【作者】 栾仲秋；

【导师】 宋立群；

【作者基本信息】 黑龙江中医药大学 ， 中医内科学， 2012， 博士

【摘要】 目的：通过动物实验观察地龙组分丝氨酸蛋白水解酶对单侧输尿管梗阻(UUO)大鼠p38MAPK信号通路及核转录因子κB的影响探讨地龙组分丝氨酸蛋白水解酶抗肾间质纤维化的作用机制方法：采用大鼠单侧输尿管梗阻法复制肾间质纤维化模型。将健康雄性Wistar大鼠60只,随机分组：假手术组12只,模型组16只,地龙低剂量组16只,地龙高剂量组16只。地龙低剂量组给-予地龙组分丝氨酸蛋白水解酶胶囊0.24g·kg1-·d-1灌胃；地龙高剂量组给予地龙组分丝氨酸蛋白水解酶胶囊0.48g·kg-1·d-1灌胃；假手术组和模型组给等量蒸馏水灌胃。分别于术后第2W、4W处死各组的6只大鼠,处死前一天收集24h尿量检测24h尿蛋白定量,处死当天收集静脉血用于血清肌酐(Scr)、尿素氮(BUN)指标的检测。取梗阻侧肾组织标本,HE染色后光镜下观察肾脏病理改变；免疫组织化学方法检测肾组织中TGF-β1、a-SMA、Col-Ⅳ的表达；Western blot检测肾组织中NF-κB、p38MAPK的蛋白表达结果：(1)从外观上看,与模型组相比,地龙治疗组在对应时间点上梗阻侧肾脏的肿大及积水程度较轻,色泽较红润。(2)在不同的时间点各组大鼠24h尿蛋白定量无统计学差异(P>0.05)。(3)地龙治疗组大鼠的Scr、BUN水平在第2、4W时均低于模型组(p<0.05或p<0.01)；相同时间点,地龙低剂量组大鼠血清Scr、 BUN水平低于地龙高剂量组。(4)免疫组化结果显示：模型组及地龙治疗组大鼠肾组织中TGF-β1、a-SMA、Col-Ⅳ的表达随梗阻时间的延长,逐渐增多；在对应的时间点,地龙高、低剂量组与模型组大鼠相比,TGF-β1、 a-SMA、Col-Ⅳ的蛋白表达显著降低(p<0.05或p<0.01)。(5)Western blot结果显示：随着梗阻时间的延长,模型组及地龙治疗组NF-κB、p38MAPK的表达逐渐增多,地龙治疗组NF-κB、p38MAPK的表达明显低于对应时间点模型组(p<0.05)。(6)实验观察到地龙低剂量组疗效优于高剂量组结论：(1)地龙组分能够降低UUO模型大鼠的血清Scr、BUN水平具有保护肾功能作用。(2)地龙组分能够改变肾脏组织形态、减轻肾间质纤维化程度,具有减轻UUO大鼠肾组织损伤作用(3)地龙组分可能是通过下调α-SMA和Col-Ⅳ的表达来抑制成纤维细胞过度活化,减少ECM的过度积聚,来延缓肾间质纤维化的进展(4)地龙组分可能是通过多靶点阻断TGF-β1→p3MAPK→NF-κB信号传导通路,来实现其延缓肾间质纤维化进程的作用更多还原

【Abstract】 Objective:To investigate the effect of earthworm component serine hydrolase on p38mitogen-actived protein kinase signaling pathway and nuelear factork kappa B of unilateral ureteralobstruction rats.and to illustrate the mechanism of the components of earthworm serine hydrolase on delaying renal interstitial fibrosis.Method:Copy the model of renal interstitial fibrosis in rats with unilateral ureteral obstructi on.The60male wistar rats were randomly divided into four groups:sham-operation group(12),UUO group (16), earthworm of low-dose group (16),earth worm of high-dose group (16). Earthworm of low-dose group was given the earthworm component of serine proteolytic enzymes capsules0.24g·kg-1·d-1to gavage.Earthworm of high-dose group was given the earthworm component of serine proteolytic enzymes capsules0.48g·kg-1·d-1to gavage.Sham operation group and UUO group were given the same dose of distilled water.6rats of each group were killed respectively at the2,4weeks after surgery.Put to death the day after the collection of24h urine detection of24urinary protein excretion, executed the same day to collect blood for serum creatinine (Scr),blood urea nitrogen(BUN)index detection. Take the obstructed kidney tissue specimens.HE staining under the light microscope to observe the pathological changes.The pathological changes and expression of TGF-β1,a-SMA and Col-IV in renal interstitium were observed.Western blot analysis the protein expression of NF-κ B and p38MAPK in renal tissue.Results:(1) From the appearance point of view, treatment group of the earthworm in the corresponding time points of the obstructed kidney enlargement and stagnant water level were lighter and more red color than the UUO group.(2) Rats in each group at different time points24hours urinary protein excretion was no significant difference (p>0.05)(3)Scr and BUN levels of Earthworm groups were lower than UUO group at the2,4weeks after surgery (p<0.05. p<0.01).The same time.Scr and BUN level of Earthworm of low-dose group were lower than Earthworm high-dose group.(4) Immunohistochemical results showed:the expressions of TGF-β1,a-SMA and Col-Ⅳ in renal tissue of UUO group and Earthworm groups increased gradually, with the extension of the time of obstruction. In the corresponding point in time,Earthworm of high-dose group and low-dose group compared with UUO group, TGF-beta1,a-SMA and Col-Ⅳ protein expression were significantly lower(p<0.05, p<0.01).(5) Western blot showed:the expressions of NF-κB and p38MAPK gradually increased with the extension of the time of obstruction in UUO group and earthworm groups. The expressions of NF-κB and p38MAPK were significantly lower than that of UUO group at the corresponding time point in earthworm groups(p<0.05).(6) Earthworm low-dose group was better than the high dose group to slow down the development of renal interstitial fibrosis.Conclusion:(1) The earthworm component could improve renal function of UUO rats through reducing serum creatinine,blood urea nitrogen level.(2) The earthworm component could reduce renal fibrosis degree(3) The earthworm component could delay the Progression of renal interstitial fibrosis,through decreasing the expressions of TGF-βl,a-SMA,Col-IV in UUO kidney tissue.(4) The earthworm component may be slow down the development of renal interstitial fibrosis, through the many targets blocking TGF-β1→p38MAPK→NF-κ B signaling pathways.更多还原