Study On Anti-Inflammatory Mechanism Of Salvia Miltiorrhiza Extract Against Endometrium Epithelial Cells Inflammation Induced By LPS

As the root cause of endometritis lied in the inflammatory changes of endometrium epithelial cells(EECs), so it was necessary to explore the damage of epithelial cell structure and function in the process of inflammation, which was helpful to better understand the nature of endometritis and to find the right medication for endometritis treatment. Salvia miltiorrhiza was one of the traditional Chinese medicines which was widely used in the treatment of endometritis in clinical, yet the report was less about Salvia miltiorrhiza modern pharmacology study on cell levels. In order to explore the protection mechanism of salvia miltiorrhiza extract(SME) on the inflammatory model induced by LPS, serial researches w ere carried out in this paper. 1.Screening the optimum inducement concentration and time of LPS on inflammation model.MTT was used to detect the proliferation of EECs after the treatment by LPS with different inducement concentration for 6h, 12 h, 24 h. And the result showed that the promote proliferation effect was most obvious when the concentration of LPS was 5μg/m L on the EECs 12 h. The optimization inflammatory model was established based on this, then the concentration of TNF-α and IL-1β were detected by ELISA kit and the result showed that the level of TNF-α and IL-1β in the LPS model group were obviously higher than the contrast group(P<0.01). Therefore, the optimal concentration and action time is 5μg/m L and 12 h. 2.Screening the optimum concentration of SME on EECs.MTT was used to detect the proliferation of EECs after the treatment by SME with six different concentrations for 24 h. And the result showed that SME did not produce obvious toxicity, and could promote the cell proliferation on EECs, so the final concentration of SME was determined, which was respectively 250μg/m L(high dose group), 100μg/m L(medium dose group) and 10μg/m L(low dose group).3.The influence of SME on protein expression of TNF-α, IL-1β, NO, PGE2, TLR4, CD14, MMP-2 and MMP-9 in goat EEC inflammation model induced by LPS.To observe the change of the indexes of EEC inflammation model induced by LPS which is treated respectively by SME in high, middle and low dose for 0h, 4h, 6h, 12 h, 18 h, 24 h and 36 h, the result reflected that TLR4, CD14,TNF-α, IL-1β,NO, PGE2,MMP-2 and MMP-9 proteins at each peak in the model group are significantly higher than the contrast group(P<0.01). And the indexes of these were signif icantly reduced with different concentrations of SME effecting on the inflammation model, and the high dose of SME was the most obvious(P<0.01).4.The influence of SME on related gene expression in goat EEC inflammation model induced by LPS.The related genes expression in the model of LPS induction inf lammation were detected by Real-time quantitative reverse transcription-PCR. And the results showed that the expression of TNF-α, IL-1β, TLR4, CD14, MMP-2 and MMP-9 m RNA in model group was significantly higher than that of control group(P<0.01). However, the inflammatory cell could reduce the expression of inflammatory cell factor TNF-α, IL-1β, TLR4, CD14 after treatment with high, medium and low dose of SME, especially the high dose SME effect is most obvious(P < 0.01). In addition, MMP-2 and MMP-9 expression gradually reduced with the extension of action time, and significantly lower than control after 24 h, but the trend gradually returned to normal after action of SME.In conclusion, EEC cell inflammatory reaction might be related that the activation TLR signaling pathways(TLR4, CD14) by LPS, then the up regulation of gene expression cytokines(TNF-α、IL-1β), inflammation mediated factors(NO, PGE2) and MMPs(MMP-2, MMP-9) expression. While the SME acted on the inflammatory cells, it could decrease the expression of inflammation mediated factor and TLR signaling pathways, then slowed the degree of inflammatory reaction. In addition, SME reduced the expression of MMP-2 and MMP-9 to inhibit endometrial cell structure degradation inflammatory damage in the ear ly inflammatory reaction, but it raised the expression of MMP-2 and MMP-9 in the late inf lammatory response, so as to improve the ability of uterus remodeling. These m ight be the mechanisms that LPS induced inflammatory reaction and anti-inflammatory effect of SME, but the specific mechanism still needed further research.