Notch信号对大鼠肝纤维化Th17/Treg平衡的影响

The effect of Notch signaling on Th17/Treg balance in rat liver fibrosis

目的建立大鼠肝纤维化模型,探讨Notch信号在肝纤维化模型中的作用及对Th17/Treg细胞平衡的影响。方法40只Wistar雄性大鼠随机分为对照组(10只)、模型组(30只)。用刀豆蛋白A(Con A)建模,8周建模成功后模型组随机分成3组(PAPT组、TGFβ组、DMSO对照组),10只/组,并对其体外心尖取血3 ml,分离外周单个核细胞(PBMC),用Notch抑制剂DAPT及TGF-β抑制剂干预培养24 h,RT-PCR测Notch信号及TGF-β相关基因表达,流式细胞术测Th17和Treg细胞频率,ELISA法测PBMC培养上清中细胞因子IL-17、IL-23、TGF-β及IL-10的表达。结果与对照组相比,干预组Notch和TGF-β信号相关基因表达显著降低(P<0.05)。Th17细胞频率及其相关因子(IL-17、IL-23)水平降低(P<0.05),Treg细胞频率及其相关因子(TGF-β、IL-10)水平明显降低(P<0.05),Th17/Treg比值较对照组上升(P<0.05)。结论 Notch信号参与肝纤维化的发生发展,阻断Notch信号能抑制Treg细胞功能,降低TGF-β1水平,为肝纤维化的治疗研究提供实验基础。

To explore the role of Notch signaling in liver fibrosis and its effect on Thl7/Treg balance, 40 male Wistar rats were recruited and randomly divided into control group （n=10） and model group （n=30）. The liver fibrosis model was built with ConA, then randomly divided into 3 subgroups as DAPT （n=10）, TGF-β inhibitor （n=10） and DMSO （n=10） after 8 weeks modeling success. 3 ml blood was gathered by apex eordis from model group; peripheral blood monouclear cells （PBMC） were separated and cultured by DAPT and TGF-13 inhibitor for 24 h. RT-PCR was performed to analyze the relative mRNA level of Notch and TGF-β signaling; the frequencies of Th17 and Treg in PBMC were measured by flow cytometry （FCM）, and the levels of IL-17, IL-23, IL-10 and TGF-β1 in the culture supernatant were measured by ELISA. Compared with DMSO group, the relative mRNA expression of Notch and TGF-β signaling in DAPT group and TGF-β inhibitor group decreased significantly （P〈0.05）, the frequency of Th17 and its cytokines （IL-17, IL-23） were lower （P〈0.05）, while the frequency of Treg and its cytokines （IL-10, TGF-β1） were lower markedly（P〈0.05）. Furthermore, the ratio of Th17/Treg increased in DAPT group and TGF-β inhibitor group compared with DMSO control group. In conclusion, blocking Notch signaling can inhibit Treg cell function and reduce the level of TGF-β1, which maybe a valuable tool of research for liver fibrosis treatment in the future.