盐酸羟考酮对癫痫大鼠炎症和神经元凋亡的影响及机制研究

Effect and mechanism of oxycodone hydrochloride on inflammation and neuronal apoptosis in epileptic rats

目的探究盐酸羟考酮通过c-Jun氨基末端激酶(JNK)/p38丝裂原活化蛋白激酶(p38 MAPK)信号通路对癫痫大鼠炎症和神经元凋亡的影响。方法构建癫痫大鼠模型,将造模成功的72只癫痫大鼠随机分为模型组、低剂量组(盐酸羟考酮0.125mg/kg)、中剂量组(盐酸羟考酮0.25mg/kg)、高剂量组(盐酸羟考酮0.5mg/kg)、茴香霉素组(茴香霉素5mg/kg)、联合组(盐酸羟考酮0.5mg/kg+茴香霉素5mg/kg),每组12只。另取12只健康大鼠作为对照组。给药结束24h后,记录各组大鼠癫痫发作频率和持续时间;酶联免疫吸附法检测血清TNF-α、白介素细胞6(IL-6)水平;苏木精-伊红染色观察海马CA1区组织病理学变化;TUNEL染色观察海马CA1区组织神经元凋亡状况;蛋白印迹法检测海马CA1区组织JNK、磷酸化JNK、p38MAPK、磷酸化p38MAPK和半胱氨酸天冬氨酸蛋白酶3(Caspase-3)蛋白表达。结果与对照组比较,模型组大鼠癫痫发作频率、持续时间、血清TNF-α、IL-6水平、海马CA1区组织神经元凋亡率、磷酸化JNK/JNK比值、磷酸化p38 MAPK/p38 MAPK比值、Caspase-3表达明显升高(P<0.05);与模型组比较,低剂量组、中剂量组、高剂量组大鼠癫痫发作频率、持续时间、血清TNF-α、IL-6水平、海马CA1区组织神经元凋亡率、磷酸化JNK/JNK比值、磷酸化p38MAPK/p38MAPK比值、Caspase-3表达依次降低,茴香霉素组大鼠癫痫发作频率、持续时间、血清TNF-α、IL-6水平、海马CA1区组织神经元凋亡率、磷酸化JNK/JNK比值、磷酸化p38MAPK/p38MAPK比值、Caspase-3表达明显升高(P<0.05);联合组大鼠癫痫发作频率、持续时间、血清TNF-α、IL-6水平、海马CA1区组织神经元凋亡率明显高于高剂量组,明显低于茴香霉素组(P<0.05)。联合组大鼠海马CA1区组织磷酸化JNK/JNK比值、磷酸化p38 MAPK/p38 MAPK比值、Caspase-3表达明显高于高剂量组,明显低于茴香霉素组(0.89±0.12vs0.25±0.05vs1.08±0.16,0.81±0.08vs 0.21±0.04vs0.94±0.12,0.79±0.12vs0.26±0.04vs0.89±0.14,P<0.05)。结论盐酸羟考酮可降低癫痫大鼠炎性反应,改善大鼠癫痫症状和病理损伤,保护神经元,其机制与抑制JNK/p38MAPK信号通路有关。

Objective To explore the effect of oxycodone hydrochloride on inflammation and neuronal apoptosis in epileptic rats through c-Jun amino terminal kinase(JNK)/p38 mitogen activated protein kinase(p38 MAPK)signaling pathway.Methods After epileptic rat model was successfully constructed,72 epileptic rats were randomly divided into model group,low-,medium-and high-dose oxycodone hydrochloride groups(0.125,0.25 and 0.5 mg/kg),anisomycin(JNK activator 5 mg/kg)and combined group(0.5 mg/kg oxycodone hydrochloride+5 mg/kg anisomycin),with 12 rats in each group.Another 12 healthy rats were selected as control group.In 24 h after the end of administration,the frequency and duration of seizures were recorded for all rats.ELISA was used to detect the serum levels of TNF-αand IL-6,HE staining was employed to observe the histopathological changes in hippocampal CA1 region,and TUNEL staining was applied to detect the apoptosis of CA1 region neurons.The expression of JNK,p-JNK,p38 MAPK,p-p38 MAPK and Caspase-3 in hippocampal CA1 region was detected by Western blotting.Results Compared with the rats from the control group,those of the model group showed higher frequency and longer duration of seizures,higher serum TNF-αand IL-6 levels,increased apoptotic rate of hippocampal CA1 neurons,and elevated p-JNK/JNK ratio,p-p38 MAPK/p38 MAPK ratio and Caspase-3 expression(P<0.05).While low-,medium-and high-dose oxycodone hydrochloride treatment reversed above changes in frequency and duration of seizures,serum TNF-αand IL-6 levels,neuronal apoptosis,p-JNK/JNK ratio,p-p38 MAPK/p38 MAPK ratio and Caspase-3 expression(P<0.05).In the anisomycin group,higher frequency and longer duration of seizures,elevated serum TNF-αand IL-6 levels,increased neuronal apoptotic rate in hippocampal CA1 region,and enhanced p-JNK/JNK ratio,p-p38 MAPK/p38 MAPK ratio and Caspase-3 expression(P<0.05).Lower frequency and shorter duration of seizures,decreased serum TNF-αand IL-6 levels,and reduced neuronal apoptotic rate in hippocampal CA1 region were observed in the combined group than the anisomycin group(P<0.05).The combined group obtained statistically lower p-JNK/JNK ratio,p-p38 MAPK/p38 MAPK ratio and Caspase-3 expression in hippocampal CA1 region than the high-dose group,and opposite results than the anisomycin group(0.89±0.12 vs 0.25±0.05 vs 1.08±0.16,0.81±0.08 vs 0.21±0.04 vs 0.94±0.12,0.79±0.12 vs 0.26±0.04 vs 0.89±0.14,P<0.05).Conclusion Oxycodone hydrochloride can reduce inflammatory response,improve epileptic symptoms and pathological damages,and protect neurons in epileptic rats,which is related to the inhibition of JNK/p38 MAPK signaling pathway.