Background & Aims

protein p53 plays a pivotal role in the regulation of cell cycle progression, DNA repair, and apoptosis [1,2]. Although its primary function is to suppress abnormal cell growth and prevent cancer, recent research suggests that p53 may also be involved in pain perception and modulation[3-5]. However, the precise mechanisms underlying p53’s role in pain regulation are still being investigated. It is important to note that p53 is a highly versatile protein with complex functions, and its role in pain regulation may vary depending on the context and specific cellular environment. This study aimed to evaluate behavioral changes and the spinal nociceptive processing in response to inflammatory pain induced by formalin, using p53-knock-out (KO) mice.

Methods

Male p53-KO and wildtype (WT) C57BL/6J mice aged 8-12 weeks and weighing 20-30 g were used. The Von Frey test and Hargreaves test were employed to assess basal pain perception. The formalin test was performed according to a previously described method[6]. The mice were lightly anesthetized with isoflurane, and a 30-gauge needle was used to subcutaneously inject 10 ?l of 5% formalin solution into the dorsal surface of the left hind paw. Subsequently, each mouse was placed individually in a mirror-backed Plexiglass chamber for a 60-minute observation of pain behaviors. After 60 minutes, the mice were euthanized with isoflurane, and their spinal cords were collected for further analysis. Immunohistochemistry was conducted to investigate the expression of c-Fos in the spinal dorsal horn. Activation or inhibition of p53 within the spinal cord was achieved by intrathecal injection of the p53 activator nutlin-3a[7] or the inhibitor PFT-µ[8] one hour prior to formalin injection.

Results

The mechanical paw withdrawal thresholds and latency of p53-KO mice did not significantly differ from WT mice at baseline. Subcutaneous injection of formalin into the hind paw induced a biphasic nociceptive response in both WT and p53-KO mice: an immediate and intense response followed by a transient quiescent period (phase 1: 1–10 minutes), and a prolonged tonic response (phase 2: 11–60 minutes). During phase 2, the duration of pain behavior, specifically licking of the formalin-injected paw, was significantly increased in p53-KO mice compared to WT mice. Additionally, the number of c-Fos-positive neurons in laminae I–II was significantly higher in p53-KO mice. Intrathecal administration of nutlin-3a significantly reduced licking time on the formalin-injected paw and the number of c-Fos-positive neurons in laminae I–II compared to vehicle controls. Conversely, PFT-µ significantly increased licking time and the number of c-Fos-positive neurons.

Conclusions

Our findings demonstrate that deletion or inhibition of p53 in mice enhances formalin-induced pain behavior, accompanied by increased activation of neurons in the spinal dorsal horn. Conversely, exogenous activation of p53 reduces formalin-induced pain behavior and results in decreased neuronal activation. Therefore, our data suggest that p53 is involved in acute inflammatory pain and may serve as a potential target for the development of analgesics.
Acknowledgement: This study was supported by the National Natural Science Foundation of China (32060186 to Liu T) and the Innovation Foundation of Jiangxi Province (YC2022-s089 to Zhong L).

References

[1] Levine A J. p53: 800 million years of evolution and 40 years of discovery [J]. Nat Rev Cancer, 2020, 20(8): 471-480.
[2] Hafner A, Bulyk M L, Jambhekar A, et al. The multiple mechanisms that regulate p53 activity and cell fate [J]. Nat Rev Mol Cell Biol, 2019, 20(4): 199-210.
[3] Muralidharan A, Sotocinal S G, Yousefpour N, et al. Long-term male-specific chronic pain via telomere- and p53?mediated spinal cord cellular senescence [J]. J Clin Invest, 2022, 132(8).
[4] Yamashita A, Matsuoka Y, Matsuda M, et al. Dysregulation of p53 and Parkin Induce Mitochondrial Dysfunction and Leads to the Diabetic Neuropathic Pain [J]. Neuroscience, 2019, 416: 9-19.
[5] Gao Y, Sun N, Wang L, et al. Bioinformatics Analysis Identifies p53 as a Candidate Prognostic Biomarker for Neuropathic Pain [J]. Front Genet, 2018, 9: 320.
[6] Tjolsen A, Berge O G, Hunskaar S, et al. The formalin test: an evaluation of the method [J]. Pain, 1992, 51(1): 5-17.
[7] Loughery J, Cox M, Smith L M, et al. Critical role for p53-serine 15 phosphorylation in stimulating transactivation at p53-responsive promoters [J]. Nucleic Acids Res, 2014, 42(12): 7666-80.
[8] Qin Q, Baudry M, Liao G, et al. A novel function for p53: regulation of growth cone motility through interaction with Rho kinase [J]. J Neurosci, 2009, 29(16): 5183-92.

Presenting Author

Ling Zhong

Poster Authors

Ling Zhong

bachelor

the First Affiliated Hospital, Jiangxi Medical College, Nanchang University

Lead Author

E-mail

TAO LIU

The First Affiliated Hospital of Nanchang University

Lead Author

E-mail

Huichao Sun

master

the First Affiliated Hospital, Jiangxi Medical Hospital, Nanchang University

Lead Author

E-mail

Qiyuan Wang

the First Affiliated Hospital, Jiangxi Medical Hospital, Nanchang University

Lead Author

E-mail

Tianfeng Zhong

Lead Author

E-mail

Mengye Zhu

Lead Author

E-mail

Daying Zhang

The First Affiliated Hospital of Nanchang University

Lead Author

E-mail

Topics

  • Mechanisms: Biological-Molecular and Cell Biology