基于TGF-β1/Foxp3/RORγt通路探讨清窍胶囊对分泌性中耳炎大鼠的作用机制

Action Mechanism of Qingqiao Capsule on Secretory Otitis Media in Rats Based on TGF-β1/Foxp3/RORγt Pathway

目的 基于TGF-β1/Foxp3/RORγt通路探讨清窍胶囊对分泌性中耳炎大鼠的作用机制。方法36只雌性SD大鼠,其中30只构建分泌性中耳炎大鼠模型,造模成功后,30只大鼠随机分为模型组、强的松组(0.003 g/100 mL)、清窍胶囊低剂量治疗组(0.36 g/100 mL)、清窍胶囊中剂量治疗组(0.54 g/100 mL)、清窍胶囊高剂量治疗组(0.72 g/100 mL),每组6只,另6只设为空白对照组。治疗组分别按不同剂量灌胃给药,模型组与空白对照组灌服相同容积的蒸馏水,1次/天,连续14天。给药结束采血,处死,采样待检。苏木精-伊红染色(Hematoxylin-eosin stain,HE)染色观察各组大鼠中耳炎症细胞的变化;酶联免疫吸附(Enzymelinked immunosorbent assay,ELISA)检测血清白介素-4(Interleukin-4,IL-4)、β-转化生长因子(Transforming growth factor beta,TGF-β)、白介素-6(Interleukin-6,IL-6)、γ-干扰素(Interferon γ,IFN-γ)、白介素-10(Interleukin-10,IL-10)、白介素-17(Interleukin-17,IL-17)水平;流式细胞技术检测脾脏和胸腺Th17/Treg细胞含量;实时荧光定量PCR检测脾脏和胸腺组织叉头盒P3(Forkhead Box P3,FOXP3)、IL-17、维甲酸相关核孤儿受体γt(Retinoic acid-related orphan receptor γt,RORγt)mRNA表达;蛋白质免疫印迹(Western Blot,WB)检测脾脏和胸腺组织RORγT、Foxp3表达。结果 与空白组比较,分泌性中耳炎大鼠黏膜层明显增厚,有大量炎性细胞浸润;且TGF-β、IL-6、IFN-γ、Th17、Treg细胞、IL-17、RORγT水平明显升高,IL-10、IL-4、Foxp3水平明显降低(P<0.01);与模型组比较,清窍胶囊高剂量组可明显改善中耳炎炎细胞浸润,明显降低模型大鼠TGF-β、IL-6、IFN-γ、IL-17水平,明显升高IL-10、IL-4水平(P<0.05),并明显降低模型大鼠Treg细胞、RORγT水平,明显升高Foxp3表达(P<0.05)。结论 清窍胶囊可有效地调节TGF-β/Foxp3/RORγt信号通路,进而调节Treg和Th17细胞介导的中耳炎的发生及发展,改善分泌性中耳炎。

Objective To explore the mechanism of Qingqiao capsule on secretory otitis media in rats based on TGF-β1/Foxp3/RORγt pathway. Methods A total of 36 female SD rats, of which 30 rats with secretory otitis media were randomly divided into model group, prednisone group(0.003 g/100 mL), low dose Qingqiao capsule group(0.36 g/100mL), medium dose Qingqiao capsule group(0.54 g/100 mL) and high dose Qingqiao capsule group(0.72 g/100 mL), with6 rats in each group and the other 6 rats were set as control group. The treatment group was given intragastric administration according to different doses, and the model group and control group were given the same volume of distilled water once a day for 14 days. After administration, the blood was collected, and then killed rats. The samples were collected for examination. Hematoxylin-eosin staining was used to observe the changes of inflammatory cells in the secretory otitis media of rats in each group;The levels of serum interleukin-4, transforming growth factor beta,interleukin-6, interferon γ, interleukin-10, interleukin-17 were detected by enzyme-linked immunosorbent assay. The content of Th17/Treg cells in thymus and spleen was detected by flow cytometry. The expression of forkhead BoxP3, IL-17 and retinoic acid-related orphan receptor γt mRNA in thymus and spleen tissue was detected by real-time fluorescence quantitative PCR, and the expression of RORγT and Foxp3 in thymus and spleen tissue was detected by Western Blot. Results Compared with the control group, the mucosal layer of secretory otitis media rats was significantly thickened with a large number of inflammatory cell infiltration;and the levels of TGF-β, IL-6, IFN-γ,Th17, Treg cells, IL-17 and RORγT in secretory otitis media were significantly higher, while the levels of IL-10, IL-4,and Foxp3 were significantly lower in secretory otitis media(P<0.01);Compared with the model group, the infiltration of secretory otitis media was significantly improved, and the levels of TGF-β, IL-6, IFN-γ, IL-17 were significantly decreased. The level of IL-10, IL-4 were significantly increased in Qingqiao capsule high-dose group(P<0.05), and the high dose group of Qingqiao capsule could significantly decrease the levels of Treg cells and RORγT and increase the expression of Foxp3 in the model rats(P<0.05).Conclusion Qingqiao capsule can effectively regulate TGF-β/Foxp3/RORγt signal pathway, and then regulate the occurrence and development of otitis media mediated by Treg and Th17 cells, and improve secretory otitis media.