The role of RIP1 and RIP3 in the development of aplastic anemia induced by cyclophosphamide and busulphan in mice.

Abstract

This study aimed to investigate the role of RIP1 and RIP3 in the pathogenesis of aplastic anemia (AA) induced by cyclophosphamide and busulphan in mice. Animals were randomly divided into three groups: the control group, the AA group, and the Nec-1 group. Mouse AA model was established by intraperitoneal injection of cyclophosphamide (40 mg/kg/d) and busulfan (20 mg/kg/d) for 12 days. The Nec-1 group mice received intraperitoneal injection of Nec-1 (2 mg/kg/d) for 12 days prior to intraperitoneal injection of cyclophosphamide (40 mg/kg/d) and busulfan (20 mg/kg/d) for 12 days. The control mice received intraperitoneal injection of equal volume of saline. At 12 h after the last intraperitoneal injection, blood and bone marrow tissues were collected from mice. Peripheral blood cells were analyzed using hematology analyzer and the histological changes of bone marrow tissues were examined using scanning electron microscopy (SEM). The levels of RIP3 and RIP3 in bone marrow were measured using Western blot analysis and the interaction of RIP1 and RIP3 proteins was investigated on the basis of immunoprecipitation analysis. ELISA was used to measure the levels of IL-6, TNF-α, and FLT-3L in bone marrow tissue supernatant. Apoptosis and necrosis of bone marrow cells were analyzed using flow cytometry. Western blot showed that the expression of RIP1 and RIP3 was significantly increases in AA mice compared to the normal controls. Immunoprecipitation detected the pro-necrotic RIP1-RIP3 complex, suggesting that RIP1 and RIP3 mediated necroptosis may involved in the damage of bone marrow cells. Compared to the AA mice, Nec-1 group mice exhibited significantly increase of peripheral blood cells and mononuclear cells in bone marrow tissues and decrease of the apoptosis/necrosis of bone marrow cells. In addition, we observed significant decrease of IL-6, TNF-α, and FLT-3L in bone marrow tissue supernatant in the Nec-1 group mice compared to AA mice. Our results suggest that Nec-1 can prevent the development of AA by inhibiting bone marrow cells necrosis and the production of inflammatory mediators. RIP1 and RIP3-mediated necroptosis may involve in the pathogenesis of AA induced by cyclophosphamide and busulfan in mice.