The Regulatory Effect Of "Jinggong Formula" And Monome Of Chinese Herb On Inflammation Of Sheep Endometrial Epithelial Cells

Sheep plays an important role in the breeding industry,but the frequent occurrence of endometritis restricts the development of sheep industry.At present,the main treatment for endometritis in sheep is antibiotic therapy,but the use of antibiotics in large quantities will cause the emergence of drug-resistant bacteria,causing adverse effects on human production and life.However,More and more researchers focus on the exploration and development of traditional Chinese medicine due to its characteristics such as easy access,no drug resistance and no rest period.Therefore,the purpose of this paper is to invent a prescription of traditional Chinese herbal which can be used for the prevention and treatment of sheep endometritis,and explore the possible anti-inflammatory mechanism of its active components,so as to provide a new method and theoretical basis for the clinical prevention and treatment of sheep endometritis.1.Sheep endometrial epithelial cells were isolated and cultured by enzyme digestion and differential digestion respectively,and CK18 expression was detected by IF method for cell identification.E.coli with different MOI were used to infect sheep endometrial epithelial cells,and the optimal MOI of E.coli in the in vitro model of endometritis was selected by observing the cell morphology,detecting the level of cell apoptosis and exploring the expression of inflammation-related genes.The results showed that the endometrial epithelial cells of sheep were isolated and purified successfully.The expression rate of CK18 was higher than 95% detected by the IF method.In addition,when the MOI of E.coli was 1:10,the cell morphology and the proportion of apoptosis did not change significantly,and the expression level of inflammatory factors showed no significant increase.When the MOI of E.coli was 1:1,there was no significant changes in cell morphology,only a small amount of vacuoles were appeared.The level of cell apoptosis was not significantly increased,while the expression of inflammatory factors was significantly increased.However,when MOI exceeded 5:1,the expression of inflammatory factors increased significantly,but the cell morphology obviously changed,with transparency,shrinkage and even shedding of cells,and the proportion of apoptosis also increased significantly.2.The semi-bionic enzyme method was used to extract the effective constituents of “Jinggong Formula”,and the regulatory effect of the “Jinggong Formula” extraction on the inflammatory response of endometrial epithelial cells induced by E.coli was detected by qPCR,Western Blot and ELISA.The results showed that the extraction of “Jinggong Formula” had no acute toxicity,and reduced the mRNA and protein expression of TLR4/NF-κB pathway related gene and the secretion of inflammatory factors in sheep endometrium epithelial cells induced by E.coli.3.QPCR,Western Blot,ELISA,IF and YF? 488-annexinv and PI double staining were used to detect the regulatory effect of Astragalin and chlorogenic acid(CGA)on the inflammatory response of endometrial epithelial cells induced by E.coli and its potential mechanism.The results showed that E.coli-infection can significantly increase the expression of related gene mRNA and protein in the TLR4/NF-κB signaling pathway,promote the phosphorylation and nuclear translocation of NF-κB,and cause cell apoptosis.However,after pretreatment with Astragalin and CGA,the mRNA and protein expression levels of genes in TLR4/NF-κB signaling pathway were significantly reduced,the phosphorylation and nuclear translocation of NF-κB were inhibited,the secretion of inflammatory factors was significantly reduced,and the proportion of apoptotic cells was also significantly decreased.The results of this study suggested that the endometrial epithelial cells of sheep were successfully isolated and purified.An in vitro model of sheep endometritis was established successfully,and the optimal MOI of E.coli was 1:1.In addition,it has been proved that the “Jinggong Formula” extraction can effectively alleviate the inflammation of sheep endometrial epithelial cells induced by E.coli.Besides,it has been demonstrated that Astragalin(100 μg/mL)and CGA(50 μg/mL)have obvious anti-inflammatory effects on E.coli induced inflammation of sheep endometrial epithelial cells,and their potential mechanism is to inhibit the expression of TLR4-mediated NF-κB signaling pathway by downregulating the expression of TLR4.