骨髓间充质干细胞外泌体调控TLR4/NF-κB/NLRP3信号轴缓解小鼠变应性鼻炎症状的功能及机制研究

The Study of the Functional and Mechanistic on the Regulation of TLR4/NF-κ B/NLRP3 Signaling Axis by Bone Marrow Mesenchymal Stem Cell Exosomes to Alleviate Allergic Rhinitis Symptoms in Mice

摘要:

目的 探究骨髓间充质干细胞外泌体（BMSC exo）缓解小鼠过敏性鼻炎（AR）的作用及其分子机制。方法 提取并鉴定BMSC exo。将40只Balb/c小鼠随机分为Control组、AR组、BMSC exo组、BMSC exo+TLR4 agonist组，除Control组外均采用卵清蛋白（OVA）法建立AR模型。造模后，三组小鼠均经尾静脉注射BMSC exo，其中BMSC exo+TLR4 agonist组再腹腔注射TLR4激动剂。治疗后记录小鼠挠鼻及打喷嚏次数；HE染色观察鼻黏膜病理损伤；ELISA检测鼻黏膜组织中IgE、IL-6、IL-4、IL-10水平；流式细胞术检测外周血Th1、Th2细胞比例；Western blot检测鼻黏膜组织中TLR4、p-NF-κB、NF-κB、NLRP3蛋白表达。结果 BMSC exo粒径约100 nm，呈典型“杯托”样结构，表达TSG101、CD81、CD9，符合外泌体特征。与Control组相比，AR组小鼠挠鼻、打喷嚏次数显著增加，鼻黏膜组织IgE、IL-6、IL-4升高，IL-10降低，Th1/Th2比值下降，TLR4、NLRP3及NF-κB磷酸化水平显著上调（P＜0.05）。与AR组相比，BMSC exo组上述指标均显著逆转（P＜0.05）。与BMSC exo组相比，额外给予TLR4激动剂后，BMSC exo的保护作用被显著削弱（P＜0.05）。结论 BMSC exo可通过抑制TLR4/NF-κB/NLRP3信号通路，纠正Th1/Th2失衡，减轻炎症反应，从而缓解小鼠过敏性鼻炎症状。

Abstract:

Objective To investigate the therapeutic effect of bone marrow mesenchymal stem cell-derived exosomes (BMSC exo) on allergic rhinitis (AR) in mice and its underlying molecular mechanism. Methods BMSC exo were extracted and characterized. Forty Balb/c mice were randomly divided into control group, AR group, BMSC exo group, and BMSC exo+TLR4 agonist group. An AR mouse model was established using ovalbumin (OVA) in all groups except the control group. After modeling, three groups of mice were intravenously injected via the tail vein with BMSC exo, and the BMSC exo+TLR4 agonist group was additionally intraperitoneally injected with a TLR4 agonist. Following treatment, the frequencies of nasal scratching and sneezing were recorded. Nasal mucosal pathological damage was observed using HE staining. The levels of IgE, IL-6, IL-4, and IL-10 in nasal mucosal tissue were measured by ELISA. The proportions of Th1 and Th2 cells in peripheral blood were detected by flow cytometry. The protein expression levels of TLR4, p-NF-κB, NF-κB, and NLRP3 in nasal mucosal tissue were determined by Western blot. Results BMSC exo had a particle size of approximately 100 nm, exhibited a typical cup-shaped morphology, and expressed the exosomal markers TSG101, CD81, and CD9, consistent with exosome characteristics. Compared with the control group, the AR group showed significantly increased frequencies of nasal scratching and sneezing; elevated levels of IgE, IL-6, and IL-4; decreased IL-10 level; reduced Th1/Th2 ratio; and significantly upregulated expression of TLR4, NLRP3, and phosphorylated NF-κB (p-NF-κB) in nasal mucosal tissue (all P<0.05). Compared with the AR group, all the above parameters were significantly reversed in the BMSC exo group (P<0.05). Compared with the BMSC exo group, co-administration of the TLR4 agonist significantly attenuated the protective effects of BMSC exo (P<0.05). Conclusion BMSC exo alleviate AR in mice by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway, reducing inflammatory responses, and restoring Th1/Th2 balance.