Study On The Mechanism Of Curcumin In Improving POF Based On The Inhibition Of Oxidative Stress In Granulosa Cells

Background:Currently,Premature Ovarian Failure（POF）is one of the common female diseases.It is also a major problem affecting fertility and quality of life for young women.mainly characterised by sporadic menstruation and even amenorrhoea and infertility.With the rapid development of society and economy,environmental factors,postponement of the reproductive age of women and increased life and mental stress,the incidence of premature ovarian failure is on the rise year by year,and presents an obvious tendency to be younger.The main pathogenesis of premature ovarian failure is follicular atresia,which is caused and accelerated by a decrease in granulosa cell function.Previous studies have shown that premature ovarian failure may be associated with an imbalance between oxidative and antioxidant stress in the ovarian microenvironment,which affects cell both proliferation and differentiation as well as apoptosis.Curcumin is a naturally occurring polyphenolic compound with a wide pharmacological spectrum of anti-inflammatory,antioxidant,anti-fibrotic,anti-tumour and neurocell protection effects.Curcumin can protect the tissue and cell damage caused by oxidative stress by reducing the oxidative stress response of ovarian tissue,providing a new option for the current research on oxidative damage diseases of female reproductive system.but The mechanisms of action are not well understood.Objective:In this research paper,KGN cell lines were used to investigated the effect of curcumin on the proliferation and apoptosis of granulosa cells with oxidative damage,and then we examined its effectiveness in ameliorating ovarian oxidative damage,and determined the effective concentrations and duration of anti-oxidation of curcumin on granulosa cells.Then proteomics analysis was performed to screen for the significantly different proteins and further to explore the potential targets and molecular mechanisms by which curcumin may act in the treatment of premature ovarian failure.Methods:1.The human ovarian granulosa cell line KGN was used to construct a cellular oxidative stress injury model using hydrogen peroxide（H₂O₂）:KGN cells were treated with different concentrations of H₂O₂ solution and acted for different times,The CCK-8 kit was tested for cell viability and determine the time and concentration of H₂O₂ to construct the oxidative model.2.Determine the effective concentration and time of curcumin’s antioxidant effect on KGN cells.On the basis of the Oxidative stress injury model,KGN cells were treated with cell culture medium containing different final concentrations of curcumin,and the cell survival rate was also calculated by adding CCK-8 reagent at different time points and detecting cell OD by enzyme meter.3.The DCFH-DA probe method was administered on a flow cytometry analyser to capture intracellular reactive oxygen species levels.Drug-treated cells were collected,than loaded with the DCFH-DA probes and flow cytometry was used for fluorescence intensity detection to detect intracellular reactive oxygen species levels.4.DIA quantitative proteomics technology was used to identify and quantify the obtained proteins and screen the differentially expressed proteins;Gene Ontology functional annotation and enrichment analysis was performed on each identified differentially expressed proteins with the aid of Blast2GO software;KEGG pathway annotation and pathway enrichment analysis of all differentially expressed proteins with the KOBAS software to search for potential targets and signaling pathways of curcumin to improve premature ovarian failure by alleviating oxidative stress damage of granulosa cells.Results:1.A model of oxidative stress injury was structured with the use of H₂O₂ to induce KGN cells,and the cell survival rate was 51.45±2.15%when the H₂O₂ concentration was 400μmol/L and the stimulation duration was0.5h,so this was used as the modeling condition for subsequent experiments.2.The results of CCK-8 assay demonstrated a significant decrease in cell survival rate in the H₂O₂ and DMSO groups compared to the Control group（P<0.001）,and a significant increase in cell survival rate in the2μmol/L-10μmol/L concentration dosing group when compared to the DMSO group in a time-dependent mode（P<0.05）.3.Flow cytometry results showed that the total apoptosis rate and fluorescence intensity of cells in the H₂O₂ and DMSO groups were significantly higher compared with the normal group（P<0.01）.Compared with the DMSO group,the total apoptosis rate and the intracellular fluorescence intensity were reduced in all the dosing groups,which was statistically significant（P<0.05）,These results indicated that curcumin could effectively reduce H₂O₂-induced apoptosis rate and intracellular ROS levels in KGN cells.4.The results of protein identification and quantitative analysis showed that a total of 6074 proteins were detected in the two groups of samples,with a total of 138 proteins that were statistically significant（P value<0.05）,of which 38 were up-regulated proteins and 100 were down-regulated proteins.5.The structural domain enrichment analysis indicated that the differentially expressed proteins were mainly enriched in Fibrinogen alpha/beta chain family、Fibrinogen beta and gamma chains,C-terminal globular domain、Small cytokines（intecrine/chemokine）.6.GO analysis indicated that the differentially expressed proteins were annotated to 326 biological processes,64 molecular functions and 22cellular components（P<0.05）;In biological processes,among the biological processes,the most significant differential protein enrichment mainly included the negative regulation of endothelial cell apoptosis,the positive regulation of xenotypic cell-cell adhesion,the induced bacterial agglutination,fibrinolysis and plasminogen activation.Its biomolecular function mainly concerning:cytokine activity、cytokine activity、guanylyltransferase activity、ubiquitin-like protein ligase activity;The cytological components of the differentially expressed proteins are mainly targeted to the fibrinogen complex,PAM complex/TIM23 mitochondrial import motor,guanosine-nucleotide exchange factor complex and intracellular transport particle B.7.KEGG pathway analysis showed that the potential pathways of curcumin in the treatment of premature ovarian failure mainly involved multiple signaling pathways,such as tight junction,biotin metabolism,viral protein interaction with the cytokines and cytokine receptors,cytokine-cytokine receptor interaction,Inositol phosphate metabolism,complement and coagulation cascade,Glycosphingolipid biosynthesis-lacto and neolacto series,Choline metabolism in cancer,Hippo signaling pathway-multiple species,JAK-STAT signaling pathway,Platelet activation Fatty acid elongation,Biosynthesis of unsaturated fatty acids,Phosphatidylinositol signaling system.Conclusions:1.Curcumin can effectively improve H₂O₂-induced oxidative stress damage of granule cells,promote the restoration of granule cell vitality,inhibit apoptosis of granule cells,and reduce the level of reactive oxygen species in cells.2.In this project,three potential functional proteins were screened out through proteomics technology:TRMT61B、OXSM、BOLA2、RNF121 and COMMD2.Among them,TRMT61B、OXSM、BOLA2 are all up-regulated expression proteins,and it is closely associated with mitochondria,the main production site of ROS,Therefore,it is speculated that it may be the main target of curcumin to improve ovarian oxidative stress.And RNF121 and COMMD2 are associated with the NF-κB signalling pathway.3.According to the results of KEGG pathway analysis and literature research,it is preliminarily believed that the main pathways involved in curcumin to improve premature ovarian failure are:biotin metabolism,complement and coagulation cascade,JAK/STAT signaling pathway and Hippo signaling pathway.