Effect of ERK1/2 Signaling Pathway Inhibitor PD98059 on the Expression of Ras, BRaf, MEK, ERK1/2 in Marrow Nucleated Red Blood Cells of CMS Patients / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of ERK1 / 2 signaling pathway inhibitor PD98059 on Ras, Raf, MEK, ERK1, ERK2 expression in order to explore a new way for basic research and clinical treatment of the chronic mountain sickness(CMS).</p><p><b>METHODS</b>Sixteen CMS patients were selected, the bone marrow was collected for isolation of monomuclear cells (MNC), the cells were sorted by using CD71 and CD235a antibody magnetic beads, then positive cells were diveded into 5 groups: blank control, DMSO and PD98059 5, 10 and 20 µmol/L, and were cultured in hypoxid condition for 72 hours. The Ras-GTP levels in supernatant was detected by ELISA, the RT-PCR was used to determine the expression of BRaf, MEK, ERK1, ERK2 mRNA in nucleated red blood cells, and the Western blot method was used to detect expression of BRaf, MEK, ERK1, ERK2 protein.</p><p><b>RESULTS</b>PD98059 had no effect on the level of Ras-GTP in each groups. Compared with the blank control group, the expression levels of BRaf, MEK mRNA in DMSO group were not statistically significant (P values were 0.826, 0.298). Compared with the PD98059 20 mol/L group, the expression level of ERK1/2 mRNA was statistically significant (P=0.001, 0.002). Compared with the blank control group, expression levels of p-BRaf, p-MEK protein in DMSO group were not statistically significant (P=0.370, 0.351). Compared with the PD98059 20 mol/L group, the difference of p-ERK1/2 protein level in other 4 groups were statistically significant (P values were <0.001, 0.007).</p><p><b>CONCLUSION</b>PD98059 can up-regulate the expressions of ERK1/2 miRNA and p-ERK1/2 protein in bone marrow nucleated red blood cells, the Ras / Raf / MEK / ERK 1/2 pathway is the main signal transduction pathway in regulating bone marrow nucleated red blood cells, suggesting that Ras/Raf /MEK /ERK 1/2 pathway may be involved in the pathogenesis of chronic mountain sickness process.</p>

Influence of GATA-1 on Expression of EpoR in Bone Marrow CD71+ Cells of Rat Model with High Altitude Polycythemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the influence of GATA-1 on expression of EpoR in bone marrow CD71+ cells of rat model with high altitude polycythemia (HAPC).</p><p><b>METHODS</b>Forty-eight male SD rats were randomly divided into normal control and HAPC model group. HAPC model was established at the altitude of 4 300 meters in the natural environment, and verified by bone marrow cell counts and hematological parameters. Myeloid CD71+ cells were separated by the density gradient centrifugation combined with magnetic activated cell sorting. The expression of EpoR on cell membrane was detected by flow cytometry and cell immunofluorescence. The expression changes of GATA-1 and EpoR mRNA and protein were detected by Q-PCR and Western blot, respectively. CD71+ cells were cultured under normoxia and hypoxia, respectively. After transfection for 96 h, the optimal interference sequence GATA-1 shRNA1 was selected. And the mRNA and protein expression level of GATA-1 and EpoR were detected by Q-PCR and Western blot respectively.</p><p><b>RESULTS</b>The animal model with HAPC was established successfully and comfirmed by the bone marrow cell counting and the hematologic parameters in comparison with that of the normal control. EpoR expression on the myeloid CD71+ cell membrane in HAPC group was significantly higher than that in normal control (P<0.05). The expression of GATA-1 and EpoR in myeloid CD71+ cells of HAPC group was higher than that in control group (P<0.05). The mRNA and protein expression of GATA-1 and EpoR in two groups positively correlated (control group, r=0.929, P<0.01, r=0.802, P<0.05; HAPC group, r=0.822, P<0.05, r=0.839, P<0.01). However, the mRNA and protein expression of EpoR at normoxia and hypoxia was significantly lower than that in negative control group after interfernce with GATA-1 shRNA1 for 96 h (P<0.05). And the expression of GATA-1 and EpoR under hypoxia was higher than that in normoxia.</p><p><b>CONCLUSION</b>The effect of GATA-1 on EpoR expression may be correlated with the pathogenesis of HAPC.</p>

Effect of PD98059 on Proliferation and Apoptosis of CD71(+),CD235a(+) Nucleated Erythrocytes in the Patients with High Altitude Polycythemia / 中国实验血液学杂志

<p><b>UNLABELLED</b>Objective： To study the effect of PD98059, a specific inhibitor of Ras/Raf/MEK/ERK signaling pathway, on the proliferation and apoptisis of bone marrow CD71(+), CD235a(+) nucleated erythrocytes in patients with high altitude polycythemia (HAPC) and the pathogenesis of HAPC.</p><p><b>METHODS</b>The CD71(+) and CD235a(+) nucleated erythrocytes in HAPC patients and controls (patients with simple obsolete stracture) were sorted by using the immunemagnetic beads, then were added with 5, 10, 20 µmol/L of PD98059 and DMSO (as control) and were cultured for 72 h under hypoxia. The cell apoptosis was detected by flow cytometry with Annexin V and PI double staining, the cell proliferation was detected by CCK8 method, at same time the erythroid colong-formation ability of bone marrow mononuclear cells (BMMNC) treated with 5, 10, 20 µmol/L of PD98059 and DMSO was observed.</p><p><b>RESULTS</b>With the increase of PD98059 concentration, the apoptosis rate of bone marrow CD71(+) and CD235a(+) nucleated erythrocytes in HAPC patients was enhanced (r=0.807,P<0.01), while the proliferation rate of CD71(+) and CD235a(+) nucleated erythrocytes in HAPC patients dereased (r=0.502,P<0.01). The erythroid colong-formation ability of BMMNC in HAPC patients decreased with the increase of PD98059 concentration (r=0.504,P<0.01). There were statistic differences among different groups at 7 and 14 d.</p><p><b>CONCLUSION</b>The MEK specific inhibitor PD98059 can inhibit the proliferation and promote the apoptosis of CD71(+) and CD235a(+) nucleated erythrocytes in HAPC patients, then inhibit the excessive accumulation of erythrocytes.</p>