蛇床子催眠活性组分对PCPA失眠大鼠松果体细胞凋亡和相关Bcl-2/Bax蛋白表达的影响

目的:观察蛇床子催眠活性组分(CHC)对PCPA致大鼠松果体细胞凋亡及其Bcl-2/Bax蛋白阳性表达的影响,探讨其改善松果体损伤的作用机制。方法:雄性SD大鼠随机分成空白对照组,模型对照组,MT阳性对照(10 mg/kg)组,蛇床子催眠活性成分高、中、低剂量(60、30、15 mg/kg)共6组,每组10只,除空白对照组外,其余各组大鼠每日腹腔注射PCPA 450 mg/kg,连续2 d,制备失眠大鼠松果体损伤模型,造模完成每日灌胃给药1次,连续7 d。分别于造模及给药前后观察各组大鼠的精神状态、皮毛光泽度、体质量变化情况;观察给药后HE染色松果体组织病理变化,原位末端凋亡(TUNEL)计数松果体组织凋亡细胞数得出凋亡指数(AI)、流式细胞术分析松果体细胞早期凋亡率,免疫组化法半定量分析凋亡相关蛋白Bcl-2、Bax表达。结果:松果体病理组织镜下观察可见模型对照组细胞数目明显减少,排列紊乱,核固缩,空泡变性增多,CHC低剂量组与模型组无明显差异,其余各给药组大鼠松果体细胞排列紊乱均有一定程度改善、空泡变性减少;松果体组织TUNEL的AI指数模型对照组显著高于空白对照组(P<0.01),MT、CHC高、中剂量组显著低于模型对照组(P<0.01)。松果体细胞早期凋亡率模型对照组显著高于空白对照组(P<0.05),CHC高剂量组显著低于模型对照组(P<0.05);Bcl-2模型对照组显著低于空白对照组(P<0.05),MT阳性对照组、CHC高、中剂量组显著高于模型对照组(P<0.01,P<0.05);Bax模型对照组显著高于空白对照组(P<0.01),MT阳性对照组、CHC高、中剂量组著低于模型对照组(P<0.05,P<0.01)。结论:CHC改善PCPA致失眠大鼠松果体组织结构损伤,减少松果体凋亡,其作用机制与降低诱导凋亡蛋白Bax、增加抑制凋亡蛋白Bcl-2表达、上调Bcl-2/Bax有关。

Effects of Hypnotic Active Components of Fructus Cnidii on Apoptosis of Pineal Cells and Expression of Related Bcl-2/Bax Proteins in PCPA Rats with Insomnia

Objective:To observe the effects of hypnotic active components of Fructus Cnidii on the apoptosis of rat pineal cells induced by PCPA and the positive expression of Bcl-2/Bax protein,and to explore the mechanism of its improvement on pineal injury.Methods:Male SD rats were randomly divided into the blank group,the model group,the melatonin(MT)group(10 mg/kg),and the hypnotic active components of Fructus Cnidii(CHC)groups of high-dose,medium-dose and low-dose(60 mg/kg,30 mg/kg,15 mg/kg),with 10 rats in each group.The insomnia model of pineal gland damage was established by daily intraperitoneal injection of PCPA for two days(450 mg/kg).After successful modeling,each group was given the corresponding intervention for seven days.Mental state,fur gloss and body weight of rats were observed before and after the treatment in each group.The pathological changes of pineal tissues were observed by HE staining after the treatment.The early apoptotic rate of pineal cells was analyzed by flow cytometry,the number of apoptotic cells in situ terminal apoptosis(TUNEL)was counted to obtain the apoptotic index(AI),and the apoptotic proteins expressions of Bcl-2 and Bax were analyzed semi-quantitatively by immunohistochemistry.Results:The pineal gland pathological tissue under histopathoscope showed decreased cell number,disordered arrangement,nucleus pycnosis and increased vacuoles degeneration in the model group;of which there were no statistical differences between the CHC group of low-dose and the model group.The disordered arrangement of rat pineal cells and vacuoles degeneration were improved in the other treatment groups.In terms of the AI index of TUNEL in pineal tissue,it was significantly higher in the model group than that in the blank group(P<0.01),and it was significantly lower in the MT group and in the CHC groups of high-dose and medium-dose than that in the model group(P<0.01).In terms of the early apoptosis rate of pineal cells,it was significantly higher in the model group than that in the blank group(P<0.05),and it was significantly lowerin the CHC group of high-dose than that in the model group(P<0.05).In terms of the expression of Bcl-2,it was significantly lower in the model group than that in the blank group(P<0.05),and it was significantly higher in the MT group and the CHC groups of high-dose and medium-dose than that in the model group(P<0.05,P<0.01).In terms of the expression of Bax,it was significantly higher in the model group than that in the blank group(P<0.01),and it was significantly lower in the MT group and the CHC groups of high-dose and medium-dose than that in the model group(P<0.05,P<0.01).Conclusion:CHC can improve the pineal tissue structure damage and reduce pineal apoptosis induced by PCPA in insomnia rats,and the action mechanism is related to the decrease of inducing apoptosis protein of Bax,the increase of inhibiting apoptosis protein of Bcl-2 and up-regulation of Bcl-2/Bax.