Research Of Astaxanthin On Inflammatory Response Mechanism Of Endometrial Cells In Dairy Cows

Endometritis is a common reproductive disease in dairy cows,it could cause serious economic losses to dairy farming industry.There are many factors contributing to endometritis in dairy cows,but the main factors are there:one is poor nutritional status of dairy cows and low autoimmunity,the other is that dairy cows are infected through pathogenic microorganisms.Preventing and controling the occurrence of endometritis in dairy cows has became to the focus of the research.The current main method is the use of antibiotics,but the overuse of antibiotics will lead to the increase of microbial resistance.In order to explore a new method to solve endometritis in dairy cows,Astaxanthin（AST）,which has strong antioxidant activity,was used to act on endometrial cells with established inflammation models to explore its effect and mechanism on cellular inflammatory response.It is mainly studied in the following three aspects:Trial 1:Establishment of an in vitro endometrial inflammation model1μg/mL lipopolysaccharide（LPS）was used to stimulate cow endometrial cells for 3 h,6 h,9 h and 12 h,respectively.The contents of interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）in cell culture medium were detected by ELISA,and the expression of Claudin1,CDH1,TJP1and other tight junction genes were detected to determine the optimal action time of LPS was 6 h,a model of endometrial cell inflammation was established.Trial 2:Effect of AST on LPS induced inflammation of bovine endometrial cells by protecting mucosal barrierWith the inflammation model of endometrial cells established in trial 1,after adding AST of1×10^-7 mol/L,1×10^-6 mol/L and 1×10^-5 mol/L into the inflammation model cells,the cells were treated for 3 h,6 h,9 h,12 h,24 h and 48 h,respectively.It was found that compared with the group without AST,adding AST of 1×10^-6 mol/L for 24 h in the cell culture medium,IL-6 and TNF-αwere the lowest（P<0.05）,and tight junction-related genes such as Claudin1,CDH1 and TJP1 were the highest（P<0.05）.The optimal concentration of AST was added to the model of endometrial cell inflammation,and the culture was continued for 24 h.It was found that the secretion levels of pro-inflammatory cytokines IL-6 and TNF-αin cell culture medium of LPS untreated group were significantly lower than those of LPS treated group（P<0.05）,and the secretion amounts of growth factors such as fibroblast growth factor（FGF）,epidermal growth factor（EGF）and transforming growth factor（TGF）were significantly higher than those of LPS treated group（P<0.05）,the intracellular levels of reactive oxygen species（ROS）were significantly decreased（P<0.05）,and the activities of oxidase superoxide dismutase（SOD）,catalase（CAT）were significantly higher in LPS-treated group than in LPS-treated group（P<0.05）.The fluorescence signals of Claudin1,CDH1,TJP1and other tight junction proteins located in the cell membrane were weakened,and their mRNA and protein expression levels were significantly reduced（P<0.05）.For inflammation model cells,Claudin1,CDH1,TJP1 protein fluorescence signal expression was up-regulated at the cell edge and nucleus of cells added with AST,and the levels of mRNA and protein expression of three tight junction proteins were significantly increased（P<0.05）.Trial 3:Effect of AST on LPS-induced bovine endometrial cell inflammation by inhibiting apoptosisWith the inflammation model of endometrial cells established in trial 1,the optimal concentration of AST was added to the inflammation model cells,and the cells were cultured for24 h.It was found that the secretion level of soluble FasL was significantly increased（P<0.05）,the apoptosis level of endometrial cells was significantly decreased（P<0.05）,and the expression of apoptosis-related genes and proteins,such as Bax,Bcl-2,Casp3,Fas and Casp8,was significantly different from that in LPS-treated group（P<0.05）,and the intracellular distribution and expression of apoptosis-related proteins were also significantly improved.In conclusion,AST can not only alleviate the inflammatory damage of endometrial cells in dairy cows by protecting the mucosal barrier,but also alleviate the LPS-induced inflammation of endometrial cells in dairy cows by inhibiting apoptosis,thereby alleviating the damage of endometrial cells caused by inflammation,playing a role of protecting endometrium and providing a new method for the prevention and treatment of endometritis in dairy cows.