基于Tim-3-Gal-9通路探讨补肾安胎冲剂对复发性流产小鼠Th1/Th2、Th17/Treg细胞平衡的影响

Effects of Bushen Antai Granules on the Balance of Th1/Th2,Th17/Treg in Recurrent Spontaneous Abortion Mice Based on Tim-3-Gal-9 Pathway

目的观察补肾安胎冲剂对复发性流产(RSA)小鼠Th1/Th2、Th17/Treg细胞平衡的影响,从Tim-3-Gal-9通路探讨其作用机制。方法以雌性CBA/J小鼠与雄性DBA/2小鼠合笼饲养建立RSA小鼠模型,将成模小鼠随机分为模型组及补肾安胎冲剂低、高剂量组,每组10只,另设10只与雄性BALB/c小鼠合笼交配的雌性CBA/J小鼠为正常妊娠组。使用相应药物干预15 d,ELISA检测小鼠血清γ干扰素(IFN-γ)、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-2、IL-4、IL-5、IL-10、IL-12、IL-17、IL-23含量,流式细胞术检测小鼠外周血CD4^(+)CD25^(+)Foxp3^(+)/CD4^(+)和CD4^(+)Foxp3^(+)细胞比例,RT-PCR和Western blot分别检测小鼠蜕膜组织Tim-3、Gal-9 mRNA和蛋白表达。结果与正常妊娠组比较,模型组小鼠流产率、胚胎丢失率显著升高,血清IL-2、IL-12、IFN-γ、TNF-α、IL-17、IL-23含量显著增加,IL-4、IL-5、IL-10含量显著减少,CD4^(+)CD25^(+)Foxp3^(+)/CD4^(+)和CD4^(+)Foxp3^(+)细胞比例显著降低,差异均有统计学意义(P<0.01),蜕膜组织Tim-3 mRNA和蛋白表达显著升高,Gal-9 mRNA和蛋白表达显著降低(P<0.01);与模型组比较,补肾安胎冲剂低、高剂量组小鼠流产率、胚胎丢失率显著降低(P<0.05,P<0.01),补肾安胎冲剂高剂量组小鼠血清IL-2、IL-12、IFN-γ、TNF-α、IL-17、IL-23含量显著减少,IL-4、IL-5、IL-10含量显著增加,CD4^(+)CD25^(+)Foxp3^(+)/CD4^(+)和CD4^(+)Foxp3^(+)细胞比例显著升高,蜕膜组织Tim-3 mRNA和蛋白表达显著降低,Gal-9 mRNA和蛋白表达显著升高,差异均有统计学意义(P<0.01)。结论补肾安胎冲剂可能通过调控Tim-3-Gal-9通路调节Th1/Th2、Th17/Treg细胞平衡,从而治疗RSA。

Objective To observe the effects of Bushen Antai Granules on the balance of Th1/Th2,Th17/Treg in recurrent spontaneous abortion(RSA)mice;To explore the mechanism of action from Tim-3-Gal-9 pathway.Methods Mouse model of RSA with female CBA/J mice and male DBA/2 mice in a cage were established.The mice were randomly divided into model group and Bushen Antai Granules low-and high-dosage group,with 10 mice in each group.In addition,10 female CBA/J mice mated with male BALB/c mice in a cage were set as the normal pregnancy group. Each group received gavage with corresponding medicine for 15 days. ELISA was used to detect the contents of interferon-γ (IFN-γ), tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12, IL-17 and IL-23 in serum. The proportion of CD4^(+)CD25^(+)Foxp3^(+)/CD4^(+) and CD4^(+)Foxp3^(+) cells in peripheral blood was detected by flow cytometry. The mRNAs and proteins expression of Tim-3 and Gal-9 in decidual tissues were detected by RT-PCR and Western blot respectively. Results Compared with the normal pregnancy group, the abortion rate and embryo loss rate of mice in the model group increased significantly, the contents of IL-2, IL-12, IFN-γ, TNF-α, IL-17 and IL-23 in serum significantly increased, the contents of IL-4, IL-5 and IL-10 significantly decreased, the proportion of CD4^(+)CD25^(+)Foxp3^(+)/CD4^(+) and CD4^(+)Foxp3^(+) cells were significantly decreased, with statistical significance (P<0.01), the expression of Tim-3 mRNA and protein in decidua tissue increased significantly, the expression of Gal-9 mRNA and protein decreased significantly (P<0.01). Compared with the model group, the abortion rate and embryo loss rate of mice in Bushen Antai Granules low- and high-dosage groups significantly decreased (P<0.05, P<0.01), the contents of IL-2, IL-12, IFN-γ, TNF-α, IL-17 and IL-23 in serum of Bushen Antai Granules high-dosage group was significantly reduced, the contents of IL-4, IL-5 and IL-10 significantly increased, the proportion of CD4^(+)CD25^(+)Foxp3^(+)/CD4^(+) and CD4^(+)Foxp3^(+) cells were significantly increased, the expression of Tim-3 mRNA and protein in decidua tissue significantly decreased, the expression of Gal-9 mRNA and protein significantly increased, with statistical significance (P<0.01). Conclusion Bushen Antai Granules is possible to regulate the balance of Th1/Th2 and Th17/Treg cells by regulating the Tim-3-Gal-9 pathway to treat RSA.