The Regulation Role Of GHRL And LPS On Proliferation And Apoptosis Of Ovarian Graniular Cell As Well As Reproduction Hormones Secretion Of Mice

Ghrelin(GHRL), a 28 amino acid acylated peptide, is found in 1999 from gastrointestinal tract of the rats. It is found that GHRL has a widely existence in animals. Increasingly, many studies proved that GHRL and it’s functional receptor GHSR-1α have a kind of regμlation effects with neuroendocrine on Hypothalamus-pituitary-gonad Axis. It`s indicates that GHRL may have an effect on reproduction, especially the development of follicle.Lipopolysacchride(LPS) is located on the outside of the cytoderm of germ and it is also the component of the cytoderm. The microorganism in cows rumen coμld produce the dissociative LPS within the metabolism and will be absorbed into the internal environment through digestive track. It is the fact that low-dose LPS is stable existence in body. The effect of low-dose LPS on organism, especially the development of follicle, is still unclear.The current study isusing GC cells as the model to determine the effects of GHRL and LPS on proliferation, apoptosis and the gene related to reproductive hormones from the point of receptor and signal pathway. Research contents and resμlts are as follows:1. The effects and mechanismof GHRL on GCs(1) The expression of GHSR-1α, the receptor of GHRL, on GC cellsObjective: verify the existence of GHSR-1αand the relationship between the concentration of GHRL and the expression of GHSR-1αon GCs. Methods: the GCs were treated with different concentration of GHRL(0,10-6,10-7,10-8mol/L).Cleave GCs and collect the protein after 24 h. Use Western Blotting to test the expression of GHSR-1α. Resμlts: Compared to control group, GHSR-1α is surely expressed in GCs. However, the expression in experiment group is not so high as it in control group. It might tell us that GC may express a kind of feedback supression against to GHRL.(2) The effects of GHRL on cell cycle and apoptosis of GCObjective: The current study is to test the change of cell cycle and apoptosis on GC with different concentration of GHRL. Method: GC is treated with different concentration of GHRL(0,10-6,10-7,10-8mol/L). Test the change of cell cycle and apoptosis with FCM after 24 h. Result: It shows us that GHRL has no significent effect on cell cycle. However, the apoptosis of GC becomes higher with the increase of GHRL(p<0.01).(3) The effects of GHRL on the genes expression related to reproduction hormoneObjective: The change is tested on expression of genes related to reproductionhormone with 10-7mol/L GHRL. Method:GC is treated with 10-7mol/L GHRL for 24 h and then the total RNA is extracted for testing the change of gene expression related to reproduction hormone with Q-PCR. Result:10-7mol/L GHRL could stimulate the expression of genes related to reproduction hormone(p<0.05), like FSHr,INH-βa,AR,LHcgr,P450 scc,StAR.(4) Effect of GHRL on the expression of Estradiol(E2) and Progesterone(P)Objective: The effect of E2 and P is tested with the treatment of differentconcentration of GHRL. Method: GC is treated with different concentration of GHRL(0,10-6,10-7,10-8mol/L). RIA is used to check the change of concentration of E2 and P expressed by GC after 24 h treatment of GHRL(p<0.01).Result:GHRLshows a positive effect on E2 and P expression(p<0.01). Besides, the positive effect of Ghrelin present a dose-dependent manner.(5) The effect of GHRL on MAPK and phosphorylation of ERKObjective:This part of study is conducted to examine the effect of GHRL on MAPK and phosphorylation of ERK. Method: GCs were treated with different concentration of Ghrelin(0,10-6,10-7,10-8mol/L) in media. After 24 h cleaved the cells and collected the total protein. Western Blotting was used to test the change of some protein like ERK,p-ERK,Akt,p-Akt,p-P38,p-P65. Resμlts: it showed us that Ghrelin may affect the GCs via the signal pathway MAPK and p-Akt.2. The effect of LPS on GC(1) The effects of LPS on GC proliferationObjective: the current study is to examine the effect of LPS on GCs, also to find the best concentration of treatment. Methods: the CCK-8 kit is used to test if LPS has the effect of proliferation on GCs with different concentration of LPS(0, 50μg/ml,100μg/ml,150μg/ml,200μg/ml). Resμlts: it is found that 50μg/ml and 100μg/ml groups have a positive effect on proliferation of GCs(p<0.01), but the 150μg/ml and 200μg/ml groups have the opposite effect(p<0.01). Both positive and negative effect show a dose-dependent manner and 50μg/ml is chosen as best treatment concentration.(2)The effect of LPS on cell cycle and apoptosis of GCsObjective: this part of study was conducted to examine the effect of LPS on cell cycle and apoptosis of GCs. Methods:50μg/ml LPS were added in media. After 24 h, the cell cycle and apoptosis are tested with Flow Cytometry(FCM). Results: FCM showed us that 50μg/ml LPS coμld stimμlate the GCs from phase S to phase G2. It indicates that LPS has a positive effect on proliferation(p<0.01). Besides, 50μg/ml LPS may restrain the apoptosis of GCs(p<0.01).(3)The effects of LPS on the genes related to reproductive hormones.Objective: the current study was conducted to find out if LPS(50μg/ml) has effects on the genes related to reproductive hormones on GCs. Methods: LPS(50μg/ml) was added in media. After 24 h, extract the total RNA and do Q-PCR to test the genes related to reproductive hormones. Results: LPS(50μg/ml) has the positive effect on the expression of the genes related to reproductive hormones(p<0.05), like FSHr, INH-βa, AR, LHcgr, P450 scc and StAR.(4)The effect of LPS on expression of Estrogen(E2) and Progesterone(P)Objective: this research is to investigate the effects of LPS(50μg/ml) on the secretion of E2 and P in GCs. Methods: the media contained 50μg/ml LPS. After 24 h, we just used the Radio Immuoassary(RIA) to testify the change of P and E2 in media. Resμlts: high dose Ghrelin and 50μg/ml LPS have significantly positive effect on the secretion of P and E2. Besides, the positive effect of Ghrelin present a dose-dependent manner(p<0.01).To sum up, this study does confirm that Ghrelin and LPS have some effect on GCs including proliferation, apoptosis and the secretion of reproductive hormones and the way Ghrelin affects the cells is related to MAPK and p-Akt.