| Objective:Osmotic pressure slow-release pump technology has the superiority of slowly regular-ly quantitatively releasing drug in the body in accordance with the conditions selected in advance. Based on this technology, the experiment in this paper builds a novel osmotic pump C-BSA glomerular nephritis model by implanting osmotic pump containing C-BSA inside animal body. By observing the changes of 24-hour urinary protein, blood biochemistry, renal function,body protection, antioxidant activity, renal pathological morphology and related signaling pathways of rats, we can compare osmotic pump model with the traditional model from the overall level and gene level, explore the pathogenesis of chronic glomerulonephritis (CGN) initially, provide a new modeling method for pharmacological studies of CGN, and also offer a new idea for new and efficient treatment options of CGN.Methods:1.40 Healthy male SD rats were given adaptive feeding for 1 week. Than,10 of them were selected randomly as the blank control group, and the left 30 rats were pre-immuned. Osmotic pump containing C-BSA was implanted into the area beneath the right kidney of the rats to induce the new C-BSA osmotic pump CGN rat model, and the experimental rats were given interventional treatment with prednisone acetate (8mg/kg). At the same time, a traditional C-BSA CGN rat model was induced by injection of C-BSA through the caudal vein.2. After finishing modeling, levels of 24h urine protein and urine creatinine of rats of each group were measured. By taking blood of the experimental rats, levels of serum creatinine (SCr), blood urea nitrogen (BUN), triglycerides (TG), total cholesterol (TC), serum total protein (TP), albumin (ALB); levels of content of nitric oxide (NO), superoxide dismutase (SOD), malondialdehyde (MDA) in serum of rats in each group were measured as well. The pathomorphological changes of renal tissues were observed with light and immunofluorescence and electron microscopes.3. Measuring the levels of TNF-α and IL-1β of kidney and blood through ELISA; measuring the expression levels of NF-κB p65,IκB, Ub and E2mRNA of kidney through RT-PCR; immunohistochemical detection of renal NF-κB, Ub, TNF-α protein expression; measuring renal NF-κB-DNA binding activity through EMSA.Results:1.24h urine protein and urine creatinine:Compared with blank control group, urine protein of osmotic pump model group and traditional model group were extremely significantly higher (P<0.01). Compared with osmotic pump model group, urine protein of prednisone acetate group dropped extremely significantly (P<0.01). Compared with blank control group, levels of urine creatinine in osmotic pump model group and traditional model group were extremely significantly reduced (P<0.01). Prednisone acetate could extremely significantly increase rat urine creatinine levels (P<0.01).2. Biochemical indexes:In comparison with the blank control group, levels of SCr, BUN, TG, and TC in serum of rats in osmotic pump model group and traditional model group increased remarkably (P<0.05 or P<0.01). Compared with osmotic pump model group, levels of BUN, TG, and TC in serum of rats in prednisone acetate group decreased remarkably (P<0.05 or P<0.01); levels of SCr showed a downward trend, but the difference was not statistically significant. Compared with blank control group, levels of TP and ALB in serum of rats in osmotic pump model group and traditional model group were significantly lower (P<0.05 or P<0.01). Compared with osmotic pump model group, levels of TP and ALB in serum of rats in prednisone acetate group rose remarkably (P<0.05 or P<0.01).3. Pathomorphological changes of renal tissues:some phenomena were observed in osmotic pump model group as follows:under light microscope, glomerular swelled significantly, glomerular balloon lumen narrowed, hyperplasia of mesangial cells and mesangial matrix, irregular thickening of the glomerular basement membrane; under electron microscope, foot process fusion was seen extensively, marked thickening of the basement membrane showed, and a large number of crumb electron dense deposit under the epithelium; under immunofluorescence, with a 4+ fluorescence intensity, there was a large number of granular IgG deposition along the capillary. Compared with osmotic pump model group, degree of renal pathological changes in traditional model group was shallower. Pathomorphological changes of renal tissues improved in prednisone acetate group.4. Body protection function and antioxidant ability:Compared with blank control group, levels of NO and SOD in serum of rats in osmotic pump model group and traditional model group decreased extremely remarkably (P<0.01), levels of MD A increased extremely remarkably (P<0.01). Compared with osmotic pump model group, levels of NO and SOD in serum of rats in prednisone acetate group increased extremely remarkably (P<0.01), levels of MDA decreased extremely remarkably (P<0.01).5. NF-κB and Ubiquitin-proteasome pathway (UPP):in ELISA, concentrations of TNF-α and IL-1β of kidney and blood in osmotic pump model group rose remarkably and were higher than traditional model group (P<0.05 or P<0.01); prednisone acetate intervention could decrease concentrations of TNF-α and IL-1β of kidney and blood. In RT-PCR, expressions of NF-κB p65, Ub and E2 mRNA in osmotic pump model group and traditional model group upregulated extremely remarkably (P<0.01), while expression of IκB mRNA down regulated extremely remarkably (P<0.01). Compared with osmotic pump model group, expressions of NF-κB p65, Ub and E2 mRNA in prednisone acetate group down-regulated extremely remarkably (P<0.01), while expression of IκB mRNA upregulated extremely remarkably (P<0.01). In Immunohistochemistry, expressions of NF-κB, Ub and TNF-α in osmotic pump model group and traditional model group upregulated remarkably. Compared with osmotic pump model group, expressions of NF-κB, Ub and TNF-α in prednisone acetate group down-regulated remarkably. In EMSA, binding activity of NF-κB-DNA in osmotic pump model group rose remarkably, binding activity of NF-κB-DNA in traditional model group rose. Prednisone acetate intervention could decrease the renal binding activity of NF-κB-DNA.Conclusion:1. By releasing C-BSA at regular hours and in a fixed quantity through osmotic controlled-release pump, some disadvantages of traditional model which needs several times of repeated intravenous injections, simulates animals, consumes a long time and is not easy to operation can be avoided, a steady and reliable C-BSA CGN rat model has been established.2. As NF-κB and UPP are be closely related to the pathogenesis of CGN, new ideas for developing strategy about prevention and treatment of CGN based on gene targeting can be proposed by research about this relationship. |
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