Significance And Mechanism Ofnecroptosis In Hemophagocyticsyndrome

Part Ⅰ: Non-lethal hemophagocytic syndromeObjective:1)To construct a non-lethal hemophagocytic syndrome mouse model by repeatedly stimulating Toll-like receptor 9 with unmethylated CpG DNA fragment;2)To detect the phenomenon of necroptosis in mouse model.Method:8-week-old C57 / BL6 mice were randomly divided into 2 groups,3 mice each group,and experimental group mice were injected intraperitoneally with 2.5ug / g body weight unmethylated CpG DNA fragment on day 0,2,4,7,and 9.Control group mice were injected intraperitoneally with 200 ul PBS solution.Mice were sacrificed on the day 9,and blood was collected for blood cell count examination.The spleens were collected and photographed.Liver RNA was extracted for reverse transcription,and m RNA expression level of inflammatory cytokine in each group of mice was detected by RT-PCR,mainly included IFN-γ,IL-10,IL-12.The liver protein was collected,and the expression level of necroptosis-related protein RIPK1,RIPK3,MLKL and CC3 was detected by western blot.At the same time,livers of mice from each group were collected for frozen section and immunofluorescence staining.Result:Compared with control group,the mice in the experimental group showed decreased blood cell counts,splenomegaly.The RT-PCR result showed that the expression level of inflammatory cytokine in liver was significantly increased.The abnormal expression of necroptosis-related protein mainly showed as decreased expression of RIPK1,increased expression of MLKL and CC3.The immunofluorescence staining of liver showed positive expression of RIPK1 and MLKL.Conclusion:Repeated stimulation of Toll-like receptor 9 by unmethylated CpG DNA fragment could construct a non-lethal hemophagocytic syndrome mouse model,which was characterized by decreased blood cell counts,splenomegaly,and increased expression of liver inflammatory cytokine.In addition,necroptosis was found in the liver of experimental group mice.The successful construction of mouse model lays the experimental foundation for subsequent research.Part Ⅱ: Lethal hemophagocytic syndromeObjective:1)To construct a mouse model of lethal hemophagocytic syndrome by repeatedly stimulating Toll-like receptor 9 with unmethylated CpG DNA fragment combined with galactosamine(DG);2)To detect the phenomenon of necroptosis in mouse model.Method:5-week-old C57 / BL6 mice were randomly divided into 2 groups,5 mice in experimental group and 3 mice in control group.Experimental group mice were injected intraperitoneally 3ug/g body weight unmethylated CpG DNA fragment and0.30 mg / g body weight galactosamine on day 0,2,4,7,9,11,14,16,18,21.Control group mice were injected intraperitoneally 200 ul PBS solution.Mice were sacrificed on day 4,10 and 21.Peripheral blood smears were taken for Giemsa staining.The spleens were collected and photographed,weighed,extracted cell for Giemsa staining.The femurs were collected,part for Giemsa staining of bone marrow cell,and part for paraffin section and HE staining.The liver protein was collected,and the expression level of necroptosis-related protein RIPK1,RIPK3,MLKL and CC3 was detected by western blot.The livers of mice in each group were collected,part for HE staining and part for immunohistochemical staining.Result:Part of experimental group mice died on the first day.Compared with control group,the mice in the experimental group showed significantly splenomegaly.Hemophagocytic phenomenon was found in tissue of peripheral blood,spleen,and bone marrow.Femurs HE staining showed decreased bone marrow cell.Western blot result showed that the abnormal expression of necroptosis-related protein mainly showed as decreased expression of RIPK1,increased expression of MLKL and CC3.HE staining of liver tissue showed monocyte infiltration and focal necrosis.Immunohistochemical staining of the liver showed positive expression of RIPK1,MLKL and CC3 at the site of focal necrosis.Conclusion:Repeated stimulation of Toll-like receptor 9 by unmethylated CpG DNA fragment combined with galactosamine could construct a lethal hemophagocytic syndrome mouse model,mainly manifested as rapid death of partial mice after injection of drugs.The survival mice had hemophagocytic phenomenon,splenomegaly,monocyte infiltration and focal necrosis of liver,and decreased bone marrow cell.In addition,necroptosis was found in the liver of experimental group mice.These results imply necroptosis participate in liver injury.Liver failure is an important cause for mice death,therefore necroptosis is expected to be a potential therapeutic target for hemophagocytic syndrome.