大黄庶虫虫丸加减对大鼠肾间质纤维化的保护作用及机制
|
摘要
目的:探讨大黄庶虫虫丸加减对梗阻性肾病大鼠肾间质纤维化的保护作用及机制。方法:采用单侧输尿管结扎(UUO)致大鼠肾间质纤维化模型,将50只SD大鼠随机分为5组,分别为假手术组,模型组,依那普利组(0. 001 g·kg~(-1)),大黄庶虫虫丸加减大、小剂量组(19,9. 5 g·kg~(-1))。术后第15天处死各组大鼠,收集血清,酶法测定血肌酐(SCr),尿素氮(BUN)水平,收集24 h尿液,连苯三酚红钼终点法测定24 h尿蛋白量(24 h-Upro);留取结扎侧肾组织行苏木素-伊红(HE)染色,马松(Masson)染色;采用免疫组化法(IHC)检测转化生长因子-β_1(TGF-β_1),纤连蛋白(FN),α-平滑肌肌动蛋白(α-SMA)表达;蛋白免疫印迹法(Western blot)检测TGF-β_1,p38丝裂原活化蛋白激酶(p38 MAPK),磷酸化p38 MAPK(p-p38 MAPK)蛋白表达。结果:与假手术组比较,模型组大鼠24 h-Upro,SCr,BUN含量明显增高(P <0. 01),光镜下肾组织损伤严重,TGF-β_1,FN,α-SMA含量明显升高(P <0. 05),TGF-β_1,p38 MAPK,p-p38 MAPK蛋白表达明显升高(P <0. 05);与模型组比较,大黄庶虫虫丸加减大、小剂量组24 h-Upro,SCr,BUN明显降低(P <0. 05),肾脏组织损伤减轻,TGF-β_1,FN,α-SMA含量明显降低(P <0. 05),TGF-β_1,p-p38 MAPK蛋白表达明显降低(P <0. 05)。结论:大黄庶虫虫丸加减方可通过降低FN,α-SMA的高表达,下调p38 MAPK信号通路中的p-p38 MAPK,TGF-β_1表达,减少细胞外基质过度沉积和肾脏固有细胞损伤来改善肾间质纤维化。
Objective: To explore the protective effect and mechanism of modified Dahuang Zhechong Wan on renal interstitial fibrosis in rats with obstructive nephropathy. Method: The unilateral ureteral ligation( UUO)-induced renal interstitial fibrosis model was adopted,50 SD rats were randomly divided into 5 groups:sham operation group,model group,enalapril group( 0. 001 g·kg~(-1)),and high and low-dose modified Dahuang Zhechong Wan group( 19,9. 5 g·kg~(-1)). Rats in each group were put to death on the 15 thday after operation. The serum levels of serum creatinine( SCr) and urea nitrogen( BUN) were collected by enzyme method. The 24-hour urine was collected for 24-hour urinary protein quantity( 24 h-Upro) by pyrogallol red molybdenum end point. The kidney tissue was removed from the ligated side. Hematoxylin-eosin( HE) staining and Masson staining were performed; the expressions of transforming growth factor-β_1( TGF-β_1),fibronectin( FN) and α-smooth actin( α-SMA) were determined by immunohistochemistry( IHC). Expressions of TGF-β_1,p38 mitogen-activated protein kinase( p38 MAPK) and phosphorylated p38 MAPK( p-p38 MAPK) were detected by Western blot.Result: Compared with Sham group,UUO group showed a significant increase in 24 h-Upro,SCr,and BUN( P <0. 01),the renal tissue damage was severe under light microscope,and TGF-β_1,FN,and α-SMA were increased obviously( P < 0. 05); TGF-β_1,p38 MAPK,and p-p38 MAPK were increased obviously( P < 0. 05). Compared with the UUO group,24 h-Upro,SCr,BUN decreased obviously( P < 0. 05); renal tissue damage was alleviated;TGF-β_1,FN and α-SMA decreased obviously( P < 0. 05); TGF-β_1 and p-p38 decreased obviously( P < 0. 05).Conclusion: Modified Dahuang Zhechong Wan may improve renal interstitial fibrosis by reducing the high expressions of FN and α-SMA,down-regulating the expressions of p-p38 MAPK and TGF-β_1 in p38 MAPK signaling pathway,and decreasing extracellular matrix over deposition and renal cell damage.
Objective: To explore the protective effect and mechanism of modified Dahuang Zhechong Wan on renal interstitial fibrosis in rats with obstructive nephropathy. Method: The unilateral ureteral ligation( UUO)-induced renal interstitial fibrosis model was adopted,50 SD rats were randomly divided into 5 groups:sham operation group,model group,enalapril group( 0. 001 g·kg~(-1)),and high and low-dose modified Dahuang Zhechong Wan group( 19,9. 5 g·kg~(-1)). Rats in each group were put to death on the 15 thday after operation. The serum levels of serum creatinine( SCr) and urea nitrogen( BUN) were collected by enzyme method. The 24-hour urine was collected for 24-hour urinary protein quantity( 24 h-Upro) by pyrogallol red molybdenum end point. The kidney tissue was removed from the ligated side. Hematoxylin-eosin( HE) staining and Masson staining were performed; the expressions of transforming growth factor-β_1( TGF-β_1),fibronectin( FN) and α-smooth actin( α-SMA) were determined by immunohistochemistry( IHC). Expressions of TGF-β_1,p38 mitogen-activated protein kinase( p38 MAPK) and phosphorylated p38 MAPK( p-p38 MAPK) were detected by Western blot.Result: Compared with Sham group,UUO group showed a significant increase in 24 h-Upro,SCr,and BUN( P <0. 01),the renal tissue damage was severe under light microscope,and TGF-β_1,FN,and α-SMA were increased obviously( P < 0. 05); TGF-β_1,p38 MAPK,and p-p38 MAPK were increased obviously( P < 0. 05). Compared with the UUO group,24 h-Upro,SCr,BUN decreased obviously( P < 0. 05); renal tissue damage was alleviated;TGF-β_1,FN and α-SMA decreased obviously( P < 0. 05); TGF-β_1 and p-p38 decreased obviously( P < 0. 05).Conclusion: Modified Dahuang Zhechong Wan may improve renal interstitial fibrosis by reducing the high expressions of FN and α-SMA,down-regulating the expressions of p-p38 MAPK and TGF-β_1 in p38 MAPK signaling pathway,and decreasing extracellular matrix over deposition and renal cell damage.
引文
[1] LIU Y H. Cellular and molecular mechanisms of renal fibrosis[J]. Nat Rev Nephrol,2011,7(12):684-696.
[2]张浩洋,庞立健,刘创,等.基于慢性复杂性疾病“络虚邪瘀”理论探讨器官纤维化共性病机及治疗[J].中医杂志,2017,58(18):1562-1565.
[3]赵先锋.中医药治疗慢性肾衰竭研究进展[J].现代中西医结合杂志,2014,23(2):217-220.
[4]齐堃,张彧,杨仁义,等.中医方药调控肾纤维化相关信号通路的研究进展[J].湖南中医药大学学报,2017,37(9):1044-1048.
[5]乔汉连.厄贝沙坦联合大黄庶虫虫丸治疗早期糖尿病肾病34例总结[J].湖南中医杂志,2017,33(10):63-64.
[6]邓艳芳.大黄庶虫虫丸与病、证关系的文献研究[D].北京:北京中医药大学,2015.
[7]陈继红,孙伟,周栋,等.大黄庶虫虫丸对肾小球硬化大鼠纤溶酶原激活物抑制物1和金属蛋白酶1组织抑制剂mRNA表达的影响[J].中西医结合学报,2008(5):512-516.
[8]吕小燕,刘强,苏娟萍,等.大黄蟅虫丸对肾间质纤维化大鼠血液流变学及微循环的影响[J].中国药物与临床,2012,12(5):585-587.
[9]鲁华,林海英. TGF-β1/p38 MAPK通路在介导单侧输尿管梗阻大鼠肾间质纤维化过程中的作用[J].中国老年学杂志,2012,32(20):4448-4450.
[10]王永胜,杨丽霞,程涛,等.糖尿病肾病的炎症致病机制与中药防治[J].中国实验方剂学杂志,2018,24(2):200-207.
[11]袁金凤,陈兰英,王慧玲,等.金匮肾气丸对腺嘌呤致肾间质纤维化大鼠的影响[J].中国实验方剂学杂志,2018,24(21):149-156.
[12]徐淑云,卞如谦,陈修.药理实验方法学[M]. 3版.北京:人民卫生出版社,2002:203-206.
[13] ZHOU T B,OU C,QIN Y H,et al. LIM homeobox transcription factor 1B expression affects renal interstitial fibrosis and apoptosis in unilateral ureteral obstructed rats[J]. AM J Physiol Renal Physiol,2014,306(12):1477-1488.
[14]程红新,杨晓萍,蒋雅红,等.纤维连接蛋白在肾间质纤维化大鼠肾组织中的表达[J].中国组织工程研究,2012,16(31):5837-5842.
[15] Eddy A A. The origin of scar-forming kidney myofibroblasts[J]. Nat Med,2013,19(8):964-966.
[16] SHEN Y,MIAO N,WANG B,et al. C-myc promotes renal fibrosis by inducing integrinαv-mediated transforming growth factor-βsignaling[J]. Kidney Int,2017,92(4):888-899.
[17]陈升平,李红霞. ERK1/2信号通路参与人滋养层细胞中转化生长因子-β1对MMP-9表达的抑制作用[J].中国细胞生物学学报,2011,33(6):658-666.
[18] Omori K,Hattori N,Senoo T,et al. Inhibition of plasminogen activator inhibitor-1 attenuates transforming growth factor-β-dependent epithelial mesenchymal transition and differentiation of fibroblasts to myofibroblasts[J]. PLo S One,2016,doi:10. 1371/journal. pone. 0148969. e Collection 2016.
[19] Das D,Holmes A,Murphy G A,et al. TGF-beta1-induced MAPK activation promotes collagen synthesis,nodule formation,redox stress and cellular senescence in porcine aortic valve interstitial cells[J]. J Heart Valve Dis,2013,22(5):621-630.
[20] Pourreyron C,CHEN M,Mcgrath J A,et al. High levels of type VII collagen expression in recessive dystrophic epidermolysis bullosa cutaneous squamous cell carcinoma keratinocytes increases PI3K and MAPK signalling,cell migration and invasion[J]. Br J Dermatol,2014,170(6):1256-1265.
[21] Ono K,HAN J. The p38 signal transduction pathway:activation and function[J]. Cell Signal,2000,12(1):1-13.
[22]黄燕如,万毅刚,孙伟,等.雷公藤多苷调节肾组织p38 MAPK信号通路改善糖尿病肾病肾小球炎症性损伤的作用和机制[J].中国中药杂志,2014,39(21):4102-4109.
[23] HONG F,WU N,GE Y,et al. Nanosized titanium dioxide resulted in the activation of TGF-β/Smads/p38 MAPK pathway in renal inflammation and fibration of mice[J].J Biomed Mater Res A,2016,104(6):1452-1461.
[2]张浩洋,庞立健,刘创,等.基于慢性复杂性疾病“络虚邪瘀”理论探讨器官纤维化共性病机及治疗[J].中医杂志,2017,58(18):1562-1565.
[3]赵先锋.中医药治疗慢性肾衰竭研究进展[J].现代中西医结合杂志,2014,23(2):217-220.
[4]齐堃,张彧,杨仁义,等.中医方药调控肾纤维化相关信号通路的研究进展[J].湖南中医药大学学报,2017,37(9):1044-1048.
[5]乔汉连.厄贝沙坦联合大黄庶虫虫丸治疗早期糖尿病肾病34例总结[J].湖南中医杂志,2017,33(10):63-64.
[6]邓艳芳.大黄庶虫虫丸与病、证关系的文献研究[D].北京:北京中医药大学,2015.
[7]陈继红,孙伟,周栋,等.大黄庶虫虫丸对肾小球硬化大鼠纤溶酶原激活物抑制物1和金属蛋白酶1组织抑制剂mRNA表达的影响[J].中西医结合学报,2008(5):512-516.
[8]吕小燕,刘强,苏娟萍,等.大黄蟅虫丸对肾间质纤维化大鼠血液流变学及微循环的影响[J].中国药物与临床,2012,12(5):585-587.
[9]鲁华,林海英. TGF-β1/p38 MAPK通路在介导单侧输尿管梗阻大鼠肾间质纤维化过程中的作用[J].中国老年学杂志,2012,32(20):4448-4450.
[10]王永胜,杨丽霞,程涛,等.糖尿病肾病的炎症致病机制与中药防治[J].中国实验方剂学杂志,2018,24(2):200-207.
[11]袁金凤,陈兰英,王慧玲,等.金匮肾气丸对腺嘌呤致肾间质纤维化大鼠的影响[J].中国实验方剂学杂志,2018,24(21):149-156.
[12]徐淑云,卞如谦,陈修.药理实验方法学[M]. 3版.北京:人民卫生出版社,2002:203-206.
[13] ZHOU T B,OU C,QIN Y H,et al. LIM homeobox transcription factor 1B expression affects renal interstitial fibrosis and apoptosis in unilateral ureteral obstructed rats[J]. AM J Physiol Renal Physiol,2014,306(12):1477-1488.
[14]程红新,杨晓萍,蒋雅红,等.纤维连接蛋白在肾间质纤维化大鼠肾组织中的表达[J].中国组织工程研究,2012,16(31):5837-5842.
[15] Eddy A A. The origin of scar-forming kidney myofibroblasts[J]. Nat Med,2013,19(8):964-966.
[16] SHEN Y,MIAO N,WANG B,et al. C-myc promotes renal fibrosis by inducing integrinαv-mediated transforming growth factor-βsignaling[J]. Kidney Int,2017,92(4):888-899.
[17]陈升平,李红霞. ERK1/2信号通路参与人滋养层细胞中转化生长因子-β1对MMP-9表达的抑制作用[J].中国细胞生物学学报,2011,33(6):658-666.
[18] Omori K,Hattori N,Senoo T,et al. Inhibition of plasminogen activator inhibitor-1 attenuates transforming growth factor-β-dependent epithelial mesenchymal transition and differentiation of fibroblasts to myofibroblasts[J]. PLo S One,2016,doi:10. 1371/journal. pone. 0148969. e Collection 2016.
[19] Das D,Holmes A,Murphy G A,et al. TGF-beta1-induced MAPK activation promotes collagen synthesis,nodule formation,redox stress and cellular senescence in porcine aortic valve interstitial cells[J]. J Heart Valve Dis,2013,22(5):621-630.
[20] Pourreyron C,CHEN M,Mcgrath J A,et al. High levels of type VII collagen expression in recessive dystrophic epidermolysis bullosa cutaneous squamous cell carcinoma keratinocytes increases PI3K and MAPK signalling,cell migration and invasion[J]. Br J Dermatol,2014,170(6):1256-1265.
[21] Ono K,HAN J. The p38 signal transduction pathway:activation and function[J]. Cell Signal,2000,12(1):1-13.
[22]黄燕如,万毅刚,孙伟,等.雷公藤多苷调节肾组织p38 MAPK信号通路改善糖尿病肾病肾小球炎症性损伤的作用和机制[J].中国中药杂志,2014,39(21):4102-4109.
[23] HONG F,WU N,GE Y,et al. Nanosized titanium dioxide resulted in the activation of TGF-β/Smads/p38 MAPK pathway in renal inflammation and fibration of mice[J].J Biomed Mater Res A,2016,104(6):1452-1461.