The Role And Mechanism Of Chemerin/CMKLR1 In Oxidative Stress And Apoptosis Of Granulosa Cells In Obese Female

Part I.Detection of oxidative stress,apoptosis and molecular level of chemerin/CMKLR1 in follicular fluid and granulosa cells in overweight or obese patientsObjective: To measure oxidative stress,apoptosis,and changes of chemerin/CMKLR1 molecular levels in follicular fluid and granulosa cells from overweight or obese women receiving IVF/ICSI(Overweight group)and BMI in the normal range(Control group).Methods: Flow cytometry was used to detect the levels of reactive oxygen species(ROS)in granulosa cells of overweight group and control group.RT-PCR was used to assess the transcription levels of antioxidant enzymes SOD1,SOD2,CAT and Gpx in granulosa cells of both groups;RT-PCR and Western Blot were used to measure the apoptosis level of granulosa cells.The chemerin content of follicular fluid was determined by ELISA.The expressions of chemerin and CMKLR1 were evaluated by RT-PCR and Western Blot.Results:(1)Compared with the control group,the levels of ROS in the overweight female granulosa cells were increased,the transcription level of SOD2 was significantly down-regulated,and the transcription level of Gpx was significantly increased.(2)Compared with the control group,the m RNA ratio of the proapoptotic factor Bax/anti-apoptotic factor Bcl2 in the overweight female granulosa cells increased significantly,and the activated caspase-3 protein level increased significantly.(3)Compared with the control group,the level of chemerin in the overweight female follicular fluid was significantly increased,and the transcription levels of chemerin and CMKLR1 in granulosa cells increased,as well as the level of CMKLR1 protein.Conclusion: In granulosa cells of overweight or obese women,oxidative stress is imbalanced,ROS accumulate abnormally,and apoptosis occurs.The level of chemerin in the follicular fluid of overweight or obese women increased,and the chemerin/CMKLR1 system of granulosa cells was activated.Part II.Detection of antioxidase,apoptosis and molecular level of chemerin/CMKLR1 in Diet-induced-obesity(DIO)mouse ovariesObjective: To observe the effects of obesity on oxidative stress,apoptosis and chemerin/CMKLR1 expression in mouse ovarian and granulosa cells by constructing a DIO mouse model,and to explore the probable molecular mechanisms.Methods: Four-week-old female C57BL/6J mice were fed food containing 60% fat to build DIO mouse models(OB group),whereas those in the control group(CHOW group)were fed a normal diet.Mice were weighed every week.Blood serum,ovaries and isolated granulosa cells were collected after 12-week treatment.Glucose(GLU),triglyceride(TG),and total cholesterol(TC)contents in serum were measured using a biochemical analyzer.Anti-Mullerian hormone(AMH)contents in serum of mice were assessed with ELISA.RT-PCR was used to detect the transcription levels of antioxidant enzymes SOD1,SOD2,CAT and Gpx in ovary and granulosa cells of two groups of mice.In situ TUNEL staining was used to observe the apoptotic follicles of mouse ovarian slice and calculate the number of apoptotic follicles in ovarian slices of the two groups.RT-PCR and Western Blot were used to evaluate the apoptosis level of ovary and granulosa cells in two groups of mice.ELISA was used to assess serum chemerin levels in two groups of mice,meanwhile RT-PCR and Western Blot to determine the expression of chemerin and CMKLR1 both in ovaries and granulosa cells between the two groups.Immunohistochemistry was used to analyze the distribution of p-AKT in mouse ovarian tissue.The difference of p-AKT between two groups of granulosa cells was compared by Western Blot.Results:(1)After 3 weeks of dietary intervention,the body weights of mice in the OB group increased significantly.At the end of the study,the mean body weight in the OB group was significantly higher than that in the chow group.Additionally,increased GLU,TC,and TG were detected in the serum of the OB group.There was no significant difference in AMH between the two groups.(2)Compared with the CHOW group,CAT and Gpx transcription levels were up-regulated in the ovary of OB mice,and SOD2 transcription levels were significantly down-regulated in granulosa cells.(3)In situ TUNEL staining of mouse ovarian sections revealed that apoptotic follicles mainly showed apoptosis in the inner circle granule cells and scattered granulosa cell apoptosis.The apoptosis of all granulosa cells in follicles and oocyte apoptosis were gradually developed.Compared with the CHOW group,the number of apoptotic follicles in the ovary of the OB group was significantly higher,the ratio of Bax/Bcl2 m RNA in ovarian tissue and granulosa cells increased significantly as well as the level of activated caspase-3 protein in granulosa cells.(4)Compared with the CHOW group,the serum level of chemerin was significantly increased in the OB group.The chemerin and CMKLR1 m RNA,the chemerin protein in the ovary increased in the OB group.And the m RNA and protein of CMKLR1 in the granulosa cells of the OB group were up-regulated.(5)Immunohistochemical staining revealed that p-AKT was mainly expressed in granulosa cells and oocytes of growing follicles.The p-AKT of granulosa cells in OB group was significantly lower than that in CHOW group.Conclusion: In DIO mouse ovarian and granulosa cells,oxidative stress is imbalanced,number of apoptotic follicles increased,apoptosis-related proteins were up-regulated.The chemerin/CMKLR1 system of ovarian tissue and granulosa cells was also activated.These phenomena may be caused by inhibition of the AKT signaling pathway in granulosa cells.Part III.Effect and mechanism exploration of chemerin on oxidative stress and apoptosis of mouse granulosa cells and its mechanismObjective: In vitro experiments were conducted to observe the effects of chemerin on oxidative stress and apoptosis in primary granulosa cells of mice(m GCs)and to explore the mechanism of these changes.Methods: Mouse GCs were isolated from 4-week-old C57BL/6J female mice and cultured in vitro.Viability of m GCs were evaluated using CCK-8 assays after treated with different doses of chemerin for different duration,with or without gonadotropin(Gn).ROS was detected using DCFH-DA probe by in situ staining and flow cytometry with different doses of chemerin treated.In situ TUNEL was conducted on m GCs to observe the number of apoptotic cells and Annexin V/PI staining was used to assess the proportion of apoptotic cells by flow cytometry after treated with different doses of chemerin.RT-PCR and Western Blot were used to evaluate the apoptosis level.P-AKT,p-AMPK,and NF-?B p-p65 were determined by Western Blot.Effect of chemerin on p-AKT protein in m GCs was also detect by Western Blot cultured with or without gonadotrophin.Detection of chemerin downstream p-AKT,p-AMPK and activated caspase-3 of m GCs by Western Blot after activation AKT signaling pathway by sc79,an AKT agonist.After transfecting si RNA to knockdown CMKLR1,the chemerin receptor in mouse granulosa cells,the cells were treated with chemerin to detect the changes of downstream signaling pathway.And the oxidative stress and apoptosis level of CMKLR1-silenced cells were detected by flow cytometry.Results:(1)Chemerin significantly suppressed mouse GC viability when used at concentrations of 100 or 1000 ng/m L for 48 h.In addition,chemerin significantly suppressed gonadotropin-stimulated mouse GC growth.(2)ROS levels were notable increased following 10 ng/m L chemerin treatment.(3)The rate of apoptosis was increased by treatment with 100 or 1000 ng/m L chemerin.Additionally,activated caspase-3 and the m RNA ratio of Bax/Bcl2 were significantly increased in mouse GCs after exposure to more than 100 ng/m L chemerin.(4)Phospho-AKT levels were reduced by treatment with 100 and 1000 ng/m L chemerin.100 or 1000 ng/m L chemerin increased the phosphorylation of AMPK? and NF-?B p65.Western blot analysis showed that treatment with 100 ng/m L chemerin inhibited gonadotropin-induced phosphorylation of AKT.(5)AKT activator treatment of chemerin-stimulated cells revealed a significant increase in p-AKT and decreased expression of activated caspase-3.(6)Knockdown of CMKLR1 blocked the chemerin-induced decreases in p-AKT,up-regulating of AMPK?,and NF-?B p65 phosphorylation compared with those in control si RNA-transfected cells and negative control mouse GCs without transfection.The proportion of apoptotic cell of chemerin stimulated CMKLR1 silenced granulosa cells significantly small than the that of non-silencing group?Conclusion: Chemerin may increase the level of ROS and increase the proportion of apoptotic cells in primary granulosa cells through three signaling pathways.The down-regulation of p-AKT is a key pathway for chemerin-induced apoptosis of primary granulosa cells in mice.Inhibition of the chemerin receptor CMKLR1 reverses the effect of chemerin on mouse primary granulosa cells.