类风湿关节炎（Rheumatoid arthritis，RA）是一种以免疫功能失调、自身抗体产生、滑膜炎性增生、关节骨侵蚀为特征的难治性自身免疫性疾病。破骨细胞（Osteoclasts，OC）异常活化引起的骨侵蚀是RA致畸、致残的关键病理进程，调节性T细胞（Regulatory T cells，Tregs）通过与OC功能互作，介导骨免疫以减缓RA骨侵蚀进程。肠黏膜免疫系统与肠道菌群构成肠道微环境，二者互相影响，共同维持肠道微环境平衡。诸多研究证实RA患者内存在肠道微环境失调，且课题组前期的实验同样表明胶原诱导性关节炎（Collagen-induced arthritis，CIA）动物模型中存在肠道微环境失调。此外，研究发现芳香烃受体（Aryl hydrocarbon receptor，AhR）信号通路在骨免疫和肠道微环境中扮演重要角色，是连接免疫与骨平衡、肠道黏膜免疫与肠道菌群的关键节点。人脐间充质干细胞（Human umbilical mesenchymal stem cells，HUMSCs）具有治疗RA的潜能，但其机制却不清楚。本研究拟基于CIA模型和体外Tregs-OC共培养体系，探究HUMSCs对骨免疫的调节作用和肠道微环境的干预作用，并探讨AhR信号通路在其中的可能作用机制。

实验一  人脐间充质干细胞通过调节骨免疫和肠道微环境治疗胶原诱导性关节炎大鼠的作用研究

目的：随着对肠道菌群与疾病相关性认识的逐渐深入，众多研究发现肠道微环境失调可影响RA；且先前研究证实Tregs介导的骨免疫可通过调节OC介导的骨破坏而影响RA的疾病进程；此外，AhR不仅是调节肠道微环境的关键因素，而且其对Tregs和OC的分化和功能均具有调节作用。HUMSCs具有极低的免疫原性，研究发现其具有治疗RA潜能，但机制尚未完全阐明。本研究拟基于骨免疫和肠道微环境探讨HUMSCs对CIA大鼠的治疗作用及可能机制。

方法：从新生儿脐带分离并培养HUMSCs，并利用流式细胞术对分离培养的HUMSCs进行鉴定。取第三代HUMSCs（1×10⁶），通过尾静脉注射干预CIA模型大鼠，并以甲氨蝶呤（Methotrexate，MTX）为阳性药，干预28天，期间采用五级评分法进行关节炎评分。至实验终点，以NuMA蛋白为标志，通过免疫荧光鉴定HUMSCs归巢组织；采用H&E染色和Micro-CT评估其对踝关节病理和骨破坏的影响；通过抗酒石酸酸性磷酸酶（Tartrate-resistant acid phosphatase，TRAP）染色检测踝关节OC数目；通过流式细胞术检测腘窝淋巴结Tregs、辅助性T细胞17（T helper 17，Th17）的百分比，并通过RT-PCR检测相关细胞因子的基因表达变化；通过流式细胞术检测肠系膜淋巴结、小肠派氏集合淋巴结（Peyer's patch，PP）和小肠固有层淋巴细胞（Lamina propria lymphocyte，LPL）中Tregs、Th17和B细胞百分比，通过RT-PCR检测肠系膜淋巴结和回肠中相关细胞因子的基因表达变化，通过免疫组化检测回肠中相关蛋白的表达；此外，采用Luminex进一步检测血清中免疫相关因子的水平；通过16S测序检测回肠内容物中菌群的变化；通过液相色谱-串联质谱法（Liquid chromatography tandem-mass spectrometry，LC-MS/MS）检测血浆中吲哚及其衍生物的水平；最后分别采用RT-PCR和免疫组化检测回肠中AhR基因及蛋白表达。

结果：成功分离和培养HUMSCs。在CIA大鼠中，HUMSCs归巢于脾脏、腹股沟淋巴结、腘窝淋巴结、肠系膜淋巴结、踝关节软骨和回肠黏膜。MTX和HUMSCs干预均降低了CIA大鼠踝关节关节炎评分、病理评分、骨体积/骨表面积比值和关节OC数目（P<0.05）。 在最接近病灶的淋巴结-腘窝淋巴结和肠道相关淋巴组织之一的肠系膜淋巴结中，MTX和HUMSCs干预升高Tregs的比例和IL-10、TGF-β1的基因表达水平并降低Th17细胞的比例和IL-17A的基因表达水平（P<0.05；P<0.01）。在其他肠道相关淋巴组织即PP结和LPL中，MTX和HUMSCs干预升高Tregs和B细胞的比例并降低Th17细胞的百分比（P<0.05；P<0.01）。回肠中，MTX和HUMSCs干预显著上调了TGF-β1的基因表达及IL-10，TGF-β1，IL-22和IgA的蛋白表达（P<0.05；P<0.01），降低IL-17A的基因和蛋白表达（P<0.05；P<0.01）。且MTX和HUMSCs干预能回调CIA造模大鼠血清中异常的IL-10，TGF-β1，IL-17A，IL-1β，TNF-α水平（P<0.05）。 CIA组回肠菌群的OTU和Chao1值升高（P<0.05），MTX和HUMSCs干预逆转此Alpha多样性异常（P<0.01）；偏最小二乘法判别分析（Partial least square discriminant analysis，PLS-DA）结果显示，CIA造模改变正常大鼠肠道微生物菌落结构，MTX和HUMSCs干预使CIA的菌落结构分布发生逆时针方向变化；造模和治疗均改变了回肠的优势菌，HUMSCs处理的CIA大鼠，拟杆菌属（Bacteroides）和芽孢杆菌属（Bacillus）的相对丰度增加。 此外，与CIA组相比，MTX和HUMSCs干预后血浆中吲哚、吲哚乙酸和吲哚-3-乳酸水平显著上调（P<0.05）。最后，与Control组相比，CIA组回肠AhR基因和蛋白表达降低；与CIA组相比，HUMSCs回肠AhR基因和蛋白表达显著增加（P<0.05；P<0.01）。

小结：HUMSCs对CIA大鼠具有治疗作用，其作用机制有以下二方面：一方面HUMSCs靶向至腘窝淋巴结和关节，通过调节腘窝淋巴结Tregs和Th17免疫状态影响骨免疫状态，降低CIA大鼠踝关节骨侵蚀。另一方面HUMSCs能归巢于肠系膜淋巴结和回肠，通过调节小肠PP结和LPL中Tregs、Th17和B细胞免疫状态改善肠黏膜免疫，而影响肠道微环境稳态，间接改善关节炎；其机制与上调AhR激动性配体吲哚及其衍生物水平导致AhR激活有关。

实验二 芳香烃受体激动剂Tapinaraf对Tregs-OC共培养体系的影响及机制探究

目的：RA以关节部位的软骨进行性损伤和继发性骨侵蚀为特点，因OC能介导骨吸收功能，故其在骨侵蚀发生和发展中扮演关键角色；且Tregs介导的免疫抑制功能可以通过骨免疫作用抑制OC骨破坏。本实验旨基于课题组前期构建的Tregs-OC共培养体系，以AhR激动剂Tapinaraf为工具药，探讨AhR信号通路在RA骨免疫中的可能作用及机制。

方法：分离小鼠胫骨和股骨骨髓进行OC诱导与分化培养，分离小鼠脾细胞进行Tregs分离与扩增培养，并建立Tregs-OC共培养体系。采用浓度梯度Tapinaraf干预OC、Tregs及Tregs-OC共培养体系。通过CCK-8法分别检测Tapinaraf对OC与Tregs的毒性；通过TRAP染色法评价单独培养和共培养下OC分化数目并计数；通过RT-PCR法检测单独培养及共培养下OC分化相关通路的基因表达和单独培养下Tregs相关细胞因子的基因表达；最后通过RT-PCR法检测单独培养下OC和Tregs及共培养下OC中AhR通路相关分子的基因表达。

结果：本实验成功诱导分化出OC，并成功扩增出高纯度Tregs。Tapinaraf在10^-3-10³nM浓度之间对OC和Tregs的存活率（Survival rate，SR）无影响，在104nM浓度下显著降低二者细胞SR（P<0.05），显示出细胞毒性。TRAP染色结果显示，Tapinaraf浓度依赖的抑制OC的分化数量：与Control组相比，OC单独培养组和Tregs-OC共培养组分别于Tapinaraf 10⁰nM和10^-1nM浓度开始显示出对OC的分化抑制作用（P<0.05）；且相同Tapinaraf浓度下，与OC单独培养组相比，Tregs-OC共培养组OC分化数量均显著降低（P<0.05；P<0.01）。 与Control组相比，Tapinaraf对OC单独培养组和Tregs-OC共培养组RANK基因的表达无影响，但分别于10⁰nM和10^-1nM浓度开始显示出对RANK下游c-fos基因表达的抑制（（P<0.05），分别于10^-3nM，10¹nM和10³nM浓度下和10^-1nM浓度开始显示出对NFATc1基因表达的抑制（P<0.05）；且相同Tapinaraf浓度下，与OC单独培养组相比，Tregs-OC共培养组OC的RANK、NFATc1、c-fos基因表达均显著降低（P<0.05；P<0.01）。 此外，对Tregs功能相关基因IL-10、TGF-β1的检测结果表明，Tapinaraf浓度依赖的增加Tregs中IL-10和TGF-β1基因的表达，分别于10⁰nM和10¹nM浓度开始显示出统计学差异（P<0.05）。 最后，Tapinaraf以浓度依赖的方式升高Tregs单独培养组、OC单独培养组和Tregs-OC共培养组OC的AhR基因及其下游基因CYP1A1的表达，均于10^-1nM浓度开始有统计学差异（P<0.05）；且相同Tapinaraf浓度下，与OC单独培养组相比，Tregs-OC共培养组OC的AhR和CYP1A1基因表达均显著升高（P<0.05；P<0.01）。

小结：在Tregs、OC单独培养及Tregs-OC共培养体系中，Tapinaraf可激活AhR及下游CYP1A1。一方面导致OC中c-fos与NFATc1表达下降，从而抑制OC的分化；另一方面，通过提高Tregs中IL-10和TGF-β1的表达，间接发挥对OC的分化的抑制作用。

实验三 人脐间充质干细胞通过芳香烃受体调节Tregs-OC共培养体系研究

目的：间充质干细胞对体外培养的Tregs和OC的影响已有报道，但对Tregs-OC共培养体系的作用和相关机制却仍未阐明。本研究旨在探究HUMSCs对Tregs-OC共培养体系的影响和作用机制。

方法：分离小鼠胫骨和股骨骨髓进行OC诱导与分化培养，分离小鼠脾细胞进行Tregs诱导与扩增培养，并建立Tregs-OC共培养体系。采用HUMSCs培养上清液干预细胞。通过CCK-8法检测Tregs和OC的增殖率；采用TRAP染色检测OC分化数量；通过RT-PCR和Luminex分别检测Tregs相关细胞因子的基因和蛋白表达；最后采用RT-PCR检测Tregs和OC中AhR通路相关分子的基因表达。

结果：HUMSCs培养上清液增加Tregs和OC前体细胞的增殖率（P<0.05）。与Control组相比，HUMSCs培养上清液干预显著降低OC的分化数量（P<0.01）；且与OC单独培养组相比，Tregs-OC共培养OC的分化数量更少，但未观察到统计学差异。 此外，HUMSCs培养上清液显著提高Tregs中IL-10、TGF-β1的基因表达水平和TGF-β1的蛋白表达水平（P<0.05；P<0.01）。

最后，HUMSCs培养上清液显著提高OC中AhR和Tregs中CYP1A1和基因表达水平（P<0.01）。

小结：HUMSCs通过AhR抑制OC的分化，也通过AhR下游CYP1A1提高Tregs中IL-10和TGF-β1的表达，发挥对OC分化的抑制作用。

结论：HUMSCs具有治疗CIA大鼠的作用；其通过调节腘窝淋巴结免疫状态发挥关节骨免疫作用，降低CIA大鼠踝关节骨侵蚀；其通过调节肠黏膜免疫状态和改变回肠肠道菌群发挥对肠道微环境的调节作用，间接改善关节炎；其机制可能与AhR激动性配体吲哚及其衍生物水平上调所引起的AhR激活有关。在Tregs-OC共培养体系中，Tapinarof介导的AhR及下游信号通路激活能抑制OC的分化，也能提高Tregs骨免疫功能，进一步发挥对OC的抑制作用。HUMSCs对OC分化的抑制作用通过激活OC中AhR和通过激活Tregs中AhR下游CYP1A1而提高Tregs骨免疫功能双重方式实现。

Rheumatoid arthritis (RA) is a refractory autoimmune disease characterized by immune dysfunction, autoantibody production, synovial inflammatory hyperplasia, and joint bone erosion. Bone erosion caused by abnormal activation of osteoclasts (OC) is a key pathological change in being teratogenic and disabling of RA. Regulatory T cells (Tregs) can slow down the bone erosion process by mediating bone immunity via interacting with OC. The intestinal mucosal immune system and gut microbiota constitute the intestinal microenvironment. And by influencing each other, they maintain the balance of the intestinal microenvironment jointly. Numerous studies have confirmed the disorders of intestinal microenvironment in patients with RA. And the previous experiments of our research team have also showed that unbalance of intestinal microenvironment exists in RA animal model, named Collagen-induced arthritis (CIA). Furthermore, studies have found that the aryl hydrocarbon receptor (AhR) signaling pathway plays an important role in bone immunity and intestinal microenvironment, and it is a key node between connecting immunity and bone balance, intestinal mucosal immunity and gut microbiota. Human umbilical mesenchymal stem cells (HUMSCs) have the potential for the treatment of RA, but the underlying mechanism is still unclear. Based on the CIA model and the Tregs-OC co-cultured system to explore, this study is aimed at exploring the effect of HUMSCs on bone immunomodulatory and intestinal microenvironment, and the possible mechanism of the AhR signaling pathway involved.

1. Effect of human umbilical mesenchymal stem cells on collagen-induced arthritis rats by regulating bone immunity and intestinal microenvironment

Objective: With the in-depth study in relationship between gut microbiota and diseases, numerous studies have gradually found that disorder of intestinal microenvironment could affect RA. And previous studies have also verified that bone immune mediated by Tregs affectes process of RA via regulating bone destruction of OC. Furthemore, AhR is not only a key factor in regulating intestinal microenvironment, but also a impotant role in regulating differentiation and function of Tregs and OC. HUMSCs are accompanied by extremely low immunogenicity and show the therapeutic potential against RA, but the underlying mechanisms have not been fully elucidated. The purpose of this study was to explore the effects and mechanisms of HUMSCs in rats with CIA based on bone immunity and intestinal microenvironment.

Methods: HUMSCs were isolated and cultured from neonatal umbilical cord and identified by flow cytometry. Third generation of HUMSCs (1×10⁶) were transplanted into rat with CIA via intravenous injection through the tail. Methotrexate (MTX) was used as a positive drug for 28 days and arthritis score was evaluated by five-level grading system every 3 days during the period. At the end of the experiment, HUMSCs homing tissues were identified by immunofluorescence with NuMA protein as the marker. H&E staining and Micro-CT were used to assess pathology and bone destruction in ankle joint. The number of OC in ankle joint was detected by tartrate-resistant acid phosphatase (TRAP) staining. The percentage of Tregs and helper T cell 17 (Th17) and the gene expressions of related cytokines were detected by flow cytometry and RT-PCR in popliteal lymph nodes, respectively. The percentages of Tregs, Th17 and B cells in the mesenteric lymph nodes, small intestine Peyer's patch (PP) and lamina propria lymphocyte (LPL) were detected by flow cytometry. The gene expressions of related cytokines in mesenteric lymph nodes and ileum were detected by RT-PCR and the expression of related proteins in ileum was detected by immunohistochemistry. Furthermore, the levels of immune-related factors in serum and the changes in gut microbiota in the ileum were detected by Luminex multi-cytokine analysis technique and 16s sequencing technology. The levels of indole and its derivatives in plasma were detected by liquid chromatography tandem-mass spectrometry (LC-MS/MS). At last, the gene and protein levels of AhR in ileum were evaluated via RT-PCR and immunohistochemistry, respectively.

Results: HUMSCs were successfully isolated and cultured. HUMSCs homed to the spleen, inguinal lymph nodes, popliteal lymph nodes, mesenteric lymph nodes, ankle cartilage, and ileum mucosa in rats with CIA. The MTX and HUMSCs treatment reduced the arthritis score, pathology score, bone volume/bone surface area ratio and OC number in the ankles (P<0.05).

The ratio of Tregs and the gene expression levels of IL-10 and TGF-β1 were increased and the ratio of Th17 and the gene expression level of IL-17A were decreased in the popliteal lymph nodes and mesenteric lymph nodes (P<0.05; P<0.01), which were the lymph tissue closest to the nidus and one of the gut-associated lymphoid tissues, respectively. The proportion of Tregs and B cells were increased and the proportion of Th17 were decreased in other gut-associated lymphoid tissues, namely, PP and the LPL after interventing of MTX and HUMSCs (P<0.05; P<0.01). In ileum, gene expression of TGF-β1 and protein expressions of IL-10, TGF-β1, IL-22 and IgA were increased (P<0.05; P<0.01) and expressions of IL-17A gene and protein were decreased after MTX and HUMSCs intervention (P<0.05; P < 0.01). MTX and HUMSCs intervention were able to respond to abnormal serum levels of IL-10，TGF-β1，IL-17A，IL-1β，TNF-α in CIA rats (P<0.05).

The OUT and Chao1 diversity of gut microbiota in ileum was increased in CIA group (P<0.05), while this anormal Alpha diversity was reversed after MTX and HUMSCs intervention (P<0.01). The partial least square discriminant analysis (PLS-DA) showed that the gut microbiota colony structure was changed after CIA model, MTX and HUMSCs intervention caused counterclockwise changes based on CIA colony structure distribution. Both CIA and treatments altered the relative abundance of the dominant bacteria in ileum compared with Control rats. The relative abundances of the genera Bacteroides and Bacillus were increased in the HUMSCs-treated rat with CIA.

Besides, the level of indole, indoleacetic acid and indole-3-lactic acid were consistently upregulated compared with CIA group (P<0.05). Finally, compared with the Control group, the expression of the ileum AhR gene and protein was decreased in the CIA group; compared with the CIA group, the levels of ileum AhR gene and protein were significantly increased in HUMSCs group (P<0.05; P < 0.01).

Summary: HUMSCs had therapeutic effect on CIA rats. Mechanisms were as follows: on the one hand, HUMSCs targeted to popliteal lymph nodes and joints. By regulating the immune status of Tregs and Th17 in popliteal lymph nodes, HUMSCs showed the bone immunity effect and reduced the bone erosion in ankle joints. On the other hand, HUMSCs targeted to mesenteric lymph nodes and ileum. By regulating the immune status of Tregs, Th17 and B cells in PP and LPL, HUMSCs improved the intestinal mucosal immunity and influenced the intestinal microenvironment homeostasis, and arthritis was indirectly improved. The mechanism was related to the upregulation of AhR agonistic ligand indole and its derivatives followed by AhR activation.

2. Effect and related mechanism of AhR agonist Tapinaraf on Tregs-OC co-cultured system

Objective: RA is characterized by the progressive damage of articular cartilage and secondary bone erosion. OC plays a key role in development of bone erosion because of function in bone resorption. And Tregs participate in bone immunity to inhibit bone destruction mediated by OC. Based on Tregs-OC co-cultured system established in the early stage, this study aimed to investigate the possible role and mechanism of AhR signaling pathway in RA bone immune by Tapinaraf as the tool medicine.

Methods: Mouse bone marrow from the tibia, femur and spleen cells were isolated and induced into OC and Tregs respectively. Then, the co-culture system of Tregs-OC was established. The AhR agonist Tapinaraf with a concentration gradient was used to intervene OC, Tregs and Tregs-OC co-culture system. Cytotoxicity in Tregs and OC was evaluated by CCK-8 and number of OC differentiation under single culture and co-culture was detected by TRAP. Gene expressions were detected by RT-PCR of OC differentiation pathway under single culture and co-culture condition and Tregs cytokines under single culture condtion. Finally, Gene levels of AhR pathway in both cells were detected by RT-PCR.

Results: In this study, OC and high-purity Tregs were successfully induced and differentiated. Tapinaraf at 10^-3-10³nM had no effect on the survival rate of OC and Tregs, while the survival rate was reduced at 10⁴nM (P<0.05) in both OC and Tregs, indicating cytotoxicity of Tapinaraf at this concentration. Then, TRAP staining showed that Tapinaraf decreased the number of OC differentiation via concentration-dependent manner. Compared with the Control group, the minimum effective concentrations of Tapinaraf were 10⁰nM and 10^-1nM in OC group and Tregs-OC group, respectively (P<0.05). Furthermore, at the same Tapinaraf concentration, the number of OC differentiation in Tregs-OC group was significantly reduced compared with that in OC group (P<0.05; P<0.01).

Compared with the Control group, Tapinaraf had no effect on the gene expression of RANK in both OC group and Tregs-OC group. But Tapinaraf inhibited c-fos gene expression from concentrations of 10⁰nM and 10^-1nM respectively (P<0.05), and Tapinaraf inhibited NFATc1 gene expression at concentrations of 10^-3nM, 10¹nM, 10³nM and from 10^-1nM respectively (P<0.05). Furthermore, at the same Tapinaraf concentration, the gene expressions of RANK, NFATc1 and c-fos in Tregs-OC group were significantly lower than that in OC group (P<0.05; P < 0.01).

In addition, the gene expressions of Tregs related gene such as IL-10 and TGF-β1 were upregulated by Tapinaraf in concentration-dependent manner at the minimum effective concentrations of 10⁰nM and 10¹nM respectively (P<0.05).

Finally, the gene expressions of AhR and CYP1A1 were enhanced by Tapinaraf in concentration dependence among Tregs group, OC group and Tregs-OC group and there were statistical differences at the concentration of 10^-1nM (P<0.05). Besides, at the same Tapinaraf concentration, the gene expressions of AhR and CYP1A1 in Tregs-OC group were significantly higher than that in OC group (P<0.05; P<0.01).

Summary: Tapinaraf activated AhR and downstream CYP1A1 in Tregs, OC and Tregs-OC co-culture system. On the one hand, the expressions of c-fos and NFATc1 in OC were reduced after intervened by Tapinaraf, which suppressed the differentiation of OC. On the other hand, Tapinaraf indirectly exerted the inhibitory effect to the differentiation of OC by increasing the expression of IL-10 and TGF-β1 in Tregs.

3. Study on the regulation of Tregs-OC co-culture system by human umbilical mesenchymal stem cells through aryl hydrocarbon receptor

Objective The effects of mesenchymal stem cells on Tregs and OC in vitro have been reported, but the effects and related mechanisms of mesenchymal stem cells on Tregs-OC co-cultured system have not been elucidated. The purpose of this study was to investigate the possible role and mechanisms of HUMSCs on Tregs-OC co-cultured system.

Methods: Mouse bone marrow and spleen cells were isolated and induced into OC and Tregs, respectively. And the Tregs-OC co-culture system was established. The HUMSCs culture supernatant was used to intervene the cells. Cell proliferation rates of both Tregs and OC were detected by CCK-8 assay. Then, number of OC differentiation was evaluated by TRAP staining. Besides, gene expression and protein level of cytokines in Tregs were detected by RT-PCR and Luminex multi-cytokine analysis technique respectively. Finally, AhR gene expression of Tregs and OC were detected via RT-PCR.

Results: The HUMSCs culture supernatant increased the cell proliferation rate of Tregs and OC (P<0.05). Compared with Control group, HUMSCs culture supernatant decreased the number of OC differentiation (P<0.01). The number of OC differentiation in Tregs-OC group was less than that in OC group, but it was no statistical difference observed.

Furthermore, HUMSCs culture supernatant increased the gene expressions of IL-10, TGF-β1 and protein level of TGF-β1 in Tregs (P<0.05; P<0.01).

Finally, the gene expressions of AhR and CYP1A1were enhanced in OC and Tregs after HUMSCs culture supernatant intervention (P<0.05).

Summary: HUMSCs inhibited the differentiation of OC through AhR. HUMSCs increased the expression of IL-10 and TGF-β1 in Tregs through CYP1A1 to exert an inhibitory effect on OC differentiation.

Conclusion: HUMSCs had therapeutic effect on CIA rats. HUMSCs exerted joint bone immunity by regulating the immune state of popliteal lymph nodes to reduce bone erosion of ankle joint in CIA rats. By regulating the intestinal mucosal immune and changing the ileal gut microbiota, HUMSCs exerted its effect on regulating the intestinal microenvironment and improve arthritis indirectly. The mechanism might be related to AhR activation caused by the up-regulation of the AhR agonistic ligand indole and its derivatives. In the Tregs-OC co-culture system, Tapinarof-mediated AhR and downstream signaling pathway activation could inhibit the differentiation of OC, which also improved the bone immune function of Tregs, and further exerting the inhibitory effect on OC. The inhibitory effect of HUMSCs to OC differentiation was achieved by activating AhR in OC and by activating downstream gene CYP1A1 of AhR in Tregs to increase bone immunity of Tregs.