| 【Objective】We used Apo E-/- mice that feed high rich fat diet to made atherosclerosis combine lupus prone animal model through intraperitoneal administering pristine. And by intraperitoneal administering IRS869(TLR9 inhibitor), we tried to find the relationship between TLR9 and Vascular Endothelial Growth Factor in Systemic lupus erythematosus combined atherosclerosis through detecting the kidney tissue and serum level of TLR9 signal way molecule and Vascular Endothelial Growth Factor.【Methods】Apo E-/- C57BL/6 mouse and C57BL/6 mouse was randomized into six groups: C57BL/6 mouse control group by intraperitoneal administering normal saline(A group), Apo E-/- mouse control group by intraperitoneal administering normal saline(B group), C57BL/6 mouse by intraperitoneal administering pristine(C group), Apo E-/- mouse intraperitoneal administering pristine(D group), C57BL/6 mouse by intraperitoneal administering pristine plus IRS869(E group), Apo E-/- mouse by intraperitoneal administering pristine plus IRS869(F group). All groups was fed high rich fat diet 12 week, then all mouse was bled to death. Anti-double strand DNA antibodies(ds-DNA) was assessed by ELISA. Renal protein of TLR9, nuclear factor-kappa B(p65), My D88 and Vascular Endothelial Growth Factor(VEGF) were assessed by Western-blot. Renal c DNA of IFN, TLR9, p65, IRF7, My D88 were assessed by real-time PCR. Ig G and C3 were assessed by immunofluorescence. And we used Massion coloration to detect the degree of kidney fibrosis.【Results】1. Immunological effect1.1 The serum level of Anti ds-DNA antibody(1) Anti-double strand DNA antibodies(Anti ds-DNA antibody) in the pristine C57BL/6 group in high than in the C57BL/6 control group(P<0.05). In the pristine Apo E-/- group in high than in the Apo E-/- control group(P<0.05). But in the pristine Apo E-/- group is not high than in the pristine C57BL/6 group(P>0.05).(2) Anti ds-DNA antibody in the pristine plus IRS869 C57BL/6 group and in the pristine plus IRS869 Apo E-/- group is lower than in the control group(P<0.05).1.2 Renal immunofluorescence(1) Direct immunofluorescence revealed that in the pristine C57BL/6 group and pristine Apo E-/- group, Ig G down into continuous distribution in the glomerular mesangial capillary loops. The distribution is seen scattered in the control. Quantitative analysis of it has statistical significance(P<0.05). Indirect immunofluorescence shows that in the pristine C57BL/6 group and pristine Apo E-/- group, complement component 3 down into obvious scatter distribution in the glomerular mesangial capillary loops. The distribution is seen scattered in the control. Quantitative analysis of it has statistical significance(P<0.05).(2) After injection of TLR9 inhibitor, the Ig G and complement component 3 in the glomerular mesangial capillary loops of pristine plus C57BL/6 group and pristine plus Apo E-/- group less than the pristine C57BL/6 group and pristine Apo E-/- group. Quantitative analysis of it has statistical significance(P<0.05).2.2.1 The protein level of TLR9 and downstream signaling molecules and VEGF in the renal(1) The Western-blot analysis, the protein level of TLR9, MyD88 and NF-κB in the pristine C57BL/6 group and pristine Apo E-/- group is high than control group. Quantitative analysis of it has statistical significance(P<0.05). Also the same occurs with pristine Apo E-/- group and pristine C57BL/6 group(P<0.05).(2) The protein level of TLR9, My D88 and NF-κB in the pristine plus IRS869 C57BL/6 group and pristine plus IRS869 Apo E-/- group is lower than pristine Apo E-/- group and pristine C57BL/6 group(P<0.05).2.2 The cDNA level of TLR9 and downstream signaling molecules in the renal(1) The Real-time PCR analysis, the cDNA level of TLR9, NF-κB,IRF7, IFN Ⅰin the pristine C57BL/6 group and pristine Apo E-/- group is high than control group. Quantitative analysis of it has statistical significance(P<0.05). The c DNA level of My D88 in the pristine Apo E-/- group is high than control group but out occur with pristine Apo E-/- group. the c DNA level of TLR9、NF-κB in the pristine Apo E-/- group is high than the pristine C57BL/6 group(P<0.05), but The c DNA level of IRF7、IFN Ⅰ is not has statistical significance(P>0.05).(2) The c DNA level of TLR9,My D88, NF-κB, IRF7, IFN Ⅰ in the pristine plus IRS869 C57BL/6 group and pristine plus IRS869 Apo E-/- group is lower than pristine Apo E-/- group and pristine C57BL/6 group. Quantitative analysis of it has statistical significance(P<0.05).3. Renal fibrosis3.1 Renal Masson dyeing(1) The glomerular collagen fibers of pristine C57BL/6 group and pristine Apo E-/-group is higher than the control group. Quantitative analysis of it has statistical significance(P<0.05). Also pristine Apo E-/- group is high than pristine C57BL/6 group(P<0.05).(2) The glomerular collagen fibers of pristine plus IRS869 C57BL/6 group and pristine plus IRS869 Apo E-/- group is higher than the pristine C57BL/6 group and pristine Apo E-/- group. Quantitative analysis of it has statistical significance(P<0.05).3.2 Protein level of VEGF(1) The Western-blot analysis, the protein level of VEGF in the pristine C57BL/6 group and pristine Apo E-/- group is high than control group. Quantitative analysis of it has statistical significance(P<0.05). Also pristine Apo E-/- group is high than pristine C57BL/6 group(P<0.05).(2) The protein level of VEGF in the pristine plus IRS869 C57BL/6 group and pristine plus IRS869 Apo E-/- group is lower than pristine Apo E-/- group and pristine C57BL/6 group(P<0.05).【conclusions】1. Pristane could induce ApoE-/- nouse to produce anti ds-DNA antibody, and pristane and Apo E gene knockout costimulatory renal glomerular Ig G and complement C3 deposition.2. By anti-ds-DNA antibody activating and binding TLR9, it activate the downstream signaling pathways, activate NF-κB nuclear translocation and activate VEGF.3. Pristane could induce the formation and increasing of renal fibrosis in Apo E-/-mouse. And Inhibition of TLR9 can reverse renal fibrosis of SLE combine atherosclerotic. |
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