安宫牛黄丸对脓毒症大鼠脑功能保护作用的机制研究

Protective effect and mechanism of Angong Niuhuang pill in sepsis-associated brain dysfunction of rats

目的观察安宫牛黄丸对脓毒症大鼠脑功能的保护作用,并初步探讨其保护机制,为安宫牛黄丸治疗脓毒症相关性脑病(SAE)提供实验依据。方法将30只雄性SD大鼠按随机数字表法分为假手术组、脓毒症模型组及安宫牛黄丸组,每组10只。采用盲肠结扎穿孔术(CLP)制备脓毒症模型;假手术组只给予开腹和关腹;安宫牛黄丸组在制模前3 d开始每日灌胃安宫牛黄丸0.3 g/kg进行预处理,制模后12 h再给药1次。于制模后24 h,对各组大鼠进行神经反射评分,检测血白细胞计数(WBC),用酶联免疫吸附试验(ELISA)检测血清神经元特异性烯醇化酶(NSE)、中枢神经特异蛋白S100β水平;随后处死大鼠取脑组织,苏木素-伊红(HE)染色后光镜下观察脑组织病理学改变;用聚合酶链反应检测脑组织白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的mRNA表达。结果与假手术组比较,脓毒症模型组和安宫牛黄丸组神经反射评分总分明显减低(分:4.43±1.40、6.57±1.90比9.40±0.84,均P<0.05),血WBC和血清NSE、S100β水平明显升高〔WBC(×10^(9)/L):8.07±1.32、5.84±0.94比3.60±0.32,NSE(μg/L):1.04±0.14、0.61±0.07比0.16±0.04,S100β(ng/L):255.62±30.25、97.72±15.41比46.88±12.03,均P<0.05〕,脑组织IL-6和TNF-α的mRNA表达量明显增高〔IL-6 mRNA(2^(-ΔΔCt)):5.668±2.195、3.605±1.014比0.997±0.329,TNF-αmRNA(2^(-ΔΔCt)):18.996±0.913、1.746±0.710比0.674±0.132,均P<0.05〕。但与脓毒症模型组比较,安宫牛黄丸组神经反射评分总分明显增高(分:6.57±1.90比4.43±1.40,P<0.05),血WBC和血清NSE、S100β水平及脑组织IL-6、TNF-α的mRNA表达均明显降低〔WBC(×10^(9)/L):5.84±0.94比8.07±1.32,NSE(μg/L):0.61±0.07比1.04±0.14,S100β(ng/L):97.72±15.41比255.62±30.25,IL-6 mRNA(2^(-ΔΔCt)):3.605±1.014比5.668±2.195,TNF-αmRNA(2^(-ΔΔCt)):1.746±0.710比18.996±0.913,均P<0.05〕。脑组织病理学观察可见脓毒症模型组大鼠海马神经细胞排列紊乱,大量神经细胞核固缩深染,组织疏松水肿明显;给予安宫牛黄丸预处理后大鼠神经细胞核固缩深染及组织疏松水肿情况较脓毒症模型组明显减轻。结论安宫牛黄丸预处理可以改善脓毒症大鼠脑功能障碍,同时降低脑内促炎细胞因子的表达,推测安宫牛黄丸可能通过抑制脑内炎症反应来发挥对脓毒症的脑保护作用。

Objective To observe the protective effect of Angong Niuhuang pill on brain function of rats with sepsis,explore its protective mechanism,and provide the experimental basis for clinical application of Angong Niuhuang pill in the treatment of sepsis-associated encephalopathy(SAE).Methods Thirty male Sprague-Dawley(SD)rats were divided into sham operation group,sepsis model group and Angong Niuhuang pill group according to random number table method,with 10 rats in each group.The sepsis model was established by cecal ligation and puncture(CLP);rats in sham operation group received open and closed abdomen.The rats in the Angong Niuhuang pill group were given Angong Niuhuang pill(0.3 g/kg)by gastric irrigation daily for 3 days before CLP,and the drugs were administrated 12 hours after modeling again.After 24 hours of CLP,the neuroreflex scores were evaluated,white blood cell count(WBC),the levels of serum neuron-specific enolase(NSE)and S100βwere detected.Then the brain tissue was harvested.After hematoxylin-eosin(HE)staining,the pathological changes of brain tissue were observed under the light microscope.The mRNA expressions of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in brain tissue were detected by polymerase chain reaction.Results Compared with the sham operation group,the total score of neuroreflex scores in the sepsis model group and the Angong Niuhuang pill group were significantly reduced(4.43±1.40,6.57±1.90 vs.9.40±0.84,both P<0.05),WBC,serum NSE,S100βwere significantly increased[WBC(×10^(9)/L):8.07±1.32,5.84±0.94 vs.3.60±0.32;NSE(μg/L):1.04±0.14,0.61±0.07 vs.0.16±0.04;S100β(ng/L):255.624±30.25,97.72±15.41 vs.46.88±12.03,all P<0.05],and the mRNA expressions of IL-6 and TNF-αin brain tissue were significantly increased[IL-6 mRNA(2^(-ΔΔCt)):5.668±2.195,3.605±1.014 vs.0.997±0.329;TNF-αmRNA(2^(-ΔΔCt)):18.996±0.913,1.746±0.710 vs.0.674±0.132,all P<0.05].Compared with the sepsis model group,the total score of neuroreflex scores in the Angong Niuhuang pill group was significantly increased(6.57±1.90 vs.4.43±1.40,P<0.05),WBC,serum NSE,S100βconcentration,and the mRNA expressions of IL-6 and TNF-αin the brain were significantly reduced[WBC(×10^(9)/L):5.84±0.94 vs.8.07±1.32,NSE(μg/L):0.61±0.07 vs.1.04±0.14,S100β(ng/L):97.72±15.41 vs.255.62±30.25,IL-6 mRNA(2^(-ΔΔCt)):3.605±1.014 vs.5.668±2.195,TNF-αmRNA(2^(-ΔΔCt)):1.746±0.710 vs.18.996±0.913,all P<0.05].Brain histopathological observation showed that the hippocampal neurons in the sepsis model group were disordered arrangement,a large number of neuronal nuclei were contracted,and the tissue was loose with obvious edema.Compared with the sepsis model group,the Angong Niuhuang pill group had less nuclear shrinkage and tissue edema.Conclusions The pretreatment of the Angong Niuhuang pill can improve the brain dysfunction of septic rats and reduce the expression of pro-inflammatory cytokines in the brain.It is speculated that the Angong Niuhuang pill can protect the brain function in sepsis by inhibiting the inflammatory reaction in the brain.