Effects Of Humanin On Oxidative Stress And Insulin Resistance In Polycystic Ovarian Syndrome And Its Mechanism

Part 1 The role of humanin in oxidative stress of polycystic ovarian syndrome and its mechanism[Purpose] To study the humanin concentration of serum and follicular fluid in women with polycystic ovarian syndrome(PCOS)and women with only male factor infertility(control)and humanin m RNA expression in ovarian granulosa cells,to investigated the effects of exogenous humanin analogue S14G-HN(HNG)administration on the levels of superoxide dismutase(SOD),catalase(CAT)and malondialdehyde(MDA)of local ovary and serum in PCOS rats,to probe the mechanism of humanin in alleviating oxidative stress by constructing ovarian granulosa cell line COV434 and primary rat granulosa cell oxidative stress model with vitamin K3(vit K3)in vitro.[Methods] A total of 44 women aged between 20-40 who came to the clinic for IVF/ICSI treatment were recruited for the study,16 women were diagnosed with PCOS using Rotterdam criteria,other 28 women with only male factor infertility were normal controls.The clinical and epidemiological characteristics were analyzed.Serum,follicular fluid and ovarian granulocyte cells were collected,ELISA and q RT-PCR were used to detect the concentration and expression differences of humanin.Sixty female Sprague-Dawley rats(21 days old)were purchased from the Animal Centre of Tongji Medical College.Forty-eight rats were subcutaneously injected with DHEA(6mg/100 g body weight)daily to establish the PCOS model.Twelve animals were injected with 0.2ml of sesame oil(control group).After 14 days’ DHEA injection,the model animals were randomly divided into four experimental groups(n=12 in each group).One group received only subcutaneous DHEA injections for another seven days(PCOS group),and the other three groups received subcutaneous DHEA injections for 7 days simultaneously with intraperitoneal injection of low,medium and high doses of HNG(2.5mg/kg,5mg/kg,10mg/kg body weight)for 14 days(PCOS+HNG 2.5mg/kg group,PCOS+5mg/kg group,PCOS+10mg/kg group,respectively).The body weight and ovarian weight of rats in each group were counted.Blood sample and ovaries were collected to probe the effect of S14G-humanin(HNG)supplementation on body weight,ovarian morphological alterations,endocrinological disorders and ovarian and systemic oxidative stress.Ovarian granule cell line COV434 and primary rat granule cells were treated with gradient(0,20,40,60,80,100,120μM)and(0,10,20,30,40,50,60μM)of vit K3.The cell viability was detected by CCK8 assay.The concentration of vit K3 with 50% cell viability was selected as the concentration to induce moderate oxidative stress injury.The COV434 was treated with vit K3 and gradient HNG(0,2.5,5,10,15,20μg/m L)simultaneously,cell viability was detected by CCK8 assay,selecting the concentration of HNG restore cell viability similar to control group as the concentration to alleviate oxidative stress injury.The COV434 cells were divided into blank control group,vit K3 and vit K3 +HNG groups,levels of SOD and MDA were detected.The expression levels of genes and proteins related to Keap1/Nrf2 /ARE signaling pathway in each group were detected by q RT-PCR and western blotting.The nuclear translocalization of Nrf2 from cytoplasm to nucleus was verified by immunofluorescence and western blotting.[Results] We found that compared with the control group,patients in the PCOS group had significantly higher levels of luteinizing hormone(LH),total testosterone(AMH),antimiillerian hormone(AMH),moreover insulin level and homeostasis model assessment of insulin resistance(HOMA-IR)index was also significantly increased compared with control group(P=0.038,0.045,0.003,0.028,0.044).Humanin concentrations were significantly lower in the follicular fluid of PCOS patients than that of controls,whereas no significant difference in serum humanin concentration was observed between these two groups(p=0.024,p=0.642).The m RNA expression of humanin in the granulosa cells of PCOS patients was also significantly lower than in control patients(p=0.021).HNG supplementation by 5.0 or 10.0 mg/kg body weight intraperitoneal injection significantly attenuated body weight,ovary weight,serum DHEA and oestradiol(E2)concentrations,follicle development,serum and ovarian superoxide dismutase(SOD)activity,catalase(CAT)activity and malondialdehyde(MDA)concentrations,in comparison with PCOS controls(all p < 0.05).In the in vitro study,we found that vit K3 at 80μM reduced cell viability by 50%,causing moderate oxidative stress injury to COV434 cells.Compared with the control group,the SOD level of 80μM vit K3 group was significantly decreased(P=0.02)and the MDA level was significantly increased(P=0.001),however,HNG(5.0 μg/m L)intervention significantly attenuated vit K3(80μM)-induced decrease of SOD activity(p=0.01)and increased MDA concentrations(p=0.04)in COV434 cells and also significantly upregulated the m RNA and protein expression of Nrf2(p=0.002,0.03)and its downstream proteins haem oxygenase-1(HO-1)(p=0.001)and NADPH quinineoxidoreductase-1(NQO1)(p=0.04)relative to the blank control group.An obvious translocation of Nrf2 from the cytoplasm to the nucleus was also observed in COV434 cells and rat primary granuloma cells following 5.0 μg/m L HNG supplementation by immunofluorescence.Western blotting showed that the expression of Nrf2 in the cytoplasm of 80μM vit K3+5μg/ m L HNG group was significantly decreased(P=0.001,0.002)and the expression in the nucleus was significantly increased(P=0.004,0.045)compared with the blank control group and 80μM vit K3 group.[Conclusions] Humanin expression was found to be significantly downregulated in the granulosa cells of PCOS patients relative to those of non-PCOS patients.Exogenous administration of HNG improve endocrine,promote follicular development and ovulation,and alleviate oxidative stress in PCOS rats.Humanin alleviates oxidative stress in ovarian granulosa cells of PCOS patients via modulation of the Keap1/Nrf2 signaling pathway.Part 2 The role of humanin in insulin resistance of polycystic ovarian syndrome and its mechanism[Purpose] In this study,we aim to compare humanin concentrations in serum and follicular fluid between polycystic ovarian syndrome(PCOS)patients with and without IR,to investigated the effect of humanin analogue S14G-HN(HNG)supplementation on IR in a dehydroepiandrosterone-induced PCOS rat model and probe its molecular mechanism,to study the glucose consumption in normal and humanin si RNA-transfected COV434 cells when treated with HNG and insulin.[Methods] Eighty-three women aged between 20-35 who visited the clinic for IVF/ICSI treatment were enrolled in the study.Forty-seven had been diagnosed as having PCOS according to the Rotterdam criteria,and the remaining thirty-six non-PCOS women were considered as controls.Both the PCOS patients and the non-PCOS controls were subdivided into groups with or without IR according to their fasting insulin(FINS)level and the homeostasis model assessment of insulin resistance(HOMA-IR)index(IR was diagnosed in a patient with a FINS level ≥15 μIU/m L or HOMA-IR index≥2.0).Blood samples were collected on days 2–4 of menstruation.Follicular fluid from the first punctured follicle from either ovary was collected during oocyte retrieval.Demographic and clinical data including the antral follicle count and the levels of reproductive hormones,anti-Müllerian hormone(AMH),lipids,fasting plasma glucose(FPG)and fasting insulin(FINS),were collected from the ART system.21-day-old female Sprague–Dawley rats(n=35)were purchased from the Animal Centre of Tongji Medical College.Twenty-eight of these rats were subcutaneously injected daily with dehydroepiandrosterone(DHEA)(6 mg/100 g body weight)for 21 days to generate the PCOS model.The remaining seven rats were injected with 0.2 m L of sesame oil(control group,n=7).After injection with DHEA for 14 days,the animals were randomly divided into four experimental groups(n = 7 per group).One group received only a subcutaneous injection of DHEA for another 7 days(PCOS group).The remaining three groups received a subcutaneous injection of DHEA for 7 days and low-,medium-and highdose injections of HNG(S14G Humanin,a humanin analogue)for 14 days(2.5,5.0 and 10.0 mg/kg body weight,respectively).Blood and bilateral ovaries were collected after overnight fasting.The serum levels of FPG and FINS were measured by a glucose oxidation method using commercial kits.Protein expression levels of IRS1,PI3 K,AKT and GLUT4 in the granulosa cells of PCOS rats was investigated by western blotting.We knocked down humanin expression in the human granulosa cell line COV434 using two reported si RNAs,glucose consumption in the COV434 cells 24 h after humanin knockdown and exogenous HNG supplementation was measured by glucose oxidase method,m RNA expression levels of IRS1/PI3K/ AKT pathway and glucose transporter 4(GLUT4)in each group were detected by q RT-PCR,western blotting and immunofluorescence were used to verify the translocation of GLUT4 from cytoplasm to cell membrane of primary rat granulocyte cells and COV434 after exogenous HNG supplementation.[Results] In this study,these four groups had similar serum humanin concentrations but significantly different follicular fluid humanin concentrations.The PCOS patients with IR had significantly lower follicular fluid humanin concentrations than those without IR(186.56 ± 51.83 pg/m L versus 250.20 ± 47.19 pg/m L,p = 0.006).In the PCOS patients,the basal total testosterone level,FINS concentration and HOMA-IR index were significantly and negatively associated with the follicular fluid humanin concentration(p = 0.017,0.012 and 0.015,respectively).The FPG and FINS levels in PCOS rats were significantly higher than those in normal rats(p < 0.001 and p < 0.001,respectively).However,alterations of the FPG and FINS levels were attenuated significantly by HNG supplementation.The phosphorylation levels of IRS1,PI3 K and AKT in the PCOS models were all significantly downregulated relative to those in the controls(p=0.008,p=0.013 and p=0.001 respectively).The two designed si RNAs described earlier were used for humanin knockdown.The humanin m RNA levels decreased to approximately 30% of those in the controls at 24 h after transfection.The effects of HNG supplementation(1,2.5,5 μg/ml)on glucose consumption in normal COV434 cells and si RNA1-and si RNA2-transfected COV434 cells were evaluated.Combined supplementation with insulin(10 ng/m L)and HNG(5 μg/m L)increased the IRS1,AKT and GLUT4 m RNA expression levels relative to the corresponding controls(all p<0.001).Immunofluorescence microscopic analysis showed that combined supplementation with insulin and HNG strengthened the GLUT4 signal and promoted GLUT4 translocation from the cytoplasm to the membrane in COV434 and primary rat granulosa cells.The cell membrane protein and cytoplasmic protein of COV434 were extracted respectively for western blotting.Compared with the control group,the expression of GLUT4 in the cell membrane was significantly increased after the exogenous addition of HNG(P=0.02),but the expression of GLUT4 in the cytoplasm was not significantly changed.[Conclusions] Downregulated humanin in the ovaries may have been involved in the pathogenesis of IR in PCOS,and exogenous supplementation with HNG improves local ovarian IR through modulation of the IRS1/PI3K/AKT signaling pathway.