天王补心丹加减改善PCPA失眠大鼠Trx系统氧化损伤的机制探讨
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摘要
目的:探讨天王补心丹加减对对氯苯丙氨酸(PCPA)所致失眠大鼠硫氧还蛋白(Trx)系统抗氧化损伤的作用机制。方法:60只雄性SD大鼠随机分为空白组、造模组。造模组以腹腔注射PCPA造模,注射剂量为150 mg·kg~(-1),间断注射7 d。造模成功后,依次分为模型组(等容生理盐水),天王补心丹加减低、中、高剂量组(8. 8,17. 6,35. 2 g·kg~(-1)·d~(-1))和艾司唑仑组(0. 1 mg·kg~(-1)·d~(-1)),每组10只。自发活动视频分析系统监测大鼠昼夜活动节律,透射电镜观察大鼠下丘脑视交叉上核(SCN)形态及细胞器完整性,酶联免疫吸附测定法(ELISA)检测大鼠血清超氧化物歧化酶(SOD),丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-Px)的水平,免疫荧光(IF)和蛋白免疫印迹法(Western blot)分别检测SCN细胞硫氧环蛋白2(Trx2),硫氧环蛋白还原酶2(TrxR2)的表达。结果:与空白组比较,模型组大鼠活动时间显著增加(P <0. 01),昼夜节律紊乱,线粒体嵴断裂,数量减少,排列紊乱,伴空泡化和髓鞘样变,SOD,GSH-Px活性下降且MDA含量显著升高(P <0. 01),Trx2的蛋白表达量显著降低(P <0. 01),TrxR2蛋白表达量显著增加(P <0. 01);与模型组比较,天王补心丹中、高剂量组能够明显改善大鼠昼夜节律紊乱,减少活动时间(P <0. 01),同时线粒体水肿程度减轻,部分嵴尚完整,SOD,GSH-Px活性升高,MDA水平明显降低(P <0. 05,P <0. 01),Trx2蛋白表达明显升高,TrxR2蛋白表达明显下降(P <0. 05)。结论:天王补心丹加减对失眠模型大鼠的改善作用与调控Trx抗氧化系统中Trx2与TrxR2的蛋白表达有关。
Objective: To explore the mechanism of modified Tianwang Buxindan on oxidative damage of Trx system in parachlorophenylalanine( PCPA) insomnia model rats. Method: Sixty male SD rats were randomly divided into blank group and model group. Insomnia model was prepared through intraperitoneal injection with PCPA( 150 mg·kg~(-1)). The discontinuous injection lasted for 7 d. After successful modeling,the rats were divided into model group( the same volume of normal saline),low,medium,high-dose Tianwang Buxindan groups( 8. 8,17. 6,35. 2 g·kg~(-1)·d~(-1)) and estazolam group( 0. 1 mg·kg~(-1)·d~(-1)),with 10 in each group. Autonomous activity video was used to detect circadian activity rhythm. Transmission electron microscopy( TEM) was used to observe supra chiasmatic nucleus( SCN) morphology and Organelle integrity. Enzyme-linked immunosorbent assay( ELISA) was used to detect the expressions of superoxide dismutase( SOD),malondialdehyde( MDA) and glutathione peroxidase( GSH-Px) in serum. Expressions of Trx2, TrxR2 in SCN cells were detected by immunofluorescence( IF) and Western blot. Result: Compared with blank group,the activity rhythm of model group was irregular,the activity time increased( P < 0. 01),the mitochondrial cristae were broken,the number was reduced,the arrangement was disorder,SOD and GSH-PX decreased,MDA increased( P < 0. 01),the expression of Trx2 decreased,while the expression of TrxR2 increased( P < 0. 01). Compared with model group,medium and high-dose Tianwang Buxindan groups could alleviate the circadian rhythm disorder and reduce the activity time( P < 0. 01),the mitochondrial edema was relieved,part of the cristae were intact,the activities of SOD and GSH-PX were increased,the level of MDA was reduced( P < 0. 05,P < 0. 01),expression of Trx2 was significantly increased,and expression of TrxR2 was significantly reduced( P < 0. 05,P < 0. 01). Conclusion:The effect of modified Tianwang Buxindan on insomnia model rats is related to the regulation of Trx2 and TrxR2 protein expressions in Trx system.
Objective: To explore the mechanism of modified Tianwang Buxindan on oxidative damage of Trx system in parachlorophenylalanine( PCPA) insomnia model rats. Method: Sixty male SD rats were randomly divided into blank group and model group. Insomnia model was prepared through intraperitoneal injection with PCPA( 150 mg·kg~(-1)). The discontinuous injection lasted for 7 d. After successful modeling,the rats were divided into model group( the same volume of normal saline),low,medium,high-dose Tianwang Buxindan groups( 8. 8,17. 6,35. 2 g·kg~(-1)·d~(-1)) and estazolam group( 0. 1 mg·kg~(-1)·d~(-1)),with 10 in each group. Autonomous activity video was used to detect circadian activity rhythm. Transmission electron microscopy( TEM) was used to observe supra chiasmatic nucleus( SCN) morphology and Organelle integrity. Enzyme-linked immunosorbent assay( ELISA) was used to detect the expressions of superoxide dismutase( SOD),malondialdehyde( MDA) and glutathione peroxidase( GSH-Px) in serum. Expressions of Trx2, TrxR2 in SCN cells were detected by immunofluorescence( IF) and Western blot. Result: Compared with blank group,the activity rhythm of model group was irregular,the activity time increased( P < 0. 01),the mitochondrial cristae were broken,the number was reduced,the arrangement was disorder,SOD and GSH-PX decreased,MDA increased( P < 0. 01),the expression of Trx2 decreased,while the expression of TrxR2 increased( P < 0. 01). Compared with model group,medium and high-dose Tianwang Buxindan groups could alleviate the circadian rhythm disorder and reduce the activity time( P < 0. 01),the mitochondrial edema was relieved,part of the cristae were intact,the activities of SOD and GSH-PX were increased,the level of MDA was reduced( P < 0. 05,P < 0. 01),expression of Trx2 was significantly increased,and expression of TrxR2 was significantly reduced( P < 0. 05,P < 0. 01). Conclusion:The effect of modified Tianwang Buxindan on insomnia model rats is related to the regulation of Trx2 and TrxR2 protein expressions in Trx system.
引文
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[2]中华医学会神经病学分会睡眠障碍学组.中国成人失眠诊断与治疗指南[J].中华神经科杂志,2012,45(7):534-540.
[3]Aggelopoulos N C,Meissl H.Responses of neurones of the rat suprachiasmatic nucleus to retinal illumination under photopic and scotopic conditions[J].J Physiol,2000,523(1):211-222.
[4]Vignatelli L,Masetti S,Amore M,et al.Insomnia cycling with a 42-day infradian period:evidence for two uncoupled circadian oscillators?[J].Sleep Med,2010,11(4):343-350.
[5]Patenaude A,Murthy M R,Mirault M E.Emerging roles of thioredoxin cycle enzymes in the central nervous system[J].Cell Mol Life Sci,2005,62(10):1063-1080.
[6]HUANG J,XU J,TIAN L,et al.A thioredoxin reductase and/or thioredoxin system-based mechanism for antioxidant effects of ambroxol[J].Biochimie,2014,97(1):92-103.
[7]Macdonald R,Barnes K,Hastings C,et al.Mitochondrial abnormalities in Parkinson's disease and Alzheimer's disease:can mitochondria be targeted therapeutically?[J].Biochem Soc Trans,2018,46(4):891-909.
[8]Ganguly G,Chakrabarti S,Chatterjee U,et al.Proteinopathy,oxidative stress and mitochondrial dysfunction:cross talk in Alzheimer's disease and Parkinson's disease[J].Drug Des Devel Ther,2017,11(11):797-810.
[9]黄攀攀,王平,李贵海,等.老年阴虚失眠动物模型的建立与评价[J].中华中医药学刊,2010,28(8):1719-1723.
[10]谢光璟,薄文集,黄攀攀,等.天王补心丹对慢性睡眠剥夺模型大鼠心肌、下丘脑视交叉上核VIP、AVP表达的影响[J].中华中医药学刊,2018,36(2):323-326.
[11]吴宝金,陈志勇.天王补心汤加减治疗阴虚火旺型失眠临床观察[J].中国中西医结合杂志,2017,37(9):1147-1148.
[12]胥丹,曲寿河,单爽,等.天王补心丹的现代临床应用[J].中国药剂学杂志:网络版,2018,16(2):26-34.
[13]王广艳,腾名子,朱君.天王补心丹加减治疗心房颤动合并冠心病阴虚火旺证的临床观察[J].中国实验方剂学杂志,2018,24(17):189-194.
[14]陈奇.中药药理研究方法学[M].2版.北京:人民卫生出版社,2006.
[15]李越峰,张泽国,徐福菊,等.白芍改善睡眠作用的药效物质基础研究[J].中国实验方剂学杂志,2014,20(15):127-130.
[16]Walsh J K,Deacon S,Dijk D J,et al.The selective extrasynaptic GABAA agonist,gaboxadol,improves traditional hypnotic efficacy measures and enhances slow wave activity in a model of transient insomnia[J].Sleep,2007,30(5):593-602.
[17]Lira A B,Rodrigues C F D S.Evaluation of oxidative stress markers in obstructive sleep apnea syndrome and additional antioxidant therapy:a review article[J].Sleep Breath,2016,20(4):1155-1160.
[18]Korcarz C E,Stein J H,Peppard P E,et al.Combined effects of sleep disordered breathing and metabolic syndrome on endothelial function:the wisconsin sleep cohort study[J].Sleep,2014,37(10):1707-1713.
[19]LU J,Holmgren A.The thioredoxin antioxidant system[J].Free Radic Biol Med,2014,66(8):75-87.
[20]Ramus S M,Cilensek I,Petrovic M G,et al.Single nucleotide polymorphisms in the Trx2/TXNIP and TrxR2genes of the mitochondrial thioredoxin antioxidant system and the risk of diabetic retinopathy in patients with type2 diabetes mellitus[J].J Diabetes Complications,2016,30(2):192-198.
[21]Mcevoy B,Sumayao R,Slattery C,et al.Cystine accumulation attenuates insulin release from the pancreaticβ-cell due to elevated oxidative stress and decreased ATP levels[J].J Physiol,2016,593(23):5167-5182.