Abstract: BACKGROUND: The “inflammatory storm” induced by microglia is an important pathological factor causing the death of neuronal cells after spinal cord injury, which is extremely unfavorable for the recovery after spinal cord injury. Dihydroquercetin belongs to the vitamin P group and has excellent biological activity. It has good anti-inflammatory, anti-lipid peroxidation and neuroprotective effects on spinal cord injury. The mechanism by which it promotes the repair of spinal cord injury needs to be further clarified.
OBJECTIVE: To investigate whether dihydroquercetin can ameliorate the inflammatory state induced after spinal cord injury through its anti-inflammatory and antioxidant properties for the purpose of protection against spinal cord injury.
METHODS: Forty-eight Sprague-Dawley rats were divided into sham operation group, spinal cord injury group, low-dose dihydroquercetin group, and high-dose dihydroquercetin group according to the random number table method. The sham operation group only underwent laminectomy and was given intraperitoneal injection of normal saline after the operation. Animal models of spinal cord injury were made in the latter three groups. After the operation, normal saline, 30 mg/kg, and 50 mg/kg dihydroquercetin were given by intraperitoneal injection respectively, and samples were collected 4 weeks after the operation. The Basso-Beattie-Bresnahan (BBB) limb motor function score and mechanical paw withdrawal threshold were used to evaluate the limb motor function and pain recovery of rats. Hematoxylin-eosin staining was used to observe the pathological changes of spinal cord tissue; Nissl staining was used to observe the number of Nissl bodies and the morphology of neurons. The activity of superoxide dismutase in spinal cord tissue was detected. Immunofluorescence was used to detect the expression of reactive oxygen species and microglia Iba1. Western blot was used to detect the protein expression of Iba1 and interleukin-1β, interleukin-6, interleukin-10, tumor necrosis factor-α, and p38 mitogen-activated protein kinase in the spinal cord.
RESULTS AND CONCLUSION: (1) In the behavioral test, the BBB limb motor function score showed that since 14 days after the operation, the BBB limb motor function score of the high-dose dihydroquercetin group was significantly better than that of the spinal cord injury group (P < 0.01); the mechanical paw withdrawal threshold was significantly lower in the high-dose dihydroquercetin than the spinal cord injury group at 21 days after the operation (P < 0.05), and the former group showed better motor function. (2) Hematoxylin-eosin staining and Nissl staining showed that compared with the spinal cord injury group, the high-dose dihydroquercetin group showed slightly swollen but structurally intact and clearer neuron morphology in the spinal cord tissue, and the number of Nissl bodies increased. (3) Superoxide dismutase detection: Compared with the spinal cord injury group, the activity of superoxide dismutase in the spinal cord tissue of the high-dose dihydroquercetin increased significantly (P < 0.05). (4) Reactive oxygen species immunofluorescence detection: Compared with the spinal cord injury group, the high-dose dihydroquercetin could significantly reduce the accumulation of reactive oxygen species after spinal cord injury (P < 0.05). (5) Detection of microglial marker Iba1: Compared with the spinal cord injury group, the high-dose dihydroquercetin could significantly reduce the expression of microglial marker Iba1 after spinal cord injury (P < 0.01). (6) Western blot detection results showed that compared with the spinal cord injury group, the expressions of inflammatory factors (interleukin-1β, interleukin-6, and tumor necrosis factor-α) and phosphorylated p38 mitogen-activated protein kinase in the high-dose dihydroquercetin group decreased significantly (P < 0.01), while the expression of anti-inflammatory factor interleukin-10 increased significantly (P < 0.01). These findings indicate that dihydroquercetin can effectively reverse the expression changes of multiple inflammatory markers induced after spinal cord injury in rats, thereby exerting neuroprotective effects and improving motor function.

Key words: spinal cord injury, inflammatory factors, microglia, Nissl body, dihydroquercetin, neurons, reactive oxygen species, anti-inflammatory factors