Effect And Molecular Mechanism Of Cistanche Deserticola Polysaccharide On Promoting The Differentiation Of Female Germline Stem Cells Through BMP4/Smad Signaling Pathway

Research background With the change of social environment and people’s lifestyle,ovarian dysfunction has become the main cause of female fertility decline and infertility.Age,disease and harmful substances in the process of disease treatment,such as radiotherapy rays and chemotherapy drugs,can cause female ovarian function decline and eventually lead to infertility.Clinically,several measures can be taken to deal with ovarian dysfunction,but the effect is limited,and none of them can fundamentally restore ovarian function.Therefore,how to restore the declined ovarian function is a difficult problem in the field of reproductive medicine.The discovery and research of female germ stem cells have brought a turning point to solve this problem.Female Germline Stem Cells(FGSCs)are derived from Primordial Germ Cells(PGCs),which have the ability of self-renewal and directional differentiation into oocytes.They can continuously replenish the depleted primordial follicle pool to restore ovarian function.Therefore,it has certain clinical application value in dealing with infertility caused by ovarian dysfunction.If FGSCs in dysfunctional ovaries can be activated to differentiate into oocytes,the clinical application value is huge.In recent years,scientists are constantly looking for FGSCs activation factors and trying to explore its differentiation mechanism.The traditional Chinese herbal medicine Cistanche deserticola Y.C.Ma has been recorded and applied in the treatment of infertility and impotence since ancient times.It is widely distributed in northwest China,and Cistanche deserticola polysaccharides(CDPs)is one of its main active ingredients.Many pharmacological studies have confirmed its effect on the proliferation of lymphocytes,fibroblasts and hematopoietic cells.Research purpose Zoological experiments explored the recovery effect of Cistanche deserticola polysaccharides on female reproductive stem cells in ovaries and cortex of mice with ovarian dysfunction.Cytological experiments explored the differentiation of female reproductive stem cells induced by Cistanche deserticola polysaccharides in vitro;subsequently,the molecular mechanism and process of Cistanche deserticola polysaccharides promoting the differentiation of female reproductive stem cells were further explored.Research methods The normal control group,model group and CDPs treatment group were set up at the animal level.The animal model of ovarian dysfunction was established by intraperitoneal injection of cyclophosphamide(120 mg/kg)on the first day and then 15 mg/kg for 2 weeks.After the successful establishment of the animal model,the mice were treated with CDPs 200mg/kg for 6 weeks.After the administration,the mice were taken at the same time as the other two groups of mice.The ovarian tissue structure,endocrine function and fertility status of the mice were observed.The effect of CDPs on ovarian dysfunction after administration.Observation index of ovarian tissue structure: H & E staining was used to observe the histological structure of mice;count the number and proportion of follicles at all levels;d NA damage(γH2AX and PARP1)and cell proliferation activity(Ki67)were detected.Immunofluorescence double staining was used to observe FGSCs in ovarian cortex.Endocrine function observation index: estrous cycle of mice;serum estrogen and progesterone levels.Fertility function observation index: mating experiment was used to observe whether the mother mice gave birth,the birth rate and the weight of the offspring mice.At the cytological level,the ovaries of newborn 3-5 d female mice were selected,and the female germ stem cells were isolated,purified and cultured by immunomagnetic bead method and two-step enzymatic method to obtain a cell line that can stably proliferate in vitro.The optimal treatment concentration of CDPs was determined to be 0.5 μg / m L.The cells were randomly divided into two groups and cultured with normal FGSCs medium and 0.5 μg/m L CDPs.The cell proliferation activity(CCK-8 method),oxidative stress(ROS,SOD and GSH levels)and apoptosis(flow cytometry)were detected and compared to determine whether CDPs have cytotoxicity.During the time period of CDPs treatment(12 h,24 h,36 h,48 h),we used cell morphology,CCK-8 method,real-time fluorescence quantitative PCR(real-time PCR,q PCR),cell immunofluorescence and Western blot to detect and analyze germ stem cells and germ cell markers and differentiation markers,and explore the changes in the expression of hormone-related markers to determine the effect of CDPs on the differentiation of FGSCs in vitro.Molecular mechanism exploration the changes of genes in FGSCs before and after CDPs treatment were detected by whole gene RNA-Seq sequencing.Gene ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)and Ingenuity Pathway Analysis(IPA)database analysis were performed on the differentially expressed genes to preliminarily grasp the key factors and signaling pathways of cell differentiation after CDPs treatment.The expression of BMP-related receptors(Bmpr1a,Bmpr2 and Bmpr1b),endogenous BMP4,BMP downstream Smad family markers(Smad1,Smad2,Smad4,Smad5 and Smad9)were detected by PCR,q PCR and/or Western blot after CDPs addition.Subsequently,Smad9 was used as a key factor in this signaling pathway for gene modification to establish a Smad9 differentially expressed cell model.At the same time of CDPs treatment,q PCR and Western blot were used to detect cell self-renewal and differentiation(Markers Oct4,Mvh,Stra8 and Sycp3)and Smad family markers(Smad1,Smad5,Smad9,Smad1/5/9 and p-Smad1/5/9).To determine the molecular mechanism and process of CDPs regulating FGSCs differentiation through BMP-Smad family signaling pathway.Research results The animal model of hypoovarian function induced by cyclophosphamide was successfully constructed based on the disorder of estrous cycle in mice and the changes of ovarian structure in mice.The ovarian tissue structure of mice showed that compared with the model group,the ovarian structure of mice in the treatment group recovered compactly,the staining of tissue cells was more uniform,the morphology of follicles at all levels was clear,the number and proportion of primordial follicles and primary follicles increased,and the structure of medulla area was no longer loose.The expression of DNA damage marker protein γH2 A and Parp1 in ovary decreased significantly(P<0.01),and the expression of cell proliferation activity marker Ki67 increased significantly(P<0.01).The results of endocrine function showed that the estrous cycle of mice in the treatment group recovered,and obvious estrous cycle and cycle rules could be observed.Compared with the model group,the content of estrogen and progesterone in serum of mice increased significantly(P<0.01).The results of fertility function showed that the mating experiment showed that the mice in the treatment group could give birth normally,and the pups could survive for a long time.The birth rate of the mother was significantly higher than that of the model group(P<0.01),and the weight of the offspring mice increased significantly(P<0.01).The ovarian cortical area of the mice in the treatment group was significantly restored,the structure of the cortical area was continuous and complete,and the cells recovered deeply stained and uniform.Compared with the model group,the number of FGSCs in this region was significantly increased(P<0.01),and the expression of germ stem cells and germ cell markers Oct4 and Mvh as well as meiosis markers Stra8 and Sycp3 in the ovary were significantly increased(P<0.01).At the cytological level,FGSCs were cultured in vitro with 0.5μg / m L CDPs.Compared with the control group,CCK-8 results showed that the cell proliferation activity of the two groups was basically flat(P>0.05),and the intracellular and / or cell supernatant ROS,antioxidant enzymes SOD,GSH content also did not increase significantly(P>0.05).Flow cytometry showed that there was no significant difference in the degree of apoptosis between the two groups,indicating that CDPs had no cytotoxicity to FGSCs.Compared with the control group,the cell diameter of the experimental group increased and the morphology tended to oocytes.After CDPs treatment for 12 h,24 h,36 h and 48 h,the relative expression levels of germ stem cell marker gene Oct4,germ cell marker gene Mvh,meiosis initiation factor Stra8 and meiosis marker Sycp3 were significantly increased(P<0.05),and the results of protein level detection were the same(P<0.05).The expression of germ cell-related hormone secretion markers Amh,Cyp17a1,Cyp19a1,Fshr and Lhr increased significantly after CDPs treatment for 24 h and 36 h(P<0.01),suggesting that the addition of CDPs promotes the expression of hormone-related markers and is closely related to the regulation of FGSCs development.Molecular mechanism exploration: After RNA-Seq compared the gene expression of the control group and the experimental group,key differentially expressed genes(DEGs)were found,of which 549 genes were up-regulated and 465 genes were down-regulated.GO,KEGG analysis and IPA signaling pathway analysis showed that DEGs were mostly enriched in functional categories related to germline cell development.Among them,TGF-β pathway and several genes in Smad family proteins and BMP signaling pathway may be activated to regulate the differentiation of FGSCs in vitro.The BMP-Smad family signaling pathway through which CDPs regulate FGSCs cell development was further studied.After CDPs treatment,the receptors Bmpr1 a,Bmpr2 and Bmpr1 b on BMP cell membrane changed to varying degrees,and the expression of the former two increased significantly(P<0.01).CDPs increased the expression of endogenous Bmp4(P<0.01).Compared with the control group,the expression of Smad1/2/5/9 in the experimental group was significantly increased(P<0.05).Compared with the CDPs group,the expression of Smad1/5/9 in the CDPs + LDN(BMP4inhibitor)group was significantly decreased(P<0.05),suggesting that CDPs affect the expression of downstream Smad family markers through BMP4.Subsequently,Smad9 was selected as the key gene to construct the cell model of gene differential expression.The addition of CDPs increased the expression of Smad1/5/9,and also increased the expression of cell self-renewal and differentiation markers(Oct4 and Mvh,Stra8 and Sycp3).The treatment of CDPs had a positive effect on the expression of Smad1,Smad5 and Smad9 in the Smad family markers,indicating that the intervention of CDPs on the differentiation of FGSCs could be regulated by Smad1/5/9 in the Smad family.Research conclusion 1.At the animal level,CDPs improved the ovarian structure and function of mice with hypoovarian function induced by chemotherapeutic drugs.2.CDPs enhanced the structural recovery of the ovarian cortex and promoted the proliferative activity of FGSCs in the cortex,thus affecting the recovery of the ovary.3.At the cellular level,CDPs did not cause oxidative stress and apoptosis of FGSCs,but promoted the maintenance and differentiation of FGSCs cell characteristics while maintaining cell proliferation activity.4.CDPs regulated FGSCs differentiation through BMP4/Smad family signaling pathways.