Mechanism of Qihuang Yiqi Shexue Prescription in Treatment of ITP Model Mice Based on Autophagy Mediated by AMPK/mTOR/ULK1 Signaling Pathway / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo explore the mechanism of Qihuang Yiqi Shexue prescription （QHYQSX） in the treatment of immune thrombocytopenia （ITP） model mice based on the autophagy mediated by the adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR）/Unc-51-like kinase 1 （ULK1） signaling pathway. MethodFifty BALB/c mice were randomly divided into normal group， model group， high- and low-dose QHYQSX groups， and prednisone group， with 10 mice in each group. The ITP model was induced by intraperitoneal injection of anti-platelet serum （APS） of guinea pig. On the 8th day of the APS injection， drugs were administered by gavage for 14 days. Peripheral blood platelet （PLT） count and hemoglobin （Hb） concentration were detected. Spleen and thymus were separated， weighed， and the organ index was calculated. Sternum was sampled for bone marrow smear， and bone marrow megakaryocytes were classified under a microscope. Thrombopoietin （TPO）， interleukin-6 （IL-6）， IL-10， tumor necrosis factor-α （TNF-α）， transforming growth factor-β1 （TGF-β1）， and interferon-γ （IFN-γ） in the serum were detected by enzyme-linked immunosorbent assay（ELISA）. AMPK， mTOR， ULK1， microtubule-associated protein light chain 3 （LC3）， Beclin1， and p62 mRNA expression levels in the spleen were detected by Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR）. The protein expression of AMPK， p-AMPK， p-mTOR， p-ULK1， LC3Ⅱ/LC3Ⅰ， Beclin1， and p62 in the spleen was detected by Western blot. ResultCompared with the normal group， the model group showed reduced peripheral blood PLT count， Hb， and TPO levels （P<0.05，P<0.01）， increased spleen and thymus indexes （P<0.01）， decreased number of bone marrow megakaryocytes （P<0.01）， elevated serum levels of IL-6， TNF-α， and IFN-γ （P<0.01）， and reduced IL-10 and TGF-β1 levels （P<0.01）. Compared with the model group， the groups with drug intervention showed increased PLT counts and TPO levels （P<0.01）， decreased spleen and thymus indexes （P<0.05， P<0.01）， elevated number of bone marrow megakaryocytes （P<0.05， P<0.01）， reduced serum levels of IL-6， TNF-α， and IFN-γ （P<0.05， P<0.01）， and up-regulated IL-10 and TGF-β1 levels （P<0.05，P<0.01）. Compared with the low-dose QHYQSX group， the high-dose QHYQSX group and the prednisone group showed different degrees of significant differences in improving PLT counts and levels of cellular inflammatory factors （P<0.05， P<0.01）. Real-time PCR and Western blot results showed that compared with the normal group， the model group showed up-regulated mRNA expression of AMPK， LC3， and Beclin1 and protein expression of p-AMPK/AMPK， LC3Ⅱ/LC3Ⅰ， and Beclin1 in the spleen （P<0.05， P<0.01）， and down-regulated mRNA expression of mTOR， ULK1， and p62 and protein expression of p-mTOR， p-ULK1， and p62 （P<0.05， P<0.01）. Compared with the results in the model group， high- and low-dose QHYQSX and prednisone could down-regulate the mRNA expression of AMPK， LC3， and Beclin1 and protein expression of p-AMPK/AMPK， LC3Ⅱ/LC3Ⅰ， and Beclin1 in the spleen （P<0.05， P<0.01）， and up-regulate the mRNA expression of mTOR， ULK1， and p62 and protein expression of p-mTOR， p-ULK1， and p62 （P<0.05， P<0.01）. ConclusionQHYQSX may inhibit excessive autophagy by regulating the AMPK/mTOR/ULK1 signaling pathway， thereby regulating immune intolerance and playing a role in the treatment of ITP.