CN108542907A - A kind of pachymic acid or Poria cocos acid derivative are used to prepare application and the pharmaceutical preparation for the treatment of polycystic ovary syndrome drug - Google Patents

Abstract

The present invention relates to application and pharmaceutical preparations that a kind of pachymic acid or Poria cocos acid derivative are used to prepare treatment polycystic ovary syndrome drug.The present invention provides pachymic acid and its a kind of new purposes of derivative, has notable curative effect to polycystic ovary syndrome, and be free from side effects.Pachymic acid is proved to can be used for treating mouse Stein-Leventhal syndrome disease in embodiments of the present invention, and a kind of drug candidate of new treatment polycystic ovary syndrome can be provided for clinic.

Description

A kind of pachymic acid or Poria cocos acid derivative are used to prepare treatment polycystic ovary syndrome medicine The application of object and pharmaceutical preparation

Technical field

The present invention relates to polycystic ovary syndrome therapy field, especially a kind of pachymic acid or Poria cocos acid derivative are for making The application of standby treatment polycystic ovary syndrome drug and pharmaceutical preparation.

Background technology

Stein-Leventhal syndrome (polyeystie ovary syndrome, PCOS) is also known as Stein-leventhal synthesis Sign, is the more typical endocrinic syndrome of the women of child-bearing age, be caused by polygenes, more inherent causes and the factors such as multi-environment under The disorder of hypothalamic-pituitary-ovarian function axis, menstrual disorder (oligomenorrhea or amenorrhoea), chronic anovulation, infertile, insulin support The different substantiality disease that anti-, hyperinsulinemia, ovary polycystic change and hyperandrogenism are characterized.Most of researchers The principal pathogenetic of this disease is thought the reason is that the factors such as diet, heredity, spirit and environment by a large amount of clinical datas, and its is main Pathogenesis is hypothalamic-pituitary-ovarian axis functional disturbance, hyperandrogenism, the imbalance of adrenal gland endocrine function, insulin Resist etc., it also strangles and swashs with proinflammatory factor, tumor necrosis factor, insulin-like growth factor, adiponectin, anti-seedling some researches show that it The cell factors such as element, leptin are related.

The pathogenesis of polycystic ovary syndrome is extremely complex, hypothalamo pituitary hypothalamic pituitary ovarium axis (hypothalamus- Pituitary-ovarianaxis, HPO) disorderly, ovarian function exception, adrenal cortex function exception, chronic inflammation and pancreas Insulin resistance all has an impact the morbidity of ovary.

It include at present three kinds for PCOS therapeutic schemes：Control body weight, drug therapy, operative treatment.First, to endomorphy type PCOS patient, it is desirable that insulin sensitivity can be increased in this way to lose weight and reduce waistline by keeping on a diet and increasing movement Property, testosterone, insulin level are reduced, its ovulation and fecundity are finally restored.Drug therapy, which includes appropriate drug administration, to be improved With the adjusting menstrual cycle, regular reasonable drug administration, such as oral contraceptive；It in addition to this, also can be by reducing blood androgen Level, such as oral dexamethasone are taken progesterone or spirolactone in ring and are combined with contraceptive, and effect can be more preferable.Secondly, for fertilizer It is fat or can be by taking insulin sensitizer, if melbine is to improve ovary ovulation function with Patients with Insulin Resistance； For the patient for thering is fertility to require, can be adjusted by taking promoting ovulation drug.Finally, invalid for conservative medication No-clay weak interbed patients with polycystic ovary syndrome, can finally take operative treatment, pass through operation stimulate ovary, ovarian hormone level meeting It is rapid to fall after rise, and then Hypothalamic Stimulation, to make the promoting sexual gland hormone of pituitary normal level.

However, currently without a kind of effective therapy, operative treatment damage to the body is very big, and drug therapy will appear Apparent side effect, and induce the generation of other diseases.

Although Stein-Leventhal syndrome is a kind of gynecological disease, diabetes, heart disease, hypertension and son also can be increased simultaneously The incidence of endometrial carcinoma.Therefore, it is difficult that PCOS first-line treatments appropriate are provided now.Melbine is quick as a kind of insulin Chemical drug object is widely used as the first-line drug of PCOS treatments.However, the long-term clinical application of melbine can lead to diarrhea, stomach The side effects such as intestines discomfort.Therefore, prolonged application drug Or Metformin In Treating PCOS may be not appropriate for, and find PCOS more suitably Medicine (improving her ovaries' function to improve Oocyte quality) is of great significance to settling the dilemma.

Pachymic acid, 3-8- acetoxyl groups -16- Alpha-hydroxies-lanostane -8,24 (31)-diene -21- acid, white powder, A kind of triterpene compound being naturally occurring in the Chinese herbal medicines such as ganoderma lucidum, Poria cocos, at present can by Spawn incubation or in Medicine extracting mode obtains pure sample.According to the literature, pachymic acid is swollen to lung cancer, cancer of pancreas, colon cancer, prostate cancer, breast cancer etc. The invasion of oncocyte and proliferation suffer from the effect of significantly inhibiting.Apparent secondary work is not observed in the process of clinical application of Poria cocos With.

Invention content

The purpose of the invention is to overcome existing polycystic ovary syndrome medicine side effect is big, therapeutic effect not Good problem, provides a kind of pachymic acid or Poria cocos acid derivative is used to prepare application and the medicine for the treatment of polycystic ovary syndrome drug Object preparation.

Concrete scheme is as follows：

A kind of pachymic acid or Poria cocos acid derivative are used to prepare the application for the treatment of polycystic ovary syndrome drug.

Further, the pachymic acid or Poria cocos acid derivative are used to prepare answering for treatment polycystic ovary syndrome drug With including inhibiting weight gain, improves impaired glucose tolerance, improve hormone disturbance, hyperlipemia and chronic inflammation, inhibit inflammation, delay Insulin resistance is solved, the damage of granular cell is inhibited, inhibits at least one of egg mother cell oxidation and apoptosis.

Further, the derivative of the pachymic acid include Poria cocos hydrochlorate, Poria cocos acid hydrate, pachymic acid solvate, Any one in pachymic acid cocrystalization compound.

The present invention also protects a kind of pharmaceutical preparation for treating polycystic ovary syndrome, including pachymic acid or pachymic acid spread out Biology.

Further, it is described treatment polycystic ovary syndrome pharmaceutical preparation be dispersant, tablet, capsule, granule, Suppository or pill.

Further, the pharmaceutical preparation of the treatment polycystic ovary syndrome further includes auxiliary material.

Advantageous effect：

The present invention provides pachymic acid and its a kind of new purposes of derivative, is used to prepare treatment polycystic ovary syndrome medicine Object has polycystic ovary syndrome notable curative effect, and is free from side effects.Moreover pachymic acid is as extraction separation in Chinese medicine Compound, in embodiments of the present invention confirm can be used for improving the mouse PCOS diseases induced by DHEA, further, this hair It is bright from ovary and egg mother cell angle, elaborate the therapeutic effect of pachymic acid.In short, pachymic acid can inhibit inflammation and alleviation PCOS mouse islets elements are resisted, and to inhibit the damage of granular cell, are inhibited egg mother cell oxidation and apoptosis, are improved more capsules indirectly Ovary Syndrome oocyte of mouse quality has preferable effect to treatment polycystic ovary syndrome.

Description of the drawings

In order to illustrate more clearly of technical scheme of the present invention, attached drawing will be briefly described below, it should be apparent that, The accompanying drawings in the following description merely relates to some embodiments of the present invention rather than limitation of the present invention.

Fig. 1 is control group, model group, pachymic acid treatment group and Or Metformin In Treating group provided by one embodiment of the present invention The morphological assessment figure for the MII phase egg mother cells developed in vivo, N normal appearances in figure；A polar bodys are degenerated；B perivitellines expand；C cells Matter fragmentation)；

Fig. 2 is embryo in the mouse model that pachymic acid and melbine provided by one embodiment of the present invention induce DHEA The spilting of an egg and later stage develop image, Bl=blastaeas in figure；The embryo of Ly=cracking；Mo=mulberry bodys)；Oct4 is (red in four groups of embryos Color) and TUNEL (green) representative immunofluorescence image, with DAPI dye core (blue), engineer's scale=20 μm；

Fig. 3 is the representative H＆E dye images of each group mouse ovarian slice provided by one embodiment of the present invention, is existed respectively Amplify X40, X100, micro-image is shot at X400 multiples, box is amplified a times region, and (* is folliculi ovarici vesiculosi；HC is hemorrhagic Tumour；Arrow indicates granular cell layer)；

Fig. 4 is control group, model group, pachymic acid treatment group and Or Metformin In Treating group provided by one embodiment of the present invention The tem study (n=3) of ovarian growth ovarian follicle.The growing follicle of each group ovary sample presentation graphics (a1, B1, c1, d1, e1, f1；Engineer's scale=10 μm).Egg mother cell (a2, b2, c2, d2, e2, f2), granular cell (a4, b4, c4, D4, e4, f4), thecacells (a6, b6, c6, d6e6, f6), ovarian stromal cell (a8, b8, c8, d8, e8, f8) engineer's scale =2 μm.A3, b3, c3, d3, e3, f3 show that egg mother cell image box is amplified region；A5, b5, c5, d5, e5, f5 from Granulocyte image box is amplified region；A7, b7, c7, d7, e7, f7 are amplified region from thecacells image box； A9, b9, c9, d9, e9, f9 are amplified region from ovarian stromal cell image box；Engineer's scale=0.5 μm.In control group-a, (ellipse) shows the size dimension and ridge appearance of normal mitochondria in pachymic acid treatment group-e and Or Metformin In Treating group-f.Line Plastochondria is in " vacuole " shape (ellipse), and " black or similar ghost image " shape (ellipse) and " tubulose/cellular " ridge are (ellipse in cross-section It is round) model group-b, c, d are appeared in, it partly appears in pachymic acid treatment group-e and Or Metformin In Treating group-f.Cell fat drips (star) appears in four groups.Show that mitochondrial apoptosis is downright bad (d8, star).

Specific implementation mode

The preferred embodiment of the present invention is described in more detail below.Although the following describe the preferred implementations of the present invention Mode, however, it is to be appreciated that may be realized in various forms the present invention without should be limited by embodiments set forth herein.

In the present invention, relational language is defined as follows：

Pachymic acid (PA) is a kind of triterpene compound in Poria cocos and ganoderma lucidum Chinese herbal medicine, is the principle active component of Poria cocos One of, it plays an important role in various pharmacological actions.Such as Chinese medicine lead phlegm ball, open palace ball and decoction for Irregular Menstruation compatibility ingredient in have Identical vital Chinese medicine, i.e. Poria cocos simply.

There is no particular limitation in source of the present invention to pachymic acid, can be commercially available, can also be according to existing Various methods are prepared, including traditional Chinese medicine extraction separation and Spawn incubation separating-purifying etc..In addition it is also necessary to explanation, The derivative of pachymic acid has the similar activity of pachymic acid, equally has the effect of same or similar treatment polycystic ovary syndrome Fruit.Such as Poria cocos hydrochlorate, Poria cocos acid hydrate, pachymic acid solvate, pachymic acid cocrystalization compound etc..

In the present invention, pachymic acid includes inhibiting weight gain to the therapeutic effect of mouse, improves impaired glucose tolerance, improves and swashs Plain disorderly, hyperlipemia and chronic inflammation, inhibit inflammation, alleviate insulin resistance, inhibit the damage of granular cell, inhibit ovum female At least one of cellular oxidation and apoptosis, correspondingly, pachymic acid or Poria cocos acid derivative are used to prepare treatment polycystic ovary synthesis Disease drug also has above-mentioned effect for general receptor, can know to this those skilled in the art, therefore not to repeat here.

In the present invention, pachymic acid or derivatives thereof is applied to prepare the pharmaceutical preparation for the treatment of polycystic ovary syndrome, drug The form of preparation includes but not limited to dispersant, tablet, capsule, granule, suppository or pill.In addition, pharmaceutical preparation can To contain auxiliary material, for example, starch, binder, sugar, can know this those skilled in the art, therefore not to repeat here.

The present invention will be described in detail by way of examples below.In the examples where no specific technique or condition is specified, It is carried out according to technology or condition described in document in the art or according to product description.Agents useful for same or instrument are not noted Bright production firm person, being can be with conventional products that are commercially available.

Experimental animal involved by following embodiment：

The mouse of experiment is the female KM mice of 5 week old, weight 20-23g, this Leco Corp.'s (certificate of Chinese Shanghai Number：SCXK2012-0002), according to the strict regulations (authentication code of Chinese Xiamen University's zooscopy committee： XMUMC2011-10-08 it) is raised and is used, standard complies fully with National Institutes of Health (National Institutes of Health, NIH) announce " management of laboratory animal criterion " (Principles of Laboratory Animal Care) relevant regulations.Illumination is controlled in strict accordance with 12h illumination and dark alternate period, temperature at 24 DEG C ± 1 DEG C, mouse takes the photograph water feed freely, and adaptable fed can be used for testing after a week.

Main agents used below include：

1) DHEA (dehydrobenzene)：Purchased from the joyful beauty bio tech ltd in Wuhan, purity >=98%, 4 DEG C is kept away Light preserves；

2) Pachymic acid (pachymic acid)：Purchased from the Shanghai bio tech ltd Shi Feng, purity >=98%, -20 DEG C It is kept in dark place；

3) Metformin (melbine)：Purchased from the joyful beauty bio tech ltd in Wuhan, purity >=98%, 4 DEG C It preserves；

4) injection soybean oil：Purchased from emerging (Tieling) Pharmacy stock Co., Ltd, 4 DEG C are kept in dark place；

5) DMSO (dimethyl sulfoxide (DMSO))：Purchased from Sigma Chemical Co. (St.Louis, MO, USA) company, room temperature is protected It deposits；

6)D-Glucose：Purchased from Sigma Chemical Co. (St.Louis, MO, USA) company, specification 100mg, room Temperature preserves；

7) blood sugar test paper：Purchased from Johson ＆ Johnson (the steady persons of outstanding talent times of Johnson ＆ Johnson ONE TOUCH-Ultra easily blood glucose meter test-paper excellent again), Room temperature is kept in dark place；

8) CHO, TG, HDL, LDL biotinylation kit：Bioengineering Research Institute is built up purchased from Nanjing, 4 DEG C are kept in dark place；

9) Isoflurane (Isoflurane)：Purchased from Shanghai Yu Yan scientific instrument Co., Ltd, specification 100mL, 4 DEG C are protected from light guarantor It deposits；

10) hyaluronidase：Purchased from Sigma Chemical Co. (St.Louis, MO, USA) company, -20 DEG C are protected from light guarantor It deposits；

11) Reverse Transcriptase kit (RevertAid First Strand cDNA Synthesis Kit)：Thermo All reagent sets of Scientific, USA, q-PCR (SYBR Green Real-time PCR Master Mix Kit)： Takara, Japan；

12) promote superfecundation hormone：PMSG (pregnant equine gonadotropin, Pregnant mare serum ) and hCG (human chorionic gonadotrophin, Human chorionic gonadotrophin) gonadotropin：Purchased from Ningbo Three lives pharmaceutcal corporation, Ltd (Ningbo Sansheng pharmaceutical company, China), -20 DEG C of preservations；

13) M2medium (M2 cell culture mediums)：Purchased from Sigma Chemical Co. (St.Louis, MO, USA) public affairs Department, 4 DEG C of preservations；

14) the anti-β-actin monoclonal antibodies in mouse source (Mouse monoclonal Anti-beta Actin antibody)：Purchased from Abcam (Abcam ab8226) company, specification：100 μ g, -20 DEG C of preservations, WB dilution ratios 1:10000；

15) the anti-IL-6 polyclonal antibodies in rabbit source (Rabbit PolyclonalAnti-IL-6Antibody)：It is purchased from Proteintech (proteintech21865-1-AP) company, specification：53 μ g/150 μ l, -20 DEG C of preservations, WB dilution ratios 1: 1000；

16) rabbit source anti-tnf-alpha polyclonal antibody (Rabbit Polyclonal Anti-TNF-Alpha Antibody)： Purchased from proteintech (proteintech 17590-1-AP) company, -20 DEG C of preservations, WB dilution ratios 1:1000；

17) the anti-OCT4 polyclonal antibodies in rabbit source (Rabbit Polyclonal Anti-OCT4Antibody)：Purchased from purchased from Proteintech (proteintech 11263-1-AP) company, specification：54 μ g/150 μ l, -20 DEG C of preservations, IF dilution ratios 1:100；

18) Alexa Fluor 546 mark donkey anti-rabbit secondary antibody (Donkey Anti-Rabbit IgG Antibodies Conjugates Alexa Fluor546)：Purchased from INVITROGEN (A10040) company, specification：500 μ l, 4 DEG C are kept in dark place, IF dilution ratios 1:400；19) FITC is coupled mouse source anti alpha-tubulin monoclonal antibodies (mouse monoclonal anti- α-tubulin antibody conjugate FITC)：Purchased from Sigma Chemical Co. (St.Louis, MO, USA) public affairs Department, specification：1.5mg/mL, -20 DEG C are kept in dark place, 4 DEG C of preservations of working solution, IF dilution ratios 1:200；

20) mouse source γ-tubulin monoclonal antibodies (Mouse monoclonal anti-γ-tubulin antibody)：Purchased from Sigma Chemical Co. (St.Louis, MO, USA) company, specification：1.3mg/mL, -20 DEG C of guarantors It deposits, 4 DEG C of preservations of working solution, IF dilution ratios 1:100；21) rhodamine label phalloidine (Phalloidin conjugates TRICT)：Purchased from Sigma Chemical Co. (St.Louis, MO, USA) company, specification：0.5mg/mL, -20 DEG C are protected from light guarantor It deposits, 4 DEG C of preservations of working solution, IF dilution ratios 1:100；

22) mountant (Vectashield mounting medium with are quenched in anti-containing core dyestuff DAPI DAPI)：Purchased from Vector Laboratories, I (Burlingame, CA, USA) company, specification：1.5 μ g/ml, stoste and point 4 DEG C of working solution of dress is kept in dark place；

23) Tunel Apoptosis in-situ detection reagent box (terminal deoxynucleotidyltransferasemediated dUTP nick-end labelingreaction mixture)： Purchased from Kai Ji Biotechnology Ltd., -20 DEG C are kept in dark place；

24) Annexin V-PE cell apoptosis detection kits：Purchased from green skies Biotechnology Ltd. (Beyotime-C1065, China), 4 DEG C of preservations；

Unless otherwise indicated, all chemicals reagents used in this experiment are purchased from Sigma Chemical.

Involved primer is synthesized in Shanghai Sheng Gong biotech firms in embodiment, and primer information is as follows：

PI-3K(mouse)：

forward,5’-AGCCAACAACAGCATGAACA-3’；SEQ ID NO:1

reverse,5’-AAGGTCCCATCAGCAGTGTC-3’；SEQ ID NO:2

GLUT4：

forward,5’-TCTCAATGGTTGGGAAGGAA-3’；SEQ ID NO:3

reverse,5’-GAGGAACCGTCCAAGAATGA-3’；SEQ ID NO:4

IRS-1：

forward,5’-AGCCTGTTGTGGACTTGGTC-3’；SEQ ID NO:5

reverse,5’-ACTCGAGCCTGTGCATTCTT-3’；SEQ ID NO:6

CYP17：

forward,5’-TGGTCATATGCATGCCAACT-3’；SEQ ID NO:7

reverse,5’-CCCTTCTTCACGAGCACTTC-3’；SEQ ID NO:8

GSK3-β：

forward,5’-TTCCTTTGGAATCTGCCATC-3’；SEQ ID NO:9

reverse,5’-TGAAACATTGGGCTCTCCTC-3’；SEQ ID NO:10

IL-6：

forward,5’-AGTTGCCTTCTTGGGACTGA-3’；SEQ ID NO:11

reverse,5’-CCTCCGACTTGTGAAGTGGT-3’；SEQ ID NO:12

TNF-α：

forward,5’-ACGGCATGGATCTCAAAGAC-3’；SEQ ID NO:13

reverse,5’-GTGGGTGAGGAGCACGTAGT-3’；SEQ ID NO:14

The preparation method of related reagent used below is as follows：

1) experimental drug object：

DHEA solution：600mgDHEA is taken to be dissolved in 10ml injection soybean oils, stirring at normal temperature is completely dissolved for 1-2 days.Dissolving Milky, milk sample is presented in liquid afterwards.4 DEG C are kept in dark place.

Poria cocos acid solution：It takes 1.1g pachymic acid powder to be dissolved in 176ml physiological saline, 5 ‰ DMSO hydrotropies is added, fully After mixing, packing (every 8ml) is kept in dark place to -20 DEG C of refrigerators.It is on-demand.

Melbine solution：It takes 11g melbine powder to be dissolved in 176ml physiological saline, 5 ‰ DMSO is added, fully Dissolving, packing (every 8ml) is preserved to 4 DEG C of refrigerators, on-demand.

2) HE staining reagents：

Hematoxylic preparation：20g alum powder is added in 200mL distilled water first, heating makes it dissolve, then The dissolving of 10mL absolute ethyl alcohols is added in 1g hematoxylin powder；It mixes above two liquid and heats and boil；0.5g mercury oxide is added Continue to heat and stir after powder, subsequent solution becomes darkviolet, cooling in ice water rapidly；It is eventually adding 8mL glacial acetic acid；Room Warm 1h filterings.

The preparation in Yihong：The Yihong 1g is added to dissolving to get to 1% Yihong the ethanol solution for taking 100mL95%.

3) IF provides reagent for oneself：

PBS buffer solution (pH7.4,1000mL)：KH is weighed respectively₂PO₄0.2g, NaCl 8g, Na₂HPO₄1.15g (or NaHPO₄·12H₂O 2.9g), drug is added in 500ml ultra-pure waters, is stirred on magnetic stirring apparatus by KCl 0.2g according to this To being completely dissolved, after be transferred in 1000mL volumetric flasks, the appropriate ultra-pure water for cleaning after beaker is poured into volumetric flask, it is rear to be added Ultra-pure water makes it be settled to 1000mL, and mixing fully adjusts PH to 7.4 afterwards, through high pressure sterilization, 4 DEG C of preservations.

Fixer (4% paraformaldehyde, 10mL)：0.4g paraformaldehyde powder is weighed, is put into 10mL PBS buffer solution, after Heating in 65 DEG C or so of water-bath is put into until powder is completely dissolved, fixer is filtered and is dispensed after cooling down by room temperature；4 It DEG C preserves 1 week, -20 DEG C 1 month storable.

Permeable membrane liquid (MPs, 10mL)：50 μ LTritonX-100 are drawn to be added in 9.95mL PBS buffer solution, (due to TritonX-100 characters are more sticky, are not easy to be drawn, and pipette tips tip is cut sub-fraction so as to the absorption of liquid), wait for that its is complete After fully dissolved, filtering packing can save 1 month in 4 DEG C.

Eluent (Washing Buffer, WBF；50mL)：50 μ L Tween20 are drawn to be added to 50mLPBS buffer solutions, (since Tween20 characters are more sticky, being not easy to be drawn, pipette tips tip is cut sub-fraction so as to the absorption of liquid), waits for it After being completely dissolved, filtering packing can save 1 month in 4 DEG C.

Confining liquid (Blocking Buffer, BBF；10mL)：Weigh 0.2g bovine serum albumins (Bovine Serum Albumin, BSA) it is added into 10mL WBF after it is completely dissolved, filtering packing can save 1 month in 4 DEG C.

Antigen retrieval buffers：Configuration A liquid (0.1M citric acid solns) first, weighs 21.01g citric acids (C₆H₈O₇·H₂O) add Enter into the 1000mL beakers equipped with about 500mL ultra-pure waters, the stirring on magnetic stirring apparatus is rear to turn until citric acid is completely dissolved It moves in 1000mL volumetric flasks, the appropriate ultra-pure water after cleaning beaker is poured into volumetric flask, the rear ultra-pure water that is added makes its constant volume To 1000mL, it is transferred to after mixing well for use in the blue mouth bottle of cleaning of 1000mL specifications.It is reconfigured B liquid (0.1M sodium citrates Solution), weigh 29.41g sodium citrates (C₆H₅Na₃O₇·2H₂O it) is added to the 1000mL beakers equipped with 500mL or so ultra-pure water In, on magnetic stirring apparatus stirring until citric acid be completely dissolved, after be transferred in 1000mL volumetric flasks, by cleaning beaker after Appropriate ultra-pure water pour into volumetric flask, the rear ultra-pure water that is added makes it be settled to 1000mL, and 1000mL rule are transferred to after mixing well It is for use in the blue mouth bottle of cleaning of lattice.The configuration of final working solution:9mL A liquid and 41mL B liquid are taken respectively, are mixed to clean beaker In, after mixed liquor is transferred in the volumetric flask of 500mL specifications, add appropriate ultra-pure water to be settled to 500mL, pay attention to now with the current.

4) surpass row's reagent：

PMSG(50U/mL)：PMSG freeze-dried powders are diluted to 50U/mL with pure PBS, are distributed into that (0.1mL/ is every immediately The injection of mouse) amount storage.- 20 DEG C can save 1 month；

hCG(50U/mL)：HCG freeze-dried powders are diluted to 50U/mL with pure PBS, are distributed into (0.1mL/ every immediately Mouse inject) amount storage.- 20 DEG C of preservations can be 1 month, pays attention to being protected from light.

5) other reagents：

Hyaluronidase (0.1%)：1mg hyaluronidases are weighed, are dissolved in 1ml M2 solution.

Methylene blue (0.1%)：40ml tetrachloroethanes is added into 95% ethyl alcohol of 54ml, mixing shakes up, water-bath (65 DEG C) in place 3min, 0.1g methylene blues are added later, mixing postcooling is eventually adding 6ml glacial acetic acids to 4 DEG C, after mixing often Temperature preserves.

Test used below, analysis method include：

1) oestrous cycle measures

Oestrous cycle information is obtained by the microexamination of vaginal cell, proestrum vaginal exfoliated is with segment angle Change based on epithelial cell；Oestrus：Vaginal exfoliated is based on complete superficial cell；The heat later stage：Vaginal prolapse is thin Born of the same parents are based on leucocyte and intermediate layer cell；Dioestrus：Vaginal exfoliated is a small amount of leucocyte and intermediate layer cell.

Concrete operations are as follows, first fix mouse, and appropriate physiological saline is dipped with thin cotton swab, fully gently inserted after wetting Enter genital tract (about 0.5cm), taken out after gently rotating, uniform, drying is gently smeared into cotton swab moistening part in glass slide center To natural drying, methylene blue staining (0.1%) is put in dyeing 5min at glass slide smearing with syringe, uses distilled water later Extra dye liquor is slowly rinsed, observes the cellular morphology of smear and the changing features of tissue after drying under the microscope, to really Determine the stage of oestrous cycle at mouse, one time a day.

2) oral glucose tolerance test (OGTT)

The female mice of fasting 8h gives the D-Glucose for being dissolved in physiological saline (2g/kg) by gavage, uses OneTouch Ultra blood glucose meters measure Glucose in Blood by Cyclic.0min after fasting time (oral glucose it Before), the blood sample collected from tail vein is given often by gavage immediately for measuring blood glucose level as initial blood glucose level The D-Glucose normal saline solution of mouse (2g/kg), measures these in 30min, 60min, 90min and 120min later The blood glucose value of mouse, and record its change of blood sugar.Data record is the absolute value of blood sugar concentration.Use GraphPad Prism 5.0 softwares calculate area (AUC) under glucose response curve.

3) serum Biochemical Indexes test and analyze

Blood sample is collected by way of extracing eyeball of mouse.After mouse fasting 8h, anaesthetized by isoflurane inhalation, immediately It carries out plucking eyeball and blood is taken to handle.After taking blood, after room temperature is static, be centrifugally separating to obtain serum, and be stored in immediately -80 DEG C with Carry out the measurement of subsequent Biochemical Indices In Serum.The sharp bio tech ltd in Hunan is entrusted to measure serum using ELISA method Middle E2, T, AMH, INS, LEP, IL-6, TNF-α are horizontal.It is measured using biochemical reagents box (Bioengineering Research Institute is built up in Nanjing) CHO in serum, TG, HDL, LDL contents.

4) ovarian histology analysis and ovarian follicle count

After collecting blood sample, the ovary of mouse (every group 3) is taken out rapidly, 4% paraformaldehyde is used in combination to fix.Then according to normal Rule Histological method is dehydrated and is embedded in paraffin, and the ovary of mouse side is cut into 5 μm of thin slice, and extends to glass slide use It is assessed in ovarian morphology.The ovary of the other side is cut entirely, serial section (8 μm of pieces), takes one to extend to load glass in every 5 On piece is counted for ovarian follicle.It is finally dyed, observed under an optical microscope and counted with h and E.

The detailed process of HE dyeing includes that dehydration embedding, slice and exhibition piece, HE dye three steps.Dehydration embedding：First by ovum Nest tissue is positioned over 12h in 4% paraformaldehyde fixer, after fixed；Distilled water flushing 3 times, graded ethanol is de- later Water, respectively places 25min in 50%, 70%, 80%, 90% graded ethanol, 2 times in 100% ethyl alcohol, each 30min；Immediately without Water-ethanol and dimethylbenzene 1:30min in 1 mixed liquor 2 times, each 20min in dimethylbenzene, finally places dimethylbenzene and paraffin 1:1 Mixed liquor 30min, 2 times, each 1h in paraffin liquid, after dehydration, with paraffin by organization embedding.Slice and exhibition piece：It repaiies first Whole embedded block, is gently held on slicer, and adjusting slice thickness is desired value (4 μm/8 μm), after cutting out thin slice, is pressed from both sides with tweezers It picks and places and opens up piece into water-bath (40 DEG C)；It takes glass slide to stretch into water-bath, can be assisted gently after paraffin piece, be erected with tweezers Directly slowly lift glass slide, it is seen that paraffin piece bonds on sheet glass；Glass slide is put into baking oven (60 DEG C) 30min to carry out at dewaxing Reason, it is necessary to assure it is hot that glass slide in dimethylbenzene is put into when dyeing.HE is dyed：Paraffin section need to be taken off with dimethylbenzene at this time Wax, ethyl alcohol hydration process：Slice is put into dimethylbenzene 2 times, each 20min, 100% ethyl alcohol 2 times, each 2min, subsequent gradient Alcohol aquation, 95%, 80%, 70% each 1min of ethyl alcohol, 1min in last distilled water；H and E dyes：According to bush Essence dyeing 5min, tap water are embathed to colourless, 1% acidic alcohol 10s, and tap water is embathed to colourless, Yihong liquid dyeing 8min, from Water, which is embathed to colourless step, can make tissue staining；Ethyl alcohol is dehydrated immediately, dimethylbenzene carries out transparent, neutral gum and carries out Sealing, respectively places 30s in 70%, 80%, 85%, 95% ethyl alcohol of detailed process, last 100% ethyl alcohol 2 times, each 1min, Diformazan benzene-alcohol 1:2min in dimethylbenzene II is placed into after 1min in 1 mixed liquor 1min, dimethylbenzene I, immediately neutral gum mounting.

Ovarian follicle method of counting refers to the description of pertinent literature, determines that the tranquillization (original) of each ovary, growth are (first in detail It is grade, secondary) and graaffian follicle quantity, primordial follicle：There is one layer of flat follicle cell in the periphery of egg mother cell, and close to white Film center；Primary growth ovarian follicle in growing follicle：Oocyte peripheral has one or more layers cube follicle cell, and occurs saturating The membrana follicularis of oolemma, connective tissue appears in ovarian follicle periphery；Secondary growth ovarian follicle in growing follicle：Antrum folliculi occurs, big ovum It steeps chamber and forms ovarian cumulus, and granular cell layer (follicle cell on ovarian follicle inner wall is intensive and is arranged in several layers) occur, membrana follicularis can Separate two layers of inner membrance and outer membrane；Graaffian follicle：Antrum folliculi bigger, ovarian cumulus clearly, thecal cells and stratum granulosum of ovarian follicle It abuts, it is separated by by one layer of basement membrane with Granulosa cells, and endo cell cytoplasm is limpid, is in polygon, karyon is rounded, many hairs Thin blood vessel appears in iuntercellular, and the pericyte for more being in fusiformis is located at outermost layer, the boundary pole unobvious with surrounding connective tissue, There is corona radiata；Atretic follicle：There is shrinkage in oolemma, and the structure of egg mother cell is very unintelligible, or even vanishes from sight, ovarian follicle wall It collapses.In brief, the folliculus with Visible Core in given slice is only calculated, to avoid true ovarian follicle quantity is over-evaluated, The Follicles number of every five slices, one slice of record, the Follicles number of last stored count slice, as each ovary Contained Follicles actual value.

5) transmission electron microscope (TEM) is analyzed

Every group of other three mouse is put to death according to the method described above, and the side of every mouse ovarian is stored in -80 DEG C It is detached for RNA in Trizol.Other side ovary is dissected into two parts on the stencil plate filled with 2.5% glutaraldehyde, is stood It is fixed i.e. in 2.5% glutaraldehyde, at least 8h is placed in 4 DEG C of refrigerators later, fixation terminates.It is then that fixer is dilute with PBS Three times are released, it is fixed after being carried out with osmium tetroxide, Gradient elution using ethanol is then used, then permeates and is embedded in 618 resins, pay attention to Embed position ovary cortex upward.Finally 3% acetic acid uranium-citric acid is observed with super transmission electron microscope (JEM-2100, TEM) Ultra-thin section after the double dyeing of lead.

6) RNA extractions and real-time quantitative PCR

This experiment using real-time quantitative PCR (qPCR) come detect PI3K in ovary tissue and adipose tissue, The mRNA relative expression levels of GLUT4, GSK3- β, IRS-1, CYP-17, TNF-α, IL-6.

The extraction operation flow of ovary tissue or adipose tissue total serum IgE:

The RNA in tissue is extracted, liquid nitrogen is added in mortar first, then ovary tissue or adipose tissue are placed on liquid respectively It clays into power in nitrogen, (notices that tissue total powder volume can not be more than Trizol volumes used in the Trizol liquid of 1ml 10%) about 50~100mg is added in and organizes powder (being taken with the spoon after precooling), it is made to be sufficiently mixed uniformly.It is being placed at room temperature for After 5min, 200 μ l of chloroform are added, EP pipe lids are covered tightly and sway 15s.Centrifuge is adjusted to 12000rpm rotating speeds immediately, centrifugation It is taken after 10min in upper strata aqueous phase to new EP pipes and (pays attention to that subnatant and the intermediate beds of precipitation can not be mixed into, otherwise need to centrifuge again Separation), mixing after 500 μ l of isopropanol is added；It is placed at room temperature for 10min, centrifuge is adjusted to 12000rpm rotating speeds, after centrifuging 10min Liquid is carefully discarded supernatant, 75% ethyl alcohol 1ml is added later, vortex makes its mixing, is centrifuged in 4 DEG C of centrifuges (12000rpm) 5min.Repetitive operation is primary.Supernatant (as possible removing residual liquid) is finally abandoned it, places 5~10min of drying at room temperature. The 30 processed water of μ l DEPC are taken, are dissolved for RNA, it if needed can 55 DEG C~60 DEG C water-bath about 10min.It can as do not used It is stored in 70% ethyl alcohol and is stored in -70 DEG C.

Points for attention in Total RNAs extraction：Whole process wears sterile gloves and mask, wears lab-gown, and words of saving your breath simultaneously can not Other articles (needing to change gloves when necessary) are touched with hand.Work is ready before operation, as consumptive material, reagent, sample, instrument are It is no complete etc.).The cleaning-sterilizing that operation console and instrument should be carried out in advance prepares, and before the formal beginning of experiment, first uses and contains RNA Ambion liquid spray operating table surface, instrument, hand and the reagent shell of enzyme degradation agent, to remove RNA enzyme.All reagents and each sample Product have to centrifuge tube cover label it is clear, be sure not to obscure.Operation is whole to pay attention to that cross contamination, liquid transfer gun head is avoided to need timely It replaces.The RNA solution finally extracted need to carry out reverse transcription reaction processing (degradation for preventing RNA) immediately.

The reverse transcription PCR of total serum IgE reacts:

The EP pipes equipped with RNA are stood 5min under the conditions of 65 DEG C first, denaturation treatment is carried out to RNA, immediately placed It is allowed to cool on ice.After reverse transcription system (20 μ l reaction systems) prepares, is gently mixed well and (paid attention to the greatest extent using liquid-transfering gun Bubble not occur in amount, if generating bubble, brief centrifugation can remove at low temperature；Or when finding that sample is more, in low The activity for being conducive to inhibit enzyme under the conditions of temperature, reacts before can delaying upper machine).The PCR instrument used is Biometra T-Gradient Thermoblock PCR equipment.EP pipes equipped with 20 μ l reverse transcription reactions systems (bubble-free and mix well) are put Intermetallic metal slot in instrument starts reverse transcription process by after selection and confirmation, and reverse transcription reaction just proceeds by.It is then right CDNA concentration and purity are measured, and Available templates can be obtained, and last reverse transcription reaction terminates.Later, if experimenter one week Within will do the upper machine testing of real-time, then cDNA templates can save in 4 DEG C；If just doing machine examination after the long period It surveys, needs -20 DEG C of long-term preservations (cDNA is not easy to be degraded under cryogenic conditions).

Real time qPCR reactions:

During the preparation of reaction system and sample-adding, in order to reduce loading errors, it is proposed that before sample-adding, first in 20 μ 80 μ l distilled waters are added in lcDNA templates, and mix well, are then respectively completed the premixed operation of cDNA templates and primer.

CDNA templates premix：+ 10 μ l SYBR dye liquors of 5+0.4 μ l ROX Reference Dye II (50X) of μ l cDNA liquid =15.4 μ l

Primer premixes：The μ l of+0.4 μ l reverse primers of+0.4 μ l forward primers of 3.8 μ l distilled waters=4.6

After the completion of the above process, upper machine carries out Real time qPCR detections, the used Real time of this experiment QPCR instrument is 7500Fast Real-Time PCR System, selects β-actin as internal reference, to carry out relative quantification detection. The experimental arrangement setting of qPCR is as follows：95 DEG C of 30s first, next 95 DEG C of 3s and 60 DEG C of 30s (completing 1 cycle), 40 in total Cycle.Subsequent 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s.

For data analysis, according to the recurring number CT values having been detected by, purpose can be finally calculated using Δ Δ CT methods The opposite mrna expression amount of gene.

7) IL-6 and TNF-α in protein immunoblotting experiment (Western blot) detection adipose tissue

The extraction of 1st step protein sample:

White adipose tissue after isolated peritonaeum is weighed, fritter is cut and is put into pipe.The precooling containing inhibitor is added Protein extraction agent (250mg tissue in 1ml extraction agents are about added).With cracking Ultrasound Instrument each 30s in ice-water bath Low frequency interval ultrasound 1min, until tissue cracking completely, lysate centrifuges 15min in the centrifuge of precooling later.Supernatant It is transferred at once in new centrifuge tube and 10X loading buffer is added and be allowed to become 1X, boil 5min at 100 DEG C, boiled and put Enter -20 DEG C to preserve for use.

2nd step protein immunoblotting tests (Western blot):

Configure gel first, including 12% separation gel and 5% concentration glue.Loading electrophoresis later, by sample from -20 DEG C Taking-up is placed in 100 DEG C and boils 5min, and then electrophoresis is arranged by sample all addition loading hole in high speed centrifugation 5min at room temperature Program is that concentration glue 80V runs 30min, and separation gel 110V runs 70min.It waits for that albumen is kept completely separate, carries out transferring film, cut corresponding position Gel, cut out two filter paper of corresponding size and be put into together in the capsule equipped with transferring film buffer solution and impregnate about 10min, cut out phase It answers the nitrocellulose filter (pvdf membrane) of size to be put in methanol to be also transferred in transferring film buffer solution after activation, by negative from positive best Pole sequence is that the sequence of filter paper-film-glue-filter paper stacks, and setting transferring film program is 15V transferring films 1h.Transferring film terminates, and prepares closing Liquid 0.5g skimmed milk powers is added in 10ml TBST, 5% confining liquid is made, pvdf membrane immersed confining liquid, at room temperature on shaking table Close 1h.Be incubated primary antibody, by pvdf membrane be put into preparation primary antibody solution (β-actin, 1:10000, Abcam ab8226；IL-6, 1:1000, proteintech21865-1-AP；TNF-α, 1:1000, proteintech 17590-1-AP) in, it is being shaken at 4 DEG C It is shaken overnight on bed.Incubation terminates, and film is washed 3 times with TBST, and every all over 10min, subsequent room temperature shaker is incubated secondary antibody, and (mouse is anti-；Rabbit-anti) 1h washes film 3 times with TBST later, every all over 10min.Finally, it is protected from light Western Light ECL industry detection reagents Leaching is obtained in darkroom with exposure in pvdf membrane surface after standing 3min or so after (Keygene biotech companies) mixing Take protein band situation.

8) oocytes collection and in vitro culture (IVM)

Mouse is put to death to minimize the pain of mouse using vertebra dislocation method, is placed in mouse web portion downward after execution It on the workbench that 75% ethanol disinfection is crossed, continues thereafter with and is carried out disinfection to its back with 75% ethyl alcohol, with eye scissors in back skin Skin does an opening, presss from both sides out back fat pad with tweezers and takes advantage of a situation and involves fallopian tubal and ovary, goes out bilateral ovum with eye scissors careful separation Ovary is positioned in preheated M2 culture mediums by nest, is cleaned up ovary with a little fresh M2 in culture dish, use is sterile Blade thoroughly minces ovary, and fresh M2 is added thereafter and fully shakes up ovary tissue, culture dish is placed under Stereo microscope, uses Mouth suction pipe picks out well-developed GV phases egg mother cell, and egg mother cell is transferred to covering after being cleaned up repeatedly with fresh M2 It in the equilibrated M2 culture solutions of paraffin oil, is placed in incubator and cultivates, condition of culture is 37 DEG C, 5%CO₂, saturated humidity.

Then the GV phase egg mother cells of collection are placed in 37 DEG C, 5%CO₂It is cultivated in incubator and observation of following up, germ-vesicle Rupture (GVBD) oocyte number measures after 2h.Then, the quantity that first polar body (PB1) is discharged is recorded after 12h.

The oocyte number of GVBD rates=generation GVBD/GV phases oocyte number × %

Oocyte number × % of the oocyte number of PB rates=generation PB1/generation GVBD

9) egg mother cell (MII) and embryo collection and morphological assessment

In order to collect MII egg mother cells, surpass row by the way that 5IU PMSG are injected intraperitoneally from different groups of mouse, then 48h Afterwards, cervical dislocation puts to death mouse after intraperitoneal injection 5IU hCG, 14h, its fallopian tubal is taken to be placed in the M2 liquid that pre-temperature is crossed.By micro- Mirror is needled ampulla of uterine tube using the syringe needle of asepsis injector, acquisition cumulus oocytes complesxes (COCs), then It transfers them in the M2 liquid containing 0.2% hyaluronidase, digestion 3-5min removes cumulus cell.Collect the exposed MII phases Egg mother cell and stereomicroscopy under the microscope.Finally, the MII phase egg mother cells for collecting health do the immunofluorescence in later stage to examine Survey spindle situation.

In order to collect embryo, as the above method carries out superfecundation to mouse.After having injected hCG, by sex ration 1:1, with 8 The healthy male KM mouse in or so week routinely mates mating overnight, the next morning 8:00 observation female mice vaginal plug, discovery have the moon The mouse of road bolt illustrates successfully to mate, and is denoted as gestation 0.5 day.Gestation takes uterus, uses cervical dislocation human first after 3.5 days Mouse is put to death, the mouse outside of belly of execution is placed on the operation console of sterilizing pan upward, sprays mouse web portion with 70% ethyl alcohol at once. Abdominal cavity is opened, stripping is coiled in intestines together, and uterus bicornis, fallopian tubal and ovary are visible.It is clamped close to uterus with tweezers Position scissors and tweezers the separation fallopian tubal and ovary of neck (after bladder), the mesentery between fallopian tubal and uterus is removed with scissors, Then it will be cut off between fallopian tubal and ovary, it is ensured that the gate oxide integrity between fallopian tubal and uterus.By uterus be placed on drop have it is pre- On the tissue culture dishes of warm good M2 culture solutions.Uterus is then rinsed, using the method rinsed from the angular cervix direction in uterus. Cornua uteri is being cut close to the position of cervix, is then being inserted into No. 26 needles close to the uterus upper end of uterus and fallopian tube interconnecting piece Head draws the M2 culture mediums of about 0.3mL, and cornua uteri is rinsed towards cervix direction.Embryo is picked under the microscope with mouth suction pipe Out.The embryo gathered is washed several times with the fresh M2 culture solutions of several drops again, removes impurity, total to each group mice embryonic, Mulberry body number, blastaea number dissolve embryo number, and development embryo number etc. before morula stage carries out express statistic, and by each group capsule Embryo picks out the immunofluorescence dyeing for carrying out next step.

10) immunofluorescence

1) ovary apoptosis (TUNEL) detects:

Using In situ terminal labeling (deoxynucleotidyltransferase-mediated dUTP nick-end Labelling, TUNEL) kit detection, it is as follows：

Paraffin section carries out dewaxing treatment：According to following procedure, 60 DEG C of baking piece 1h, dimethylbenzene dewaxes 2 times, graded ethanol water It closes (100% → 95% → 80% → 75%), each 5min；Slide is put into slide slot, PBS buffer solution is added and is allowed to no mistake Tissue block rinses three times, each 5min.

Wax circle is drawn later：To prevent from being added in structural fluid loss, wax circle need to be drawn around tissue block with groupization pen, draw The water around sample can be blotted and (pay attention to untouchable sample) as possible with clean blotting paper before circle, the hour circle of drawing a circle should not mistake Greatly, it is advisable for block edge 2-3 millimeters away from tissue.

Penetrating processing：With the proportional arrangement Proteinase K working solutions of 45 μ l PBS+5 μ l Proteinase K, it is added dropwise On 50 μ l to each sample, 30min is reacted under the conditions of 37 DEG C.Working solution matching while using.Slide is put into slide slot after reaction, PBS buffer solution is added to be allowed to not cross tissue block, rinses three times, each 5min.

The positive piece of system：By the proportional arrangement DNaseI working solutions of 40 μ l DNaseI+10 μ l DNaseI Buffer.It chooses 50 μ l DNaseI working solutions, 37 DEG C of reaction 30min are added in one sample.Slide is put into slide slot after reaction, PBS is added Buffer solution is allowed to not cross tissue block, rinses three times, each 5min.

Linkage flag：Now match TdT enzyme reaction solutions：With 45 μ l Equilibration Buffer+1 μ lbiotin-11- The proportional arrangement TdT enzyme reaction solutions of UTP+4 μ l TdT Enzyme, preserve, pay attention to being protected from light on ice.Then blotting paper is all by sample The water enclosed blots, and is careful not to touch tissue, and each sample is added dropwise 50 μ l TdT reaction solutions, is put into wet box, and 37 DEG C are protected from light, React 1h (selecting a sample to be not added with TdT enzymes, to do negative control).Slide is put into slide slot after reaction, PBS buffer solution is added It is allowed to not cross tissue block, rinse three times, each 5min.By 5 μ l Streptavidin-Fluorescein+45 μ l Labeling Buffer ratio mixed configuration Streptavidin-Fluorescein reaction solutions, the balance around blotting sample with blotting paper After liquid, each sample is added dropwise 50 μ l reaction solutions, and 37 DEG C are protected from light in reacting 30min in wet box.Slide is put into slide slot after reaction In, PBS buffer solution is added and is allowed to not cross tissue block, rinses three times, each 5min.

DAPI contaminates core and mounting：Nucleus is contaminated with DAPI dyeing liquors, in being protected from light 10min, rear cover in wet box under room temperature Upper coverslip finally uses FV1000 confocal laser scanning microscope, CLSMs (Olympus, Japan) to be observed.

2) egg mother cell (GV phases) early apoptosis (TUNEL) detects:

In order to assess the apoptosis of egg mother cell, Annexin-V probes (Beyotime, China) are used according to producer's explanation. The GV phase egg mother cells taken out from separate groups of mice are directly placed into the good Annexin-V-FITC fluorescence of pre-temperature in advance first Probe drop (Annexin-V-FITC fluorescence probes：M2 culture solutions are 1：40) in 37 DEG C, 5%CO₂Incubator in be incubated 30min.Culture is rinsed three times after terminating with M2 culture solutions, these egg mother cells are then transferred to fixer 30min, fixed After, washing finally to be transferred to drip in advance three times in WBF has in the clean glass slide of DAPI mountants (2.5 μ l), can use Glass needle gently mixing is gently capped clean coverslip along one time, and useful binders close coverslip to prevent from covering later Slide slides and moisture loss.It is kept in dark place to -20 DEG C after label is clear, whole operation process, which should be noted, to be protected from light, and mounting terminates After be put into avoid light box, can be taken off when needing, observed using laser confocal microscope.

3) egg mother cell (MII) spindle dyeing in vivo:

In order to assess the spindle and chromatin state of the MII phase egg mother cells from ampulla of uterine tube, it will utilize and exempt from Epidemic disease fluorescent staining method dyes spindle and chromosomal marker, and detailed process can be in view of described in previous studies.First MII phases egg mother cell room temperature in 4% paraformaldehyde fixes 30min, then in permeable membrane solution (0.5%Triton X-100) Permeabilization 30min.1h then is closed with the PBS containing 1% bovine serum albumin(BSA) (BSA), with first antibody (mouse anti alpha- Tubulin-FITC antibody；1:200；Sigma, the U.S.) it is incubated overnight at 4 DEG C.Then, with containing 0.1% Tween-20 and 0.01% The PBS of Triton X-100 is washed three times, after each 5min, by egg mother cell and another antibody (phalloidine-TRITC；1: 100；Sigma, USA) it is incubated at room temperature 30min.Egg mother cell is finally total to dye with DAPI (Vector, Switzerland) Color is sealed on glass slide.Then, it is checked using FV1000 confocal laser scanning microscopies.

4) embryo Oct4 and apoptosis (TUNEL) detection in vivo:

It is as follows：Pretreatment：It will be solid from the blastaea taken out in separate groups of mice the respectively paraformaldehyde with 4% Determine 30min, handles 30min in permeable membrane liquid (Mps), be then transferred in confining liquid (BBS) and close 1h.Add primary antibody：Blastaea is set In anti-Oct4 polyclonal antibodies (1:100, proteintech11263-1-AP) it is stayed overnight for 4 DEG C in；Rinsing：Second day, eluent (WBF) it rinses 3 times, each 5min；Add secondary antibody：Sample is transferred to Alexa Fluor 546 and marks donkey anti-rabbit secondary antibody (1:400, INVITROGEN (A10040)) 1h is incubated in working solution；Rinsing：After incubation, rinsed 3 times with WBF, each 5min；Then Reaction solution is prepared according to tunel kits, carries out tunel dyeing；It rinses again：After incubation, with WBF rinse 3 times, every time 5min；Mounting：Net glass slide center be added dropwise containing on a small quantity can anti-fluorescent quenching DAPI, by sample transfer so far, gently With glass needle mixing, it is then capped clean coverslip mounting；Last useful binders close coverslip to prevent coverslip from sliding Dynamic and moisture loss.It is put into slide avoid light box after the completion of mounting, -20 DEG C of preservations are observed simultaneously under laser confocal microscope It counts.

11) statistical analysis

Each experiment is at least repeated 3 times above in our current research, using SPSS softwares (IBM Corp, USA) software to reality It tests the data obtained to be analyzed, statistical method is using one-way analysis of variance Bonferroni methods of inspection, and data are with Value ± standard error (means ± SEM) expression, * P<0.05, * * P<0.01vs control groups；ΔP<0.05, Δ Δ P<0.01vs models Group；P<0.05 is identified as statistically difference, and n herein indicates the number of egg mother cell.

Experimental method

The female KM mice of raising after a week is being randomly divided into four groups of (control group, model group (DHEA), pachymic acids (Pachymic acid) treatment group, melbine (Metformin) treatment group), every group of 30-40 is only.Control group mice is subcutaneously noted It penetrates soybean oil (emerging (Tieling) Pharmacy stock Co., Ltd) and physiological saline gavage is given once daily and be once used as blank control, even It is 21 days continuous, while DHEA (6mg/100g are subcutaneously injected in model group mouse daily；The joyful beauty bio tech ltd in Wuhan) It is dissolved in injection soybean oil, identical physiological saline is given for manufacturing PCOS disease models, and by gavage.And Poria cocos The identical DHEA solution of the mouse subcutaneous injection of sour treatment group and Or Metformin In Treating group, for manufacturing disease model, same to time-division It is not given containing pachymic acid (5mg/100g by gavage mode；The Shanghai bio tech ltd Shi Feng) and melbine (50mg/100g；The joyful beauty bio tech ltd in Wuhan) physiological saline solution liquid.All mouse for participating in experiment exist (the natural light dark cycle of simulation 12h) raising under controlled temperature (24 DEG C) and illumination condition, and maintain and provide sufficient food And water.

During entire experiment, all female mices are weighed once for every two days, and record every group of mouse weight variation.From It 14 days, selects 3 at random from every group of mouse, observes the variation of its sexual cycle, every morning 8:00~9:00 carries out vaginal smear Terminate to experiment, observes that the vaginal epithelial cell of mouse occurs keratinization for continuous 8 days and successfully indicated as PCOS models. Every group randomly selects some animals (about 15/group) for acquiring blood sample, while a portion acquires white fat after its peritonaeum Fat tissue, ovary tissue are changed with detecting adipose tissue and ovary；The GV phases ovum that another part is used to collect these mouse is female thin Born of the same parents carry out the experiments such as vitro maturation (IVM), to detect egg mother cell A-stage.The blood sample collected centrifuges in time Upper serum is taken, the serum specimen and tissue collected, timely processing is put in -80 DEG C of refrigerators for subsequent experimental, after being used for Phase serum factor detects and tissue PCR detections.Remaining mouse is divided into two parts, and a part about 10 after fasting 8h, carries out OGTT is tested, after OCTT is tested, Superovluation, and MII phase egg mother cells are collected, for MII phase Oocyte qualities Assessment；Another part is put into healthy male mice and carries out 1:1 mating processing is used for the quality evaluation of embryo to collect embryo.

Experimental result

1) influence of the pachymic acid to mouse weight and sugar tolerance

The weight of all mouse is similar when treating beginning.From the 11st to 21 day for the treatment of, the body weight table of model group mouse Reveal the trend (P significantly risen<0.01), and compared with the control group, pachymic acid treatment group and Or Metformin In Treating group mouse Weight all increased, but increase slower, and the weight of pachymic acid treatment group is closer to the weight of control group.Tables of data Bright, after model group mouse is by being subcutaneously injected DHEA, weight shows to dramatically increase, meanwhile, thus pachymic acid can obviously improve Caused mouse weight increases.

By carrying out OGTT to study the influence that pachymic acid is metabolized mouse glucose.It was found that when just starting between group on an empty stomach Blood glucose level is similar.Compared with model group mouse, after oral glucose when 30 and 60min, pachymic acid and Or Metformin In Treating Group mice serum glucose level is significantly reduced.Pachymic acid treatment group mouse and control group mice are to glucose response situation It is similar.Statistical analysis is shown, compared with model group mouse, pachymic acid and Or Metformin In Treating group mouse are in 0 to 120min AUC value is remarkably decreased.In addition, alleviating between medicine group and control group mice without finding significant difference, show to be drawn by DHEA The impaired glucose tolerance risen is alleviated by pachymic acid and melbine.

2) pachymic acid disorderly, hyperlipemia and chronic inflammation influence to mouse hormone in vivo

Hormone abnormality is the most common features of PCOS, it and estradiol, testosterone, anti-Miao Le Shi hormones, insulin and leptin Etc. correlations.Therefore concentration of these hormones in four groups of mice serums is measured respectively.It was found that compared with the control group, model group In these hormonal readinesses all increased.And in addition to other than reducing on insulin level, pachymic acid is in terms of improving these exceptions All it is better than melbine.In addition to These parameters, also measured were in serum participate in hyperlipidemia formed total cholesterol and TG, HDL, LDL is horizontal.It was found that after pachymic acid and Or Metformin In Treating, CHO, TG, LDL raising caused by being induced by DHEA are eased, Meanwhile low-level HDL gets a promotion.These may participate in lipid-metabolism the result shows that pachymic acid, similar melbine.Meanwhile Also the TNF-α in serum, IL-6 (Systemic inflammation evaluation index) content are had detected., it is surprising that compared with the control group, The two inflammatory factors reduce in model group, while finding that TNF-α value is even more less than model group in pachymic acid treatment group, although This difference does not reach statistical significance.In short, compared with the control group, some rush in model group and two kinds of medication therapy groups The serum-concentration of inflammatory cytokine reduces.These statistics indicate that, pachymic acid can improve hormone disturbance, high blood caused by PCOS Fat disease and chronic inflammation.

3) influence of the pachymic acid to mouse ovarian GV phases oocyte number and potentiality of development

In order to study influence of the drug to mouse GV phase egg mother cell quantity and quality, it is female to have detected each group mouse GV phases ovum Cell quantity, apoptosis and potentiality of development.The results show that the GV phase egg mother cell quantity and Oocyte quality of model group mouse are bright It is aobvious to be less than control group (P<0.01).However, compared with model group, Oocyte Apoptosis, GVBD rates and PB1 rates are in PA groups (2.2% Vs 10.3%, oocyte number/n；91.0%vs 83.6%, GVBD；69.6%vs 55.3%, PB1) and Met processing it is small Mouse (3.8%vs 10.3%, oocyte number/n；91.8%vs 83.6%, GVBD；73.8%vs 55.3%, PB1) it obtains It significantly improves, while control group, the quantity of every mouse recycling egg mother cell does not have difference in pachymic acid and melbine group.

Next, the GV phase egg mother cells after evaluation of markers annexin-V, only morphologically normal egg mother cell by with In analysis.Compared with model group, the fatality ratio of the GV phase egg mother cells of Liang Zu treatment groups significantly reduces (P<0.01, PA；P< 0.01, Met).Detection finds that the phosphatidylserine detected by annexin-V cannot be displaced in inner membrance, and only be existed The egg mother cell with green florescent signal could survive and that apoptosis does not occur on oolemma.The GV phases with early apoptosis Egg mother cell is characterized in there is clearly green in oocyte membrane, the GV phase ovum of model group (37.1%, n=116) Mother cell early apoptosis rate is significantly higher than control group (9.4%, n=138).Wherein, pachymic acid group (12.6%, n=132) and two The GV phase egg mother cell early apoptosis rates of first biguanides group (12.6%, n=159) significantly reduce compared with model group.The result shows that Pachymic acid can be by the early apoptosis of inhibition GV phase egg mother cells, so as to improve the GV phase Oocyte qualities of PCOS mouse.

4) pachymic acid influences the quantity of the abnormal MII phases egg mother cell of mouse

In order to study the influence of pachymic acid, dehydrobenzene, melbine to the egg mother cell of cylinder mature, select from defeated The MII phase egg mother cells taken out in oviduct are tested.First, MII phase egg mother cells are detected by stereoscopic microscope observing Morphological abnormalities.It was found that normal morphology (Fig. 1) is presented in most of MII phases egg mother cells in control group, and handled from pachymic acid Group (21.1%, a；9.5%, b；4.8%, c；N=124, n indicate the number of egg mother cell) and melbine processing group (13.1%, a；9.7%, b；7%, c；N=114) the oocyte number with abnormal morphology feature, these include (a) pole extremely Body is degenerated, and (b) perivitelline expands, (c) cytoplasm fragmentation, substantially less than model group (22.5%, a；7.1%, b；17.3%, c；n =98) (Fig. 1).The MII phases egg mother cell developed in these results all display bodies can be protected by pachymic acid.

5) influence of the pachymic acid to the embryonic development of mouse

In order to further assess influence of the pachymic acid to DHEA treated the mouse spilting of an egg and embryonic development exception, compare In four processing groups in randomly selected Mice Body embryo morphological feature, apoptosis situation and Oct4 expression.As shown in Fig. 2, Most of embryo is blastaea in control group, and abnormal embryo includes cleaved fragment (LY) and mulberry body (MO).Embryo is different with model group Reason condition (26.9%, LY；34.2%, MO；N=85 it) compares, in pachymic acid processing group (11.1%, LY；36.1%, MO；N= And melbine processing group (14.7%, LY 72)；22.4%, MO；N=89 the embryo in) is alleviated extremely.Meanwhile passing through Confocal scanning microscope has checked embryo's total cell number, apoptosis situation and Oct4 expressions.It was found that with model group (34 ± 1.9) average embryo total cell number is compared, pachymic acid treatment group (37 ± 1.4) or Or Metformin In Treating group (43 ± 1.8；P< 0.01) the significant increase of total cell number.Significant change also has occurred in these embryos in the expression of Oct4.In addition, and model Group apoptosis points (10 ± 0.8) are compared, and apoptosis significantly improves through pachymic acid (4 ± 0.6；P<Or melbine 0.01) (7±0.8；P<0.05).Meanwhile compared with control group embryo apoptosis rate (31%), the apoptosis rate of pachymic acid treatment group (36%) It significantly reduces, the improvement of Or Metformin In Treating group (81%) apoptosis is not apparent.This shows that DHEA can develop the spilting of an egg and blastaea It causes to damage, and developmental embryo Oct4 abnormal expressions and apoptosis is caused to increase, these exceptions can be treated by pachymic acid To improvement.

6) influence of the pachymic acid to follicular development

It is that Growth of Oocytes develops most important place based on ovary, has studied PCOS mouse and treatment group's mouse ovarian The variation of form and follicular development.Fig. 3 shows the ovarian follicle and thecacells and ovum of control group mice ovary different phase Bubble stratum granulosum is shown normally.However, there is Graafian follicle, and the interstitial group with unusual appearance in the ovary from model group mouse It knits (arrow, Fig. 3,400X).Although equally existing folliculi ovarici vesiculosi in pachymic acid treatment group and Or Metformin In Treating group mouse and going out The anomaly sxtructure (Fig. 3,40X and 100X) of haemocele, but compared with model group ovary (Fig. 3,400X), pachymic acid treatment group or The granular cell number of vesicular follicle is significantly more than model group in the mouse of Or Metformin In Treating and form is more normal, shows Fu Siberian cocklebur acid and melbine can reduce the apoptosis of the granular cell around ovary tube chamber.

Next, studying pachymic acid, dehydrobenzene and melbine to ovarian follicle shape by counting Follicles quantity At effect.It was found that although secondary and antral follicles quantity do not have significant difference between each group, it is noticeable It is, compared with the control group, the quantity decline of the primordial follicle and primary follicle of model group mouse ovarian, moreover, atretic follicle Quantity increases.However these damages occurred in model group can be improved by pachymic acid and melbine.These are the result shows that Poria cocos The follicular development tool of acid pair has a certain impact.

7) influence of the pachymic acid to organelle in mouse growth ovarian follicle

Ultra microstructure is detected by transmission electron microscope, has further looked at the various types that around growing follicle and inside includes The microphoto of cell, including egg mother cell, granular cell, endo cell and stroma cell.As shown in figure 4, with control group phase Than the ovarian follicle membrane structure of model group ovary is generally impaired serious, and pachymic acid processing group and melbine processing group do not go out Now so serious damage.Next, the Ultrastructural observation of egg mother cell is shown, the quantity and appearance of each group mitochondria are all sent out Variation is given birth to.Fig. 4 a3, a5, a7, a9 shows normal mitochondria in control group egg mother cell, with many parallel film layers (blue) Normal mitochondria ridge tissue appearance.However, in model group exception egg mother cell, mitochondria, which has, changes sparse/scarce ridge Anomaly sxtructure, including there is the mitochondria (red, Fig. 4 .c, d, e, f) and " black or similar ghost image " appearance of " vacuole " appearance Mitochondria (yellow, Fig. 4 .c5, f5, c7, b9).The example of above-mentioned all abnormal Mitochondrial Shapes rarely occurs in pachymic acid treatment In group and Or Metformin In Treating group.In addition, pachymic acid treatment group egg mother cell Mitochondria quantity is significantly more than other groups.It calculates The injury of mitochondria rate of every group of egg mother cell.Model group injury of mitochondria rate is apparently higher than control group, however, pachymic acid can be with Improve this damage (P well<0.01).In addition, observing particle and cumulus cell in this research, both cells exist Apoptosis is serious in the growing follicle of model group ovary, meanwhile, abnormalities, endoplasmic reticulum severe dilation is presented.Interesting It is that a kind of abnormal mitochondria newly is found in the ovary of model group and melbine processing group, is referred to as " tubulose/honeycomb Shape " (orange, Fig. 4 .f7, c9, d9, f9) has thicker and expansion mitochondrial cristae appearance.Finally, to the ultra micro of stroma cell Structure is observed, and is found compared with the control group, and the stroma cell lactones drop volume bigger and density of model group induction significantly increase Add, while mitochondria also shows that " cellular ".It is all these the result shows that, various types are thin in model group ovarian growth ovarian follicle Born of the same parents damage, and this damage is extremely related with mitochondria and liposome.For these lesions, pachymic acid and melbine are all It can be effectively improved, wherein pachymic acid improves injury of mitochondria and achieves noticeable achievement.

8) pachymic acid to PI3K in ovary, GLUT4, GSK3- β, IRS-1, CYP-17, TNF-α expression influence

Real-time fluorescence quantitative PCR is used to carry out quantitative analysis to every group of ovary important gene, has selected 6 genes, each Gene code participates in the albumen in glucose uptake or insulin signaling pathway.It is measured using while GAPDH as in standard Ginseng.As a result significant variation has occurred there are four showing in these genes, compared with model group, the gene of pachymic acid treatment group (GLUT4, GSK3- β, IRS-1 and CYP-17；P<0.01) performance is dramatically different, and the IRS-1 of model group is shown singularly Increase.

9) pachymic acid can reduce the proinflammatory cytokine in adipose tissue

In mouse adipose tissue, the mRNA level in-site that TNF-α and IL-6 are detected by q-PCR changes, and is exempted from by protein Epidemic disease blotting observing protein level changes.It was found that TNF-α and the mRNA level in-site of IL-6 are significant in model group mouse adipose tissue Higher than control group (TNF-α, P<0.05；IL-6；P<0.01).And these pro-inflammatory cytokine levels are in pachymic acid treatment group (TNF-α, P=0.09；IL-6；P<0.01) it is significantly restored to close to normal level in.And melbine handles mouse adipose group Knit (TNF-α, P=0.9；IL-6；P=1 it) is not significantly improved.Correspondingly, compared with model group, pachymic acid treatment These inflammatory factors in group and Or Metformin In Treating group mouse adipose tissue are also declined on the expression of protein, Although being not statistically significant.

The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above Detail can carry out a variety of simple variants to technical scheme of the present invention within the scope of the technical concept of the present invention, this A little simple variants all belong to the scope of protection of the present invention.

It is further to note that specific technical features described in the above specific embodiments, in not lance In the case of shield, it can be combined by any suitable means.In order to avoid unnecessary repetition, the present invention to it is various can The combination of energy no longer separately illustrates.

In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally The thought of invention, it should also be regarded as the disclosure of the present invention.

SEQUENCE LISTING

<110>Xiamen University

<120>A kind of pachymic acid or Poria cocos acid derivative are used to prepare application and the drug for the treatment of polycystic ovary syndrome drug

Preparation

<130> XMDX-18015-CNI

<160> 14

<170> PatentIn version 3.5

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Claims (6)

1. a kind of pachymic acid or Poria cocos acid derivative are used to prepare the application for the treatment of polycystic ovary syndrome drug.

2. pachymic acid according to claim 1 or Poria cocos acid derivative are used to prepare treatment polycystic ovary syndrome drug Using, it is characterised in that：The pachymic acid or Poria cocos acid derivative is used to prepare answering for treatment polycystic ovary syndrome drug With including inhibiting weight gain, improves impaired glucose tolerance, improve hormone disturbance, hyperlipemia and chronic inflammation, inhibit inflammation, delay Insulin resistance is solved, the damage of granular cell is inhibited, inhibits at least one of egg mother cell oxidation and apoptosis.

3. pachymic acid according to claim 1 or 2 or Poria cocos acid derivative are used to prepare treatment polycystic ovary syndrome medicine The application of object, it is characterised in that：The derivative of the pachymic acid includes Poria cocos hydrochlorate, Poria cocos acid hydrate, pachymic acid solvation Any one in object, pachymic acid cocrystalization compound.

4. a kind of pharmaceutical preparation for treating polycystic ovary syndrome, including the derivative of pachymic acid or pachymic acid.

5. the pharmaceutical preparation for the treatment of polycystic ovary syndrome according to claim 4, it is characterised in that：The more capsules for the treatment of The pharmaceutical preparation of ovarian syndrome is dispersant, tablet, capsule, granule, suppository or pill.

6. the pharmaceutical preparation for the treatment of polycystic ovary syndrome according to claim 4, it is characterised in that：The more capsules for the treatment of The pharmaceutical preparation of ovarian syndrome further includes auxiliary material.