健脾滋肾泻火方通过LMP2/JAK/STAT信号通路改善原发免疫性血小板减少症的机制研究

Mechanism of Jianpi Zishen Xiehuo Recipe(健脾滋肾泻火方)in Improving Primary Immune Thrombocytopenia by LMP2/JAK/STAT Signaling Pathway

目的:基于LMP2/JAK/STAT信号通路探讨健脾滋肾泻火方治疗原发免疫性血小板减少症(ITP)的可能作用机制。方法:收集脾肾亏虚、火伤血络证ITP患者及正常者外周血单个核细胞(PBMC),将细胞分为正常对照组、模型对照组、强的松5 mg/kg组、健脾滋肾泻火方20 g/kg组。给予相应的药物干预后,收集细胞采用CCK8法检测不同浓度含药血清对细胞活力的影响,RT-PCR法检测Janus激酶/信号转导与转录激活因子(Jak/Stat)信号通路、免疫蛋白酶体亚单位β1i(Lmp2)、干扰素-γ(Ifnγ)、白细胞介素-4(Il4)mRNA的表达,Western blot法检测JAK1、STAT1蛋白表达。结果:与5%、10%含药血清组相比,20%含药血清组的细胞活力明显减低(P<0.05或P<0.01);与正常对照组比较,模型对照组PBMC中IfnγmRNA表达显著上调,Il4 mRNA表达显著下调(P<0.01);Lmp2、Jak1、Stat1 mRNA及JAK1、STAT1蛋白表达显著上调(P<0.01);与模型对照组比较,5 mg/kg强的松10%含药血清组、20 g/kg健脾滋肾泻火方10%含药血清组中Ifnγ、Lmp2、Jak1、Stat1 mRNA表达及JAK1、STAT1蛋白表达显著下调,Il4 mRNA表达明显上调(P<0.05或P<0.01)。结论:健脾滋肾泻火方可能通过抑制ITP患者PBMC中LMP2的表达,进而抑制JAK/STAT信号通路,调节Th1/Th2免疫平衡,恢复免疫耐受,减少血小板破坏。

Objective:To explore the possible mechanism of Jianpi Zishen Xiehuo Recipe(健脾滋肾泻火方)in treating primary immune thrombocytopenia(ITP)based on the large multifunctional peptidase 2/Janus kinase-signal/signal transducer and activator of transcription(LMP2/JAK/STAT)signaling pathway.Methods:Peripheral blood mononuclear cells(PBMCs)were collected from normal subjects and patients with ITP of Pi(脾)-Shen(肾)deficiency and huoshagnxueluo(火伤血络)syndromes.The cells were divided into a normal group,a model group,a prednisone 5 mg/kg group,and a Jianpi Zishen Xiehuo Recipe 20 g/kg group.After the corresponding drug intervention,the cells were collected and cell viability of different concentrations of serum containing drugs was detected by using the cell counting kit-8(CCK8).Real-time fluorescence quantitative polymerase chain reaction(RT-PCR)was used to determine the mRNA expression of JAK/STAT signaling pathway,immunoproteasome subunits β1i(LMP2),interferon-γ(IFN-γ),and interleukin-4(IL-4).The Western blot was used to determine the protein expression of JAK1 and STAT1.Results:Compared with serum containing 5% and 10% drugs,respectively,the serum containing 20% drug significantly reduced the cell viability(P<0.05 or P<0.01).As compared with the normal control group,the mRNA expression of Ifnγwas up-regulated significantly in PBMCs of the model control group,while that of Il4 was down-regulated significantly(P<0.01).The mRNA expression of Lmp2,Jak1,and Stat1 and the protein expression of JAK1 and STAT1 were up-regulated in the model control group(P<0.01).As compared with the model control group,the mRNA expression of Ifnγ,Lmp2,Jak1,and Stat1 and the protein expression of JAK1 and STAT1 were down-regulated significantly in the 5 mg/kg 10% prednisone group and the 20 g/kg 10% Jianpi Zishen Xiehuo Recipe group,and the mRNA expression of Il4 was up-regulated in the 20 g/kg 10% Jianpi Zishen Xiehuo Recipe group(P<0.05 or P<0.01).Conclusion:Jianpi Zishen Xiehuo Recipe may inhibit the expression of LMP2 in PBMCs of patients with ITP,thereby inhibiting the JAK/STAT signaling pathway,regulating Th1/Th2 immune balance,restoring the immune tolerance,and reducing platelet damage.