Effect Of Electroacupuncture On PI3K/AKT Signaling Pathway In Female Letrozole-induced Rat Models Of Polycystic Ovary Syndrome With Insulin Resistance

[Objective]: This study was to establish a rat model of polycystic ovary syndrome with insulin resistance(PCOS-IR)using letrozole combined with high fat diet,and to investigate the mechanism of electroacupuncture treatment on the regulation of reproductive endocrine and metabolic abnormalities in PCOS-IR rats from PI3K/AKT signaling pathway.[Method]: 1.Twenty-six female 21-day-old SD rats were divided into a control group(n=8)and a PCOS-IR group(n=18).The PCOS-IR group was fed with 45% high-fat diet for 16 weeks.Letrozole was feed from the third week of the experiment until the end of modeling and the control group was fed with conventional feed.PCOS-IR rats model was established by observing the estrous cycle,ovarian and inguinal adipose tissue changes,comparing body mass,organ mass and organ index changes,detecting of serum testosterone and estrogen levels,glucose tolerance and insulin tolerance test,blood lipid levels,fasting insulin and insulin sensitivity index.(Success rate 66.7%)2.18 successfully prepared PCOS-IR rats were randomly divided into electroacupuncture group(n=6),metformin group(n=6,positive control)and model group(n=6),After 2 weeks of treatment respectively with acupuncture and metformin,the estrous cycle,ovarian and inguinal adipose histological changes were observed.Weekly body mass,organ quality,and organ index changes were recorded;serum testosterone and estrogen levels,glucose tolerance test,lipid levels,fasting insulin,insulin sensitivity and insulin resistance indexes were measured.3.18 successfully prepared PCOS-IR rats were randomly divided into electroacupuncture group(n=6),metformin group(n=6,positive control)and model group(n=6),After 2 weeks of treatment respectively with acupuncture and metformin,the immature oocytes of each group were collected and cultured in vitro.The mitochondrial function of mature oocytes in each group was detected by mitochondrial fluorescent probe technique.The proportion of mitochondrial distribution of oocytes,mitochondrial membrane potential,and changes in reactive oxygen species in mitochondria were observed in each group.4.Real-time quantitative PCR,immunohistochemistry and Western blotting were used to detect the expression of m RNA and protein of IRS1,PI3K,AKT and GLUT4 in PI3K/AKT signaling pathway in ovarian tissue of control group,model group,electroacupuncture group and metformin group.[Result] : 1.The body weight and organ wet weight of the PCOS-IR group were significantly different from those of the control group.The weight of rats in the PCOS-IR model group were significantly higher than that in the control group since the 6th week.The diameter and area of the inguinal adipocytes in the PCOS-IR model group were significantly higher than those in the control group.Pituitary,uterine weight and pituitary index,uterus index,ovarian index and adrenal index in the PCOS-IR model group were significantly lower than in the control group,while the inguinal fat weight and index were significantly higher than the control group.2.The reproductive endocrine indexs of rats in the PCOS-IR group was abnormal.The rats in the PCOS-IR model group showed an abnormal estrous cycle which was continuously in the estrous interval.The ovarian tissue showed multiple follicular cystic dilatation,less granular cell layer,thickening of the tunica albuginea;serum testosterone level was significantly increased,serum estrogen level dropped significantly.3.The metabolic phenotype indicators of rats in the PCOS-IR group were abnormal.The glucose tolerance and insulin tolerance were abnormal in the PCOS-IR group.The blood glucose,fasting insulin and AUC were significantly increased at 15 min,30min,60 min,90min,120 min,and the insulin sensitivity index was significantly decreased.The Insulin resistance indexes triglyceride and Low-density lipoprotein levels are significantly elevated.4.After treatments,the weight gain of the electroacupuncture group was significantly lower than that of the PCOS-IR group;the uterus and pituitary weight,the uterus index and pituitary index of the electroacupuncture group and metformin group were significantly higher than those of the PCOS-IR model group.The ovarian index of the acupuncture group and the metformin group was significantly higher than that of the PCOS-IR model group.The morphological indexes of the ovaries in the electroacupuncture group and the metformin group were significantly improved.The vesicles became smaller and the granule cell layer increased under high magnification.The diameter and area of inguinal adipocytes in the group and metformin group were smaller than those in the model group.The intervention effect was similar to the metformin treatment group.5.After treatments,the testosterone levels in the electroacupuncture group were significantly lower than those in the PCOS-IR model group,and the estrogen level was significantly increased.The blood glucose values of the 60-minute and 90-minute glucose tolerance tests Insulin resistance indexes in the electroacupuncture group were significantly lower than those in the PCOS-IR model group and superior to the metformin treatment group.6.The mitochondrial membrane potential of oocytes in PCOS-IR model was lower than that in the control group.The mitochondrial membrane potential value increased after the electroacupuncture treatment;PCOS-IR model rat oocyte mitochondria Reactive oxygen species was higher than the control group,and the mitochondrial reactive oxygen species was significantly decreased after the electroacupuncture treatment.7.The expressions of IRS-1,PI3K,AKT and GLUT4 m RNA and PI3K,AKT and GLUT4 proteins in ovarian tissue of PCOS-IR model group were significantly lower than those of the control group.After intervention treatment,compared with the PCOS-IR model group,the expression of IRS-1 and GLUT4 m RNA in the ovarian tissue of the electroacupuncture group was significantly increased.At the same time,the expression of AKT and GLUT4 protein in the ovarian tissue of the electroacupuncture group was significantly increased.The expression of PI3K m RNA,PI3K and p-AKT protein in ovarian tissue of rats in electroacupuncture group also increased.The electroacupuncture group was superior to the metformin group.[Conclusion] Letrozole combined with 45% high fat diet can establish an effective animal model of PCOS with endocrine and metabolic phenotype abnormalities.Electroacupuncture treatment could improve the reproductive endocrine and glycolipid metabolism phenotype of polycystic ovary syndrome rats with insulin resistance by affecting PI3K/AKT signaling pathway,and regulate the mitochondrial function of oocytes in rats with insulin resistance.Electroacupuncture is a more effective way to improve insulin resistance polycystic ovary syndrome.