MEK/ERK信号通路在环磷酰胺诱导的白细胞减少症模型大鼠造血功能中的作用研究
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摘要
目的探讨丝裂原活化蛋白激酶激酶/细胞外信号调节激酶(MEK/ERK)信号通路在环磷酰胺诱导的白细胞减少症模型大鼠造血功能中的作用。方法采用随机数字表法将60只SD大鼠分为ARRY-162组、模型组和空白对照组。ARRY-162组和模型组连续3 d腹腔注射环磷酰胺30 mg/(kg·d)造模,对照组注射等体积生理盐水。自造模当天起,ARRY-162组给予ARRY-1623 mg/(kg·d)尾静脉注射,模型组和对照组给予生理盐水尾静脉注射处理。连续干预7 d,采用全自动分析仪分析外周血白细胞(WBC)、红细胞(RBC)、血小板(PLT)、网织红细胞(REC)数量,苏木素-伊红(HE)染色观察骨髓组织病理改变,流式细胞仪检测骨髓细胞凋亡情况,蛋白免疫印迹分析Bax、Bcl-2、MEK1/2、ERK1/2蛋白表达情况。结果与对照组比较,模型组大鼠外周血WBC、RBC、PLT数量及REC比率均显著减少,差异有统计学意义(P<0.05);骨髓组织增生程度低,造血细胞减少,现大量脂肪组织增生,造血组织结构被破坏;骨髓细胞凋亡率明显升高(P<0.05);骨髓组织Bcl-2蛋白水平显著降低,Bax、p-MEK1/2、p-ERK1/2蛋白水平明显增高(P<0.05)。与模型组比较,ARRY-162组大鼠外周血WBC、RBC、PLT数量及REC比率均明显增多(P<0.05);骨髓细胞凋亡率明显降低(P<0.05);骨髓组织增生明显好转,组织损伤程度减轻;骨髓组织Bcl-2蛋白水平显著升高,Bax、p-MEK1/2、pERK1/2蛋白水平明显降低(P<0.05)。结论 MEK/ERK信号通路在环磷酰胺诱导的白细胞减少症大鼠的造血功能下降中具有重要作用,MEK抑制剂ARRY-162可有效改善白细胞减少症大鼠的造血功能。
Objective To investigate MEK/ERK signaling pathway in the hematopoietic function of leukopenia rats induced by cyclophosphamide.Methods 60 SD rats were randomized into 3 groups,namely ARRY-162,model and blank control.ARRY-162 group and model group were intraperitoneal administered with 30 mg/(kg·d) of cyclophosphamide for 3 d and the control with equal volume of NS.For 7 d since the beginning,,the ARRY-162 group was injected 3 mg/(kg·d) of ARRY-162;the model and the control with equal volume of NS,all in the tail vein.After 7 d of intervention,all rats' peripheral cell counts of white blood cell(WBC),red blood cell(RBC),platelet(PLT) and reticulocyte(REC) were analyzed and bone marrow biopsied.Flow cytometry was used to detect the apoptosis of bone marrow cells;Western blot to analyze the expression of Bax,Bcl-2,p-MEK1/2 and p-ERK1/2.Results For model group,the numbers of peripheral WBC,RBC,PLT and the ratio of REC were significantly less than control(P < 0.05);signs of bone marrow hypoplasia were found,i.e.,reduced hematopoietic cells,destroyed hematopoietic tissue,hyperplastic adipose tissue,and increased apoptosis rate of bone marrow cells compared with control(P <0.05).The model group had decreased Bcl-2 and increased Bax,p-MEK1/2 and p-ERK1/2 expression compared with control(P < 0.05).Oppositely,the ARRY-162 group saw increased numbers of WBC,RBC,PLT and the ratio of REC,decreased apoptosis rate of bone marrow cells,improved bone marrow proliferation,and less bone marrow injury compared with model group(P < 0.05).The ARRY-162 group had increased Bcl-2 and decreased levels of Bax,p-MEK1/2 and p-ERK1/2 compared with the model group(P < 0.05).Conclusion MEK/ERK signaling pathway plays an important role in the decrease of hematopoietic function in leukopenia rats induced by cyclophosphamide.MEK inhibitor ARRY-162 can effectively improve hematopoiesis of rats with leucocyte reduction.
Objective To investigate MEK/ERK signaling pathway in the hematopoietic function of leukopenia rats induced by cyclophosphamide.Methods 60 SD rats were randomized into 3 groups,namely ARRY-162,model and blank control.ARRY-162 group and model group were intraperitoneal administered with 30 mg/(kg·d) of cyclophosphamide for 3 d and the control with equal volume of NS.For 7 d since the beginning,,the ARRY-162 group was injected 3 mg/(kg·d) of ARRY-162;the model and the control with equal volume of NS,all in the tail vein.After 7 d of intervention,all rats' peripheral cell counts of white blood cell(WBC),red blood cell(RBC),platelet(PLT) and reticulocyte(REC) were analyzed and bone marrow biopsied.Flow cytometry was used to detect the apoptosis of bone marrow cells;Western blot to analyze the expression of Bax,Bcl-2,p-MEK1/2 and p-ERK1/2.Results For model group,the numbers of peripheral WBC,RBC,PLT and the ratio of REC were significantly less than control(P < 0.05);signs of bone marrow hypoplasia were found,i.e.,reduced hematopoietic cells,destroyed hematopoietic tissue,hyperplastic adipose tissue,and increased apoptosis rate of bone marrow cells compared with control(P <0.05).The model group had decreased Bcl-2 and increased Bax,p-MEK1/2 and p-ERK1/2 expression compared with control(P < 0.05).Oppositely,the ARRY-162 group saw increased numbers of WBC,RBC,PLT and the ratio of REC,decreased apoptosis rate of bone marrow cells,improved bone marrow proliferation,and less bone marrow injury compared with model group(P < 0.05).The ARRY-162 group had increased Bcl-2 and decreased levels of Bax,p-MEK1/2 and p-ERK1/2 compared with the model group(P < 0.05).Conclusion MEK/ERK signaling pathway plays an important role in the decrease of hematopoietic function in leukopenia rats induced by cyclophosphamide.MEK inhibitor ARRY-162 can effectively improve hematopoiesis of rats with leucocyte reduction.
引文
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[19]Orouji E,Ziegler B,Umansky V,et al.Leukocyte count restoration under dabrafenib treatment in a melanoma patient with vemurafenib-induced leukopenia:case report[J].Medicine,2014,93(28):e161-e167.
[20]Burotto M,Chiou VL,Lee J,et al.The MAPK pathway across different malignancies:A new perspective[J].Cancer,2015,120(22):3446-3456.
[2]Stillwell TJ,Benson RC,Deremee RA,et al.Cyclophosphamide‐induced bladder toxicity in wegener's granulomatosis[J].Arthritis Rheum,2014,31(4):465-470.
[3]Ahlmann M,Hempel G.The effect of cyclophosphamide on the immune system:implications for clinical cancer therapy[J].Cancer Chemother Pharmacol,2016,78(4):661-671.
[4]Shalit I,Kletter Y,Halperin D,et al.Immunomodulatory effects of moxifloxacin in comparison to ciprofloxacin and G-CSF in a murine model of cyclophosphamide-induced leukopenia[J].Eur J Haematol,2015,66(5):287-296.
[5]Kim EK,Choi EJ.Compromised MAPK signaling in human diseases:an update[J].Arch Toxicol,2015,89(6):867-882.
[6]赵厚德,郝智慧,郑海发,等.大鼠白细胞及骨髓有核细胞减少模型的研究[J].中国比较医学杂志,2003,13(5):291-293.
[7]朱彤,饶井芬,冯友繁,等.生血宁片对肿瘤放化疗白细胞减少症患者外周血Fas/Fas L表达的影响[J].临床和实验医学杂志,2018,17(6):598-601.
[8]郑丹,沙启明,王剑青,等.去甲斑蝥素对环磷酰胺诱导的白细胞减少症模型大鼠骨髓造血功能的影响[J].中国实验血液学杂志,2015,23(3):826-831.
[9]Schirmer JH,Bremer JP,Moosig F,et al.Cyclophosphamide treatment-induced leukopenia rates in ANCA-associated vasculitis are influenced by variant CYP450 2C9 genotypes[J].Pharmacogenomics,2016,17(4):367-374.
[10]Wang ZY,Lin JH,Muharram A,et al.Beclin-1-mediated autophagy protects spinal cord neurons against mechanical injury-induced apoptosis[J].Apoptosis,2014,19(6):933-945.
[11]Badalzadeh R,Mokhtari B,Yavari R.Contribution of apoptosis in myocardial reperfusion injury and loss of cardioprotection in diabetes mellitus[J].J Physiol Sci,2015,65(3):201-215.
[12]Sui X,Kong N,Ye L,et al.p38 and JNK MAPK pathways control the balance of apoptosis and autophagy in response to chemotherapeutic agents[J].Cancer Lett,2014,344(2):174-179.
[13]Hu X,Olsson T,Johansson I,et al.Dynamic changes of the anti-and pro-apoptotic proteins Bcl-w,Bcl-2,and Bax with Smac/Diablo mitochondrial release after photothrombotic ring stroke in rats[J].Eur J Neurosci,2015,20(5):1177-1188.
[14]Gross A,Jockel J,Wei M C,et al.Enforced dimerization of BAX results in its translocation,mitochondrial dysfunction and apoptosis[J].Embo J,2014,17(14):3878-3885.
[15]Liu X,Dong J,Cai W,et al.The Effect of Thymoquinone on Apoptosis of SK-OV-3 Ovarian Cancer Cell by Regulation of Bcl-2 and Bax[J].Int J Gynecol Cancer,2017,27(8):1596-1601.
[16]Cervellini I,Galino J,Zhu N,et al.Sustained MAPK/ERK activation in adult Schwann cells impairs nerve repair[J].J Neurosci,2018,38(3):2255-2217.
[17]Lim W,Park S,Bazer FW,et al.Apigenin Reduces Survival of Choriocarcinoma Cells by Inducing Apoptosis via the PI3K/AKT and ERK1/2MAPK Pathways[J].J Cell Physiol,2016,231(12):2690-2699.
[18]Ayub A,Yip WK,Seow HF.Dual treatments targeting IGF-1R,PI3K,m TORC or MEK synergize to inhibit cell growth,induce apoptosis,and arrest cell cycle at G1 phase in MDA-MB-231 cell line[J].Biomed Pharmacother,2015,75(3):40-50.
[19]Orouji E,Ziegler B,Umansky V,et al.Leukocyte count restoration under dabrafenib treatment in a melanoma patient with vemurafenib-induced leukopenia:case report[J].Medicine,2014,93(28):e161-e167.
[20]Burotto M,Chiou VL,Lee J,et al.The MAPK pathway across different malignancies:A new perspective[J].Cancer,2015,120(22):3446-3456.