Protective effect of the total flavonoids from Clinopodium chinense against LPS-induced mice endometritis by inhibiting NLRP3 inflammasome-mediated pyroptosis

Ethnopharmacological relevance: Clinopodium chinense (Benth.) O. Kuntze (C. chinense) is a Chinese herbal medi-cine used in treating gynecological hemorrhagic diseases for hundreds of years. Flavonoids are one kind of the major components in C. chinense. The flavonoids of C. chinense (TFC) have a vital role in treating endometritis but the underlying therapeutic mechanisms of TFC against endometritis have been rarely reported.Aim of the study: To elucidate the therapeutic effect and possible mechanisms of TFC against lipopolysaccharide (LPS)-induced endometritis in vivo and LPS-induced primary mouse endometrial epithelial cells (MEECs) injury in vitro.Materials and methods: The holistic phytochemicals of the TFC and TFC-contained serum were screened and identified using UPLC-Q-TOF-MS. The model of endometritis was established by intrauterine injection of LPS (5 mg/mL) into female BALB/c mice, and the model mice were treated with TFC for 7 days. The value of MPO was measured by Myeloperoxidase assay kit, the pathological changes in the endometrium were evaluated using H&E staining and transmission electron microscope (TEM), the secretions of IL-18, IL-1 beta and TNF-alpha were determined by ELISA kits, the mRNA expressions of IL-18, IL-1 beta and TNF-alpha were determined by RT-PCR assay, and the protein levels of TLR4, IKB alpha, p-IKB alpha, p65, p-p65, caspase-1, ASC, NLRP3 and GSDMD were measured by Western blot. Subsequently, MEECs were isolated from the uterus of pregnant female mice, injured by LPS for 24 h and incubated with the TFC-contained serum. Finally, cell viability, LDH release, hoechst 33342/PI staining, immunofluorescence staining, scanning electron microscope observation, ELISA assay, RT-PCR detection and Western blot analysis were carried out to further validate the therapeutic effect and the underlying mechanisms of TFC.Results: A total of 6 compounds in the plasma of mice after being intragastric administrated of TFC were iden-tified. The results in vivo showed that TFC significantly reduced MPO value and alleviated pathological injury of the endometrium. Furthermore, TFC significantly decreased the serum IL-18, IL-1 beta and TNF-alpha levels, and the mRNA levels of IL-18, IL-1 beta and TNF-alpha. TFC also inhibited the expressions of TLR4, p-IKB alpha, p-p65, caspase-1, ASC, NLRP3 and GSDMD. Besides, compared with the model group in MEECs cells, TFC-contained serum pre-vented pyroptosis, decreased the levels of IL-18 and IL-1 beta, and inhibited the mRNA expressions of IL-18, IL-1 beta and GSDMD. TFC-contained serum also reversed the activation of NLRP3 inflammasome caused by nigericin, and restrainted the translocation of NF-kappa B into nuclear.Conclusions: TFC protects mice endometritis from the injury of LPS via suppressing the activation of NLRP3 inflammasome and pyroptosis, the underlying mechanisms of which were related to restraining the TLR4/NF-kappa B/ NLRP3 pathway activation.