疏肝理肺方对应激条件下哮喘大鼠脑海马形态的影响

Effects of shuganlifei method on the cerebral hippocampus formation in rats with asthma under the condition of stress

目的:探讨束缚应激哮喘大鼠应用疏肝理肺方药后脑海马形态结构的变化。方法:实验于2002-03/2003-01在上海中医药大学国家重点实验室完成。选择SD雄性大鼠56只,随机分为7组,正常组、哮喘组、应激组、哮喘应激组、地塞米松组、理肺组和疏肝理肺组各8只。正常组和应激组大鼠给予1mL生理盐水腹腔注射,其余各组分别腹腔注射等量抗原液致敏,致敏第8天开始,每日将应激组、哮喘应激组、地塞米松组、理肺组和疏肝理肺组大鼠双后肢用绷带立式固定2h给予束缚应激刺激,共进行3周;同时每日分别给予地塞米松组、理肺组和疏肝理肺组大鼠地塞米松(0.75mg/片)1g/(L·kg)、理肺方(麻黄、桂枝、炙甘草、细辛、干姜、半夏、五味子及白芍均9g)28g/(mL·kg)和疏肝理肺方(在理肺方基础上增加柴胡9g及炙枳实9g)36g/(mL·kg)灌胃。在致敏第15天开始,每日给予哮喘组、哮喘应激组、地塞米松组、理肺组和疏肝理肺组大鼠卵蛋白20g/L雾化吸入3min,共雾化2周。造模及治疗结束后,麻醉大鼠抽取腹主动脉血,测定血浆皮质酮水平应用放射免疫法。抽血后,开颅取大鼠脑,采用光镜及透射电镜检测大鼠脑海马的形态结构。结果:56只大鼠全部进入结果分析,无脱失。①各组大鼠血浆皮质酮水平比较:哮喘组和应激组大鼠的血浆皮质酮水平均较正常组显著升高犤(532.48±115.28),(323.32±100.40),(235.27±46.64)μg/L,(P<0.01,0.05)犦。疏肝理肺方组大鼠较哮喘应激组显著降低犤(309.50±46.52),(569.89±72.32)μg/L(P均<0.01)犦。疏肝理肺方组较理肺组(528.60±130.72)μg/L显著降低(P<0.01)。②各组大鼠光镜下脑海马形态结构的比较:正常组大鼠脑海马锥体细胞排列规则,细胞核圆而大,核仁清晰。哮喘应激组、地塞米松组和理肺组大鼠相似脑海马锥体细胞排列不规则,在CA3区可见大量的锥体细胞核固缩。疏肝理肺组大鼠脑海马锥体细胞排列比较规则,在CA3区核固缩神经元明显减少。③各组大鼠电镜下脑海马形态结构的比较:正常组大鼠脑海马锥体细胞核形态规则,核膜光滑,核异染色质分布均匀,线粒体丰富,嵴排列规则。哮喘应激组和理肺组大鼠相似,脑海马锥体细胞核形态不规则,核异染色质明显增多,胞浆疏松,多数线粒体扩张,出现空泡、嵴排列紊乱。地塞米松组大鼠脑海马锥体细胞形态不规则,细胞器变性。疏肝理肺组大鼠脑海马锥体细胞核形态较规则,核膜光滑,核异染色质分布较均匀,胞浆疏松,线粒体丰富但致密。结论:慢性束缚应激会诱发或加重哮喘,导致哮喘大鼠血浆皮质酮水平升高,从而损伤脑海马神经元形态。疏肝理肺方可以通过抑制应激条件下哮喘大鼠的血浆皮质酮分泌,对脑海马神经元形态起一定的保护作用。

AIM： To explore the changes of the formation of cerebral hippocampus in rats with binding stress asthma after treated with shuganlifei method. METHODS： The experiment was conducted in the key laboratory of Shanghai University of Traditional Chinese Medicine between March 2002 and January 2003. Fifty-six SD male rats were selected and assigned randomly into 7 groups： normal group, asthma group, stress group, asthma stress group, desamethasone group, regulating lung group and shuganlifei group with 8 rats in each group. The rats in normal group and stress group were treated with 1 mL saline through intraperitoneal injection. Those in other groups were treated with equivalence antigen fluid induced sensitization respectively through intraperitoneal injection. From the 8^th day after sensitization, the both hindlimb of the rats in stress group, asthma stress group, desamethasone group, regulating lung group and shuganlifei group were fixed by band every day vertically for 2 hours and gave the binding stress stimulation, totally for 3 weeks; Meanwhile, the rats in desamethasone group, regulating lung group and shuganlifei group were given desamethasone （0.75mg/each）1 g/（L ·kg） and regulating lung method （ephedra, cassia twig, asarum, dried ginger, pinellia tuber, Chinese magnoliavine fruit and white peony root, all 9 g） 28 g/（mL ·kg） and shuganlifei method （based on the regulating method, 9 g Chinese thorowax root and 9 g immature bitter orange were added） 36 g/（mL ·kg） for gastric perfusion. From the 15^th day after sensitization, the rats in the asthma group, asthma stress group, desamethasone group, regulating lung group and shuganlifei group were given 20 g/L egg albumin absorbing for 3 minutes, totally for 2 weeks. At the ending of building models and treatment, the blood of abdominal aorta was separated from anesthesia rats. The level of plasm corticosterone was tested by radio-immunity method. After exacting the blood, the brain of rats was obtained by bittern the cranium. The form structure of cerebral hippoeampal was detected by light microscope and transmission electron microscope. RESULTS： Totally 56 rats were involved in the result analysis with outdrop. ① The comparison of the level of plasm corticosterone in every group： It increased significantly in the asthma group and the stress group than that in the normal group [（532.48±115.28）, （323.32±100.40）, （235.27±46.64） μg/L, （P 〈 0.01, 〈 0.05 ）]. It decreased significantly in the shuganlifei group than that in the asthma stress group [（309.50±46.52）, （569.89±72.32） μg/L（P all 〈 0.01 ）]. It decreased significantly in the shuganlifei group than that in the regulating lung group [（528.60±130.72） μg/L, （P〈 0.01 ）]. ② The comparison of the formation structure of cerebral hippoeampus under light microscope in the rats in each group： The pyramidal cells of cerebral hippocampus in the normal group were regularly arranged and the nucleus was round and big with clear nucleolus. The pyramidal cells in the asthma stress group, the desamethasone group and the regulating lung group were irregularly arranged. There were a large number of karyopyknosis of pyramidal cells in the CA3 area. The pyramidal cells arrangement of cerebral hippocampus in the shuganlifei group was more regular, and the pyknosis neurons in CA3 area decreased significantly. ③ The comparison of the formation structure of cerebral hippocampus under electron microscope in the rats in each group： The nucleus form of pyramidal cells in cerebral hippocampus in the normal group was regular with smooth karyotheca, the even distribution of nucleus heterochromatin, plenty mitochondrion and regular crest arrangement. It was similar between the asthma stress group and regulating lung group. The nucleus form of pyramidal cells in cerebral hippocampus was irregular, and the nucleus heterochromatin significantly increased with rarefaction cell plasm and most of extended mitochondrion, vacuoles and disorder crest arrangement. The nucleus form of pyramidal cells in cerebral hippocampus in desamethasone was irregular with the organell degeneration. The nucleus form of pyramidal cells in cerebral hippocampus in the shuganlifei group was regular with smooth karyotheca, even distribution of nucleus heterochromatin, cell plasm of rarefaction plenty mitochondrion but compact. CONCLUSION： The chronic binging stress can evoke or aggravate asthma, induce the increase of the level of plasm corticosterone in the rats with asthma, in order that the form of cerebral hippocampus neuron was injured. The shuganlifei method can play certain protective effects on the form structure of cerebral hippocampus neuron by the means of inhibiting the excretion of plasm corticosterone in the rats with asthma under the condition of stress.