A traditional Chinese medicine preparation for treating hashimoto's thyroiditis and a preparation method thereof

Abstract

The application provides a traditional Chinese medicine preparation for treating Hashimoto's thyroiditis, and relates to the technical field of traditional Chinese medicines.The traditional Chinese medicine preparation is prepared from the following components: sea sponges, walnuts, water red flower seeds, turmeric, rhizomes of lilies, bupleurum, euphorbia, sall tree seeds, red vine, turmeric and licorice; in-vitro studies prove that the traditional Chinese medicine preparation can effectively reduce the content of inflammatory factors, inhibit thyroid cell apoptosis, and thus relieve the pathological process of Hashimoto's thyroiditis; in-vivo experiments further prove that the traditional Chinese medicine preparation can significantly reduce the level of the model animal's Hashimoto's thyroiditis marker antibody, effectively regulate thyroid function, and obviously improve the damage of the pathological structure of the thyroid tissue; in addition, the traditional Chinese medicine preparation can regulate the inflammatory microenvironment, help balance the specific immune state, reduce the incidence of complications and the risk of adverse reactions; in conclusion, the application provides a safe and effective new traditional Chinese medicine preparation for the clinical treatment of Hashimoto's thyroiditis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, specifically to a traditional Chinese medicine preparation for treating Hashimoto's thyroiditis and its preparation method. Background Technology

[0002] The thyroid gland is an important endocrine organ in the human body, playing a crucial role in regulating growth, intellectual development, and metabolism, and significantly influencing multiple systems and glands. Hashimoto's thyroiditis (HT) is characterized by diffuse thyroid enlargement, lymphocytic infiltration, and elevated thyroid autoantibodies. Persistent inflammation can damage thyroid follicular cells, causing interfollicular fibrosis, ultimately leading to hypothyroidism and even increasing the risk of malignant tumors. Reports indicate that the incidence of thyroid nodules in Hashimoto's thyroiditis patients is as high as 24.3%, with a malignancy rate ranging from 7.9% to 58.3%. Therefore, effectively protecting thyroid follicular cell function, slowing the progression of chronic inflammation, and preventing hypothyroidism or the development of thyroid malignancies are pressing challenges that need to be addressed.

[0003] Currently, the clinical treatment of Hashimoto's thyroiditis mainly aims to improve thyroid function and regulate the body's immune balance. Mainstream therapies include oral levothyroxine replacement therapy, selenium supplementation, and glucocorticoid therapy. However, these approaches all have certain limitations: levothyroxine overdose can induce arrhythmias, and this therapy only supplements hormones to maintain thyroid function, failing to reduce autoantibody levels or halt disease progression; the clinical efficacy of selenium supplements varies greatly among individuals, and overdose carries toxicity risks; while glucocorticoids can rapidly reduce thyroid inflammation, long-term use has significant side effects, making them unsuitable for long-term disease management.

[0004] Traditional Chinese medicine (TCM) theory holds that Hashimoto's thyroiditis originates in the liver, spleen, and kidneys. Deficiency of spleen qi leads to stagnation and accumulation of dampness and phlegm, which then stagnates in the neck. Liver qi stagnation and impaired qi circulation result in internal blood stasis, causing foul-smelling qi that accumulates in the neck over time. The pathogenesis is primarily characterized by qi stagnation, phlegm accumulation, and blood stasis that persists for a long time. The treatment principle focuses on tonifying qi and strengthening the spleen, dispelling dampness, resolving phlegm, and dispersing blood stasis. TCM possesses unique advantages and characteristics in treating Hashimoto's thyroiditis, alleviating clinical symptoms and improving patients' quality of life. Therefore, addressing the aforementioned shortcomings of existing medications for Hashimoto's thyroiditis, developing a scientifically formulated, mild, multi-syndrome-compatible TCM preparation with minimal side effects has significant clinical importance and application value. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] In view of the shortcomings of the prior art, the present invention provides a traditional Chinese medicine preparation for treating Hashimoto's thyroiditis and its preparation method.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] On one hand, the present invention provides a traditional Chinese medicine preparation for treating Hashimoto's thyroiditis, the preparation being made from the following raw materials in parts by weight: 5-20 parts of pumice, 5-20 parts of oyster shell, 1-10 parts of water lily seed, 4-18 parts of turmeric, 4-20 parts of lily bulb, 5-20 parts of bupleurum, 3-20 parts of safflower seed, 3-15 parts of salsa seed, 2-15 parts of red vine, 1-12 parts of turmeric, and 1-10 parts of licorice.

[0010] Specifically, the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 8-15 parts of pumice, 8-15 parts of oyster shell, 2-8 parts of water lily seed, 5-12 parts of turmeric, 9-18 parts of lily bulb, 6-12 parts of bupleurum, 6-12 parts of safflower seed, 5-9 parts of salsa seed, 3-12 parts of red vine, 2-10 parts of turmeric, and 2-6 parts of licorice.

[0011] Specifically, the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 10 parts pumice, 10 parts oyster shell, 3 parts water lily seed, 8 parts turmeric, 15 parts lily bulb, 8 parts bupleurum, 8 parts safflower seed, 8 parts salsa seed, 9 parts red vine, 5 parts turmeric, and 3 parts licorice.

[0012] On the other hand, the present invention also provides a method for preparing the above-mentioned traditional Chinese medicine preparation, comprising the following steps:

[0013] (1) Grind each component into fine powder and pass it through a 100-mesh sieve.

[0014] (2) Mix the fine powders of each component to obtain a drug mixture powder, add starch and water, and make pills.

[0015] (3) The prepared pills are dried to obtain the Chinese medicine water pills.

[0016] Specifically, the amount of starch and water added should be such that the resulting pills are not sticky to the touch.

[0017] Specifically, the dry weight of the Chinese medicine pills obtained in step (3) is 0.1g-0.3g, preferably 0.15-0.25g.

[0018] This invention provides the application of the aforementioned traditional Chinese medicine preparation in the preparation of a drug for treating Hashimoto's thyroiditis.

[0019] The pharmacological effects of each Chinese herbal raw material in the Chinese herbal preparation of this invention are as follows:

[0020] Pumice: Derived from the dried skeleton of the bryozoan *Costazia aculeata* Canu et Bassler (family Pyrenidae). It has a salty and neutral flavor; it enters the liver meridian and has the effects of clearing the lungs and resolving phlegm, softening hard masses and dissipating nodules. It is mainly used for cough with phlegm-heat, phlegm accumulation, goiter, scrofula, hernia, and sores.

[0021] Clam shells: derived from the shells of the cockles *Arca subcrenata* Lischke, *Arca granosa* Linnaeus, or *Arca inflata* Reeve. Salty and neutral in nature; they enter the liver and spleen meridians, and have the effects of resolving phlegm, removing blood stasis, softening hard masses, and dissipating nodules. They are mainly used for scrofula, goiter, abdominal masses, and persistent cough with phlegm.

[0022] Water Red Flower Seed: The dried, ripe fruit of *Persicaria orientalis* (L.) Spach, a plant in the Polygonaceae family. It has a salty taste and slightly cold nature; it enters the liver and stomach meridians, and has the effects of dispersing blood stasis and eliminating masses, relieving pain, and promoting diuresis and reducing swelling. It is mainly used for abdominal masses, goiter, indigestion, stomach distension and pain, and edema with ascites.

[0023] Turmeric: Derived from the dried tuberous roots of *Curcuma wenyujin* YHChen et C.Ling, *Curcuma longa* L., *Curcuma kwangsiensis* SGLee et CFLiang, or *Curcuma phaeocaulis* Val. (family Zingiberaceae). It has a pungent and bitter taste, and is cold in nature; it enters the liver, heart, and lung meridians, and has the effects of promoting blood circulation and relieving pain, regulating qi and relieving depression, clearing the heart and cooling the blood, and promoting bile secretion and reducing jaundice. It is mainly used for stabbing pain in the chest and ribs, chest pain, amenorrhea, dysmenorrhea, breast distension and pain, and delirium due to febrile diseases.

[0024] Lily: Derived from the fleshy scales of plants in the genus *Lilium lancifolium* Thunb., *Lilium brownnii* FE Brown var. *viridulium baker*, or *L. Pumilum* DC. It has a sweet taste and cold nature; it enters the heart and lung meridians, and has the effects of nourishing yin and moistening the lungs, clearing the heart and calming the mind. It is mainly used for dry cough due to yin deficiency, hemoptysis due to overwork, restlessness and palpitations, insomnia and dreaminess, and mental confusion.

[0025] Bupleurum: Derived from the dried root of *Bupleurum chinense* DC or *Bupleurum corzonerifolium* Willd, both belonging to the Apiaceae family. It tastes bitter and pungent, and is slightly cold in nature; it enters the liver, gallbladder, and lung meridians, and has the effects of dispersing and reducing fever, soothing the liver and relieving depression, and raising yang qi. It is mainly used to treat colds with fever, alternating chills and fever, chest and rib pain, irregular menstruation, uterine prolapse, and rectal prolapse.

[0026] Tiankui seeds: These are the dried tuberous roots of *Semiaquilegia adoxoides* (DC.) Makino, a plant in the Ranunculaceae family. They are sweet and bitter in taste, and cold in nature; they enter the liver and stomach meridians, and have the effects of clearing heat and detoxifying, reducing swelling and dissipating nodules. They are mainly used for carbuncles, boils, mastitis, scrofula, and snake and insect bites.

[0027] Sal seeds: These are the dried, mature seeds of *Aesculus chinensis* Bge., *Aesculus chinensis* Bge. var. *chekiangensis* (Huet Fang) Fang, or *Aesculus wilsonii* Rehd., all belonging to the genus *Aesculus* of the family Aesculaceae. They are sweet in taste and warm in nature; they enter the liver and stomach meridians, and have the effects of soothing the liver and regulating qi, harmonizing the stomach and relieving pain. They are mainly used for stagnation of liver and stomach qi, chest and abdominal distension, and stomach pain.

[0028] Red vine: The dried stem of *Sargentodoxa cuneata* (Oliv.) Rehd. & EHWilson in CS Sargent, a plant in the Lardizabalaceae family. It has a bitter and neutral taste; it enters the liver and large intestine meridians, and has the effects of clearing heat and detoxifying, promoting blood circulation and removing blood stasis. It is mainly used for intestinal abscesses and abdominal pain, rheumatic pain, traumatic injuries, dysmenorrhea, dysentery, hematuria, and infantile malnutrition with parasitic intestinal pain.

[0029] Turmeric: Derived from the dried rhizome of Curcuma longa L., a plant belonging to the genus Curcuma in the ginger family (Zingiberaceae). It has a bitter taste and is neutral in nature; it enters the spleen and liver meridians, and has the effects of promoting blood circulation, regulating qi, and relieving pain. It is mainly used for chest and rib pain, chest pain, dysmenorrhea, amenorrhea, abdominal masses, rheumatic shoulder and arm pain, and swelling and pain from falls.

[0030] Licorice: Derived from the dried roots and rhizomes of the legumes *Glycyrrhiza uralensis* Fisch., *Glycyrrhiza inflata* Bat., or *Glycyrrhiza glabra* L. It has a sweet and neutral flavor; it enters the spleen, stomach, and lung meridians, and has the effects of tonifying the spleen and replenishing qi, clearing heat and detoxifying, resolving phlegm and relieving cough, relieving spasms and pain, and harmonizing other medicines. It is used for spleen and stomach weakness, fatigue, palpitations, shortness of breath, cough with excessive phlegm, abdominal pain, spasms and pain in the limbs, carbuncles and boils, and to alleviate the toxicity and harshness of other drugs.

[0031] (III) Beneficial Effects

[0032] Iodine plays a crucial role in the growth, development, and functional maintenance of the thyroid gland and is a contributing factor to Hashimoto's thyroiditis. High iodine levels can directly damage thyroid cells and also bind with antigens to generate iodinated antigens, enhancing their immunogenicity. In response to this, when selecting medications for our inventive formula, we carefully chose non-toxic, iodine-free softening and dispersing herbs such as pumice and oyster shell, which can resolve phlegm, remove blood stasis, soften and disperse nodules, offering greater safety and applicability to patients at all stages of Hashimoto's disease. This choice of medication directly aligns with the aforementioned treatment principles, fully demonstrating the scientific nature and targeted approach of this prescription. Furthermore, Traditional Chinese Medicine believes that Hashimoto's thyroiditis is primarily caused by "phlegm and blood stasis leading to hardening." Pumice in this formula is particularly effective at resolving old and stubborn phlegm; oyster shell possesses both "phlegm-resolving" and "blood stasis-removing" effects. These two herbs, with their heavy texture, directly enter the lung, liver, and stomach meridians, specifically targeting tangible phlegm and blood stasis, making them the principal herbs. This formula addresses the clinical characteristics of Hashimoto's thyroiditis patients, primarily characterized by mood swings and neck fullness and discomfort. The selection and combination of subsequent assistant and adjuvant herbs focus on "resolving phlegm and soothing the liver." Traditional Chinese medicine believes that emotional activity mainly depends on the liver's function of regulating qi. When liver qi flows smoothly, the body's energy mechanisms are unobstructed, resulting in a peaceful mood; conversely, stagnation of liver qi leads to depression, irritability, frequent sighing, and hypochondriac pain—the most common emotional problems among Hashimoto's thyroiditis patients. Therefore, this formula includes liver-regulating herbs such as Bupleurum, Curcuma longa, and Polygonum hydropiper, as well as blood-activating herbs like Polygonum hydropiper. Bupleurum soothes the liver, relieves stagnation, and regulates liver qi; Curcuma longa promotes qi circulation, relieves stagnation, invigorates blood, and relieves pain. Together, they regulate the body's overall qi flow, especially targeting qi stagnation along the liver meridian. When liver qi flows smoothly, phlegm and blood stasis have no source. Water lily seeds disperse blood stasis, eliminate masses, and relieve pain; *Polygonum multiflorum* seeds soothe the liver, regulate qi, and harmonize the stomach. These four herbs work together to assist the principal herb in dispersing nodules, resolving phlegm, and regulating qi, aiming to reduce the incidence of Hashimoto's thyroiditis accompanied by depression. Hashimoto's disease is a chronic autoimmune inflammation, with local microcirculatory disturbances and fibrous tissue hyperplasia being the pathological basis for its goiter and firm texture. The "entering the collaterals" properties of *Smilax glabra* and *Curcuma longa* in the formula perfectly match the chronic and difficult-to-cure nature of this disease. Lily bulbs can prevent excessive coldness from stimulating yin in herbs like pumice, and can also improve the yin deficiency and dryness-heat that may occur in goiter; *Smilax china* seeds clear heat and detoxify, reduce swelling and disperse nodules, and can clear the "stagnant heat" generated by qi stagnation and phlegm accumulation; these four herbs serve as adjuvant herbs. Licorice root can strengthen the spleen and replenish qi, eliminating the source of phlegm production, and also harmonizes the properties of all the herbs in the formula. The formula works synergistically to regulate qi, resolve phlegm, disperse nodules and reduce swelling, while also clearing away stagnant heat and harmonizing the middle jiao. This allows for multi-target intervention in the pathological chain of goiter, which involves clearing heat, liver stagnation, qi stagnation, phlegm accumulation, and blood stasis.

[0033] Pharmacodynamic studies were conducted using in vitro and in vivo models to simulate Hashimoto's thyroiditis. In vitro results showed that this traditional Chinese medicine preparation significantly reduced IFN-γ-induced thyroid cell apoptosis, while also decreasing levels of inflammatory factors IL-6 and TNF-α, as well as thyroid function markers, thereby improving IFN-γ-induced thyroid cell inflammation and damage. In vivo results showed that this traditional Chinese medicine preparation significantly reduced TSH, TgAb, and TPOAb levels in the model group animals and effectively improved thyroid follicular structural damage. Furthermore, after intervention with this traditional Chinese medicine preparation, Th1, Th2, and Th17 related indicators all showed varying degrees of anti-inflammatory trends, suggesting that this traditional Chinese medicine preparation can broadly regulate the proportion of immune cells, thereby modulating the inflammatory microenvironment and improving the local specific immune status of the thyroid gland. A comparative analysis was conducted on the adverse reactions of existing treatments and the high correlation between Hashimoto's thyroiditis and depression. The results showed that this traditional Chinese medicine preparation not only did not cause arrhythmia but may also have a certain regulatory effect on the abnormal emotional manifestations associated with the disease.

[0034] The above results indicate that this traditional Chinese medicine preparation has clear comprehensive efficacy advantages in anti-inflammation, regulation of thyroid metabolism, elimination of immune imbalance, and reduction of adverse reactions. Attached Figure Description

[0035] Figure 1 This describes the apoptosis status after drug intervention. * P<0.05, ** P<0.01.

[0036] Figure 2 The levels of cytokines IL-6 and TNF-α in cell supernatant after drug intervention. * P<0.05, ** P<0.01.

[0037] Figure 3 The results showed the thyroid function-related indicators in the cell supernatant after drug intervention, compared with the control group. # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01; compared with the traditional Chinese medicine preparation group, @ P<0.05, @@ P<0.01.

[0038] Figure 4 The serum levels of thyroid hormones TSH, TgAb, and TPOAb in each group of mice were measured. * P<0.05, ** P<0.01.

[0039] Figure 5 The serum levels of cytokines IL-17 and IFN-γ in each group of mice were measured. * P<0.05, ** P<0.01.

[0040] Figure 6 The heart rate of mice in each group was monitored. * P<0.05, ** P<0.01.

[0041] Figure 7 The levels of 5-HT in the prefrontal cortex of mice in each group. * P<0.05, ** P<0.01.

[0042] Figure 8 The results show the histopathological examination of thyroid tissue in each group of mice. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] Example 1

[0045] A traditional Chinese medicine preparation for Hashimoto's thyroiditis with nodules is made from the following raw materials in parts by weight: 5g of pumice, 5g of oyster shell, 3g of water lily seed, 4g of turmeric, 4g of lily bulb, 5g of bupleurum, 3g of safflower seed, 3g of salsa seed, 2g of red vine, 1g of turmeric, and 1g of licorice.

[0046] Preparation steps of traditional Chinese medicine preparations:

[0047] (1) Grind each component into fine powder and pass it through a 100-mesh sieve.

[0048] (2) Mix the fine powders of each component to obtain a drug mixture powder, add starch and water, and make pills.

[0049] (3) The prepared pills are dried to obtain the 0.15-0.25g Chinese medicine water pills.

[0050] Example 2

[0051] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 20g of pumice, 20g of oyster shell, 10g of water lily seed, 18g of turmeric, 20g of lily bulb, 20g of bupleurum, 20g of safflower seed, 15g of salsa seed, 15g of red vine, 8g of turmeric, and 5g of licorice.

[0052] Example 3

[0053] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 5g of pumice, 20g of oyster shell, 10g of water lily seed, 18g of turmeric, 20g of lily bulb, 20g of bupleurum, 20g of safflower seed, 15g of salsa seed, 15g of red vine, 8g of turmeric, and 5g of licorice.

[0054] Example 4

[0055] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 12g of pumice, 12g of oyster shell, 5g of water lily seed, 5g of turmeric, 9g of lily bulb, 6g of bupleurum, 6g of safflower seed, 5g of salsa seed, 3g of red vine, 2g of turmeric, and 3g of licorice.

[0056] Example 5

[0057] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 15g of pumice, 15g of oyster shell, 8g of water lily seed, 12g of turmeric, 12g of lily bulb, 12g of bupleurum, 12g of safflower seed, 9g of salsa seed, 6g of red vine, 3g of turmeric, and 6g of licorice.

[0058] Example 6

[0059] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 10g of pumice, 10g of oyster shell, 3g of water lily seed, 8g of turmeric, 15g of lily bulb, 8g of bupleurum, 8g of safflower seed, 8g of salsa seed, 9g of red vine, 5g of turmeric, and 3g of licorice.

[0060] Example 7

[0061] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 12g of pumice, 5g of oyster shell, 8g of water lily seed, 8g of turmeric, 12g of lily bulb, 6g of bupleurum, 8g of safflower seed, 5g of salsa seed, 3g of red vine, 2g of turmeric, and 4g of licorice.

[0062] Example 8

[0063] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 5g of pumice, 5g of oyster shell, 2g of water lily seed, 15g of turmeric, 20g of lily bulb, 16g of bupleurum, 15g of safflower seed, 10g of salsa seed, 6g of red vine, 5g of turmeric, and 8g of licorice.

[0064] Experiment 1

[0065] 1.1 Experimental Cells

[0066] Human normal thyroid cells (Nthy-ori 3-1 cells, batch number: CL-0817) were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0067] 1.2 Preparation of experimental drugs and drug-containing serum

[0068] 1.2.1 Laboratory Animals

[0069] SPF-grade male SD rats, weighing 180-220 g, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. in Shandong Province. The animals were housed in a constant temperature (22±2)℃, constant humidity (50%±10%), 12-hour light-dark cycle environment with free access to water and food. Experiments were conducted after 7 days of acclimatization.

[0070] 1.2.2 Experimental Drugs

[0071] Traditional Chinese medicine preparation: The formula described in Example 6 of this invention is prepared into a decoction.

[0072] Control preparation 1: Prepared into a decoction according to the following formula: 12g of pumice, 5g of oyster shell, 8g of water lily seed, 8g of salvia miltiorrhiza, 12g of ophiopogon japonicus, 6g of cyperus rotundus, 8g of senna seed, 5g of salvia miltiorrhiza, 3g of angelica sinensis, 2g of chuanxiong rhizome, and 4g of licorice.

[0073] Control preparation 2: Wuhai Yingliu Pills, purchased from Jilin Huangzhihua Pharmaceutical Co., Ltd., National Medicine Approval Number Z22026192, was prepared as a traditional Chinese medicine suspension.

[0074] 1.2.3 Experimental grouping and drug administration methods

[0075]

[0076] Each treatment group was administered the medication by gavage once daily for 7 consecutive days. Two hours after the last gavage, the animals were anesthetized, blood was collected from the abdominal aorta, the serum was separated, inactivated at 56°C for 30 minutes, filtered, and dispensed for later use.

[0077] 1.3 Experimental Grouping and Processing Methods

[0078] Cell concentration adjusted to 2×10 51.0 mL of cells per well is seeded into each well of a 24-well plate. Once the cells have adhered, proceed with the following steps:

[0079]

[0080] 1.4 Apoptosis after drug intervention

[0081] Cells from each group were washed twice with PBS after culture, centrifuged at 1000 rpm for 5 min, and the PBS was discarded. Subsequent assays were performed according to the instructions of the apoptosis assay kit.

[0082] 1.5 Levels of cytokines IL-6 and TNF-α in cell supernatant after drug intervention

[0083] Collect the supernatant of cells from each group after culture, centrifuge at 3000 rpm for 5 min, transfer the supernatant to a new tube, and perform the detection according to the kit instructions.

[0084] 1.6 Thyroid function-related indicators in cell supernatant after drug intervention

[0085] Collect the supernatant of cells from each group after culture, centrifuge at 3000 rpm for 5 min, transfer the supernatant to a new tube, and perform the detection according to the kit instructions.

[0086] 1.7 Statistical Methods

[0087] All experimental data were analyzed using GraphPad Prism 8.0 statistical software, and one-way ANOVA was used for comparisons between groups. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered extremely statistically significant.

[0088] 2 Results

[0089] 2.1 Apoptosis after drug intervention

[0090] The results are shown below. Figure 1 Compared with the blank group, the apoptosis rate in the model group was significantly increased, and the difference was statistically significant (P<0.01), indicating that the model group was successfully established and induced apoptosis. Compared with the model group, the apoptosis rate in the traditional Chinese medicine preparation group was significantly reduced after intervention (P<0.01).

[0091] 2.2 Levels of cytokines IL-6 and TNF-α in cell supernatant after drug intervention

[0092] The results are shown below. Figure 2Compared with the blank group, the levels of IL-6 and TNF-α in the cell supernatant of the model group were significantly increased, and the difference was statistically significant (P<0.01). Compared with the model group, the levels of IL-6 and TNF-α in the traditional Chinese medicine preparation group were significantly reduced after intervention (P<0.01); the control preparation group 2 reduced the level of TNF-α (P<0.05), but its regulatory effect was not as good as that of the traditional Chinese medicine preparation group.

[0093] 2.3 Status of thyroid function-related indicators TPOAb and TgAb in cell supernatant after drug intervention

[0094] Compared with the control group, the levels of TPOAb and TgAb in the model group were significantly increased (P<0.01), indicating significant autoimmune damage to the thyroid gland in the model group. Compared with the model group, the levels of the above indicators in each treatment group were reduced to varying degrees, and the differences were statistically significant (P<0.05, P<0.01). Among them, the traditional Chinese medicine preparation group had the most significant and stable effect on reducing each indicator, and the overall regulatory effect was better than that of control preparation group 1 and control preparation group 2. Figure 3 .

[0095] 2.4 Conclusion

[0096] In Hashimoto's thyroiditis, excessive apoptosis of thyroid follicular cells is a core pathological link leading to glandular destruction and hypothyroidism. This study detected apoptosis and disease marker antibodies in cells treated with different drugs. The results showed that the traditional Chinese medicine (TCM) preparation significantly reduced cell apoptosis and the levels of disease marker antibodies TPOAb and TgAb, thereby protecting thyroid function. Hashimoto's thyroiditis is an autoimmune disease characterized by chronic inflammation of the thyroid tissue. The inflammatory response can induce apoptosis of thyroid follicular cells, ultimately leading to glandular destruction. Th1 cells mainly secrete factors such as TNF-α; Th2 cells mainly secrete factors such as IL-6. IFN-γ regulates the local immune microenvironment, further activating Th1 and Th2 cells, forming an inflammatory amplification loop. Therefore, we selected IL-6 and TNF-α indicators for detection. After intervention with the TCM preparation, the levels of TNF-α and IL-6 in the model cells were significantly reduced, indicating that this TCM preparation can inhibit IFN-γ-induced inflammatory damage to normal human thyroid cells. This effect is a key mechanism for its intervention in the progression of Hashimoto's thyroiditis and warrants further in-depth research.

[0097] Through comparative experiments using drug substitution, this traditional Chinese medicine composition, through the rational combination of its various ingredients, achieved a synergistic therapeutic effect on Hashimoto's thyroiditis. The effect of this technique is not a simple additive effect of the individual components, but rather an overall effect produced under specific compatibility relationships.

[0098] Experiment 2

[0099] 2.1 Laboratory Animals

[0100] SPF-grade CBA / J mice, female, 6-8 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were housed in a constant temperature (22±2)℃, constant humidity (50%±10%), 12-hour light-dark cycle environment with free access to water and food. Experiments were conducted after 7 days of acclimatization.

[0101] 2.2 Experimental Drugs

[0102] Porcine thyroglobulin: purchased from Hefei Bomei Biotechnology Co., Ltd., product number: JT5894.

[0103] Tripterygium wilfordii polyglycoside tablets: purchased from Guizhou Hanfang Pharmaceutical Co., Ltd., national approval number: Z52020369.

[0104] 2.3 Modeling methods and experimental grouping

[0105] Six mice were randomly selected as the normal control group, and the remaining mice were used for model establishment. Mice underwent initial immunization by subcutaneous injection of 100 μg of antigen emulsion at multiple sites per mouse. PTg was thoroughly mixed with an equal volume of complete Freund's adjuvant to a final concentration of 2 mg / mL. Mice in the normal control group received PBS buffer instead of antigen emulsion injection twice weekly for 2 weeks. Mice underwent a second immunization by subcutaneous injection of 100 μg of antigen emulsion at multiple sites per mouse. PTg was thoroughly mixed with an equal volume of incomplete Freund's adjuvant to a final concentration of 2 mg / mL. Mice in the normal control group received PBS buffer instead of antigen emulsion once weekly for 3 weeks. Throughout the modeling process, the mice were continuously fed high-iodine solution (0.64 g / L). Starting from week 6 of modeling, the control group and the model group were administered the same volume of physiological saline by gavage. The high-dose group (3.66 g / kg / d), medium-dose group (1.83 g / kg / d), and low-dose group (0.92 g / kg / d) of traditional Chinese medicine preparations were administered the corresponding concentration of traditional Chinese medicine preparation suspension by gavage. The Tripterygium wilfordii polyglycoside tablet group (6.25 mg / kg / d) was administered once a day for 10 weeks.

[0106] 2.4 Serum levels of thyroid hormones TSH, TgAb, and TPOAb in each group of mice

[0107] Mice were fasted for 12 hours but allowed free access to water the day before treatment. Blood was collected by enucleation and stored overnight at 4°C. Serum was collected and the levels of TSH, TgAb, and TPOAb in the serum were detected by ELISA, following the instructions.

[0108] 2.5 Serum levels of cytokines IL-17 and IFN-γ in each group of mice

[0109] Mice were fasted for 12 hours but allowed free access to water the day before treatment. Blood was collected by enucleation and stored overnight at 4°C. Serum was collected and the levels of IL-17 and IFN-γ in the serum were detected by ELISA, following the instructions.

[0110] 2.6 Heart rate monitoring in each group of mice

[0111] Mice were anesthetized by intraperitoneal injection of sodium pentobarbital at a dose of 55 mg / kg body weight. After the animals were anesthetized, their heart rate (HR) was collected using an RM6240 multichannel physiological signal analyzer.

[0112] 2.7 5-HT levels in the prefrontal cortex of mice in each group

[0113] Mouse prefrontal lobe tissue was isolated, flash-frozen in liquid nitrogen, and transferred to a -80°C freezer for testing.

[0114] 2.8 Histopathological examination of thyroid tissue in mice of each group

[0115] Thyroid tissue was harvested, fixed in 10% formalin, routinely sectioned, and stained with hematoxylin-eosin (HE) for histopathological observation.

[0116] 2.9 Statistical Methods

[0117] All experimental data were analyzed using GraphPad Prism 8.0 statistical software, and one-way ANOVA was used for comparisons between groups. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered extremely statistically significant.

[0118] 3 Results

[0119] 3.1 Serum levels of thyroid hormones TSH, TgAb, and TPOAb in each group of mice

[0120] Compared with the normal control group, the serum TSH, TgAb, and TPOAb levels of mice in the model control group were significantly increased (P<0.01); compared with the model control group, the serum TSH, TgAb, and TPOAb levels of mice in the high-dose group of this traditional Chinese medicine preparation and the Tripterygium wilfordii polyglycoside tablet group showed a significant decreasing trend (P<0.01, P<0.05), as shown in Figure 4.

[0121] 3.2 Serum cytokine IL-17 and IFN-γ levels in each group of mice

[0122] Compared with the control group, the serum IL-17 and IFN-γ levels in the model group mice were significantly increased (P<0.01). Compared with the model group, the serum IL-17 and IFN-γ levels in the high-dose group of this traditional Chinese medicine preparation and the group treated with Tripterygium wilfordii polyglycoside tablets were significantly decreased (P<0.01). Compared with Tripterygium wilfordii polyglycoside tablets, the high-dose traditional Chinese medicine preparation showed superior inhibitory activity. Figure 5 .

[0123] 3.3 Heart rate monitoring of mice in each group

[0124] Compared with the control group, the heart rate of mice in the model group was not significantly increased (P>0.05). Compared with the model group, there were no abnormal heart rates in any of the traditional Chinese medicine preparation groups (P>0.05). Figure 6 .

[0125] 3.4 5-HT levels in the prefrontal cortex of mice in each group

[0126] Compared with the control group, the 5-HT content in the prefrontal cortex of mice in the model group was significantly decreased (P<0.01). However, after intervention with traditional Chinese medicine preparations, the content of 5-HT in all groups with different doses of traditional Chinese medicine preparations rebounded, with the high-dose group showing a significant difference (P<0.05). Figure 7 .

[0127] 3.5 Pathological examination of thyroid tissue in each group of mice

[0128] The control group mice had intact thyroid follicles, regular epithelial cell arrangement, relatively uniform follicular lumen size, and abundant colloid. Figure 8 A). In the model group mice, the thyroid follicle structure was disordered, with epithelial cells proliferating into clumps or cords, follicular cavities shrinking and varying in shape, and colloid distribution uneven (…). Figure 8 B). Compared to the model group, the other treatment groups showed varying degrees of improvement in the above pathological characteristics, among which the high-dose group of traditional Chinese medicine preparations ( Figure 8 C) The thyroid tissue structure was relatively clear, the oval or round structure of the follicles was basically restored, the shape was relatively regular, the follicular lumen area increased, the colloid content was rich and the staining was uniform, and the hyperplasia and enlargement of follicular epithelial cells were significantly improved. No significant improvement was observed in the medium and low dose groups of the traditional Chinese medicine preparation and the high dose group of Tripterygium wilfordii polyglycoside tablets. Figure 8 DF).

[0129] 4. Conclusion

[0130] By establishing a mouse model of Hashimoto's thyroiditis, this study investigated the regulatory effects of this traditional Chinese medicine preparation on thyroid function, immunity, and tissue structure. Experimental results showed that this traditional Chinese medicine preparation exhibited good therapeutic effects at multiple levels. Regarding thyroid function, TPOAb and TgAb are hallmark antibodies of Hashimoto's thyroiditis, and their titers were positively correlated with the degree of thyroid damage. TSH is an important indicator of thyroid function. Thyroid hormone results showed that this traditional Chinese medicine preparation could effectively reduce TSH, TgAb, and TPOAb levels, thereby regulating the synthesis and release of thyroid hormones. In terms of immunological indicators, a core immune disorder in Hashimoto's thyroiditis is the disruption of the balance between Th17 cells (promoting inflammation) and regulatory T cells (suppressing inflammation). IFN-γ, a major Th1 cytokine, can directly induce thyroid cell apoptosis, while IL-17, a core effector molecule secreted by Th17 cells, can continuously induce the expression of other inflammatory factors, promote inflammation progression, and promote thyroid follicular cell apoptosis, ultimately leading to the destruction of thyroid tissue structure. The results showed that the traditional Chinese medicine preparation significantly reduced IFN-γ and IL-17 levels after intervention, suggesting that it may reduce immune damage to thyroid tissue by inhibiting Th1 and Th17 cell-mediated immune responses and correcting immune imbalance. Regarding the pathological morphology of thyroid tissue, the high-dose group of the traditional Chinese medicine preparation significantly improved thyroid tissue cell damage. Although existing treatments can reduce Hashimoto's thyroiditis marker antibodies and related inflammatory factors, the side effects of cardiac arrhythmias, such as those associated with Tripterygium wilfordii glycoside tablets, are unavoidable. However, the data from this study showed that the traditional Chinese medicine preparation had no significant effect on the heart rate of the animals.

[0131] In recent years, clinical findings have shown a gradual increase in the incidence of mood disorders in patients with Hashimoto's thyroiditis. Therefore, we selected 5-HT, an important neurotransmitter involved in mood regulation, for testing. The results indicated that after intervention with this traditional Chinese medicine preparation, the relevant indicator of mood abnormalities, 5-HT, was improved, suggesting that this preparation may have a certain positive effect on mood regulation related to Hashimoto's thyroiditis.

[0132] In summary, this traditional Chinese medicine preparation can improve the pathological state of Hashimoto's thyroiditis through multiple pathways and targets. It shows good effects in regulating thyroid function, regulating immune balance, and repairing tissue structure, demonstrating a significant comprehensive therapeutic effect. Furthermore, this preparation also shows a positive effect in alleviating the depressive mood often associated with Hashimoto's thyroiditis, a topic of clinical concern in recent years. Overall, this traditional Chinese medicine preparation has a significant comprehensive therapeutic effect, and no obvious adverse reactions were observed.

[0133] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A traditional Chinese medicine preparation for treating Hashimoto's thyroiditis, characterized in that, The traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 5-20 parts of pumice, 5-20 parts of oyster shell, 1-10 parts of water lily seed, 4-18 parts of turmeric, 4-20 parts of lily bulb, 5-20 parts of bupleurum, 3-20 parts of safflower seed, 3-15 parts of salsa seed, 2-15 parts of red vine, 1-12 parts of turmeric, and 1-10 parts of licorice.

2. The traditional Chinese medicine preparation according to claim 1, characterized in that, The traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 8-15 parts of pumice, 8-15 parts of oyster shell, 2-8 parts of water lily seed, 5-12 parts of turmeric, 9-18 parts of lily bulb, 6-12 parts of bupleurum, 6-12 parts of safflower seed, 5-9 parts of salsa seed, 3-12 parts of red vine, 2-10 parts of turmeric, and 2-6 parts of licorice.

3. The traditional Chinese medicine preparation according to claim 1, characterized in that, The traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 10 parts pumice, 10 parts oyster shell, 3 parts water lily seed, 8 parts turmeric, 15 parts lily bulb, 8 parts bupleurum, 8 parts safflower seed, 8 parts salsa seed, 9 parts red vine, 5 parts turmeric, and 3 parts licorice.

4. The method for preparing the traditional Chinese medicine preparation for treating Hashimoto's thyroiditis according to any one of claims 1 to 3, characterized in that, The preparation process steps of the traditional Chinese medicine preparation are as follows: (1) Grind each component into a fine powder and pass it through a 100-mesh sieve; (2) Mix the fine powders of each component to obtain a drug mixture powder, add starch and water, and make into pills; (3) The prepared pills are dried to obtain the Chinese medicine water pills.

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