| Background Polycystic ovary syndrome(PCOS)is one of the most common endocrine diseases in women of reproductive age.It is easy to cause ovulation disturbance and infertility.There is a high risk of glycemic lipid metabolism disorder.The proportion of Non-alcoholic fatty liver disease(NAFLD)is high.Insulin resistance(IR)is a common manifestation of PCOS and a key pathogenesis factor of NAFLD.IR is one of the important causes of NAFLD,which leads to impaired lipolysis inhibition function,and then increases the flow of free fatty acids into the liver,aggravating hepatic steatosis and liver injury.In addition,metabolic disorders such as hyperandrogen and obesity,which are common in PCOS,will also promote the development of NAFLD.Electroacupuncture(EA)is one of the important methods for clinical treatment of PCOS,which can improve ovulation disorder and glucose lipid metabolism disorder and enhance insulin sensitivity.Our previous study found that letrozole plus high-fat diet induced obese PCOS rat models,and showed the characteristics of liver fatty degeneration,and electroacupuncture intervention on PCOS rats liver lipid accumulation has a significant improvement effect.However,it is still unclear whether the letrozole plus high-fat diet induced hepatic adipose change model of PCOS rats is stable and how electroacupuncture improves hepatic adipose change in PCOS rats.Objective1.To verify the rat model of letrozole and high-fat diet induced PCOS with hepatic fatty degeneration;2.Based on Akt/GSK3β pathway,to explore the mechanism of electroacupuncture improving lipid metabolism in PCOS rats.Methods1.Construction of rat models Thirty female SD rats(21 days of age)were matched with body weight and randomly divided into control group and high fat diet group with 15 rats each: control group(after 1 week of adaptation,continued to eat ordinary diet);In the high-fat diet group(after 1 week of adaptation,rats were given high-fat diet),rats in the two groups were given different diets at 28 days of age,which was recorded as the first day of the experiment.Subsequently,rats were fed high-fat diet(HFD)and normal diet(ND)for35 consecutive days,and were randomly divided into the following four groups:Letrozole(LET)and electroacupuncture(EA)for intervention,respectively.HFD+LET,HFD+LET+EA,Control,Control+EA.Rats in HFD+LET group and HFD+LET +EA group were given letrozole solution 1.0 mg/kg once a day for 25 consecutive days.Control group and Control+EA group were given equal volume solvent(0.5% CMC-Na)for 25 days.At the same time,HFD +LET +EA group and Control+EA group were treated with electroacupuncture,and the selected points were Zusanli and Tianshu points(about 5mm on both sides of the rat navel,and about 5mm on the lateral tibial anterior tubercle of the rat hind limb).Electroacupuncture was performed 5 ～ 6 times a week,up to 20 times.The fixed device was used to fix the rats during electroacupuncture treatment,and the time of each treatment was 15-25 min.Since 1 rat in CON group had irregular estrous cycle,and 1 rat in model group had no typical PCOS phenotype and estrous cycle,a total of 2 rats were excluded,and the final 4 groups were divided as follows:(1)HFD+LET group: induced PCOS combined with liver fatty transformation model(n = 7);(2)HFD+LET+EA group: electroacupuncture was used to treat PCOS with hepatic adipose lesion(n = 7);(3)Control group: The control group was given equal volume solvent(0.5% CMC-Na)by gavage(n = 7);(4)Control+EA group: equal volume solvent(0.5% CMC-Na)gavage +electroacupuncture treatment(n = 7);2.Mechanism analysis of electroacupuncture intervention During the experiment,the rats were weighed and smeared regularly to evaluate the changes in body weight and estrous cycle.On the 61 st day of the experiment,heart blood was collected under isoflurane anesthesia,and liver and adipose tissue samples(including paradadal fat,mesenteric fat,inguinal fat and retroperitoneal fat)were collected.The samples were weighed respectively to evaluate the effect of electricity on adipose tissue.Blood samples of rats were collected to detect hormone levels,liver function and blood lipids,and to evaluate glucose and lipid metabolism.HE staining and oil red O staining were performed on rat liver tissue to evaluate the changes of liver morphology.The expression of Akt/GSK3β pathway related proteins was detected by western blot analysis of liver tissues.In addition,real-time fluorescence quantitative polymerase chain reaction(PCR)was performed on liver tissues to detect the expressions of inflammatory factors TNF-α,IL-1β,IL-6,Mcp1 and lipid metabolism-related molecules PPAR-α,PPAR-γ and Acc,so as to explore the mechanism of electroacupuncture improving liver lipid metabolism in PCOS rats.Results1.Evaluation of rat model:Compared with the control group,the body weight,liver weight,adipose tissue weight(including parametrial fat,mesenteric fat,inguinal fat)and total cholesterol of the letrozole combined with high-fat diet induced rat model were significantly increased(p < 0.001).The levels of alanine aminotransferase(ALT),free fatty acid(FFA)and Liver triglyceride(Liver TG)were significantly increased(p < 0.05,p <0.001,p < 0.01),and the level of testosterone was increased(p < 0.01).In the glucose tolerance test,the blood glucose levels of the rats at 15,60,90 and 120 minutes after intragastric administration of glucose were significantly higher than those of the control group(p < 0.05,p < 0.01,p < 0.01 and p < 0.001),reflecting the metabolic problems of PCOS complicated with fatty liver.The estrus cycle of rats was observed by vaginal smear,and the results showed that the model group did not show a normal estrus cycle.HE staining showed that the number of white vacuolated adipocytes in the liver of the model group increased,and the adipose tissue was significantly enlarged.Oil red O staining showed that the red lipid droplets were arranged disorderly,and the density was significantly increased compared with the control group,indicating the fatty change of the liver.2.Efficacy evaluation of electroacupuncture:(1)Body weight and tissue weight: compared with the control group,the weight of body weight,liver,parametrial fat,mesenteric fat and inguinal fat were significantly decreased in the letrozole plus high-fat diet group after EA treatment(p<0.001);(2)Glucose tolerance test and insulin resistance index: the fasting blood glucose and fasting insulin in the EA treatment group were significantly decreased(P<0.001,P<0.05),the glucose levels at 15,30,60,and 120 minutes of glucose tolerance were significantly decreased(p<0.001,p<0.01,p<0.05,p<0.01),and the area under the curve(AUC)of blood glucose was significantly decreased(p<0.001);(3)Estrus cycle: after EA treatment,the estrus cycle of rats began to fluctuate to some extent,but failed to return to the regular estrus cycle.(4)In the EA group,HE staining showed that the number of lipid droplets in the liver was significantly reduced or disappeared,and oil red O staining showed that the degree of abnormal lipid accumulation in the liver was reduced.(5)Liver inflammatory factors: EA significantly reduced the levels of inflammatory factors TNF-αand IL-6 in HFD+LET group rats(p<0.05,p<0.001),thereby improving liver inflammation.(6)At the gene and protein levels,EA up-regulates the protein expression of p-Akt(Ser473),p-GSK 3β(Ser9)and GLUT4 and down-regulates the protein expression of GSK3β,which is involved in regulating the PI3K/Akt signaling pathway.Conclusion1.Letrozole plus high-fat diet can induce PCOS with hepatic adipose change in rat models;2.Electroacupuncture can improve lipid metabolism,reduce liver fat accumulation and liver inflammation in PCOS rats through Akt/GSK3β pathway,providing ideas and basis for clinical treatment of PCOS complicated with NAFLD. |
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