Fujisawa Hiroki, Numazawa Takumi, Kawamura Minoru, Naiki Mitsuru
Department of Pharmacological Research, Institute of Bio-Active Science, Nippon Zoki Pharmaceutical Co., Ltd, 442-1, Kato-shi, Hyogo, Japan.
Life Sci. 2017 Feb 15;171:39-44. doi: 10.1016/j.lfs.2016.12.018. Epub 2016 Dec 28.
Neurotropin® (NTP), an analgesic for chronic pain, has antihyperalgesic effects in specific alternation of rhythm in temperature (SART)-stressed rats. Previous studies have shown that SART stress induces hyperalgesia, as well as post-translational modification of proteins (including substrates for calpain, a calcium-dependent cysteine protease) in the mesencephalon of rats. To better understand the mechanism of action of NTP, we investigated whether SART stress activates calpain in the mesencephalon of rats and whether NTP inhibits this activation.
Wistar rats were exposed to SART stress for 5days. NTP (200NU/kg/day) was administered intraperitoneally every day from the onset of SART stress. The mechanical pain threshold was measured using the Randall-Selitto test on the 6th day. Thereafter, the rat mesencephalon was immediately collected and calpain activity was examined using western blot analysis with a calpain cleavage site-specific antibody.
SART stress induced hyperalgesia and increased the calpain activity in the mesencephalon of rats. In contrast, NTP treatment attenuated the hyperalgesia and prevented the increase in calpain activity in the mesencephalon of SART-stressed rats. Interestingly, a negative correlation was identified between calpain activity and mechanical pain threshold in SART-stressed rats treated with or without NTP. Furthermore, NTP inhibited calpain activity on mammalian uncoordinated-18 in rat mesencephalon homogenate and Ac-LLY-AFC as substrates in an in vitro cell-free system.
Our data demonstrate that NTP treatment prevents SART stress-induced calpain activation in the mesencephalon of rats and suggests that NTP-mediated antihyperalgesia is associated with an inhibition of calpain activity in the mesencephalon.
神经妥乐平（Neurotropin®，NTP）是一种用于治疗慢性疼痛的镇痛药，对特定温度节律改变应激（SART）的大鼠具有抗痛觉过敏作用。先前的研究表明，SART应激会诱导大鼠中脑出现痛觉过敏以及蛋白质的翻译后修饰（包括钙蛋白酶的底物，一种钙依赖性半胱氨酸蛋白酶）。为了更好地理解NTP的作用机制，我们研究了SART应激是否会激活大鼠中脑的钙蛋白酶，以及NTP是否会抑制这种激活。
将Wistar大鼠暴露于SART应激5天。从SART应激开始每天腹腔注射NTP（200NU/kg/天）。在第6天使用Randall-Selitto试验测量机械性疼痛阈值。此后，立即收集大鼠中脑，并使用针对钙蛋白酶切割位点特异性抗体的蛋白质印迹分析检测钙蛋白酶活性。
SART应激诱导大鼠中脑出现痛觉过敏并增加了钙蛋白酶活性。相比之下，NTP治疗减轻了痛觉过敏，并防止了SART应激大鼠中脑钙蛋白酶活性的增加。有趣的是，在接受或未接受NTP治疗的SART应激大鼠中，钙蛋白酶活性与机械性疼痛阈值之间存在负相关。此外，在体外无细胞系统中，NTP抑制了大鼠中脑匀浆中哺乳动物不协调蛋白-18上的钙蛋白酶活性，并以Ac-LLY-AFC作为底物。
我们的数据表明，NTP治疗可防止SART应激诱导的大鼠中脑钙蛋白酶激活，并提示NTP介导的抗痛觉过敏与抑制中脑钙蛋白酶活性有关。
