In the current study, we identified 661 variants in 507 genes associated with CPSP. These results were obtained after bioinformatics analysis and require further functional validation. These genes are associated with numerous functions; however, many are related to neuroinflammation, cognitive function, nervous system development, and key metabolic enzymatic activities.
Identified Genes Of Interest
Caspase 5 gene (CASP5), which encodes for a proinflammatory caspase related to inflammasome formation, was significantly associated with CPSP. Recently, Islam et al. found that blood CASP5 levels were markedly increased in patients with neuropathic pain, suggesting that inflammation is involved in the occurrence and development of neuropathic pain (Islam et al., 2021), which is consistent with the results of our study. The number of CASP5 variants in the patients with severe CPSP was significantly higher than that in the control group (Supplementary file S1). According to other studies, the upregulation of CASP5 can promote the progress of OA and increase the sensory input to the central nervous system, thus aggravating pain (An, Hu, Li, & Hu, 2020). Thus, it can be reasonably inferred that the CASP5 variants in patients with OA may lead to increased CASP5 expression levels after arthroplasty, which subsequently increases the risk of severe CPSP. CASP1 of the caspase family was also significantly related to CPSP in our study. Some studies have shown that NLRP3-CASP1 mediates the promotion of microglia in neuroinflammation. However, whether it regulates the occurrence or development of pain remains to be further verified (Wang et al., 2017).
Acid-sensitive ion channel 1 gene (ASIC1), which is widely expressed in the nervous system, was also significantly associated with CPSP. ASIC1 is a proton-gated cation channel that is activated by extracellular acidification. It has been reported that MiR-485-5p is involved in the development of OA and that reducing the expression of MiR-485-5p can cause inflammatory pain by upregulating ASIC1expression. Therefore, MiR-485-5p/ASIC1 may be a potential target for the treatment of inflammatory pain in patients with OA (Tsai et al., 2020; M. Xu et al., 2020). Furthermore, ASIC1 is also expressed in microglia. In inflammation, ASIC1 promotes microglia migration and releases inflammatory cytokines, such as NOS and COX-2; this indicates that ASIC1 is involved in neuroinflammatory response (Yu et al., 2015). In our study, the ASIC1 variant was observed in the control group. It can be speculated that this variant interferes with the correct transformation of ASIC1 into a protein with normal physiological function, thus reducing the risk of CPSP in the control group. The above hypothesis can be evaluated through further functional verification. These findings suggest that blocking ASIC1 may be a powerful method for the prevention and treatment of chronic pain after lower extremity arthroplasty.
Another significantly associated gene, A disintegrin and metalloproteinase 12 (ADAM12), is expressed in T cells in the brain. As a costimulatory molecule for T-helper 1 cell activation, ADAM12 mediates tissue inflammation and may play a role in the formation and development of pain. The polymorphism of ADAM12 has also been significantly associated with knee OA (Liu et al., 2021; Lv et al., 2017). Many of the genes significantly correlated with CPSP in this study are associated with the pathogenesis of arthritis, including BCAP29, IDO2, IGFBP1, TLR10, and TNC. How these genes regulate and affect chronic pain after arthroplasty requires further exploration (Evangelou et al., 2011; Hasegawa, Yoshida, & Sudo, 2020; J. Li et al., 2018; Merlo & Mandik-Nayak, 2016; Zhang et al., 2018).
ALOXE3 encodes epidermal lipoxygenase-3 (eLOX3) and is expressed in the spinal cord. A study demonstrated that peripheral inflammation significantly increases the level of eLOX3 in the spinal cord and the metabolites of eLOX3. These metabolites promote inflammatory pain via inflammatory cytokines (Gregus et al., 2013).
PLCE1 encodes phospholipase C ϵ-1, which can stimulate the expression of many inflammatory cytokines, including TNF-α, IL-4, and IFN-γ. It can also activate the NF-κB signaling pathway to promote inflammation and may play an important role in inflammatory pain (W. Li et al., 2019). The above genes are closely related to inflammation, indicating that the formation of CPSP may be attributed to uncontrolled local inflammation. Therefore, adequate perioperative anti-inflammatory and analgesic therapy may help reduce the risk of CPSP.
CPSP is usually multifactorial and is associated with not only postoperative inflammation but also neuropathic factors, such as peripheral nerve injury, and pain regulation disorder in the central nervous system during operation (Sahin, Beyaz, Karakus, & Inanmaz, 2021). AKAP12/SSeCKS is a substrate of protein kinase C, which is upregulated when neurons are injured. It is involved in the regulation of astrocyte activation through the SSeCKS–ERK pathway and aggravating neuropathic pain (X. H. Li et al., 2015). MZF1 encodes for a myeloid zinc-finger transcription factor that regulates the transcription process of related proteins. Many studies have shown that MZF1 plays an important role in neuropathic pain. MZF1 can regulate the expression of voltage-gated K+ channel and TRPV1 genes, thus enhancing the excitability of dorsal root ganglion (DRG) neurons, thereby strengthening the transmission of pain signals and promoting the development of neuropathic pain (F. Li & Wang, 2021; Xing et al., 2019; X. Zhao et al., 2013). ZNF382 encodes for zinc-finger protein 382, which is a transcription factor highly expressed in the nervous system. ZNF382 is persistently downregulated in injured DRG neurons, losing its binding to the silencer upstream of the Cxcl13 promoter, which promotes the transcription of Cxcl13. This eventually contributes to the development and maintenance of neuropathic pain (Ma et al., 2021). These genes may promote neuropathic pain by regulating the function of key cells or the expression level of key molecules in the nervous system. The susceptibility genes identified in this paper have been previously associated with pain. The mechanisms through which these genes regulate pain indicate that the underlying mechanism of chronic pain after arthroplasty is complex and multifactorial.
Combined with the above-related factors leading to CPSP, TET1 from the current study is of particular interest. The TET enzyme encoded by TET1 is essential for brain function. TET1 participates in the regulation of DNA demethylation, gene expression, synaptic transmission, and memory formation. Its mutation is related to human cognitive dysfunction. After injury, TET1 is upregulated so that DNA demethylation at the CpG site of BDNF and mGluR5 promoters is mediated by the TET1 increase, which increases the transcription of BDNF and mGluR5 and promotes the development of abnormal neuropathic pain. TET1 also regulates the function of astrocytes through Ca2+ signaling, thus affecting neuronal development and cognitive function (Greer et al., 2021; Hsieh et al., 2017; Hsieh et al., 2016; W. Xu et al., 2021). Moreover, TET enzyme participates in the development and repair of the nervous system as well as the occurrence and development of neuropathic pain; this may be a powerful therapeutic target for chronic pain.
Although many of our findings are not specifically associated with pain, these genes are involved in the development of the nervous system or the repair process after injury, which is likely to play a role in neuropathic pain. For example, DCC, which encodes the DCC receptor, is highly expressed in dopamine cells and interacts with Netrin-1, which plays an important role in regulating synaptic development. Its polymorphism is closely related to the susceptibility to emotional disorders, psychosis, and addiction (Vosberg, Leyton, & Flores, 2020). Similar to our results, polymorphism of DCC showed a significant correlation with chronic pain in another study, further supporting the correlation between nervous system development and pain (Johnston et al., 2019). Similarly, the nebulin family member LASP1 plays an important role in formation and maintenance of synaptic in the hippocampus in rats (Myers, Yu, Kremerskothen, Butt, & Zheng, 2020). Genetic variants and the expression level of LASP1 have also been associated with many neurological diseases, such as schizophrenia, autism, and bipolar disorder (Giusti et al., 2014). In patients with chronic pain, the expression of these genes is upregulated, indicating a potential association between mood and pain. Additionally, AP1S1 encodes an adaptor protein complex that is related to synaptophysin and the vesicular acetylcholine transporter, which is very important for spinal cord development; Slitrk2 mediates excitatory synapse formation and transmission; and GPR50 encodes the G protein coupled receptor 50 that is associated with synaptic plasticity (Han et al., 2019; Q. Li et al., 2020; Montpetit et al., 2008). CLIP3 regulates astrocyte proliferation and myelination and participates in regeneration after nerve injury (X. Chen, Chen, Hao, Zhang, & Zhang, 2018; Deng et al., 2012). In patients with CPSP, variants in these susceptibility genes may indicate that the patients are more likely to develop an intraoperative nerve injury. These patients may have difficulty repairing the injured neurons or remodeling synapses after injury because of related gene mutations, resulting in severe chronic pain.
Polymorphisms not only affect patients’ sensitivity to pain, but also the large variation in individual efficacy of analgesic drugs. Some genes may encode for the key metabolic enzyme for these drugs. Variants in these genes alter enzyme activity, preventing drug metabolism or transformation into active forms in the body, leading to great differences in the demand for analgesic drugs in different patients. For example, AMACR, encoding α-methylacyl-CoA racemase, catalyzes key steps in ibuprofen metabolism (Lloyd et al., 2013), while AOX1 encodes xanthine dehydrogenase, which metabolizes aza- and oxo-heterocycles representing the scaffold of many drugs like morphine and fentanyl (Garattini & Terao, 2012) and CYP1A1 encodes cytochrome oxidase and participates in steroid catabolism (Ugartondo et al., 2021). In addition to exogenous drugs, some active molecules in the human body also have analgesic effects, such as endogenous palmitoylethanolamide (PEA), which reduces pain by activating PPAR-α. The NAAA encodes for the hydrolase of PEA, and variants in NAAA may affect the level of PEA, thus affecting the activation of corresponding receptors, causing the biological transformation from acute pain to chronic pain (Fotio et al., 2021).
Correlation Between Enrichment Analysis Results And Pain
As mentioned in the results, the enrichment analysis found many pathways, molecular functions, and biological processes associated with CPSP. The most significant findings are discussed in this study. Cell adhesion molecules mediate the migration of leukocytes to the injured tissues and the release of opioids locally (mainly opioids β-endorphins), which produce an analgesic effect, and block adhesion molecules (Machelska et al., 2004). Other studies have shown that the serum level of soluble intercellular adhesion molecule-1 (sICAM-1) is significantly correlated with the pain intensity of patients, suggesting that sICAM-1 can be used as a biomarker of pain intensity (Luchting et al., 2017). The cGMP signaling pathway plays an important role in the processing of pain by sensory neurons and dorsal horn neurons (Schmidt, Bottcher, Gross, & Schmidtko, 2021). Phosphatidylinositol bisphosphate (PIP2) is located at the key convergence point of multiple receptors, ion channels, and signal pathways that promote chronic pain. Downregulating PIP2 in neurons can weaken receptor signals, which is a potential new method for the treatment of pain (Loo, Wright, & Zylka, 2015). In OA, the degradation of chondrocytes affects the synthesis and secretion of ECM and the degradation of ECM further damages the chondrocytes. Chondrocytes release various proinflammatory cytokines that stimulate inflammation, such as IL-1, IL-6, IL-17, TNF-α, and PGE2, which not only induce pain but also stimulate chondrocytes to secrete protease, thus hydrolyzing ECM and aggravating OA symptoms (Lee et al., 2013). The stimulation and destruction of chondrocytes also occurs in the process of arthroplasty. Surgery intensifies inflammation of the operated joint, resulting in severe acute pain. Intervention of the ECM–receptor interaction may become a therapeutic target to reduce acute postsurgical pain and reduce the transformation of acute pain to CPSP. PI3K and its downstream Akt are widely expressed in the spinal cord, especially in the lamina I–IV of the dorsal horn, where the primary afferent nerve fibers mostly terminate. At present, many studies have confirmed that the PI3K/Akt pathway plays a key role in the development and maintenance of chronic pain (S. P. Chen et al., 2017). Our study suggested that the PI3K/Akt pathway also plays a role in chronic pain after arthroplasty and may become a powerful therapeutic target.