【摘要】 目的:基于靶向调控CD8~+T细胞自噬途径探讨银甲丸加减治疗生殖道沙眼衣原体持续感染的疗效及机制。方法:36只BALB/c小鼠，除空白组以外，其余组小鼠阴道内均接种衣原体建立沙眼衣原体持续性感染模型。造模成功后分组分别给予银甲丸加减方低、中、高剂量和阿奇霉素连续灌胃4周。HE染色观察小鼠子宫、输卵管组织形态学变化；实时荧光定量聚合酶链反应检测下生殖道沙眼衣原体基因表达量；流式细胞术检测小鼠淋巴结OX40、LAG-3阳性细胞占比；免疫印迹检测小鼠脾CD8~+T细胞中自噬相关蛋白Beclin-1、Atg5、LC3Ⅱ/LC3Ⅰ、p62及Ras/Raf/MEK/ERK通路蛋白表达。结果:与模型组比较，各治疗组炎性细胞浸润均有不同程度的减少，其中中药高剂量组的子宫及输卵管仅见极少量炎性细胞浸润，输卵管未见明显炎性渗出及增粗。与模型组比较，各治疗组Beclin-1、Atg5、LC3Ⅱ/LC3Ⅰ表达均降低(P<0.01),p62、Ras、P-Raf、P-MEK和P-ERK的蛋白表达均上调(P<0.01),且淋巴细胞中OX40、LAG-3阳性细胞占比均降低(P<0.01);其中中药高剂量组Beclin-1、Atg5、LC3Ⅱ/LC3Ⅰ蛋白表达以及OX40、LAG-3阳性细胞占比均最低(P<0.01)。结论:银甲丸加减方可能通过Ras/Raf/MEK/ERK信号通路，抑制CD8~+T细胞自噬活性，从而增强CD8~+T细胞对沙眼衣原体包涵体的靶向清除功能，延缓生殖道沙眼衣原体持续性感染进程。更多还原

【Abstract】 Objective:To investigate the efficacy and mechanism of modified Yinjia formula in the treatment of persistent genital tract chlamydia trachomatis infections based on the targeted regulation of CD8~+T cell autophagy pathway.Methods:Thirty-six BALB/c mice, except for the blank group, were inoculated intravaginally with chlamydia to establish a model of persistent chlamydia trachomatis infection.After successful modeling, patients were divided into groups and given low, medium and high doses of Yinjia modifiedformula and azithromycin by continuous gavage for 4 weeks, respectively.HE staining was used to observe the histomorphologic changes of uterus and fallopian tube tissues of the mice.Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression level of chlamydia trachomatis genes in the low reproductive tract.Proportion of OX40 and LAG-3 positive cells in mouse lymph nodes was detected by flow cytometry.Immunoblotting was used to detect the expression of autophagy-related protein Beclin-1,Atg5,LC3Ⅱ/LC3Ⅰ,p62,and Ras/Raf/MEK/ERK pathway protein in mouse splenic CD8~+T cells.Results:Compared with the model group, the inflammatory cell infiltration was reduced to different degrees in all treatment groups.Among them, only a very small amount of inflammatory cell infiltration was observed in the uterus and fallopian tubes in the high-dose group of traditional Chinese medicine, and no significant inflammatory exudation and thickening was observed in the fallopian tubes.Compared with the model group, the expression of Beclin-1,Atg5,LC3Ⅱ/LC3Ⅰ was decreased in all treatment groups(P<0.01),while the protein expression of p62,Ras, P-Raf, P-MEK and P-ERK was up-regulated(P<0.01).Additionally, the proportion of OX40 and LAG-3 positive cells in lymphocytes was decreased(P<0.01).Among them, Beclin-1,Atg5,LC3Ⅱ/LC3Ⅰ protein expression as well as the percentage of OX40 and LAG-3 positive cells were the lowest in the high-dose group of traditional Chinese medicine(P<0.01).Conclusions:The modified Yinjia formula may inhibit the autophagic activity of CD8~+T cells through the Ras/Raf/MEK/ERK signaling pathway, thereby enhancing the targeted clearance function of chlamydia trachomatis inclusion bodies by CD8~+T cells and delaying the process of persistent chlamydia trachomatis infection in the reproductive tract.更多还原