Experimental Study On The Mechanism Of Gadd45b In MB-MSCs Repairing Epirubicin- Induced GC Damage

Background:In recent years,the survival rate of cancer patients has been increasing gradually because patients of breast cancer,lymphoma and leukemia show a younger trend and chemotherapy effectiveness is improved.However,these treatments impair patients’ fertility and may lead to premature ovarian failure(POF).Health care and the preservation of fertility of cancer patients are paid more attention to.It is necessary to do research on the fertility protection of cancer patients who accept chemotherapy with gonadal toxicity.Different kinds of technologies are still in the research stage and have not been widely used in clinical practice.With the development of biotechnology,stem cell research is deepening,stem cell transplantation makes the repair of the structure and function of chemotherapy damaged tissue possible,which is also a research hot spot in recent years.Endometrial mesenchymal stem cells isolated from menstrual blood(MB-MSCs)is a new type of stem cells with many advantages such as the ease of collection via non-invasive methods,no ethical concerns and no autoimmune rejection.Therefore,this study is mainly on the interaction between MB-MSCs and human granulosa cells(GCs),and the exploration of the potential mechanism of MB-MSCs imparing chemotherapy-induced GC damage.Methods:GCs were isolated and cultured via density gradient centrifugation method.Then they were identified using cell immunohistochemistry(IHC)and the specific antibody of GCs is follicle-stimulating hormone receptor(FSHR).2.The most suitable inducing concentration of epirubicin on GC damage is determined through Cell Counting Kit-8(CCK8)assays.3.Three groups were set up in this study: 1)Control group: GCs;2)G+E group: GCs were treated with 25 μg/L epirubicin;3)G+E+M group: GCs were treated with 25 μg/L epirubicin and then co-cultured with MB-MSCs with cell culture insert.Each group was intervened for48 h.The estradiol,progesterone,anti-Müllerian hormone(AMH),inhibin A,and inhibin B levels were determined using enzyme-linked immunosorbent assay(ELISA).The proliferation of GCs was detected by CCK8 assays.The cell cycle distribution was detected by propidiumiodide(PI)single staining.The differentially expressed genes of GCs were analyzed with Affymetrix Human Transcriptome Array2.0 gene chip and verified with Western blot analysis.Results:GCs were successfully isolated and cultured via density gradient centrifugation method.IHC was performed to detect the specific antibody FSHR of GCs and the result indicated that over 90% of the cell membrane and cytoplasm of GCs were stained brown.CCK8 assays was performed to determine the cytotoxicity of epirubicin on GCs.The most suitable duration was set to 24 h,and the corresponding half-inhibitory concentration(IC50)was 25 μg/L.Compared with Control group,the levels of estradiol,progesterone,anti-Müllerian hormone,inhibin A,and inhibin B secreted by GCs were decreased and the inhibition ratio of the proliferation of GCs was increased.The ratio of G2/M phase was increased(P<0.05).However,when compared to G+E group,MB-MSCs repaired epirubicin-induced GC damage in G+E+M group.The levels of estradiol,progesterone,anti-Müllerian hormone,inhibin A,and inhibin B were increased and the inhibition ratio of the proliferation of GCs was decreased.The ratio of G2/M phase was decreased(P<0.05).In order to further investigate the molecular mechanism of MB-MSCs repairing epirubicin-induced GC damage,microarray and bioinformatics analysis were performed and the result showed that the cell cycle pathway was involved in 18 differentially expressed genes between G+E and control groups and 7 differentially expressed genes between G+E+M and G+E groups,including Cyclin B1(fold change> 1.5,P < 0.05),CDC2(fold change> 1.5,P < 0.05),and Gadd45b(fold change> 1.5,P < 0.05).Western blot analysis was for the verification of the differentially expressed genes and we found that compared with Control group,Cyclin B1 and CDC2 were downregulated and Gadd45 b was upregulated in G+E group(P<0.05).However,compared with G+E group,Cyclin B1 and CDC2 were upregulated and Gadd45 b was downregulated in G+E+M group(P<0.05).Conclusions: MB-MSCs repaired epirubicin-induced damage to GCs,which might be related to the inhibition of Gadd45 b protein expression.