Chemerin isoform analysis in human biofluids using an LC/MRM-MS-based targeted proteomics approach with stable isotope-labeled standard.

Targeted proteomics has advantages over earlier conventional technologies for protein detection. We developed and validated an LC/MRM-MS-based targeted proteomic method combined with immunoaffinity precipitation for the enrichment and detection of low abundance chemerin isoforms in human biofluids. After tryptic digestion, each chemerin isoform was characterized by isoform-specific peptides, and the absolute quantification was achieved by using stable isotope-labeled peptides as internal standards. In serum, follicular fluid and synovial fluid, a total of 6 chemerin isoforms were identified and quantified, among which a novel natural isoform 153Q was discovered for the first time. The relative content of the six chemerin isoforms in human serum was 157S ≫ 156F ≫ 158K > 154F ≥ 155A > 153Q in the ratio of 25:17:5:2.5:2.2:1, respectively. The absolute contents were in the range of 88-3.5 ng/mL. This distribution remained consistent among the 3 biofluids analyzed. Total chemerin were found to be increased in both polycystic ovary syndrome (serum and follicular fluid) and rheumatoid arthritis (serum) patients. However, chemerin isoform analysis revealed that only 156F & 157S were increased in the former, while 155A, 156F & 157S were increased in the latter. This demonstrates the potential of this method in detailed characterization of changes in chemerin isoforms that may be of clinical relevance.

Alterations of Sphingolipid Metabolism in Different Types of Polycystic Ovary Syndrome.

The roles of sphingolipids in polycystic ovary syndrome (PCOS) are still unknown. This study aimed to investigate the sphingolipid characteristics for different types of PCOS using liquid chromatography-mass spectrometry (LC-MS). A total of 107 women with PCOS and 37 healthy women as normal controls were studied. PCOS patients were further classified into non-obesity with insulin resistance (IR) (NOIR), obesity with IR (OIR), and non-obesity and non-IR (NIR) subgroups. A total of 87 serum sphingolipids, including 9 sphingosines, 3 sphinganines, 1 sphingosine-1-phosphate (S1P), 19 ceramides (Cers), 1 ceramide-1-phosphate, 44 sphingomyelins (SMs), 4 hexosylceramides, and 6 lactosylceramides (LacCers) were analyzed using an improved sphingolipidomic approach based on LC-MS. Notable elevations in the levels of S1P, Cer, and SM were observed in PCOS patients when compared with healthy women, and SM species with long saturated acyl chains showed potential as novel biomarkers of PCOS. In addition, the level of LacCer was only elevated in NIR, and there was almost no change in NOIR and OIR. This study is the first to report the comprehensive sphingolipidomic profiling of different subgroups of PCOS with or without IR or obesity and suggests that serum sphingolipids might be useful as diagnostic biomarkers for different types of PCOS.

[Electroacupuncture Combined with Clomiphene Promotes Pregnancy and Blastocyst Implantation Possibly by Up-regulating Expression of Insulin Receptor and Insulin Receptor Substrate 1 Proteins in Endometrium in Rats with PCOS].

OBJECTIVE: To observe the effect of electroacupuncture (EA) intervention combined with clomiphene critate (CC) on the blastocyst implantation and pregnancy rate and expression of insulin receptor (INSR) and insulin receptor substrate 1 (IRS 1) proteins in the endometrium in rats with polycystic ovary syndrome (PCOS), so as to reveal its mechanisms underlying improvement of PCOS. METHODS: One hundred and twenty-five female SD rats were randomly divided into normal control, PCOS model, medication (CC), EA and EA+CC groups (n=25 in each group, 15 for checking blastocyst implantation, and 10 for Western blot). The PCOS model was established by subcutaneous injection of Dehydroepiandrosterone (DHEA) and fed with high-fat diet. Rats of the normal control group were treated by subcutaneous injection of sesame oil and fed with the normal forage. EA stimulation was applied to "Zhongwan" (CV 12), "Guanyuan" (CV 4) and bilateral "Tianshu" (ST 25) for 30 min, 3 times a week, 5 weeks altogether. Rats of the CC and EA+CC groups were fed with CC (100 mg·kg-1·d-1) for 2 days after regular restriction (30 min, 3 times a week, 5 weeks altogether). The pregnancy was determined by vaginal smear tests and the number of blastocyst implantation determined by examination of the uterus after execution. The expression of INSR and IRS 1 proteins in the endometrium was detected by Western blot. RESULTS: The pregnancy rate and the number of blastocyst implantation were significantly lower in the model group than in the normal control group (P<0.05), and remarkably increased after EA and EA+CC interventions (P<0.05). The effects of EA+CC were obviously superior to those of simple EA and simple medication in raising the pregnancy rate and the number of blastocyst implantation (P<0.05). No significant differences were found between the EA and CC groups in the pregnancy rate and the number of blastocyst implantation (P>0.05). The relative expression levels of both INSR and IRS 1 proteins were markedly lower in the model group than in the normal control group (P<0.05), and significantly higher in both EA and EA+CC groups than in the model group (P<0.05). No significant differences were found between the model and the CC groups and between the EA and EA+CC groups in the expression levels of INSR and IRS 1 proteins (P>0.05). CONCLUSIONS: EA intervention can improve pregnancy rate and the number of blastocyst implantation in PCOS rats, which may be related to its effects in up-regulating the expression of INSR and IRS 1 proteins in the endometrium.

[Effects of Electroacupuncture on mRNA Expressions of Insulin-receptor Substrates 1 and 2 in the Endometrium of PCOS Rats and Insulin Sensitivity].

Objective To observe the effects of electroacupuncture (EA) on gene expressions of insulin receptor substrate 1 (IRS1) and insulin receptor substrate 2 (IRS2) in the endometrium and insulin sensitivity (IS) of polycystic ovary syndrome (PCOS) rats, thereby providing basic evidence of clinical ap- plication of EA for improving the pregnancy rate of PCOS patients and reducing the abortion rate. Methods Dehydroepiandrosterone (DHEA) was subcutaneously injected to 24-day-old female SD rats to induce P- COS model. Besides, rats were fed with high-fat diet. Rats in the normal group were subcutaneously injected with sesame oil and fed with common forage. PCOS model rats were randomly divided into the model group and the EA group. All rats were intervened from 80 days old. Of them, EA was started to rats in the EA group, three times per week for 5 successive weeks. Rats in the normal group and the model group were only bound every day, but with no acupuncture, three times per week for 5 successive weeks. Blood was collected from caudal vein before and after treatment. Fasting blood glucose (FPG) was detected by oxidase method. Fasting insulin (FINS) level was determined by Roche electrochemical luminescence method. Homeostasis model assessment of insulin resistance ( HOMA-IR) index was also calculated. All rats were killed by decapitation by the end of intervention, and their endometria were collected. mRNA expressions of IRS1 and IRS2 in the endometrial tissue were detected by Real-time fluorescence quantitative PCR method. Results Pre-post-treatment changes of FPG level were not significantly different among the three groups (P >0. 05). Before treatment FINS level and HOMA-IR index were significantly higher in the model group than in the normal group (P <0. 05). After treatment, they were significantly lower in the EA group than in the model group (P <0. 05). Compared with the normal group, mRNA expressions of IRS1 and IRS2 in the endometrial tissue were decreased in the model group (P <0. 05). Compared with the model group, mRNA expressions of IRS1 and IRS2 in the endometrial tissue were increased in the EA group (P < 0. 05). Conclusion EA could improve IS and elevate mRNA expressions of IRS1 and IRS2 in the endome- trial tissue of PCOS rats, which might be one of mechanisms for improving endometrial IS.

[Effective Ingredients of Yangjing Zhongyu Decoction Regulated Androgen Biosyntheses by Mitogen-Activated Protein Kinase Pathway in Porcine Granulose Cells].

OBJECTIVE: To study the molecular mechanism of Yangjing Zhongyu Decoction (YZD) n-butanol extracts (ZDC) and ethyl acetate extracts (YSYZ) in reducing androgen in porcine granulose cells by mitogen-activated protein kinase (MAPK) pathway. METHODS: Porcine granulose cells were isolated and cultured. They were inoculated by MAPK inhibitor PD98059 at different concentrations, and then they were divided into the blank control group (0), 1, 3, 10, and 25 micromol/L groups. After 24-h culture the cytochrome P450c17a (CYP17) mRNA expression level was detected using Real-time fluorescent quantitative PCR. Contents of androgen (testosterone) in the supernate were detected using RIA and optimal PD98059 concentration screened. After intervened by 10 micromol/L PD98059 for 24 h, the culture solution was intervened by effective ingredients of with or without YZD or YSYZ at various concentrations (0, 1 , 5, 25, 50 mg/mL) at various time points (3, 6, 18, 24 h). Expression levels of p-ERK1/2, c-Fos and CYP17 were detected by Western blot. Testosterone content in the supernate was determined by radioimmunoassay (RIA). RESULTS: Ten pLmol/L PD98059 could obviously decrease p-ERK1/2 protein expression and increase CYP17 mRMA expression, and elevate testosterone content in the supernate (P < 0.05). ZDC and YSYZ at 25 ng/mL could increase p-ERK1/2 protein expression and c-Fos levels, and reduce CYP17 protein expression, and lower testosterone content in the supernate after 6-h intervention (P < 0.01). CONCLUSION: Effective ingredients of YZD could reduce androgen production in porcine granulose cells through increasing activities of MAPK.

[Effect of low-frequency electroacupuncture intervention on oxidative stress and glucose metabolism in rats with polycystic ovary syndrome].

OBJECTIVE: To observe the effect of low-frequency electroacupuncture (EA) treatment on oxidative stress and metabolism disorder in polycystic ovary syndrome (PCOS) rats, so as to study its underlying mechanisms in improving PCOS. METHODS: Thirty female SD rats were randomized into control group, model group and EA group (n = 10 in each group). The PCOS model was established by subcutaneous injection of testosterone propionate (1 mg/100 g, 10 mg/mL) for 8 weeks. EA (2 Hz) was applied to "Zhongwan" (CV 12) , "Guanyuan" (CV 4) and bilateral "Sanyinjiao" (SP 6) or "Housanli" (ST 36) for 30 min, once daily (except weekends) for 5 weeks. Serum testosterone (T) , sex hormone-binding globulin (SHBG) and fasting serum insulin (FINS) contents were detected by ELISA, serum malondialdehyde (MDA) and superoxide dismutase (SOD) levels were assayed by thiobarbituric acid (TBA) and xanthine oxidation (XTO) methods, respectively, and fasting blood glucose (FBG) was determined by roche glucose meter. In addition, homeostasis model assessment for insulin resistance (HOMA-IR) and modified B cell function index (MBCI) were calculated. RESULTS: Compared with the control group, serum T, FBG, FINS, HOMA-IR, and serum MDA levels were significantly increased in the model group (P < 0.01), while serum SHBG, MBCI and SOD levels were considerably decreased in the model group (P < 0.01). Following EA treatment, all the increased serum T, FBG, FINS, HOMA-IR, and serum MDA levels, and the decreased serum SHBG, MBCI and SOD levels were reversed obviously (P < 0.05, P < 0.01). CONCLUSION: EA treatment may normalize insulin sensitivity, ameliorate insulin resistance and hyperinsulinemia in PCOS rats, probably by regulating the function of pancreatic islets ß cell and by reducing oxidative stress and free androgen.

Effectiveness of abdominal acupuncture for patients with obesity-type polycystic ovary syndrome: a randomized controlled trial.

OBJECTIVE: To assess the effectiveness of abdominal acupuncture at the endocrine and metabolic level in patients with obesity-type polycystic ovary syndrome (PCOS). METHODS: Eighty-six women from the First Affiliated Hospital of Guangzhou Medical College with a diagnosis of PCOS (body-mass index [BMI] ≥25 kg/m(2)) were randomly assigned to receive 6 months of abdominal acupuncture (once a day) or oral metformin (250 mg three times daily in the first week, followed by 500 mg three times daily thereafter). BMI, waist-to-hip ratio (WHR), ovarian volume, menstrual frequency, homeostasis model assessment for insulin resistance (HOMA-IR), and Ferriman-Gallwey score were measured at the beginning of the study and after 6 months of treatment. Luteotrophic hormone (LH), testosterone, follicle-stimulating hormone (FSH), fasting blood glucose, 2-hour Postprandial blood glucose, fasting insulin, 2-hour postprandial blood insulin, total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were also assessed. RESULTS: According to the results at baseline and 6 months, BMI, WHR, Ferriman-Gallwey score, ovarian volume, luteotrophic hormone, ratio of luteotrophic hormone to follicle-stimulating hormone, testosterone, LDL-C, triglycerides, total cholesterol, fasting blood glucose, 2-hour postprandial blood glucose, fasting insulin, 2-hour postprandial blood insulin, and HOMA-IR were reduced significantly in the two groups (p<0.05). Menstrual frequency and HDL-C (p<0.05) increased significantly in both groups; follicle-stimulating hormone also increased in both groups, but the change was not significant (p>0.05). The acupuncture group showed considerable advantages over the metformin group in terms of reduced BMI and WHR and increases in menstrual frequency (p<0.05). CONCLUSION: Abdominal acupuncture and metformin improved the endocrine and metabolic function of patients with obesity-type PCOS. Abdominal acupuncture may be more effective in improving menstrual frequency, BMI, and WHR, with few adverse effects.

[Effects of Yangling Zhongyu Decoction on the secretion of ovarian granule cells in polycystic ovarian syndrome rat model].

OBJECTIVE: To observe the effects of Yangjing Zhongyu Decoction (YJZYD) on the serum estradiol (E2), testosterone (T), 17-hydroxyprogesterone (17-OHP), ovarian follicle stimulating hormone receptor (FSHR), insulin-like growth factor I (IGF-1), steroid hormone acute regulator protein (StAR) mRNA expressions in female rats. METHODS: Fifty PCOS rats were equally divided into 5 groups, i. e., the control group (C, normal PNA rats), the model group (M), the low dose YJZYD group (Y1), the medium dose YJZYD group (Y2), and the high dose YJZYD group (Y3), 10 in each. The levels of serum hormones were detected using radioimmunoassay. The morphological changes of the ovary were observed using HE method. The expressions of FSHR, IGF-1, and StAR mRNA were detected using RT PCR. RESULTS: Compared with Group C, serum T and 17-OHP significantly increased (P < 0.01), E2 significantly decreased (P < 0.01), the expressions of FSHR, IGF-1, and StAR mRNA significantly decreased in Group M (P < 0.01). Compared with Group M, the serum T level significantly decreased (P < 0.01), 17-OHP decreased (P < 0.05), and E2 significantly increased in Group Y3 (P < 0.01). The expressions of FSHR, IGF-1, and StAR mRNA increased in Group Y1, Y2, and Y3. The increase was most obvious in Group Y3 (P < 0.01). CONCLUSIONS: YJZYD could lower the hyperandrogenemia of PCOS rats. It also could increase the ovarian expressions of FSHR, IGF-1, and StAR mRNA, improve the ovarian functions, and promote the follicular development.

[Effect of abdominal acupuncture therapy on the endocrine and metabolism in obesity-type polycystic ovarian syndrome patients].

OBJECTIVE: To observe the effect of abdominal acupuncture on the endocrine and metabolic level in obesity-type polycystic ovarian syndrome (PCOS) patients. METHODS: Eighty-six PCOS patients were randomly and equally divided into medication group and abdominal acupuncture group. Patients of medication group were treated with metformin (250 mg/time, t.i. d. in the 1st week, and 500 mg/time, t.i.d. thereafter) for 6 months,and those of abdominal acupuncture group were treated by abdominal acupuncture [Zhongwan (CV 12), Liangmen (ST 21), etc., once daily for 6 months]. Changes of the body height, body Mass index (BMI), waist-hip ratio (WHR), Ferriman-Galleey score (FGS), menstrual frequency (MF) and ovarian volume (OV) were determined. Serum luteotrophic hormone (LH), free testosterone (T), follicle-stimulating hormone (FSH) contents were detected with radioimmunoassay. Fasting blood glucose (FBG), fasting insulin (FIN), total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), Homa insulin resistance index (IRI) were detected with chromatometry respectively. RESULTS: After the treatment, BMI, WHR, FGS and OV were reduced significantly in both medication and abdominal acupuncture groups (P < 0.05), while MF of the two groups increased evidently (P < 0.05), and the effects of abdominal acupuncture group were significantly superior to those of medication group in down-regulating BMI, WHR and upregulating MF (P < 0.05). Regarding the reproductive hormons, serum LH, LH/FSH and T levels in the two groups decreased significantly (P< 0.05), and the effect of abdominal acupuncture was superior to that of medication group in reducing serum T level (P < 0.05). Following the treatment, FBG, BG and FIN and INS contents 2 h after meal,and Homa IR in both medication and abdominal acupuncture groups all decreased considerably (P < 0.05), but without significant differences between them (P > 0.05). Regarding the blood lipid levels after the treatment, serum TC, TG, and LDL-C levels of the two groups decreased significantly (P < 0.05), while serum HDL-C level increased remarkably (P<0.05), without significant differences between the two groups in these indexes (P > 0.05). CONCLUSION: Abdominal acupuncture treatment can improve the endocrine and metabolic function of patients with obesity-type PCOS.