[A research on blockage of receptor-interacting protein 2 expression by small interfering RNA in murine macrophages].
OBJECTIVE
To provide evidence that blocking the receptor-interacting protein 2 (Rip2) expression can decrease inflammatory cytokine production by macrophage and protect mice from endotoxin lethality.


METHODS
Murine Rip2 small interfering RNA (siRNA) plamids were constructed and transfected into macrophages and Rip2 expression was assessed with RT-PCR and Western blot. Cell proliferation was assayed with MTT; TNFalpha concentration was assayed with ELISA and high-mobility group box 1 protein (HMGB1) level with semi quantitative Western blot after lipopolysaccharide (LPS) stimulation. LPS challenge was given after the plasmids were injected into mice and the survival rate was calculated. Rip2 and HMGB1 expression in liver was assessed with Western blot and serum TNFalpha level with ELISA.


RESULTS
Rip2 siRNA plasmids could block the mRNA and protein expression of Rip2 and promote cell proliferation. Blocking of Rip2 could attenuate LPS-induced TNFalpha and HMGB1 production. HMGB1 expression in liver were decreased to (40.21 +/- 11.03) pg/g and serum TNFalpha level was decreased to (300.43 +/- 59.26) ng/L (P < 0.05). The survival rate of endotoxemic mice was also improved (P < 0.05).


CONCLUSIONS
The results demonstrate that Rip2 siRNA plasmids can block the expression of Rip2, and decrease the production of TNFalpha and HMGB1, thus protect mice from lethal endotoxemia.
Liu
Hong-Chun
HC
Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
Cao
Zhong-Wei
ZW
Jin
Jian-Jun
JJ
Wang
Ji-Yao
JY
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D024243:HMGB1 Protein / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D024243:HMGB1 Protein; D008070:Lipopolysaccharides; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C506255:Ripk2 protein, mouse