| Background and objectivePolycystic ovary syndrome (PCOS) is one of the most common endocrine diseases in women at adolescent and reproductive age. The incidence of PCOS is3%to8%and its etiology is still elusive. PCOS is not only involved in the reproductive system, it is also a complex syndrome concerned to multiple systems. The most common menifestations of PCOS are insulin resistance (IR), high androgens (HA), abnormal glucose metabolism, abnormal lipid metabolism, and disfunction of hypothalamic-pituitary-ovarian axis, etc. IR is the basis of PCOS that it could promote the production of HA and there is an interaction between IR and HA, hyper-luteinizing hormone, etc., which eventually develops to the disfunction of hypothalamic-pituitary-ovarian axis and abnormal growth of follicles.The aromatase cytochrome P450(P450arom), an encoding product of CYP19gene, is a rate-limiting enzyme in the formation of estrogen. Some researches indicated that the aromatase played an important role in the pathogenesis of PCOS.Liver depression, liver fire, and spleen deficiency played an essential role in the pathogenesis of PCOS in Traditional Chinese medical (TCM) throry. The liver qi stagnation was the most basic pathological change in PCOS. In70cases of infertility, the type of liver depression accounted for the largest proportion, namely51.7%. Meanwhile, there were also different changes of liver depression in other patterns of syndrome in infertility. Loss of secretion by liver was an important ovulation disorder in PCOS.DanZhi XiaoYao Power (DZXYP) is a classic recipe derived from XiaoYao Power in "Formularies of the Bureau of People’s Welfare Pharmacies". It might soothe the liver, invigorate spleen and clear away heat. The recipe was formerly used for women’s blood deficiency and over-strain, blood-heat fighting, virgin blood-heat Yin deficiency. It embodies the theory that "Liver is females prenatal". It was used to treat PCOS and IR in Type2diabetes recently. However, there are no reports about DZXYP treatment of metabolic syndrom in PCOS.PCOS rat model would be reproduced and proteomics technology research based on the two dimensional electrophoresis was used to analyze effect and mechanism of DZXYP in prevention of PCOS.1The preparation and administration by groups in PCOS animal modelsAccording to random number table, animals were randomly divided into the PCOS model group (PCOS group), PCOS+Diane-35group (PCOS+Diane-35group), PCOS+DZXYP group (PCOS+DZXYP) group and normal control group (Control group), with10rats in each group. The PCOS model was subcutaneously implanted with levorotation18-methyI norethindrone and subcutaneously injected with hCG. Intragastric administration was performed after35days. At the beginning and end of the experiment, samples were weighed and the body weight gain difference (BWGD) was calculated. At end of the experiment,5mL blood was drewn from left ventricle and stored at-80℃. The ovary was separated, and the ovary weight (OW) was recorded. The ovary tissue was used for the observation of pathological section and other detection.2Histopathological examinationThe routine dehydration, transparency, waxdip, embedding,5μm slice, routine hematoxylin-eosin (HE) dyeing in ovary group were carried out. Images were analysed by a microscope for a histopathological examination.3Detection of blood sugar, blood fat, E2, T and FINSThe biochemistry analyzer was used to detect triglyceride (TG), total cholesterol (TC), low density lipoprotein (LDL), high-density lipoprotein (HDL) and blood sugar in each group of rats.The testosterone (T), estradiol (E2) and fasting insulin (FINS) in serum was detected with the Siemens ADVIA Centaur XP fully automated immunoassay system and its chemical-emitting ancillary reagents.4Protein chips used for the detection of FSH and LHThe Rat Pituitary Magnetic Bead Panel (RPTMAG-86K) kit was use to read the fluorescence intensity onn each hole of the liquid chip detection system, and to determine the concentrations of FSH and LH according to the standard fluorescence intensity.5The dielectrophoresis, mass spectrum identification and analysis for the differential proteins regulated by DZXYP in rats with PCOSThe dielectrophoresis followed the guideline for programs of IPGPhorⅢ class electrostatic focusing system and the instruction for use of IPGstrip. After the silver nitrate dyeing, the acquired gelatin was contrasted to find out the differential protein. The Voyager DE STR MALDI-TOF-MS (ABI) was used for identification, to gain the fingerprint spectrum of peptide mass. The soft ware mascot distiller was used to filter the baseline peak and recognize the signal peak. The MOlecular Weight Search (MOWSE) was applied to search the matching relevant proteins and inquiry its function.6The protein expression of β-arrestin2, PCNA in rats preformed by Immunohistochemistry techniqueThe immunohistochemistry was carried out with routine steps. The optical microscope with image analysis system (Image Pro Plus5.0Media Cybernetics, USA) was used to detect the photodensity at the positive region to determine the dyeing extent of immunohistochemistry. Following the steps in the instruction of Stept Avidin-Biotin complex technoluogy kit to examine the distribution and expression of (3-arrestin2(β-arr2) and proliferating cell nuclear antigen (PCNA).7The mRNA expression of β-arrestin2in rats detected by Real-time quantitative polymerase chain reaction (RQ-PCR)The RQ-PCR general operating procedures in this laboratory were adopted. The relative quantification of each primer (order information) was determined by specification curve. The amount of mRNA was standardized by the inner reference β-actin. The amount of relative expression was obtained by the2-△△Ct method.8Detection of β-arrestin2protein expression in in rats detected by Western blottingThe general operating procedures in this laboratory were adopted. The imaging was exposed in the KODAK Image Station4000MM Digital Imaging System (kodak, version4.0). Molecular Imaging Software Version4.0(Kodak) was used to perform the gray analysis of the protein bands for the images. The inner reference was used to standardize the protein levels in each samples. The samples were repeatedly added to each group and tested for three times. The β-arrestin2protein expression was detected in rats with PCOS and the regulation of DZXYP to β-arrestin2. 9Detection of P450arom, extracellular signal-regulated kinase1/2(ERK1/2), insulin receptor substrate1(IRS-1), Akt and the expression of their phosphorylation protein in the DZXYP intervened ovary in rats with PCOS by western blottingThe general operating procedures in this laboratory were adopted. The imaging was exposed in the KODAK Image Station4000MM Digital Imaging System (kodak, version4.0). Molecular Imaging Software Version4.0(Kodak) was used to perform the gray analysis of the protein bands for the images. The inner reference was used to standardize the protein levels in each samples.The regulations of DZXYP to the expression of P450arom, ERK1/2and p-ERK1/2, IRS-1and p-IRS-1, Akt and p-Akt protein in rats with PCOS were detected.10Statistical analysisData were expressed as X±s. SPSS13.0software package was used to perform the statistical analysis.The homogeneity of variance should be performed first. If the variance was homogeneous, the AN OVA method should be used to performed the test and the LSD method was used for the multiple comparison between groups. If the variance was not homogeneous, the Welch method should be used for the test, and the Games Howell method was adopted for the multiple comparison. The nonparametric test with multiple independent samples was used for the measurement data. The nonparametric test with two independent samples was used for the comparison between two groups. P value being less than0.05was considered to be significant difference.Results1. The OW and Histological effects of DZXYP to rats with PCOS1.1The OW effects of DZXYP to rats with PCOS In the PCOS group, the OW of rat was significantly higher than that in control group, with a statistical significance (P=0.000). In Diane-35group and DZXYP group, OW decreased significantly, with a statistical significance (P=0.000). There was a statistical significance between DZXYP group and Diane-35group (P=0.014). In DZXYP group, OW was lighter than it was in Diane-35group, close to the OW level in control group OW (Table1).1.2The histological effects of DZXYP to PCOS in rats ovaryIn the PCOS group, the ovary structure of rats was in disorder, with obviously increased numbers of follicles with cystic dilatation, decreased number of granulosa cells layer, and loose arrangement. In control group, there were follioles at different developmental stage and multiple corpus luteum, with relatively complete granulosa cells, and a regular arrangement. In DZXYP group, the follicle expansion was notably improved, and there were near normal antral follicles, and granulosa cells with a regular arrangement.1.3. The PCNA expression effect of DZXYP to PCOS in rats ovary granulosa cellsIn the PCOS group, the layer number of granulosa cells was obviously decreased, most of which was2to3layers, with a loose arrangement. In control group, the granulosa cells were relatively complete, with a regular arrangement, most of which was8to9layers. In DZXYP group, there was significantly improved follicle expansion, with a relatively regularly granulosa cells, most of which was4～6layers of PCNA marked granulosa cells. In Diane-35group, there was still seldom antral follicles structure, while its number of granulosa cells layers, about3to4layers, was slightly more than that in PCOS group.2. The effect of DZXYP to serum levels of reproductive hormonesIn comparison with the control group, at the end of experiment, in rats with PCOS, the serum levels of both LH and T increased significantly(P=0.001and P=0.045), and the FSH level decreased notably (P=0.000), resulting in a conspicuous increase of LH/FSH (P=0.007). There were no significant differences of E2among each treated groups.DZXYP caused the significantly increased serum levels of FSH in rats with PCOS, the notably decreased levels of LH, resulting in a reduction of LH/FSH. Among them there was a statistical significance (P=0.004, P=0.041, P=0.036). DZXYP could notably decreased T level (P=0.002). In comparison with Diane-35group, there was a statistical significance (P=0.024). Diane-35could significantly increase the serum level of FSH in rats with PCOS (P=0.029), without a remarkable effect on the levels of LH, LH/FS, and T (P=0.614, P=0.086, and P=0.299).3. The effect of DZXYP to four items of blood-lipid tests in rats with PCOSIn comparison with normal control group, at the end go experiment, levels of TG, TC, LDL increased notably in PCOS group, and the level of HDL decreased significantly, with a statistical significance of difference (P=0.000, P=0.002, P=0.000, P=0.002).DXZYP significantly decreased the serum levels of TG, TC, and LDL, and increased the level of HDL, with a statistical significance of difference (P=0.000, P=0.023, P=0.000and0.049).Diane-35could decreased the serum levels of TG and increase HDL levels in rats with PCOS, with a statistical significance of difference (P=0.005and P=0.016), while no obvious effect on TC and LDL.4. The effect of DZXYP to IR in rats with PCOSAt the end of experiment, the serum levels of FBG, FINS and HOMA in rats with PCOS significantly increased (P=0.000, P=0.002, and P=0.000). In comparison with model group, DZXYP caused a significant decrease in levels of INS (P=0.035) and HOMA (P=0.007) while had no effect on FBG(P=0.099). In Diane-35group, levels of FBG, FINS and HOMA-IR in rats with PCOS were not improved significantly.5. The effect of DZXYP to the BWGDs in rats with PCOSIn comparison with the control group, the BWGD in rats with PCOS increased obviously (P=0.003). In DZXYP group, the BWGD notably decreased (P=0.013), while after using Diane-35, there was no significant changes of BWGD in rats with PCOS (P=0.096).6. Dielectrophoresis detection and mass spectrum identification for DZXYP intervened differential protein in rats with PCOSThe dielectrophoresis results was shown in Fig.3A. Comparing the PCOS group and PCOS+DZXYP group with control group separately to perform the mass spectrometric analysis (Fig.3A). Eight differential protein points were successfully indentified (See Table6). Among them, there were4decreased expression, and4increased expression. Moreover, there of them were identified as peroxiredoxin3(PRDX3) and glutathione peroxidase1(GPX1) individually.7. Verification of β-arrestin2expression7.1Immunohistochemical analysis for the effect of DZXXP to β-arr2in rats with PCOSIn the PCOS group, the (3-arr2expression was significantly lower than it was in control group (P=0.000). Nonetheless, in comparison with PCOS group, DZXYP and Diane-35notably increased its expression (P=0.000and P=0.018). In comparison with Diane-35, the effect of DZXYP was more obvious. There was a statistical significance in the difference of expression (P=0.003). 7.2Western blot analysis for the effect of DZXYP to β-arrestin2in rats with PCOSIn comparison with control group, the β-arrestin2expression in the PCOS group decreased significantly (P=0.000). After the treatment with DZXYP and Diane-35, the protein expression level of (3-arrestin2increased (P=0.000and P=0.022). However, the effect of DZXYP to the expression level of this protein was more notable than that of Diane-35, with a statistical significance of difference (P=0.000).7.3RQ-PCR detection for the effect of DZXXP top-arr2in rats with PCOSIn comparison with the control group, the β-arrestin2expression in the PCOS group decreased significantly (P=0.000). After the treatment with DZXYP, the mRNA expression level of P-arrestin2increased (P=0.000). Nevertheless, After the treatment with Diane-35, there was no remarkable changes in the mRNA expression of β-arr2(P=0.057).8.Westerm blot detection for the effect of DZXYP to the P450arom and the phosphorylation of ERK1/2In comparison with the control group, the expression of P450arom in rats with PCOS significantly decreased, while the phosphorylation of ERK1/2phosphorylation increased, with a statistical significance of difference (P=0.000and P=0.000). DZXYP significantly increased the expression of P450arom, while notably reduced increased the phosphorylation level of ERK1/2, with a statistical significance of difference (P=0.005and P=0.000). Althouth Diane-35also significantly reduced he phosphorylation level of ERK1/2, the effect of DZXYP was even more notable than that of Diane-35. Diane-35had no remarkable effect on the expression of P450arom (P=0.831). 9. Westerm blot detection for the effect of DZXYP to the phosphorylation of both IRS-1serine and AKT ser473In comparison with control group, the expression of p-IRS-1increased while the expression of p-AKT ser473decreased, with a statistical significance of difference (P=0.000and P=0.000). DZXYP significantly reduced the phosphorylation level of IRS-1in rats ovary with PCOS, while notably increased that of AKT ser473, with a statistical significance of difference (P=0.004and P=0.009). Diane-35significantly increased that of AKT ser473(P=0.024), while had no remarkable effect on that of IRS-1(P=0.078).Conclusions1.DZXYP ameliorates IR, abnormal blood lipid and weight gain of PCOS rats. The LH and HA level and OW were decreased. The abnormal growth of follicles in PCOS rats was improved effectively.2. DZXYP could promote the uncoupling of GPCR with protein G to suspend the signal transduction of LHR, and restore its receptor’s desensitization via β-arr2. DZXYP could reduce the ERK1/2phosphorylation and further increase the level of aromatase450through upregulation of β-arr2. DZXYP increase the expression of|3-arr2which is a scaffold protein. The formation of InsR/Akt/β-arr2/Src protein complex activates the PI3-K pathway through AKT473serine phosphorylation. DZXYP decreases the level of IRS-1Ser307phosphorylation and increased the AKT phosphorylation and improve IR to ameliorate PCOS. |
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