摘要
研究背景和目的
作为青春期及育龄女性最常见的妇科内分泌疾病,多囊卵巢综合征(polycystic ovary syndrome, PCOS)发病率3%-8%。其病因不详。PCOS不仅涉及生殖系统,而且是一个复杂的多系统综合征,相关的代谢失调包括胰岛素抵抗(insulin resistance, IR)、高雄激素血症(high androgens, HA),糖代谢异常、脂代谢异常等。
目前认为PCOS是一种表现为IR、代偿性高胰岛素血症、高雄激素血症、下丘脑—垂体—卵巢轴功能异常的常见慢性内分泌疾病。IR或高胰岛素血症、高雄激素血症和卵泡发育异常构成PCOS的主要病理特征。其中,IR是PCOS基础病理特征,促进高雄激素血症产生,又与HA,高促黄体激素(luteinizing hormoneLH)血症等相互作用,影响下丘脑—垂体—卵巢轴功能和卵泡发育异常。
如何改善PCOS患者胰岛素抵抗,脂代谢紊乱,肥胖等代谢紊乱症状,目前并没有非常有效的治疗方法。疏肝、健脾、清热的经典方剂丹栀逍遥散(DanZhi XiaoYao Power, DZXYP)是《太平惠民和剂局方》逍遥散加丹皮、山栀而来,又名加味逍遥散。临床研究表明,丹栀逍遥散可以改善肝经郁热型PCOS患者临床症状和体征,有效率达77%和88.33%；同时,该药能够显著增强Ⅱ型糖尿病患者胰岛素敏感性而降低胰岛素抵抗水平。降低湿热内蕴型肥胖伴非酒精性脂肪性肝病患者体重指数,腰臀比,血脂,并且改善胰岛素抵抗,预防高血压,糖尿病。DZXYP是否可以治疗PCOS性激素紊乱,排卵障碍的同时,改善其代谢紊乱表现,目前尚无相关报道。其作用机制不清楚。
我们拟建立PCOS大鼠模型,观察DZXYP改善PCOS大鼠代谢紊乱,性激素紊乱和卵泡发育异常的疗效。选用基于双向电泳的蛋白质组学技术研究分析PCOS发病机制及DZXYP的作用靶标,为DZXYP用于临床治疗PCOS提供依据,为深入研究PCOS发病机制及DZXYP作用机理奠定基础。
方法
1PCOS动物模型制备与分组给药
采用随机数字表法将动物分为PCOS模型组(PCOS组)、PCOS十达英35组(PCOS+Diane-35组)、PCOS+丹栀逍遥散组(PCOS+DZXYP)组和正常对照组(Control组),每组10只。皮下埋植左旋18-甲基炔诺酮硅胶棒和皮下注射hCG制造PCOS模型；35日龄开始,灌胃方式给药。实验开始及结束分别称重并计算体重差(body weight gain difference, BWGD),实验结束时,左心室采血5mL,-80℃保存备用；分离卵巢记录卵巢重量(ovary weight, OW);取卵巢组织用于病理切片观察和其他检测。
2组织学检查
卵巢组织常规脱水、透明、浸蜡、包埋,5μm切片,常规苏木精-伊红(hemotoxylin and eosin, HE)染色,显微镜分析图像,进行组织病理学检查。
3检测血糖、血脂、E2、T和胰岛素
生化分析仪检测各组大鼠的甘油三酯(triglyceride, TG)、总胆固醇(total cholesterol, TC)、低密度脂蛋白(low density lipoprotein, LDL)、高密度脂蛋白(high-density lipoprotein, HDL)和空腹血糖(fasting blood glucos,FBG)。
血清睾酮(testosterone, T),雌二醇(estradiol, E2)和空腹胰岛素水平(fasting insulin, FINS)用西门子ADVIA Centaur XP全自动免疫分析系统及其化学发光配套试剂进行检测。
4蛋白液相芯片检测FSH和LH
选用Rat Pituitary Magnetic Bead Panel (RPTMAG-86K)试剂盒,在LiquiChip液相芯片检测系统上读取各孔荧光强度,并根据标准品的荧光强度得到样品的FSH和LH浓度。
5双向电泳、质谱鉴定及分析DZXYP调控PCOS大鼠的差异蛋白
双向电泳参照IPGPhorⅢ等电聚焦系统程序指南和IPGstrip使用说明书进行操作,硝酸银染色后所得凝胶经对比找出差异蛋白,应用Voyager DE STR MALDI-TOF-MS(ABI)进行鉴定,获取肽质量指纹谱。利用软件mascot distiller过滤基线峰,识别信号峰。利用MOlecular Weight Search (MOWSE)寻找匹配的相关蛋白质,查询其功能。
6免疫组织化学技术检测大鼠β-arrestin2, PCNA蛋白表达
按免疫组化常规步骤进行,使用安装了图像分析系统(Image Pro Plus5.0Media Cybernetics, USA)的光学显微镜来检测阳性区域的光密度确定免疫组化染色的程度,按链霉亲和素-生物素-过氧化物酶复合物技术试剂盒说明书操作,检测β-抑制蛋白2(β-arrestin2,3-arr2)、增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)分布表达。
7Western blot检测β-arrestin2蛋白在大鼠卵巢的表达
按本实验室常规操作步骤进行,在KODAK Image Station4000MM Digital Imaging System中(kodak,版本4.0)曝光成像。图像用Molecular Imaging Software Version4.0(Kodak)进行蛋白条带灰度分析。每个样本的蛋白水平用β-actin作为内参进行标准化。每组样品重复上样检测3次。
8RQ-PCR检测β-arrestin2mRNA在大鼠卵巢的表达
按本实验室RQ-PCR常规操作步骤进行,每个引物(序列信息)的相对定量由标准曲线测定。mRNA的量由内参照β-actin标准化。相对表达量通过2-△△Ct法获取。
9Western blot检测P450arom,细胞外信号调节激酶1/2(extracellular signal-regulated kinase1/2, ERK1/2)、胰岛素受体底物1(insul in receptor substrate1, IRS-1)、Akt及其磷酸化蛋白在DZXYP干预的PCOS大鼠卵巢的表达
按本实验室常规操作步骤进行,在KODAK Image Station4000MM Digital Imaging System中(kodak,版本4.0),曝光成像。图像用Molecular Imaging Software Version4.0(Kodak)进行蛋白条带灰度分析。检测DZXYP对P450arom。ERK1/2和p-ERK1/2、IRS-1和p-IRS-1、Akt和p-Akt蛋白在PCOS大鼠中表达的调控。
10统计学分析
数据用X±s表示。采用SPSS13.0软件包进行统计分析,首先对数据进行方差齐性检验,方差齐则应用ANOVA法进行检验,并用LSD法进行组间两两比较；如果方差不齐,则应用Welch法进行检验,应用Games Howe11法进行两两比较；计量资料用多个独立样本非参数检验,两组间比较用2独立样本非参数检验；P<0.05为差异有统计学意义。
结果
PCOS模型及DZXYP作用效果鉴定
1. DZXYP对PCOS大鼠卵巢重量和组织学影响
1.1DZXYP对PCOS大鼠卵巢重量(OW)影响
PCOS组大鼠OW显著高于对照组(P=0.000)。和PCOS组比较,Diane-35组和DZXYP组OW显著减少,差异有统计学意义(P=0.000和P=0.000)。DZXYP组和Diane-35组比较OW更轻,差异有统计学意义(P=0.014)。
1.2DZXYP对PCOS大鼠卵巢组织学影响
PCOS组大鼠卵巢组织结构紊乱,囊性扩张卵泡数目明显增多,颗粒细胞层数减少,排列松散。对照组可见不同发育阶段的卵泡及多个黄体,颗粒细胞较完整,排列整齐。DZXYP组卵泡扩张显著改善,可见接近正常的窦状卵泡,颗粒细胞排列较为整齐。Diane-35组卵巢有较大囊性扩张卵泡,颗粒细胞层较PCOS稍多,但排列松散。
1.3. DZXYP对PCOS大鼠卵巢颗粒细胞PCNA表达影响
PCOS组颗粒细胞层数明显减少,多为2-3层,排列疏松。对照组颗粒细胞较完整,排列整齐,多为8-9层。DZXYP组卵泡扩张显著改善,颗粒细胞排列较为整齐,PCNA标记颗粒细胞多为4-6层。达英-35组正常窦状卵泡结构仍然很少见,但颗粒细胞层较PCOS组稍多,约3-4层。
2. DZXYP对血清生殖激素水平的影响
和对照组比较,PCOS大鼠血清LH和T在实验末期显著升高(P=0.001和P=0.045),FSH水平显著降低(P=0.000),LH/FSH显著升高(P=0.007)。各处理组间E：没有显著性差异
DZXYP使PCOS大鼠血清FSH水平显著增加,LH水平显著降低,LH/FSH降低,差异有统计学意义(P=0.004,P=0.041,P=0.036)。DZXYP显著降低T水平(P=0.002)。
Diane-35使PCOS大鼠血清FSH水平显著增加(P=0.029)。对LH、LH/FS和T水平无明显影响(P=0.614,P=0.086和P=0.299)。
3. DZXYP对PCOS大鼠血脂四项的影响
和正常对照组相比,PCOS组大鼠实验末期TG、TC、LDL水平显著升高,HDL水平显著降低,差异有统计学意义(P=0.000,P=0.002,P=0.000,P=0.002)。
DXZYP显著降低PCOS大鼠血清TG、TC、LDL水平,升高HDL水平,差异有统计学意义((P=0.000,P=0.023,P=0.000和0.049)。
Diane-35降低PCOS大鼠TG水平(P=0.005),升高HDL水平(P=0.016),差异有统计学意义,对TG、TC和LDL无显著性作用。
4. DZXYP对PCOS大鼠IR的影响
PCOS大鼠血清FBG、FINS和HOMA在实验末期显著增高(P=0.000,P=0.002,P=0.000)。与模型组相比,DZXYP显著降低INS(P=0.035)和HOMA (P=0.007)水平,对FBG无显著影响(P=0.099)。Diane-35组PCOS大鼠FBG.FINS和HOMA-IR水平没有显著改善(P=0.252,P=0.242,P=0.088)。
5. DZXYP对PCOS大鼠体重差的影响
和对照组比较,PCOS大鼠BWGD显著增加(P=0.003)。DZXYP组BWGD显著减少(P=0.013),而使用Diane-35后,PCOS大鼠的BWGD没有显著变化(P=0.096)。
DZXYP防治PCOS的蛋白质组学研究
6.双向电泳检测及质谱鉴定DZXYP干预PCOS大鼠的差异蛋白
PCOS组和PCOS±DZXYP组分别与对照组对比,进行质谱分析。成功鉴定8个差异蛋白点。其中4个表达水平下调,4个表达水平上调。其中3个分别被鉴定为过氧化氧化还原蛋白3(peroxiredoxin3, PRDX3)和谷胱甘肽过氧化酶1(glutathione peroxidase1, GPX1)。
7.分析DZXXP对PCOS大鼠β-arr2表达的影响
7.1免疫组化分析DZXXP对PCOS大鼠卵巢β-arr2表达的影响
PCOS组β-arr2表达较对照组显著降低(P=0.000),与PCOS组相比,DZXYP和Diane-35显著增加其表达(P=0.000和P=0.018),和Diane-35比较,DZXYP的作用更明显,表达的差异有统计学意义(P=0.003)。
7.2Western blot分析DZXYP对PCOS大鼠卵巢β-arr-2表达影响
与对照组相比,PCOS组β-arrestin2表达显著降低(P=0.000),DZXYP和Diane-35处理后β-arr2蛋白水平表达增加(P=0.000和P=0.022)。而DZXYP对该蛋白水平表达的影响较Diane-35更显著,差异有统计学意义(P=0.000)。
7.3RQ-PCR检测DZXXP对PCOS大鼠卵巢β-arr2表达的影响
对照组相比,PCOS组β-arrestin2表达显著降低(P=0.000),DZXYP处理后β-arr2的mRNA表达增加(P=0.000)。PCOS大鼠经Diane-35处理后β-arr2mRNA表达无显著性变化(P=0.057)。β-arrestin2介导的信号通路在DZXYP防治PCOS中的作用
8. Westerm blot方法检测DZXYP对P450arom和ERK1/2磷酸化的影响
PCOS大鼠P450arom表达较对照组显著下降,ERK1/2磷酸化增加,差异有统计学意义(P=0.000和P=0.000),DZXYP显著增加P450arom表达,显著降低ERK1/2磷酸化水平,差异有统计学意义(P=0.005和P=0.000)。虽然Diane-35也显著降低ERK1/2磷酸化水平(P=0.001),但是DZXYP的作用较Diane-35更明显,差异有统计学意义(P=0.038)。Diane-35对P450arom表达没有显著作用(P=0.831)。
9. Westerm blot方法检测DZXYP对IRS-1丝氨酸和AKT ser473磷酸化影响
和对照组比较,PCOS大鼠p-IRS-1表达增加,p-AKT ser473表达下降,差异有统计学意义(P=0.000和P=0.000),DZXYP显著降低PCOS大鼠卵巢IRS-1的磷酸化水平,显著增加AKT ser473的磷酸化水平,差异有统计学意义(P0.004和P=0.009)。Diane-35显著增加AKT ser473的磷酸化水平,差异有统计学意义(P=0.024),对IRS-1丝氨酸磷酸化没有显著影响(P=0.078)。
结论
1.DZXYP有效改善大鼠PCOS症状：改善IR,脂代谢紊乱、体重增加等代谢紊乱症状。改善高LH水平和HA。减轻卵巢重量,有效改善PCOS大鼠卵泡发育异常。
2. DZXYP作用机制涉及以下几方面：①DZXYP可能通过β-arr2促进G蛋白偶联受体(G-protein-coupled receptors, GPCRs)与G蛋白解偶联,中止LH/CG受体(LH receptor, LHR)信号转导,恢复其受体脱敏作用；②DZXYP通过β-arr2信号通路减少ERK1/2磷酸化,进而增加芳香化酶450水平；③通过增加β-arr2表达,作为支架蛋白,与IR下游信号分子形成InsR/Akt/β-arr2/Src蛋白复合物,激活PI3-K通路,磷酸化AKT473-9氨酸,从而发挥胰岛素增敏的作用。④另外,DZXYP降低IRS-1Ser307磷酸化水平,增加AKT磷酸化,改善胰岛素抵抗。
Background and objective
Polycystic 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 models
According 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 examination
The 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 FINS
The 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 LH
The 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 PCOS
The 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 technique
The 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 blotting
The 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 blotting
The 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 analysis
Data 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.
Results
1. The OW and Histological effects of DZXYP to rats with PCOS
1.1The 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 ovary
In 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 cells
In 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 hormones
In 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 PCOS
In 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 PCOS
At 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 PCOS
In 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 PCOS
The 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-arrestin2expression
7.1Immunohistochemical analysis for the effect of DZXXP to β-arr2in rats with PCOS
In 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 PCOS
In 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 PCOS
In 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 ERK1/2
In 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 ser473
In 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).
Conclusions
1.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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