CN114949087B

CN114949087B - Traditional Chinese medicine composition with protection effect on thyroid follicular cells damaged by TPOAb and application thereof

Info

Publication number: CN114949087B
Application number: CN202210503055.9A
Authority: CN
Inventors: 司富春; 司高; 宋雪杰; 司季青
Original assignee: Henan University of Traditional Chinese Medicine HUTCM
Current assignee: Henan University of Traditional Chinese Medicine HUTCM
Priority date: 2022-05-10
Filing date: 2022-05-10
Publication date: 2023-04-21
Anticipated expiration: 2042-05-10
Also published as: CN114949087A

Abstract

The invention relates to the field of traditional Chinese medicines, and in particular belongs to the field of prevention and treatment of thyroiditis diseases. The invention discloses a traditional Chinese medicine composition with a protective effect on TPOAb damaged thyroid follicular cells, which comprises immature bitter orange, green tangerine peel, almond and rhizoma alismatis. The invention also provides application of the traditional Chinese medicine composition in preparation of a medicament for treating hashimoto thyroiditis. According to the application, 4 medicinal materials of immature bitter orange, green tangerine orange peel, almond and alismatis rhizome with higher protection effect on a TPOAb damaged thyroid follicular cell model are formed into a novel traditional Chinese medicine prescription capable of effectively treating HT according to a specific proportion, and clinical observation shows that the developed effective prescription has a curative effect of obviously reducing the thyroid autoantibody level of an HT patient, so that the novel prescription is provided for treating HT.

Description

Traditional Chinese medicine composition with protection effect on thyroid follicular cells damaged by TPOAb and application thereof

Technical Field

The invention relates to the field of traditional Chinese medicines, in particular to the field of prevention and treatment of thyroiditis diseases.

Background

Hashimoto thyroiditis (Hashimoto thyroiditis, HT), also known as hashimoto's disease or chronic lymphocytic thyroiditis, is characterized by massive lymphocyte infiltration in thyroid tissue and the presence of high titres of thyroid autoantibodies in serum, and is the most common autoimmune disease at present. Serum detection of thyroid peroxidase antibodies (TPOAbs) and thyroglobulin antibodies (TgAbs) with high titer levels is the primary basis for clinical diagnosis of HT. The pathogenesis of HT has not been fully elucidated so far, and is mainly thought to be caused by the destruction of immunological tolerance due to genetic, environmental and other factors. As autoimmune diseases, this destruction is mainly due to destruction of thyroid follicular cells by a series of cell and antibody mediated immune processes, and antibody-dependent cell-mediated cytotoxicity (ADCC) is considered to be an important cellular damage mechanism for HT. TPO is a hemoglobin-like protein synthesized by thyroid follicular cells and containing 10% saccharification, is an important enzyme for catalyzing thyroid hormone, and is closely related to thyroid tissue immune injury. TPOAb can directly fight TPO and cause thyroid tissue or cell injury through ADCC (advanced cellular cytotoxicity) pathway, so that hypothyroidism is related to autoimmunity and is caused by insufficient secretion of thyroid hormone, and thus later-stage hypothyroidism often occurs in HT patients.

The current incidence of HT increases rapidly year by year, but lacks effective therapeutic agents. Currently, thyroid hormone replacement therapy, glucocorticoid immunoregulation therapy, trace element selenium auxiliary therapy and the like are mostly adopted by Western medicine for treating the disease, but the treatment methods are also radical treatment according to symptoms rather than pathogenesis of HT, the curative effect is limited, the disease is easy to relapse, and particularly, available effective medicines for early asymptomatic treatment are lack. There is therefore an urgent need to find new therapeutic methods, medicaments to prevent and treat HT. In recent years, reports on treatment of HT by traditional Chinese medicine are obviously increased, and the traditional Chinese medicine has remarkable curative effects on improving symptoms and reducing thyroid autoantibodies, but traditional Chinese medicine treatment prescription drugs for HT are limited to self-fitting prescription and empirical prescription, and further scientific research is difficult to develop, so that the prescription drugs with direct protection effect on thyroid follicular cells damaged by TPOAb are developed, and the important technical problems to be solved by the technicians in the field are provided for effective prescription drugs for early treatment of HT.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine composition with a protective effect on TPOAb damaged thyroid follicular cells and application thereof in treatment of hashimoto thyroiditis.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a traditional Chinese medicine composition with a protective effect on thyroid follicular cells damaged by TPOAb, which comprises the following components in percentage by mass of 1-3: 1:1 to 3: fructus Aurantii Immaturus, pericarpium Citri Reticulatae viride, semen Armeniacae amarum, and Alismatis rhizoma.

Preferably, the dosage form of the traditional Chinese medicine composition comprises one of decoction, granules, powder, pills and capsules.

The invention also provides application of the traditional Chinese medicine composition in preparation of a medicine for protecting thyroid follicular cells damaged by TPOAb.

The invention also provides application of the traditional Chinese medicine composition in preparation of a medicament for treating hashimoto thyroiditis.

By adopting the technical scheme, the invention has the following beneficial effects:

the invention starts from an ADCC mechanism of hashimoto thyroiditis, establishes an in vitro TPOAb damaged thyroid follicular cell model, and screens out 153 prescriptions (205 prescriptions in the book of the golden starvation, 48 prescriptions in the repeated prescriptions of the golden starvation) carried by the classic medical and miscellaneous diseases part of the traditional Chinese medicine 'typhoid' by using techniques such as cell co-culture, MTT method, flow cytometry, immunofluorescence labeling, enzyme-linked immunosorbent assay (ELISA), western Blot (Western Blot) and the like from the traditional Chinese medicine's Shengzhang's scene (153 prescriptions in the book of the golden starvation) without drug listed in the repeated prescriptions of the golden reissues, 153 prescriptions in total) and 128 single medicines (128 prescriptions in the book of the golden starvation originally related to 155 traditional Chinese medicines, 128 medicines after special medicines and shell medicines are removed), so as to form a new traditional Chinese medicine prescription medicine capable of effectively treating HT, thereby reducing the level of antibodies of the thyroid by the HT, and the invention has obvious curative effect on patients.

Drawings

FIG. 1 is a graph showing the quantitative relationship between the damage rate of Nthy-ori3-1 cells at various concentrations of TPOAb;

FIG. 2 is a graph showing the time dependence of TPOAb on the damage rate of the Nthy-ori3-1 cell model;

FIG. 3 shows changes in cell morphology before and after TPOAb injury to thyroid follicular cells;

FIG. 4 shows the flow-through double-staining method for detecting the apoptosis rate of TPOAb on cell models;

FIG. 5 shows the immunofluorescence detection of TPOAb binding to the Nthy-ori3-1 cell model;

FIG. 6 is T4 secretion levels of TPOAb damaged thyroid follicular cell model;

FIG. 7 shows Western Blot analysis of TPO protein expression from a cell model group;

FIG. 8 dose-response relationship of protection effect of immature bitter orange on TPOAb injury Nthy-ori3-1 cell model;

FIG. 9 dose-response relationship of green tangerine peel to the protective effect of TPOAb injury Nthy-ori3-1 cell model;

FIG. 10 dose-response relationship of almond to the protective effect of TPOAb injury Nthy-ori3-1 cell model;

FIG. 11 dose-response relationship of alisma to the protective effect of TPOAb damaged Nthy-ori3-1 cell model;

FIG. 12 is a dose-to-effect relationship of qi regulating and phlegm resolving formulation on the protective effect of TPOAb injury Nthy-ori3-1 cell model;

FIG. 13 aging relationship of protection effect of immature bitter orange on TPOAb injury Nthy-ori3-1 cell model;

FIG. 14 aging relationship of green tangerine peel to the protective effect of TPOAb injury Nthy-ori3-1 cell model;

FIG. 15 time dependence of almond on the protective effect of TPOAb injury Nthy-ori3-1 cell model;

FIG. 16 aging relationship of Alismatis rhizoma to the protective effect of TPOAb damaged Nthy-ori3-1 cell model;

FIG. 17 shows the aging relationship of the qi-flowing and phlegm-resolving prescription on the protection effect of TPOAb injury Nthy-ori3-1 cell model.

Detailed Description

The invention provides a traditional Chinese medicine composition with a protective effect on thyroid follicular cells damaged by TPOAb.

In the invention, the traditional Chinese medicine composition comprises the following components in percentage by mass of 1-3: 1:1 to 3: fructus Aurantii Immaturus, pericarpium Citri Reticulatae viride, semen Armeniacae amarum, and Alismatis rhizoma.

In the invention, the dosage form of the traditional Chinese medicine composition comprises one of decoction, granules, powder, pills and capsules.

In the invention, the preparation method of the traditional Chinese medicine composition comprises the following steps:

1) Mixing fructus Aurantii Immaturus, pericarpium Citri Reticulatae viride, semen Armeniacae amarum, and Alismatis rhizoma with ethanol water solution, soaking, extracting, and filtering to obtain fructus Aurantii Immaturus extractive solution, pericarpium Citri Reticulatae viride extractive solution, semen Armeniacae amarum extractive solution, and Alismatis rhizoma extractive solution;

2) The optimal concentration of 4 extracts of immature bitter orange, green tangerine peel, almond and alisma orientale is prepared according to a multiple of 0.01-2.

In the invention, the mass volume ratio of the mixture of the immature bitter orange and the ethanol water solution is 1-1.5 g: 10-15 ml, preferably 1g: 11-14 ml; the soaking and extracting time is 5-8 days, the soaking and extracting are preferably carried out in a sealing state, and the sealing is realized through sealing by a preservative film; the filtration is preferably a negative pressure filtration. After the immature bitter orange filtrate is obtained, the invention preferably further comprises the steps of evaporation, drying, dissolution, filtration and split charging.

In the invention, the mass volume ratio of the green tangerine peel to the ethanol aqueous solution is 1-1.5 g: 10-15 ml, preferably 1g: 11-14 ml; the soaking and extracting time is 5-8 days, the soaking and extracting are preferably carried out in a sealing state, and the sealing is realized through sealing by a preservative film; the filtration is preferably a negative pressure filtration. After the green tangerine peel filtrate is obtained, the invention preferably further comprises the steps of evaporation, drying, dissolution, filtration and split charging.

In the invention, the mass volume ratio of the almond to the ethanol water solution is 1-1.5 g: 10-15 ml, preferably 1g: 11-14 ml; the soaking and extracting time is 5-8 days, the soaking and extracting are preferably carried out in a sealing state, and the sealing is realized through sealing by a preservative film; the filtration is preferably a negative pressure filtration. The invention preferably further comprises the steps of evaporating, drying, dissolving, filtering and sub-packaging after the almond filtrate is obtained.

In the invention, the mass volume ratio of the alisma to the ethanol aqueous solution is 1-1.5 g: 10-15 ml, preferably 1g: 11-14 ml; the soaking and extracting time is 5-8 days, the soaking and extracting are preferably carried out in a sealing state, and the sealing is realized through sealing by a preservative film; the filtration is preferably negative pressure filtration, and the invention also preferably comprises the steps of evaporation, drying, dissolution, filtration and split charging after the alisma filtrate is obtained.

In the invention, the optimal action concentration of the 4 extracts of the immature bitter orange, the green tangerine peel, the almond and the alisma is determined according to the dose-effect relationship of the 4 extracts on the TPOAb injury Nthy-ori3-1 cell model.

In the invention, the preparation is carried out according to the optimal action concentration of 4 extracts of immature bitter orange, green tangerine orange peel, almond and alisma orientale, and the preparation multiple is preferably 0.03-0.5, and more preferably 0.125-0.25.

The invention also provides application of the traditional Chinese medicine composition in preparing a medicine for protecting thyroid follicular cells damaged by TPOAb.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

TPOAb damaged thyroid follicular cell model establishment

1. Experimental method

1.1 cell model establishment

(1) Cell treatment: taking normal thyroid follicular epithelial cells Nthy-ori3-1 (available from Ji Ni European biotechnology Co., ltd. In Guangzhou) in logarithmic growth phase, sequentially performing digestion and centrifugation according to cell passage process to obtain single cell suspension, counting by counting plate, and collecting the single cell suspension with a concentration of 1×10 ⁴ The density of cells/well was seeded in 96 well cell culture plates at 200 μl/well. The 96-well plate inoculated with cells was placed in 5% CO ₂ After culturing in an incubator at 37℃and saturated humidity for 24 hours, the cells were used as target cells for model establishment.

(2) And (3) establishing a model: after 24 hours, the 96-well plate was removed, the upper cell culture solution was aspirated, 200. Mu.L/Kong Xi of PBS was added for 2 times to wash out the FBS remaining in the wells, a blank control group and a model group were set, 100. Mu.L/well of medium without FBS was added as the blank control group to the first three columns, 0.3125ng/mL, 0.625ng/mL, 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 15ng/mL, 20ng/mL of medium containing TPOAb was sequentially added as the model group to the last three columns, 100. Mu.L/well of human promyelocytic leukemia cells HL-60 (purchased from the center of cell resource of basic medical institute of China academy of medicine) were set as the model group, and the effective target ratio was carried out in a ratio of 12:1, and centrifugation was carried out at 1000rpm for 3 minutes in a centrifuge to allow the effector cells to be in full contact with the target cells, and to continue culturing in the incubator.

(3) Detection of injury rate of TPOAb injured thyroid follicular cell model: after 24 hours, the 96-well plate was removed and washed twice again with PBS at 200. Mu.L/well to wash out HL-60 cells in the well, and then the prepared MTT working solution was added at 100. Mu.L/well. After incubation for 4h in incubator, 150 μl/well of DMSO was added, and the mixture was placed on a shaker for 30min to make the DMSO color uniform, and absorbance (i.e. OD value) was measured at two wavelengths (570 nm,630 nm) of an enzyme-labeled instrument, and the cell damage rate (%) = (1-model group OD value/control group OD value) ×100%, thereby calculating the damage rate of the model at different antibody concentrations.

The experiment is repeated for three times, ageing detection and the quantitative effect detection of TPOAb under the action of different concentrations are carried out after the result is stable, and ageing and quantitative effect relation curves are drawn.

1.2 observation of model cell morphology changes with inverted microscope

Taking Nthy-ori3-1 cells in logarithmic growth phase according to 5×10 ⁴ Inoculating cells/hole density into a 24-hole plate, culturing in an incubator, sucking supernatant PBS for two times after 24 hours, adding 500 mu L/hole of RPMI 1640 culture medium into a blank group, adding 500 mu L/hole of antibody working solution with the concentration of TPOAb of IC50 prepared by the RPMI 1640 culture medium into a model group, adding 500 mu L/hole of HL-60 cells after 2 hours of action into the incubator, enabling the final target ratio to be 12:1, centrifuging at 1000rpm for 3 minutes, enabling effector cells to fully contact target cells, and transferring to the incubator for continuous culture. After 24h of culture, the cell supernatant was aspirated, the plate was washed twice with PBS, morphological changes of each group of cells were observed under a mirror, and cell morphology of each group of cells amplified to 200-fold was recorded by photographing respectively.

1.3 flow CFSE/PI double-staining method for detecting apoptosis rate of cell injury of TPOAb injured thyroid follicular cell model

Taking 1-5×10 of Nthy-ori3-1 cells in logarithmic growth phase ⁶ After CFSE staining pretreatment, RPMI 1640 medium was resuspended as single cell suspension, counted, and complete medium with 10% fbs was resuspended in 24-well plates, 5×10 ⁴ After 24h of culture, PBS is used for washing the plate twice, 1640 culture medium 500 mu L/hole is added into a blank group, 500 mu L/hole is added into a model group, the mixture is added into a 1640 culture medium containing TPOAb, after 2h of action on a cell culture box, 500 mu L/hole is added into HL-60 cells, the final effective target ratio is 12:1, and centrifugation is carried out at 1000rpm for 3min to ensure that effector cells and target cells are fully contacted, and the mixture is transferred into the culture box for continuous operationCulturing. After 24 hours, collecting suspended cells in each hole to a corresponding 15mL centrifuge tube of each group, washing each hole twice by PBS, collecting the cells to each corresponding tube, adding digestive juice without EDTA into each hole to digest target cells and collecting the cells to each corresponding centrifuge tube, pre-cooling by PBS, centrifugally washing twice, re-suspending by 500 mu L of PBS, adding 10 mu L of PI into each tube, staining for 5-10min at room temperature in a dark place, and detecting in a machine in 30 min. The FlowJo software performs the result analysis to calculate the ratio of pi+ cells in cfse+ cells.

1.4 immunofluorescence labeling technique positioning analysis of cellular model TPO antigen antibody binding

Taking 5×10 Nthy-ori3-1 cells in logarithmic growth phase ⁴ Inoculating to an immunofluorescence special cell culture dish, placing in an incubator for culturing for 24 hours, carefully sucking out culture solution, washing twice with PBS, dropwise adding a sufficient amount of 4% paraformaldehyde for fixing for 15 minutes at room temperature, washing 3 times with PBST, 3 minutes/time, dropwise adding normal rabbit serum, sealing for 30 minutes at room temperature, removing sealing liquid, respectively setting a blank group and a model group, dropwise adding a sufficient amount of PBS as a control, dropwise adding a sufficient amount of TPOAb as a primary antibody in the model group, and incubating overnight in an environment of 4 ℃. Taking out the PBST of the culture dish, immersing and washing for 3 times, dripping FITC-labeled rabbit anti-goat IgG antibody diluted by PBS, incubating for 1 hour at 37 ℃ in a dark place, immersing and washing by PBST after dripping DAPI, incubating for 5min in a dark place, immersing and washing for 4 times by PBST again, 5 min/time, preparing 90% glycerol sealing sheet by PBS, observing and photographing under a fluorescence microscope within 30min, and preventing fluorescence quenching.

1.5ELISA technique for detecting T4 secretion level of TPOAb damaged thyroid follicular cell model

Taking 5X 10 of Nthy-ori3-1 cells ⁴ After the cells are inoculated to a 24-hole plate and cultured for 24 hours in an incubator, the plates are washed twice by PBS, a blank group and a model group are arranged, after the cells are acted for 2 hours in the incubator, 500 mu L/hole of HL-60 cells are added, the final effect target ratio is 12:1, centrifugation is carried out at 1000rpm for 3 minutes to ensure that effector cells are fully contacted with target cells, the cells are transferred to the incubator for continuous culture for 24 hours, cell supernatants are collected to a sterile 15ml centrifuge tube, centrifugation is carried out at 2000rpm for 20 minutes, the supernatants are taken and split into 1.5ml EP tubes after centrifugation, and the products are preserved at the temperature of minus 80 ℃ for standby.

The preparation of the reagent used for detection is carried out according to the operation requirement of ELISA kit, the kit is balanced for half an hour at room temperature before the experiment, and the washing liquid, biotin antigen working liquid and avidin-HRP working liquid required by the experiment are diluted and prepared according to the requirement. Taking out the enzyme-labeled coated plate, adding 50 mu L/hole of standard diluent into blank holes, adding 50 mu L/hole of prepared standard with each concentration into standard holes, adding 50 mu L/hole of each sample into sample holes, arranging 3 compound holes in each group, placing a sealing plate membrane and sealing plate in an incubator for incubation for 30min, adding 50 mu L/hole of avidin-HRP working solution into the plates except blank holes after washing the plates, washing for 5 times in the same step after incubation for 30min, sequentially adding 50 mu L/hole of color developer A, B after completion, gently shaking and uniformly mixing, and developing for 10min at 37 ℃ in a dark place. Finally, 50. Mu.L of stop solution per well is added, and the absorbance (OD value) of each well is detected at the wavelength of 450 by an enzyme label instrument within 10min. The measurement results were calculated analytically using logistic curves (four parameters) in elidacalc software.

1.6WesternBlot method for detecting TPO expression level in cell model

And (3) after the cells are treated with 1.2, adding effector cells for culturing for 24 hours, taking out a culture dish, washing 3 times with PBS, placing on ice, adding 400 mu L/dish of prepared precooled RIPA lysate, carrying out ice bath pyrolysis for 30 minutes, shaking every 5 minutes to enable the lysate to fully cover the bottom of the dish, collecting each group of cell lysate to a precooled 1.5mL EP tube by using a cell scraper after the pyrolysis is finished, carrying out low-temperature high-speed centrifugation at 15000rpm for 30 minutes at 4 ℃, slowly sucking the supernatant after centrifugation, namely, packaging the extracted protein into the precooled 1.5mL EP tube, carrying out protein concentration measurement according to the requirements of a BCA kit instruction, and respectively calculating the loading volume of each group of proteins according to the measured protein concentration by taking each group of 20 mu g/group as the total protein loaded. The constant temperature test tube heater is started in advance, the denaturation is carried out for 10min at 99 ℃, and the denatured protein is preserved at-20 ℃ for standby. Performing SDS-PAGE gel electrophoresis according to a conventional Westernblot experimental operation method, transferring to a nitrocellulose membrane, sealing at room temperature for 2 hours or overnight at 4 ℃, adding a primary antibody, incubating at room temperature for 4 hours, washing for 30 minutes, adding a secondary antibody, incubating at room temperature for 2 hours, washing for 30 minutes, and mixing ECL luminescent reagent A and reagent B according to the following formula 1:1 are added on a nitrocellulose membrane (which is used in the prior art) in a mixing mode, and a gel imaging analyzer is placed for exposure imaging. Experiments were repeated 3 times.

1.7 statistical analysis

Statistical analysis of experimental results using IBM SPSS22.0 software with mean ± standard deviation

The regression analysis and the one-way analysis of variance are performed.

2. Experimental results

2.1 dose-response relationship of thyroid follicular cell model damaged by TPOAb

Based on an ADCC mechanism, a TPOAb damaged thyroid follicular cell model is established, the MTT method is used for detecting the damage rate of target cells Nthy-ori3-1 under different concentrations of TPOAb, and the result shows that the higher the concentration of TPOAb is, the higher the damage rate of target cells is in the establishment of the model, and the obvious dose-effect dependence relation is provided (table 1). And fit dose-response curves using IBM SPSS22.0 software regression analysis with the antibody concentration on the X-axis and the damage rate of the nth-ori 3-1 cells on the Y-axis (fig. 1). The quantitative effect data of the TPOAb on the Nthy-ori3-1 cell injury is processed and analyzed by SPSS22.0 software, and the concentration of the antibody, namely the IC50 value, when the injury rate of the TPOAb on the thyroid follicular cell model reaches 50 percent is 5.212ng/mL.

TABLE 1 quantitative relationship between the damage rates of the Nthy-ori3-1 cells at various concentrations of TPOAb

2.2 aging relationship of TPOAb damaged thyroid follicular cell model

And (3) selecting the IC50 value as the concentration of the antibody when the damage rate of TPOAb to the thyroid follicular cell model reaches 50% as the action concentration, and performing aging detection. The results showed that the damage rate of TPOAb to the Nthy-ori3-1 cell model also increased with the time of action at five different time points, showing a clear time-effect dependence (see Table 2). The time-effect relationship curve was fitted using IBM SPSS 21.0 software regression analysis with the time of action of the antibody as X-axis and the damage rate to cells as Y-axis (fig. 2). From the results, it can be seen that the effect of TPOAb on Nthy-ori3-1 cell loss increases most rapidly at 24h, and the effect on Nthy-ori3-1 cell loss after 24h is slow although the effect is also increasing, so that the effect 24h is selected as the effect time of TPOAb on Nthy-ori3-1 cell model.

TABLE 2 timeliness of TPOAb to the damage rate of the Ny-ori 3-1 cell model

2.3 observation of model cell morphology changes with inverted microscope

The concentration of TPOAb is selected as IC50 value to establish cell model, and each group of cell morphology is observed when the cell is amplified under a lens by 200 times, and the record is photographed. The blank group Nthy-ori3-1 cells are in a polygonal shape, so that the growth condition is good, the cells are distributed tightly, the edges are clear, the gaps are bright, the adhesion capability is good, and obvious nuclear division phases can be seen by amplified observation; the model group has obvious cell morphology change, rough edges, poor adhesion, lower transmittance than that of the blank group, more cell fragments, a large number of cells have a vacuole-like structure, and nuclei have been dissolved in the cells and are filled with a large amount of particulate matters (figure 3).

2.4 flow CFSE/PI double-staining method for analyzing injury apoptosis rate of TPOAb injury thyroid follicular cell model

After CFSE and PI double staining of the cell model, FCM technology was used to analyze the ratio of pi+ cells in cfse+ cells in the thyroid autoantibody injured cell model, so as to verify the injury rate of TPOAb to thyroid cells under the ADCC mechanism, and the result shows that the ratio of pi+ cells in cfse+ cells in the cell model reaches 42.4 when the concentration of TPOAb is at the IC50 value (table 3, fig. 4). This suggests that FCM detection of TPOAb damage rates to cell models can reach 40+% at IC50 concentrations, consistent with MTT technology detection of cell damage rates, where the damage to cell models by TPOAb may be related to target apoptosis.

TABLE 3 flow double staining method for detecting apoptosis rate of injury of TPOAb to cell model

2.5 immunofluorescence labelling technique analysis of TPO antigen antibody binding to cell model

The specific binding of TPO antibody antigen is crucial to the success of the establishment of the cell model, TPO antigen is expressed on the surface of the Nthy-ori3-1 cell membrane, so that the TPO antigen-antibody complex is subjected to fluorescent localization by using immunofluorescence labeling technology, and the result shows that the TPO antigen-antibody complex can be localized to TPOAb on the cell membrane, which suggests that the specific binding of TPO antigen antibody exists on the cell model (figure 5).

2.6ELISA technique for detecting T4 secretion level of TPOAb damaged thyroid follicular cell model

The results of the ELISA technique for detecting the change of the cell secretion T4 level of the model group show that the cell secretion T4 content of the model group is lower than that of the blank group (P < 0.01), and the model group shows the state of the A-type cell model, and the results are shown in Table 4 and figure 6.

TABLE 4T 4 secretion content of TPOAb injured thyroid follicular cell model

2.7Western Blot method for detecting TPO protein expression of cell model group

TPO is an important enzyme for catalyzing thyroid hormone, and in order to study the change of TPO protein expression in the cell model, blank groups and model histones are respectively extracted after 24 hours of cell model establishment, and the TPO expression level of two groups of proteins is detected by a Western Blot method. The results showed that the model group TPO expression level was lower than that of the blank group, indicating that TPO protein was expressed in a cell model established under the mediation of TPOAb, and that the decrease in the secretion level of T4 was possibly associated with the low expression of TPO (FIG. 7).

Example 2

Prescription drug screening with protective effect on TPOAb damaged thyroid follicular cell model

1. Experimental method

1.1 pharmaceutical preparation

The preparation method of the water decoction medicine carried by the original book comprises the steps of weighing each single medicine according to the original formula proportion in the original book, taking a proper amount of ultrapure water (2 cm for different medicines according to the situation), soaking in a clean beaker for 1h, sealing a preservative film, boiling the soaked medicines with strong fire for 60min after the soaking, closing the electric furnace when the medicine liquid of each medicine is concentrated to about 50mL, filtering the medicine liquid into a 50mL centrifuge tube by six layers of medical gauze, centrifuging at 3000rpm/min for 30min to remove medicine residues, taking an upper layer of medicine liquid, filtering into a sterile centrifuge tube by a microporous filter head with the aperture of 0.22 mu m, drying and measuring the concentration, subpackaging into the sterile 1.5mL, and preserving at-20 ℃ for standby.

The preparation method of the single medicine comprises weighing 20g of each medicine according to the preparation method of the alcohol extraction medicine, soaking a proper amount of 95% ethanol (1 cm of the medicine is flooded with ethanol according to the situation) in a clean beaker, sealing the fresh-keeping film, placing the fresh-keeping film in a dark place, drying and cooling the fresh-keeping film, shaking and stirring twice daily, soaking for 7 days, filtering the medicine liquid into a funnel bottle under negative pressure, pouring the filtered medicine liquid into a rotary steaming bottle, performing low-temperature rotary steaming by a rotary steaming instrument, closing the rotary steaming instrument when the medicine liquid is rotary steamed to 5-8ml, pouring the medicine liquid into an evaporating dish, performing drying treatment after evaporating in a water bath for 1-2 days, and storing at-20 ℃ for standby.

1.2 screening of prescription drugs with protective action on TPOAb damaged thyroid follicular cell model by MTT method

1.2.1 prescriptions prescreening

Taking Nthy-ori3-1 cells as a study object, carrying out experiments on 265 first prescriptions and 175 single medicines of the typhoid fever theory by using an MTT method, eliminating prescriptions and single medicines with inhibition and toxicity on the cells, and primarily screening and screening the standard: comprehensively considering factors such as medicines, reagents, equipment, operation and the like, and the inhibition rate of the prescription medicine concentration to the Nthy-ori3-1 cells at 100 mug/mL is more than 0.

Taking Nthy-ori3-1 cells in logarithmic growth phase at 1×10 ⁴ The cell/well concentration was inoculated into a 96-well plate at 200. Mu.L/well, cultured for 24 hours, and observed in a mirror to grow cellsIn the state, the cell supernatant is sucked in an ultra clean bench, 200 mu L/hole of complete culture solution containing 10% FBS is added into the first three columns as blank groups, 200 mu L/hole of prepared working solution with the drug concentration of 100 mu g/mL is respectively added into the other columns as drug adding groups, three compound holes are arranged for each drug, and the drugs are placed in an incubator after being marked. After the medicines act for 24 hours, the supernatant is sucked, the prepared MTT working solution is added, 100 mu L/hole is added, the culture is continued for 4 hours, the supernatant is sucked, 150 mu L/hole is added, a decolorizing shaker is placed for 30 minutes, the medicines are fully dissolved and placed under the dual wavelength (570 nm,630 nm) of an enzyme label instrument to measure the light absorption value (namely OD value), and the inhibition rate (%) = (1-model group OD value/control group OD value) ×100% of each medicine or single medicine is calculated, so that the inhibition rate of each medicine or single medicine on Nthy-ori3-1 cells is calculated at the concentration of 100 mu g/mL.

1.2.2 Compound medicine sieves

Under the condition that the prescription with inhibiting and toxic effects on Nthy-ori3-1 cells is eliminated by primary screening, the prescription with protective effects on TPOAb damaged thyroid follicular cell models is screened by using an MTT method.

Taking Nthy-ori3-1 cells in logarithmic growth phase at 1×10 ⁴ The concentration of cells/well was inoculated into 96-well plates at 200. Mu.L/well, and after 24 hours, TPOAb-damaged thyroid follicular cell model was established. After the cell supernatant was aspirated, 200. Mu.L/Kong Xi of PBS was used twice, and a blank group, a model group, and a model dosing group were each set, wherein 100. Mu.L/well of serum-free culture solution was added to the blank group, 100. Mu.L of serum-free culture solution containing TPOAb at an IC50 value was added to the model group, 100. Mu.L/well of different-concentration drug working solutions containing TPOAb at an IC50 value, prepared without serum-containing culture medium, were each added to the model dosing group, and 8 drug concentration gradients were set, respectively: 1.5625. Mu.g/mL, 3.125. Mu.g/mL, 6.25. Mu.g/mL, 12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL, three wells per concentration, placing the mixture in an incubator for incubation for 2 hours after sample addition, adding 100. Mu.l/well of HL-60 cells as effector cells at an effective target ratio of 12:1, centrifuging at 1000rpm for 3min to make the effector cells fully contacted with the target cells, continuously culturing for 24h, then using PBS200 mu L/Kong Xi for two times again, washing out HL-60 cells in the wells, and then adding prepared MTT working solution at 100 mu L/well. After incubation in incubator for 4h, 150. Mu.L/well DMSO was added and placedThe color of DMSO is uniform after 30min on a decoloring shaking table, the absorbance (OD value) is measured under the dual wavelength (570 nm,630 nm) of an enzyme-labeled instrument, the cell damage rate is calculated as in example 1, and the cell damage rate of the model group is calculated>At 40%, the establishment of the cell model was considered successful, and the calculation of the cytoprotection ratio was performed, wherein the cytoprotection ratio (%) = (OD value of model drug-added group/OD value of model control group-1) ×100%. The experiment is repeated three times by comprehensively considering factors such as medicines, reagents, operation and the like. And finally screening out a prescription drug with high and stable protection rate on the TPOAb damaged thyroid follicular cell model, performing aging detection, and drawing a dose-effect and aging relation curve.

1.3 flow CFSE/PI double-dyeing method for analyzing apoptosis rate of cell model after effective prescription action

Taking 1-5×10 cells from Nthy-ori3-1 in logarithmic growth phase ⁶ After CFSE staining, the cells were inoculated into 24-well plates, 5X 10 ⁴ culturing cells/holes in an incubator for 24 hours to establish a model, setting a blank group, a model group and a model dosing group respectively, collecting suspension cells of each hole to a corresponding 15ml centrifuge tube after 24 hours of action, washing each hole twice by PBS, adding pancreatin digestive juice without EDTA to digest target cells and collect the target cells to each corresponding centrifuge tube, pre-cooling the PBS, centrifugally washing twice, re-suspending by 500 mu L of PBS, adding 10 mu LPI to each tube, dyeing at room temperature in a dark place for 5-10min, and performing on-machine detection within 30 min. The FlowJo software performs the result analysis to calculate the ratio of pi+ cells in cfse+ cells.

1.4ELISA technique for detecting T4 secretion level of cell model after effective prescription action

The Nthy-ori3-1 cells were taken according to a 5X 10 protocol ⁴ The density of cells/holes is inoculated to a 24-well plate for culture in an incubator, and a blank group, a model group and a model dosing group are respectively arranged. After 24h, the cell supernatant was collected to a sterile 15ml centrifuge tube, centrifuged at 2000rpm for 20min, and the supernatant was taken out and split into sterile 1.5ml EP tubes, labeled and stored at-80℃for further use. The sample detection step was performed as in example 11.5.

1.5 statistical analysis

And (3) representing.

2. Results

2.1 screening results of prescription drugs

The MTT method is used for carrying out primary screening on 153 first prescriptions and 128 single medicine alcohol extracts in the ' Jinkui ' abbreviation ' on Nthy-ori3-1 cells, removing prescription medicines with obvious inhibition and toxic effects, and carrying out primary screening on 33 prescriptions and 20 single medicines which possibly have protective effects on a thyroid follicular cell model damaged by TPOAb. 51 traditional Chinese medicines (donkey-hide gelatin, croton, japanese ampelopsis root, white honey, white peony root, white atractylodes rhizome, lily, pinellia tuber, fritillaria, bupleurum, red bean, ligusticum wallichii, soybean roll, jujube, haematitum, angelica, ledebouriella root, tetrandra root, poria cocos, aconite root, licorice root, dried ginger, kudzu vine root, cassia twig, talcum, astragalus root, polished round-grained rice, balloonflower root, orange peel, medicated leaven, ephedra, ophiopogon root, mirabilite, tree peony bark, oyster, stephania tetrandrae root, ginseng, chinese yam, dried rehmannia root, ginger, gypsum, pricklyash peel, wild jujube, evodia rutaecarpa, schisandra chinensis, asarum, almond, alisma orientalis, rhizoma anemarrhenae, immature bitter orange and bamboo leaf) related to 33 prescreened prescription medicines are compared with 20 single medicines, 10 traditional Chinese medicines including the same traditional Chinese medicines are removed, and the remaining 61 single medicine alcohol extracts are acted on a thyroid gland injury follicular cell model for rescreening, and experiments are repeated 3 times. Finally, 4 single medicines with higher protection and stable action on Nthy-ori3-1 cells are determined: fructus Aurantii Immaturus, pericarpium Citri Reticulatae viride, semen Armeniacae amarum, and Alismatis rhizoma. The prescription comprising immature bitter orange, green tangerine peel, almond and alisma orientale is named as a prescription for regulating qi and resolving phlegm according to the efficacy of the medicines.

2.2 Dose-response relationship of 4 traditional Chinese medicines to protection effect of TPOAb injury Nthy-ori3-1 cell model

After the 4 single medicines screened above are applied to the TPOAb damaged thyroid follicular cell model, the result shows that the protection effect of each medicine on cells is in a relationship of increasing and then decreasing along with the increasing of the concentration, and the optimal protection concentration exists. The dose-response curves were plotted using graphpadprism8.4.0 software with drug concentration on the X-axis and cytoprotection on the Y-axis (see table 5, fig. 8-11).

Table 5 4 dose-effect relationship of effective Chinese medicine for protecting cell model under different concentration actions

From the dose-response relation table and the graph, the optimal protection rate of the immature bitter orange on the TPOAb damaged Nthy-ori3-1 cell model is 31.06%, the optimal protection rate of the green tangerine peel on the TPOAb damaged Nthy-ori3-1 cell model is 26.06%, the optimal protection rate of the green tangerine peel on the TPOAb damaged Nthy-ori3-1 cell model is 12.5 mug/mL, the optimal protection rate of the almond on the TPOAb damaged Nthy-ori3-1 cell model is 29.00%, the optimal protection rate of the alisma on the TPOAb damaged Nthy-ori3-1 cell model is 25 mug/mL, and the optimal protection rate of the alisma on the TPOAb damaged Nthy-ori3-1 cell model is 29.32%, and the optimal protection rate of the corresponding medicine action concentration is 12.5 mug/mL. Thus, the optimal action concentrations of the immature bitter orange, the green tangerine orange peel, the almond and the alisma on the TPOAb damage Nthy-ori3-1 cell model are 25 mug/mL, 12.5 mug/mL, 25 mug/mL and 12.5 mug/mL respectively.

2.3 dose-response relation of qi regulating and phlegm resolving prescription on protecting thyroid follicular cell model of TPOAb injury

The medicinal liquid is prepared from 4 medicines of immature bitter orange, green tangerine orange peel, almond and rhizoma alismatis with different multiples of the optimal action concentration, and acts on a cell model to obtain the dose-effect relationship of the qi-regulating and phlegm-resolving prescription on the protection effect of the TPOAb damaged thyroid follicular cell model (see Table 6 and figure 12).

TABLE 6 dose-to-effect relationship of qi regulating and phlegm resolving prescription to the protection rate of the nth-ori 3-1 cell model

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According to the dose-effect relation table and the graph, the optimal action concentration multiple of the composition of the immature bitter orange, the green tangerine orange peel, the almond and the alisma orientale in the qi-flowing and phlegm-resolving prescription is 0.25 times, and the optimal action concentration of each single medicine of the immature bitter orange, the green tangerine orange peel, the almond and the alisma orientale is 6.25 mug/mL, 3.125 mug/mL, 6.25 mug/mL and 3.125 mug/mL respectively, and the mass ratio is 2:1:2:1.

2.4 aging relationship of qi regulating and phlegm resolving formula for protecting thyroid follicular cell model of TPOAb injury

And (5) selecting the medicine to perform time-effect detection on the medicine concentration when the cell model is at the optimal protection rate. The results show that the protection rate of the qi-flowing and phlegm-resolving prescription and the single medicines of the immature bitter orange, the green tangerine peel, the almond and the alisma on the cell model is gradually increased and gradually decreased along with the prolongation of the action time at five different time points, wherein the protection rate of the qi-flowing and phlegm-flowing prescription and the immature bitter orange, the green tangerine peel and the alisma on the cells reaches the highest peak at 24 hours, and the protection rate of the almond on the cells reaches the highest peak at 36 hours. Drawing time-dependent curve by prism8.4.0 software with the action time of the drug as X axis and the protection rate of cell model as Y axis (see Table 7, figures 13-17)

TABLE 7 aging relationship of qi regulating and phlegm resolving formula to cell model protection

2.5 flow CFSE/PI double-staining method for detecting apoptosis rate of cell model after effective prescription action

The cells after the effective prescription is collected, and the proportion of PI+ cells in CFSE+ cells in each group of collected cells is detected by using FCM technology, so that the proportion of PI+ cells in the model dosing group is obviously smaller than that in the model dosing group (table 8). This suggests that the cell viability in each model dosing group is higher than that in the model group, the rate of apoptosis of cell damage is reduced after drug action, and the protective effect of the drug on the cell model may be related to inhibiting apoptosis.

Table 8 flow-type double-staining method for detecting apoptosis rate change of cell model after five formulations act

2.6ELISA technique for detecting T4 secretion level of cell model after effective prescription action

In order to detect the influence of effective prescription on the secretion content of T4 in a cell model, the supernatant after the prescription acts on the cell model is collected and detected by using ELISA technology, and the results show that the secretion content of T4 in the cell model is increased after the prescription for regulating qi and resolving phlegm and 4 single medicines act (Table 9), wherein the prescription for regulating qi and resolving phlegm is particularly obvious (P < 0.05).

TABLE 9 Effect of effective formulations on T4 secretion levels in TPOAb injured thyroid follicular cell models

Example 3 clinical observations of Qi-regulating phlegm-resolving prescription for treating Qi-stagnation and phlegm-coagulation type hashimoto thyroiditis

1. Research method

1.1 clinical data

The observed cases are 60 cases of patients with the initial diagnosis of the hashimoto thyroiditis caused by qi stagnation and phlegm coagulation, which are collected and treated by a third affiliated hospital of Henan traditional Chinese medicine university at 9-2021 of the year 2020, and all the patients accord with the diagnosis standard of the hashimoto thyroiditis which is proposed in the guidelines for diagnosis and treatment of Chinese thyroiditis prepared by the endocrinology of China medical society: all diffuse thyromegaly with a tough texture, especially with isthmus cone She Zhongda, serum TPOAb and TgAb positive, diagnosis can be established, including those diagnosed by aspiration cytology through fine needle puncture (FNAC), whether thyroid function is altered or not. Case inclusion criteria: meets the Western diagnosis standard of thyroiditis, and the syndrome differentiation of traditional Chinese medicine is qi stagnation and phlegm coagulation; age 18-70 years; new diagnosis or disease course < half a year, untreated; recent patients with no serious infection or serious complications; a person willing to collaborate and sign study informed consent. Case exclusion criteria: (1) those that do not meet inclusion criteria; those with thyroid malignancy; combining severe infections and severe barycenter, brain, liver, kidney function impairment; those with mental and psychological abnormalities; patients cannot cooperate or adhere to the therapist.

Patients were divided into 30 cases of control group and treatment group by random number table method, wherein the treatment group comprises male 4 cases and female 26 cases, the average age is 42.86+ -13.28 years, the control group comprises male 3 cases and female 27 cases, and the age is 40.28 + -11.32 years. The comparison of the two groups of general data such as gender, age and the like has no statistical significance (P > 0.05) and comparability.

1.2 treatment regimen

The control group was treated with oral selenium yeast tablet, taken 1 time a day in the morning at a dose of 200 μg. Treatment group: based on the control group, the traditional Chinese medicine composition is adopted for treatment, and the traditional Chinese medicines are purchased from a third affiliated hospital of Henan traditional Chinese medicine university, decocted with water and taken at a temperature of 1 dose per day, divided into 2 times, and about 150-180 mL each time. The treatment course is 1 week 4, the total treatment course is observed for 3, the clinical observation table is filled in before and after the treatment, and the follow-up is carried out after the treatment course is ended for 3 months. Neither group took other medications during the treatment period.

1.3 observations index

1.3.1 therapeutic efficacy observations

(1) Thyroid hormone level determination: all patients were tested for FT before and after treatment ₄ 、FT ₃ 、TSH；

(2) Thyroid autoantibody level assay: TPOAb, TGAb were tested once each before and after treatment for all patients;

(3) And (3) evaluation of traditional Chinese medicine syndrome integral: the integral evaluation of the Chinese medicine symptoms is carried out before and after the treatment of all patients.

1.3.2 Security observations

(1) General physical examination: vital sign examinations (blood pressure, heart rate, respiration, body temperature), etc.

(2) Three general treatments (blood general, urine general, stool general), liver function, kidney function, electrocardiogram, etc. were performed 1 time before and after each treatment.

(3) Adverse reaction: the medicine has adverse reaction and toxic and side effects.

1.4 evaluation of efficacy

The curative effect is evaluated according to the relevant standards in the national traditional Chinese medicine administration ' traditional Chinese medicine disease diagnosis curative effect standard ' and the ' traditional Chinese medicine new medicine clinical research guiding principle (trial).

(1) And (3) curing: the clinical symptoms and signs of the traditional Chinese medicine disappear or basically disappear, the integral of symptoms is reduced by more than 95%, the thyroid gland volume is normal, the nodules disappear, the thyroid function is recovered to be normal, and TPOAb and TGAb turn to yin.

(2) The effect is shown: the clinical symptoms and signs of the traditional Chinese medicine are obviously improved, the integral of symptoms is reduced by more than 70%, the volume of thyroid gland is reduced, the texture is softened, the nodule is obviously softened and becomes smaller, the thyroid gland function is obviously improved, and the titer of TPOAb and TGAb is reduced by more than or equal to 50%.

(3) The method is effective: the clinical symptoms and signs of the traditional Chinese medicine are all improved, the integral of symptoms is reduced by more than 30%, the volume of thyroid is reduced, the texture is softened, the nodule is softened, the thyroid function is improved, and the titer of TPOAb and TGAb is reduced by more than or equal to 20% but less than 50%.

(4) Invalidation: the clinical symptoms and signs of the traditional Chinese medicine are not obviously improved, or the illness is aggravated, and the integral of symptoms is reduced by less than 30 percent.

Note that: the integral calculation formula of the traditional Chinese medicine symptoms (nimodipine method [ (integral before treatment-integral after treatment)/(integral before treatment ] ×100%).

1.5 statistical analysis

Statistical analysis was performed using SPSS22.0 statistical software. The gender of the counting data is checked by X2, and the curative effect of the orderly classified metering data is checked by rank sum. The normal metering data adopts t test, and the metering data which does not accord with normal distribution adopts non-parameter test. P <0.05 is significantly different, P <0.01 is extremely significantly different, and P >0.05 is no difference.

2. Results

2.1 integral comparison of the symptoms of traditional Chinese medical science before treatment

Table 10 comparison of the integral of the symptoms of traditional Chinese medical science before treatment for the control group and the treatment group patients

( And (3) injection: the syndrome integration of the patients in the control group and the treatment group is t-tested, and P is more than 0.05 )

From table 10, it can be seen that the traditional Chinese medicine syndrome integral of the control group and the treatment group patients have no obvious difference before treatment, and are comparable.

2.2 thyroid function before treatment and antibody comparison

Table 11 comparison of thyroid function and antibodies before treatment in control and treatment patients

(note: control and treatment patients pre-treatment FT ₃ 、FT ₄ The P values of TSH, TGAb, TPOAb are respectively detected by t test>0.05)

From table 11, it can be seen that there was no significant difference in thyroid function level and antibody level before treatment between the control group and the treated group patients, and the comparison was made.

2.3 observation of the efficacy of the integration of the symptoms of the traditional Chinese medical science after the treatment

Table 12 control group and treatment group patients show the integral change condition of the traditional Chinese medicine symptoms before and after treatment

( And (3) injection: the comparison between the treated delta control group and the treated delta control group is that P is more than 0.05, and the delta control group has no statistical significance; p <0.05 after treatment and before treatment of the treatment group, has statistical significance; after the treatment, the comparison between the treatment group and the control group is P <0.05, which has statistical significance )

As can be seen from table 12, the Chinese medicine syndrome integral of the control group and the treatment group is reduced after treatment, but P is more than 0.05 compared with that before treatment, and the Chinese medicine syndrome integral has no statistical significance; the degree of decline of the treatment group after integral treatment is larger than that before treatment, which indicates that the treatment group is superior to the control group in improving the Chinese medical syndrome score.

2.4 observation of thyroid Functions and antibody therapeutic Effect after treatment

TABLE 13 observation of thyroid function and antibody efficacy after treatment

( And (3) injection: the self comparison after delta treatment is P >0.05, and the method has no statistical significance; p <0.05 after treatment, has statistical significance; comparison P <0.05 between groups after treatment, has statistical significance )

As can be seen from table 13, the thyroid function of the patients after treatment is not significantly changed in the control group and the treatment group, and the two antibodies in the control group have no obvious decrease trend although they decrease after treatment, and have no difference from the prior treatment, which indicates that the antibody level cannot be well reduced by the treatment of simply supplementing trace element selenium; the two antibodies of the treatment group are obviously reduced, which indicates that the qi-regulating and phlegm-resolving formula can reduce the level of thyroid autoantibodies; the comparison among the groups also has statistical significance, which indicates that the curative effect of the combination of the Chinese medicinal groups and the trace element selenium in the qi-flowing regulating and phlegm-resolving prescription is better than that of the treatment of the pure supplement of the trace element selenium.

2.5 evaluation of clinical efficacy after treatment

Table 14 clinical efficacy assessment

( And (3) injection: clinical efficacy assessment after treatment is tested by rank sum, P is less than 0.05, and has obvious difference )

As can be seen from table 14, in terms of clinical efficacy, both the control group and the treatment group have efficacy after treatment, but the treatment group has higher effective rate than the control group, i.e. the treatment effect of combining the traditional Chinese medicine group with trace element selenium in the formula of regulating qi and resolving phlegm is better than the treatment effect of simply supplementing trace element selenium.

From the ADCC mechanism of hashimoto thyroiditis, the invention establishes an in vitro TPOAb damaged thyroid follicular cell model, and uses techniques such as cell co-culture, MTT method, flow cytometry, immunofluorescence labeling, enzyme-linked immunosorbent assay (ELISA), western immunoblotting (Western Blot) and the like to screen out 4 single medicinal materials such as immature bitter orange, green tangerine peel, almond and rhizoma alismatis which have higher protection effect and stable effect on the TPOAb damaged thyroid follicular cell model from 265 prescriptions and 175 single medicinal materials carried by classical medical books of Shang Han miscellaneous diseases theory of Zhi Sheng Zhang. While the weight ratio of fructus aurantii immaturus, green tangerine peel, almond and rhizoma alismatis is 2:1:2:1 has stronger protective effect on the cells of the Nthy-ori3-1 damaged by TPOAb, and obviously reduces thyroid autoantibodies after the effect of the traditional Chinese medicine composition and has obvious curative effect on HT patients.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The traditional Chinese medicine composition for protecting the thyroid follicular cells damaged by TPOAb is characterized by comprising the following components in percentage by mass: 1: 1-3: fructus Aurantii Immaturus, pericarpium Citri Reticulatae viride, semen Armeniacae amarum, and Alismatis rhizoma.

2. The traditional Chinese medicine composition according to claim 1, wherein the dosage form of the traditional Chinese medicine composition comprises one of decoction, granules, powder, pills and capsules.

3. Use of the traditional Chinese medicine composition according to claim 1 or 2 for preparing a medicament for protecting thyroid follicular cells damaged by TPOAb.

4. The use of a Chinese medicinal composition according to claim 1 or 2 in the preparation of a medicament for the treatment of hashimoto thyroiditis.