[Effects of N-butylphthalide on the expressions of calpain 1 and CaMK II in hippocampus in rats with acute severe carbon monoxide poisoning].

Objective: To investigate the effects of N-butylphthalide (NBP) on cognitive function in acute severe carbon monoxide (CO) poisoning rats and its mechanism.

Methods: 120 health Sprague-Dawley (SD) rats were randomly divided into three groups (n = 40): normal control group (NC group), CO poisoning group (CO group) and NBP treatment group (NBP group). The acute severe CO poisoning model was established in a hyperbaric oxygen chamber by intoxicated with 1 000 ×10 CO for 40 minutes, followed with 3 000 ×10 CO for another 20 minutes, and then received hyperbaric oxygen therapy 1.5 hours once a day until sacrificed. Rats in NBP group were administered orally NBP 60 mg/kg for 2 times daily until death. NC group and CO group were treated with equal amount of pure olive oil. Four rats in each group were taken from 1, 3, 7, 14, 30 days after model setup, respectively. The cognitive function score was assessed by Morris water maze test. The changes in ultrastructure of hippocampus were observed under transmission electron microscope. The expressions of calpain 1 and Ca/calmodulin dependent protein kinase II (CaMK II) in hippocampus of brain tissue were detected by immunofluorescence staining, and the localization of the two target proteins in neurons was observed by immunofluorescence double staining.

Results: Compared with NC group, the escape latency at 1 day after poisoning in CO group was significantly prolonged (s: 55.6±3.2 vs. 44.5±3.5, P < 0.05), and the times of the platform crossing was significantly decreased (times: 1.3±0.8 vs. 6.6±1.2, P < 0.05); the ultrastructure of hippocampus was obviously injured; the protein expressions of calpain 1 and CaMK II in brain tissue were significantly increased at 1 day after CO poisoning [calpain 1 (A value): 41.24±5.21 vs. 6.44±1.13, CaMK II (A value): 56.19±5.04 vs. 9.84±1.53, both P < 0.05], and the protein expression of calpain 1 reached the peak at 3 days (A value: 59.34±6.11), the protein expression of CaMK II reached the peak at 1 day (A value: 56.19±5.04). Compared with CO group, the cognitive function was significantly improved in NBP group in the late stage of poisoning [7-30 days, escape latency (s): 40.3±1.9 vs. 49.1±3.1 at 7 days, 30.1±2.9 vs. 39.4±3.1 at 30 days; times of the platform crossing (times): 2.8±1.0 vs. 1.0±0.9 at 14 days, 3.2±0.8 vs. 1.0±0.9 at 30 days, all P < 0.05]; the degree of injury of hippocampal neuron was relatively slight; the protein expression of calpain 1 in brain tissue was significantly decreased from 3 days after CO poisoning (A value: 39.63±3.03 vs. 59.34±6.11, P < 0.05), and the protein expression of CaMK II was significantly decreased from 1 day after CO poisoning (A value: 42.22±3.84 vs. 56.19±5.04, P < 0.05). Immunofluorescence double staining suggested that calpain 1 and CaMK II protein could not only coexist in the same cell, but also could be expressed separately in different cells. Linear regression analysis showed that the expression of calpain 1 and CaMK II was positively correlated (R = 0.852, P = 0.002).

Conclusions: NBP treatment could maintain ultrastructure integrity of hippocampus, balance the expression levels of calpain 1 and CaMK II proteins, and significantly improve cognitive impairment induced by CO poisoning, thus play a protective role against hippocampus damage in rats with acute severe CO poisoning.