Clinopodium chinense extract alleviates ferroptosis of endometrial epithelial cells via regulating TLR4/SLC7A11 signaling pathway in lipopolysaccharide-induced endometritis.

BACKGROUND

Clinopodium chinense (Benth.) O. Kuntze (C. chinense) is one of the traditional Chinese medicine that has been used in treating gynecological bleeding. Recent research showed that total extract of C. chinense (TEC) played an important role in the treatment of endometritis, but the mechanism of which need to be further revealed. Ferroptosis has served as a pathogenic mechanism in endometritis. The aim of this experiment was to clarify whether TEC ameliorates endometritis by attenuating ferroptosis and to elucidate its underlying mechanism.

PURPOSE: The purpose of this research is to investigate the therapeutic effect and underlying mechanism of TEC suppressing ferroptosis of endometrial epithelial cells in endometritis.

METHODS

The mouse model of endometritis was established via intrauterine injection of lipopolysaccharide (LPS, 5 mg/ml) into female mice. Primary mouse endometrial epithelial cells (MEECs) were isolated from pregnant female mice and subjected to in vitro experiments. High-resolution mass spectrometry (HRMS) identified the bioactive ingredients in TEC. Transcriptomic and proteomic analyses revealed the potential targets of TEC against endometritis. Specifically, HE staining evaluated the pathological changes in the uterus. Immunohistochemistry staining assayed the expression of CD138, SLC7A11 and GPX4. The mitochondrial morphology was observed by transmission electron microscope (TEM). Prussian blue staining and Ferro orange staining evaluated the content of Fe2+. The contents of ROS, Fe2+, LDH, GSH, MPO and pro-inflammatory cytokine levels including IL-1β, IL-6, TNF-α were detected using kits. Molecular docking evaluated the binding activities between active compounds of TEC and potential targets. Mechanistically, the expression of TLR4, p-p53, SLC7A11, ACSL4 and GPX4 were evaluated using western blotting or immunofluorescence staining.

RESULTS

The nine active compounds in TEC were detected. Transcriptomic and proteomic analyses showed that TEC may alleviate endometritis by attenuating ferroptosis. The pharmacological results demonstrated that TEC alleviated endometrial pathological damage, decreased CD138 expression, reduced MPO activity and pro-inflammatory cytokine levels. TEC also diminished mitochondrial shrinkage and increased membrane integrity in uterine tissue. And, TEC decreased ROS, Fe2+, GSH contents, and suppressed ferroptosis of endometrial epithelial cells. Furthermore, the molecular docking technique revealed that hesperidin, didymin and quercetin, which were the major active components of TEC could bind to TLR4 and SLC7A11. Western blotting analysis further verified that TEC down-regulated TLR4, p-p53 and ACSL4 expression, up-regulated SLC7A11 and GPX4 expression.

CONCLUSIONS

TEC alleviates LPS-induced inflammatory damage in both mouse uterine tissues and MEECs by suppressing ferroptosis, the underlying mechanisms of which were related to inhibiting TLR4/SLC7A11 signaling pathway.