Abstract:

Objective：To observe the effect of CD40 siRNA on expression of IFN-γ, IL-17,  IL-4 and anti-dsDNA antibody of systemic lupus erythematosus (SLE) animal model MRL/Lpr mice and to discuss its therapy on MRL/Lpr mice. Methods: In the study, 16 female MRL/Lpr mice were randomly divided into control group (n=4), empty vector group (n=4), CD40siRNA1 group (n=4) and CD40-siRNA2 group (n=4). The vectors expressing siRNA against CD40 were injected by tail veil into MRL/Lpr mice, while MRL/Lpr mice in control group and empty vector group were injected with the same dose of PBS and pGFP-V-RS vector respectively.  The injection was given six times and every one day. The mice were sacrificed  14 d after injection, and the spleen tissue was weighed. The pGFP-V-RS was labeled by green fluorescent protein（GFP) and the tissue sections were observed whether siRNA expressed in the spleen. The expression levels of IFN-γ, IL-17, IL-4 and anti-dsDNA antibody in the sera were detected by ELISA method on the 1st day before the first time and the 2nd, 5th, 8th, 11th, and 14th days after last  injection, and the expression levels of CD40 mRNA in spleen tissue of MRL/Lpr mice were detected by RT-PCR and the expression levels of CD40 protein in spleen tissue of MRL/Lpr mice were detected by immunohistochemistry method. Results: The expression vector of CD40-siRNA could express in the spleen of MRL/Lpr. The spleens in CD40-siRNA1 group ［(78.85 ±5.61) mg］ and CD40-siRNA2 group ［(80.25±4.07) mg］ were lower than those in control  ［(141.88±7.81) mg］ and empty vector group ［(153.10±7.60) mg］. The levels of IL-17, IFN-γ and anti-dsDNA antibody were lower and the levels of IL-4 was higher in CD40-siRNA1 group and CD40-siRNA2 group on the 2nd, 5th and 8th days after last injection than on the 1st day before the first time (P<0.05). The levels of IFN-γ in CD40-siRNA1 group were (118.74±10.32) ng/L, (115.24±8.26) ng/L and (113.71±5.02) ng/L in turn, the levels of IFNγ in CD40siRNA2 group were (117.83±6.83) ng/L, (114.07±0.97) ng/L and (112.67±9.66) ng/L in turn. The levels of IL-17 in CD40-siRNA1 group were (7.05±0.41) ng/L, (6.34±0.76) ng/L and (5.83±0.43) ng/L in turn, the levels of IL-17 in CD40-siRNA2 group were (7.07±0.22) ng/L, (6.35±0.49) ng/L and (6.12±0.80) ng/L in turn. The levels of anti-dsDNA antibody in CD40-siRNA1 group were (7.51±0.29) ng/L, (6.74±0.45) ng/L and (6.32±0.39) ng/L  in turn, the levels of anti-dsDNA antibody in CD40-siRNA2 group were (8.19±0.38) ng/L, (7.14±0.50) ng/L and (6.48±0.29) ng/L in turn. The levels of IL-4 in CD40siRNA1 group were (26.51±1.81)ng/L  (27.80±1.72) ng/L  and (28.08±2.21) ng/L  in turn, the level of IL-4 in CD40-siRNA2 group were (26.28±2.03) ng/L, (28.15±2.95) ng/L and (28.37±1.71) ng/L in turn. The expression  levels of IL-17 and IFN-γ antibody increased gradually and the levels of IL-4 decreased gradually in CD40-siRNA1 group and CD40-siRNA2 group on the 11th and 14th days after last injection, then reached to the levels of control group and empty vector group (P>0.05). Though the levels of anti-dsDNA antibody in CD40-siRNA1 group and CD40-siRNA2 group on the 11th day was higher than on the 8th day, there was more significance than those in control group and empty vector group (P<0.05). There was no significance between the 4 groups on the 14th day. The levels of CD40 mRNA and protein were lower in CD40-siRNA1 group and CD40-siRNA2 group than in control group and empty vector group on the 14th day after last injection (P<0.05). Conclusion: CD-40 si-RNA can reduce the concentration of IL-17, IFN-γ and of anti-dsDNA antibody in serum, and at the same time, it can elevate the concentration of IL-4 and suppress CD40 mRNA and protein of spleen in MRL/Lpr. Meanwhile after suppressing CD40 mRNA and protein, it can reduce inflammatory response of the mice and the disease activity of MRL/Lpr, suggesting that CD-40 siRNA has therapy effect on SLE.

Key words: Antigens, CD40, RNA, small interfering, Lupus erythematosus, systemic, Cytokines, Mice, inbred MRL Lpr