Studies Of Lipopolysaccharide-induced Apoptosis In Goat Endometrial Epithelial Cells

Despite their importance in the breeding industry,ruminants often suffer huge economic losses due to endometritis,which have negatively affected its development.There are mainly gram-negative bacteria that cause intrauterine infection,such as Escherichia coli.These pathogens cause infection by secreting lipopolysaccharide(LPS).The processes of apoptosis and autophagy are both part of programmed cell death,the two are vastly different,but they are not completely independent processes.Many studies have shown that the roles of apoptosis and autophagy also influence and restrict each other in some cases.As an important autophagyrelated protein,the role of ATG7 in apoptosis of goat endometrial epithelial cells has not yet been studied.In this experiment,mice were perfused with LPS to construct an endometritis model.The goat endometrial epithelial immortalized cell line(g EECs)was used as the research object.,and the mechanism of ATG7 in goat endometrial epithelial cell apoptosis was explored under the action of LPS by silencing and overexpressing the ATG7 gene.Below are key research findings:1.The expression of ATG7 in the endometritis model of miceThe mouse uterus was perfused with 20 μL of 5 mg/m L LPS at 24 h postpartum to establish a mouse model of endometritis.Uterine tissue was removed 48 h postpartum.Sections were made and HE stained.The expression of ATG7 was detected by immunohistochemistry and Western blot.The results of the study are as follows:(1)The results of HE staining showed that the endometrium of LPS-treated mice had obvious inflammatory response;(2)In the immunohistochemical experiment,compared with the blank control group,the expression of ATG7 in the endometrium of LPS-treated mice increased;The results of Western blot showed that compared with the blank control group,the expression of ATG7 protein in the endometrium of LPS-treated mice was significantly increased(p < 0.001).2.Expression of ATG7 in LPS-induced apoptosis of g EECsIn g EECs,blank control groups and LPS-treated groups were separated.Total cell RNA and protein were isolated from cells stimulated with 2 μg/m L LPS for 6 hours,12 hours,and24 hours,respectively.To detect cell apoptosis,we used RT-q PCR and Western blot to detect changes in the m RNA and protein expression of apoptosis-related genes Bcl-2 and Bax,while TUNEL detected apoptosis rate.We also observed changes in m RNA and protein expression of ATG7 during apoptosis.The results of the study are as follows:(1)When g EECs were treated with 2 μg/m L LPS for 6 hours,12 hours,24 hours,and 24 hours,the expression of Bcl-2 m RNA and protein significantly decreased(p < 0.001).Bax m RNA and protein levels were unprecedentedly high(p < 0.001).An increased apoptosis rate was detected in TUNEL.(2)When g EECs were stimulated with 2 μg/m L LPS for 6 h,12 h,24 h,and 24 h,the m RNA and protein expressions of ATG7 were significantly changed.Therefore,select 2 μg/ml LPS to stimulate cells 24 h as the follow test conditions.3.The role of ATG7 in LPS-induced apoptosis of g EECs.Total RNA was extracted from goat endometrial epithelial tissue,reverse transcribed,and ATG7 with enzyme cleavage site was amplified with designed primers.The linearized p CD513 B and ATG7 were ligated,digested and sequenced to determine whether ATG7 was fully integrated into the backbone vector p CD513 B.According to the nucleotide sequence of the goat ATG7,an oligonucleotide was designed to interfere with the target site combination,and a Negative sequence was designed as a negative control.The interference sequence was synthesized by PCR.The linearized p CD513B-U6 and interfering sequences were ligated,identified by PCR and sequencing.Using Lipofectamine 2000,the viral particles were packaged into 293 T cells.Cell supernatant should be filtered and centrifuged for high transfection efficiency before being transferred to new centrifuge tubes,and g EECs were infected.The RT-q PCR and Western blot were performed to measure the efficiency of overexpression/interference.A 2 μg/m L concentration of LPS was used to stimulate the g EECs cells expressing/interfering with ATG7.We used RT-q PCR,Western blot,and TUNEL to examine ATG7’s role in the apoptosis of g EECs treated with LPS.Based on the findings of the study,the following conclusions were drawn:(1)Having constructed the ATG7 interference and overexpression lentiviral vectors,the lentivirus has been packaged.Infection of g EECs allowed the interference/overexpression efficiency to be verified and used for further experiments;(2)ATG7 overexpression may increase apoptosis through modulation of Bcl-2 m RNA and protein synthesis,enhancement of Bax m RNA and protein synthesis,and inhibition of Bcl-2m RNA and protein synthesis.At the same time,an increased apoptosis rate was detected in TUNEL.As a result of the interference with ATG7,the expressions of Bcl-2 m RNA and protein were increased,while those of Bax were decreased.And an decreased apoptosis rate was detected in TUNEL.To summarize,two micrograms of LPS was administered for 24 hours to induce g EECs.It was determined that g EECs exhibited significantly higher expression of ATG7 following LPS-mediated apoptosis.Additionally,ATG7 overexpression was found to contribute to goat epithelial cells’ apoptosis induced by LPS.Also,it has been determined that ATG7 interference inhibits goat endometrial epithelial cells’ ability to undergo LPS-mediated apoptosis.This study laid the theoretical foundation for follow-up uterineitis treatment.