[Mechanism of Liuwei Dihuang Pills in treatment of mice with diminished ovarian reserve based on proteomics].

This study aims to investigate the efficacy and possible mechanism of Liuwei Dihuang Pills in the treatment of diminished ovarian reserve(DOR) by using proteomic techniques. Firstly, cyclophosphamide(60 mg·kg~(-1)) combined with busulfan(6 mg·kg~(-1)) was injected intraperitoneally to establish the mouse model of DOR. After drug injection, the mice were continuously observed and the success of modeling was evaluated by the disturbance of the estrous cycle. After successful modeling, the mice were administrated with the suspension of Liuwei Dihuang Pills by gavage for 28 days. At the end of the gavage, four female mice were selected and caged together with males at a ratio of 2∶1 for the determination of the pregnancy rate. Blood and ovary samples were collected from the remaining mice on the next day after the end of gavage. Hematoxylin-eosin(HE) staining and transmission electron microscopy(TEM) were then employed to observe the morphological and ultrastructural changes in the ovaries. The serum levels of hormones and oxidation indicators were measured by enzyme-linked immunosorbent assay. Quantitative proteomics techniques were used to compare the ovarian protein expression before and after modeling and before and after the intervention with Liuwei Dihuang Pills. The results showed that Liuwei Dihuang Pills regulated the estrous cycle of DOR mice, elevated the serum levels of hormones and anti-oxidation indicators, promoted follicle development, protected the mitochondrial morphology of ovarian granulosa cells, and increased the litter size and survival of DOR mice. Furthermore, Liuwei Dihuang Pills negatively regulated the expression of 12 differentially expressed proteins associated with DOR, which were mainly involved in lipid catabolism, inflammatory response, immune regulation, and coenzyme biosynthesis. These differentially expressed proteins were significantly enriched in sphingolipid metabolism, arachidonic acid metabolism, ribosomes, ferroptosis, and cGMP-PKG signaling pathway. In summary, the occurrence of DOR and the treatment of DOR with Liuwei Dihuang Pills are associated with multiple biological pathways, mainly including oxidative stress response, inflammatory response, and immune regulation. "Mitochondria-oxidative stress-apoptosis" is the key to the treatment of DOR by Liuwei Dihuang Pills. YY1 and CYP4F3 may be the key upstream targets that trigger mitochondrial dysfunction and ROS accumulation, and the metabolism of arachidonic acid is the main signaling pathway of drug action.

TKF, a mexicanolide-type limonoid derivative, suppressed hepatic stellate cells activation and liver fibrosis through inhibition of the YAP/Notch3 pathway.

BACKGROUND: Liver fibrosis is a common scarring response and may ultimately lead to liver cancer, unfortunately, there is currently no effective antifibrotic drug approved for human use. Limonoids exhibit a broad spectrum of biological activities; however, the potential role of limonoids against fibrosis is largely unknown. PURPOSE: This study investigates the antifibrotic activities and potential mechanisms of TKF (3-tigloyl-khasenegasin F), a natural mexicanolide-type limonoid derivative. STUDY DESIGN/METHODS: Two well-established mouse models (CCl4 challenge and bile duct ligation) were used to assess anti-fibrotic effects of TKF in vivo. Human hepatic stellate cell (HSC) line LX-2 and mouse primary hepatic stellate cells (pHSCs) also served as in vitro liver fibrosis models. RESULT: TKF administration significantly attenuated hepatic histopathological injury and collagen accumulation and suppressed fibrogenesis-associated gene expression including Col1a1, Acta2, and Timp1. In LX-2 cells and mouse pHSCs, TKF dose-dependently suppressed HSC activation and the expression levels of fibrogenic markers. Mechanistic studies showed that TKF inhibited Notch3-Hes1 and YAP signalings in vivo and in vitro. Furthermore, YAP inhibition or knockdown downregulated the Notch3 expression; however, Notch3 inhibition or knockdown did not affect the level of YAP in activated HSC. We revealed that TKF inhibited Notch3-Hes1 activation and downregulated hepatic fibrogenic gene expression via inhibiting YAP. CONCLUSION: The therapeutic benefit of TKF against liver fibrosis results from inhibition of YAP and Notch3-Hes1 pathways, indicating that TKF may be a novel therapeutic candidate for liver fibrosis.