Isorhamnetin (ISO) is an active flavonoid compound mainly isolated from the fruits of Hippophae rhamnoides L. and the leaves of Ginkgo biloba L. Previous studies have revealed the antifibrotic action of ISO in the liver and lungs, although its potential protective effects against renal fibrosis and the underlying mechanisms are still poorly understood. Given that many actions of ISO could be similarly attained by hydrogen sulfide (H₂S), we speculated that ISO may work through the induction of endogenous H₂S. To test the hypothesis, we established the unilateral ureteral obstruction (UUO) renal fibrosis rat model and transforming growth factor-β1(TGF-β1)-induced fibrosis in cultured renal tubular cells. ISO treatment inhibited epithelial–mesenchymal transition (EMT) formation, decreased extracellular matrix (ECM) deposition, and relieved renal fibrosis. Further analysis revealed that ISO stimulated the expression of the H₂S-synthesizing enzyme cystathionine lyase (CSE) and cystathionine beta-synthase (CBS), and promoted H₂S production in vivo and in vitro. The elevated H₂S attenuated oxidative stress and elevated the thiol level. It induced Keap1 sulfhydration, disrupted Keap1-Nrf2 interaction, and promoted the entry of Nrf2 into the nucleus. Finally, we found that circulating H₂S mainly derived from the liver, and not the kidney. Collectively, our study revealed that ISO alleviated renal fibrosis by inducing endogenous H₂S and regulating Keap1-Nrf2 interaction through sulfhydration of Keap1. Endogenous H₂S could be an important mediator underlying the pharmacological actions of ISO. Due to the multifunctional properties of H₂S, the H₂S-inducing nature of ISO could be exploited to treat various diseases.

Background/Objectives: As a non-medicinal part resource of Ziziphus jujuba, this study focuses on the total flavonoids from Ziziphus jujuba mesocarp (TFZJM), aiming to optimize the extraction process and explore its sedative and hypnotic effects. Methods: The extraction process of TFZJM was optimized by using single-factor experiments and the Box-Behnken response surface design method. The material basis of TFZJM was analyzed using Ultra-Performance Liquid Chromatography-Quadrupole-Time of Flight-Mass Spectrometry (UPLC-Q-TOF-MS). The mouse insomnia model was induced by intraperitoneal injection of PCPA, and the effects of TFZJM on this model and its potential mechanism were evaluated using multiple methods, such as sleep enhancement induced by pentobarbital sodium, HE staining of tissue sections, ELISA, RT-PCR, WB, and serum metabolomics. Results: The results showed that by optimizing the extraction conditions, a solid-liquid ratio (SLR) of 1:25 g·mL⁻¹, ethanol concentration of 60%, extraction time of 60 min, and extraction rate of 1.98% were achieved. The common chemical basis of the 10 flavonoid components was identified using UPLC-Q-TOF-MS analysis. Compared with the model group, the high-dose TFZJM (TFZJM-H) group had the most significant effect, followed by the medium-dose (TFZJM-M) and low-dose (TFZJM-L) groups. Conclusions: Metabolomic analysis revealed that TFZJM regulates pathways related to the metabolism of phenylalanine, tyrosine, cytochrome P450, and alanine. This lays the foundation for further exploration of the active substances and mechanisms of action of TFZJM in sedation and hypnosis.