| Pyometra is a common reproductive disease of the female dogs.Hormonal disorder and bacterial infections have been considered as the most basic predisposing factors.An increase level in progesterone and a decrease level in estrogen were observed in biches during the late estrus and dioestrus period when uterus was susceptible to infection,leading to endometritis,even pyometra.The endometrium is the target organ for hormone response.The combined effect of estrogen and progesterone causes many changes in the morphology and biological characteristics of endometrial cells.Recent studies have found that canine endometrial stromal cells（ESCs）play a key role in the development of pathology.Under normal physiological conditions,ESCs provide necessary nutrition support and information processing for epithelial cells,and cooperate in maintaining the stability of the intrauterine environment.However,when endometrial cells are abnormally activated,they will produce a large amount of inflammatory cytokines.The immunoregulatory effects of hormones on canine ESCs in an inflammatory environment are still poorly understood.In this experiment,epidemiological and follow-up investigations were conducted on the cases of canine pyometra,and then the ESCs were isolated and cultured in vitro to study the local function of the endometrium.LPS-induced cells were used to construct an inflammation model.Combined the concentrations of the serum estradiol（E2）and progesterone（P4）in the pyometra dogs obtained from clinical case analysis,we explored the regulatory effects of the two hormones on the uterine immune mechanism in the inflammatory state.The study aims to further clarify the molecular mechanism of uterine inflammation and the influence of estrogen and progesterone on innate mucosal immunity.The main research contents and results are as follows:1.Clinical case analysis of canine pyometra and changes of steroid hormonesThe purpose of this test is to systematically analyze the clinical pathogenesis of canine pyometra,explore the characteristics of steroid hormone changes,and provide clinical data for in-depth study of the pathogenesis of canine pyometra.From November 2018 to December 2019,a total of 44 outpatient medical records were collected from a number of pet hospitals in Nanjing,systematically sort out the clinical symptoms of the disease,diagnostic methods and results,and histopathologe of the uterus and ovaries was analysed,serum concentrations of estradiol and progesterone in pyometra dogs were detected,and the relationship between the age,breed,reproductive history,seasons and the onset of pyometra were analyzed The results show that pyometra mostly occur in medium and large dogs（59.09%）;the average age of pyometra dogs is 8.8 years old.Among them,poodles,golden retrievers,and hybrid dogs are high-risk breeds;70.45%of pyometra dogs have no reproductive history;pyometra dogs mostly show abnormalities in white blood cells,red blood cells and platelets,accompanied by liver and kidney dysfunction;pyometra dogs with ovarian cysts,open cervix pyometra are mainly endometrial cystic hyperplasia,and closed cervix pyometra are mainly glandular squamous metaplasia;peripheral blood serum detection showed that progesterone was extremely significantly higher than that in the normal group（P<0.01）,and estrogen had no differences（P>0.05）,suggesting that changes in steroid hormone levels may be closely related to canine pyometra.2.Effect of estradiol and progesterone on canine endometrial stromal cells cultured in vitroTo explore the regulation of E2 and P4 on the expression of TLR4 and related inflammatory factors in canine ESCs cultured in vitro.The collagenase digestion method was used to isolate and obtain the ESCs of healthy dogs and perform immunofluorescence identification;according to the serum E2 and P4 concentrations in peripheral blood of dogs with pyometra in the previous chapter,0.2 n M E2and 0.1μM P4were treated alone or cotreated with ESCs to detect cells proliferation by MTT;the expesssion of TLR4 and inflammatory factors were detected by q RT-PCR.The results showed that E2 had no prominent effect on the growth of ESCs in vitro.P4 treatment alone and co-treatment with E2and P4 could significantly promoted cell proliferation in vitro;Co-treatment of E2 and P4significantly increased the expression of TNF-αand IL-6（P<0.05）,but had no significant effect on the expression of IL-1βand IL-8（P>0.05）.This test proves that the proliferation of canine ESCs in vitro is regulated by E2 and P4,and P4 increases the expression of TLR4and inflammatory factors,and indicates that P4 induces endometrial inflammation.3.Effect of estradiol and progesterone on the inflammatory response of canine endometrial stromal cells induced by LPSThis experiment aims to establish an inflammation model of canine ESCs in vitro through lipopolysaccharide（LPS）induction,and explore the regulatory effects of E2 and P4on canine ESCs TLR4,inflammation-related inflammatory factors and NF-κB pathway.The concentration of bacterial lipopolysaccharide（LPS）most suitable for establishing an inflammation model was screened by MTT;cells were co-treatedwith 0.2n M E2,0.1μM P4and 1μg/m L LPS,q RT-PCR was used to detect the m RNA expression of TLR4 and inflammatory factors at 6 h,12 h,24 h in ESCs induced by LPS,and western blotting was used to detect the changes of key proteins in the NF-κB signaling pathway.The results showed that compared with the control group,TLR4,inflammatory factor expression and pathway protein phosphorylation levels in all inflammation model groups were increased（P<0.01）.Compared with the model group,E2 treatment could reduce the expression of IL-6and IL-8（P<0.05）,and P4 or E2+P4 cotreatment could inhibit TLR4,IL-1β,TNF-α,IL-6,IL-8 in varying degrees,with a time-dependent relationship.Both P4 and E2+P4 cotreatment inhibited the phosphorylation of pathway proteins IκBαand p65（P<0.05）.This test proves that E2 and P4 can down-regulate the expression of TLR4 and related inflammatory factors,inhibit the activation of ESCs signaling pathways induced by LPS,suppressing immune response The test provides information for studying the local immune regulation of canine uterus. |
