p300 on COX-2 expression in this study even though detailed information on p300’s role in chronic neuropathic pain has yet to be revealed.
C646 is capable of specifically inhibiting the HAT domain of p300. It can produce 86% inhibition of p300 in vitro specifically  and can work well in the central nervous system in vivo . p300 regulates the downstream DNA expression, not only via binding to transcription factors, but also through its acetyltransferase activity . Inhibition of the histone acetyltransferase renders nucleosomal DNA less accessible to the transcriptional machinery, thereby usually favoring transcriptional silencing . The blockage of p300 by C646 may lead to the inhibition of histone acetylation on the COX-2 promoter region. Inhibition of p300 with shRNA or C646 can reverse neuropathic pain and reduce COX-2 levels significantly; and their suppressive effects were similar, which is in line with the report by Santer et al. .
While our results show that the inhibition of histone acetylation suppresses the activity of p300 and COX-2, reducing hyperalgesia in this CCI model, there are a few studies showing that histone deacetylase (HDAC) inhibitors reduce pain hyperalgesia as a result of an increased histone acetylation [51, 52]. Most of these studies focus on the relationship between HDAC and metabotropic glutamate2 (mGlu2) receptor or gamma-aminobutyric acid (GABA) synaptic function. Since individual HAT or HDAC binds on the promoter of specific genes, it may play different roles depending on the downstream target gene is pro-hyperalgesia or anti-hyperalgesia. As for p300 in the present study, it was found combined to the promoter of COX-2, one of the well-described painful genes. Moreover, even though GABAA receptors have an important role in inflammatory pain mechanisms in Complete Freund’s Adjuvant (CFA) model , spinal GABAergic associated mechanism is found unnecessary for the development of neuropathic pain in CCI model . These may explain the difference existed in the findings of others studies and ours. As the main focus of this study was to explore the potential effect of p300 and its possible epigenetic role in neuropathic pain, two different approaches were used to accomplish the goal; to down-regulate p300 with specific shRNA, and to chemically inhibit the acetyltransferase activity of p300 using C646. We did not determine the optimal dose to reverse neuropathic pain in CCI rats, nor did we compare the inhibiting efficiency with other p300 inhibitors in an equivalent dose. In addition, behavioral changes were used as the main markers for measuring the pain and treatment effects; we did not examine other signs related to antinociception or neuropathic pain. Therefore, further studies will expand this observation of neuropathic pain and p300 acetyltransferase activity into other target molecules in neuropathic pain, multi-time point and long-term model after CCI.