The Role And Mechanism Of Chitosan Oligosacchrides In Restoring Ovarian Dysfunction Caused By Oxidative Stress

It is an urgent scientific and clinical issue to elucidate the underlying mechanism of oocyte maturation and maturation disorder in the medical community!The surrounding granulosa cells（GCs）provide the only living microenvironment,and the function of GCs directly influences the state of the oocyte maturation.Oxidative stress can cause GCs dysfunction and oocyte maturation disorders.Chitosan oligosaccharides（COS）is a natural alkaline amino oligosaccharide without toxic side effects,has distinctive physiological characteristics of regulating inflammation,antioxidant and anti-cancer.Our research group found that COS significantly increased the number of follicles and delayed ovarian aging in the infertility-damaged mice.It’s not clear whether COS improving GCs and oocyte function is related to the anti-oxidative role and the regulated signaling pathways.This study using human ovarian GCs line（KGN cells）,mice primary GCs and mice germinal vesicle（GV）oocytes explored whether COS had protective effects on ovarian GCs and oocytes in vitro and in vivo under oxidative stress circumstance,and analyzed the significance of Hypoxia-inducible factor-1α-Vascular endothelial-derived growth factor-A（HIF-1α-VEGF-A）signaling pathway.The study observed the protective role of COS in human and animal oxidative damage of H₂O₂and3-NPA;showed that COS promoted oocyte maturation and improving oocyte quality in vitro;protected ovarian function from oxidative stress damage via improving mitochondria function via HIF-1α-VEGF-A signaling pathway.These results provided scientific basis and knowledge for further understanding the mechanism of GCs and oocytes dysfunction and for finding methods to improve oocyte maturation disorder.Part I:COS alleviates oxidative damage in human granulosa cell line KGNTo study the protective role and mechanism of the COS from H₂O₂‐stimulated oxidative damage in KGN,PCR,we used Western blot,Senescence-associatedβ-galactosidase（SA-β-gal）staining,flow cytometry and immunofluorescence microscopy techniques.We found that:（1）The effect of COS on oxidative stress level in normal KGN cells.KGN cells treated with COS at 100μg/ml concentration for24h significantly increased cell viability,the production of estrogen（E₂）and progesterone（P₄）,the cellular glutathione（GSH）content,anti-inflammatory factors IL-10 and the level of Transforming growth factor beta-1（TGF-β1）,and reduced the expression of oxidative stress-related indicators such as 8-hydroxy-2′-deoxyguanosine（8-OHd G）and 4-hydroxynonenal（4-HNE）,the pro-inflammatory factor IL-6,also accompanied with a decrease in hypoxia inducible factor-1α（HIF-1α）and vascular endothelial-derived growth factor-A（VEGF-A）m RNA and protein levels.（2）The protective effects of COS on oxidative damage in H₂O₂‐stimulated KGN.（1）COS reversed the decrease in cell viability,mitochondrial quantity and function defects,GSH level,secretion of estrogen,progesterone and IL-10,and increase in cell apoptosis,reactive oxygen species（ROS）content,8-OHd G and 4-HNE,HIF-1αand VEGF-A levels,IL-6 content,TGF-β1levels,and SA-β-gal expression caused by H₂O₂.COS ameliorated the decrease in mitochondrial quantity and mitochondrial membrane potential（MMP）induced by H₂O_2,also caused significant inactivation of HIF-1α-VEGF-A pathway.（2）Using the HIF-1αprotein inhibitor KC7F2 reversed the increase in the level of VEGF-A,IL-6and SA-β-gal expression,the decrease in E₂and P₄,Mito Tracker staining,MMP,GSH content,cell viability,and TGF-β1 protein induced by H₂O₂.Moreover,KC7F2inhibited the increase in 4-HNE and 8-OHd G levels,ROS release,and cell apoptosis rate induced by H₂O₂.（3）COS and KC7F2 existed a synergistic effects on all above experimental parameters.The above experiments of human GCs in vitro demonstrated that COS reduced the oxidative stress level in normal KGN cells,meanwhile reduced the oxidative damage in H₂O₂-induced KGN cells.The mechanism may be related to stabilizing mitochondrial function via the HIF-1α-VEGF-A signaling pathway.Part II:COS improves GCs function in 3-NPA miceTo investigate the protective role and mechanism of the COS from 3-NPA oxidative damage GCs,we used PCR,Western blot,SA-β-gal staining,ELISA and immunofluorescence microscopy techniques.We found that（1）3-NPA significantly increased both HIF-1αand VEGF-A m RNA and protein expression,COS reversed the decrease in cell viability,mitochondrial quantity and functional defects,production of estrogen and progesterone,GSH content,TGF-β1 and IL-10,and the increase in IL-6 level,SA-β-gal protein expression,ROS content,oxidative stress-related genes 8-OHd G and 4-HNE,HIF-1αand VEGF-A levels in mice GCs induced by 3-NPA.COS significantly inactivated the HIF-1α-VEGF-A signaling pathway.（2）Using the HIF-1αprotein inhibitor KC7F2 reversed the increase in VEGF-A,IL-6 and SA-β-gal expression,the decrease in E₂and P₄,Mito Tracker staining,MMP,GSH content,cell viability,and TGF-β1 protein levels induced by3-NPA.Moreover,KC7F2 inhibited the increase in 4-HNE and 8-OHd G levels,ROS release induced by 3-NPA.（3）COS and KC7F2 synergistically interacted for all of these results.The above results demonstrated that COS reduced the 3-NPA-induced oxidative damage of mice primary GCs in vitro,the mechanism may be related to stabilizing mitochondrial function via the HIF-1α-VEGF-A signaling pathway.Part III:COS improves oocytes quality in 3-NPA miceTo investigate the protective effect and mechanism of COS on oxidative injury oocytes,in vitro maturation（IVM）of mice GV oocytes,immunofluorescence staining of the spindle,PCR,ROS staining,GSH content detection,mitochondrial number and MMP detection to examine 3-NPA Kunming mice GV stage oocytes after IVM were used.We found that COS significantly increased the polar body 1（PB1）extrusion rate,GSH content,mitochondrial number and MMP,also decreased ROS levels and abnormal spindle rate in 3-NPA mice oocytes.After using the HIF-1αprotein inhibitor KC7F2,the increase in ROS levels and abnormal morphology spindle rate,the decrease in Mito Tracker staining,MMP,and GSH content induced by 3-NPA were all reversed.COS and KC7F2 showed synergistically effects on above observation targets.The above experiments demonstrated that COS reduced the3-NPA-induced oxidative damage to improve oocyte maturation disorder and quality decline,the mechanism may be related to stabilizing mitochondrial function via the HIF-1α-VEGF-A signaling pathway.