Data Sheet 15_Acupuncture attenuates experimental autoimmune thyroiditis by modulating intestinal microbiota and palmitic acid metabolism.zip

IntroductionAutoimmune thyroiditis (AIT) is a common and chronic autoimmune disease. Recent evidence indicates that serum metabolites and dysbiosis of the intestinal microbiota are associated with AIT, with the underlying mechanism involving the apoptosis of thyroid follicular epithelial cells. Acupuncture, a traditional Chinese therapy, has demonstrated potential regulatory effects on various immune-related diseases. Clinical symptoms in AIT patients have shown improvement following acupuncture intervention. However, the mechanism underlying its therapeutic effects remain poorly understood.
MethodsIn this study, we investigated the mechanisms of acupuncture (Acu) treatment in rats with established experimental autoimmune thyroiditis (EAT) and evaluated the relationship between microbiota and serum metabolites after Acu treatment. After six weeks of acupuncture and Selenium yeast intervention (used as a positive control), enzyme-linked immunosorbent assay was used to employed to assess the expressions of serum thyroid function and inflammatory markers. Pathological changes in the thyroid gland were observed using hematoxylin-eosin staining and electron microscopy. Thyroid apoptosis was evaluated through TUNEL staining, immunohistochemistry and Western blot analysis. Additionally, changes in intestinal microbiota and serum metabolic profile were analyzed by 16S ribosomal RNA (16S rRNA) sequencing and LC-MS metabolomics, aiming to identify potential therapeutic targets for acupuncture intervention in AIT.
ResultsThe results revealed that Acu could effectively improve thyroid dysfunction and histopathological changes in EAT rats. Following Acu treatment, the content of B-cell lymphoma-2 (Bcl-2) increased, while the levels of Bax and the proportion of cleaved caspase-3 in thyroid tissue decreased. This may be associated with the amelioration of intestinal microbiota dysbiosis and metabolic disorders in EAT rats. Acu mitigated EAT-induced metabolic disorders by regulating the metabolism of palmitoleic acid, and adjusted intestinal microbiota dysbiosis by increasing the abundance of Prevotella. Furthermore, the microbiota (Prevotella) and metabolites (Cyclohexanecarboxylic acid, Tetradecanedioic acid) may serve as co-targets for both Acu and Selenium yeast treatment in EAT.
DiscussionAcu improves the apoptosis of thyroid follicular epithelial cells in rats in EAT model, and its mechanism may be related to intestinal microbiota and metabolism.

引言
自身免疫性甲状腺炎（Autoimmune Thyroiditis, AIT）是一种常见的慢性自身免疫性疾病。现有研究证据表明，血清代谢异常与肠道菌群失调均与自身免疫性甲状腺炎相关，其潜在分子机制涉及甲状腺滤泡上皮细胞凋亡。针灸作为中国传统疗法，已被证实对多种免疫相关性疾病具有潜在调控作用。自身免疫性甲状腺炎患者经针灸干预后，临床症状已得到改善，但目前其治疗作用的具体分子机制仍尚未明确。

方法
本研究以造模成功的实验性自身免疫性甲状腺炎（Experimental Autoimmune Thyroiditis, EAT）大鼠为对象，探究针灸（Acupuncture, Acu）治疗的作用机制，并评估针灸干预后肠道菌群与血清代谢物之间的关联。本研究采用硒酵母干预作为阳性对照，经6周针灸与硒酵母干预后，通过酶联免疫吸附测定法检测血清甲状腺功能指标与炎症标志物的表达水平；采用苏木精-伊红染色与电子显微镜观察甲状腺组织的病理形态学变化；通过TUNEL染色、免疫组化与蛋白质印迹法评估甲状腺细胞凋亡情况。此外，通过16S核糖体RNA（16S ribosomal RNA, 16S rRNA）测序与液相色谱-质谱代谢组学分析肠道菌群与血清代谢谱的变化，以期筛选出针灸治疗自身免疫性甲状腺炎的潜在靶点。

结果
结果显示，针灸可有效改善实验性自身免疫性甲状腺炎大鼠的甲状腺功能异常与组织病理学变化。针灸干预后，甲状腺组织中B细胞淋巴瘤-2（B-cell lymphoma-2, Bcl-2）的表达量升高，而Bax以及裂解型半胱氨酸天冬氨酸蛋白酶3（cleaved caspase-3）的表达水平与占比均显著降低。该作用可能与改善实验性自身免疫性甲状腺炎大鼠的肠道菌群失调与代谢紊乱相关。针灸可通过调控棕榈油酸代谢缓解实验性自身免疫性甲状腺炎诱导的代谢紊乱，并通过增加普雷沃菌属（Prevotella）的丰度改善肠道菌群失调。此外，普雷沃菌属（Prevotella）与代谢物环己烷羧酸（Cyclohexanecarboxylic acid）、十四烷二酸（Tetradecanedioic acid）或可作为针灸与硒酵母治疗实验性自身免疫性甲状腺炎的共同靶点。

讨论
针灸可改善实验性自身免疫性甲状腺炎大鼠模型中甲状腺滤泡上皮细胞的凋亡情况，其作用机制或与肠道菌群及代谢调控相关。