大麻二酚对大鼠切口痛模型术后镇痛效果及机制研究

Study of postoperative analgesic effects and mechanism of cannabidiol on incision pain model in rats

为探究大麻二酚(cannabidiol, CBD)对大鼠足底切口痛模型术后镇痛效果及机制,试验选取体重为200~220 g的雄性SD大鼠32只,随机分为4组,分别为对照组(CON组,大鼠腹腔注射生理盐水0.5 mL)、阴性对照组(CBD组,大鼠按体重腹腔注射CBD 30 mg/kg)、模型组(大鼠进行右侧足底切开术)和CBD干预组(即CBD+模型组,大鼠在足底切口痛模型建立前1小时按体重腹腔注射30 mg/kg的CBD),术前1天和术后6小时、1天、3天、5天、7天进行机械和电热痛阈值的测定,随后处死大鼠,采集血液和脊髓样本,ELISA法检测血清肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)、白细胞介素1β(IL-1β)和脊髓5-羟色胺(5-HT)、多巴胺(DA)质量浓度,Western-blot检测脊髓Toll样受体4(TLR4)、磷酸化核转录因子κB(p-NF-κB)蛋白表达情况,免疫组织化学法检测p-NF-κB表达量及定位情况。结果表明:与CON组比较,模型组在术后6小时、1天、3天、5天、7天机械痛阈值和电热痛阈值均极显著降低(P<0.01),CBD+模型组在术后6小时、1天、3天显著或极显著降低(P<0.01或P<0.05)。与模型组比较,CBD+模型组在术后6小时、1天、3天、5天、7天机械痛阈值和电热痛阈值均极显著升高(P<0.01)。与CON组比较,模型组大鼠的体重增长率极显著降低(P<0.01),血清IL-6、IL-1β、TNF-α质量浓度极显著升高(P<0.01),脊髓5-HT、DA的质量浓度极显著升高(P<0.01),脊髓TLR4、胞浆和胞核p-NF-κB蛋白相对表达量极显著升高(P<0.01),且p-NF-κB主要定位在脊髓背角内。而与模型组比较,CBD+模型组大鼠的体重增长率极显著升高(P<0.01),血清IL-6、IL-1β、TNF-α质量浓度极显著降低(P<0.01),脊髓5-HT、DA的质量浓度极显著降低(P<0.01),脊髓TLR4、胞浆和胞核p-NF-κB蛋白相对表达量极显著或显著降低(P<0.01或P<0.05)。此外,CBD组与CON组相比,所有指标差异均不显著(P>0.05)。说明CBD可通过抑制TLR4/NF-κB信号通路激活,下调下游炎症因子水平,也可通过抑制神经递质(5-TH和DA)释放,最终改善大鼠术后疼痛反应。

In order to explore the postoperative analgesic effects and mechanism of cannabidiol(CBD) on plantar incision pain model in rats, in the experiment, 32 male SD rats weighing 200-220 g were selected and randomly divided into four groups: control group(CON group, the rats were injected intraperitoneally with 0.5 mL of normal saline), negative control group(CBD group, the rats were injected intraperitoneally with CBD 30 mg/kg by body weights), model group(the rats underwent right plantotomy) and CBD intervention group(CBD+model group, the rats were injected intraperitoneally with 30 mg/kg of CBD at body weights 1 h before the plantar incision pain model was established).Mechanical and electrothermal pain thresholds were measured preoperatively and at 6 h, 1 day, 3 days, 5 days, and 7 days postoperatively;the rats were subsequently executed and blood and spinal cord samples were collected. ELISA method was used for the determination of mass concentrations of serum tumor necrosis factor-α(TNF-α), interleukin 6(IL-6), interleukin 1β(IL-1β), spinal 5-hydroxytryptamine(5-HT) and dopamine(DA). Western-blot was performed to detect spinal Toll-like receptor 4(TLR4) and phosphorylated nuclear transcription factor κB(p-NF-κB) protein expression, and immunohistochemistry was performed to detect p-NF-κB expression and localization. The results showed that mechanical pain thresholds and electrothermal pain thresholds were highly significantly lower in the model group at 6 h, 1 day, 3 days, 5 days, and 7 days postoperatively compared with the CON group(P<0.01);pain thresholds were significantly or highly significantly lower in the CBD+model group at 6 h, 1 day, and 3 days postoperatively(P<0.01 or P<0.05). Compared with the model group, the CBD+model group showed a highly significant increase in mechanical pain thresholds and electrothermal pain thresholds at 6 h, 1 day, 3 days, 5 days, and 7 days postoperatively(P<0.01). Compared with the CON group, the rat body weight growth rate was highly significantly lower in the model group(P<0.01);the mass concentrations of serum IL-6, IL-1β, and TNF-α were highly significantly higher(P<0.01);the mass concentrations of spinal 5-HT and DA were highly significantly higher(P<0.01);and the relative expression of spinal TLR4, cytoplasmic and cytosolic p-NF-κB protein was highly significantly higher(P<0.01), and p-NF-κB was mainly localized within the dorsal horn of the spinal cord. Compared with the model group, the weight growth rate of rats in the CBD+model group was significantly higher(P<0.01);the mass concentrations of serum IL-6, IL-1β and TNF-α were significantly reduced(P<0.01);the mass concentration of 5-HT and DA in the spinal cord was significantly reduced(P<0.01);the relative expression of spinal TLR4, cytoplasm and nucleus p-NF-κB proteins was very significantly or significantly reduced(P<0.01 or P<0.05). In addition, the difference between CBD and CON groups was not significant for all indexes(P>0.05). The results suggested that CBD could downregulate the level of downstream inflammatory factors by inhibiting TLR4/NF-κB signaling pathway activation, and also ultimately improve the postoperative pain response in rats, by inhibiting neurotransmitters’(5-TH and DA) release.