| Objective: This study established membranous nephropathy rat model by cationic bovine serum albumin(C-BSA), and the soft capsule of Jiangzhitongluo and benazepril were applied to the intervention. Through testing the 24-hour urinary protein and serum biochemical index of rats, observing the renal pathological changes in rats by light microscope, immunofluorescence, electron microscope, detecting the expression of ?-actinin-4 protein and ZO-1 protein in kidney by immunohistochemical staining and ?-actinin-4 m RNA and ZO-1 m RNA by real-time PCR, explore the renal protective effect and the influence of the expression of ?-actinin-4 and ZO-1 on membranous nephropathy rat of the soft capsule of Jiangzhitongluo, to provide theoretical basis for clinical treatment of membranous nephropathy.Methods: 60 healthy SD rats, by feeding a week with standard diet, whose 24-hour urinary protein is within the normal range, were randomly divided into two groups. The normal group(NG) was 10, while the marked group is 50. After preimmunization one week, the marked group was injected C-BSA via caudal vein to copy MN rat model three times every week. The 24-hour urinary protein was tested after continuous injection for four weeks. After confirming the success of modeling, the rats of the marked group were divided into five groups, as follows, model group(MG), Benazepril treatment group(BG), Jiangzhitongluo treatment group of low dosage(JLG), Jiangzhitongluo treatment group of middle dosage(JMG) and Jiangzhitongluo treatment group of high dosage(JHG), as every group including 10 rats. The rats of BG were given the grinding Benazepril at the dosage of 10mg/kg/d by intragastric administration. The rats of JLG, JMG and JHG were given a gavage of the soft capsule of Jiangzhitongluo in dose of 25mg/kg/d, 50mg/kg/d and 100mg/kg/d respectively, otherwise the rats of NG and MG were given the equal dose of normal saline. Four weeks later, the rats of every group whose 24-hour urinary protein had been tested already, were drew blood from abdominal aorta, and then the serum biochemical index such as serum total protein(TP), albumin(ALB), total cholesterol(TC), triglyceride(TG), urea nitrogen(BUN), creatinine(Scr) were determined. The fresh kidney tissues were kept, which were used for observing the renal pathological changes by light microscope, immunofluorescence, electron microscope, detecting the expression of ?-actinin-4 protein and ZO-1 protein in kidney by immunohistochemical staining and ?-actinin-4 m RNA and ZO-1 m RNA by real-time PCR.Result: 1 General conditionThe rats of NG showed glossy hair, good spirit and quick response. After molding, the rats of the marked group appeared hair coarse, spirit inanimate, resonse dull, less ingestion, even scrotum and abdomen puffy. After drug given, the rats of the treatment groups had the better condition than the MG, whose status had no changes. 2 Comparison of 24-hour urinary total protein in every group of ratsAfter molding, 24-hour urinary total protein in the marked group was significantly higher than NG, and difference was statistically significant(P<0.05). After the gastrolavage, 24-hour urinary protein in the treatment groups was effectively lower than MG, and difference was statistically significant(P<0.05); 24-hour urinary protein in the JLG was obviously elevatory compared with the other treatment groups, and difference was statistically significant(P<0.05); But there was no difference between the two of the BG, the JMG and the JHG(P>0.05). 3 Comparison of serum total protein, albumin, total cholesterol and triglyceride in every group of ratsAfter molding, compared with NG, the levels of TP and ALB in the marked group were clearly decreased, while the levels of TC and TG were markly increased, and difference was statistically significant(P<0.05); After the four weeks, gastrolavage, the levels of TP and ALB in the treatment group were apparently rised, while the levels of TC and TG were evidently reduced compared with those in the MG, and difference was statistically significant(P<0.05); the levels of TP and ALB in the JLG were significantly lower, while the levels of TC and TG were evidently higher than those in the other treatment groups, and difference was statistically significant(P<0.05); Otherwise there was unconspicuous difference of the levels of TP, ALB, TC and TG among the BG, the JMG and the JHG(P>0.05). 4 Comparison of blood urea nitrogen and serum creatinine in every group of ratsTaking the BUN and Scr into account, there were no clear changes among the six groups, as well as there was no significant difference among them(P>0.05). 5 The renal pathological changes in every groupThe rats in the NG had no obvious pathological changes in kidney. While the rats in the MG observed by light microscope appeared that glomerular volume, capillary endothelial nuclei were significantly increased, glomerular basement membrane showed diffused thickening, mesangial matrix proliferations. By immunofluorescence appeared that lg G and C3 in granular form deposited diffusively along the capillary loops and mesangial area. By electron microscope appeared irregular thickening of glomerular basement membrane, a large number of electron dense deposit under the epithelium, widely symphysic or disappeard foot process, and spikes. Referring the pathological changes in renal tissues, the rats in the treatment group displayed slighter compared with the MG, the JLM were between the MG and the other treatment groups, and there was no markly difference among the other treatment groups. 6 The expression and distribution of ?-actinin-4 and ZO-1 in kidney by immunohistochemistry?-actinin-4 had a main expression in glomeruli, showing linear distribution along the capillary loops, and the expression in MG was higher than that in the NG; ZO-1 expressed mainly in glomeruli, showing linear distribution along the capillary loops, and the expression in MG was less than that in the NG. Compared with the NG, the expression levels of ?-actinin-4 in the marked group were raised, otherwise ZO-1 were lower(P<0.05). ?-actinin-4 in the treatment groups had a more decreased expression while ZO-1 more increased than the MG(P<0.05). The expression of ?-actinin-4 in JLG was raised, while ZO-1 was cut in contrast to that in the other treatment groups(P<0.05). The expression levels of ?-actinin-4 and ZO-1 in the BG, JMG and JHG had no obvious changes, and there was no statistically significant difference between each pair(P>0.05). 7 The expression of ?-actinin-4 and ZO-1 in kidney by real-time PCRCompared with the NG, the expression levels of?-actinin-4 m RNA in the marked group were raised, otherwise ZO-1 m RNA were lower, and difference was statistically significant(P<0.05). ?-actinin-4 m RNA in the treatment groups had a more decreased expression while ZO-1 m RNA more increased than the MG, and difference was statistically significant(P<0.05). The expression of ?-actinin-4 m RNA in JLG was raised, while ZO-1 m RNA was cut in contrast to that in the other treatment groups, and difference was statistically significant(P<0.05). The expression levels of ?-actinin-4 m RNA and ZO-1 m RNA in the BG, JMG and JHG had no obvious changes, and there was no statistically significant difference between each pair(P>0.05).Conclusions:1 The soft capsule of Jiangzhitongluo can increase membranous nephropathy rats, serum total protein and albumin markedly, reduce the membranous nephropathy rats, 24-hour urinary protein, total cholesterol and triglyceride notly.2 The soft capsule of Jiangzhitongluo can restrain the deposition of immune complex in membranous nephropathy glomerular basement membrane and pinching of basement membrane of rats remarkly, reduce the pathological damage to kidney such as foot process fusion.3 The soft capsule of Jiangzhitongluo can lower the expression of ?-actinin-4 protein and m RNA in nephridial tissue of membranous nephropathy rats, while rise up the expression of ZO-1 protein and m RNA, the effect may have something to maintain the stability of sertoli cell and delay the evolve of membranous nephropathy. |
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