Effects Of NO With Different Doses On Formalin-induced Nociceptive Behavioral Responses And Bcl-2/Bax Expressions In Spinal Dorsal Cord In Rats

Objective To investigate the effects of multiple application of nitric oxide (NO) precursor L-Arginine (L-Arg) with different doses and nitric oxide synthase (NOS) inhibitor Nω-nitro-L-arginine methylester(L-NAME) on formalin-induced nociceptive behavioral responses and Bcl-2/Bax expressions at spinal level, bcl-2 and bax are two major genes in the regulation of apoptosis, bcl-2 protects apoptosis and bax promotes apoptosis, both the ratio of expression could be used as an indicator of apoptosis. Analyze of the effects of long-term application of NO on inflammatory pain and cell apoptosis, and to explore its possible mechanisms on modulation of pain.Methods 24 healthy SD rats about 120 g were divided into four groups at random. We intrathecally administrated the NO precursor L-Arg 10μg/d (low L-Arg group) or 250μg/d (high L-Arg group) and NOS inhiboter L-NAME 2700μg/d (L-NAME group), and normal saline (control group) once daily for four days. All rats were subcutaneously injected formalin (2%,100μL) into the right hindpaw at the fourth day. Then observe the nociceptive behavioral responses for 1 h. Four hours after injection of formalin, neuronal NOS (nNOS), c-fos, bcl-2 and bax expressions in spinal dorsal horn were detected with immunocytochemistry; Bcl-2 and Bax protein expressions were detected with western-blot.Results (1) In formalin test, the subcutaneous formalin injection evoked biphasic licking or lifting behaviors to the injected paw. Our results showed that there were no differences in acute phase of formalin test in the four groups, whereas the licking and lifting times in tonic phase were significantly decreased in low dose of L-Arg group and increased in high dose of L-Arg group or L-NAME groups as compared with control group. (2) The positive expressions of nNOS or c-Fos in spinal dorsal horn were significantly decreased in low dose of L-Arg group and increased in high dose of L-Arg group as compared with control group, whereas, the expression of nNOS was decreased and c-fos was increased in L-NAME group as compared with control group. The results of the immunocytochemistry showed that the positive expressions of Bcl-2 and Bax were also significantly increased in low L-Arg group than normal saline group; whereas, the positive expressions of Bcl-2 were decreased and Bax were increased in high L-Arg group than that in normal saline group; the positive expression of Bcl-2 and Bax were both increased in L-NAME group. (3) But the ratio of Bcl-2/Bax with immunocytochemistry and the ratio of Bcl-2/Bax with western-blot were all increased in low L-Arg group compared with normal saline group and were all decreased in high L-Arg group or L-NAME group compared with normal saline group.Conclusion (1) These results suggest that multiple administration of NO have double sided effects, low dose of NO produces antinociception but high dose of NO produces pronociception, lack of NO also produces pronociception. (2) Low concentration of NO can protect but high concentration of NO can promote the apoptosis of neurons and lacks of NO also promote the apoptosis of neurons. (3) Therefore, we could suggest that low dose of NO can protect apoptosis from nociceptive stimulus then produce antinociception, whereas, high dose of NO can promote apoptosis and then produce pronociception, lack of NO can also promote apoptosis and produces pronociception. Specific evidence will go to explore in the future.