A Study On Lipopolysaccharide-induced Porcine Granulosa Cells Proliferation Is Mediated By TNF?-ERK1/2 Signaling Pathway-regulated Serpin E1 And Serpin B2

Granulosa cells,between the membrane cells and the egg,are follicular cells within the basement membrane of the ovarian follicles that can convert the androgen into estradiol.Together with its secretion of other growth factors and nutrients,it plays an important role in promoting the development of the egg.Dysfunction of the granulosa cells will directly lead to declines in animal reproductive performance.As a component of the cell wall of gram-negative bacteria,LPS can directly act on granulosa cells and affect their functions,and seriously affect the reproductive performance of animals.In order to further explore the impact of LPS on the development and function of porcine granulosa cells,this study explores a new method for the isolation and cultivation of porcine granulosa cells in vitro,and on this basis,the effects of LPS on the viability of granulosa cells and the molecular regulation mechanisms.1.Isolation and purification of granulosa cellsTo overcome the problems of low adhesion rate and cell viability in culture when granulosa cells were obtained via direct extraction of follicular contents.Following the conventional cell extraction step,FICOLL separation solution was applied to further purify the granulosa cells.Such purified cells were characterized by Western Blot,immunofluorescence staining,and ELISA analytical methods.The results demonstrated that the ability of the purified cells by FICOLL to attach to the plate,cell morphology and stereoscopic form were significantly improved.Not only the cells expressed FSHR and LHCGR genes,but also the genes involved in steroidogenesis such as CYP11A1,CYP19A1,and St AR.For hormone secretion,E2 secretion was significantly increased,while P4 secretion was significantly decreased.These results indicated that the purified porcine granulosa cells are more in line with the requirements of granulosa cells in vitro.2.The molecular mechanism of porcine granulosa cells proliferation by LPS treatmentPrevious studies have demonstrated that long term exposure of granulosa cells to LPS leads to inflammatory response and inhibition of E2 secretions.However,the effects of acute exposure to LPS on the viability of granulosa cells have been rarely studied and remain largely unknown.In this study,purified porcine granulosa cells were used to reveal the potential molecular mechanism of the viability after LPS treatment through CCK-8,cell flow cytometry,Western Blot,and quantitative gene detection.Results showed that granulosa cells proliferation and the TNF-? secretion were significantly increased on exposure to LPS(500ng/ml,1000ng/ml,2000ng/ml).To prove whether TNF-a mediates LPS to promote the proliferation of porcine granulosa cells,we used the TNF-? shears enzyme inhibitor ADAM17 and the addition of recombinant TNF-?(5ng/ml,10ng/ml)alone.The results show that ADAM17 can significantly inhibit the proliferative effect of LPS;and granulosa cells proliferation induced by LPS can be mimicked by the treatment with recombinant TNF-?.This result proves that TNF-a mediates the promoting effect of LPS on the proliferation of porcine granulosa cells.To further explore its molecular mechanism,we found that the expression of Serpin E1 and Serpin B2 in granulosa cells increased due to treatment with LPS or recombinant TNF-?,and with blocking the ERK1/2 signaling pathway,cell proliferation was completely inhibited and the expression of Serpin E1 or Serpin B2 induced.Further si RNA experiments found that completely inhibit the proliferation of porcine granulosa cells by knocking down Serpin E1 or Serpin B2 in the presence of LPS or recombinant TNF-?.Moreover,mitochondrial membrane potential(MMP)polarization and ATP levels in granulosa cells was increased by LPS or recombinant TNF-? treatment.Taken together,the porcine granulocytes purified by FICOLL separation solution in this study have better vigor and better reflect the characteristics of granulosa cells,so they are more in line with the in vitro studies of porcine granulosa cells.TNF-?-mediated upregulation of Serpin E1 and Serpin B2 through activation of the ERK1/2 pathway plays a crucial role in LPS-stimulated granulosa cells proliferation,and the elevation of granulosa cells MMP possibly synergistically influences this process.