| Thymol is a monoterpenoid compound,and the anti-inflammatory effect is one of the important functions of its various pharmacological activities.Endometritis is a reproductive system disease caused by the infection of endometrium by pathogenic microorganisms,which brings great difficulties to the treatment of economic animals.As a substitute to antibiotics,the anti-inflammatory effect and mechanism of thymol on endometritis have been rarely reported.Therefore,this study investigated the inhibitory effect and mechanism of thymol on LPS-induced inflammatory response in goat endometrial epithelial cells(gEECs),and provided a scientific theoretical reference for the treatment of endometritis with Chinese herbal medicine.1.Effect of thymol on the proliferation of gEECs.The viability of gEECs was assessed by MTT assay.The morphological changes of gEECs were observed by Wright-Giemsa staining method,and the permeability of cell membrane was determined by LDH assay.The results showed that the safe concentration of thymol on gEECs was 100 ug/mL,and there was no significant effect of thymol on cell viability and morphology.But thymol above this concentration can significantly increase the membrane permeability of gEECs,causing cell damage and cytotoxicity.Therefore,thymol at concentrations of 100,50,and 25 μg/mL were selected for subsequent experiments.2.Effect of thymol on the secretion of inflammatory factors in LPS-induced gEECs.The experiment was divided into three groups: Control group(control);Inflammation model group(LPS);LPS + thymol group.The concentration of LPS was 5 μg/mL,and the concentrations of thymol were 100,50,and 25 μg/mL,respectively.The LPS-induced generation of NO was detected by NO kit,whereas LPS-induced production of inflammatory factors were measured by corresponding ELISA kits according to manufacturers respectively.The results showed that thymol inhibited LPS-induced production of inflammatory cytokines and MMPs to a significant extent,including TNF-α,IL-1β,IL-6,IL-10,NO,PGE2,MMP-2 and MMP-9.The results showed that thymol had a significant inhibitory effect on LPS-induced inflammatory response and reduced structural damage caused by excessive inflammatory response in gEECs.3.Effects of thymol on TLR4/NF-κB signaling pathway in LPS-induced gEECs.Gene expression of IL-1β,CD14、TLR4、TRIF(TICAM)and MyD88 were determined by RT-PCR respectively.Protein and gene expression of NF-κBp65 were determined by RT-PCR and WB respectively.Thymol prominently inhibited the expression of LPS-induced IL-1β,CD14,TLR4,TRIF(TICAM),and intensified the inhibitory effect as the concentration increased.However,thymol has no effect on the gene expression of MyD88.In addition,thymol down-regulated the gene and protein expression of NF-κB p65 in LPS-induced gEECs.The results revealed that the anti-inflammatory mechanism of thymol possibly involved in the inhibition of TRIF-dependent TLR4/NF-κB signaling pathway.In summary,thymol exerts anti-inflammatory activity by regulating the secretion of inflammatory factors.What’s more,it can also inhibit the TRIF-dependent TLR4/NF-κB signaling pathway,and has no significant toxicity to gEECs.Therefore,thymol may be a potential therapeutic agent against endometritis. |
