The Molecular Mechanism Study Of EGCG Catechin To Regulate Progesterone Production In Human Granulosa Cells

BackgroundThe incidence of infertility diseases in China is as high as 10-20%,and the resulting reproductive health problems are becoming an important factor related to social harmony.There are many causes of infertility.The main causes of female infertility are caused by steroid hormone production disorder and the follicular development disorder.However,there is currently a lack of detailed and thorough understanding of hormone production.Estrogen,progesterone and testosterone are steroid sex hormones.Progesterone is the precursor of steroid hormone synthesis and is an important factor regulating embryo implantation.It also plays a key role in maintaining normal female reproductive functions,including follicle formation,oocyte maturation,ovulation and embryo implantation.In the process of steroid hormone synthesis,steroidogenic acute regulatory Protein(StAR)is the rate-limiting enzyme for its synthesis.This is because cholesterol is the substrate of steroid hormone synthesis,and the transport of cholesterol into mitochondria under the action of StAR is the first step in steroid hormone synthesis.Epigallocatechin-3-gallate(EGCG)is the most abundant and biologically active catechins extracted from green tea.The health benefits of EGCG have been extendedly studied.Ovarian steroidogenesis plays a pivotal role in maintaining normal reproductive function.Increasing evidence has demonstrated that EGCG can reduce the incidence of various diseases due to its antioxidant and anti-inflammatory properties.The beneficial effects of EGCG on reproduction have been reported in many animal models.EGCG acts as an antioxidant to enhance the quality of gametes in both males and females.EGCG also improves the maturation of oocyte and improves the quality of embryo which contributes to the increase of fertilization and clinical pregnancy rates.Interestingly,the most beneficial effects are observed only treatment with lower concentrations of EGCG as opposite actions can be induced by higher concentrations of EGCG treatments.To date,the effect of EGCG on the steroidogenesis in human granulosa cells remains unclear.Thus far,it has been reported that EGCG could influence steroidogenesis in granulosa cells,but contradictory results are obtained in different studies.To the best of our knowledge,whether EGCG affects the steroidogenesis in human granulosa cells is completely unknown and the underlying mechanism remains to be defined.In the present study,we examine the physiological concentrations of EGCG on the steroidogenesis in a steroidogenic human granulosa-like tumor cell line,KGN.To make the experiments more technically feasible,we used KGN cells as our in vitro model.The KGN cell line is derived from human ovarian granulosa cell tumors and preserves many normal physiological characteristics of granulosa cells including steroidogenesis.These results provide a better understanding of the function of EGCG on steroidogenesis.which may provide new idea for study of mechanism of reproductive disorders,and also provide new approaches for individualized diagnosis and therapeutic treatment strategies.Aim:To explore the effect of catechin EGCG in regulating the expression of StAR and the synthesis of progesterone in human granulosa cells,and its molecular mechanism.Methods:The research object of this subject is human primary granulosa cells and KGN granulosa cell lines.The research aim is to explore the regulation effect and molecular mechanism of physiological concentration of catechin EGCG on StAR and progesterone.Different concentrations of physiological concentration levels(1-10 μM)of EGCG were used to treat human primary granular cells and KGN granular cell lines,and attention was paid to its regulatory effect on the rate-limiting enzyme StAR for progesterone synthesis.After treatment with 5μM EGCG,use 67-KDa laminin receptor(67LR)SiRNA to knock down the mRNA expression and observe the protein expression level of StAR.In order to further explore the signal pathway of EGCG acting on granular cells to,observe the cAMP response element binding proteint(CREB)at different time points.Apply cREB siRNA to knock down CREB mRNA expression,and further explore the effect of the activation of pCREB on the downstream during this process.On this basis,in order to explore the effect of 67LR-mediated catechin EGCG on granular cell activation of CREB signaling pathway to regulate the expression of StAR,we used 67LR siRNA to knock down its mRNA expression level in the cell and observe the effect on CREB expression.Finally,we explores the effect of GCG on the production of progesterone in human granulosa cells and the related molecular mechanism,using 67LR and CREB siRNA to treat KGN cells,respectively,to detect changes in the level of progesterone in the cell culture medium.Results:1.Treatment with 5μM and 10μM EGCG could promote the expression of StAR expression in both human granulosa cell line KGN and human primary granulosa cells.This up-regulation is dose-dependent.EGCG does not affect the expression levels of other steroidogenesis related enzymes such as P450 side-chain cleavage enzyme,3β-hydroxysteroid dehydrogenase,and aromatase.2.We firstly identify the expression of 67-kDa laminin receptor(67LR)in human granulosa cells.In human granulosa cell line KGN,knock down the expression of LR could abolish EGCG-induced StAR expression.Moreover,activation of PKA/CREB signaling pathway was participated for EGCG-induced up-regulation of StAR expression.Inhibition of PKA by H89 could abolish EGCG-induced StAR expression.Meantime this treatment up-regulates the basal expression of StAR.Knock down the expression of CREB could abolish EGCG-induced StAR expression.This demonstrates that EGCG induced StAR expression requires the 67LR-mediated activation of the PKA-CREB signaling pathway.3.Moreover,P4 levels in culture medium of granulose cell were examined after EGCG treatment.The result shows EGCG induces progesterone production by the 67LR-mediated activation of the PKA-CREB signaling pathway.Conclusion:1.Physiological concentration of catechin EGCG could increase the expression of StAR in human granulosa cells.Its effect is achieved by binding to the cell membrane receptor 67LR to activate the PKA/CREB signaling pathway.2.In human granule cells,catechin EGCG can promote progesterone production.3.Catechin EGCG could activate the PKA/CREB signal pathway by binding to its receptor 67LR to promote the production of progesterone.