Sodium Houttuyfonate Inhibits Inflammation By Blocking The MAPKs/NF-κB Signaling Pathways In Cow Endometrial Epithelial Cells

Endometritis of cows are inflammation which infected by pathogenic microorganism. E.coli is one of the major pathogenic bacteria, but pathogenesis of endometritis is not very clear. Sodium houttuyfonate (SH) is a sodium bisulfite addition compound of active ingredients cordate houttuynia cordate houttuynia element, which has strong stability and is used to treat inflammatory diseases in clinically. But the molecular mechanism of SH combat inflammation disease is still not clear. We assume that the NF-κB as a key to mediate, we want to study its anti-inflammatory molecular mechanisms in cell level. The curative effect of endometritis model of mouse and cow endometritis were observed in vivo. To further study the anti-inflammatory activity and its pharmacological mechanism of SH.In this study, we applied primary cultured cells in vitro, separated and identified the cow endometrial epithelial and stromal cells. And the training for primary cell morphological observation of different dyeing methods and projection electron microscope. On this basis, we tried to assess the anti-inflammatory effects of SH on LPS-induced bovine endometrial epithelial cell (bEEC) inflammation. We investigated the cytotoxic effect of SH on bEEC viability using the MTT and LDH assays. Quantification of TNF-α, IL-1β、IL-6and IL-8protein levels and mRNA were carried out using ELISA and qRT-PCR, respectively. The protein expression of MAPKs/NF-κB signaling passway, ERK, JNK, p38,IκBα and p65were assessed by western blotting. The treatment effect of SH to mice and cows endometritis was using clinical diagnosis.Our studies showed that the high purity bEECs was isolated by using tissue culture and composite enzyme digestion technology, which provides enough cells for subsequent experiments. bovine endometrial cells was showed clear structure, cytoplasm and cell membrane by different dyeing methods. Cultured endometrial epithelial cells can be clearly seen microvillus in vitro by ultrastructure observation; Intracellular organelles clearly visible. Cytotoxicity experiments have shown that SH was not cytotoxic to bEECs at concentrations up to120μg/mL. SH (15,30or60μg/mL) significantly suppressed the LPS-induced bEECs TNF-α, IL-1β, IL-6and IL-8, in a dose-dependent manner. SH inhibited IμBα degradation, p65phosphorylation and MAPKs’phosphorylation in LPS-induced bEECs. In vivo, curative effect experiments confirmed that SH can significantly reduce the inflammatory reaction of endometrium in mice model, the uterus index was not significant difference between the model group and the normal group, microscopy showed that uterus tissue microstructure was basic normal, SH has a good therapeutic effect in experimental endometritis model mice. Conception rate was reached86%with SH-treated in clinical endometritis, oestrus accounted for91.7%of the total numbers. Fecundability is50%and cow estrus capita accounted for67%of the total numbers with the treatment of terramycin. In additional, SH treatment effect is better than terramycin.