背景:补肾方(BSF)是有效的中药(TCM)慢性乙型肝炎(CHB)根据我们以前的研究。然而,BSF治疗不同阶段CHB患者的特殊有效性和免疫调节机制仍有待探索。
目的:比较BSF在初治患者和PEG-IFN-α治疗患者中的治疗效果,探讨免疫调节的潜在机制。
方法:超高效液相色谱-四极静电场-轨道阱高分辨率质谱和TCMSP数据库用于测定BSF的主要成分。在这项回顾性研究中纳入了256名患者,并将其分为治疗组(T组,BSF+PEG-IFN-α)和对照组(C组,PEG-IFN-α单一疗法)。在每一组中,患者被进一步分组为亚组,即T1/C1组(初治患者,T1=34,C1=94)和T2/C2组(PEG-IFN-α治疗的患者,T2=56,C2=82)。血清HBV标志物,血清HBVDNA水平,从记录中获得血清ALT/AST和中医症状。生物信息学分析用于获得BSF治疗CHB患者的潜在免疫调节机制。在T2和C2组患者中,我们使用36例患者的外周血单个核细胞,通过流式细胞术分析外周血滤泡辅助性T(Tfh)细胞和B细胞亚型的特征.大鼠含BSF血清的制备。体外,建立CXCR5+细胞与HepG2.2.15细胞共培养体系,探讨BSF的免疫调节作用。
结果:在BSF中共检测到14种主要活性化合物,这被认为是CHB治疗的关键。我们的发现表明,T2组表现出HBsAg下降≥1-log10IU/ml和HBeAg血清清除率的较高百分比,与C2组相比(35.7%vs.15.9%,P=0.033;33.9%vs.11.0%,P=0.002)。此外,T2组显示HBsAg下降≥1-log10IU/ml和HBeAg血清清除率的较高百分比,与T1组相比(35.7%vs.14.7%,P=0.031;33.9%vs.2.9%,P=0.000)。T1组和T2组中医临床证候总有效率明显高于C1组和C2组(85.3%vs.61.7%,P=0.012;89.1%vs.63.4%,P=0.000)。生物信息学分析表明,BSF治疗CHB患者的免疫调节机制主要与B细胞的生长和刺激有关,T细胞分化,和B细胞受体的信号通路。此外,Tfh细胞的频率及其IL-21水平,BSF处理后,B细胞表达的IL-21R均增加。此外,在CXCR5+细胞和HepG2.2.15细胞的共培养系统中,含BSF的血清治疗后,HBsAg和HBeAg水平降低,Tfh细胞频率上调,B细胞频率下调。
结论:BSF有较高的百分比的HBsAg下降和HBeAg血清清除在PEG-IFN-α治疗的患者相比,初治患者。潜在的免疫调节机制可能与通过IL-21/IL-21R信号通路促进Tfh细胞与B细胞的相互作用有关。
BACKGROUND: Bushen Formula (BSF) is the effective traditional Chinese medicine (TCM) for chronic hepatitis B (CHB) according to our previous researches. However, the special effectiveness of BSF treating CHB patients in different stages and the immunoregulatory mechanisms remain to be explored.
OBJECTIVE: To compare the therapeutic effects of BSF in both treatment-naive patients and Peg-IFN-α-treated patients, and explore the potential mechanism of immunomodulation.
METHODS: Ultra-high performance liquid chromatography-quadrupole electrostatic field-orbital trap high resolution mass spectrometry and the TCMSP database were used to determine the main components of BSF. Two hundred and sixty-six patients were enrolled in the retrospective study, and they were divided into the treatment group (T-Group, BSF plus Peg-IFN-α) and the control group (C-Group, Peg-IFN-α monotherapy). Within each group, patients were further grouped into subgroups, namely T1/C1 groups (treatment-naive patients, T1 = 34, C1 = 94) and T2/C2 groups (Peg-IFN-α-treated patients, T2 = 56, C2 = 82). Serum HBV markers, serum HBV DNA levels, serum ALT/AST and TCM symptoms were obtained from the record. Bioinformatics analysis was employed to obtain the potential immunoregulatory mechanisms of BSF treating CHB patients. Among patients in T2 and C2 group, peripheral mononuclear cells from 36 patients were used to analyze the characteristics of peripheral follicular helper T (Tfh) cells and B-cell subtypes by flow cytometry. Preparation of BSF-containing serum in rats. In vitro, the co-culture system of CXCR5+ cells and HepG2.2.15 cells was built to investigate the immunoregulatory effects of BSF.
RESULTS: A total of 14 main active compounds were detected in BSF, which were deemed critical for the treatment of CHB. Our findings indicated that the T2-Group exhibited the higher percentage of HBsAg decline ≥ 1-log10 IU/ml and rate of HBeAg seroclearance compared to the C2-Group (35.7% vs. 15.9%, P = 0.033; 33.9% vs. 11.0%, P = 0.002). Additionally, the T2-Group demonstrated the higher percentage of HBsAg decline ≥ 1-log10 IU/ml and rate of HBeAg seroclearance compared to the T1-Group (35.7% vs. 14.7%, P = 0.031; 33.9% vs. 2.9%, P = 0.000). The total effective rate based on TCM clinical syndrome in T1-Group and T2-Group were significantly greater than those in C1-Group and C2-Group (85.3% vs. 61.7%, P = 0.012; 89.1% vs. 63.4%, P = 0.000). Bioinformatics analysis indicated that the immunoregulatory mechanisms of BSF treating CHB patients were mainly linked to the growth and stimulation of B-cell, T-cell differentiation, and the signaling pathway of the B-cell receptor. Furthermore, the frequencies of Tfh cells and its IL-21 level, and the IL-21R expressed by B-cell were all increased after BSF treatment. Additionally, in the co-culture system of CXCR5+ cells and HepG2.2.15 cells, HBsAg and HBeAg levels were decreased after BSF-containing serum treatment,as well as the up-regulating of Tfh cell frequencies and down-regulating of B-cell frequencies.
CONCLUSIONS: BSF have the higher percentage of HBsAg decline and HBeAg seroclearance in Peg-IFN-α-treated patients compared with treatment-naive patients. The potential immunoregulatory mechanism may correlate with promoting the interaction between Tfh cells and B-cell through IL-21/IL-21R signaling pathway.