肾衰Ⅱ号方对5/6肾切除大鼠残余肾组织凋亡相关蛋白表达的影响
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摘要
目的观察肾衰Ⅱ号方对5/6(ablation/infarction,A/I)肾切除慢性肾衰竭(CRF)大鼠残余肾组织凋亡相关蛋白表达的影响。方法 SD雄性大鼠采用5/6(A/I)肾切除术制备CRF大鼠模型,随机分为模型组、肾衰Ⅱ号方组、西药组,每组15只,另取15只大鼠作为假手术组。各组给予相应干预,60 d后Western blot检测凋亡相关蛋白p53、Bax、Cleaved-parp的表达,免疫组化检测p53、Bax蛋白的表达,透射电镜观察肾组织超微结构。结果与假手术组比较,模型组大鼠肾皮质和肾髓质p53、Bax、Cleaved-parp蛋白表达明显升高(P<0.05);与模型组比较,各给药组大鼠肾皮质和肾髓质p53、Bax、Cleaved-parp蛋白表达明显降低(P<0.05)。免疫组化结果显示,p53、Bax蛋白表达主要位于肾小管,模型组大鼠炎性细胞浸润明显,蛋白表达较假手术组明显升高,各给药组较模型组炎性细胞浸润减轻,p53和Bax蛋白表达降低。透射电镜结果显示,假手术组大鼠肾皮质和肾髓质未见细胞凋亡,超微结构正常,模型组大鼠核固缩,细胞凋亡出现,超微结构紊乱,各给药组较模型组超微结构改善,少见细胞凋亡发生。结论肾衰Ⅱ号方可抑制5/6(A/I)肾切除CRF大鼠细胞凋亡,其作用机制可能与下调p53、Bax、Cleaved-parp蛋白表达相关。
Objective To observe the effects of Shenshuai Ⅱ Prescription(SSP) on the apoptosis-related protein expressions in the chronic renal failure(CRF) rats induced by 5/6(ablation/infarction, A/I) nephrectomy. Methods SD male rats were induced CRF rat model by 5/6(A/I) nephrectomy and were randomized into model group, SSP group and Western medicine group, with 15 rats in each group. Another 15 rats were recruited as sham-operation group.After 60 days of intervention, the apoptosis-related protein expressions of p53, Bax and Cleaved-parp were tested by Western blot, the protein expressions of p53 and Bax were tested by immunohistochemistry and renal tissue ultrastructure was observed by transmission electron microscope. Results Compared with sham-operation group, the expressions of p53, Bax, and Cleaved-parp in the renal cortex and the medulla increased significantly in model group(P<0.05). Compared with the model group, the protein expressions of p53, Bax and Cleaved-parp in renal cortex and renal medulla significantly decreased(P<0.05). The results of immunohistochemistry showed that the protein expressions of p53 and Bax were mainly located in renal tubules. The inflammatory cell infiltration was obvious in the model group, and the protein expressions were significantly higher than those in the sham-operation group. The inflammatory cell infiltration was reduced in the medicine-administered groups compared with the model group, and the protein expressions of p53 and Bax decreased. Transmission electron microscopy showed that there was no apoptosis in the renal cortex and renal medulla of the sham-operation group, the ultrastructure was normal, the model group was pyknosis, apoptosis occurred, and the ultrastructural disorder was disordered. The ultrastructure of each medicine-administered group was improved compared with the model group, and apoptosis was rare. Conclusions SSP can inhibit apoptosis in CRF rats induced by 5/6(A/I) nephrectomy, which can be associated with down-regulation of protein expressions of p53, Bax, and Cleaved-parp.
Objective To observe the effects of Shenshuai Ⅱ Prescription(SSP) on the apoptosis-related protein expressions in the chronic renal failure(CRF) rats induced by 5/6(ablation/infarction, A/I) nephrectomy. Methods SD male rats were induced CRF rat model by 5/6(A/I) nephrectomy and were randomized into model group, SSP group and Western medicine group, with 15 rats in each group. Another 15 rats were recruited as sham-operation group.After 60 days of intervention, the apoptosis-related protein expressions of p53, Bax and Cleaved-parp were tested by Western blot, the protein expressions of p53 and Bax were tested by immunohistochemistry and renal tissue ultrastructure was observed by transmission electron microscope. Results Compared with sham-operation group, the expressions of p53, Bax, and Cleaved-parp in the renal cortex and the medulla increased significantly in model group(P<0.05). Compared with the model group, the protein expressions of p53, Bax and Cleaved-parp in renal cortex and renal medulla significantly decreased(P<0.05). The results of immunohistochemistry showed that the protein expressions of p53 and Bax were mainly located in renal tubules. The inflammatory cell infiltration was obvious in the model group, and the protein expressions were significantly higher than those in the sham-operation group. The inflammatory cell infiltration was reduced in the medicine-administered groups compared with the model group, and the protein expressions of p53 and Bax decreased. Transmission electron microscopy showed that there was no apoptosis in the renal cortex and renal medulla of the sham-operation group, the ultrastructure was normal, the model group was pyknosis, apoptosis occurred, and the ultrastructural disorder was disordered. The ultrastructure of each medicine-administered group was improved compared with the model group, and apoptosis was rare. Conclusions SSP can inhibit apoptosis in CRF rats induced by 5/6(A/I) nephrectomy, which can be associated with down-regulation of protein expressions of p53, Bax, and Cleaved-parp.
引文
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[12]YOGOSAWA S,YOSHIDA K.Tumor suppressive role for kinases phosphorylating p53 in DNA damage-induced apoptosis[J].Cancer Sci,2018,109:3376-3382.
[13]WANG J,PANG J,LI H L,et al.lncRNA ZEB1-AS1 as suppressed by p53 for renal fibrosis in diabetic nephropathy[J].Mol Ther Nucleic Acids,2018,12:741-750.
[14]REHMAN M U,TAHIR M,QUAIYOOM K A,et al.Diosmin protects against trichloroethylene-induced renal injury in Wistar rats:plausible role of p53,Bax and caspases[J].Br J Nutr,2013,110:699-710.
[15]CHIPUK J E,KUWANA T,BOUCHIEⅡR-HAYES L,et al.Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis[J].Science,2004,303:1010-1014.
[16]CHENG J Z,CUI R,CHEN C H,et al.Oxidized low-density lipoprotein stimulates p53-dependent activation of proapoptotic Bax leading to apoptosis of differentiated endothelial progenitor cells[J].Endocrinology,2007,148:2085-2094.
[17]MEI S Q,LIN L,WEI Q,et al.Double knockout of Bax and Bak from kidney proximal tubules reduces unilateral urethral obstruction associated apoptosis and renal interstitial fibrosis[J].Sci Rep,2017,7:44892-44898.
[18]LIU X X,DENG Y F,XU Y F,et al.MicroRNA-223 protects neonatal rat cardiomyocytes and H9c2 cells from hypoxia-induced apoptosis and excessive autophagy via the Akt/mTOR pathway by targeting PARP-1[J].J Mol Cell Cardiol,2018,118:133-146.
[19]BRENNER B M,MEYER T W,HOSTETTER T H.Dietary protein intake and the progressive nature of kidney disease:The role of hemodynamically mediated glomerular injury in the pathogenesis of progressive glomerular sclerosis in aging,renal ablation,and intrinsic renal disease[J].N Engl J Med,1982,307:652-659.
[20]EDDY A A.Experimental insights into the tubulointerstitial disease accompanying primary glomerular lesions[J].J Am Soc Nephrol,1994,5:1273-1287.
[21]NATH K A.Tubulointerstitial changes as a major determinant in the progression of renal damage[J].Am J Kidney Dis,1992,20:1-17.
[2]XU Y,LIU Y Q,GUO H L,et al.Apoptosis-associated speck-like protein containing a c ARD deletion ameliorates unilateral ureteral obstruction induced renal fibrosis and endoplasmic reticulum stress in mice[J].Mediators Inflamm,2018,2018:6909035.
[3]ZHOU J,ZHONG J Y,HUANG Z X,et al.TAK1 mediates apoptosis via p38 involve in ischemia-induced renal fibrosis[J].Artif Cells Nanomed Biotechnol,2018,2018:1-10.
[4]杨婧,王琛,邵命海,等.肾衰Ⅱ号方对5/6肾切除大鼠肾血流量和肾内氧耗影响及其作用机制[J].中国中西医结合肾病杂志,2011,12(7):578-581.
[5]秦军燕,王琛,邵命海,等.肾衰Ⅱ号方对慢性肾功能衰竭大鼠肾组织AngⅡ及nNOS蛋白表达的影响[J].中国中西医结合杂志,2012,32(9):1280-1284.
[6]程勤丽,徐亚赟,杨靖,等.益气养血祛瘀泄浊法对5/6(A/I)慢性肾衰竭模型大鼠蛋白尿及残余肾功能的保护作用[J].中国中西医结合肾病杂志,2017,18(7):570-573.
[7]DENG A H,TANG T,SINGH P,et al.Regulation of oxygen utilization by angiotensinⅡin chronic kidney disease[J].Kidney Int,2009,75:197-204.
[8]朱敏杰,郝海英,陈洁,等.葛根素对肾缺血再灌注大鼠肾脏组织细胞凋亡的影响及其机制探讨[J].中国实验方剂学杂志,2016,22(23):127-132.
[9]吴坚,邹玺,刘沈林,等.健脾养正消癥方通过激活PI3K/AKT信号通路减轻顺铂所致的肾毒性作用[J].中国实验方剂学杂志,2015,21(17):106-110.
[10]杨婧,严睿俊,王琛.肾衰Ⅱ号方对慢性肾脏病患者肾功能及炎症因子的影响[J].中国中医药信息杂志,2014,21(12):15-18.
[11]周圆,王琛,何立群,等.肾衰Ⅱ号方治疗CKD3-4期患者的临床疗效观察[J].上海中医药大学学报,2011,25(4):37-40.
[12]YOGOSAWA S,YOSHIDA K.Tumor suppressive role for kinases phosphorylating p53 in DNA damage-induced apoptosis[J].Cancer Sci,2018,109:3376-3382.
[13]WANG J,PANG J,LI H L,et al.lncRNA ZEB1-AS1 as suppressed by p53 for renal fibrosis in diabetic nephropathy[J].Mol Ther Nucleic Acids,2018,12:741-750.
[14]REHMAN M U,TAHIR M,QUAIYOOM K A,et al.Diosmin protects against trichloroethylene-induced renal injury in Wistar rats:plausible role of p53,Bax and caspases[J].Br J Nutr,2013,110:699-710.
[15]CHIPUK J E,KUWANA T,BOUCHIEⅡR-HAYES L,et al.Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis[J].Science,2004,303:1010-1014.
[16]CHENG J Z,CUI R,CHEN C H,et al.Oxidized low-density lipoprotein stimulates p53-dependent activation of proapoptotic Bax leading to apoptosis of differentiated endothelial progenitor cells[J].Endocrinology,2007,148:2085-2094.
[17]MEI S Q,LIN L,WEI Q,et al.Double knockout of Bax and Bak from kidney proximal tubules reduces unilateral urethral obstruction associated apoptosis and renal interstitial fibrosis[J].Sci Rep,2017,7:44892-44898.
[18]LIU X X,DENG Y F,XU Y F,et al.MicroRNA-223 protects neonatal rat cardiomyocytes and H9c2 cells from hypoxia-induced apoptosis and excessive autophagy via the Akt/mTOR pathway by targeting PARP-1[J].J Mol Cell Cardiol,2018,118:133-146.
[19]BRENNER B M,MEYER T W,HOSTETTER T H.Dietary protein intake and the progressive nature of kidney disease:The role of hemodynamically mediated glomerular injury in the pathogenesis of progressive glomerular sclerosis in aging,renal ablation,and intrinsic renal disease[J].N Engl J Med,1982,307:652-659.
[20]EDDY A A.Experimental insights into the tubulointerstitial disease accompanying primary glomerular lesions[J].J Am Soc Nephrol,1994,5:1273-1287.
[21]NATH K A.Tubulointerstitial changes as a major determinant in the progression of renal damage[J].Am J Kidney Dis,1992,20:1-17.