Effect Of Progesterone On Lipolysis Of Lipid Droplets And Prostaglandin E2 Synthesis In Mice Cervical Epithelial Cells

Progesterone（P4）is the most important steroid hormone during pregnancy.There are two main ways by which it acts on cells:I)the classical pathway,in which the ligand-bound receptor enters the nucleus and controls transcriptional translation of genes.II)the non-classical pathway,which regulates intracellular material metabolism and transport through cell signaling pathways.Lipid droplet（LD）is an organelle composed of a single layer of phospholipid membrane with a neutral lipid as the core.It is found in almost all cell types.With the development of the research on LDs,many functional proteins inserted in the membrane of LDs have been found to control the relationship between LDs and other organelles in cells,and participate in the metabolism and transport of intracellular substances,as well as the process of signal transduction.Some researchers have found that P4 can promote the synthesis of PGE₂ and inhibit the synthesis of PGF_2αin bovine endometrial epithelial cells,but the origin of substrate,arachidonic acid（AA）,for prostaglandin synthesis is less known.The purpose of this study was to demonstrate whether P4 can promote the lipolysis of LDs in mice cervical epithelial cells（MCECs）and produce AA to provide substrates for the synthesis of PGE₂.In this experiment,the LDs in MCECs were used as the research object.To avoid the influence of basal content and functional content of P4 in luteal phase,the cervix from estrous mice was collected for in vitro culture.The cultured mouse cervical cells were labeled with immunofluorescent CK18 and progesterone receptor（PR）to identify the presence of epithelia.The results showed that the primary cultured cells were really epithelia for our requirement.The MCECs were treated with various levels of P4（0,5,10 and 20nM）for 24h.（1）The confocal microscopy was used to observe the changes in LDs（labeled with BODIPY 493/503）numbers in MCECs after treatment with various levels of P4.The results showed that P4could promote lipolysis of LDs in MCECs.The lipolytic effect of P4（10nM）was most significant（P<0.01）.At 20nM,the effect of P4 on LDs lipolysis was reduced.（2）FAs（labeled with BODIPY 558/568（RedC12））in MCECs were co-localized to further analyze the dynamic changes of lipolysis of LDs in cells.The confocal results showed that FAs were released from LDs with the increase of P4 concentration and the content of FAs in cytoplasm was increased.At the 10nM of P4,the co-localization coefficient of FAs and LDs was decreased significantly（P<0.01）.（3）To determine and analyze the PKA/ERK1/2 pathway by western blotting（WB）,the results showed that,with the increase of P4 concentration,10nM of P4 was able to stimulate significantly the increase levels of PKA/ERK1/2 protein expression（P<0.05）,but 20nM of P4 had no significant effect.（4）By WB analysis,the result indicated that the expression levels of downstream proteins p-HSL and cPLA2 were increased,and extremely significant at 10nM of P4（P<0.01）.10nM of P4 was used to treat cervical epithelial cells of mice to observe the dynamic changes of HSL and cPLA2 in the cells.（5）By laser confocal co-localization,it was found that the red fluorescence reaction of HSL and cPLA2 was increased after 24h,and they accumulated towards the surface of LDs.The co-localization coefficient was increased significantly（P<0.01）.（6）After measuring the concentrations of AA and PGE₂ in MCECs with ELISA,the results showed that the levels of AA and PGE₂ were both increased significantly with the breakdown of LDs.（P<0.01）.（7）In order to demonstrate the role of the PKA/ERK1/2 signaling pathway in lipolysis of LDs,PKA inhibitor（H89）at 20μM was used to treat cells,the results indicated that downstream protein expression was significantly reduced after PKA was inhibited（P<0.01）,the metabolites AA and PGE₂ were also decreased significantly（P<0.05）.In summary,P4（no more than 10nM）could activate the PKA/ERK1/2 signaling pathway,enhance the expression levels of p-HSL and cPLA2 in the MCECs and promote their aggregation on the surface of the LDs.The neutral lipids from the dissolved LDs and the phospholipids on the membrane provide a large amount of arachidonic acid,substrates for the synthesis of PGE₂.