Application of Tempol in preparation of medicine for treating polycystic ovarian syndrome
阅读说明：本技术 Tempol在制备治疗多囊卵巢综合征药物中的应用 (Application of Tempol in preparation of medicine for treating polycystic ovarian syndrome ) 是由 李天鹤 阴赪宏 刘瑞霞 张婷婷 于 2020-12-07 设计创作，主要内容包括：本发明公开了Tempol在制备治疗多囊卵巢综合征药物中的应用,通过动物实验首次发现Tempol可以降低哺乳动物雄激素水平、恢复哺乳动物排卵功能、改善卵巢多囊性改变、缓解肠道菌群的变化和缓解血清代谢物的差异性改变,进而能够明显改善PCOS,且具有疗效显著的优点,本发明的提出为PCOS的治疗提供了一种新的技术手段,对临床上PCOS的治疗和/或辅助治疗具有十分重要的意义,应用前景广阔。(The invention discloses an application of Tempol in preparation of a medicine for treating polycystic ovarian syndrome, and animal experiments show that Tempol can reduce the androgen level of mammals, recover the ovulation function of the mammals, improve the change of polycystic ovary, relieve the change of intestinal flora and relieve the difference change of serum metabolites for the first time, so that PCOS can be obviously improved, and the invention has the advantage of obvious curative effect.)
The application of Tempol and/or intestinal flora regulated by Tempol in preparing a medicine for treating polycystic ovarian syndrome;
preferably, the intestinal flora comprises Ideonella, Ruminococcus _ 2.
2. The use according to claim 1, wherein the Tempol is used in a dose of 30 mg/kg;
preferably, the continuous use period of the Tempol is 12 days.
3. A pharmaceutical composition comprising Tempol;
preferably, Tempol is used in the pharmaceutical composition at a dose of 30 mg/kg;
preferably, the continuous use period of the Tempol is 12 days.
4. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant.
5. A method of non-therapeutically reducing androgen levels in a mammal, restoring ovulation function in a mammal, ameliorating changes in ovarian polycystic properties, alleviating changes in gut flora, or alleviating differential changes in serum metabolites in vitro comprising administering Tempol to a mammal;
preferably, the Tempol is used in a dose of 30 mg/kg;
preferably, the continuous use period of the Tempol is 12 days.
6. The method of claim 5, wherein the androgen comprises testosterone, androstenedione, dihydrotestosterone, or a combination thereof;
preferably, the androgen is testosterone.
7. The method of claim 5, wherein the indicator of ovulation function comprises an estrus cycle.
8. The method of claim 5, wherein the indicator of change in ovarian polycystic activity comprises the number of corpus luteum and the number of cystic follicles in ovarian tissue.
9. A method of screening a candidate agent for preventing or treating polycystic ovary syndrome, comprising the steps of:
(1) providing a compound to be tested and a positive control compound, wherein the positive control compound is Tempol;
(2) detecting the influence of the compound to be tested in the step (1) on androgen level, ovarian polycystic change and/or ovulation function of the non-human animal model in a test group, and comparing the influence with corresponding experimental results in a positive control group and a negative control group;
wherein, if the reduction degree of the androgen level, the improvement degree of the ovarian polycystic change and/or the recovery degree of the ovulation function of the test compound on the non-human animal model is obviously higher than that of the negative control group, the test compound is suggested to be a candidate drug for treating polycystic ovarian syndrome.
Use of Tempol in screening a medicament for the treatment of polycystic ovarian syndrome, wherein said use comprises the screening method of claim 9.
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of Tempol in preparation of a medicine for treating polycystic ovarian syndrome.
Background
Polycystic ovary syndrome (PCOS) is a disease with complex etiology and diverse clinical manifestations of endocrine and metabolic disorders characterized by chronic anovulation (ovulation dysfunction or loss) and hyperandrogenism (excess production of male hormones in women), mainly characterized clinically by irregular menstrual cycle, infertility, hirsutism and/or acne, widely recognized as a combination of ovulation dysfunction, excess androgen levels and ovarian Polycystic changes, excluding specific diseases that may lead to similar phenotypes, and also commonly occurring mood disorders such as depression, lack of confidence, etc. The etiology and specific pathogenesis of the disease are not clarified, and researches suggest that the disease may be related to certain genetic variation, environmental factors, metabolic abnormalities and the like. Currently, approximately 4% to 8% of women worldwide suffer from polycystic ovarian syndrome, which often results in anovulatory fertility.
At present, the main reason of polycystic ovarian syndrome is not clear, so that the symptomatic treatment is mainly used, and mainly comprises the following steps: (1) aiming at amenorrhea or menstrual disorder caused by persistent anovulation or thin egg discharge of patients with polycystic ovarian syndrome, the clinical treatment mainly comprises long-term oral contraceptive, and the common medicament is Daying-35; (2) aiming at the prominent signs of part of high androgen, such as hirsutism, acne and the like, the blood androgen level is mainly reduced, and commonly used medicines comprise short-acting oral contraceptives, spironolactone and the like; (3) aiming at insulin resistance, an insulin sensitizer is mainly used, and main medicines comprise metformin, thiazolidinediones and the like (pioglitazone and rosiglitazone); (4) ovulation-promoting treatment is mainly used clinically for part of patients with fertility requirements, and common medicines Comprise Clomiphene (CC), gonadotropin, metformin, thiazole and the like.
The most prominent problem is that the medicines can bring various toxic and side effects to the body of a patient, and many medicines have large toxic and side effects and often have dose dependence, namely, the toxic and side effects are more serious when the medicine is taken, the harm to the body is larger, and the symptoms of the polycystic ovarian syndrome can appear after the medicine is stopped. Therefore, there is an urgent need in the art to develop new products with less toxic and side effects that can effectively treat polycystic ovarian syndrome, so as to improve the condition of patients with polycystic ovarian syndrome and improve the quality of life of patients. Tempol (4-hydroxy-2, 2,6, 6-tetramethylpiperidine-1 oxygen free radical) is an azacyclohexane nitroxide, has the characteristics of strong stability, high cell membrane permeability and no toxicity, and at present, the application of Tempol in the preparation of a medicine for treating polycystic ovarian syndrome is not reported.
Disclosure of Invention
Aiming at the technical problems existing in the existing treatment of polycystic ovarian syndrome, the invention aims to provide the application of Tempol in the preparation of a medicine for treating polycystic ovarian syndrome.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an application of Tempol and/or intestinal flora regulated and controlled by Tempol in preparing a medicine for treating polycystic ovarian syndrome;
preferably, the intestinal flora comprises Ideonella, Ruminococcus _ 2.
Further, the dosage of the Tempol is 30 mg/kg;
preferably, the continuous use period of the Tempol is 12 days.
In the present invention, the Tempol includes Tempol and/or a pharmaceutically acceptable salt thereof.
In the present example, the abundance of the intestinal flora Ideonella, Ruminococcus _2 was significantly increased in PCOS model rats.
Further, the medicament comprises Tempol and/or an agent that reduces the abundance of Ideonella, Ruminococcus _ 2.
Furthermore, the medicine can also comprise a pharmaceutically acceptable carrier and/or an auxiliary material.
Further, the carrier and/or adjuvant includes pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the designed dosage form.
Further, the appropriate dose of the drug to be administered may be variously prescribed depending on factors such as formulation method, administration mode, age, body weight, sex, morbid state, diet, administration time, administration route, excretion rate and response sensitivity of the patient, and the skilled physician can easily determine the prescription and the dose prescribed to be effective for the desired treatment in general.
In a second aspect of the invention, a pharmaceutical composition is provided.
Further, the pharmaceutical composition comprises Tempol;
preferably, Tempol is used in the pharmaceutical composition at a dose of 30 mg/kg;
preferably, the continuous use period of the Tempol is 12 days.
Further, the pharmaceutical composition also comprises an agent for reducing the abundance of Ideonella and Ruminococcus _ 2.
Further, the pharmaceutical composition can also comprise a pharmaceutically acceptable carrier and/or an auxiliary material.
Further, the carrier and/or adjuvant includes pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the designed dosage form.
Further, the appropriate dose of the pharmaceutical composition may be prescribed in various ways depending on factors such as the formulation method, the administration mode, the age, body weight, sex, disease state, diet, administration time, administration route, excretion rate and response sensitivity of the patient, and the skilled physician can easily determine the prescription and the dose prescribed to be effective for the desired treatment.
In a third aspect of the invention, there is provided a method for non-therapeutically reducing androgen levels in a mammal in vitro, restoring ovulation function in a mammal, ameliorating changes in ovarian polycystic properties, alleviating changes in gut flora, or alleviating differential changes in serum metabolites.
Further, the method comprises administering to the mammal Tempol;
preferably, the Tempol is used in a dose of 30 mg/kg;
preferably, the continuous use period of the Tempol is 12 days.
Further, the androgen includes testosterone, androstenedione, dihydrotestosterone, or a combination thereof.
Preferably, the androgen is testosterone.
Further, the reduction of androgen levels in a mammal refers to the reduction of androgen levels in the serum or blood of a mammal;
preferably, said lowering of androgen levels in a mammal is a lowering of androgen levels in serum of a mammal.
Further, the index of ovarian polycystic change comprises the number of corpus luteum and the number of cystic follicles in ovarian tissue.
Further, the indicator of ovulation function includes an estrus cycle.
Further, the method for detecting androgen includes (but is not limited to): radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (FIA), chemiluminescence immunoassay (CLIA), time-resolved fluorescence assay (TRFIA).
Radioimmunoassay (RIA) is a method of in vitro microanalysis using an isotope labeled with an unlabeled antigen that competitively inhibits the reaction with an antibody, and is also called competitive saturation assay, which is a laboratory assay for detecting an antigen (e.g., the level of a hormone in serum) without the use of a bioassay.
Enzyme-linked immunosorbent assay (ELISA) refers to a technique of adsorbing known antigen or antibody on the surface of a solid phase carrier and reacting the antigen and antibody labeled by enzyme on the surface of the solid phase, can be used for detecting macromolecular antigen, specific antibody and the like, and has the advantages of rapidness, sensitivity, simplicity, convenience, easy standardization of the carrier and the like.
Immunofluorescence (FIA) is the first developed method of labeling immunological techniques, and the method of labeling or detecting the corresponding antigen with a fluorescent antibody is called fluorescent antibody method, and the method of labeling or detecting the corresponding antibody with a known fluorescent antigen label is called fluorescent antigen method. The problem of non-specific staining of the method is not completely solved, the objectivity of result judgment is insufficient, and the technical procedure is complicated.
Chemiluminescence immunoassay (CLIA) refers to directly labeling an antigen or an antibody with a chemiluminescence agent, reacting with the corresponding antibody or antigen in a sample to be detected and a magnetic particle antigen or antibody, separating the chemiluminescence agent labeling substance in a binding state (a precipitation part) and a free state through a magnetic field, then adding a luminescence promoter to perform a luminescence reaction, and performing quantitative or qualitative detection through detection of luminescence intensity.
The time-resolved fluorescence analysis method (TRFIA) is a novel nonradioactive microanalysis technology established by taking lanthanide with unique fluorescence characteristics and a chelating agent thereof as a tracer, lanthanide is adopted to mark an antigen or an antibody, fluorescence is measured by using a time-resolved technology according to the luminescence characteristics of lanthanide chelate, and simultaneously two parameters of detection wavelength and time are used for signal resolution, so that the interference of non-specific fluorescence can be effectively eliminated, and the sensitivity of analysis is greatly improved.
In the embodiment of the present invention, the method for detecting androgen is preferably a radioimmunoassay.
Further, methods for detecting ovulation function include (but are not limited to): estrus cycle observation, ovulation observation, measurement of estrogen level.
In an embodiment of the invention, the method of detecting ovulation function is preferably oestrus cycle observation.
Further, the mammal includes a female mammal;
preferably, the mammal comprises a mammal with polycystic ovary syndrome;
preferably, the mammal comprises a human or non-human mammal;
more preferably, the non-human mammal comprises a rodent, such as a mouse, rat.
In a fourth aspect of the invention, there is provided a method of screening for a candidate agent for the prevention or treatment of polycystic ovary syndrome.
Further, the method comprises the steps of:
(1) providing a compound to be tested and a positive control compound, wherein the positive control compound is Tempol;
(2) detecting the influence of the compound to be tested in the step (1) on androgen level, ovarian polycystic change and/or ovulation function of the non-human animal model in a test group, and comparing the influence with corresponding experimental results in a positive control group and a negative control group;
wherein, if the reduction degree of the androgen level, the improvement degree of the ovarian polycystic change and/or the recovery degree of the ovulation function of the test compound on the non-human animal model is obviously higher than that of the negative control group, the test compound is suggested to be a candidate drug for treating polycystic ovarian syndrome.
Further, in the step (2), comparing the test results of the test group and the positive control group, comparing the reduction degree of the androgen level, the improvement degree of the change of the ovarian polycystic property and/or the recovery degree of the ovulation function (A1) of the non-human animal model by the test compound in the test group with the reduction degree of the androgen level, the improvement degree of the change of the ovarian polycystic property and/or the recovery degree of the ovulation function (A2) of the non-human animal model by Tempol in the positive control group, and if A1/A2 is more than or equal to 80%, indicating that the test compound is a candidate drug for treating polycystic ovarian syndrome.
Further, the method further comprises the following steps: and (3) further determining the treatment effect of the compound to be tested screened in the step (2) on the polycystic ovarian syndrome.
Further, the determination of the further therapeutic effect comprises the detection of the composition of the intestinal flora of the faeces of the three groups of experimental animals.
Further, the determination of the further treatment effect further comprises the detection of serum metabolites of three groups of experimental animals by using a non-targeted metabonomics method.
Further, the analysis method for detecting the composition of the intestinal flora of the three groups of experimental animal wastes adopts a Linear Discriminant Analysis (LDA) effect size (LEfSe) method.
Further, the analysis method for detecting serum metabolites of three groups of experimental animals by using the non-targeted metabonomics method adopts Principal Component Analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA).
Further, the treatment effect of the compound to be tested screened in the step (2) is evaluated, and the compound has a remarkable treatment effect: A1/A0 is more than or equal to 2, and the medicine has better treatment effect: A1/A0 is more than or equal to 3, and the better treatment effect is A1/A0 is more than or equal to 4, wherein A1 is the reduction degree of the androgen level, the improvement degree of ovarian polycystic change and/or the recovery degree of ovulation function of the to-be-tested compound (test group) in the non-human animal model, and A0 is the corresponding experiment result in a negative control group (model group).
In a fifth aspect, the invention provides an application of Tempol in screening drugs for treating polycystic ovarian syndrome, which is characterized in that the application comprises the screening method in the fourth aspect.
The invention has the following advantages and beneficial effects:
(1) animal experiments show that Tempol can restore the ovulation function of a PCOS model rat, adjust functional cells in ovarian tissues and serum hormones thereof to restore to normal levels, relieve the abundance of intestinal flora and the change of serum metabolites, and prove that Tempol has a protective effect on the PCOS model rat induced by DHEA.
(2) The invention provides a new technical means for the treatment of PCOS, and Tempol can obviously improve PCOS, has the advantage of obvious curative effect, has very important significance for the treatment and/or auxiliary treatment of PCOS clinically, and has wide application prospect.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a graph showing the results of observing the estrous cycle of a rat for 10 consecutive days, wherein, A is a graph: oil + PBS group, panel B: DHEA + PBS group, panel C: DHEA + Tempol group;
fig. 2 is a graph showing the results of HE staining of rat ovarian tissue, in which, a graph: oil + PBS group, panel B: DHEA + PBS group, panel C: DHEA + Tempol group, # ovarian corpus luteum, # cystic follicles;
fig. 3 is a statistical plot of the number of corpus luteum, number of cystic follicles and testosterone levels in serum in rat ovarian tissue, wherein panel a: corpus luteum number, panel B: number of cystic follicles, panel C: testosterone levels in serum, P <0.05, # compared to Oil + PBS group, P <0.05, # compared to DHEA + PBS group;
FIG. 4 is a graph of the results of statistical analysis of biomarker levels for the Oil + PBS, DHEA + PBS, and DHEA + Tempol groups, where A is: histogram, graph B: a branching diagram;
fig. 5 is a graph of the results of relative abundance at the gate level, where, a-graph: a gate level abundance map; and B, drawing: histograms, P <0.05 compared to Oil + PBS group, # 0.05 compared to DHEA + PBS group;
fig. 6 is a graph of the results of relative abundance at genus level, in which, a graph: genus level abundance maps; and B, drawing: histograms, P <0.05 compared to Oil + PBS group, # 0.05 compared to DHEA + PBS group;
fig. 7 is a graph of the results of the determination of serum metabolites of three groups using a non-targeted metabolomics approach, wherein panel a: scatter plot of Principal Component Analysis (PCA) model, panel B: an orthogonal partial least squares-discriminant analysis (OPLS-DA) scoring scattergram;
FIG. 8 is a heat map of hierarchical cluster analysis of differential metabolites, wherein the abscissa represents different experimental groups, the ordinate represents differential metabolites, and different colors represent relative expression amounts of metabolites at corresponding positions.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.
EXAMPLE 1 construction of animal models
3 weeks old SD rats, divided into three groups: control group (Oil + PBS group), PCOS model group (DHEA + PBS group), Tempol treatment group (DHEA + Tempol group). The construction method of the rat model of the control group, 6-11 rats in each group, is as follows: injecting 200 μ L oleum Sesami subcutaneously for 21 days, and injecting PBS intraperitoneally for 12 days; the construction method of the PCOS model group rat model is as follows: subcutaneously injecting Dehydroepiandrosterone (DHEA) (Solarbio, D8950)6mg/100g, 200 μ L, and after 21 days of continuous injection, injecting PBS intraperitoneally for 12 days; the construction method of the Tempol treatment group rat model is as follows: dehydroandroandrosterone (DHEA) was injected subcutaneously at 6mg/100g, 200. mu.L, and Tempol (30mg/kg) was injected intraperitoneally for 21 consecutive days (Selleck Chemicals LLC # S2910, Houston, TX, USA) after 21 consecutive days. The weight was measured once per week during molding.
On day 33 of modeling, three groups of experimental animals were divided into 2 groups, and 3 rats in each group were smeared with vaginal cells at the same time every afternoon for 10 consecutive days, and the morphology of the cells was observed after staining with the lewy stain (leageene, 1029a 20). Blood and ovarian tissue samples were taken after anesthesia of the remaining rats.
Example 2 Observation of the estrous cycle in rats, Observation of the morphology of ovarian tissue and measurement analysis of serum hormones 1, Observation of the estrous cycle in rats
Rats are animals with a perennial estrus and have a relatively stable sexual cycle, which is divided into: the typical change of the vaginal mucosa of each period is generated in the prophase estrus (P), the anaphase estrus (E), the anaphase estrus (M) and the anaphase estrus (D), and the period can be judged according to the cytological characteristics of the vaginal cell smear. In this example, 3 rats in three experimental animals of a control group (Oil + PBS group), a PCOS model group (DHEA + PBS group), and a Tempol treatment group (DHEA + Tempol group) were smeared with vaginal cells at the same time every afternoon for 10 consecutive days, and after staining with lawy's staining solution (leageee, 1029a20), cell morphology was observed and recorded and counted, respectively.
2. Ovarian tissue HE staining
Fresh ovarian tissue was fixed in 4% paraformaldehyde for 24h prior to HE staining. Washing the obtained material in sterilized and precooled PBS, washing blood as thoroughly as possible to avoid the influence of red blood cells on the result when observing slices, dehydrating the material for 1h by 50 percent and 70 percent alcohol respectively, preserving the sample in 70 percent alcohol at 4 ℃ until embedding paraffin, taking out tissue blocks from 70 percent alcohol, dehydrating the tissue blocks in 80 percent, 95 percent and 100 percent alcohol for 1h respectively, and clearing the tissue blocks in 100 percent alcohol/dimethylbenzene (1:1) for 30 min; and then the tissue blocks are transparent in xylene, the specific time is determined according to the size of the tissue blocks, the tissue blocks are preferably completely transparent, the embedding machine can directly receive the heated and filtered paraffin liquid, and the tissue blocks are respectively wax-permeated in xylene/paraffin (1:1), paraffin and paraffin for 2 hours. The method comprises the steps of firstly spreading paraffin with a certain thickness at the bottom of an embedding box, placing the embedding box on a hot table, placing tissues into the hot table by using forceps, adjusting the direction, placing the embedding box on a cold table, slightly solidifying the bottom, continuously pouring the paraffin, placing a slicing support, freezing the cold table, pouring the paraffin, and freezing until the whole paraffin block is molded. And (5) placing the paraffin blocks on a cooling table, and taking out the paraffin blocks by using the slicing support after the paraffin is completely solidified. And (4) tightly fixing the slicing knife rest, and then clamping the wax block on the slicer fixing device by using the slicing support. When slicing, the thickness is adjusted to 5 μm, the wax sheet is cut into pieces, the wax sheet is shot by a small tweezers on the right hand, the wax sheet is gently separated along the blade edge by a brush pen on the left hand, the cut surface faces downwards, the slices are placed into water with the temperature of 42 ℃, after flattening, the continuous wax sheets are gently separated by the tweezers, and then the continuous wax sheets are fished up by a glass slide, dried and placed into a slice box. Baking the slices at 37 ℃ overnight, dewaxing, performing HE dyeing, and recording the experimental results.
3. Assay for serum hormones
Blood samples were taken after anaesthetizing three experimental animals of the control group (Oil + PBS group), PCOS model group (DHEA + PBS group), and Tempol treatment group (DHEA + Tempol group), and serum testosterone levels in the blood of rats of each group were measured by radioimmunoassay (XH6080), and the experimental results were recorded and counted.
4. Results of the experiment
Results plot of estrous cycle in rats showing regular estrous cycle in the Oil + PBS group rats (see fig. 1A); the PCOS model rats in DHEA + PBS group lose regular periodic changes in the estrous cycle, with a late/intermittent (M/D) estrous period of about 6-7 days (see FIG. 1B); relative to the DHEA + PBS group PCOS model rats, the DHEA + Tempol group PCOS model rats restored to some extent a relatively regular estrus cycle (see fig. 1C). The results show that the PCOS model rat treated by Tempol can recover the ovulation function of the rat to a certain extent and recover the ovulation dysfunction of the rat induced by DHEA.
Ovarian tissue HE staining results showed that there was a decrease in the number of yellow bodies and an increase in the number of cystic follicles in ovarian tissue of the DHEA + PBS group PCOS model rats, and an increase in the number of yellow bodies and a decrease in the number of cystic follicles in ovarian tissue of the Tempol treated PCOS model rats (see fig. 2A-C and fig. 3A, B). The serum testosterone levels in the PCOS model rats in the DHEA + PBS group were significantly up-regulated and the testosterone levels in the PCOS model rats after Tempol treatment were significantly down-regulated and returned to normal levels, as compared to the Oil + PBS group rats (see FIG. 3C). The Tempol is shown to have protective effect on DHEA-induced PCOS rats.
Example 3 statistical analysis at biomarker level
1. 16S rRNA Gene sequencing
Total bacterial DNA was extracted from feces using a DNA isolation kit (MO BIO Laboratories, Carlsbad, Calif., USA). The quality and quantity of DNA was evaluated as the ratio of 260nm/280nm and 260nm/230 nm. The DNA was then stored at-80 ℃ until further processing. The V3-V4 region of the bacterial 16S rRNA gene was amplified using common primers (SEQ ID NO.1-SEQ ID NO.2) in combination with the adapter sequence and barcode sequence. The thermal cycling conditions were: initial denaturation at 95 ℃ for 5min, followed by 15 denaturation at 95 ℃ for 1min, 50 ℃ for 1min and 72 ℃ for 1min, and final extension at 72 ℃ for 7 min. The first step PCR product was purified by VAHTSTM DNA clean beads. The second round of PCR was performed in a 40. mu.L reaction, including 20. mu.L of 2 XPh. mu.sion HF-MM, 8. mu.L of ddH2O, 10. mu.M of each primer and 10. mu.L of the PCR product of the first step. The thermal cycling conditions were: initial denaturation at 98 ℃ for 30s, then at 98 ℃ for 10s, 65 ℃ for 30s, 72 ℃ for 30s, and finally extension at 72 ℃ for 5min, and finally quantification and pooling of all PCR products with Quant-iTTM-dsDNA-HS reagents. Purified pooled samples were paired using Illumina Hiseq 2500 platformHigh-throughput sequencing analysis of bacterial rRNA genes was performed.
The sequence of the primer is as follows:
a forward primer: 5'-ACTCCTAGGAGCAGCA-3', respectively;
reverse primer: 5 '-GGACTACHVGGGTWTCTAAT-3';
wherein, H is A/C/T, V is A/C/G, and W is A/T.
Biomarker levels were statistically analyzed for the Oil + PBS, DHEA + PBS, and DHEA + Tempol groups using the Linear Discriminant Analysis (LDA) effect size (LEfSe) method. Both the green group (dha + dha) and the pba + LDA group (pba + LDA) were enriched in the pba + dha group. Only taxa satisfying the four LDA significance thresholds were revealed, the length of the histogram representing the effect of different species. Cladogram visualizes the output of the LEfSe algorithm. Significantly different taxonomy nodes are colored and the branch regions are shaded according to the size of the effect of the taxon. Of the first ten bacteria, the ileal bacteria are most abundant in phylum and genus. Significant changes in abundance at the phylum and genus levels. (blue) Oil + PBS group enriched taxa; (orange) DHEA + PBS group enriched taxa; (green) DHEA + PBS group enriched taxa; (yellow) respective nodes of the inter-group taxonomy group. N-6 rats per group, values are expressed as mean ± SEM.
2. Results of the experiment
To clarify the effect of DHEA and Tempol on rat phenotype, analysis was performed using the Linear Discriminant Analysis (LDA) effect size (LEfSe) method. The results showed that the genus desulfovibrioceae was enriched in the Oil + PBS group, while the genera muribacteriaceae and Alloprevotella increased in abundance in the DHEA + PBS group and DHEA + Tempol group, respectively (see fig. 4A and B), indicating that Tempol can remodel the microbial structure of the intestinal microbiota of PCOS rats.
The overall composition of the three groups of intestinal flora was further compared by analyzing the degree of similarity of the bacterial classifications at the phylum and genus levels. At the phylum level, the phyla Firmicutes and bacteroidides are the dominant group of bacteria among the three groups (see fig. 5A). DHEA treatment increased the abundance of Actinobacteria, bacteroides, patescibacter, Tenericutes and Verrucomicrobia and decreased the abundance of episilonbacterota and Proteobacteria. In addition, tempol intervention can alter DHEA-induced changes in actinobacilla, Patescibacteria, Proteobacteria and tenericites (see fig. 5B). At the genus level, Lachnospiraceae _ NK4a136_ group and mutibacteriaceae are dominant (see fig. 6A). DHEA significantly increases the abundance of Thauera, Staphylococcus, Ideonella, Corynebacterium and Ruminococcus _2, and decreases the abundance of Ruminococcus _ 1. The Temopol treatment can mitigate these changes (see fig. 6B).
Example 4 statistical analysis at serum metabolite level
1. Serum metabolomics analysis
After the rats were sacrificed under anesthesia, blood was taken and serum was isolated for metabolome testing. Serum metabolites were determined in three groups of rats using a non-targeted metabolomics approach.
LC-MS/MS analysis: UHPLC separations were carried out using an ExionLC Infinity series UHPLC system (AB Sciex, Boston, MA, USA) equipped with a UPLC BEH amide column (2.1 × 100mm, 1.7 μm, water) with a mobile phase of 25mmol/L ammonium acetate and 25mmol/L aqueous ammonia hydroxide (pH 9.75) (a) and acetonitrile (B). The elution gradient was: 0-0.5 min, 95% B; 0.5-7.0 min, 95% -65% B; 7.0-8.0 min, 65-40% B; 8.0-9.0 min, 40% B; 9.0-9.1 min, 40% -95% B; 9.1-12.0 min, 95% B, and the column temperature is 25 ℃. The temperature of the automatic sample injection was 4 ℃ and the sample injection volumes were 2. mu.L (pos) and 2. mu.L (neg), respectively. In the LC/MS experiments, MS/MS spectra were obtained on an information-dependent basis using a triple 5600 mass spectrometer (AB-Sciex, USA). In this mode, the acquisition software (analysis TF 1.7, AB Sciex) continuously evaluates the full scan measurement MS data according to pre-selected criteria while collecting and triggering MS/MS spectra acquisition. In each cycle, 12 precursor ions with an intensity > 100 were selected for MS/MS analysis at a Collision Energy (CE) of 30 eV. Cycle time was 0.56s, and ESI source conditions were set as: gas 1 was 60psi, gas 2 was 60psi, curtain gas was 35psi, source temperature was 600 ℃, de-agglomeration potential was 60v, and Ion Spray Voltage Float (ISVF) was 5000v or-4000 v.
Data preprocessing and annotation: MS raw data (. wiff) files were converted to mzXML format using proteomwizard and processed with R-package XCMS (version 3.2). This process includes peak deconvolution, alignment, and integration. Minfrac and cut-off are set to 0.5 and 0.3, respectively. The internal MS2 database was used for metabolite identification.
The raw data for positive ion mode contained 3 Quality Control (QC) samples and 13 experimental samples, from which 1848 peaks were extracted, and individual peaks were filtered to remove noise, leaving only Peak area data for no more than 50% of the null value in a single set or no more than 50% of the null value in all sets. And carrying out standardization processing on the data. Normalization was performed using the total ion current for each sample.
And (3) carrying out intuitive, reliable and statistically significant analysis on the sample on the whole by adopting a Principal Component Analysis (PCA) and an orthogonal partial least squares-discriminant analysis (PLS-DA) method. Each point in the score map represents a corresponding sample, the discrete points in the load map represent the variables separated by the score map, and the higher the dispersion, the greater the contribution to the score map. And calculating each variable through t test to obtain a P value, wherein if the P is more than 0.01 and less than 0.05, the significant difference is considered, and if the P is less than 0.01, the significant difference is considered. Therefore, the mass-to-charge ratios of substances with difference among groups can be obtained, the matched molecular formula is obtained through the obtained accurate mass number, and the matched information is obtained through database search.
2. Results of the experiment
The serum metabolites of the three groups were measured, and Principal Component Analysis (PCA) showed significant differences in the major metabolic components among the three groups (see FIG. 7A), and the orthogonal partial least squares-discriminant analysis (OPLS-DA) score also showed significant differences among the three groups (see FIG. 7B). In addition, 52 serum metabolites (including 26 lipids and lipid molecules) were identified, and the abundance of these metabolites differed between the control and PCOS rats. These differences were mostly alleviated by Tempol (see fig. 8).
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.