Objective: To explore the effects and molecular mechanism of the selective JAK1inhibitor SHR0302 and Ruxolitinib on myeloproliterative neoplasms (MPN) cell line SET2 and primary cells in vitro. Methods: Cell proliferation was detected by CCK8 kit. Colony forming experiment was conducted to evaluate erythroid burst colony formation unit (BFU-E) of primary cells from MPN patients. Multi-factor kits were used to detect six inflammatory cytokines. Phosphorylated proteins of Jak-Stat signaling pathway were tested by Western blot. Results: At different time points after treated with SHR0302 and Ruxolitinib, the inhibition of cell proliferation was dose dependent by both drugs (P<0.01) . The inhibitory rates of 2.5 μmol/L SHR0302 and 0.1 μmol/L Ruxolitinib on SET2 cells for 72 h were comparable, i.e. (59.94±0.60) % and (64.00±0.66) %, respectively, suggesting that the inhibitory effect of SHR0302 was weaker than that of Ruxolitinib. Similarly, both SHR0302 and Ruxolitinib inhibited BFU-E in primary marrow cells from MPN patients in a dose-dependent manner. SHR0302 1.0 μmol/L produced similar degree of inhibition compared to Ruxolitinib 0.2 μmol/L. Except IL-12, the expression of other 5 cytokines (IL-6, TNF-α, IL-1β, IL-2, IL-8) was significantly inhibited by 1.6 μmol/L SHR0302 in SET2 cells at 24 h (P<0.01) , while Ruxolitinib 1.0 μmol/L had the same effect. Several phosphorylated molecules of Jak-Stat signaling pathway were significantly inhibited by SHR0302 in SET2 cells only for 3 h. P-stat1 (Tyr701) , p-stat3 (Tyr705) were down-regulated when treated with SHR0302 1.0 μmol/L (P<0.05) , p-jak1 (tyr1022/1023) and p-stat5 (Tyr694) were inhibited at 5.0 μmol/L (P<0.05) . Ruxolitinib significantly inhibited the downstream STAT protein at 0.1 μmol/L. Again, the inhibitory effect of SHR0302 on protein expression was weaker than that of Ruxolitinib. Conclusion: SHR0302 can effectively inhibit the proliferation of MPN cell line and patients' primary cells, as well as the expression of inflammatory factors. The molecular mechanism is possibly related to the down-regulation of phosphorylated proteins of Jak-Stat signaling pathway. Overall, the anti-proliferative and anti-inflammatory effects of SHR0302 are weaker than those of Ruxolitinib.
目的： 探究选择性JAK1抑制剂SHR0302和芦可替尼对骨髓增殖性肿瘤（MPN）细胞株SET2细胞和MPN患者原代细胞的影响以及分子作用机制。 方法： CCK8法检测不同浓度SHR0302和芦可替尼对SET2细胞增殖能力的影响；集落形成实验检测SHR0302和芦可替尼对MPN患者原代细胞红系爆式集落形成单位（BFU-E）的作用；多因子检测试剂盒MSD检测SHR0302和芦可替尼对SET2细胞IL-6、TNF-α、IL-1β、IL-2、IL-8、IL-12p70蛋白表达水平的影响；以Western blot法检测SHR0302和芦可替尼对SET2细胞JAK-STAT信号通路分子磷酸化水平的影响。 结果： 0.1、1.0、2.5、5.0 μmol/L SHR0302作用SET2细胞24、48、72 h，细胞增殖抑制率随药物浓度的增大而增高（P<0.001）；0.01、0.05、0.10 μmol/L芦可替尼作用SET2细胞48、72 h，细胞增殖抑制率亦随药物浓度的增大而增高（P<0.001），且均在72 h抑制作用最显著。2.5 μmol/L SHR0302、0.1 μmol/L芦可替尼作用SET2细胞72 h，细胞增殖抑制率分别为（59.94±0.60）%、（64.00±0.66）%，提示SHR0302的增殖抑制作用弱于芦可替尼。与对照组比较，0.1、1.0、5.0 μmol/L SHR0302和0.1、0.2、0.4 μmol/L芦可替尼均浓度依赖性地抑制MPN患者BFU-E形成，1.0 µmol/L SHR0302对MPN患者BFU-E集落形成抑制程度与0.2 µmol/L芦可替尼相当。除IL-12外，1.6 μmol/L SHR0302作用SET2细胞24 h可明显抑制炎性介质蛋白IL-6、TNF-α、IL-1β、IL-2、IL-8表达（P<0.01），与1.0 μmol/L芦可替尼对炎性介质抑制作用相当。不同浓度SHR0302作用SET2细胞3 h后JAK-STAT信号通路显著抑制，在1.0 μmol/L时可显著抑制p-STAT1（Tyr701）、p-STAT3（Tyr705）蛋白磷酸化，在5.0 μmol/L时可使p-JAK1（Tyr1022/1023）、p-STAT5（Tyr694）蛋白磷酸化水平明显下调（P<0.05），而芦可替尼在0.1 μmol/L即可明显抑制下游STAT蛋白磷酸化水平。 结论： SHR0302能有效抑制MPN细胞株和患者原代细胞的增殖以及炎性因子表达，其机制可能与下调p-JAK1及其下游p-STAT1、p-STAT3、p-STAT5蛋白磷酸化水平相关，但其抗增殖、抗炎作用弱于芦可替尼。.
Keywords: Cell proliferation; Inflammatory cytokine; JAK-STAT signaling pathway; Ruxolitinib; Selective JAK1 inhibitor SHR0302.