CN110251549B

CN110251549B - Application of epimedium total flavone extract in preparing medicine for preventing and treating hashimoto thyroiditis

Info

Publication number: CN110251549B
Application number: CN201910548638.1A
Authority: CN
Inventors: 李清林; 葛明华; 郑传铭
Original assignee: Zhejiang Cancer Hospital
Current assignee: Zhejiang Cancer Hospital
Priority date: 2019-06-24
Filing date: 2019-06-24
Publication date: 2021-12-03
Anticipated expiration: 2039-06-24
Also published as: CN110251549A

Abstract

The invention relates to a new application of a traditional Chinese medicine, in particular to an application of epimedium total flavone extract in preparing a medicine for preventing and treating hashimoto thyroiditis, and belongs to the technical field of traditional Chinese medicines. Application of herba Epimedii total flavone extract in preparing medicine for preventing and treating hashimoto thyroiditis is provided. Animal experiment results prove that the epimedium total flavone extract has obvious effect of treating hashimoto thyroiditis on hashimoto thyroiditis rats; the epimedium total flavone extract has good safety and good curative effect, and can effectively treat hashimoto thyroiditis. The epimedium total flavone extract is a traditional Chinese medicine extract with good effect of improving clinical symptoms, small toxic and side effects and good clinical application prospect.

Description

Application of epimedium total flavone extract in preparing medicine for preventing and treating hashimoto thyroiditis

Technical Field

The invention relates to a new application of a traditional Chinese medicine, in particular to an application of epimedium total flavone extract in preparing a medicine for preventing and treating hashimoto thyroiditis, and belongs to the technical field of traditional Chinese medicines.

Background

Hashimoto thyroiditis (hashimoto thyroiditis) is also called chronic lymphocytic thyroiditis and is a chronic autoimmune disease with autologous thyroid tissue as antigen. Since the first report of Hashimoto of Kyushu university in Japan was based on histological features, it was also named Hashimoto's thyroiditis, the most common thyroid inflammation in clinical practice. The age of good hair is 40-50 years. There are also many cases in children.

At present, the western medicine mainly adopts thyroxine to replace treatment methods such as a treatment method, an immunotherapy method and an operation to relieve local symptoms of the thyroid gland, but has poor curative effect on the general symptoms of patients. Among them, the most conventional therapy is hormone replacement therapy, whereas hormones have no immunoregulatory effect, cannot lower thyroid autoantibody titers, and patients need to take medicine for life. Secondly, the patient needs continuous physical examination, the dosage of the hormone is adjusted according to the indexes of the thyroid gland function, the adverse reaction is more, and the stopping of the medicine is easy to repeat. Therefore, the search for new effective drugs for hashimoto thyroiditis has obvious social and economic values.

Disclosure of Invention

The invention aims to provide application of epimedium total flavone extract in preparing a medicament for preventing and treating hashimoto thyroiditis.

The technical scheme adopted by the invention for solving the technical problems is as follows:

application of herba Epimedii total flavone extract in preparing medicine for preventing and treating hashimoto thyroiditis is provided. Pharmacological research shows that the inventor uses the epimedium total flavone extract to prevent and treat hashimoto thyroiditis, obtains satisfactory effect and discovers the new application of the epimedium total flavone extract.

The epimedium total flavone extract is extracted and purified from traditional Chinese medicine epimedium according to a conventional method in the field. The extraction process can be performed by using the methods for extracting traditional Chinese medicine flavone known in the prior art, such as but not limited to cold soaking, percolation, ultrasonic extraction, Soxhlet extraction, hot reflux extraction, supercritical extraction and the like, and by using the combination of extraction methods such as cold soaking-percolation, cold soaking-ultrasonic extraction, cold soaking-Soxhlet extraction, cold soaking-hot reflux extraction and the like. The epimedium total flavone extract is purified mainly according to the known traditional Chinese medicine flavone purification methods, such as but not limited to column chromatography, solvent extraction, solid phase extraction, ion exchange and the like.

Preferably, the medicine for preventing and treating hashimoto thyroiditis is a preparation prepared by taking epimedium total flavone extract as an active ingredient and adding pharmaceutically acceptable auxiliary materials. The epimedium total flavone extract can be directly prepared into various traditional Chinese medicine oral conventional medicaments for preventing and treating hashimoto thyroiditis or be prepared into various traditional Chinese medicine oral conventional medicaments with pharmaceutically acceptable drug carriers according to conventional methods in the field.

The pharmaceutical formulation may be in any pharmaceutically acceptable dosage form including: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, buccal agents, granules, pills, powders, ointments, pellets, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, patches; oral dosage forms are preferred, such as: capsule, tablet, oral liquid, granule, pill, powder, pellet, and unguent. The oral dosage forms may contain conventional excipients such as binders, fillers, diluents, tabletting agents, lubricants, disintegrating agents, coloring agents, flavoring agents and wetting agents, and the tablets may be coated if necessary. Suitable fillers include cellulose, mannitol, lactose and other similar fillers; suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycolate; suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulfate and the like.

Animal experiment results prove that the epimedium total flavone extract has obvious effect of treating hashimoto thyroiditis on hashimoto thyroiditis rats; the epimedium total flavone extract has good safety and good curative effect, and can effectively treat hashimoto thyroiditis. The epimedium total flavone extract is a traditional Chinese medicine extract with good effect of improving clinical symptoms, small toxic and side effects and good clinical application prospect.

Drawings

FIG. 1 is a photograph of thyroid pathology (HE staining, 200-fold) of rat in HE staining observation Hashimoto thyroiditis model;

FIG. 2 is a photograph of thyroid pathology in rat with HE-stained Hashimoto's thyroiditis model (HE-stained 400-fold);

FIG. 3 shows the effect of total flavonoids of epimedium on the content of FT3, FT4, TOP-Ab, ATGA, T3 and T4 in HT rat serum (x + -s, n-6);

FIG. 4 shows the effect of total flavonoids of Epimedium herb on the levels of IFN-. gamma.IL-17, IL-6, IL-10, IL-23 and TGF-. beta.in the serum of HT rats (x. + -. s, n ═ 6);

fig. 5 is a flow scatter plot of peripheral blood Treg cells from each group of rats, note: a blank control group; b, model group; c, herba Epimedii total flavone extract low dose group; d, preparing a middle-dose group of epimedium total flavone extract; e, a high-dose group of epimedium total flavone extracts; f, a levothyroxine sodium tablet positive drug group;

fig. 6 is a cell flow scattergram of Th17 peripheral blood of rats in each group, note: a blank control group; b, model group; c, herba Epimedii total flavone extract low dose group; d, preparing a middle-dose group of epimedium total flavone extract; e, a high-dose group of epimedium total flavone extracts; f, a levothyroxine sodium tablet positive drug group;

fig. 7 is a photograph of icariin expression on FoxP3 (n 6, x 200 times) in rat thyroid tissue of hashimoto thyroiditis;

fig. 8 is a photograph of icariin expression on FoxP3 (n 6, x 400 times) in rat thyroid tissue of hashimoto thyroiditis;

fig. 9 is a photograph of ROR γ t expression of epimedium flavonoids on thyroid tissue of hashimoto thyroiditis rat (n-6, x 200 times);

fig. 10 is a photograph of ROR γ t expression of epimedium flavonoids on thyroid tissue of hashimoto thyroiditis rat (n-6, x 400 times);

FIG. 11 is a photograph of IL-17 expression of epimedium flavone on thyroid tissue of rats with hashimoto thyroiditis (n-6, x 200 times);

FIG. 12 is a photograph of IL-17 expression of epimedium flavone on thyroid tissue of Hashimoto's thyroiditis rat (n-6, x 400 times);

FIG. 13 is a photograph of IL-23 expression of epimedium flavone on thyroid tissue of rats with hashimoto thyroiditis (n-6, x 200 times);

FIG. 14 is a photograph of IL-23 expression of epimedium flavone on thyroid tissue of rats with hashimoto thyroiditis (n-6, x 400 times);

FIG. 15 is a photograph of IL-6 expression of epimedium flavone on thyroid tissue of rats with hashimoto thyroiditis (n-6, x 200 times);

FIG. 16 is a photograph of IL-6 expression of epimedium flavone on thyroid tissue of rats with hashimoto thyroiditis (n-6, x 400 times);

FIG. 17 is a photograph of IL-10 expression of epimedium flavone on thyroid tissue of rats with hashimoto thyroiditis (n-6, x 200 times);

FIG. 18 is a photograph of IL-10 expression of epimedium flavone on thyroid tissue of rats with hashimoto thyroiditis (n-6, x 400 times);

FIG. 19 is a photograph of IFN-. gamma.expression of epimedium flavonoids in thyroid tissues of rats with hashimoto thyroiditis (n.6, x.200 times);

FIG. 20 is a photograph of IFN-. gamma.expression of epimedium flavonoids in thyroid tissues of rats with hashimoto thyroiditis (n.6, x 400 times);

FIG. 21 shows the statistics of relative expression levels of thyroid-associated genes in rats: (

n＝3)；

FIG. 22 is a graph showing the expression of a thyroid-associated protein in rat: (

n＝3)；

FIG. 23 shows the relative expression statistics of thyroid-associated proteins in rats: (

n＝3)；

FIG. 24 is a photograph of rat thyroid primary cells (100X);

FIG. 25 is a photograph of the activity of drug-containing serum on rat thyroid cells, note: a blank control group; b, model group; c, epimedium flavone low-dose group; d, preparing a middle-dose group of epimedium flavone; e, a high-dose group of epimedium flavone; f, a levothyroxine sodium tablet positive drug group; FIG. 3, FIG. 4, FIG. 21, FIG. 23, Note: normal group ratioIn comparison, the method has the advantages that,^▲P<0.05,^▲▲P<0.01; comparison with model groups:^★P＜0.05，^★★P＜0.01。

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.

In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified. The main reagent sources used in the following examples are as follows:

example 1 therapeutic Effect of Epimedium herb Total Flavonoids extract of the present invention on Hashimoto's thyroiditis rats

Preparation of epimedium total flavone extract:

roasting epimedium: taking herba Epimedii raw medicinal materials, removing impurities, picking up leaves, spraying clear water, slightly moistening, shredding, and drying. Heating refined adeps Caprae Seu Ovis, melting, adding herba Epimedii shreds, parching with slow fire until the surface shows uniform oily luster and slight yellow color, taking out, and cooling. 20kg of refined mutton tallow oil is used for every 100kg of epimedium.

Preparing a total flavone extract: taking a proper amount of roasted epimedium, respectively adding 70% ethanol in an amount which is 10 times the amount of the roasted epimedium, and extracting for 2 times, 2 hours each time. The liquid medicine is concentrated to 1mL liquid medicine containing 1g crude drug, and the technology of purifying the total icariin by adopting HPD300 macroporous resin is determined by investigation as follows: diluting the medicinal liquid with water to obtain medicinal liquid containing 0.1g crude drug per 1mL, passing through HPD300 macroporous adsorbent resin column (resin column diameter height ratio of 1:11) at flow rate of 4BV/h, washing with 5 times of column volume of water, eluting with 7 times of column volume of 50% ethanol at flow rate of 4BV/h, collecting eluate, recovering ethanol under reduced pressure, filtering, adding 3% gelatin solution into the filtrate until no precipitate is generated, adding ethanol to make ethanol content reach 80%, standing overnight, filtering, recovering ethanol under reduced pressure to obtain herba Epimedii total flavone extract (total icariin content of about 80%).

Purpose of the experiment

1, HE staining and observing thyroid pathological changes of a Hashimoto thyroiditis model rat;

ELISA testing the levels of FT3, FT4, TOP-Ab, ATGA, T3, IFN-. gamma., IL-17, IL-6, IL-10, IL-23 and TGF-. beta.and T4 in rat serum;

3. influence of herba Epimedii total flavone extract on Treg and Th17 cells in peripheral blood of HT rat;

4. immunohistochemical observation of the expression of FoxP3, ROR gamma t, IL-17, IL-23, IL-6, IL-10 and IFN-gamma in the thyroid gland of the hashimoto thyroiditis model rat;

qRT-PCR detection of relative expression levels of FoxP3, ROR gamma t, TGF-beta 1, IL-17, IL-23, IL-6, IL-10 and IFN-gamma genes of the thyroid gland of the rat;

WB detecting the expression of the thyroid FoxP3, ROR gamma t, TGF-beta 1, IL-17, IL-23, IL-6, IL-10 and IFN-gamma protein of the rat;

7. establishing a rat thyroid epithelial cell injury model caused by Dextran Sodium Sulfate (DSS);

8. drug treatment and cell proliferation detection;

9. detecting the proliferation and toxicity of thyroid epithelial cells of rats;

detecting the influence of the serum containing the medicine on the apoptosis by an Annexin V-FITC/PI method.

2 method of experiment

2.1 animal model preparation and group administration

SPF grade female wistar rats weighing 150-180 g. Provided by Shanghai Sphere-BiKai laboratory animals Co., Ltd, the animal production license number SCXK (Shanghai) 2013-0016.

Feeding conditions are as follows: constant temperature, temperature of 22 +/-2 ℃, humidity of 50-60%, light and shade alternation every 12 hours of illumination, and air exchange times of 15-20 times/hour. Is bred by animal experiment research center of Zhejiang Chinese medicine university. The license number SYXK (Zhe) 2013-.

60 rats were acclimatized for one week and treated as follows: the mice were divided into 6 groups of 10, 60 total:

blank control group A

Group of B models

C high dose group of Epimedium general flavone extract (240mg/kg)

D middle dose group of epimedium total flavone extract (120mg/kg)

E Low dose group of Epimedium general flavone extract (60mg/kg)

F positive medicine group

Dosage to be administered

Group A was given 0.4ml of physiological saline once a day for 14 consecutive days.

Group B was given 0.4ml of physiological saline once a day for 14 consecutive days.

Group C was administered with 240mg/kg of epimedium total flavone extract solution once a day for 14 consecutive days.

Group D was administered 120mg/kg of a total flavonoids extract solution of Epimedium herb once a day for 14 consecutive days. (administration dose: 400mg/kg)

Group E was administered with 60mg/kg of epimedium total flavone extract solution once a day for 14 consecutive days.

Levothyroxine sodium tablets were given treatment in group F at 10. mu.g/dose for 1/day for 14 consecutive days.

2.2 model preparation

1) Primary immunization: the prepared thyroglobulin PTG antigen is dissolved by normal saline and then is mixed with complete Freund's adjuvant according to the ratio of 1:1 after being fed for 1 week, the final concentration is 0.25mgPTg of adjuvant-containing antigen per milliliter, the solution with the content of PTg mg/mL is dissolved by PBS buffer solution, the mixture is fully emulsified by the equal volume of the complete Freund's adjuvant CFA, the first dose is 100 mug/mouse, and the injection is injected subcutaneously on the back of the rat. The normal control group rats were not treated at all.

2) And (3) boosting immunity: the PTg PBS solution and IFA were mixed in equal volumes and emulsified thoroughly, at a dose of 100. mu.g/mouse, by booster immunization once a week from week 2 after the initial immunization, and by multi-point subcutaneous injections at the toes, back, etc. of the rats to week 6.

2.3 HE staining for tumor changes

And (3) detecting the time point: the rats were dissected 6 weeks after model preparation, thyroid tissue was fixed in 10% neutral formaldehyde, and embedded sections were obtained.

1) The slices were dewaxed conventionally with xylene, washed with various grades of ethanol to distilled water: xylene (I)5min → xylene (II)5min

2) Absolute ethyl alcohol 2min → 95% ethyl alcohol 1min → 80% ethyl alcohol lmin → 75% ethyl alcohol lmin → distilled water washing 2min

3) Staining with hematoxylin for 5min, washing with tap water for 5min

4) Differentiation with ethanol hydrochloride for 30s

5) Soaking in tap water for 15min

6) Placing in eosin solution for 2min, and washing with tap water

7) Conventional dehydration, transparency, mounting: 95% ethanol (I)1min → 95% ethanol (II)3min → absolute ethanol (I)5min → absolute ethanol (II)5min → xylene (I) transparent 5min → xylene (II) transparent 5min → neutral resin sealing

8) Image acquisition and analysis: the relevant parts of the analysis sample are collected by taking pictures through a microscope (observing under a 200-fold and 400-fold microscope).

2.4 ELISA testing of rat serum levels of FT3, FT4, TOP-Ab, ATGA, T3, T4, IFN-. gamma., IL-17, IL-6, IL-10, IL-23 and TGF-. beta.

And (3) detecting the time point: after 2 weeks of administration, fasting for 12h (without water), taking blood from heart, standing for 30min, centrifuging at 3000r/min for 10min, and preparing serum for use.

(1) Preparing: the kit was taken out of the refrigerator and allowed to re-warm and equilibrate at room temperature for 20 min.

(2) Preparing liquid: the 20-fold concentrated washing solution is diluted into the original-fold washing solution by using distilled water.

(3) Adding a standard substance and a sample to be detected: fixing a sufficient number of enzyme-labeled coated plates on a frame, respectively arranging a standard product hole, a sample hole to be detected and a blank contrast hole, recording the position of each hole, and adding 50 mu L of a standard product into the standard product hole; firstly adding 10 mu L of sample to be detected into a sample hole to be detected, and then adding 40 mu L of sample diluent (namely, the sample is diluted by 5 times); blank control wells were not added.

(4) In addition to blank wells, 100. mu.L of detection antibody labeled with horseradish peroxidase (HRP) was added to each of the standard wells and the sample wells.

(5) And (3) incubation: the reaction wells were sealed with a sealing film and incubated in a 37 ℃ water bath or incubator for 60 min.

(6) Washing the plate: discarding liquid, drying on absorbent paper, filling each hole with cleaning solution, standing for 1min, throwing off cleaning solution, drying on absorbent paper, and repeating the plate washing for 5 times (or washing plate with plate washer according to the instruction).

(7) Color development: adding 50 μ L of developer A solution and 50 μ L of developer B solution into each well, mixing with a plate mixer for 30s (or shaking gently with hand for 30s), and developing at 37 deg.C in dark for 15 min.

(8) And (4) terminating: the ELISA plate was removed and 50. mu.L of the stop solution was added to each well to stop the reaction.

(9) And (3) determination: the wells were zeroed and the absorbance (OD) of each well was measured at a wavelength of 450nm within 15min after termination.

2.5 Effect of Epimedium Total Flavonoids extract on Treg cells in peripheral blood of HT rat

After 3 days of administration, 100 μ L of peripheral blood of each rat group is injected into a test tube, 25 μ L of heparin sodium solution is injected into each tube, then 10 μ L of each of CD4-FITC (5 μ g/mL) and CD25-ApC (2 μ g/mL) is added into each tube, the tubes are placed into a refrigerator at 4 ℃ for incubation for 20min, 2mL of each erythrocyte lysate is added into each tube, the mixture is fully mixed (5 min at 37 ℃), then the mixture is centrifuged (1500r/min) for 5min, supernatant is discarded, Hanks is washed for 2 times, 20 μ L of PE-Foxp3 is added, and the mixture is incubated for 50min at 4 ℃ and protected from light. Perm Buffer (200. mu.L) was washed twice and then resuspended in 400. mu.L Hanks Buffer. Resuspension cells were injected into labeled flow tubes at 400. mu.L machine final volume (machine final concentration 5X 106 cells) and resuspended in Hanks' solution in each tube, and 100. mu.L resuspended cells were injected into labeled flow tubes in each tube, and the change in the amount of Treg in peripheral blood of rats in each group was detected by up-flow.

2.6 Effect of Epimedium Total Flavonoids extract on Th17 cells in HT rat peripheral blood

After 3 days of administration, 100 mu L of fresh heparin anticoagulation blood is taken, added with 860 mu L of culture medium IRPMI 1640, mixed evenly, and then added with 10 mu L of IPMA working solution, 10 mu L of Ionomycin working solution and 20 mu L of BFA working solution, wherein the concentrations of PMA, Ionomycin and BFA in the system are respectively 50ng/mL, 750ng/mL and 10 mu L/mL. Stimulating and culturing at 37 deg.C in 5% CO2 incubator for 3 hr; gently mixing the cultured cell suspension, adding the mixture into 1 flow test tube, and centrifuging; discarding the supernatant, shaking and mixing, adding 2 mu LFITC-Anti rat-CD4, and incubating for 15min in dark; adding 100 μ L of fixing solution, and standing in dark for 15 min; washing with 2mL of 1 XPBS, and centrifuging; discarding the supernatant, shaking, mixing, adding 100 μ L membrane-breaking agent, avoiding shaking, mixing gently, and keeping out of the sun for 5 min; adding 2 μ L of PE-Anti rat-IL-17 into the tube, mixing gently, and incubating for 15min in dark; add 2mL of 1 XPBS to wash once, centrifuge, discard the supernatant, and detect on the flow cytometer.

2.7 immunohistochemical visualization of FoxP3, ROR γ t, IL-17, IL-23, IL-6, IL-10 and IFN- γ expression in the thyroid glands of rats which are the hashimoto thyroiditis model.

Paraffin wax slices were baked at 60 deg.C for 1 hr, and dewaxed with xylene (xylene I10min, xylene II 10min), gradient alcohol (100% 3min, 100% 3min, 95% 3min, 90% 3min, 85% 3min, 75% 3min, distilled water 3min, PBS 3 min).

Antigen retrieval: placing the slices in 0.1mol/L citric acid solution repairing solution with pH of 6.0, boiling with microwave oven 100 fire power for three minutes to slightly boil, maintaining 50 fire power for 7 minutes, stopping heating, and naturally cooling for 20-30 minutes. Rinse 5min × 3/time with PBS.

Immune hybridization: 3% H₂O₂Incubate for 10min to eliminate endogenous peroxidase activity. PBS wash, 5min X3 times. Blocking solution (5% BSA) was added dropwise and the mixture was left in a wet box at room temperature for 30 minutes. The blocking solution was wiped off with filter paper without washing. FoxP3, ROR gamma t, IL-17, IL-23, IL-6, IL-10 and IFN-gamma primary antibody with appropriate concentration are dripped into the wet box to be incubated overnight at 4 ℃, the primary antibody is washed off by PBS for 5 minutes and 3 times, and PBS outside the sample is wiped off by filter paper. Adding biotinylated second antibody working solution dropwise, and incubating in a wet box at room temperature for 20minThe secondary antibody was washed off with PBS 5 minutes × 3 times, and the PBS outside the specimen was wiped off with a filter paper. And (3) dripping horseradish enzyme labeled streptavidin working solution, incubating in a wet box for 20 minutes at room temperature, washing with PBS for 5 minutes multiplied by 3 times, and wiping the PBS outside the sample with filter paper. DAB color developing agent color development, and tap water fully washes. And performing hematoxylin counterstaining, dehydrating, and sealing with transparent neutral gum. Sections were randomly selected under 10X 20 and 10X 40 times microscope for thyroid gland without repeating 6 fields.

2.8 qRT-PCR detection of relative expression levels of FoxP3, ROR gamma t, TGF-beta 1, IL-17, IL-23, IL-6, IL-10 and IFN-gamma genes in rat thyroid

The sequence information of the primers used for qRT-PCR is as follows:

RNA extraction

1) Sample treatment: thyroid tissue (1 ml per 200 mg) and thyroid epithelial cells (1X 10 per cell)⁷1ml of each cell) was placed in a Trizol homogenizer tube, and the lysed sample was allowed to stand at room temperature for 5-10min to completely separate the nucleoprotein from the nucleic acid.

2) The mixture was placed in a clean bench, incubated for 5min, 12000rpm, and centrifuged for 10 min.

3) The supernatant was aspirated into a new 1.5mL centrifuge tube, 200. mu.L of chloroform was added thereto, shaken, allowed to stand at room temperature for 2min, at 4 ℃ and 12000rpm, and centrifuged for 10 min.

4) The supernatant was aspirated into a new 1.5mL centrifuge tube, 600. mu.L of isopropanol was added, mixed well, left to stand at room temperature for 15min, 4 ℃, 12000rpm, centrifuged for 15min, and the supernatant was discarded.

5) The precipitate was rinsed by adding 1mL of 75% absolute ethanol (750. mu.L of absolute ethanol and 250. mu.L of DEPC water), centrifuged at 12000rpm for 5min at 4 ℃ and the supernatant discarded.

6) Adding 1mL of anhydrous ethanol, rinsing the precipitate, centrifuging at 4 deg.C and 12000rpm for 5min, discarding the supernatant, and drying at room temperature for 10 min.

7) 40 μ L of DEPC water was added to dissolve the RNA, and the RNA was stored in a freezer at-80 ℃ for further use.

Reverse transcription reaction

The following reaction system was prepared for the reverse transcription reaction. Reaction conditions are as follows: 42 ℃ for 15 min; 85 ℃ for 5 min.

qPCR reaction

1) The following reaction system was prepared for real-time fluorescent quantitative PCR reaction. Thoroughly and uniformly mixing the solution in the tube by using a vortex oscillator,

and (5) carrying out short-time low-speed centrifugation. Reaction conditions are as follows: denaturation at 95 ℃ for 10 min; 95 ℃ for 15 s; 60 ℃ for 60 s; and (4) 40 times of circulation.

2) Sample application: and (2) adding the mixed liquid in the step (1) into a pore plate, wherein each gene of each sample ensures 3 multiple pores.

3) And (3) PCR reaction: the Real time PCR instrument uses a bio-red Real-time fluorescent quantitative PCR instrument, and the PCR program is optimized. And (3) placing the well-spotted 96-well plate in the step (2) on a Realtime PCR instrument for PCR reaction.

4) Reaction conditions are as follows: denaturation at 95 ℃ for 10 min; 95 ℃ for 15 s; 60 ℃ for 60 s; and (4) 40 times of circulation.

2.9 WB detection of FoxP3, ROR gamma t, TGF-beta 1, IL-17, IL-23, IL-6, IL-10 and IFN-gamma protein expression in rat thyroid

Protein sample preparation

1) Thyroid tissue (1 ml per 200 mg) and thyroid epithelial cells (1 ml per 1X 107 cells) RIPA lysate (containing PMSF and protease inhibitor) were ground using a hand homogenizer. Placing on ice for cracking for 30 min;

2) centrifuging at 12000rpm for 5min, and collecting supernatant.

Determination of Total protein concentration of tissue after drug treatment (BCA method)

1) BCA protein concentration determination kit Solambio Cat pc0020

2)96 test plate Costow 3599

3) BCA reagent: cu reagent: preparing 8ml of BCA working solution according to the volume ratio of 50: 1;

4) diluting the standard substance to 150 μ L with PBS, and preparing to concentration of 0.5 mg/ml;

5) samples were diluted 2-fold and 8-fold, and 20 μ L was added to a 96-well plate;

6) add 200. mu.L of BCA working solution, 20 minutes at 37 ℃ and measure the wavelength with a microplate reader A562 nm.

SDS-PAGE electrophoresis and membrane transfer

1) A clean 1.5mm glass plate is taken and mounted on a glue making frame according to the instruction.

2) Preparing 10% separation glue and 5% concentrated glue

Adding the above materials, mixing, pouring separation gel, and sealing with water.

Adding the above materials, mixing, and filling concentrated gel.

3) SDS-PAGE concentrated gel with 5% concentration is prepared and injected into the upper end of the separation gel, and a sample comb with the thickness corresponding to that of the glass plate is carefully inserted to avoid air bubbles.

4) After polymerization of the layering gel, the sample comb was carefully removed and 1 × Tris-Gly running buffer was added.

5) Sucking a proper amount of sample supernatant, adding the sample supernatant into a sample hole, adding a pre-stained protein Marker into a hole beside the sample, and adding 1 xSDS loading buffer solution into a hole without the sample supernatant to keep the gel surface balance.

6) The power supply is turned on, the voltage is initially set to 80V, and when the protein sample enters the separation gel, the voltage can be increased to 120V. Referring to the location of the prestained Marker, the electrophoresis was stopped when the band of interest entered the optimal separation zone of the gel (about 2/3 of the gel).

7) Precooling the membrane transferring liquid at 4 ℃ in advance.

8) The transfer cassette was opened on the tray, the inner face near the cathode side was covered with a porous fibrous pad which had been soaked with the transfer membrane buffer, and three layers of filter paper soaked with the transfer membrane buffer were placed thereon, taking care to remove air bubbles.

9) Carefully prying the glass plate open, placing the glue in a tray containing the membrane transferring solution, cutting the separation glue containing the target strip, soaking the separation glue in the membrane transferring solution, and placing the separation glue on filter paper.

10) The PVDF membrane soaked by methanol and the membrane transferring liquid is paved on the gel, no air bubbles can be reserved between the gel and the membrane, and the sizes of the membrane, the filter paper and the gel are approximately the same.

11) And placing three layers of filter paper soaked with the membrane transferring liquid on the PVDF membrane, and paying attention to the air bubble removal.

12) And placing a second sponge cushion to enable the whole transfer printing interlayer to sequentially form a fiber cushion-filter paper-gel-NC membrane-filter paper-fiber cushion layer, closing the transfer printing clamp, placing the transfer printing clamp into a transfer groove, and filling the transfer printing groove with the transfer printing liquid.

13) And (5) turning on a power supply, stabilizing the current for 200mA, and performing 120 min.

14) After the transfer of the membrane was completed, the PVDF membrane was taken out and labeled, and the membrane was washed with TBST for 10min X3 times.

Immunoblotting

Blocking and antigen-antibody reaction

1) And (3) sealing: putting the PVDF membrane into an incubation box, adding a confining liquid containing 5% skimmed milk powder, and oscillating for 1.5-2 h by using a shaking table;

2) after the sealing is finished, washing the membrane for 10min multiplied by 3 times by TBST;

3) the membrane was placed in a solution containing Anti-diluent Anti-IL17A (1: 1000); Anti-IL6(1: 50); Anti-IL23(1: 2000); Anti-INF γ (1: 1000); Anti-ROR γ t (1: 2000); Anti-Foxp3(1: 2000); incubating the cells in an incubation box of GAPDH (1:1000) overnight at 4 ℃ with shaking;

4) taking out the mixture the next day, oscillating the mixture for 30min at room temperature, absorbing and discarding the primary antibody, and washing the mixture for 10min multiplied by 3 times by TBST;

5) diluting the secondary antibody with 5% skimmed milk powder confining liquid, and oscillating and reacting for 1-2 h in a table at room temperature;

6) after the secondary antibody reaction was completed, the secondary antibody was recovered. And washing the membrane with TBST for 5-10min multiplied by 3 times.

Sensitization, development and fixation by X-ray film in darkroom

1) Mixing the reagent A and the reagent B on the preservative film in equal volume; after 1min, the membrane protein surface is downward and fully contacted with the mixed solution; after 1min, the film was transferred to another plastic wrap, the residual liquid was removed, wrapped and placed in an X-ray film holder.

2) In a dark room, respectively putting 1 × developing solution and fixing solution into a plastic tray; taking out the X-ray film under a red light, and cutting the film into a proper size (the size is 1cm larger than the length and the width of the film) by using a paper cutter;

3) opening the X-ray film holder, placing the X-ray film on the film, once the X-ray film is placed on the film, the X-ray film holder cannot be moved, closing the X-ray film holder, and starting timing; properly adjusting the exposure time according to the intensity of the signal, generally 1min or 5min, and optionally pressing for multiple times at different times to achieve the best effect;

4) after exposure, opening the X-ray film clamp, taking out the X-ray film, quickly immersing the X-ray film in a developing solution for development, and stopping development immediately after an obvious strip appears. The developing time is generally 1-2 min (20-25 ℃), and when the temperature is too low (lower than 16 ℃), the developing time needs to be properly prolonged;

5) after the development is finished, immediately immersing the X-ray film into the fixing solution, wherein the fixing time is generally 5-10min, and the film is transparent; after washing off the residual fixer with tap water, the plate was dried at room temperature.

It should be noted that: when the film needs to be moved for development and fixation, one corner of the film is held as far as possible, and the fingernail does not scratch the film, otherwise, the result is influenced.

2.10 Effect of IL-23 on IL-17 and IFN-. gamma.secretion by peripheral blood mononuclear cells of HT rats (6 groups 3. multidot.2 indices. multidot.2 time points)

Before and after administration, peripheral blood from rats was collected and mononuclear cells from peripheral blood were isolated, and 1mL of each cell suspension (cell concentration about 2X 106/mL) was added to each of two cell culture dishes, and 5. mu.L of IL-23(50ng/mL) was added to 1 of the cell suspensions, and mixed well, and the other 1 was not added and clearly marked. 37 ℃ and 5% CO₂The culture was carried out in a sterile incubator for 72 hours. Collecting culture supernatant, and respectively filling the supernatant with IL-23 and without IL-23 into 2 sterile EP tubes, and freezing and storing at-20 ℃ for later use. And (3) detecting the contents of IL-17 and IFN-gamma of the culture supernatant by adopting an ELISA kit.

2.11 preparation of rat thyroid epithelial cell suspension

1. Thyroid epithelial tissue of normal SD rat was immediately washed with Hank's solution containing penicillin and streptomycin at 37 ℃ for 5 times to wash blood and other adherent substances.

2. Placing the tissue into a culture dish containing green and streptomycin PBS solution, removing fat connective tissue with ophthalmologic scissors, and separating thyroid epithelial tissue.

3. Placing the thyroid epithelial tissue into another sterile culture dish, washing with PBS containing double antibody for 3 times, transferring into a sterile bottle containing double antibody solution, cutting the thyroid epithelial tissue into fragments with the size of 1-2mm, and soaking the samples in PBS in the whole operation process.

4. The minced tissue pieces were filtered through a sterile 70 μm cell sieve and repeatedly rinsed with PBS to minimize the contamination of blood cells and small fat cells.

5. Collecting small pieces of tissue, placing into a sterile culture dish, adding 0.2% collagenase II to completely cover the cut synovial membrane tissue, placing in a 37-degree cell culture box, incubating for 2.5h, adding 0.25% trypsin, and incubating for 0.5 h.

6. The digestion was stopped by adding medium containing 10% FBS, and the digested small tissue pieces were filtered through a 70um cell sieve, and the small tissue pieces were repeatedly rinsed with PBS. Cells attached to the filter and dish bottom were washed with PBS and the filtrate was transferred to a 10ml sterile centrifuge tube. Centrifuging at 1000rpm, discarding the supernatant, and taking the precipitate. (can use PBS heavy suspension centrifugation 3 times, try to reduce the pancreatic enzyme concentration)

7. Resuspended in 1640 containing 10% FBS, counted and plated in culture flasks. Medium change is carried out for 3-4 days.

8. The purification was performed by limiting dilution monoclonal culture.

9. Cells were divided into 5 groups: respectively, normal control group, model control group, herba Epimedii total flavone extract low dose, herba Epimedii total flavone extract medium dose, and herba Epimedii total flavone extract high dose group. Viable cells were counted using trypan blue solution.

2.12 model establishment of injury of rat thyroid epithelial cells caused by Dextran Sodium Sulfate (DSS)

After the thyroid epithelial cells of rats are cultured to a logarithmic growth phase, the solutions are fully changed, and Dextran Sodium Sulfate (DSS) with different concentrations of 1 mug/ml, 2 mug/ml, 5 mug/ml, 10 mug/ml and 15 mug/ml are respectively added for 12-72h (12, 24, 36 and 72 h). The culture was continued for 24h after the total liquid change. The growth of epithelial cells was measured by CCK-8 colorimetry to determine the optimal concentration of Dextran Sodium Sulfate (DSS) fluid damage and the optimal time of administration. Cell viability ═ 100% (dosed cells OD-blank OD/control cells-blank OD).

2.13 rat thyroid epithelial cell proliferation and toxicity detection

The influence of the epimedium total flavone extract drug-containing serum on the proliferation of thyroid epithelial cells of normal rats is detected by adopting a CCK-8 colorimetric method, and the influence of the epimedium total flavone extract drug-containing serum on the toxicity of thyroid epithelial cells of the rats is detected by adopting an LDH detection kit.

2.14 drug treatment and cell proliferation assays

Taking the thyroid epithelial cells of a rat in logarithmic growth phase,

adding herba Epimedii total flavone extract containing drug serum with optimal concentration into the first group, incubating for optimal action time, and adding DSS solution with optimal concentration, incubating for optimal time;

the second group is added with 10% normal fetal calf serum, and then added with DSS solution with the optimal concentration for incubation for the optimal time (determined according to the experimental result);

the third group of cells was cultured normally and was not treated. CCK-8 is adopted to detect the proliferation condition of the cells.

2.15 Annexin V-FITC/PI method for detecting influence of drug-containing serum on apoptosis

Adding medicated serum into cells of each group (normal control group, model control group, herba Epimedii total flavone extract low dose group, herba Epimedii total flavone extract middle dose group, herba Epimedii total flavone extract high dose group and levothyroxine sodium group), culturing for 48h, centrifuging to collect suspension cells, centrifuging at 2000rpm for 5min, and discarding culture medium (the digestion time of adherent cells with pancreatin is not too long, about lmin, to prevent false positive). Cells were washed twice with cold PBS (2000rpm, centrifuged for 5min to collect cells) and the experiment was performed using the Annexin-V FITC/PI double staining kit. 5 μ Lannexin-V FITC staining solution and 10 μ L PI staining solution were added to the cell suspension (cell concentration was about 1X 106/mL), mixed gently, incubated at 4 ℃ in the dark for 30min, and apoptosis was detected by flow cytometry at L h.

3 results and analysis

3.1 HE staining for thyroid tissue changes

As can be seen from fig. 1 and 2, the normal group: the thyroid gland structure of a normal rat is complete, thyroid follicular cells (TEC) are arranged in order, the size of the thyroid follicular cells is uniform and uniform, the size of the thyroid follicular cells is circular or elliptical, the thyroid follicular cells are rich in colloid in a follicular cavity, the thyroid follicular cells are dyed in light red, and the thyroid follicular cells and the thyroid cells are not infiltrated by lymphocytes and plasma cells; model group: compared with a normal group, the thyroid follicular structure of the mouse is seriously damaged, part of the thyroid follicular structure is atrophied or disappears, the atrophied follicular form is incomplete, the colloid in the cavity is rare, the staining is light red, the periphery of the follicular is obviously infiltrated by lymphocytes, and the inside of the follicular is infiltrated by plasma cells; high dose group of epimedium total flavone extract: most of thyroid structures of rats are complete, follicular forms are basically regular, sizes of the follicles are consistent, occasionally lymphocyte infiltration occurs among TECs, plasma cells do not occur in the follicles, and the content of colloids is slightly less; the traditional Chinese medicine composition comprises the following components in percentage by weight: the thyroid structure of a rat is complete, the shape of the follicular is basically regular, the size of the follicular is inconsistent, partial lymphocytes and plasma cells are arranged in the follicular and among the TECs, few patients are damaged, and the content of colloid is low; low dose group of total flavonoids of epimedium: the thyroid structure of a rat is damaged, thyroid follicles are different in size, the colloid content is low, the nucleus staining is deep, and lymphocytes and plasma cells are infiltrated in the thyroid follicles and among TECs; a positive drug group: the thyroid structure of the rat is complete, the shape of the follicular is basically regular and is inconsistent in size, occasionally lymphocyte infiltration among TECs is observed, plasma cells are not observed in the follicular, the content of colloid is slightly less, and lymphocyte and plasma cell infiltration are not observed.

3.2 ELISA testing of rat serum levels of FT3, FT4, TOP-Ab, ATGA, T3, T4, IFN-. gamma., IL-17, IL-6, IL-10, IL-23 and TGF-. beta.

Table 1 effect of total flavonoids of epimedium herb extract on the content of FT4, ATGA, T3, T4, FT3 and FT4 in serum of HT rats (x ± s, n ═ 6)

Note that, compared with the normal group,^▲P<0.05,^▲▲P<0.01; comparison with model groups:^★P＜0.05，^★★P＜0.01

as is clear from Table 1 and FIG. 3, after the model group was modeled, the serum contents of ATGA, TPO-Ab and T3 were significantly increased, and the contents of indexes T4, FT3 and FT4 were significantly decreased, and they were significantly different (P <0.05 or P <0.01) from those of the normal group. After administration for two weeks, indexes ATGA, TPO-Ab and T3 are reduced to different degrees compared with model groups, wherein the high-dose group of epimedium total flavone extract and the treatment group of levothyroxine sodium tablet are obviously reduced, and the significant difference is shown (P <0.05 or P < 0.01). After the administration, the indexes T4, FT3 and FT4 are increased compared with the model group in each administration group, wherein the increase degree of the epimedium total flavone extract high-dose group and the levothyroxine sodium tablet treatment group is the largest, and the indexes have significant difference (P < 0.05).

After the epimedium flavone and levothyroxine sodium tablets are administrated for two weeks, the reduction degree of ATGA, TPO-Ab and T3 in the serum of rats is sequentially low, medium and high dose groups of epimedium total flavone extracts and a levothyroxine sodium tablet treatment group. The rising degree of T4, FT3 and FT4 in rat serum is low, medium and high dosage group of herba Epimedii total flavone extract, and levothyroxine sodium tablet treatment group.

TABLE 2 influence of Epimedium herb Total Flavonoids extract on the levels of IFN-. gamma.IL-17, IL-6, IL-10, IL-23 and TGF-. beta.in HT rat serum (x. + -. s, n ═ 6)

as can be seen from Table 2 and FIG. 4, the contents of IFN-. gamma.IL-17, IL-6, IL-23 and TGF-. beta.in the serum of the model group were significantly increased, and the content of IL-10 was significantly decreased, showing significant difference (P <0.05), as compared with the normal group. After each administration group is administrated for two weeks, indexes IFN-gamma, IL-17, IL-6, IL-23 and TGF-beta are reduced to different degrees compared with model groups, wherein the high-dose group of epimedium total flavone extract is obviously reduced, and has significant difference (P is less than 0.01). Index IL-10 after administration, each administration group was increased compared with the model group, wherein the increase degree of the epimedium total flavone extract high dose group and the levothyroxine sodium tablet treatment group was the largest, and the significant difference was found (P < 0.05).

3.3 Effect of Epimedium total flavone extract on Treg and Th17 cells in HT rat peripheral blood

As can be seen from table 3 and fig. 5, the proportion of Treg cells in the model group was significantly decreased compared to the blank control group, and there was statistical significance (P < 0.01). The proportion of Treg cells of each dose group of the epimedium total flavone extract and the levothyroxine sodium tablet positive drug group is obviously increased, and the Treg cells have statistical significance (P is less than 0.01 or P is less than 0.05) compared with a model group, and the proportion of the Treg cells is increased along with the increase of the administration dose of the epimedium total flavone extract.

As can be seen from Table 3 and FIG. 6, the proportion of Th17 cells was significantly increased in the model group compared to the blank control group (P < 0.01). The proportion of Th17 cells in the peripheral blood of rats in various dose groups of epimedium total flavone extracts and levothyroxine sodium tablet positive medicines is remarkably reduced compared with that in a model group (P is less than 0.01 or P is less than 0.05), and the proportion of Th17 cells is reduced along with the increase of the administration dose of the epimedium total flavone extracts.

TABLE 3 proportion of Treg and Th17 cells in peripheral blood of rats in each group

Note: compared with the blank control group, the composition of the composition,^▲▲p is less than 0.01; comparing with model group, P <0.05, P <0.01

3.4 immunohistochemical Observation of FoxP3, ROR gamma t, IL-17, IL-23, IL-6, IL-10 and IFN-gamma expression in the thyroid gland of rat in Hashimoto's thyroiditis model

From the immunohistochemistry results shown in tables 4-10 and FIGS. 7-20, the thyroid tissues of rats in each group showed positive expression of FoxP3, ROR γ t, IL-17, IL-23, IL-6, IL-10 and IFN- γ. Compared with the normal group, the ROR gamma t, IL-17, IL-23, IL-6 and IFN-gamma positive expression of the model group rats is obviously increased (P < 0.01). After administration, compared with the model group, the rats ROR gamma t, IL-17, IL-23, IL-6 and IFN-gamma positive expression, positive staining area and accumulated optical density value of each administration group are obviously reduced, and the reduction degree is from high to low, and the rats are a levo-thyroxine sodium tablet positive medicine group, an epimedium flavone high dose group, an epimedium flavone medium dose group and an epimedium flavone low dose group in sequence.

FoxP3 and IL-10 index showed significantly lower positive expression in the model group (P <0.01) than in the normal group, and significantly increased expression in each administration group after administration.

TABLE 4 Effect of Epimedium flavonoids on FoxP3 expression in thyroid tissue of Hashimoto's thyroiditis rats (for example, Epimedium flavonoids)

n＝6)

Note: compared with the normal control group, the composition has the advantages that,^▲：P<0.05，^▲▲：P<0.01; compared with the model control group,^★：P<0.05，^★★：P<0.01。

TABLE 5 Effect of Epimedium flavonoids on ROR γ t expression in thyroid tissue of Hashimoto thyroiditis rats (R)

n＝6)

TABLE 6 Effect of Epimedium flavonoids on IL-17 expression in thyroid tissue of Hashimoto's thyroiditis rats ((

n＝6)

TABLE 7 Effect of Epimedium flavonoids on IL-23 expression in thyroid tissue of Hashimoto's thyroiditis rats(

n＝6)

TABLE 8 Effect of Epimedium flavonoids on IL-6 expression in thyroid tissue of Hashimoto's thyroiditis rats (II)

n＝6)

TABLE 9 Effect of Epimedium flavonoids on IL-10 expression in thyroid tissue of Hashimoto's thyroiditis rats ((

n＝6)

TABLE 10 Effect of Epimedium flavonoids on IFN- γ expression in thyroid tissue of Hashimoto thyroiditis rats (S)

n＝6)

3.6 qRT-PCR detection of relative expression levels of FoxP3, ROR gamma t, TGF-beta 1, IL-17, IL-23, IL-6, IL-10 and IFN-gamma genes in rat thyroid

As can be seen from FIG. 21 and Table 11, the expression levels of the genes ROR γ t, IL-17, IL-23, IL-6 and IFN- γ were significantly increased and the expression levels of IL10, TGF- β and Foxp3 were significantly decreased in the model group (P <0.01) compared to the control group; compared with the model group, the low-dose group of the epimedium total flavone extract, the medium-dose group of the epimedium total flavone extract, the high-dose group of the epimedium total flavone extract and the positive medicine group ROR gamma t, IL-17, IL-23, IL-6 and IFN-gamma gene expression level are gradually reduced, and the expression level of IL10, TGF-beta and Foxp3 genes is gradually increased (P <0.01 or P <0.05), so that the dosage dependence is presented.

TABLE 11 statistical table of relative expression of thyroid-associated genes in rats

Note:^▲▲indicates a comparison with the normal control group of p<0.01；^★Comparison of representation to model set p<0.05；^★★Comparison of representation to model set p<0.01

3.7 WB detection of FoxP3, ROR gamma t, TGF-beta 1, IL-17, IL-23, IL-6, IL-10 and IFN-gamma protein expression in rat thyroid

As can be seen from fig. 22, fig. 23 and table 12, the protein expression levels of ROR γ t, IL-17, IL-23, IL-6 and IFN- γ were significantly increased and the protein expression levels of IL10, TGF- β and Foxp3 were significantly decreased in the model group (P <0.01) compared to the control group; compared with the model group, the low-dose group of the epimedium total flavone extract, the medium-dose group of the epimedium total flavone extract, the high-dose group of the epimedium total flavone extract and the positive medicine group ROR gamma t, IL-17, IL-23, IL-6 and IFN-gamma are gradually reduced in protein expression level, and IL10, TGF-beta and Foxp3 are gradually increased in protein expression level (P <0.01 or P <0.05) to show dose dependence.

TABLE 12 statistical table of relative expression of thyroid-associated proteins in rats

3.8 Effect of IL-23 on IL-17 and IFN-. gamma.secretion by peripheral blood mononuclear cells of HT rats

As can be seen from tables 13 and 14, before administration, the levels of IL-17 and IFN-gamma expression of the model group and each administration group which are not stimulated by IL-23 are obviously increased compared with the blank control group, and the statistical significance exists (P is less than 0.01); after IL-23 stimulation, the IL-17 and IFN-gamma expression levels of the model group and each administration group are obviously increased compared with those of a blank control group, and the statistical significance exists (P is less than 0.01). After IL-23 stimulation, the level of IL-17 and IFN-gamma expression was higher in each group than in the same group of cells not stimulated by IL-23.

After administration, compared with a blank control group, the model group without IL-23 stimulation has obviously increased IL-17 and IFN-gamma expression levels and has statistical significance (P <0.01), compared with the model group, each administration group has obviously reduced IL-17 and IFN-gamma expression levels, the levothyroxine sodium group has the lowest expression, and the IL-17 and IFN-gamma expression levels gradually reduce with the increase of the administration concentration of the serum containing the medicine, and both the groups have statistical significance (P <0.01 or P < 0.05). After IL-23 stimulation, compared with the model group, the expression levels of IL-17 and IFN-gamma are obviously reduced on average, the expression of the levothyroxine sodium positive medicine group is the lowest, and the expression levels of IL-17 and IFN-gamma are gradually reduced along with the increase of the administration concentration of the serum containing the medicine, and both have statistical significance (P is less than 0.01 or P is less than 0.05).

TABLE 13 Effect of IL-23 stimulation on IL-17 and IFN-y expression levels in peripheral blood mononuclear cells in rats of various groups before administration: (

pg/ml，n＝3)

TABLE 14 Effect of IL-23 stimulation on the expression levels of IL-17 and IFN-y in peripheral blood mononuclear cells after administration to groups of rats: (

pg/ml，n＝3)

Note: compared with the blank control group, the composition of the composition,^▲P＜0.05，^▲▲p is less than 0.01; comparing with model group, P <0.05, P <0.01

3.9 rat thyroid epithelial Primary cell preparation

Rat thyroid tissue was digested and purified to obtain paving stone-like irregularly adherent cells, as shown in fig. 24. The cell survival rate after trypan blue staining was (97.43 + -5.62%).

3.10 model establishment of rat thyroid epithelial cell injury caused by dextran sodium sulfate

Compared with a blank control group, after rat thyroid epithelial cells are induced by DSS with different concentrations and different action time, OD values of the cells of each administration group are reduced. As can be seen from Table 15, the DSS showed statistical differences (p <0.05, p <0.01) at concentrations greater than 15. mu.g/ml and at effects above 24h, with the 20. mu.g/ml DSS showing the most significant differences (p <0.01) at 24 h.

TABLE 15 OD values of DSS at different concentrations on rat thyroid cells at different times (S-D)

n＝4)

Note: compared with the blank control group, the composition of the composition,^▲P＜0.05，^▲▲P＜0.01

3.11 rat thyroid epithelial cell proliferation and toxicity detection

After the epimedium total flavone extract containing serum groups with different concentrations are applied to cells, the OD value of the cells of each administration group is slightly reduced compared with that of a blank control group, but the OD value is not statistically different (P is more than 0.05). Compared with the blank control group, the LDH activity of each group of cells is increased along with the increase of the administration concentration, wherein the epimedium total flavone extract high-dose group is remarkably increased and has statistical difference (P < 0.05). There was no statistical difference between the groups administered with the total flavonoids of epimedium (P > 0.05).

TABLE 16 proliferative and toxic Effect of different concentrations of drug-containing serum on rat thyroid cells: (

n＝4)

Note: compared with the blank control group, the composition of the composition,^▲P＜0.05，^▲▲p is less than 0.01 and P is less than 0.05 and 0.01 compared with the low dose group of epimedium total flavone extract.

3.13 drug treatment and cell proliferation assay

Compared with a blank control group, the OD value of the model group cells is obviously reduced, and the statistical difference (P <0.01) exists; compared with the model group, the cell OD value of the epimedium total flavone extract group is obviously increased, and the statistical difference is realized (P is less than 0.05).

TABLE 17 Effect of drug-containing serum on rat thyroid cell Activity: (

n＝4)

3.14 Effect of drug sera on apoptosis

As can be seen from Table-18 and FIG. 25, the apoptosis rate of the model group was significantly increased compared to that of the blank control group, and there was a statistical significance (P < 0.01). The apoptosis rate ratio of each dose group of epimedium flavone and the levo-thyroxine sodium tablet positive drug group is obviously reduced, and compared with a model group, the doses of epimedium flavone have statistical significance (P is less than 0.01 or P is less than 0.05), and the apoptosis rate is reduced along with the increase of the administration dose of the epimedium flavone.

Table 18 effect of drug sera on apoptosis (%,

n＝3)

In conclusion, the epimedium total flavone extract has obvious effect of treating hashimoto thyroiditis on the rats with hashimoto thyroiditis. Animal experiment results prove that the epimedium total flavone extract has good safety and good curative effect, can effectively treat hashimoto thyroiditis, has good effect of improving clinical symptoms, has small toxic and side effects, and has good clinical application prospect.

Example 2 Epimedium total flavone extract tablet or other health products

The herba Epimedii total flavone extract tablet comprises

Composition (I)	The dosage of each tablet
		Epimedium total flavone extract	100mg
Microcrystalline cellulose	37.5mg
		Silica gel micropowder	10.5mg
Stearic acid	17.5mg
		Lactose	10.5mg
Magnesium stearate	1.05mg

Adding the epimedium total flavone extract (prepared by the method of example 1) into microcrystalline cellulose, aerosil, stearic acid and lactose according to the prescription amount, uniformly mixing, preparing granules, adding magnesium stearate, uniformly mixing and tabletting to obtain the compound preparation (each tablet contains 80mg of epimedium total flavone). The dosage for adult is 2-4 tablets/time, and 2-3 times daily.

Example 3 Epimedium herb Total Flavonoids extract Capsule or other health products

The epimedium total flavone extract capsule comprises the following components

Composition (I)	The dosage of each tablet
		Epimedium total flavone extract	100mg
Microcrystalline cellulose	37.45mg
		Silica gel micropowder	10.5mg
Stearic acid	17.5mg
		Lactose	10.5mg

The epimedium total flavone extract (prepared by the method of example 1) is added with microcrystalline cellulose, aerosil, stearic acid and lactose according to the prescription amount, evenly mixed, prepared into granules, filled into a No. 0 hard capsule shell and polished to obtain the compound preparation (each capsule contains 80mg of epimedium total flavone). The dosage for adult is recommended to be 2-4 granules/time, and 2-3 times daily.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.

Sequence listing

<110> Zhejiang province people hospital

Application of epimedium total flavone in preparing medicine or health product for preventing and treating hashimoto thyroiditis

<130> W-HLY19-031

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence (Rat IL6-F)

<400> 1

ttccagccag ttgccttctt 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (Rat IL6-R)

<400> 2

ctggtctgtt gtgggtggta 20

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence (Rat IL17-F)

<400> 3

gtgaaggcag cggtactcat 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (Rat IL17-R)

<400> 4

gggtgaagtg gaacggttga 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Rat IL23-F)

<400> 5

caaaggatcc gccaaggtct 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Rat IL23-R)

<400> 6

tggaacggag aagagaacgc 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence (Rat INF-gamma-F)

<400> 7

ggcaaaagga cggtaacacg 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (Rat INF-gamma-R)

<400> 8

tctgtgggtt gttcacctcg 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (Rat IL10-F)

<400> 9

aagggttact tgggttgcca 20

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence (Rat IL10-R)

<400> 10

tggccttgta gacacctttg t 21

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Rat ROR gamma t-F)

<400> 11

cctcctgcca ccttgagtat 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence (Rat ROR. gamma.t-R)

<400> 12

tctgagccct gttctggttc 20

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence (Rat TGF-beta-F)

<400> 13

ctgctgaccc ccactgatac 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence (Rat TGF-beta-R)

<400> 14

agccctgtat tccgtctcct 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (Rat Foxp3-F)

<400> 15

gcccacacct cctcttcttc 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence (Rat Foxp3-R)

<400> 16

ctgtcctgga gaagtgcctg 20

<210> 17

<211> 22

<212> DNA

<213> Artificial sequence (Rat beta-actin-F)

<400> 17

cagggtgtga tggtgggtat gg 22

<210> 18

<211> 22

<212> DNA

<213> Artificial sequence (Rat beta-actin-R)

<400> 18

agttggtgac aatgccgtgt tc 22

Claims

1. The application of epimedium total flavonoids in preparing the medicine for treating hashimoto thyroiditis is characterized in that: the preparation method of the epimedium total flavonoids comprises the following steps:

(1) roasting epimedium: taking herba Epimedii raw medicinal materials, removing impurities, picking up leaves, spraying clear water, slightly moistening, shredding, and drying; heating refined adeps Caprae Seu Ovis for melting, adding herba Epimedii shreds, parching with slow fire until the surface shows uniform oily luster and slight yellow color, taking out, and cooling; 20kg of refined mutton tallow oil is used for every 100kg of epimedium;

(2) preparing a total flavone extract: taking a proper amount of roasted epimedium, respectively adding 70% ethanol in an amount which is 10 times the amount of the roasted epimedium, and extracting for 2 times, wherein each time is 2 hours; the liquid medicine is concentrated to 1mL liquid medicine containing 1g crude drug, and the technology of purifying the total icariin by adopting HPD300 macroporous resin is determined by investigation as follows: diluting the medicinal liquid with water to obtain medicinal liquid containing 0.1g crude drug per 1mL, passing through HPD300 macroporous adsorbent resin column at flow rate of 4BV/h, washing with 5 times of column volume of water, eluting with 7 times of column volume of 50% ethanol at flow rate of 4BV/h, collecting eluate, recovering ethanol under reduced pressure, filtering, adding 3% gelatin solution into the filtrate until no precipitate is generated, adding ethanol to make ethanol content reach 80%, standing overnight, filtering, recovering ethanol under reduced pressure to obtain herba Epimedii total flavone extract.

2. Use according to claim 1, characterized in that: the medicine for treating hashimoto thyroiditis is a preparation prepared by taking epimedium total flavonoids as active ingredients and adding pharmaceutically acceptable auxiliary materials.