Mapping The Single Cell Transcriptome And Investigation On The Mechanism Of Metformin Reversing Cisplatin-induced Ovarian Damage

Objective Cisplatin is a first-line antitumor drug,which can lead to early menopause and loss of fertility in women.However,the mechanism of cisplatin-induced ovarian damage has not been fully clarified,and there is a lack of effective intervention strategies.This study aims to reveal the changes in ovarian cell composition and gene expression after cisplatin intervention by single-cell transcriptome sequencing.On this basis,to explore the protective effect of metformin on cisplatin-induced ovarian reserve and function in mice,draw the single-cell transcriptome atlas,and clarify the potential mechanism of metformin against cisplatin-induced ovarian damage by reprogramming various ovarian cells and their gene expression.Methods120 female C57BL/6 mice with normal estrous cycle were selected(randomly divided into4 groups,N=30/group).The control group(Con)was treated with normal saline by gavage and intraperitoneal injection;the Cisplatin group(Cis)was given 5.0mg/kg cisplatin intraperitoneal injection once;Cisplatin group + metformin group(Cis +Met)was treated with the same dose of cisplatin.Metformin(250mg/kg)was pretreated by gavage for one week and continued until the end of the experiment;Metformin group(Met)was given the same dose and cycle by gavage.During this period,the general condition and estrous cycle of mice in each group were monitored,the ovarian and serum samples of mice were collected,and the follicular count and hormone level were detected to evaluate the ovarian function of mice.At the same time,cluster analysis was carried out on the captured mouse ovarian single cells,including the number and proportion of various cells,the expression of genes and transcription factors in each cell;The granulosa cell differentiation trajectories of different follicular development stages were constructed and analyzed by single-cell quasi temporal algorithm;Integrate the ligand-receptor information of ovarian cells,predict the communication relationship between ovarian cells based on cellphone DB,and use the co-culture system to culture mouse ovarian primary granulosa cells and RAW264.7 macrophage lines were tested in vitro.Results1.The general condition of mice intervened by cisplatin was poor,the proportion of mice in regular estrous cycle decreased,the level of serum FSH increased,the level of E2 decreased,and the degree of ovarian fibrosis increased;Metformin can improve the estrous cycle and the expression levels of FSH and E2 in mice intervened by cisplatin,and reverse ovarian fibrosis.2.The number of ovarian cells after cisplatin intervention changed significantly,in which the proportion of T cells,plasma cells,monocytes,primordial follicular granulosa cells,and lymphatic endothelial cells decreased,while the proportion of other ovarian somatic cells increased;Metformin can positively reverse the proportion of immune cells(T cells,plasma cells,monocytes,dendritic cells),follicular granulosa cells and lymphatic endothelial cells,and negatively reverse the proportion of other cells to regulate the microenvironment of ovarian cells.3.Cisplatin up-regulated the expression of genes and transcription factors including immune cell chemotaxis,cytokine secretion,apoptosis,and oxidative stress;Metformin can reverse the expression of pro-inflammatory genes,reduce the secretion of inflammatory factors in the ovary and improve the inflammatory microenvironment of follicular growth and development by regulating the interaction between immune cells secreting cytokines and other cells(ligand-receptor interaction).At the same time,metformin had a positive regulatory effect on apoptosis,oxidative stress,ovarian fibrosis,and other related pathways and genes.4.Cisplatin induced apoptosis of follicular granulosa cells(primordial follicular granulosa cells and cumulus granulosa cells);Pseudo time series analysis showed that cisplatin could accelerate the activation of primordial follicles and interfere with the normal differentiation and development track from primordial follicles to growing follicles;Metformin can reduce the apoptosis of cumulus granulosa cells,reverse the differentiation and development trajectory of follicles,recruit M2 macrophages to chemotaxis to atresia follicles,exert the "cell burial" effect on granulosa cells and reduce the apoptosis of granulosa cells induced by inflammatory cytokines.Conclusion1.Metformin attenuates cisplatin-induced ovarian reserve,endocrine function and alleviates fibrosis in mice.2.The single cell transcriptome map of metformin against cisplatin induced ovarian injury is drawn,which reveals the changes of cell composition,gene expression and intercellular communication of cisplatin induced ovarian damage.On this basis,it is clear that metformin can reprogram the genes,transcription factors and cell interactions of ovarian cells intervened by cisplatin.3.Metformin can regulate the cytokines secreted by various ovarian cells,eliminate the accumulation of proinflammatory factors among ovarian cells,and improve the immune and inflammatory microenvironment of the ovary.