甲基莲心碱对七氟烷麻醉后大鼠认知障碍与NLRP3炎症小体表达的影响

Effects of neferine on cognitive impairment and NLRP3 inflammatory body expression in rats after sevoflurane anesthesia.

目的探讨甲基莲心碱对七氟烷麻醉后大鼠认知障碍与NLRP3炎症小体表达的影响。方法选择30只6~8周成年雄性Sprague-Dawley大鼠为研究对象,将其按照实验目的分为3组:对照组(定期吸入空气,不加七氟烷,流速1 L/min),麻醉组(接受1.5%七氟烷麻醉,流量1 L/min,持续6 h)和观察组大鼠麻醉诱导后,腹膜内注射莲心碱(20 mg/kg,每2 d 1次,持续4周),每组各10只。通过蛋白质印迹法分析大鼠海马组织中凋亡蛋白Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2基因(Bcl-2)、半胱氨酸蛋白酶-3(caspase-3)的蛋白表达。通过酶联免疫吸附试验(ELISA)分析大鼠海马组织中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6和IL-18的水平。通过TUNEL分析大鼠海马中的凋亡细胞。通过莫里斯水迷宫试验比较实验大鼠穿越平台的逃逸潜伏期。通过逆转录-聚合酶链反应(RT-PCR)和免疫组织化学染色评估海马中炎症小体NLRP3的表达。通过使用特定的检测试剂盒对大鼠海马组织中丙二醛(MDA)及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽(GSH)进行分析。结果麻醉组较对照组Bax、caspase-3的蛋白表达、TNF-α、IL-1β、IL-6和IL-18的含量、海马中的凋亡细胞数量均升高,Bcl-2蛋白表达降低,差异均有统计学意义(P<0.05);观察组较麻醉组Bax和caspase-3的蛋白表达、TNF-α、IL-1β、IL-6和IL-18的含量、海马中的凋亡细胞数量均降低,Bcl-2蛋白表达升高,差异均有统计学意义(P<0.05)。第1天各组大鼠穿越平台的逃逸潜伏期比较,差异无统计学意义(P>0.05),第3天和第5天麻醉组较对照组的逃逸潜伏期升高,差异有统计学意义(P<0.05),观察组较麻醉组的逃逸潜伏期降低,差异有统计学意义(P<0.05)。麻醉组较对照组大鼠穿越平台的次数、花费时间和目标象限覆盖的距离百分比均降低,差异均有统计学意义(P<0.05);观察组较麻醉组穿越平台的次数、花费时间和目标象限覆盖的距离百分比均升高,差异均有统计学意义(P<0.05)。麻醉组较对照组NLRP3 mRNA表达和NLRP3阳性细胞数量、MDA水平升高,SOD、CAT和GSH水平降低,差异均有统计学意义(P<0.05);观察组较麻醉组NLRP3 mRNA表达和NLRP3阳性细胞数量、MDA水平降低,SOD、CAT和GSH水平升高,差异均有统计学意义(P<0.05)。结论甲基莲心碱可改善七氟烷麻醉后大鼠的记忆和认知功能障碍,并且这些作用与抑制氧化应激,神经炎症反应及降低NLRP3的表达有关。

Objective To investigate the effect of neferine on cognitive impairment and NLRP3 inflammasome expression in rats after sevoflurane anesthesia.Methods Thirty adult male Sprague-Dawley rats of 6-8 weeks were selected as the research objects,and they were divided into control group(regular inhalation of air,no sevoflurane,flow rate 1 L/min),anesthesia group(receiving 1.5%sevoflurane anesthesia,flow rate 1 L/min,duration 6 h)and observation group after induction of anesthesia,rats were injected intraperitoneally with liensinine(20 mg/kg,once every 2 days for 4 weeks)according to the experimental purpose,each group with 10 rats.The protein expression of apoptosis protein Bcl-2 related X protein(Bax),B lymphoma-2 gene(Bcl-2)and caspase-3(caspase-3)in rat hippocampus was analyzed by Western blotting;the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6 and IL-18 in rat hippocampus were analyzed by enzyme-linked immunosorbent assay(ELISA);the apoptotic cells in the rat hippocampus were analyzed by TUNEL;the Morris water maze test was used to compare the escape latency of experimental rats across the platform;the expression of inflammasome NLRP3 in the hippocampus was evaluated by reverse transcription-polymerase chain reaction(RT-PCR)and immunohistochemical staining.The analysis of malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT)and glutathione(GSH)in rat hippocampal tissues was carried out by using a specific detection kit.Results Compared with the control group,the protein expression of Bax and caspase-3,the levels of TNF-α,IL-1β,IL-6 and IL-18,the number of apoptotic cells in the hippocampus of rats in the anesthesia group were increased,and the protein expression of Bcl-2 was decreased,the differences were statistically significant(P<0.05).Compared with the anesthesia group,the protein expression of Bax and caspase-3,the levels of TNF-α,IL-1β,IL-6 and IL-18,the number of apoptotic cells in the hippocampus of rats in the observation group were decreased,and the protein expression of Bcl-2 was increase,the differences were statistically significant(P<0.05).There was no difference in the escape latency of rats across the platform on the 1st day(P>0.05).Compared with the control group,the escape latency of the 3rd and 5th day in the anesthesia group were increased,the difference was statistically significant(P<0.05).Compared with the anesthesia group,the escape latency in the anesthesia group was decreased,the difference was statistically significant(P<0.05).Compared with the control group,the number of times to cross the platform,time spent,and the percentage of distance covered by the target quadrant in the anesthesia group were decreased,the differences were statistically significant(P<0.05).Compared with the anesthesia group,the number of times,time spent and the percentage of distance covered by the target quadrant in the observation group were increased,the differences were statistically significant(P<0.05).Compared with the control group,the NLRP3 mRNA expression and the number of NLRP3 positive cells,the level of MDA in the anesthesia group were increased,and the levels of SOD,CAT and GSH were decreased,the differences were statistically significant(P<0.05).Compared with the anesthesia group,the NLRP3 mRNA expression and the number of NLRP3 positive cells,the level of MDA were decreased,the levels of SOD,CAT and GSH were increased in observation group,the differences were statistically significant(P<0.05).Conclusion Neferine can improve the memory and cognitive dysfunction of rats after sevoflurane anesthesia,and these effects are related to inhibiting oxidative stress,neuro-inflammatory response and reducing the expression of NLRP3.