益气健脾生津方对气阴两虚证原发性干燥综合征患者Tfr/Tfh细胞平衡及自噬水平影响的研究

Effects of Yiqi Jianpi Shengjin recipe on Tfr/Tfh cells balance and autophagy in patients with primary Sjögren's syndrome and qi-yin deficiency syndrome

目的观察益气健脾生津方对气阴两虚证原发性干燥综合征患者Tfr/Tfh细胞平衡及自噬表达水平的影响,探讨益气健脾生津方可能治疗原发性干燥综合征的干预机制。方法收集安徽省中医院风湿免疫科气阴两虚证的原发性干燥综合征患者60例,随机数字表法分为实验组和对照组,每组各30例。对照组给予硫酸羟氯喹0.2 g bid;实验组在对照组治疗基础上给予益气健脾生津方口服,均连服12周。流式细胞仪检测外周血Tfh细胞、Tfr细胞比例;酶联免疫吸附试验(ELISA)检测IL-10、IL-21、IL-6水平;免疫印迹法(Western blot)检测Atg5、Beclin1、LC3-Ⅱ/Ⅰ蛋白表达水平;荧光定量PCR法(qPCR)检测Atg5、Beclin1、LC3 mRNA表达水平;统计治疗前后炎症指标、干燥三项、ESSPRI、ESSDAI及中医证候积分。结果1)与治疗前相比,治疗后2组角膜荧光染色评分下降,泪膜破裂时间延长,静态唾液流率增加,相较于对照组,实验组角膜荧光染色评分显著下降,静态唾液流率显著增加(P<0.05);2)与治疗前相比,治疗后2组第4、8、12周中医症候评分、ESSDAI和ESSPRI动态降低,相较于对照组,实验组在治疗后各项积分明显下降(P<0.05);3)与治疗前相比,治疗后2组外周血IL-21、IL-10均降低,相较于对照组,实验组IL-21、IL-10表达降低,IgG降低(P<0.05);4)与治疗前相比,治疗后2组外周血Tfh细胞、Tfr细胞比例、Tfr/Tfh比值均降低,相较于对照组,实验组Tfr细胞显著降低(P<0.01);5)与治疗前相比,治疗后2组外周血CD4+T细胞Atg5、Beclin1、LC3-Ⅱ/Ⅰ蛋白显著降低,外周血CD4+T细胞Atg5、Beclin1、LC3 mRNA显著降低,相较于对照组,实验组自噬蛋白、mRNA表达均显著降低(P<0.01)。结论益气健脾生津方有效重建Tfr/Tfh细胞平衡,调节细胞因子,抑制自噬活性,具备减轻临床症状、延缓疾病活动、改善泪腺、涎腺分泌的功能。

This study performed to observe the effect of Yiqi Jianpi Shengjin recipe on Tfr/Tfh cell balance and autophagy expression in primary Sjögren’s syndrome(pSS)patients,and to explore the intervention mechanism of Yiqi Jianpi Shengjin recipe in treating pSS.We collected 60 cases of pSS with deficiency of both qi and yin from the Department of Rheumatology and Immunology of Anhui Provincial Hospital of TCM and randomly grouped them into experimental group and control group,with each group consisting of 30 cases.The control group was given a dose of hydroxychloroquine sulphate 0.2 g bid,the experimental group received an additional oral dose of Yiqi Jianpi Shengjin formula on top of the control group's treatment,and both continued to take it for 12 weeks.IL-10,IL-21 and IL-6 levels were measured by enzyme-linked immunosorbent assay,Tfh and Tfr cells ratios were measured by Flow cytometry,Western blot was used to detect the protein expression of Atg5,Beclin1 and LC3-Ⅱ/Ⅰ,and qPCR was used to detect the RNA expression of Atg5,Beclin1 and LC3.The indexes of inflammation,dryness,ESSPRI,ESSDAI and TCM syndrome integral before and after treatment were statistically analyzed.Data showed that the scores of corneal fluorescence stainin g in the experimental group were significantly lower and the static saliva flow rate was significantly higher compared with the control group.The experimental group showed a marked reduction in TCM,ESSPRI and ESSDAI score after treatment compared to control group.The expression of IL-21,IL-10 and IgG in the experimental group was markedly lower compa red with the control group.Tfr cells were significantly lower in the experimental group as compared to the control group.T he expression and ratio of autophagy protein and mRNA in the experimental group were significantly lower compared with t he control group.In conclusion,Yiqi Jianpi Shengjin recipe can effectively reestablish the balance of Tfr/Tfh cells,regulate cytokines,inhibit autophagy and activity,relieve clinical symptoms,delay disease activity,and improve the secretion of lacrimal gland and salivary gland.