This study aimed to determine the expression of TMEM100 in NP and to explore the possible role of TMEM100 in pain relief. We found that the expression of TMEM100 was significantly reduced in the DRG of rats with peripheral NP by creating two different pain models: CCI and TNI. We established an AAV6 vector encoding recombinant fluorescent TMEM100 and transfected it into the DRG proximal to the peripheral nerve injury. We found that the expression of TMEM100 in the DRG of transfected rats was significantly higher than that of model rats alone, and the pain behavior of rats was significantly improved. Moreover, we discovered that reversing the expression of TMEM100 inhibited NP and microglia (Iba-1), astrocytes (GFAP), and inflammatory mediators (IL -1β, TNF-α, and IL-6). Overall, the findings of this study direct that TMEM100 is an important pain-regulating protein that plays an important role in NP and may alleviate pain by reducing inflammatory mediators.
TMEM100 is a two-transmembrane protein that is widely distributed in various tissues. It has been reported that TMEM100 is expressed in blood vessels, notochords, and other tissues and is related to kidney development, angiogenesis, and lung cancer metastasis (10, 35, 36). The expression of TMEM100 has recently been found in the nervous system (14). However, the expression and role of TMEM100 in NP are still unclear.
To further determine the association between TMEM100 and pain, we established two pathological pain models: CCI and TNI. The CCI model is a well-established (30) and the most used pain model in research. Pain is induced by the compression of four thread knots of the SN trunk, and rats may experience paresthesia, mechanical allodynia, and caloric allodynia in the operated limb, similar to the characteristics of human NP (37). The TNI model is an optimized derivative type of spared nerve injury (SNI). It has the typical characteristics of SNI class and some advantages. Lee et al. (31) found that simultaneous transection of the tibial and sural nerves or a single TNI resulted in more severe pain threshold changes. The tibial nerve may play an important role in the pain process (38). Therefore, researchers believed single TNI to be a more stable and efficient model of peripheral NP than classic SNI (39).
We performed behavioral tests on rats with two different pain models and evaluated the expression of TMEM100 in each group. We found that the pain production was accompanied by changes in the TMEM100 expression in the DRG of rats in the two pain models. The expression of TMEM100 was significantly reduced in the two groups, so we hypothesized a close relation of TMEM100 in the generation or regulation of pain. Interestingly, although the expression of TMEM100 was significantly decreased in both CCI and TNI groups compared to the normal group, the decrease in TMEM100 was more pronounced in the CCI model. Studies have demonstrated that (9) the down-regulation of TMEM100 may be related to the proliferation of astrocytes and microglia after nerve injury. By detecting astrocyte-specific marker (GFAP) and microglia-specific marker (Iba-1), we discovered that there were different degrees of elevation in both pain models; in the CCI model, the elevation of GFAP and Iba-1 was more pronounced than that of TNI, which explained the decrease of TMEM100 in NP, and lower expression of TMEM100 in CCI compared to TNI model.
The function of TMEM100 is implicated in many aspects of biology. For example, TMEM100 is involved in the control of developmental proliferation and differentiation (40). It plays a role in cell development and differentiation through pathways such as TGF-BMP in the enteric nervous system. It has essential functions in maintaining vascular integrity as well as in the formation of blood vessels. Meanwhile, TMEM100 acts as a tumor suppressor in various tumor cells to inhibit metastasis and proliferation (41). Pan et al. (24) demonstrated that TMEM100 is crucial for the secretion of inflammatory factors and found that TNF-α had an inhibitory effect on the expression of TMEM100, while decreased TMEM100 expression could significantly reduce the secretion of inflammatory factors such as IL-1β and IL-6. This is consistent with the findings of our study that the expression of TNF-α, IL-1β, and IL-6 in DRG of CCI and TNI rats decreased after overexpression of TMEM100. The release of inflammatory mediators (TNF-α, IL-1β, and IL-6) is closely related to the pathogenesis of NP. These inflammatory mediators contribute to central spinal cord sensitization, thereby enhancing the development of NP (42, 43).
Activation of glial cells and interactions between these cells and neurons may be involved in nociception in the central and peripheral nervous systems (44). Glial cells, including astrocytes and microglia, are involved in the induction and maintenance of NP (45). The vital role astrocytes play upon activation may be related to the production of cytokines after injury (46). It has been suggested that upregulation of GFAP, a marker of astrocyte activation following injury, has a role in the maintenance of NP (47). One study found that up-regulation of GFAP persisted from 3–21 days after nerve injury (48). Our study showed that GFAP levels increased in the groups of CCI and TNI models injected with empty virus (AAV-GFAP group). In contrast, the group injected with a virus carrying TMEM100 (AAV-TMEM100) exhibited attenuated GFAP levels in CCI and TNI models.
Furthermore, the activation of microglia has a key role in the central sensitization of NP (49). The pathological condition of NP results in microglia activation: microglia release many pro-inflammatory cytokines, such as TNF-α, along with glutamate release, excess reactive oxygen, and apoptosis. In this study, we detected different degrees of elevation of Iba-1, a marker of microglia activation, among the rats of AAV-GFP group in the CCI and TNI models. The levels of Iba-1 in the AAV-TMEM100 group were significantly decreased. Yu et al. (9) proved through in vitro experiments that overexpression of TMEM100 in astrocytes and microglia cell lines significantly inhibited their proliferation and found through animal experiments that TMEM100 may play a role in the control of satellite glial cells (SGCs) proliferation. It is believed that glial cell proliferation in animals after nerve injury may be the reason for the down-regulation of TMEM100 expression, which is consistent with our findings.
After we injected AAV6-TMEM100 into sciatic nerve of rats, the expression of TMEM100 in both CCI and TNI rats was significantly increased, and the expression of pain behavior was significantly improved, which also reflected the potential therapeutic mechanism of analgesic effect of TMEM100. Therefore, AAV-mediated DRG-targeted delivery of TMEM100 has the potential to be translated into clinical use for treating patients with NP, although long-term safety requires further study.