Effect of Suan Zao Ren (Semen Ziziphi Spinosae) Extract on the TXNIP/NLRP3 Pathway in Insomniac Rats

酸枣仁提取物对失眠大鼠TXNIP/NLRP3通路的影响

Objectives This study aimed to investigate the therapeutic effects of Suan Zao Ren(Semen Ziziphi Spinosae,SZS)extract on insomnia induced by p-chlorophenylalanine(PCPA)in rats and its influence on the thioredoxin-interacting protein(TXNIP)/nucleotide-binding domain Leucine-rich repeat and pyrin domain-containing receptor 3(NLRP3)inflammasome pathway,and to preliminarily explore themechanism by which SZS extract improves insomnia.Methods Fifty male Sprague–Dawley(SD)rats were used,with 8 rats in the blank group and 42 rats in the modeling group.The modeling group was induced by intraperitoneal injection of PCPA at a dose of 500mg·kg1 for six consecutive days,with daily cage exchange.After 6 days,40 successfully modeled rats were randomly divided into five groups:themodel group(equal volume of distilled water),the positive group(0.75 mg·kg1),and low-,medium-,and high-dose SZS extract groups(1.5,3,and 6 g·kg1,respectively),with 8 rats in each group.Treatments were administered for seven consecutive days.Enzyme-linked immunosorbent assay was used to measure levels of 5-hydroxytryptamine(5-HT)and gamma-aminobutyric acid(GABA)in the rat cerebral cortex.The thiobarbituric acid(TBA)method was used to determine malondialdehyde(MDA)levels,and the hydroxylamine method was used to determine superoxide dismutase(SOD)levels.The 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)method was used to measure total antioxidant capacity(TAOC)in the cerebral cortex.Pathological changes in the cerebral cortex were observed,and Western blot was used to detect the protein expressions of TXNIP,NLRP3,apoptosis-associated speck-like protein containing a Caspase activation and recruitment domain(CARD),and cysteine–aspartate-specific protease 1(Caspase-1)in the cerebral cortex.Results Compared with the blank group,the model group showed a significantly prolonged sleep latency(p<0.001)and a significantly shortened sleep duration(p<0.001).There were no changes in serum MDA and SOD levels.MDA levels in the cerebral cortex were significantly increased(p<0.001),while SOD and TAOC levels were significantly decreased(p<0.001).The 5-HT level was increased(p<0.05),and the GABA level was significantly decreased(p<0.001).SZS extract improved these conditions to varying degrees.Light microscopy showed no significant changes in cortical neurons but transmission electron microscopy revealed intact mitochondrial structures in the blank group,while the model group showed swollen and unclear mitochondria with reduced organelles.After 7 days of treatment,these conditions improved in the SZS extract groups.Compared with the blank group,the expressions of the four proteins in the model group were increased,and the expressions of these proteins were decreased in the SZS extract groups compared with the model group.Conclusion SZS extract may exert an antioxidant effect to treat insomnia by downregulating the expression of TXNIP/NLRP3 proteins and regulating oxidative stress levels in the cerebral cortex.

目的:探讨酸枣仁提取物对对氯苯丙氨酸(p-chlorophenylalanine,PCPA)诱导失眠大鼠的治疗效果及其对硫氧还蛋白相互作用蛋白(thioredoxin-interacting protein,TXNIP)/核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3(nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3,NLRP3)炎症通路的影响,并初步探讨酸枣仁提取物改善失眠的机制。方法:50只SD大鼠随机分为空白组8只,建模组42只。建模组连续6天通过腹腔注射500 mg·kg-1对氯苯丙氨酸建立失眠模型,并每日更换笼舍。6天后,40只成功建模的大鼠随机分为五组:模型组(等体积蒸馏水)、佐匹克隆组(0.75 mg·kg-1)及高(6 g·kg-1)、中(3 g·kg-1)、低(1.5 g·kg-1)剂量酸枣仁提取物组,每组8只大鼠。治疗持续7天。采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)测定大鼠大脑皮层中5-羟色胺(5-hydroxytryptamine,5-HT)和γ氨基丁酸(γaminobutyric acid,GABA)的水平;采用硫代巴比妥酸法(thiobarbituric acid method,TBA)测定丙二醛(malondialdehyde,MDA)水平;采用羟胺法测定超氧化物歧化酶(superoxide dismutase,SOD)水平;使用2,2′-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐[2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid),ABTS]测定脑皮层总抗氧化能力(total antioxidant capacity,TAOC);观察脑皮层的病理变化,并采用蛋白免疫印迹法(western blot,WB)检测脑皮层中TXNIP、NLRP3、凋亡相关斑点样蛋白质(apoptosis-associated speck-like protein containing a CARD,ASC)及半胱氨酸天冬氨酸蛋白酶1(cysteine-aspartate-specific protease,Caspase-1)的蛋白表达水平。结果:与空白组相比,模型组睡眠潜伏期显著延长(P<0.001),睡眠持续时间显著缩短(P<0.001),血清中MDA和SOD水平无明显变化;脑皮层中MDA水平显著增加(P<0.001),而SOD和TAOC水平显著降低(P<0.001);5-HT水平显著增加(P<0.05),GABA水平显著降低(P<0.001)。与模型组相比,酸枣仁提取物在不同程度上改善了这些情况。光学显微镜观察未见皮层神经元显著变化,但透射电子显微镜显示空白组的线粒体结构完整,而模型组线粒体肿胀且结构不清晰,细胞器减少。经7天治疗后,酸枣仁提取物组的这些情况有所改善。与空白组相比,模型组四种蛋白的表达水平显著增加(P<0.01);与模型组相比,酸枣仁提取物组的蛋白表达水平显著降低(P<0.05)。结论:酸枣仁提取物可能通过下调TXNIP/NLRP3通路相关蛋白的表达并调节脑皮层氧化应激水平发挥抗氧化作用以治疗失眠。