阅读说明：本技术 调控ApoC3表达量的制剂在制备预防或治疗胰岛素抵抗型多囊卵巢综合征药物中的应用 (Application of preparation for regulating and controlling expression level of ApoC3 in preparation of medicine for preventing or treating insulin-resistant polycystic ovarian syndrome ) 是由 李荔 曾静 胡克 莫蕙 张红梅 李小芳 繆华章 茹雪媚 周嘉禾 钟明琳 叶喜阳 于 2021-02-22 设计创作，主要内容包括：本发明公开了调控ApoC3表达量的制剂在制备预防或治疗胰岛素抵抗型多囊卵巢综合征药物中的应用。本发明通过发现,PCOS患者卵巢组织及颗粒细胞呈增殖状态,PCOS患者卵巢组织ApoC3高表达,PCOS模型大鼠卵巢组织ApoC3高表达,与PCOS卵巢局部胰岛素抵抗密切相关,进一步发现,利用胰岛素增敏剂可以下调卵巢中APOC3蛋白表达,改善模型大鼠卵巢组织的IR抵抗情况,因此可以将调控ApoC3表达量的制剂在制备预防或治疗胰岛素抵抗型多囊卵巢综合征药物中的应用。(The invention discloses application of a preparation for regulating and controlling the expression level of ApoC3 in preparing a medicament for preventing or treating insulin-resistant polycystic ovary syndrome. According to the invention, the ovarian tissue and granular cells of a PCOS patient are in a proliferation state, the ovarian tissue ApoC3 of the PCOS patient is highly expressed, and the ovarian tissue ApoC3 of a PCOS model rat is highly expressed and is closely related to local insulin resistance of the PCOS ovary, and further, the application of an insulin sensitizer to the preparation of a medicine for preventing or treating insulin-resistant polycystic ovarian syndrome can be used for regulating the expression level of ApoC3, so that the APOC3 protein in the ovary can be down-regulated, and the IR resistance condition of the ovarian tissue of the model rat can be improved.)

1. Application of a preparation for regulating and controlling the expression level of ApoC3 in preparing a medicament for preventing or treating insulin-resistant polycystic ovarian syndrome.

2. The use of claim 1 wherein the agent that modulates the expression of ApoC3 is an agent that reduces the expression of ApoC 3.

3. The use of claim 2 wherein the agent that reduces the expression of ApoC3 is an insulin sensitiser.

4. The use according to claim 3, wherein the insulin sensitiser is metformin or berberine.

The technical field is as follows:

the invention belongs to the field of biological medicines, and particularly relates to application of a preparation for regulating and controlling the expression level of ApoC3 in preparation of a medicine for preventing or treating insulin-resistant polycystic ovarian syndrome.

Background art:

firstly, the more serious glycolipid metabolic disturbance exists in the PCOS patient with the insulin resistance type, the early intervention is performed, and the method is important for improving the reproductive health of the PCOS patient.

Insulin Resistance (IR) and hyperinsulinemia are the main pathological bases of patients with Polycystic Ovary Syndrome (PCOS), play a key role in the occurrence and development of PCOS, and 50-70% of PCOS patients have IR and hyperinsulinemia through domestic and overseas researches. The insulin resistant state (PCOS-IR) of patients with PCOS not only affects their fertility, but is also associated with a range of health problems, including thin or closed menstrual cycle, abnormal uterine bleeding, spontaneous abortion, hypertensive disorders of pregnancy, type 2 diabetes, cardiovascular disorders, endometrial cancer, and the like. The U.S. endocrine Association issued PCOS treatment guidelines in 2013 indicate that: infertility is a prominent clinical problem in patients with PCOS; there are also many obstetrical complications such as gestational diabetes, preeclampsia, etc. in PCOS-IR patients, who are at 2-6 times higher risk of type 2 diabetes, cardiovascular disease, endometrial cancer than PCOS patients who are not associated with IR. The international evidence-based guideline for PCOS in 2018 suggests: PCOS is a disease affecting public health. Our earlier studies also found that: the PCOS patient with the insulin resistance type has more serious glycolipid metabolic disturbance, and the insulin resistance of the PCOS patient is intervened as soon as possible, so that the method has important significance for improving the reproductive health of women.

II, apolipoprotein C-III can be used as a molecular marker for diagnosing the insulin resistance state of a PCOS patient.

The ApoC3 gene is located in chromosome 11A 1/CIII/AIV gene cluster, has a full length of 3.1kb and encodes ApoC3 protein. ApoC3 protein is a glycoprotein with 79 amino acids, which is mainly synthesized in the liver and a small amount in the small intestine of human body, and mainly regulates the decomposition and metabolism of triacylglycerol lipoprotein (TRLS). ApoC3 acts as a lipoprotein lipase (LPL) inhibitor and affects lipid metabolism by inhibiting the activities of lipase, hepatic lipase, cholesterol-egg phosphatidyl transferase. Many researchers have suggested that ApoC3 is excessive, and that it inhibits the binding of lipoprotein to low-density lipoprotein receptor, and also inhibits the binding of lipoprotein to TRLs and their remnant, and inhibits the effective binding of receptor to very low-density lipoprotein (VLDL) and chylomicron (DM), and causes lipid metabolism disorder, visceral fat accumulation, obesity, hypertriglyceridemia, type 2 diabetes, and the like.

Third, PCOS patients have ovarian local IR, and the IR is closely related to high-expression ApoC3, so that ovulation dysfunction is caused.

Over the last 20 years, more and more studies have shown that insulin resistance and compensatory hyperinsulinemia are one of the important characteristic changes of PCOS, and that abnormalities in insulin metabolism or insulin signaling pathway are central to the pathogenesis of PCOS. PCOS-IR can increase the responsiveness of the pituitary to gonadotropin-releasing hormone, leading to increased luteinizing hormone and androgen secretion, disrupting normal function of the hypothalamus-pituitary-ovarian axis, and is critically associated with hyperandrogenism, reproductive disorders, and metabolic syndrome. Several studies have shown that in women with PCOS, loss of insulin activity in classical target organs (adipose tissue and skeletal muscle, etc.) leads to insulin resistance. PCOS has both ovarian local IR, such as ovarian granulosa cell IR, affecting ovarian local glucose metabolism, cholesterol uptake, steroid hormone production, and cell division proliferation. The research shows that the change of insulin signal transduction molecules after the receptor exists in the ovarian granulosa cells of a PCOS patient results in the change of downstream kinase activity, the reduction of insulin signal transduction and the promotion of local insulin resistance of the ovary. It can be seen that the complex mechanism of insulin activity deficiency can also be expressed in the ovarian part, which is the main content of our study. The signal transduction pathways of insulin in cells include a metabolic pathway regulating glucose metabolism, a mitogenic pathway causing cell division and proliferation, and the like. Local IR of ovary shows that the metabolic action promoting pathway of insulin in cell is damaged, and the division promoting pathway is amplified, so that the volume of ovary is increased, follicle development is stopped, ovulation is disturbed, and the local hyperandrogenism state of ovary is caused, and reproductive disorder is caused. We found earlier that: ovarian tissue and granulosa cells are in a proliferative state in patients with PCOS.

The invention content is as follows:

the invention aims to provide application of a preparation for regulating and controlling the expression level of ApoC3 in preparing a medicament for preventing or treating insulin-resistant polycystic ovary syndrome.

Preferably, the agent for regulating the expression level of ApoC3 is an agent for reducing the expression level of ApoC 3.

Further preferably, the agent for reducing the expression level of ApoC3 is an insulin sensitizer.

Preferably, the insulin sensitizer can be metformin or berberine.

According to the invention, the ovarian tissue and granular cells of a PCOS patient are in a proliferation state, the ovarian tissue ApoC3 of the PCOS patient is highly expressed, and the rat ovarian tissue ApoC3 of a PCOS model is highly expressed and closely related to the local insulin resistance of the PCOS ovary, and further, the application of an insulin sensitizer to the preparation of a medicine for preventing or treating insulin resistance polycystic ovarian syndrome can be used for down-regulating the APOC3 protein expression in the ovary and improving the IR resistance condition of the rat ovarian tissue of the model, so that the preparation for regulating and controlling the ApoC3 expression level can be applied to the preparation of the medicine for preventing or treating insulin resistance type polycystic ovarian syndrome.

Description of the drawings:

FIG. 1 shows the immunohistochemical method and Western blot detection of PCNA expression, where A is the immunohistochemical detection of PCNA expression, B is the Westernblot detection of PCNA expression, and C is the Westernblot detection of caspase3 expression.

FIG. 2 shows that ApoC3 protein is highly expressed (HE X40) in ovarian tissue of patients with PCOS-IR, ApoC3 is highly expressed in A.PCOS-IR ovarian granulosa cells, and ApoC3 is lowly expressed in B.PCOS-NIR ovarian tissue;

fig. 3 shows PCOS model group and control group rat vaginal epithelial cells (a. model group, b. control group);

FIG. 4 shows that the insulin and HOMA-IR of model rats are higher than those of control rats (p < 0.01);

FIG. 5 is a graph showing that the IR resistance of ovarian tissues of model rats improved by insulin sensitizers (high, medium and low doses of berberine and metformin), with a concomitant decrease in the expression of rat ovarian ApoC 3.

The specific implementation mode is as follows:

the following examples are intended to further illustrate the invention, but not to limit it.

Example 1:

materials and methods

1. Collection of clinical specimens

Due to the high heterogeneity of PCOS, the diagnostic criteria are divergent and the project group adopted the diagnostic criteria recommended by the European society for human reproduction and embryo and American society for reproductive medicine (ESHRE/ASRM) Lutecan specialist conference in 2003:

(1) dilute or no ovulation;

(2) the clinical manifestations of hyperandrogenism and/or hyperandrogenism;

(3) the ultrasound shows that the ovary is a polycystic ovary (more than 12 follicles with the diameter of 2-9 mm are arranged on one side or two sides of the ovary, and/or the volume of the ovary is more than 10 ml);

(4) and 2 of the 3 above, and excluding other hyperandrogenic diseases such as congenital adrenal cortical hyperplasia, cushing's syndrome, androgen-secreting tumors.

Since diagnosis of PCOS requires exclusion of other related hyperandrogenism diseases, screening of PCOS patients was performed by professional researchers following the special red specialist conference.

The case inclusion criteria were as follows:

(1) the diagnosis standard of polycystic ovarian syndrome is met;

(2) women between 18-45 years of age;

(3) during the treatment period, other western medicines are not used for treatment;

(4) informed consent, volunteer subjects, and observers can be tracked.

The case exclusion criteria were as follows:

(1) those who do not meet the diagnostic criteria for polycystic ovarian syndrome;

(2) women under 18 or over 45 years old, or women in gestation or lactation;

(3) serious cardiovascular diseases, liver diseases, kidney diseases, blood system diseases, lung diseases or serious diseases affecting the survival of the patients, such as tumors or AIDS and the like;

(4) patients with mental or legal disabilities;

(5) patients with endocrine dysfunction such as adrenal gland, thyroid gland, and pituitary gland;

(6) patients suspected of having or determined to have a history of alcohol, drug abuse;

the PCOS insulin resistance group (experimental group) was selected for 30 cases, and the PCOS non-insulin resistance group (control group) was selected for 30 cases, with the age and weight of the patients matched with those in the PCOS insulin resistance group; two groups of patients underwent laparoscopic wedge resection of the ovaries, and the tissues of the ovaries that were removed in a wedge shape were taken for study. Before collecting the samples, the patients were informed of the consent of the ethical committees of hospitals, and the approval numbers of the ethical committees were: 201401011, and signs a surgical consent with the patient. When an ovarian tissue sample is collected in a laparoscopic surgery, the size of the ovarian tissue sample is one tenth of that of the whole ovarian tissue, a tissue similar to a pyramid-like structure is taken on the surface of the ovarian tissue, and the tissue comprises granular cells, theca cells, oocytes, interstitial cells and other various structures. The operation will be performed by a gynecologic endocrine master physician qualified for type IV laparoscopic practice at the women and young health care institute of guangdong province, to ensure the accuracy and representativeness of the clinical specimen collection.

2. Ovarian histopathology and immunohistochemistry

A. Tissue slice preparation

Paraffin embedding of tissue blocks

The endometrial tissue obtained by biopsy was fixed with 4% formaldehyde solution (preparation method: 40% formaldehyde solution and water at a ratio of 1: 9). The fixed time is preferably 12-24 h. Washing the tissue block in a fixed container with running water for 24 hr, sequentially dehydrating with 70% alcohol and 80% alcohol for 2 hr, 90% alcohol for 1 hr, 95% alcohol for 40min, anhydrous alcohol I for 40min, and anhydrous alcohol II for 30 min. And the tissue can be placed in 70% alcohol overnight. The dehydrated tissue is directly immersed in xylene clearing agent (xylene I, xylene II), and each clearing time is 10 min. Then, immersing the tissue into soft wax I for about 1h by adopting paraffin wax with the temperature of 56-58 ℃, and then immersing into soft wax II for 40 min; hard wax I for 40min and hard wax II for 1 h. (the general wax dipping time is 3-4h), the melting point of the soft wax is 45-50 ℃, and the melting point of the hard wax is 55-60 ℃. And finally, injecting the wax liquid into an embedding hard tool, quickly and flatly placing the tissue block into the wax liquid, righting and paving the tissue block, and then moving the tissue block to a cooling table to solidify the tissue block and the wax liquid together. (hard wax fixation type)

b Paraffin section

The excessive paraffin around the specimen is cut off before slicing (the process is called as 'block trimming'), but the paraffin cannot be kept too little, otherwise, the tissue is easy to damage, and the slicing is difficult when the specimen is sliced continuously. The slice thickness is typically 5 μm. Coating a layer of protein glycerol on a clean glass slide, and then baking for 1-2 hours in a constant temperature box at 60 ℃ until the glass slide is dried; if the tissue is stripped, a layer of egg white can be coated for fixation. The pasted sheet is placed in a clean glass sheet box and stored in a refrigerator at 4 ℃ for standby.

B. Immunohistochemical moiety

a dewaxing and hydration

Prior to deparaffinization, the tissue sections should be left to bake for 20min at room temperature for 60min or in an incubator at 60 ℃. Then placing the tissue slices in xylene to soak for 10min, and soaking for 10min after replacing xylene; then soaking in absolute ethanol, 95% ethanol and 70% ethanol for 5min respectively, and washing with PBS for 5min twice. Then preparing fresh 3% H2O2 with distilled water or PBS, sealing at room temperature for 5-10 min, washing with PBS for 3 times, each time for 2 min.

b embedding

Antigen retrieval for formalin fixed paraffin embedded tissue sections. Boiling and heat repairing, heating in an electric furnace or a water bath, heating 0.01M sodium citrate buffer solution (pH6.0) to about 95 ℃, placing the tissue slices into the heated tissue slices for heating for 10-15 min, and cooling the tissue slices to room temperature by using tap water after heating so as to take out the slices. After washing with PBS for 5min, 5% BSA blocking solution was added dropwise and blocked at room temperature for 15 min. And (5) throwing off redundant liquid without washing. Then, primary antibody was added dropwise at 4 ℃ overnight (4 ℃ overnight followed by rewarming at room temperature for 45 min). The next morning was washed 3 times with PBS for 2min each. Then, a poly-HRP-labeled anti-rabbit/mouse IgG secondary antibody is added dropwise, and incubation is carried out for 1h at room temperature. Then washed 3 times with PBS for 2min each.

c color development

And (3) developing with a DAB developing kit or a self-prepared developer (the developing degree is mastered under a mirror), and stopping with distilled water immediately when the background color is light when the target signal is observed to be dark. Counterstaining with hematoxylin for 20s, washing with distilled water for 2 times and 2min each time. Then, the slices are put into 50%, 70%, 80%, 90%, 95% and 100% ethanol in sequence for dehydration for 2min each time. The sections were then placed in 100% xylene for 10min to clear. Finally, 50ul of neutral resin is used for sealing, and the product is stored at room temperature.

3. As a result:

human tissue sample caspase3 and PCNA detection

(1) And selecting 30 PCOS patients and 30 non-PCOS ovarian tissues for research, extracting total protein, and detecting the protein expression amount of caspase3 by using Western blot, wherein the result shows that the expression amount of clear caspase3 in the PCOS group is lower than that in the normal group, and the apoptosis amount in the PCOS ovarian tissues is less than that in the normal group. PCNA expression is detected by an immunohistochemical method and a Westernblot respectively, and immunohistochemical results show that: the expression of PCNA in patients with PCOS was increased compared to the normal group (reddish brown), because PCNA was correlated with the degree of cell proliferation, and an increase in the expression amount of PCNA indicates an increase in cell proliferation in PCOS compared to the normal group. Western blot detection tissues show that the expression level of PCNA in PCOS tissues is higher than that of normal groups, and the result is consistent with that of immunohistochemistry. GAPDH is an internal reference, two groups of caspase3 and PCNA are counted in gray scale, and the PCOS group is obviously higher than the control and has statistical significance. Error bars represent standard errors, P < 0.05. FIG. 1 shows a schematic view of a

(2) And early-stage preliminary experiments are used for carrying out immunohistochemical research on ovarian tissues of PCOS patients: ApoC3 protein was found to be highly expressed in ovarian tissue from patients with PCOS-IR, as shown in FIG. 2, where A shows that ApoC3 protein was highly expressed in both nuclei and cytoplasm of ovarian granulosa cells in ovarian tissue from patients with PCOS-IR, and the staining was dark brown-yellow; b shows that the ApoC3 in the nucleus and cytoplasm of the ovarian granular cells of the PCOS-NIR patient is low in expression. Immunohistochemical results suggest: ApoC3 may be involved in PCOS ovarian local insulin resistance.

Example 2: establishment of PCOS-IR rat model

Materials and methods

1. Laboratory animal

80 21-day-old female SD rats were raised in a clean-grade manner at a constant temperature of 25 ℃ (50% humidity) from the center of medical laboratory animals in Guangdong province. The 12-hour illumination (6: 00-18: 00) and 12-hour darkness (18: 00-6: 00) are performed alternately. Food and water were taken freely.

2. Experimental materials and instruments

DHEA, sesame oil, common feed (processed by Guangdong province medical laboratory animal center), glucose, 0.9% physiological saline, an Elisa kit, a Rui-Ji's staining kit, a glucometer (Sanuo), blood glucose test paper, an optical microscope, a centrifuge and the like.

3. Method of producing a composite material

3.1 model making method for rat animal model selecting 21-day-old SD female rat, dividing into model group and blank control group, 15 control groups and 65 model groups randomly according to random number table method. After grouping, 0.2ml (6mg/100g) of Dehydroepiandrosterone (DHEA) solution dissolved in sesame oil for injection was subcutaneously injected into the back of the neck of the rat in the model group, and 0.2ml of sesame oil for injection was subcutaneously injected into the back of the neck of the rat in the blank control group for 49 days continuously. Rats were weighed every three days.

3.2 method for evaluating success of mold making

3.2.1 vaginal epithelial cells in rats

And observing the vaginal shedding cytological changes of the rats continuously and daily by a Rui-Ji's staining method 10 days before the molding is finished, wherein the vaginal epithelial cells are in a keratinized state for 10 days, and the rats are PCOS rats successfully induced.

The specific experimental method comprises the following steps: dipping a little physiological saline water by using a sterile cotton swab, inserting the cotton swab into a vagina of a rat, slightly twisting, taking out the cotton swab, smearing mucus with vaginal epithelial cells on a glass slide along the same direction, naturally drying, manufacturing two glass slides for each rat every day, uniformly smearing 3-4 drops of a Rui-Ji's staining reagent A for 30-60s, adding 6-10 drops of a reagent B, staining for 10min, washing with tap water, naturally drying, and observing the vaginal epithelial cells under an optical microscope of 40 times and 100 times respectively.

3.2.2 rat fasting blood glucose and fasting insulin detection: 10 mice were randomly selected from each of the rats of the successful PCOS model and the blank group, fasted at 8 nights, and orbital venous blood of the rats was taken at 8 next morning for Fasting blood-glucose (FBG) and insulin-insulin (FINS) detection. Blood glucose levels were measured by the glucose oxidase method (roche glucometer), and Insulin (INS) was measured by the Enzyme linked immunosorbent assay (ELISA). According to the dosage of 2g/kg, rats are intragastrically filled with 50% glucose, a drop of blood is taken from the tail of the rat 30min, 60min and 120min after the glucose administration, the blood sugar of the rat is monitored by a glucometer, an OGTT curve is drawn, and the area under the curve is calculated. And taking 1ml of blood from orbital vein at 60,120min, placing in a drying tube containing anticoagulant, placing on ice, centrifuging for 15min (2500 rpm) after the sampling is finished on the day, carefully collecting supernatant, and detecting serum insulin by ELISA method. HOMA-IR (HOMEOSTASIS model assessment of insulin resistance, HOMA-IR) was calculated as FBG (mmol/L). times.FINS (m U/L)/22.5.

4. Results of animal experiments

PCOS-IR rat ovarian tissue ApoC3 protein is highly expressed, and the use of insulin sensitizer can improve the model rat IR state, and the ovarian tissue ApoC3 protein expression is reduced.

1. We successfully established a PCOS-IR rat model by the subcutaneous injection of dehydroepiandrosterone (dissolved in sesame oil) method. The results showed that the vaginal epithelial cells of the model group rats were in a cornified state for 10 days, with no apparent estrous cycle, while the control group rats had an estrous cycle. The PCOS-IR rat model was suggested to be successfully established, as shown in FIG. 3.

2. The fasting insulin value and HOMA-IR index of the rats in the model group (PCOS-IR group) are obviously higher than those in the control group; the fasting insulin level, 1h and 2h of insulin after glucose administration of the rats in the model group are far higher than those of the rats in the control group, and p is less than 0.01, which prompts the successful establishment of a PCOS-IR rat model, and is shown in figure 4.

3. Western Blot to detect the expression of APOC3 protein in the ovarian tissues of rats in each group:

experimental methods

60 PCOS-IR rats were randomized into groups of 12 rats: a model group, a berberine low-dose group, a berberine middle-dose group, a berberine high-dose group and a metformin hydrochloride positive control group.

The control group adopts insulin sensitizer metformin hydrochloride (500mgbid), and the equivalent dose of the rat is converted according to the conversion coefficient method of the animal kilogram body weight dose. The rats in the treatment group are respectively subjected to intragastric administration intervention with berberine of high dose (162mg/kg), medium dose (81mg/kg) and low dose (40.2mg/kg), 2ml of metformin hydrochloride solution (45mg/kg) is administered once a day for one time, and the metformin group is administered with 2ml of metformin hydrochloride solution (45mg/kg) for one day. The administration was continued for 28 days.

High expression of ApoC3 protein in PCOS-IR rat ovarian tissue, and improved IR resistance in rat ovarian tissue model using insulin sensitizers (high, medium and low doses of berberine and metformin), with a concomitant decrease in rat ovarian ApoC3 expression, as shown in FIG. 5.

APOC3 protein: the results show that compared with the normal group, the APOC3 protein expression level of the model group is remarkably up-regulated (P <0.01), and the APOC3 protein expression can be down-regulated by the high-dose and low-dose groups (P <0.05, P < 0.01). Medium doses down-regulated APOC3 protein expression, but showed no significant difference (P > 0.05). (Note: 1. Normal group; 2. Positive drug (metformin) group; 3. model group; 4. Low dose berberine group; 5. Medium dose berberine group; 6. high dose berberine group).