Inhibition Of Nuclear Factor Kappa B By MiRNA Plasmid Carried By Novel Polyethyleneimine Nanogel Particles In Kupffer Cells Of Rats

Objective:A novel gene carrier, named polyethyleneimine nanogel particles (PEI-NP), is planned to deliver the mircroRNA (miRNA) into Kupffer cells (KCs) for knockdown the nuclear transcription factor κB (NF-κB) P65.Methods:The capacity of PEI-NPs to carry miRNA was tested by gel electrophoresis. RAW264.7 cell line was transfected with the cationic polymers which mix together PEI-NP and miRNA. Then, the cytotoxicity of RAW264.7 cells was test by cell counting kit-8 (CCK-8), the cell apoptosis and transfection efficiency were detected by flow cytometer, the gene and protein expression of NF-κB P65 were tested respectively by real time quantitative polymerase chain reaction (qRT-PCR) and western blot (WB), the distribution of cationic polymers in cytoplasm was observed by transmission electron microscope (TEM). The sprague dawley rats were injected the cationic polymers via portal vein. After that, the gene and protein expression of NF-κB P65 in liver tissue were detected respectively by Q-PCR and WB, the distribution of cationic polymers in liver tissue was observed by TEM, the protein expression of NF-κB P65 in liver KCs was tested by immunohistochemistry (IHC).Results:PEI-NPs were able to completely combine with miRNA at the least mass ratio of 20. The max transfection efficiency of 48 h was 33.63%±1.94% in vitro. The protein expression of NF-κB P65 of 48h was significantly inhibited. The cationic polymers were distributed in RAW264.7 cytoplasm and only in KCs of liver tissue after transfection. The protein expression of NF-κB P65 in liver KCs was lower than control group.Conclusion:KCs are successfully transfected with the cationic polymers in vitro and in vivo. The expression of NF-κB P65 is knockdown significantly.