The latest statistics from the International Diabetes Federation in 2019 shows that the number of diabetic patients worldwide has increased year after year, with an average growth rate of 51%. The number of patients is expected to reach 700 million by 2045 . Diabetes can bring about various injuries in the body thanks to its complications, which include DNP caused by peripheral neuropathy. The symptoms of DNP are obvious, including hyperalgesia, touch-induced pain, spontaneous pain and hyperalgesia. Most of them can develop into chronic pain, lasting for several years . Thus, it is essential to study the pathogenesis and pretreatment of DNP. In this study, the blood glucose concentration of the model group was increased and accompanied by hyperalgesia, suggesting that the DNP model was successfully established. Compared with DNP group, P2Y14 shRNA and UC.25+ shRNA treatment can increase the mechanical hyperalgesia and thermal hyperalgesia thresholds of rats, indicating that P2Y14 shRNA and UC.25+ shRNA can alleviate the pain behavior of DNP rats, but the specific mechanism needs further study.
Adenosine triphosphate (ATP) is a kind of important excitatory neurotransmitters, which plays a key role in the cellular metabolic pathways, and also acts as significant signal molecules in pain signal transduction from the periphery to the central nervous system . Purinergic receptors participate in the regulation of many physiological and pathological activities, and play a significant role in inflammatory pain and neuropathic pain. When peripheral injury or injurious stimulation occurs, ATPs as well as those metabolites are released to extracellular environment and bind to purinergic receptors located on the cell membrane to produce biological effects. P2Y14 receptor is one of the subtypes of P2Y receptors, which can perform specific physiological functions by modulating different signal transduction pathways in cells . Studies have confirmed that in neuropathic pain models, the expression of P2Y14 receptors was obviously increased, and the application of P2Y14 antisense locked nucleotides can relieve the pain . The upregulation of P2Y12 receptors in satellite glial cells of dorsal root ganglion can be increase mechanical or thermal hyperalgesia in diabetic rats, thereby mediating the DNP process . The activation of P2Y13 receptors in spinal microglia can promote the transmission of pain in diabetic rats, and the application of P2Y13 receptor specific antagonist MRS2211 can significantly relieve pain. P2Y12, P2Y13, and P2Y14 receptors belong to Gi/o-coupled receptors with strong sequence homologies, and it has been proved that P2Y12 and P2Y13 receptors exist in spinal microglia . Therefore, we speculated that the P2Y14 receptor in spinal cord may participate in the development of DNP. The data showed that the expression of P2Y14 receptor protein and mRNA in the DNP group was remarkably increased than that in the Control group. P2Y14 shRNA treatment could reduce the expression of P2Y14 receptor. Combined with the results of rat pain behavior, it indicated that the abnormal pain in rats was related to the up-regulation of P2Y14 receptor. Inhibiting the expression of P2Y14 receptor could alleviate the pain behavior of rats, implying that P2Y14 receptor may participate in the DNP process. At the same time, UC.25+ shRNA treatment could reduce the expression level of P2Y14 receptor and alleviate the pain behavior of DNP rats, indicating that UC.25+ may affect the transmission of pain in rats through P2Y14 receptor.
There are astrocytes, oligodendrocytes and microglia in the central nervous system. Recent research have found that microglia play a significant role in pain relief, and the maintenance of pain signal transduction pathways depends on the activation of microglia . Microglia are extremely sensitive to the change in the surrounding environment and can respond quickly. When the nerves are injured or noxiously stimulated, microglia are rapidly activated, releasing a large number of cytokines to act on neurons and alter the synaptic effects . Furthermore, it is found that inhibiting the activation of microglia can effectively relieve the pain . It has been confirmed that injury can activate a variety of purinergic receptors on microglia, cause a large amount of Ca2 + influx, promote microglia proliferation and accelerate pain transmission . OX42 (CD11b monoclonal antibody) is a marker for identifying the activation of microglia . To verify the activation of microglia during DNP, the expression of OX42 was observe by immunofluorescence. These data showed that the expression of OX42 was increased obviously during DNP, indicating that microglia were activated in diabetic rats after nerve injury. At the same time, it was observed that OX42 and P2Y14 receptors were co-expressed in microglia, indicating that P2Y14 receptors can be expressed in microglia. The co-expression of OX42 and P2Y14 in the DNP group was obviously higher than that in the Control group. P2Y14 shRNA treatment can reduce the co-expression intensity. The results of binding protein showed that the activation of microglia is accompanied by the up-regulation of P2Y14 receptors. The inhibition of P2Y14 receptors diminishes the activation of microglia and reduces the transmission of pain. UC.25+ shRNA treatment can also reduce the co-expression of OX42 and P2Y14 receptor, suggesting that UC.25+ may decrease the activation of microglia by inhibiting the expression of P2Y14 receptor, thereby alleviating DNP.
Inflammation is a defensive response, and any factor that can cause tissue damage may lead to chronic inflammation, which features excessive secretion of pro-inflammatory cytokines . Diabetes can abnormally produce inflammatory cytokines and activate inflammatory signal pathways, which is a low-grade systemic inflammatory state. When peripheral nerves are injured, the secretion of pro-inflammatory cytokines increases. The up-regulated cytokines will further enhance inflammatory signals and promote the development of pathological pain. The activation of microglia can also promote the secretion of inflammatory factors. This study found that the expression levels of inflammatory factors in the DNP group were obviously higher than those in the Control group, indicating that DNP rats released more inflammatory factors and produced a stronger inflammatory response; Compared with the DNP group, P2Y14 shRNA treatment can down-regulate the expression of inflammatory factors, indicating that the P2Y14 receptor is related to the release of inflammatory factors. UC.25+ shRNA treatment can reduce the secretion of inflammatory factors, suggesting that UC.25+ may reduce the inflammatory response by inhibiting the P2Y14 receptor.
Mitogen-activated protein kinases (MAPKs) can transfer extracellular stimuli into intracellular signalling, and participate in cell signal transduction. Nerve injury or noxious stimulation not only activates microglia, but also activates multiple signal transduction pathways in the cell, such as p38 MAPK . Studies have shown that during inflammatory pain, the activation of p38 MAPK can promote the synthesis and release of inflammatory factors, and p38 MAPK inhibitors can relieve pain; activation of the P2Y12 receptor in spinal microglia can over-release inflammatory factors and activate the p38 MAPK pathway, and participate in maintenance of various types of pain . Our data indicate that the phosphorylation of p38 MAPK was obviously raised during DNP, and P2Y14 shRNA treatment could inhibit its phosphorylation, indicating that the upregulation of P2Y14 receptors could activate signaling pathways, and down-regulation of the expression of P2Y14 receptors could relieve the phosphorylation of p38 MAPK. UC.25+ shRNA treatment can also reduce the phosphorylation of p38 MAPK, indicating that UC.25+ may inhibit the p38 MAPK signaling pathway mediated by the P2Y14 receptor to relieve pain in rats.
Recent studies have verified that lncRNAs can participate in the development of diseases . There are many abnormally expressed lncRNAs in damaged neurons and spinal dorsal horn after peripheral nerve injury. The dysregulation of lncRNAs may be associated with neuropathic pain . Our results indicate that the expression of UC.25+ in DNP rats was obviously higher than that in the Control group, and treatment with UC.25+ shRNA could reduce the expression of UC.25+ and P2Y14 receptors, the secretion of inflammatory factors and the phosphorylation of p38 MAPK, resulting in the alleviation of the pain behavior of the rates. These data suggest that UC.25+ shRNA may inhibit the expression of P2Y14 receptor to alleviate the DNP process, and UC.25+ can participate in DNP mediated by P2Y14 receptor by regulating the expression of P2Y14 receptor.
In order to go a step further investigate the potential mechanism of UC.25+ regulating the P2Y14 receptor, the screening results through multiple databases showed that the P2Y14 promoter region contained the action element of STAT1, suggesting that the transcription factor STAT1 may participate in the regulation of P2Y14 receptor expression. The signal transducer and activator of transcription (STAT) family has seven subtypes, which involves STAT1. Before activation of cells, STAT is located in the cytoplasm and is in an inactive monomer state . When the cells are stimulated, STAT is quickly activated to form a dimer and enter the nucleus. The activated STAT dimer can recognize the promoter of the target gene, not only induce but increase the transcription efficiency, and participate in the regulation of gene expression. Recent research has shown that high sugar and inflammation can promote the development of diabetic nephropathy through STAT signals ; STAT activation is related to the process and sustain of neuropathic pain injury models . In this experiment, the RIP results showed that UC.25+ can enrich STAT1, indicating that there is an interaction between UC.25+ and STAT1. The overexpression or low-expression of UC.25+ in HUVEC cells respectively increased or decreased the expression of STAT1, suggesting that UC.25+ could positively regulate the expression of STAT1. In addition, ChIP results showed that STAT1 could specifically bind to the acting element of P2Y14 gene promoter. Similarly, overexpression or low-expression STAT1 in HUVEC cells enhanced or reduced the expression of P2Y14, implying that STAT1 could positively regulate the expression of P2Y14 receptor. In summary, UC.25+ can enrich STAT1 and positively regulate the expression of STAT1. As a transcription factor of the P2Y14 receptor, STAT1 positively regulates the expression of the P2Y14 receptor, thereby promoting the occurrence and development of P2Y14 receptor-mediated DNP.