MO007
INDUCTION OF IMMUNOSUPPRESSIVE MICRO-RNA IN SPLEEN ATTENUATES SEPSIS INDUCED ACUTE KIDNEY INJURY

INTRODUCTION AND AIMS: Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host immune response to infection, and known as major causes of mortality and morbidity in most intensive care units. Mortality rates in sepsis were higher in patients with more organ failure, including acute kidney injury (AKI) . However, therapies to treat or prevent sepsis and AKI are largely ineffective. Several studies have revealed that some micro-RNAs (miRNAs) had roles in regulation of systemic inflammation including sepsis and related organ failure. Here, we investigated the therapeutic potential of a kind of miRNA (miR-X) which is exogenously admitted to the mouse sepsis model to attenuate sepsis related organ failures by targeting TLR (toll like receptor)-NFkB pathway.

METHODS: As in vitro study, miR-X was transfected into RAW264.7 cells using lipofectamine. 12h after transfection, Each group was treated with 1ug/ml of lipopolysaccharide for 6h, then RNA was isolated using Trizol Reagent. Supernatant were collected at the same time. For in vivo study, 8-12 week-old C57BL/6-N male mice were used. Before 7 days of sepsis onset, miR-X along with GFP expression plasmid or empty plasmid, mixed with polyethyleneimine (PEI), were injected to mice via tail vein. Sepsis was induced by cecal ligation and puncture (CLP). Kidneys and spleens were harvested after 24 h of CLP. Blood samples were collected at the same time. Real-time PCR was performed to detect the miR-X expression and inflammation related genes. Kidney injury was evaluated by serologically, histologically, and immunohistochemical KIM-1 expression. Apoptosis was observed by staining of cleaved caspase-3. Macrophage infiltration was evaluated by Ly6B staining.

RESULTS: In vitro study, over-expression of miR-X was confirmed in plasmid transfected RAW264.7 cells. These cells were tolerant toward the stimulation of LPS, and less productive inflammatory cytokines such as IL-6, and TNF-a. Injected plasmid was mainly detected in splenic macrophages. Compare with empty plasmid group and non-treated group, miR-X expression plasmid group showed better survival rate, less inflammatory serum cytokine production, less severe renal dysfunction, renal tubular injury, and lower number of infiltrated macrophages in renal tissue. Furthermore, less apoptotic cells were observed in spleen of miR-X expression plasmid group, than that of empty plasmid group and non-treated CLP group.

CONCLUSIONS: Over-expression of MiR-X in splenic macrophage negatively regulates excessive immune response in septic mice by targeting TLR-NFkB pathway, and leads less severe AKI. At the same time, our data suggests that spleen might be a good target to regulate the production of inflammatory cytokines and the onset of organ injury in sepsis.