第一部分 中药小RNA干预Graves病的实验研究

【背景】

Graves病（Graves’ disease，GD）是甲状腺功能亢进症的最常见类型。当前抗甲状腺药物（antithyroid drugs，ATDs）、放射性碘和手术是常规治疗手段，但均存在一定局限性。因此探索GD的新型治疗策略将对临床有所裨益。

钠/碘同向转运体（sodium iodide symporter，NIS）是甲状腺激素合成的关键蛋白。研究表明，GD患者甲状腺组织中NIS mRNA表达水平显著升高，且NIS自身抗体在GD患者中阳性率高于正常人群，提示NIS 可能是GD的潜在治疗靶点。

小RNA（small RNA，sRNA）是一类长度不超过200 nt的非编码RNA，能够直接调控基因表达。本草sRNA数据库收录了大量来源于中药的sRNA序列，其中部分序列已在多种疾病模型中证实具有显著疗效，显示出中药sRNA在疾病干预中的应用前景。因此，本研究旨在从该数据库中筛选出可靶向NIS的sRNAs并验证其疗效。

【方法】

本研究基于本草sRNA数据库，运用生物信息学方法筛选可靶向NIS的sRNA，并通过体内外实验验证其疗效。体外实验中，将候选sRNA转染至甲状腺细胞后，检测细胞NIS mRNA的表达水平及细胞活力变化。体内实验部分，通过肌肉注射表达TSHR的重组腺病毒构建GD小鼠模型。sRNA干预后测定甲状腺功能指标并分析甲状腺组织的病理改变。

【结果】

本研究筛选出一条可靶向NIS的小核酸（NIS-sRNA-1）。细胞实验结果显示，NIS-sRNA-1可显著下调NIS基因的转录水平。动物实验中，NIS-sRNA-1干预组小鼠的甲状腺功能指标下降，NIS表达水平降低，但与阴性对照组相比均无显著统计学差异；组织学分析显示甲状腺滤泡结构趋于恢复，滤泡增生缓解。

【结论】

本草sRNA数据库中的NIS-sRNA-1，在体外可显著下调NIS的转录水平，体内实验显示其具有缓解甲亢的趋势，提示其具有治疗GD的潜在价值，未来还需扩大样本量深入研究。

【关键词】 Graves病；中草药来源的小RNA；靶向治疗；钠/碘同向转运体

第二部分 基于逆向网络药理学探讨Graves病合并骨质疏松症的发病机制及中药组方预测

【背景】

Graves病（Graves’ disease，GD）可加速骨转换、降低骨密度，是导致继发性骨质疏松症的内分泌疾病之一。研究显示，长期GD患者骨折风险显著升高。然而，目前尚无针对GD合并骨质疏松症的公认治疗方案。中医“异病同治”理论以及中药多成分、多靶点等特点，在多系统疾病治疗中展现出独特的优势。逆向网络药理学可以从疾病相关靶点出发，系统筛选相关活性成分及中药，为中药干预提供参考方案。因此，本研究构建GD小鼠模型，评估长期GD状态对骨微结构和骨代谢的影响，并基于逆向网络药理学探讨GD合并骨质疏松症的潜在机制，筛选具有干预潜力的中药活性成分及组方，旨在为GD合并骨质疏松症的中药治疗提供理论依据和参考。

【方法】

通过肌肉注射表达TSHR的重组腺病毒构建GD小鼠模型，采用ELISA测定骨代谢相关指标，micro-CT检测骨微结构改变。从GeneCards数据库获取GD和继发性骨质疏松症相关基因，利用STRING平台构建交集基因的蛋白互作网络（protein-protein interaction，PPI），随后进行GO功能注释和KEGG通路富集分析。应用Cytoscape软件筛选关键靶点，并逆向筛选对应的活性成分与中药。选取度值较高的靶点与成分对进行分子对接，并整理度值≥4的中药的性味归经特征。

【结果】

GD模型小鼠表现出骨微结构受损，骨小梁数量减少甚至消失。逆向网络药理学分析共筛选出122个GD与继发性骨质疏松相关的交集靶点，其主要富集于PI3K-AKT等信号通路。进一步筛选出12个关键靶点，并逆向匹配到槲皮素、木犀草素等30种潜在活性成分，368种中药。度值排名前5的关键靶点与活性成分的分子对接结果良好且稳定。度值≥4的中药多具有苦味、寒性，主要归肝经与肺经。

【结论】

GD可导致骨微结构受损。逆向网络药理学分析预测，木蝴蝶、菊花等苦味寒性药及其活性成分如木犀草素、槲皮素等，可能通过多靶点、多通路机制发挥对GD合并骨质疏松症的干预作用。

【关键词】 Graves病；骨质疏松症；逆向网络药理学

第三部分 甲状腺功能正常体检人群维生素D与甲状腺激素敏感性的相关性

【背景】

维生素D是一种脂溶性类固醇，多项研究表明维生素D水平与甲状腺激素以及促甲状腺激素水平存在关联，但相关结果不尽一致，这种差异可能与甲状腺激素敏感性的变化有关。近期研究显示，在甲状腺功能处于正常范围的群体中，甲状腺激素敏感性的下降也可能增加高尿酸血症、糖尿病等代谢性疾病的发病风险。因此，本研究旨在探讨甲状腺功能正常人群的维生素D水平与甲状腺激素敏感性的相关性。

【方法】

本研究为横断面研究，纳入2023年12月至2024年2月在北京协和医院体检的5,894例甲状腺功能正常者。维生素D缺乏定义为25（OH）D＜20 ng/ml。通过甲状腺反馈分位数指数（thyroid feedback quantile-based index，TFQI）、促甲状腺激素指数（TSH index，TSHI）、促甲状腺激素-T4抵抗指数（thyrotroph thyroxine resistance index，TT4RI）和游离三碘甲状腺原氨酸（free triiodothyronine，FT3）/游离甲状腺素（free thyroxine，FT4）比值评估甲状腺激素敏感性。采用多元线性回归与多因素Logistic回归分析25（OH）D水平与甲状腺激素敏感性的关联。

【结果】

维生素D缺乏组TFQI更低（P=0.033），FT3/FT4比值更高（P＜0.001）。在调整性别、年龄等混杂因素后，多元线性回归分析表明，血清25（OH）D水平与TFQI呈正相关，与FT3/FT4呈负相关（均P＜0.05）。Logistic回归显示，非缺乏组中枢和外周甲状腺激素敏感性受损的发生风险增加[TFQI（OR=1.16，95%CI：1.01~1.34）；FT3/FT4（OR=1.23，95%CI：1.05~1.45）]（均P＜0.05）。

【结论】

在甲状腺功能正常人群中，维生素 D水平越高，甲状腺激素敏感性受损的风险越高。

【关键词】 维生素D；甲状腺激素敏感性；甲状腺功能正常；体格检查

Part I  Experimental Study on the Intervention of Graves' Disease by sRNA derived from Chinese Herbal Medicine

Background:

Graves’ disease (GD) is the most common form of hyperthyroidism. The conventional treatment approaches include antithyroid drugs (ATDs), radioactive iodine, and surgery. However, these treatments have certain limitations. Therefore, exploring novel therapeutic strategies for GD holds significant clinical value.

The sodium iodide symporter (NIS) is a key protein involved in thyroid hormone synthesis. Studies have shown that NIS expression is significantly elevated in patients with GD, and the prevalence of NIS autoantibodies is higher than that in the general population, suggesting that NIS may represent a potential therapeutic target for GD.

Small RNA (sRNA), which is non-coding RNAs no longer than 200 nucleotides, plays a crucial role in the post-transcriptional regulation of gene expression. The herbal sRNA database contains a vast collection of sRNA sequences derived from traditional Chinese medicines. Some of these sRNAs have shown promising therapeutic effects in various disease models, highlighting the potential of herbal sRNAs in disease intervention. This study aimed to screen for sRNAs that target NIS from the database and to assess their therapeutic efficacy.

Methods：

Based on the herbal sRNA database, bioinformatics approaches were employed to screen for sRNA targeting NIS, which were subsequently validated both in vitro and in vivo. In vitro, after transfecting the candidate sRNAs into thyroid cells, the expression level of NIS mRNA and the changes in cell viability were detected. In vivo, a GD mouse model was established by intramuscular injection of recombinant adenoviruses expressing the TSHR. After the intervention with sRNAs, the thyroid function indexes were measured, and the pathological changes of the thyroid tissue were analyzed.

Results:

A specific sRNA targeting NIS (NIS-sRNA-1) was identified, which significantly downregulated NIS gene transcription in vitro. Furthermore, intervention with NIS-sRNA-1 in the GD group led to a reduction in thyroid function indices and a decrease in NIS expression. However, no statistically significant differences were observed when compared to the negative control group. Histological analysis revealed partial restoration of the thyroid follicular structure, with a reduction in follicular hyperplasia.

Conclusion:

NIS-sRNA-1 from the herbal sRNA database significantly downregulated NIS transcription in vitro, and in vivo experiments showed a trend toward alleviating hyperthyroidism, suggesting its potential therapeutic value for GD. Further research with a larger sample size is required for deeper exploration.

Keywords

Graves’ disease; sRNA from Chinese herbal medicine; Targeted therapy; Sodium iodide symporter

Part II  Exploring Pathogenesis of Graves’disease combined Secondary Osteoporosis and Predicting Traditional Chinese Medicine Prescription Based on Reverse Network Pharmacology

Background:

Graves’ disease (GD) can accelerate bone turnover and decrease bone mineral density, and it is one of the endocrine diseases leading to secondary osteoporosis. Studies have shown that the fracture risk in patients with long-term GD is significantly increased, and abnormal bone metabolism may persist even after the restoration of thyroid function. However, currently, there is no recognized treatment regimen for GD complicated with osteoporosis. The traditional Chinese medicine (TCM) principle of “same treatment for different diseases” and the characteristics of TCM, such as multiple components and multiple targets, have demonstrated unique advantages in the treatment of multi-system diseases. Reverse network pharmacology, which begins with disease-related targets to identify active compounds and candidate herbs, provides a systematic strategy for TCM intervention. Therefore, in this study, a GD mouse model was established to evaluate the effects of prolonged GD on bone microstructure and bone metabolism. In addition, reverse network pharmacology was utilized to explore the potential mechanisms underlying GD complicated with osteoporosis, and to screen active compounds and herbal formulae with potential therapeutic effects, aiming to provide a theoretical basis and reference for the treatment with relevant TCM herbs.

Methods：

A GD mouse model was established via intramuscular injection of recombinant adenoviruses encoding the TSHR. The indices related to bone metabolism were determined by ELISA, and the changes in bone microstructure were detected by micro-CT. Genes associated with GD and secondary osteoporosis were retrieved from the GeneCards database. A protein–protein interaction (PPI) network of intersecting genes was generated through the STRING platform, followed by GO functional annotation and KEGG pathway enrichment analyses. Key targets were identified using Cytoscape, and reverse screening was performed to identify corresponding active compounds and TCMs. Molecular docking was conducted on target-compound pairs with higher degree value. TCMs with degree value ≥ 4 were analyzed for their properties, flavors, and meridian tropism.

Results:

GD model mice exhibited impaired bone microstructure, with reduced or absent trabecular bone. Reverse network pharmacology identified 122 common targets related to GD and secondary osteoporosis, mainly enriched in pathways such as PI3K-AKT. Twelve key targets were selected, along with 30 potential active ingredients, including quercetin and luteolin, and 368 TCMs matched to these targets. Molecular docking revealed stable interactions between the top 5 key targets and their corresponding compounds. TCMs with degree value ≥ 4 were primarily bitter and cold in nature, acting mainly on the liver and lung meridians.

Conclusion:

GD can lead to damage to the bone microstructure. Reverse network pharmacology analysis predicted that bitter cold herbs such as Dendrobium and Chrysanthemum, along with their active components such as luteolin and quercetin, may exert their intervention effects on GD-related osteoporosis through a multi-target and multi-pathway mechanism.

Keywords：

Graves’ disease; Osteoporosis; Reverse network pharmacology

Part III  Association Between Vitamin D and Thyroid Hormone Sensitivity in Euthyroid Adults Undergoing Health Examination

Background：

Vitamin D is a lipid-soluble secosteroid. Numerous studies have demonstrated an association between vitamin D levels and thyroid-stimulating hormone (TSH) as well as thyroid hormones, although the findings are not entirely consistent. These discrepancies may be attributed to variations in thyroid hormone sensitivity, which reflects the body's responsiveness to thyroid hormones. Recent studies have suggested that even in euthyroid individuals, impaired sensitivity to thyroid hormone may elevate the risk of various metabolic disorders, including hyperuricemia and diabetes. Therefore, the aim of this study is to investigate the relationship between vitamin D levels and thyroid hormone sensitivity in euthyroid individuals.

Methods：

This cross-sectional study included 5,894 euthyroid individuals who underwent health examinations at the Department of Health Management, Peking Union Medical College Hospital, from December 2023 to February 2024. Vitamin D deficiency was defined as serum 25-hydroxyvitamin D [25(OH)D] levels < 20 ng/mL. Thyroid feedback quantile-based index (TFQI), TSH index (TSHI), thyrotroph thyroxine resistance index (TT4RI), and the ratio of free triiodothyronine (FT3)/free thyroxine (FT4) were calculated to assess thyroid hormone sensitivity. Multivariable linear regression and Logistic regression analyses were employed to evaluate the relationship between 25(OH)D levels and thyroid hormone sensitivity.

Results：

The vitamin D deficiency group exhibited a lower TFQI (P=0.033) and a higher FT3/FT4 ratio (P<0.001). After adjusting for confounding factors such as sex and age, multivariable linear regression analysis revealed that serum 25(OH)D levels were positively correlated with TFQI and negatively correlated with FT3/FT4 (all P<0.05). Furthermore, Logistic regression showed that the non-deficient group had an increased risk of impaired central and peripheral thyroid hormone sensitivity [TFQI (OR=1.16, 95% CI: 1.01~1.34); FT3/FT4 (OR=1.23, 95% CI: 1.05~1.45)] (all P<0.05).

Conclusion：

In euthyroid individuals, people with higher vitamin D levels have a higher risk of impaired thyroid hormone sensitivity.

Keywords：

Vitamin D; Thyroid hormone sensitivity; Euthyroid; Physical examination