摘要
目的:探索"补肾填精"中药复方改善脑瘫儿运动发育落后的分子机制,为临床上采用中医药治疗脑瘫患儿提供科学依据.方法:对处于妊娠期16 d Wistar大鼠进行腹腔注射细菌脂多糖(Lipopolysaccharides,LPS)模拟宫内感染,并将孕鼠置于低氧环境下(92%氮气+8%氧气)处理2 h,以此建立妊娠期大鼠宫内感染合并缺氧模型.通过动物神经行为学测试,在子代大鼠中筛选获得幼年脑瘫大鼠.脑瘫模型幼鼠和正常幼鼠分别设置治疗组和对照组,共4个组别,每组动物10只;另取10只正常幼鼠不做任何处理,作为空白对照组.按1 g·(25 mL)-1 ddH2O的质量体积比配制免煎颗粒药.通过灌胃给予治疗组大鼠"补肾填精"中药复方水剂,给药剂量为5 mL·kg-1老鼠体重,每日1次,连续灌服15 d.分别于给药后第5 d、第10 d和第15 d对大鼠的运动能力进行观察和评估.给药15 d后将大鼠处死,大鼠眼眶采血,ELISA检测各组动物外周血中炎症因子的水平,取脑组织,对mTOR相关信号通路分子进行免疫印迹检测.结果:悬吊试验结果显示,与空白对照组相比,脑瘫对照组的悬吊时间明显缩短(P＜0.001);与脑瘫对照组相比,脑瘫治疗组悬吊时间明显延长(P＜0.001);而正常治疗组与正常对照组相比,悬吊时间差异无统计学意义(P=0.064).斜坡实验结果显示,与空白对照组相比,脑瘫对照组转头时间明显延长,差异有统计学意义(P＜0.001);与脑瘫对照组相比,脑瘫治疗组转头时间明显缩短,差异有统计学意义(P＜0.001);而正常治疗组与正常对照组相比,转头时间差异无统计学意义(P=0.863).对各组别大鼠的体重进行监测,与空白对照组及正常对照组相比,正常治疗组体重无明显差异(P＞0.05);与空白对照组相比,脑瘫治疗组体重明显减轻(P＜0.001);与脑瘫对照组相比,脑瘫治疗组体重明显增加(P=0.031).ELISA检测各组动物外周血中炎症因子的水平发现,与脑瘫对照组相比,脑瘫治疗组外周血中促炎因子TNF-α和IL-1β显著下降(P＜0.001),但对IL-6的水平影响不大(P＞0.05),而抗炎因子IL-4和IL-10显著升高(P＜0.001).WB方法检测各组大鼠神经组织中PI3K/AKT/mTOR信号通路相关蛋白表达水平发现,与脑瘫对照组相比,脑瘫治疗组PI3K表达增加,磷酸化AKT和mTOR表达增加,LC3-Ⅱ与LC3-Ⅰ比值降低,自噬相关蛋白p62表达增加(P＜0.01).结论:"补肾填精"中药复方可通过调控mTOR信号通路介导的神经细胞自噬,并抑制炎症的发生达到改善脑瘫幼年大鼠运动功能的效应.
Abstract
Objective:To explore the molecular mechanism of"Bu Shen Tian Jing"compound of traditional Chinese medicine to improve motor development in cerebral palsy(CP)of children.Methods:The cerebral palsy(CP)animal model was established in pregnant Wistar rats at 16 days of gestation by intraperitoneal injection of bacterial lipopolysac-charide(LPS)combined with a treatment of hypoxic living environment(8%oxygen + 92%nitrogen)for 2 h.The offspring CP rats were screened by neurobehavioral testing.The CP young mice and healthy mice were set up as treatment group and control group,respectively,with a total of 4 groups and 10 animals in each group.Another 10 normal young mice were taken as a blank control group without any treatment.The''Bu Shen Tian Jing''decoction-free granules was dissolved in water at a mass-to-volume ratio of 1 g·(25 mL)-1.Animals were treated with medicine or aqueous solvents by intragastric administration with a dosage of 5 mL·kg-1 body weight once a day for 15 days.The exercise capacity of rats was observed and evaluated on the 5th,10th and 15th days after administration,respectively.At the end of the animal experiment,the rats were sacrificed and the peripheral blood was collected in eyes socket.The inflammatory factors in the peripheral blood were detected by ELISA assay,and protein levels associated with the mTOR signaling pathway in the brain tissue were detected by western blotting(WB).Results:Compared with the blank control group,the suspension time of the CP control group was significantly shortened(P＜0.001);compared with the CP control group,the suspension time of the CP treatment group was significantly longer(P＜0.001),while in normal group,the suspension time in the treatment group and control group had no significant differences(P=0.064).The results from the slope experiment showed that compared with the blank control group,the head turning time of the CP control group was significantly longer,which had a significant difference(P＜0.001);Compared with the CP control group,the head turning time of the CP treatment group was significantly shortened,which had a statistically significant difference(P＜0.001).However,there was no statistically significant difference in normal group in treatment group and control group(P=0.863).The body weight of rats in each group was monitored every day.It was demonstrated that the body weight in normal treatment group had no significant difference compared with the blank control and normal control group(P＞0.05),while compared with the blank control group,the CP treatment group had a significant weight loss(P＜0.001).And CP treatment group had a significant increase in body weight compared with the CP control group(P=0.031).ELISA testing was used to detect the expression level of inflammatory factors in the peripheral blood of each group of animals.Compared with the CP control group,the pro-inflammatory factors TNF-α and IL-1β in the CP treatment group were significantly decreased(P＜0.001),with IL-6 had not significant(P＞0.05);while the anti-inflammatory factors IL-4 and IL-10 were significantly increased(P＜0.001).WB was used to detect the protein expression level associated with the PI3K/ATK/mTOR signaling pathway in the brain tissue.It was demonstrated that compared with the CP control group,the expression levels of PI3K,phosphorylated-AKT,phosphorylated-mTOR and autophagy-related protein p62 were increased in the CP treatment group;with the LC3-Ⅱ/Ⅰratio decrease(P＜0.01).Conclusion:The"Bu Shen Tian Jing"compound can improve the motor function of young rats with CP by regulating the mTOR signaling pathway-mediated autophagy of nerve cells and inhibiting the occurrence of inflammation.
基金项目
江西省中医药管理局科技计划项目(2022B1057)
江西省卫生健康委员会科技计划资助项目(202311918)
国家科技期刊平台
NSTL
万方数据