目的 :1. 探索艾曲波帕（Eltrombopag, EPAG）联合环孢素 A（Cyclosporine A，CsA）与 EPAG 单药治疗难治/复发再生障碍性贫血（Aplastic anemia，AA）患者的疗效、安全性，克隆演变与长期预后，及预测疗效及复发的相关因素。2. 探索 EPAG 对经干扰素-γ（Interferon-γ，IFN-γ）诱导的人骨髓间充质干细胞细胞系是否具有增殖调节的作用及可能机制。方法:1. 回顾性收集自 2018 年 1 月至 2021 年 7 月，在本中心接受 EPAG+CsA 或 EPAG单药治疗的复发/难治 AA 患者，通过查阅病历及电话随访的形式收集患者基线特征、 EPAG 的使用剂量、临床疗效、不良反应和临床结局，在所有患者中及不同年龄亚组中评估 EPAG+CsA 和 EPAG 单药在疗效、安全性、长期预后、克隆演变等方面的差异，并对可能影响两种方案疗效的因素进行分析。2. 以人类 BM-MSC 细胞系 HS-5 细胞为研究对象，以 IFN-γ、 不同浓度的 EPAG或阿伐曲波帕（Avatrombopag, AVA）单独或共同诱导 HS-5 细胞，通过 CCK-8 法检测不同诱导处理对细胞增殖的影响，通过流式凋亡实验检测不同诱导处理对细胞凋亡的影响。通过转录组测序的方法检测 IFN-γ、 EPAG 单独或共同诱导后 HS-5 细胞差异化表达的基因，并对基因筛选后进行功能富集分析，寻找可能的机制分子，并通过 Western blotting 的方法从蛋白表达水平对关注的分子进行验证。结果:1. 纳入 103 例难治/复发 AA 患者，其中 77 例接受 EPAG+CsA 治疗， 26 例接受EPAG 单药治疗，两组在基线性别、年龄、SAA/NSAA 比例、既往接受ATG+CsA/CsA 治疗比例、诊断至开始 EPAG 治疗的时间、基线血常规、生化指标、EPAG 治疗时长及总剂量等无显著差异（P>0.05）。在相似的随访时间（EPAG+CsA 组 25 （6-48）个月， EPAG 单药组 19.5 （6-44）个月， P=0.627）内，EPAG+CsA 和 EPAG 单药治疗的患者在随访 3/6/12 个月及随访期末的总体缓解率（Overall response rate，ORR）分别为 37.7% vs. 30.8%（P=0.526），62.3% vs. 38.5%（P=0.034）、65.1% vs. 48.0%（P=0.140）及 61.0% vs. 34.6%（P=0.019）。两组随访 3/6/12 个月/随访期末的完全缓解率 （Complete response rate， CRR）均无显着差异（P 均>0.05）。与 EPAG 单药治疗组相比， EPAG+CsA 组的累积无复发生存（Relapse-free survival，RFS）曲线在随访时间增长时逐渐出现一定的优势（P=0.095）。EPAG+CsA 组患者的主要不良反应包括消化不良、牙龈增生、肌酐升高等， EPAG 单药组的主要不良反应包括消化不良、皮肤瘙痒、ALT 升高等，两治疗组各不良反应发生率无显著差异（P>0.05）。EPAG+CsA 组 3 例（3.9%）无效死亡， EPAG 单药组 1 例（3.8%）无效死亡。两组总生存曲线 （Overall survival， OS）无显著差异（P=0.953），克隆演变率（7.8% vs 3.8%，P=0.676）无显著差异。EPAG+CsA 组中，有效患者的基线网织红细胞计数较高（61.5（11.5-230.5）×10~9 /L vs. 21.6（6.6-56.6）×10~9/L，P=0.039），而 EPAG 单药组未发现显著影响有效率的因素。两组中均未发现显著影响复发的因素。将患者分为年龄<60 岁和≥60 岁亚组进一步分析。 在<60 岁年龄亚组，基线特征匹配的情况下，EPAG+CsA 组 ORR 显著高于单药组（6 个月：62.3% vs.35.3%，P=0.047；随访期末：62.3% vs. 29.4%，P=0.016）。对于随访 3 个月和12 个月的 ORR，EPAG+CsA 组的 ORR 数值更高，但无显著差异。两组在3/6/12 个月及随访期末的 CRR 无统计学差异（P>0.05）。EPAG+CsA 组 RFS曲线显著优于 EPAG 单药组（P=0.047），两组 OS、克隆演变率无显著差异。≥60 岁亚组中，除性别比例外，其他基线特征匹配的患者， EPAG+CsA 与 EPAG单药组第 3/6/12 个月/随访期末 ORR、 CRR、 RFS、 OS、克隆演变率无显著差异（P>0.05）。2. IFN-γ 可使 HS-5 细胞的增殖显著下降。确定 EPAG 对 HS-5 细胞的工作浓度为 0.5 μg/mL 及 4.0 μg/mL，A VA 对 HS-5 细胞的工作浓度为 0.2μg/mL 及 4.0μg/mL。 EPAG 仅在 0.5μg/mL 可以改善 1000ng/mL IFN-γ 对 HS-5 细胞的增殖抑制作用，而 AVA 在各个浓度剂量组均无法改善 1000ng/mL IFN-γ 对 HS-5细胞的增殖抑制作用。使用 EPAG 进行后续实验，流式凋亡实验显示 IFN-γ可显著升高 HS-5 细胞的晚期凋亡/死亡比例，EPAG 则可在一定程度上改善IFN-γ 提高的 HS-5 细胞晚期凋亡/死亡比例。通过转录组测序结果筛选出的差异性表达基因 ROMO1、 GPX4、 PRDX5、 NDUFB7、 EDF1 及 SF3B5， 这些主要与氧化磷酸化、耐受氧化应激及铁死亡相关。进一步在蛋白水平验证，发现 IFN-γ 单独诱导 HS-5 细胞， 可显著下调谷胱甘肽过氧化物酶 4 （Glutathione peroxidase 4，GPX4）的表达水平，而 GPX4 为抑制细胞铁死亡的关键蛋白；EPAG 单独作用时， HS-5 细胞中的 GPX4 水平无显著变化。而 EPAG 与 IFN-γ 共同诱导的 HS-5 细胞中，GPX4 蛋白水平相对 IFN-γ 诱导组显著上调。结论:1. EPAG+CsA 相比 EPAG 单药在难治/复发 AA 患者中存在疗效优势，且在<60岁患者中的优势更加突出；两种疗法的安全性、生存率、克隆演变率无显著差异；具有更高的基线网织红细胞计数可能在接受 EPAG+CsA 治疗后有更高的缓解率。 2. 不同于 AVA， EPAG 可改善被 IFN-γ 抑制的 HS-5 细胞增殖能力，其作用机制可能与 EPAG 上调了被 IFN-γ 抑制的铁死亡相关基因 GPX4 相关。

Objectives : 1. Enrolled patients with refractory/relapsed aplastic anemia (AA) under eltrombopag (EPAG)+cyclosporine A (CsA) or EPAG monotherapy in our center. Compare the efficacy, safety, clonal evolution and long-term prognosis between 2 therapies, and further explore whether there are factors that could predict the efficacy and recurrence of the two regimens, respectively. 2. Preliminarily explore whether EPAG can regulate the proliferation of IFN-γ induced cell line of human bone marrow mesenchymal stem cell (BM-MSC), and the possible mechanism to explain it. Methods: 1. From January 2018 to July 2021, patients with relapsed/refractory AA who received EPAG+CsA or EPAG monotherapy in our center were retrospectively enrolled, and their baseline characteristics, EPAG dosage, clinical efficacy, adverse events (AE), long-term survival and clonal evolution, etc. were reviewed and compared in all patients and in different age subgroups. Factors that could possibly affect the efficacy or recurrence of the two regimens were analyzed. 2. BM-MSC cell line HS-5 cell was selected for our research. HS-5 was induced by interferon-γ (IFN-γ), different concentrations of EPAG and avatrombopag (AVA) alone or together. The effects of different treatments on cell proliferation were detected by CCK-8, and that on cell apoptosis were detected by flowmetry apoptosis assay. Transcriptome sequencing was used to identify differentially expressed genes in HS-5 induced by IFN-γ and/or EPAG alone or together, and Western blotting(WB) was performed to validate for protein expression levels.Results: 1. 103 patients were enrolled in the study. Seventy-seven patients received EPAG+CsA and 26 received EPAG only. No significant difference was found in baseline characteristics of patients between EPAG+CsA and EPAG monotherapy group, including age at EPAG initiation, SAA/NSAA ratio, previous ATG+CsA/CsA treatment ratio, time of previous IST treatment, patient baseline data before EPAG, EPAG exposure and follow-up. With the comparable median follow-up time (25 (6-48) months vs. 19.5 (6-44) months for EPAG+CsA and EPAGmonotherapy respectively, P=0.627), the OR rate (ORR) for patients treated with EPAG+CsA and EPAG alone was 37.7% vs. 30.8% (P=0.526), 62.3% vs. 38.5% (P=0.034), 65.1% vs. 48.0% (P=0.140), 61.0% vs. 34.6% (P=0.019) at the end of follow-up. No significant difference was found in CR rate (CRR) at 3~rd /6~th/12~th month/end of follow-up between the two groups. EPAG+CsA group seemed to have a superior cumulative relapse-free survival compared with EPAG monotherapy group, although not statistically significant (P=0.095). The major AE of EPAG+CsA group were dyspepsia, gum hypertrophy and elevated creatinine, and dyspepsia, skin pruritus and elevated ALT for EPAG monotherapy group. The rates of each AE at different grade were comparable. Three NR patients (3.9%) died in EPAG+CsA group and 1 NR patient (3.8%, P=1.000) died in EPAG monotherapy group, and the overall survival curves between 2 groups were comparable (P=0.953). No significant difference was detected in clonal evolution rate (7.8% vs. 3.8%, P=0.676) between 2 groups either. For EPAG+CsA group, responders had a significantly higher baseline reticulocyte count (P=0.039) compared with nonresponders, while for EPAG monotherapy group, no significant predictor was found for ORR. No significant predictor was found for relapse in either EPAG+CsA or EPAG monotherapy group. Patients were divided into 2 subgroups (<60 year-old/≥60 year-old) for further analysis. For the subgroup of <60 year-old where baseline characteristics before EPAG were comparable, the ORR at 6~th month/end of follow-up was significantly higher in patients with EPAG+CsA compared with those with EPAG monotherapy. and for ORR at 3~rd month/12~th month, ORR of EPAG+CsA was mathematically higher. The CRR were comparable between 2 regimens in this subgroup (P>0.05). The cumulative relapse-free survival (RFS) curve was significantly superior in patients with EPAG+CsA than those with EPAG only (P=0.047) in this subgroup. For the subgroup of ≥60 year-old where baseline characters were comparable except for male proportion, no significant difference was found between EPAG+CsA and EPAG monothearpy in ORR, CRR, cumulative RFS or clonal evolution (P>0.05). 2. IFN-γ significantly reduced the viability of HS-5 cells. The working concentrations were determined for EPAG as 0.5 μg/mL and 4.0 μg/mL, and 0.2 μg/mL and 4.0μg/mL for AVA. After 48 hours of induction with IFN-γ and 0.5 μg/mL EPAG together , the viability of HS-5 cells was significantly elevated compared with the cells treated with IFN-γ only . However, EPAG at higher concentration did not have the ability of rescuing the negative effect of IFN-γ on HS-5’s viability. Meanwhile, CCK-8 assay indicated that AVA did not have the same effect as EPAG in rescuing the negative effect of IFN-γ on HS-5 cell viability. Apoptosis cytometry indicated that EPAG could partially reduce the proportion of late apoptotic/dead cells which was increased by IFN-γ. Together with the results of transcriptome sequencing and verification of WB, we found that the key inhibitor of ferroptosis GPX4 was downregulated in cells treated with IFN-γ only, and the level of GPX4 was up-regulated in cells treated with both EPAG and IFN-γ compared with those treated with IFN-γ only. Conclusions: 1. Compared with EPAG monotherapy, EPAG+CsA had an advantage in efficacy towards refractory/relapsed AA patients, and the advantage was more prominent in patients <60 year-old; the AE rates of 2 therapies were similar, and so was longterm survival. There were also no significant differences in clonal evolution rate between 2 groups; refractory/relapsed AA patients with higher baseline reticulocyte count levels were more likely to respond to EPAG+CsA. 2. Unlike AVA, EPAG could improve the viability of HS-5 cells which was inhibited by IFN-γ, and the possible mechanism involved the restoration of expression level of key ferroptosis inhibitor GPX4, which was downregulated by IFN-γ.