Study On The Change Of ANGPTL4 Expression In Mice With Endometritis

Objective: Endometritis is a common reproductive disorder of female animals.Generally due to its inflammatory products and bacterial toxins on the sperm production harm,so that the fertilized egg implantation were blocked or early embryo death was found,clinical manifestations of repeated female infertility or pregnancy abortion were observed.Endometritis is one of the important causes of female infertility and its pathogenesis is complex.Demand of dairy and meat products of consumption are larger and larger in today’s society.Endometritis leads to increase in female foetus time interval,milk production decline,and elimination rate,which greatly affects the production performance of a female,and brings direct economic losses to the breeding.Angiopoietin like protein 4(ANGPTL4)is a novel class of secretarial glycoproteins whose function is like angiopoietin and were identified and named by Kim et al in 2000.Studies at home and abroad have shown that ANGPTL4 is closely related to inflammation and is an important pro-inflammatory factor.But the current study did not show whether ANGPTL4 is associated with endometritis or not,therefore in the present study,mice model of endometritis was used to detect mRNA and protein expression levels of ANGPTL4,and to detect the inflammatory pathway gene hypoxia-inductive factor-1α in mice with endometritis(HIF-1α)and nuclear factor p65(NF-κBp65)mRNA expression in order to discuss the relationship between ANGPTL4 and endometritis and its inflammatory pathway.It also provides a new train of thought for the prevention and treatment of endometritis.Methods:In the first experiment,24 Kunming mice aged 8 weeks were randomly divided into four groups.The endometritis model was established by injecting saline,1mg/mL,3 mg/mL and 5 mg/mL lipopolysaccharide(LPS)50 μl each into the uterus of the mice by uterine perfusion.The success of the model was confirmed by clinical observation and necropsy,making histomathological sections and HE staining.According to the results of paraffin section microscopic examination,1 mg/mL LPS was selected as the optimal concentration for subsequent experiments.In the second experiment,20 Kunming mice aged 8 weeks were randomly divided into two groups.50 μL normal saline and 50 μL 1 mg/mL lipopolysaccharide(LPS)were injected into the uterus,respectively.The uterus of the two groups was collected 24 hours after infusing,and the clinical manifestations were observed.Uterine histopathological changes of the two groups were analyzed by autopsy.The mRNA expression level of ANGPTL4 in the uterus of the two groups was detected by fluorescence quantitative PCR.Western blotting was used to detect the protein expression of ANGPTL4 in the uterus of mice.In the third experiment,the experimental groups were the same as the second experiment.The uterus of mice in the two groups were infused with 50 μL normal saline and 50 μL 1 mg/mL LPS,respectively.The uterus of mice in the two groups were collected24 hours later,fluorescence relative quantitative PCR was used and 2(-△△ Ct)method was used to detect the mRNA expression levels of tumor necrosis factor α(TNF-α),interleukin-6(IL-6),ANGPTL4,HIF-1α and NF-κBp65 in uterine mice were detected in normal saline group and 1 mg/mL LPS group.Results: in the first experiment,after 24 hours of uterine perfusion,it was observed that spirit,appetite and reaction ability of mice in the normal saline group were normal and no changes were observed.The size of uterus was normal at necropsy,and the endometrium was intact in tissue sections.Mice in 1 mg/mL LPS group were depressed,had decreased appetite,rough hair,diarrhea,decreased body weight and reaction ability,and had obvious resistance to touching abdomen.At necropsy,uterine redness and swelling were visible.The endometrium was shed and incomplete,and a large number of neutrophils were observed.Mice in the 3 mg/mL LPS group were highly depressed,anorexia,severe diarrhea,emasement,and basically had no normal reaction ability.The uterine swelling was obvious at necropsy,and the endometrium was seriously broken and showed snowflake fragments in tissue sections.Mice in the 5 mg/mL LPS group died,uterine swelling was observed at necropsy,and the endometrium was severely shed in tissue sections.In the second experiment,24 hours after uterine perfusion,no significant changes were observed in the spirit,appetite and reaction ability of mice in the normal saline group.The size and color of the uterus were normal at necropsy,and the endometrium was intact in tissue sections.Mice in 1 mg/mL LPS group were depressed,had decreased appetite,increased drinking water,diarrhea,decreased reaction ability,and had obvious resistance to touching the abdomen.At necropsy,uterine redness and swelling were visible.Compared with the blank group,the expression of ANGPTL4 mRNA in uterus of mice in 1 mg/mL LPS group was significantly increased(P<0.05);Compared with the blank group,the expression level of ANGPTL4 protein in uterus of 1 mg/mL LPS group was significantly increased(P<0.001).In the third experiment,compared with the blank group,the expressions of IL-6mRNA,ANGPTL4 mRNA and TNF-α mRNA in uterus of mice in 1 mg/mL LPS group were significantly increased(P<0.01);The expression of HIF-1α mRNA was increased(P<0.05);mRNA expression of NF-κBp65 was significantly increased(P<0.001).Conclusions: The results of this study suggest that the up-regulated expression of ANGPTL4 in mouse endometritis may be related with the inflammation of mouse endometritis.The up-regulated expression of pathway genes HIF-1α and NF-κBp65 in mice with endometritis may be related to the up-regulated expression of ANGPTL4.