Effect of recombinant human thrombopoietin on endotoxin-induced thrombocytopenia in mice

Abstract

To investigate the effect of recombinant human thrombopoietin (rhTPO) on thrombocytopenia (TCP) induced by endotoxin lipopolysaccharide (LPS) in mice. Sixty male C57BL/6 mice were divided into normal saline (NS) control group (NS group), sepsis-induced TCP model group (LPS group) and rhTPO treatment group (LPS+rhTPO group) by random number table with 20 mice in each group. Sepsis-induced TCP model was reproduced by one intraperitoneal injection of LPS 30 mg/kg, and the mice in NS group were given the same amount of NS. In LPS+rhTPO group, 2.7 kU/kg rhTPO was subcutaneously injected into mice immediately after intraperitoneal injection of LPS, once every 24 hours. The mice in NS group and LPS group were injected subcutaneously with the same amount of NS. The observation period of each group lasted for 72 hours. The inner canthus blood was harvested before and every 24 hours after modeling, and the platelet count (PLT) was measured by animal blood cell counter. The eyeball blood of mice was harvested at 72 hours after modeling, and the proportion of CD61+CD62p+ cells in platelet-rich plasma was detected by flow cytometry, by which the platelet activation was reflected. Lung and spleen tissues of mice were harvested, and the positive expression of CD41 was determined by immunohistochemistry, by which the platelet sequestration in organs was reflected. Bone marrow cells from unilateral femur of mice were harvested, and the proportion of CD41+CD61+ cells was determined by flow cytometry to reflect the proliferation of bone marrow megakaryocytes. There was no significant difference in PLT among the groups before modeling. With the extension of the time after modeling, PLT in LPS group was decreased continuously, and increased slightly at 72 hours, but it was still significantly lower than that in NS group (×109/L: 308.60±21.70 vs. 1 152.72±50.27, P < 0.05); PLT in LPS+rhTPO group was increased continuously with the extension of modeling time, and it was significantly higher at 72 hours than that in LPS group (×109/L: 926.78±48.85 vs. 308.60±21.70, P < 0.05). At 72 hours after modeling, the proportion of CD61+CD62p+ cells in platelet-rich plasma of LPS group was significantly higher than that of NS group [(25.07±2.55)% vs. (4.17±0.38)%, P < 0.05], while the value in LPS+rhTPO group was significantly lower than that of LPS group [(15.92±1.26)% vs. (25.07±2.55)%, P < 0.05]. The proportion of CD41+CD61+ cells in bone marrow megakaryocytes of LPS group was significantly higher than that of NS group [(11.84±0.80)% vs. (3.60±0.42)%, P < 0.05], and the proportion of CD41+CD61+ cells in LPS+rhTPO group was significantly higher than that in LPS group [(30.96±2.49)% vs. (11.84±0.80)%, P < 0.05]. Immunohistochemistry showed that the positive expressions of CD41 in lung and spleen tissues of LPS group increased significantly than NS group [A value: 828.94±119.30 vs. 447.09±16.19 in lung tissue, (280.15±16.71)×103 vs. (0.65±0.26)×103 in spleen tissue, both P < 0.05], while the positive expressions of CD41 in lung and spleen tissues of LPS+rhTPO group decreased significantly than LPS group [A value: 542.78±2.95 vs. 828.94±119.30 in lung tissue, (129.40±13.49)×103 vs. (280.15±16.71)×103 in spleen tissue, both P < 0.05]. The rhTPO in endotoxin-induced TCP may stimulate the proliferation of bone marrow megakaryocytes, inhibit platelet activation and affect platelet sequestration in organs, so as to increase platelet levels.