Effect Of Fisetin On Inflammation And Oxidative Stress Of LTA-induced Endometritis

Bovine endometritis is one of the common reproductive diseases in breeding,affecting the estrous cycle and reducing reproductive efficiency.Bacterial infections are common,including E.coli and Staphylococcus aureus,which can cause subclinical or chronic endometritis.Lipoteichoic acid(LTA)is found in the cell wall of Staphylococcus aureus and plays a key role in inducing disease.Bovine endometrial epithelial cells(BEECs)are the primary defense line of endometrial resistance,and inflammatory reactions occur under various stimuli,affecting the reproductive performance of cattle.Antibiotics are widely used in clinical practice,but overuse will lead to drug resistance,drug metabolism and other problems,need to find new alternative drugs.In recent years,a number of studies have found that fisetin,as a natural flavonoid,has antioxidant,anti-inflammatory and other biological activities,which can be used to prevent pneumonia,mastitis and other diseases.Based on the above,in this study,BEECs were used to construct an in vitro model and mice were tested in vivo to investigate whether fisetin can alleviate LTA-induced inflammation and oxidative stress responses.Specific research contents are as follows.(1)Establishment of bovine endometrial epithelial cell culture with in vitro oxidative stress and inflammation modelBovine endometrial epithelial cells were cultured and their morphology was "paving stone" after adherent.Immunofluorescence results showed that keratin 18(CK-18)expression in epithelial cells was positive,and the cell line was pure,which laid the foundation for subsequent experiments.The concentration of LTA was detected by CCK-8.25 μg/m L will significantly influence the cell vitality;Reactive oxygen species(ROS)and Western blot,RT-q PCR reaction further findings: 25 μg/m L of LTA induced cells after 48 h ROS,TNF-α,IL-1β expression level increased significantly.Composite showed that the concentration of 25 μ g/m L of LTA induced 48 h to build cells in vitro cell model of inflammation and oxidative stress.(2)The effect of fisetin on the regulation of oxidative stress and inflammatory response in LTA-stimulated bovine endometrial epithelial cellsFirst detected by CCK 8 fisetin affected the cell vitality,found that fisetin concentration acuity 100 μg/m L will significantly reduce the cell vitality,when the test is divided into 6 groups(n=3),respectively in the control group,the LTA group,LTA+fisetin(12.5,25,50 μg/m L)group,the fisetin group(50 μg/m L),The regulatory effects of fisetin were analyzed by RT-qPCR and Western blot.It was found that fisetin at different concentrations could inhibit the expression of TLR2 induced by LTA and the activation of NF-κB in the downstream,reduce the secretion of TNF-α and IL-1β,promote the expression of Nrf2 and NQO1 and SOD in the downstream,and reduce the accumulation of ROS and MDA.These results indicated that fisetin alleviates LTA-induced inflammation and oxidative stress in bovine endometrial epithelial cells.(3)The effect of fisetin on the regulation of oxidative stress and inflammatory response in the endometrium of mice under LTA stimulationKunming mice were tested in vivo.5 mg/kg LTA solution was injected into the uterus by uterine perfusion method for 24 h,and then different doses of fisetin solution(25 mg/kg and 50 mg/kg)were injected intraperitoneally for 24 h.The mice were killed at the neck and uterine samples were collected.H&E staining,RT-q PCR,Western blot method for sample analysis.The results showed that the uterus was enlarged and shortened,and more neutrophils infiltrated the endometrial epithelial cells and deep structures after LTA perfusion.Activation of TLR2-mediated NF-κB pathway increases the expression of MDA and pro-inflammatory cytokines TNF-α and IL-1β.After different doses of fisetin,the uterine morphology of mice gradually returned to normal,and neutrophil infiltration in endometrial epithelial cells decreased.Inhibit the activation of NF-κB pathway and reduce the production of TNF-α and IL-1β.Promote the expression of Nrf2,increase the expression level of antioxidant enzymes such as NQO1 and SOD,and reduce the accumulation of MDA.In conclusion,fisetin alleviates LTA induced endometrial inflammation and oxidative damage in mice.In conclusion,fisetin can relieve LTA-induced endometrial inflammation and oxidative stress by inhibiting the activation of NF-κB signaling pathway,reducing the secretion of pro-inflammatory cytokines TNF-α and IL-1β,increasing the expression of Nrf2 and downstream antioxidant enzymes such as NQO1 and SOD,and reducing the accumulation of ROS and MDA.