Panx-1 is a homomeric membrane semi-channel Located in the plasma membrane and endoplasmic reticulum[15], mediating a variety of physiological functions such as intracellular ATP release, intracellular Ca2+ transfer, blood flow regulation and cellular immune response. ATP-mediated post-SCI inflammatory response is one of the important mechanisms of secondary injury. Studies have shown that after a few minutes of hypoxia in rats, the level of ATP in nerve cells significantly decreased, accompanied by the outflow of ATP into the intercellular space[16]. The low concentration of ATP can act as a chemokine of microglia [17], guiding microglia to the damaged site, and the expression of ATP-specific P2 × 7 receptor on microglia is up-regulated [18]. These results suggested that ATP activation of P2 × 7 receptor caused the opening of P2 × 7 plasma membrane pores, released a large number of inflammatory mediators, induced inflammatory response after SCI [19], and leads to secondary damage. At the same time, ATP can also activate MAPK or other signaling pathways through P2 × 7 receptor [20], mediating cell inflammation and apoptosis, which is the important mechanism of ischemia-reperfusion injury. Ca2+ is closely related to neuronal cell apoptosis. The increased concentration of Ca2+ in neurons can trigger arachidonic acid metabolism cascade and activate the xanthine oxidation system through oxidative stress [21], causing the formation of a large number of free radicals, forming a vicious cycle, and finally activating the Ca2+ dependent endonuclease, initiating the neuronal apoptosis process, leading to neuronal apoptosis [22].
In this study, we used modified Allen’s method and NYU impact-III hammer to establish the rat SCI model, and applied the BBB score of hind limb motor function to observe the degree of nerve injury and recovery of rats after SCI. The results showed that although the BBB score of SCI group gradually recovered to different degrees with the extension of injury time, it was still significantly lower than that of Sham group and NC group. In the process of BBB score gradually rising after SCI, BBB score significantly decreased at day 2 after injury, even lower than the level of day1 after injury. It was not until day 3-day 5 after the injury that the function of the hind limb of the rats began to recover significantly. This indicated that the rats after SCI can repair the damage spontaneously under the action of the central pattern generator (CPG) neural network [23]. However, BBB score of day 2 declined again after SCI, which should be considered as the peak of spinal cord edema caused by SCI secondary injury. Huang T [24] has observed that the BBB score of motor function basically fluctuated in a stable state and was much lower than the normal value in 14–28 days after SCI, which further explained the severity and irreversibility of SCI on nerve cell injury. This phenomenon was closely related to the excessive proliferation of glial cells with scar formation, the excessive secretion of neurosuppressive factors, and the obvious inhibition of axonal regeneration.
After SCI, the expression of Panx-1 in rats significantly increased with the time extension compared with that in NC group and Sham group, reached a peak at day 2, and then decreased slowly. Moreover, the changes of Panx-1 in gene level and protein level were basically the same on day 1, day 2, day 3 and day 5 after SCI. This proved that the expression of Panx-1 in rat spinal cord had a certain regularity and adjustability after SCI, which makes it possible to regulate the expression of Panx-1 by genetic engineering technology.
After SCI, the expression of Panx-1 reached a peak at day 2, which was consistent with the significant decline of BBB score at the same time. In order to further study the correlation between Panx-1 protein expression changes and BBB score of hind limb motor function, we carried out correlation analysis. And the results showed that Panx-1 protein expression changed with time at day 1, day 2, day 3 and day 5, which had significant negative correlation with BBB score. This was consistent with the fact that secondary injuries of SCI such as ischemia, hypoxia, cell apoptosis, immune inflammatory response, excitatory toxicity, free radical damage and lipid peroxidation reached the peak of spinal cord edema 2–3 days after the injury, so the BBB score of day 2 after SCI declined significantly. The characterization of Panx-1 protein expression was negatively correlated with BBB score, which proved that Panx-1 has an important effect on the recovery of nerve function after SCI and Panx-1 might be one of the important mechanisms of secondary injury after SCI.
In conclusion, Panx-l proteins are distributed in the nervous system where they formed homomeric semi-channels, regulating the balance of Ca2+ and ATP in and out of the cell, and mediating cell apoptosis and inflammatory response in the activated state. The stimulation of SCI significantly up-regulated Panx-l expression in rats, and reached the peak at day 2 after SCI, while the motor function of hind limbs of rats significantly deteriorated, showing a significant negative correlation. Therefore, Panx-l might be one of the important mechanisms of the secondary injury after SCI. it is of great significance to further study how to block Panx-l semi-channel mediated cell apoptosis and inflammatory response for the treatment of the secondary injury of SCI, and it is a possible therapeutic target to improve the neurological function after SCI.