Chitosan Oligosaccharide Improves Ovarian Granulosa Cells Inflammation And Oxidative Stress In Patients With Polycystic Ovary Syndrome

Objective:Polycystic ovary Syndrome（PCOS）is the most common reproductive endocrine disorder among women of reproductive age,which is the main cause of anovulatory infertility.Granulosa cells（GCs）from the ovary are known to be the most closely related cells to oocytes,and the reduced proliferation ability and/or increased level of apoptosis of GCs may be the key factors leading to abnormal follicular development.Previous studies have shown that changes in the ovarian microenvironment,such as oxidative stress and inflammation,would lead to developmental disorders of PCOS follicles by impairing the physiological state of the cumulus GCs.Therefore,optimizing the ovarian microenvironment may be one of the important means to improve the development potential of PCOS oocytes.Chitosan oligosaccharide（COS）was recognized as a natural antioxidant,which had anti-aging and anti-inflammation effects.This study firstly confirmed the relationship between the expression of inflammation factors and oxidative stress factors and PCOS by using human ovary GCs.Subsequently,we investigated the proliferation and apoptosis level of GCs in PCOS and non-PCOS patients,respectively.Moreover,KGN cell line was used to verify the improved effects of chitosan oligosaccharide on GCs inflammation and oxidative stress,as well as on GCs proliferation and apoptosis.Finally,the primary GCs were cultured with the optimal concentration and optimal time to further confirm our findings.Methods:First,120 clinical data of ART patients（60 cases of PCOS and 60 cases of control group）were collected from Maternal and Child Health Hospital of Jiangxi Province,and the follicular fluid samples were collected and the GCs in the follicular fluid were extracted for:1)WB and Q-PCR were used to detect the protein and m RNA expressions of inflammatory factors and oxidative stress related factors in GCs in PCOS and control group;2)the proliferation and apoptosis of GCs in PCOS and control group were detected by EDU,TUNEL and flow cytometry,respectively.Subsequently,KGN cells were selected and cultured with different concentrations of（0,100,200,300μg/m L）COS for 24 hs and 36 hs,respectively.The expression of inflammatory cytokines in GCs was detected,while the proliferation of GCs was detected by CCK8.H₂O₂ was used to establish oxidative stress injury to simulate the survival microenvironment of GCs in PCOS patients.COS dope experiment was performed,WB was used to detect the protein expression of oxidative stress related factors,flow cytometry was used to detect the apoptosis of GCs,and ROS Assay Kit was used to detect the reactive oxygen level.Finally,the optimal concentration and culture time of COS obtained from the above experiments were used to observe the influence of COS on the expression of GCs inflammatory and oxidative stress related factors protein in PCOS patients,and to detect the improvement effect of COS on reactive oxygen species in GCs.Results:（1）The clinical data of the patients showed that there were significant differences in BMI,age,basal AMH,basal LH,basal P,basal T,left sinus follicle number,right sinus follicle number,MⅡegg rate,and high-quality embryo rate between the control group and the PCOS group（P<0.05）,there were no significant differences in basal FSH,basal E2,normal fertilization rate and cleavage rate between control group and PCOS group（P>0.05）.（2）The results of RT-PCR and WB showed that the expression of pro-inflammatory factors and oxidative stress related factors extracted in GCs in PCOS group were significantly higher than those in control group（P<0.01 or P<0.001）,while the expressions of anti-inflammatory factors were significantly lower than those in control group（P<0.01 or P<0.001）.（3）The results of EDU,TUNEL and flow cytometry showed that the proliferation ability of GCs in PCOS group was significantly lower than that in control group（P<0.05）,while the level of apoptosis was higher than that in control group（P<0.05 or P<0.01）.（4）The KGN experiments showed that COS could promote the expression of anti-inflammatory factors TGF-β1 and IL-10 in KGN cells（P<0.05,P<0.01 or P<0.001）,and inhibit the expression of pro-inflammatory factors TNFαand IL-6（P<0.05 or P<0.01）.CCK-8 results showed that COS could promote the proliferation of GCs（P<0.05,P<0.01 or P<0.001）.（5）When H₂O₂ was used to induce oxidative stress injury in KGN cells,it was found that COS could reverse oxidative stress injury in KGN cells induced by H₂O₂and inhibit the expression of oxidative stress related factors HIF-1αand VEGFA（P<0.05,P<0.01 or P<0.001）.Flow cytometry showed that COS could suppress the apoptosis of GCs（P<0.01 or P<0.001）and decrease the ROS level of GCs（P<0.05or P<0.01）.（6）The results showed that COS could promote the expression of anti-inflammatory factors TGF-β1 and IL-10（P<0.05 or P<0.01）,inhibit the expression of pro-inflammatory factors TNFα,IL-6 and oxidative stress related factors HIF-1αand VEGFA in the primary GCs of PCOS patients（P<0.05,P<0.01or P<0.001）,and weaken the level of reactive oxygen species（P<0.05 or P<0.01）.Conclusions:（1）In PCOS patients,the proliferation of granulosa cells was decreased,the level of apoptosis was increased,and there was inflammation and oxidative stress in the granulosa cell microenvironment.（2）Chitosan oligosaccharide can improve the inflammation and oxidative stress injury induced by H₂O₂ in KGN cells,promote the proliferation of granulosa cells,and inhibit the apoptosis of granulosa cells.（3）Chitosan oligosaccharide can improve the inflammation and oxidative stress of ovarian granulosa cells in PCOS patients.