Effect of Integrated Treatment with Traditional Chinese Medicine on Hashimoto’s Thyroiditis Patients

What is Known and Objective . To explore the efects of traditional Chinese medicine (TCM) combined with levothyroxine (L-T4) on thyroid autoantibodies, infammation, and sleep quality in Hashimoto’s thyroiditis patients. Methods . Patients were randomly divided into group A and group B. Group A was treated with L-T4 alone, while group B was treated with integrated TCM and L-T4. TCM symptoms were quantifed before and after treatment as well as PSQI. Blood samples were taken to detect clinical indicators and thyroid autoantibodies. Cytokines in serum and thyroid tissues were analyzed by ELISA and RT-PCR. Results and Discussion . Totally, 196 patients were enrolled in the study, and there were no diferences between group A and group B at the baseline. TCM treatment efectively reduced the levels of TGAb and TPOAb and was a protective factor for the improvement of Hashimoto’s thyroiditis antibody titers, p < 0 . 05. Te serum expressions of IL-17A, IL-6, and IFN-c in group B after treatment were lower than those in group A and before, while the IL-10 level was raised from the baseline, p < 0 . 05. Similar results were found in the comparison of IL-17A, IFN-c , and IL-10 in thyroid tissues of group A, B, and control, p < 0 . 05. Besides, with integrated treatment, all TCM symptoms except for poor memory were improved as well as sleep quality and mood, p < 0 . 05. However, these changes were not observed before and after treatment with L-T4 in group A. What is New and Conclusion . Te integrated treatment with TCM had a signifcant efect on thyroid autoantibodies, infammation, and sleep quality in Hashimoto’s thyroiditis patients and provided a new and efective method for future treatment.


What Is Known and Objective
Hashimoto's thyroiditis (HT), also known as chronic lymphocytic thyroiditis (CLT), is a common clinical autoimmune thyroid disease (AITD) [1].In recent years, the incidence of HT has increased.Te latest fow survey data show that [2,3] at present, the prevalence of HT has increased to 5-10%, and the course of disease is prolonged, which is common in women.Te immune mechanism of the disease is complex.Te increase of serum TGAb and TPOAb is its clinical marker, which often coexists with other autoimmune diseases and tumor diseases, and the harm to health cannot be ignored.At present, L-T4 is the main drug used in western medicine to treat HT.L-T4 can increase the level of thyroxine in patients with clinical hypothyroidism and subclinical hypothyroidism and reduce TSH.Te curative efect is positive, but it is easy to cause insomnia, palpitation, spontaneous sweating, and even myocardial ischemia.Long-term use has the risk of osteoporosis and fracture.
Chinese herbal medicine, as traditional Chinese medicine, has unique advantages in those aspects of medical treatment, prophylaxis, and health protection and has been paid more and more attention and acceptance from researchers at home and abroad.HT belongs to the category of "gall disease" in traditional Chinese medicine.It is caused by internal emotional injury, diet and water, and soil loss, resulting in circulation of vital energy stagnation, phlegm coagulation, and blood stasis in front of the neck.Te infammatory process in the early stage of the disease is consistent with the theory of "excess circulation is fre" in TCM [4].Te pharynx where the thyroid located is the part of the liver meridian, so "gall disease" is closely related to the liver.Te traditional Chinese medicine "Shu Gan Qing Huo Decoction" is cold in nature and belongs to the liver meridian.It has the functions of clearing heat and detoxifcation, cooling blood and eliminating spots, and purging fre and calming shock.Previous study used "Shu Gan Qing Huo Decoction" to treat HT in the early stage and found that it may relieve symptoms and reduce thyroid autoantibodies [5].
Currently, there is no specifc treatment for HT, and oral medication, selenium, and L-T4 tablets, as well as surgery and low-intensity laser methods, are not confrmed clear therapeutic efects, or the long-term toxicity and side efects are unpredictable.Terefore, this topic organically combines the above two methods by the randomized controlled study, scientifcally summarizes their respective advantages, evaluates the efcacy of integrated treatment with TCM, and explores a new scheme for HT intervention.

2.1.
Patients.Hashimoto's thyroiditis patients were collected from the outpatient department of Endocrinology, Huadong Hospital Afliated to Fudan University, Shanghai, China, during January 2019 to December 2021.Tey were divided randomly into two groups by a random number table: group A was treated with L-T4 alone, while group B was combined with Chinese herbal medicine after 2-week L-T4 treatment.Te estimated sample size was about 200, based on the calculated formula: n � z 2 σ 2 /d 2 and the pre-experiment.Total treatment course of the two groups was 12 weeks, and the primary outcomes were thyroid function and cytokines before and after treatment.Te secondary outcomes were TCM symptoms, PSQI, and HAMA scores.Te calculation formula for BMI was weight divided by the square of height (BMI � kg/m 2 ).Te clinical study was approved by the Ethics Committee of Huadong Hospital afliated to Fudan University (Number: 20190108), and all subjects were registered anonymously.

Diagnostic Criteria.
Te diagnostic criteria of HT in all patients were referred to Chinese guidelines for the diagnosis and treatment of thyroid diseases issued by the endocrinology branch of the Chinese Medical Association [6].Te details were as follows: (1) goiter, tough texture, isthmic pyramidal lobe enlargement or asymmetry, or accompanied by nodules; (2) serum TPOAb and or TGAb were positive; (3) thyroid ultrasound examination showed that the thyroid gland showed difuse uneven changes, or with isthmus thickening, uneven hypoechoic areas of varying degrees, or with thyroid nodules; (4) thyroid fne-needle biopsy revealed difuse lymphocyte and plasma cell infltration, lymphoid follicles, and fbrous tissue hyperplasia in the thyroid gland.HT can be diagnosed if both (1) (2) (3) and /or (4) were present.
Hypothyroidism was a systemic metabolic syndrome caused by decreased synthesis and secretion of thyroid hormone or weakened action of peripheral tissues.
According to the degrees of hypothyroidism, it can be divided into clinical hypothyroidism and subclinical hypothyroidism.Clinical hypothyroidism: the laboratory examinations showed an increase in serum TSH and a decrease in FT4 or TT4.Subclinical hypothyroidism: there may be no obvious hypothyroidism in clinical performance, but laboratory tests showed elevated TSH and normal FT4 or TT4.

Treatment. Levothyroxine (L-T4
) preparation: the L-T4 tablets were produced by the German Merck company, with 50 μg per tablet, taken in the morning, once a day.Te thyroid levels were remeasured after 6 and 12 weeks of treatment, and individualized L-T4 doses were formulated according to the thyroid function to ensure that there was no overdose.Te prescription of traditional Chinese medicine "Shu Gan Qing Huo recipe": bupleurum root 6 g, white peony root 6 g, jiaobaizhu 9 g, white Poria cocos 9 g, Ligusticum chuanxiong 6 g, tangerine peel 6 g, Prunella vulgaris 9 g, Scutellaria baicalensis 9 g, and indigo naturalis 3 g.Te granules were packed in two bags according to the daily dose, one bag each time, twice a day, and decocted with warm water half an hour after breakfast and dinner.

Clinical Observations
2.5.1.Symptoms.Te main TCM symptoms of Hashimoto's thyroiditis patients, including weakness, irritability, poor appetite, neck discomfort, and poor memory, were divided into 4 grades according to diferent degrees of symptoms.None marked as 0, mild as 1, moderate as 2, and severe as 3 points.

Sleep Quality.
Te Pittsburgh sleep quality index (PSQI) is a self-reported questionnaire used to evaluate sleep quality and disorders.It consists of seven components [7], the score range of each question is 0-3, and the total score is 0-21.Te higher the score, the worse the sleep quality.

Anxiety.
Te Hamilton anxiety scale (HAMA) was used to assess the severity of patients' anxiety symptoms.All options were scored 0-4 points with a total of 14 items.Level 2 Journal of Clinical Pharmacy and Terapeutics 0 was asymptomatic, and level 4 was extremely severe.All participants had 15 minutes to complete the questionnaire under the guidance of a trained researcher.

2.7.
Real-Time qPCR.Te total RNA was isolated from thyroid fne-needle aspiration biopsy samples with Trizol reagent (Invitrogen Life Technologies Inc., Carlsbad, CA, USA), and GAPDH was used as the internal control.A quantity of 1 μg RNA was reverse-transcribed using the MMLV reverse transcription system (Promega, Madison, WI, USA) as previously described [9].Real-time reverse transcription-polymerase chain reactions (RT-PCR) were performed using TB Green Premix Ex Taq (RR420A; Takara) through the StepOne Real-Time PCR System (Applied Biosystems, Foster City, California).Relative expression of mRNA was calculated after normalization to GAPDH, using the comparative 2 −ΔΔCt method as described previously [10].Before treatment, the quantifcations of all symptoms in group A and group B were not statistically signifcant.After treatment with integrated TCM and L-T4, all symptoms were improved except for poor memory, p < 0.05.See Table 2 for details.Te efective rates of group B in treating TCM syndromes of "irritability," "poor appetite," and "neck discomfort" were 37.5%, 38.7%, and 56.3%, respectively, signifcantly higher than those of group A, 5%, 10%, and 20%, p < 0.05.Te total efective rate of clinical efcacy was 78.43% (80/102) in group B and 55.32% (52/94) in group A. Terefore, the TCM treatment group was superior to the control group (p < 0.05).

Tyroid Function and Antibodies. With traditional
Chinese medicine and L-T4, the average value of FT4 in both groups was increased and TSH decreased when compared with before treatment, p < 0.05 (Figure 2(a)).Tat is, the hypothyroidism of patients in the two groups could be recovery after treatment.In addition, the paired t test showed that after treatment the thyroid autoantibodies in group B were far less than before, though no statistical diference in value except for TPOAb (Figure 2(b)).However, the proportion of patients with one improved thyroid antibody titer in group A was 52.5%, while that in group B was 84.4%, p � 0.006.Te proportion of both thyroid antibody tilters improved was 15.0% in group A and 53.1% in group B, p � 0.001.Of note, the improvement of antibody titers referred to a decrease by 10% from the baseline.As shown in Table 3, the risk factors related to the improvement of one antibody titer were L-T4 doses (OR � 1.006, p � 0.008) and group B/group A (OR � 0.198, p � 0.021) and that of two antibody titers improvement were L-T4 doses (OR � 1.005, p � 0.007) and group B/group A (OR � 0.185, p � 0.008).In other words, when compared with group A, group B was a protective factor for the improvement of Hashimoto's thyroiditis antibody titer.It can be seen that the therapeutic of integrated TCM and L-T4 on Hashimoto's thyroiditis patients was obvious.As can be seen from Table 4, the cytokine levels of IL-17A, IFN-c, IL-6, and IL-10 were no statistical diferences at the baseline, p > 0.05.When after treatment, the levels of proinfammatory factors in group B were lower than those in group A, p < 0.05.While as an anti-infammatory cytokine, the level of IL-10 was increased, although there was no signifcance between the two groups.Besides, through the paired t test, all the cytokines in group B had dramatically changed compared with those before treatment, p < 0.05, indicating that "Shu Gan Qing Huo Decoction" can alleviate the infammation of Hashimoto's thyroiditis patients.

mRNA Expressions of Cytokines in Tyroid Tissues.
For Hashimoto's thyroiditis with solid nodules of more than 1 cm, according to the 2020 Chinese guidelines for ultrasound malignant risk stratifcation of thyroid nodules (C-TIRADS), fne-needle aspiration biopsy (FNAB) was performed for the patients with C-TIRADS grading 3 and above   who were willing to undergo.After treatment of 3 months, 12 patients (4 men and 8 women) were recruited in group A (A, n � 12) and 11 patients (2 men and 9 women) in group B (B, n � 11).12 of the 196 patients (2 men and 10 women) before treatment were taken as the control group (C, n � 12).Te pathological diagnosis of all the patients was Hashimoto's thyroiditis or combined with nodular goiter.Te real-time qPCR results of FNAB are revealed in Figure 3. Te expression levels of IL-17A and IFN-c in group B were much lower than those in group A and C, p < 0.05.On the contrary, as a protective cytokine, the level of IL-10 after treatment with TCM and L-T4 was higher than group A (p > 0.05) and C (p < 0.05).However, it was found that the expressions of the four cytokines in comparison between groups A and C were of no signifcance.It was further proved from histology that integrated treatment with TCM played an important role on improving the infammation of Hashimoto's thyroiditis.

Discussion
In view of the complexity of HT pathogenesis, human beings have not yet recognized the essence of HT.Recent studies have shown that HT development depends on an immune defect in an individual with genetic susceptibility together with environmental factors [2].Unfortunately, there is still a lack of efective therapeutic drugs for HT.Although thyroid function can be rectifed by L-T4, it had no efect on autoantibodies, which can also be proved by our study.Te use of the traditional Chinese medicine "Shu Gan Qing Huo Decoction" confrmed that the integrated treatment could efectively reduce the titers of TGAb and TPOAb in Hashimoto's thyroiditis patients with subclinical and clinical hypothyroidism, p < 0.05.
Bossowski and Otto Buczkowska pointed out that serum anti-thyroid antibodies, especially anti-TG and anti-TPO, and more rarely TSH stimulation blocking antibody (TSBAb), were positively correlated with the increase of thyroiditis and the development of hypothyroidism [3].TGAb and TPOAb can directly or synergistically participate in the autoimmune response of thyroid tissues.Tere was evidence that TGAb and TPOAb levels were signifcantly correlated with the degree of cell infltration in HT thyroiditis and HT low-density imaging detected by ultrasound [12].Terefore, TGAb and TPOAb have become characteristic clinical markers of HT.Previous studies have reported that TCM treatment can notably decrease thyroid antibodies, as well as reduce goiter [4,13].It can be seen in our results that the TCM group was a protective factor for the improvement of Hashimoto's thyroiditis antibody titers.We have reason to speculate that prolonging the treatment time of TCM and L-T4 may have a more obvious efect on Hashimoto's thyroiditis.
In addition, researchers have found that the incidence of HT was related to T17 cells [14], and the transforming growth factor (TGF)-β, IL-6, IL-10, and IFN-c all played a promoting role in the diferentiation and formation of T17 [15,16].In the study, the analysis of cytokines by 6 Journal of Clinical Pharmacy and Terapeutics ELISA from patient serum revealed that after combining with TCM treatment, the proinfammatory factors decreased signifcantly when compared with the L-T4 group and before, p < 0.05.Similar results were obtained in histology by RT-PCR detection from FNAB and strongly suggested that "Shu Gan Qing Huo Decoction" can alleviate the infammation of Hashimoto's thyroiditis patients.As a secondary end point, the curative efect standard of TCM syndrome was assessed in our study.We can see from Table 2 that all symptoms were improved after integrating TCM, except for poor memory, p < 0.05.In addition, we investigated the PSQI and HAMA before and after medication.PSQI questionnaire contained seven component scores: subjective sleep quality, sleep latency, sleep duration, habitual sleep efciency, sleep disturbance, use of sleeping medication, and daytime dysfunction [7].Te score range of each question was 0-3 in PSQI and 0-4 in HAMA.Te results showed that the scores of both questionnaires in the TCM treatment group were apparently lower than the L-T4 treatment group and baseline, p < 0.05.Te PSQI score even increased after intervened with L-T4 alone, although no statistical signifcance, indicating that TCM treatment had a certain role on improving patients' sleep quality and mood.
Combination treatment can comprehensively regulate the local lesions and systemic symptoms of patients, making up for the asynchronous phenomenon of functional and immune relief caused by the single use of L-T4.However, there were some limitations in our study.Firstly, as a prospective study, the intervention course was only 12 weeks due to case dropout and loss of follow-up; thus, the evaluation of long-term efcacy of TCM may not be as expected.Secondly, there were various clinical examination errors caused by multiple individuals and multiple batch tests in this study, which may lead to low statistical power.Tirdly, because of diferent compliance, it was not possible to ensure that each patient followed our instructions to take the medications, and we needed to expand the sample size as much as possible.Fourthly, the number for FNAB was relatively small, making it difcult to avoid biased experimental results and inadequate analysis.

What Is New and Conclusion
In conclusion, our study revealed that integrated treatment of TCM and L-T4 had a signifcant efect on thyroid autoantibodies, infammation, and sleep quality in Hashimoto's thyroiditis patients.It was drafted with reference to the guiding principles for clinical research of new Chinese medicine (trial) [11].Signifcant efect: symptoms basically disappeared, signs basically returned to normal, serum TSH level, and anti-thyroid autoantibodies were close to normal; Efective: the symptoms were signifcantly improved, the related signs such as goiter were alleviated, and the serum TSH level and anti-thyroid autoantibody examination were improved; inefective: symptoms and signs are not improved, the serum TSH level is increased or decreased, and anti-thyroid autoantibodies are not changed or increased.Te total efective rate of clinical efcacy in the TCM group was superior to the control group, p < 0.05, providing a new method for the disease treatment.
Signifcant efect: the clinical symptoms and signs of TCM were signifcantly improved, and the score reduction rate was ≥70% and <90%.Efective: the clinical symptoms and signs of TCM are improved, and the score reduction rate is ≥30% and <70%.Inefective: the clinical symptoms and signs of TCM have not improved signifcantly, or even worsened, and the score reduction rate is less than 30%.
[11]acteristics.Te fow diagram of this study is shown in Figure1.A total of 263 subjects were enrolled in our study, of which 52 were excluded according to in-and ex-criteria and 15 were dropped out of the trial; most of them were caused by bad compliance.Finally, there were 102 in group A (17 males and 85 females) and 94 in group B (15 males and 79 females), with an average age of 53.60 ± 16.64 years and 49.81 ± 13.60 years, respectively, p > 0.05.At the baseline, there was no signifcant diference between the two groups in BMI, FPG, HbA1c, ALT, Cr, TC, TG, LDL-C, and HDL-C, p > 0.05.Tus, the infuence of confounding factors was excluded in the subsequent statistical results.Te clinical characteristics of the participants are summarized in Table1.Te average daily dose of L-T4 was 30.0 μg in group A and 28.12 μg in group B at the terminal point, p > 0.05.3.2.Symptoms' Assessment.Te curative efect standard is drafted with reference to the guiding principles for clinical research of new Chinese medicine (Trial)[11].Clinical recovery: the clinical symptoms and signs of TCM disappear or basically disappear, and the score reduction rate is ≥90%.

Table 1 :
Quality and Cytokine Levels.Before treatment, the PSQI and HAMA scores in group A and group B were no statistical diferences, p > 0.05.After intervention, the PSQI in group B decreased to 4.66 ± 2.31, while that in group A was 8.80 ± 4.62, p < 0.05.Similarly, HAMA dropped to 6.25 ± 2.70 in group B and 8.83 ± 5.06 in group A, p < 0.05 (shown in Table 4).A marked decline of PSQI and HAMA scores in group B after treatment compared with before was found by the paired t test.Te results Clinical characteristics of participants between two groups.

Table 2 :
Comparison of symptom quantifcations before and after treatment.

Table 3 :
Analysis of risk factors related to the improvement of the antibody titer.

Table 4 :
Comparison of cytokine levels before and after treatment.Note.Compared with that before treatment of group A, * p < 0.05; compared with that before treatment of group B, # p < 0.05; compared with that after treatment of group A, ∆ p < 0.05.