Effect of Suan Zao Ren (Semen Ziziphi Spinosae) Extract on the TXNIP/NLRP3 Pathway in Insomniac Rats
Authors: Xu Zijing , Xiong Wei , Wang Qian , Li Shuyu , Jia Dexian 
Published: September 2024

Abstract
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 the mechanism 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 500 mg·kg−1 for six consecutive days, with daily cage exchange. After 6 days, 40 successfully modeled rats were randomly divided into five groups: the model group (equal volume of distilled water), the positive group (0.75 mg·kg−1), and low-, medium-, and high-dose SZS extract groups (1.5, 3, and 6 g·kg−1, 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.