Based On IL-6/STAT-3/miR-155/PPAR-? Pathway To Explore The Mechanism Of Myo-inositol To Treat PCOS-IR

1.BackgroundPolycystic ovary syndrome(PCOS)is one of the most common female endocrine and metabolic disorders,which can be accompanied by insulin resistance(IR)and obesity.Myo-inositol,a drug used to treat nonalcoholic fatty liver,has been shown to reduce IL-6 and FFA,and might therefore function through the IL-6/STAT-3/miR-155/PPAR-y pathway.In the past decade,myo-inositol has been introduced into the treatment of PCOS.Myo-inositol is a sugar alcohol and a glucose isomer which is found in many foods,including cereals and fruits.It is also the precursor of numerous secondary messengers and is once considered to be a B vitamin.It reduces the IR status of women with PCOS and improves their ovarian function and androgen levels.Myo-inositol has also displayed good curative effects on abnormal lipid metabolism.Numerous studies have shown that myo-inositol can be used to treat PCOS-IR and improve pregnancy outcomes,although the mechanism is unclear.Many studies have shown that a high-fat diet(HFD)can induce a model of IR,Increased FFA and IL-6 are found in nonalcoholic fatty liver and PCOS-IR models induced by HFD,free fatty acids(FFA)promotes the expression of interleukin 6(IL-6),so it can be speculated that elevated FFA and elevated IL-6 contributed by HFD play important roles in the mechanism of IR.PCOS is a proinflammatory state,and chronic low-grade inflammation plays a vital role in its pathogenesis.Serum inflammatory factors(including IL-6)are significantly higher in women with PCOS than in those without this disease.IL-6 is the main proinflammatory cytokine involved in chronic inflammation and is intrinsically linked to the condition.Therefore,various inflammatory diseases may share the same or similar signal pathways.Thus,IL-6 is an early marker of low-grade chronic inflammation and a key molecule in PCOS.The IL-6-mediated JAK2/STAT-3 signaling pathway plays an important role in PCOS.The signal transducer and activator of transcription 3(STAT-3)signaling pathway stimulates the expression of micro RNA 155(miR-155)and inhibits the phosphorylation of STAT-3,which inhibits the expression of miR-155.The activation of STAT-3 increases the expression of its downstream molecule miR-155.Studies of the extracellular vesicles involved in diabetes have shown that miR-155 participates in IR and the pathogenesis of diabetes by targeting peroxisome proliferators activated receptor gamma(PPAR-?).The expression of both IL-6 and miR-155 is increased in PCOS patients.It has also been demonstrated experimentally that IL-6 promotes STAT-3 activation in PCOS.However,the expression of PPAR-?,which is targeted by miR-155,a downstream molecule of STAT-3,is reduced in patients with PCOS.Because HFD increases the expression of FFA in the blood of PCOS patients,and FFA increases the expression of IL-6,FFA and IL-6 can activate STAT-3.Therefore,we speculate that the IR caused by abnormalities in lipid metabolism may be caused by an increase in FFA,affecting the IL-6/STAT-3/miR-155/PPAR-y pathway.Both ovarian autophagy and ovarian angiogenesis are significantly elevated in rat models of PCOS and in patients with PCOS.Ovarian autophagy and ovarian angiogenesis have significant effects on follicular development and ovulation,and the IL-6/STAT-3/miR-155/PPAR-y pathway also plays an important role in ovarian autophagy and angiogenesis Therefore,the IL-6/STAT-3/miR-155/PPAR-y pathway may regulate IR,ovarian autophagy,and ovarian vascular abnormalities in PCOS.Does myo-inositol affect insulin resistance,ovarian autophagy and ovarian angiogenesis?Does the IL-6/STAT-3/miR-155/PPAR-? pathway participate in IR,ovarian autophagy,and abnormal angiogenesis in PCOS?Whether the mechanism underlying the action of myo-inositol is related to this pathway is unclear.Answering these questions will not only clarify the mechanism by which myo-inositol treats PCOS,but will provide a theoretical basis for PCOS-IR and other IR-related diseases.2.ObjectivesRats were continuously treated with letrozole for 4 weeks and HFD for 8 weeks to establish the PCOS-IR model,after treated with myo-inositol,the expression of IL-6,STAT-3,miR-155,and PPAR-y in ovary tissue,adipose tissue and liver tissue was detected to explore the detailed mechanism of myo-inositol to improve insulin resistance in polycystic ovary syndrome-insulin resistance(PCOS-IR),and then to explore the effect and mechanism of myo-inositol for ovarian autophagy and abnormal ovarian angiogenesis in PCOS.3.MethodsTwenty female Sprague-Dawley rats(3-4 weeks old)were used to construct a rat model of PCOS-IR.The rats were randomly divided into two groups(n=10 each)rats in PCOS group were continuously treated with letrozole for 4 weeks and HFD for 8 weeks to establish the PCOS-IR model,and control group was treated with normal diet.The morphology of the ovaries was determined after hematoxylin and eosin(HE)staining.The expression of sex hormones,insulin,FFA,and IL-6 were detected.PPAR-y mRNA,and GLUT4 mRNA was detected with fluorescent real-time quantitative PCR,the expression of PPAR-y,and GLUT4 in ovary was detected with immunofluorescence.And then forty-five female Sprague-Dawley rats(3-4 weeks old)were used for the following experiment.The female SD rats were randomly divided into 3 groups(n=15 in each group and rats in each group kept in two cages in the same environment),negative control group(chow diet,treated with physiological saline),PCOS-IR group(HFD&letrozole,treated with physiological saline),and PCOS-IR+Myo-inositol(HFD&letrozole,treated with Myo-inositol).The morphology of the ovaries was determined after hematoxylin and eosin(HE)staining.The expression of sex hormones,insulin,triglycerides,cholesterol,FFA,IL-6,and vascular endothelial growth factor(VEGF)were detected.CD31 was used to mark vascular endothelial cell.Autophagosomes and extracellular vesicles were observed with electron microscopy.The expression of IL-6,STAT-3,and phosphorylated-ST AT-3(p-STAT-3)in ovary tissue was detected with immunohistochemistry.The expression of IL3,PPAR-y,and GLUT4 in ovary tissue was detected with immunofluorescence.The expression of miR-155,miR-21,PPAR-y mRNA,and GLUT4 mRNA was detected with RT-PCR.The expression of IL-6,STAT-3,p-STAT-3,PPAR-y,GLUT4,LC3,mTORC1,and mTORC2 was detected with western blot.4.Results1.After the administration of HFD and letrozole,ovarian HE staining showed polycystic ovarian changes in the rats in the PCOS model group.The HOMA-IR index of the control group was 2.09±0.64,whereas that of the PCOS model group was 4.55±0.49,indicating a significant difference between the two groups(P<0.01).Serum luteinizing hormone(LH),testosterone,the LH/follicle-stimulating hormone(FSH)ratio,FFA,and IL-6 were significantly increased in the PCOS model group.The immunofluorescence results indicated that the expression of PPAR-y and GLUT4 was significantly elevated in the PCOS model group.The expression of PPAR-y mRNA and GLUT4 mRNA in the ovarian tissues of the two groups was detected with RT-PCR,and was lower in the PCOS group than in the control group(P<0.01).2.Average weekly food intake and body weight were significantly higher in the PCOS group than in myo-inositol group or blank control group(P<0.01).Serum LH,testosterone,and the LH/FSH ratio were significantly higher in the PCOS rat model group than them in negative control group or myo-inositol group(P<0.01),whereas the serum estradiol(E2)level was slightly lower in the PCOS rats than it in negative control group or myo-inositol group(P<0.01).However,there was no significant difference in the FSH levels of the three groups(P=0.157).The HOMA-IR indices of rats in negative control group and myo-inositol group were 2.15 and 2.27,respectively,whereas that of the PCOS model group was 4.22(P<0.01).3.Polycystic changes were observed in the PCOS rats.In contrast,the follicles developed normally in myo-inositol group and blank control group.The serum levels of triglycerides,VEGF and IL-6 were significantly higher in PCOS group than them in negative control or myo-inositol group(P<0.01),whereas their serum cholesterol levels did not differ significantly(P=0.216).4.RT-PCR was used to detect the expression of miR-155,miR-21,IL-6 mRNA,and PPAR-y mRNA in the ovaries.The ovarian expression of PPAR-y mRNA was significantly higher in negative control and myo-inositol group than it in PCOS group,whereas the ovarian expression of miR-155,miR-21,and IL-6 mRNA in the rats was significantly lower in negative control group and myo-inositol group than them in PCOS group(P<0.01).5.The results of immunohistochemistry,the immunofluorescence assays,and the results of western blot suggested that the expression of IL-6 and p-STAT-3 in the ovarian tissues of PCOS group was significantly higher than them in the ovarian tissues of myo-inositol group or negative control group.Myo-inositol supplementation did not affect STAT-3 expression(P<0.01).However,the expression of PPAR-y and GLUT4 in the ovarian tissues of PCOS group was significantly lower than them in the ovarian tissues of myo-inositol group or negative control group(P<0.01).6.Immunofluorescence microscopy was used to detect the expression of LC3 in ovarian tissues,and the expression of LC3 was significantly enhanced in the ovary of PCOS group.The LC3-II/LC3-I ratio was significantly higher in the ovarian tissues of PCOS group than them in the ovarian tissues of the myo-inositol or control group.The ovarian expression of mTORCl and mTORC2 was significantly lower in the ovarian tissues of the PCOS group than them in the ovarian tissues of blank control group or myo-inositol group.7.The results of immunofluorescence and immunohistochemistry revealed clear proliferation of the blood vessels in the ovary of PCOS group,however,in myo-inositol group and blank control group,the proliferation of blood vessels in the ovary was not clear.8.HE staining showed that the volumes of white fat cells in the PCOS-IR group were greater and they contained less cytoplasm than them in blank control or myo-inositol group.western blot was used to detect the protein expression of IL-6,STAT-3,p-STAT-3,and PPAR-y in the adipose tissues.The expression of IL-6 and p-STAT-3 in the adipose tissues of PCOS group was significantly elevated.However,there was no significant difference in the expression levels of STAT-3 in the adipose tissues of the three groups.The expression of PPAR-y was significantly reduced in the adipose tissues of PCOS group(P<0.01).9.The expression of miR-155 and miR-21 in the adipose tissue and serum extracellular vesicles was significantly higher in PCOS group than them in myo-inositol group or negative control group(P<0.01).The serum levels of VEGF and FFA were also significantly higher in PCOS group than them in myo-inositol group and negative control group(P<0.01).10.HE staining of liver tissues showed that many fat vacuoles accumulated around the liver cells in the PCOS model rats,whereas there were significantly fewer fat vacuoles around the liver cells in myo-inositol group and blank control group.The local expression of PPAR-y and GLUT4 was significantly lower in the liver of PCOS group than them in the liver of myo-inositol group or blank control group(P<0.01).5.Conclusions1.HFD and letrozole successfully induced a rat model of PCOS-IR.FFA,IL-6 and miR-155 played important roles in the establishment of the PCOS-IR model.The reduced expression of PPAR-y and GLUT4 in the ovarian tissues is involved in the ovarian IR in PCOS-IR model rats.2.High fat diet and letrozole could induce insulin resistance in ovary tissue,myo-inositol supplementation could reduce FFA in serum,reduce the expression of FFA,IL-6,p-STAT-3,and miR-155,and increase the expression of PPAR-y,thereby improving insulin resistance in ovary.3.More autophagy in ovary was found in PCOS-IR rats,and myo-inositol could reduce autophagy in ovary by up-regulation of MTORC1 and MTORC2.4.More abnormal angiogenesis in ovary was found in PCOS-IR rats,and myo-inositol could reduce abnormal angiogenesis in ovary by up-regulation of MTORCl and down-regulation of VEGF.5.High fat diet and letrozole could induce insulin resistance in adipose tissue,myo-inositol supplementation could reduce FFA in serum,reduce the expression of FFA,IL-6,p-STAT-3,and miR-155,and increase the expression of PPAR-y,thereby improving insulin resistance in adipose6.High fat diet and letrozole could induce insulin resistance in liver tissue,myo-inositol supplementation could improve insulin resistance in liver tissue,and its mechanism is related to the down-regulation of of miR-155 in exocrine body and up-regulation of PPAR-? in liver tissue.