抗纤灵方对肾纤维化大鼠TGF-β_1/Smad信号通路表达的影响
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摘要
目的:研究抗纤灵方对5/6肾切除所致肾纤维化大鼠的治疗效果,并探讨其分子作用机制。方法:将50只SD大鼠随机分为正常组(n=10),假手术组(n=10),5/6肾切除肾纤维化造模组(n=30)。将造模成功的大鼠随机分为模型组、抗纤灵组、氯沙坦钾组。氯沙坦钾组、抗纤灵方组每日分别给予氯沙坦钾(33. 3 g·kg-1)及抗纤灵方(21 g·kg-1)灌胃治疗,其余组都用等体积的生理盐水。持续8周灌胃后处死,检测各组大鼠血肌酐(SCr),尿素氮(BUN),24 h尿蛋白(24 h-Pro);苏木素-伊红(HE)染色观察肾脏组织病理学改变;马松(Masson)染色观察肾脏纤维化程度;蛋白免疫印迹法(Western blot)及实时荧光定量聚合酶链式反应(Real-time PCR)分别检测肾脏转化生长因子-β_1(TGF-β_1),Smad2,Smad3,Smad7蛋白和mRNA在肾脏组织中的表达。结果:与正常组和假手术组比较,模型组大鼠的SCr,BUN,24 h-Pro上升,纤维化面积和肾脏损伤评分增加,TGF-β_1,Smad2,Smad3 mRNA与蛋白表达升高,Smad7 mRNA与蛋白表达显著下降(P <0. 01)。与模型组相比,氯沙坦钾组、抗纤灵方组可减少纤维化面积,降低肾脏损伤评分,SCr,BUN,24 h-Pro,TGF-β_1,Smad2,Smad3 mRNA与蛋白的表达水平,Smad7mRNA与蛋白表达显著上调(P <0. 01),但低于正常组与假手术组的水平。结论:中药抗纤灵方可通过抑制TGF-β_1/Smad通路来延缓肾纤维化。
Objective: To study the therapeutic effect of Kangxianling Decoction on renal fibrosis induced by 5/6 nephrectomization in rats,and discuss its action mechanism. Method: Totally 50 SD rats were randomly divided into normal control group(n = 10),sham-operation group(n = 10),and 5/6 nephrectomized renal fibrosis model group(n = 30). After successful modeling,rats were randomly divided into the model group,Kangxianling group,and Losartan Potassium group. Rats in the Losartan Potassium group were administered with Losartan Potassium by gastrogavage; rats in the Kangxianling group were administered with Kangxianling by gastrogavage.Equal volume of distilled water was administered to rats in the other groups. Rats were sacrificed after 8 weeks of consecutive medication,and then serum creatinine(SCr),urea nitrogen(BUN),24 hour urine protein(24 hPro) were measured in each group. Hematoxylin-eosin(HE) staining was used to observe the renal pathological changes and the score of Kidney damage was made. Masson staining was used to observe the degree of renal fibrosis. Western blot and real-time fluorescent quantitative polymerase chain reaction(Real-time PCR) were used to detect the protein and mRNA expression levels of transforming growth factor(TGF-β_1),Smad2,Smad3,and Smad7 in kidneys. Result: As compared with the normal control and sham-operation group,the renal tissue fibrosis,score of kidney damage,SCr,BUN and 24 h-Pro levels were higher in model group(P < 0. 01),with increased protein and mRNA expression levels of TGF-β_1,Smad2,and Smad3(P < 0. 01),and significantly reduced protein and mRNA expression levels of Smad7 in the model group(P < 0. 01). As compared with the model group,the renal tissue fibrosis,the score of kidney damage,SCr,BUN,24 h-Pro level were significantly decreased(P < 0. 01),protein and mRNA expression levels of TGF-β_1,Smad2,Smad3 were reduced(P <0. 05),while protein and mRNA expression levels of Smad7 were elevated in the Losartan Potassium group and Kangxianling group(P < 0. 01),but lower than those of normal control group and sham-operation group.Conclusion: Kangxianling decoction could alleviate renal fibrosis by inhibiting TGF-β_1/Smad signal pathway.
Objective: To study the therapeutic effect of Kangxianling Decoction on renal fibrosis induced by 5/6 nephrectomization in rats,and discuss its action mechanism. Method: Totally 50 SD rats were randomly divided into normal control group(n = 10),sham-operation group(n = 10),and 5/6 nephrectomized renal fibrosis model group(n = 30). After successful modeling,rats were randomly divided into the model group,Kangxianling group,and Losartan Potassium group. Rats in the Losartan Potassium group were administered with Losartan Potassium by gastrogavage; rats in the Kangxianling group were administered with Kangxianling by gastrogavage.Equal volume of distilled water was administered to rats in the other groups. Rats were sacrificed after 8 weeks of consecutive medication,and then serum creatinine(SCr),urea nitrogen(BUN),24 hour urine protein(24 hPro) were measured in each group. Hematoxylin-eosin(HE) staining was used to observe the renal pathological changes and the score of Kidney damage was made. Masson staining was used to observe the degree of renal fibrosis. Western blot and real-time fluorescent quantitative polymerase chain reaction(Real-time PCR) were used to detect the protein and mRNA expression levels of transforming growth factor(TGF-β_1),Smad2,Smad3,and Smad7 in kidneys. Result: As compared with the normal control and sham-operation group,the renal tissue fibrosis,score of kidney damage,SCr,BUN and 24 h-Pro levels were higher in model group(P < 0. 01),with increased protein and mRNA expression levels of TGF-β_1,Smad2,and Smad3(P < 0. 01),and significantly reduced protein and mRNA expression levels of Smad7 in the model group(P < 0. 01). As compared with the model group,the renal tissue fibrosis,the score of kidney damage,SCr,BUN,24 h-Pro level were significantly decreased(P < 0. 01),protein and mRNA expression levels of TGF-β_1,Smad2,Smad3 were reduced(P <0. 05),while protein and mRNA expression levels of Smad7 were elevated in the Losartan Potassium group and Kangxianling group(P < 0. 01),but lower than those of normal control group and sham-operation group.Conclusion: Kangxianling decoction could alleviate renal fibrosis by inhibiting TGF-β_1/Smad signal pathway.
引文
[1]麻志恒,钟利平,余柯娜,等.抗纤灵水煎剂对慢性肾衰模型小鼠PI3K-AKT-mTOR mRNA表达的影响[J].中国实验方剂学杂志,2016,22(20):96-100.
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[7] Wolf G. Molecular mechanisms of angiotensinⅡin the kidney:emerging role in the progression of renal disease:beyond haemodynamics[J]. Nephrol Dial Transplant,1998,13(5):1131-1142.
[8]史晓伟,张定华,罗向霞,等.补肾益气化浊通络法对糖尿病肾病小鼠肾脏细胞外基质的影响[J].中国实验方剂学杂志,2018,24(7):114-120.
[9]王颖,麻志恒,钟利平,等.抗纤灵方对5/6肾切除小鼠p38MAPK/NF-κBp65介导的炎症因子的影响[J].中国中西医结合杂志,2017,37(3):365-369.
[10]钟利平,麻志恒,余柯娜,等.抗纤灵方通过PI3K/AKT/m TOR信号通路干预肾纤维化机制研究[J].中国实验方剂学杂志,2015,21(18):126-129.
[11]吉晶,何立群.抗纤灵方对肾纤维化大鼠肾功能及肾组织ECM表达的影响[J].中国实验方剂学杂志,2019,25(1):63-68.
[12]吉晶,何立群.抗纤灵方对5/6肾切除大鼠肾纤维化及ACE-AngⅡ-AT1R轴的影响[J].中国实验方剂学杂志,2019,25(1):57-62.
[13]陈建,曾莉,陈刚,等.抗纤灵方对AngⅡ诱导肾纤维化小鼠肾组织α-SMA和Ⅰ型胶原的影响[J].中华中医药杂志,2017,32(2):739-742.
[14]麻志恒,钟利平,余柯娜,等.抗纤灵对慢性肾功能衰竭小鼠模型肾组织相关炎症因子的干预作用[J].武汉大学学报:医学版,2017,38(3):357-360.
[15] MA S K, Joo S Y, Kim C S, et al. Increased phosphorylation of PI3K/Akt/m TOR in the obstructed kidney of rats with unilateral ureteral obstruction[J].Chonnam Med J,2013,11(3):108-112.
[16] ZHENG L,ZHANG C,LI L,et al. Baicalin ameliorates renal fibrosis via inhibition of transforming growth factorβ1production and downstream signal transduction[J].Mol Med Rep,2017,15(4):1702-1712.
[17] Masszi A,Kapus A. Smaddening complexity:the role of Smad3 in epithelial-myofibroblast transition[J]. Cells Tissues Organs,2011,193(1/2):41-52.
[18] Hills C E, Squires P E. The role of TGF-βand epithelial-to mesenchymal transition in diabetic nephropathy[J]. Cytokine Growth Factor Rev,2011,22(3):131-139.
[19]张聪,谌贻璞,杨彦芳,等.益肾软坚散对慢性马兜铃酸肾病大鼠模型肾间质纤维化的保护作用[J].中国中西医结合杂志,2005,25(8):714-718.
[20]黄东华,何红涛,檀银川.肾络通汤对慢性肾小球肾炎患者TGF-β1,MMP-9和TMP-1因子的影响[J].中国实验方剂学杂志,2017,23(15):196-201.
[21]丁武杰,方敬爱,孙艳艳,等.益肾胶囊对糖尿病肾病大鼠肾组织JAK/STAT信号通路及α-SMA及FN的影响[J].中国中西医结合肾病杂志,2013,14(6):484-487.
[22]马晓燕,闫渊,崔峥.肾衰饮对TGF-β1诱导的下游因子CTGF及FN的调控作用[J].中华中医药学刊,2015,33(7):1543-1545.
[23] TANG N,ZHANG Y P,YING W,et al. Interleukin-1βupregulates matrix metalloproteinase-13 gene expression via c-Jun N-terminal kinase and p38 MAPK pathways in rat hepatic stellate cells.[J]. Mol Med Rep,2013,8(6):1861-1865.
[24] Quiroga B,Arroyo D,De G A. Present and future in the treatment of diabetic kidney disease[J]. J Diabetes Res,2015,doi:10. 1155/2015/801348.
[25]何伟明,刘不悔,高坤,等.中医药防治肾纤维化的研究现状及实践[J].南京中医药大学学报,2018,34(2):112-117.
[26] Schnaper H W,Hayashida T,Poncelet A C. It's a Smad world:regulation of TGF-βsignaling in the kidney[J]. J Am Soc Nephrol,2002,13(4):1126-1128.
[27] Runyan C E,Schnaper H W,Poncelet A C. Smad3 and PKCdelta mediate TGF-beta1-induced collagen I expression in human mesangial cells[J]. Am J Physiol Renal Physiol,2003,285(3):F413.
[28] Bottinger E P,Bitzer M. TGF-βsignaling in renal disease[J]. J Am Soc Nephrol,2002,13(10):2600-2610.
[29]冉龙娇,梁健,邓鑫. MicroRNA调控TGF-β/SMAD通路调节肝纤维化机制[J].世界华人消化杂志,2017,25(2):166-171.
[30]陈超,武德梅,孙建洁.转化生长因子β与肾脏纤维化的关系[J].医学综述,2014,20(11):1961-1962.
[31]周栋,傅海燕.细胞外基质的产生、重塑与肾脏纤维化[J].江苏医药,2015,41(8):936-938.
[2]刘煜敏,张悦,何立群,等.抗纤灵抗大鼠肾间质纤维化的实验研究[J].中国中西医结合杂志,2007,27(10):901-904.
[3] Gross N J. Anticholinergic agents in asthma and COPD[J]. Eur J Pharmacol,2006,533(1/3):36-39.
[4]高艳红,张昱,李澎,等.基于TGF-β1/Smad信号通路的加味黄芪赤风汤抗肾脏纤维化机制研究[J].中国中西医结合杂志,2016,36(12):1486-1490.
[5]武贵群,高翔,梅长林,等. TGF-β1/Smad通路与肾脏纤维化研究进展[J].中国中西医结合肾病杂志,2012,13(5):459-461.
[6]宋小乐. TGF-β1/Smad信号转导通路对肾间质纤维化的影响[J].南昌大学学报:医学版,2011,51(6):92-95.
[7] Wolf G. Molecular mechanisms of angiotensinⅡin the kidney:emerging role in the progression of renal disease:beyond haemodynamics[J]. Nephrol Dial Transplant,1998,13(5):1131-1142.
[8]史晓伟,张定华,罗向霞,等.补肾益气化浊通络法对糖尿病肾病小鼠肾脏细胞外基质的影响[J].中国实验方剂学杂志,2018,24(7):114-120.
[9]王颖,麻志恒,钟利平,等.抗纤灵方对5/6肾切除小鼠p38MAPK/NF-κBp65介导的炎症因子的影响[J].中国中西医结合杂志,2017,37(3):365-369.
[10]钟利平,麻志恒,余柯娜,等.抗纤灵方通过PI3K/AKT/m TOR信号通路干预肾纤维化机制研究[J].中国实验方剂学杂志,2015,21(18):126-129.
[11]吉晶,何立群.抗纤灵方对肾纤维化大鼠肾功能及肾组织ECM表达的影响[J].中国实验方剂学杂志,2019,25(1):63-68.
[12]吉晶,何立群.抗纤灵方对5/6肾切除大鼠肾纤维化及ACE-AngⅡ-AT1R轴的影响[J].中国实验方剂学杂志,2019,25(1):57-62.
[13]陈建,曾莉,陈刚,等.抗纤灵方对AngⅡ诱导肾纤维化小鼠肾组织α-SMA和Ⅰ型胶原的影响[J].中华中医药杂志,2017,32(2):739-742.
[14]麻志恒,钟利平,余柯娜,等.抗纤灵对慢性肾功能衰竭小鼠模型肾组织相关炎症因子的干预作用[J].武汉大学学报:医学版,2017,38(3):357-360.
[15] MA S K, Joo S Y, Kim C S, et al. Increased phosphorylation of PI3K/Akt/m TOR in the obstructed kidney of rats with unilateral ureteral obstruction[J].Chonnam Med J,2013,11(3):108-112.
[16] ZHENG L,ZHANG C,LI L,et al. Baicalin ameliorates renal fibrosis via inhibition of transforming growth factorβ1production and downstream signal transduction[J].Mol Med Rep,2017,15(4):1702-1712.
[17] Masszi A,Kapus A. Smaddening complexity:the role of Smad3 in epithelial-myofibroblast transition[J]. Cells Tissues Organs,2011,193(1/2):41-52.
[18] Hills C E, Squires P E. The role of TGF-βand epithelial-to mesenchymal transition in diabetic nephropathy[J]. Cytokine Growth Factor Rev,2011,22(3):131-139.
[19]张聪,谌贻璞,杨彦芳,等.益肾软坚散对慢性马兜铃酸肾病大鼠模型肾间质纤维化的保护作用[J].中国中西医结合杂志,2005,25(8):714-718.
[20]黄东华,何红涛,檀银川.肾络通汤对慢性肾小球肾炎患者TGF-β1,MMP-9和TMP-1因子的影响[J].中国实验方剂学杂志,2017,23(15):196-201.
[21]丁武杰,方敬爱,孙艳艳,等.益肾胶囊对糖尿病肾病大鼠肾组织JAK/STAT信号通路及α-SMA及FN的影响[J].中国中西医结合肾病杂志,2013,14(6):484-487.
[22]马晓燕,闫渊,崔峥.肾衰饮对TGF-β1诱导的下游因子CTGF及FN的调控作用[J].中华中医药学刊,2015,33(7):1543-1545.
[23] TANG N,ZHANG Y P,YING W,et al. Interleukin-1βupregulates matrix metalloproteinase-13 gene expression via c-Jun N-terminal kinase and p38 MAPK pathways in rat hepatic stellate cells.[J]. Mol Med Rep,2013,8(6):1861-1865.
[24] Quiroga B,Arroyo D,De G A. Present and future in the treatment of diabetic kidney disease[J]. J Diabetes Res,2015,doi:10. 1155/2015/801348.
[25]何伟明,刘不悔,高坤,等.中医药防治肾纤维化的研究现状及实践[J].南京中医药大学学报,2018,34(2):112-117.
[26] Schnaper H W,Hayashida T,Poncelet A C. It's a Smad world:regulation of TGF-βsignaling in the kidney[J]. J Am Soc Nephrol,2002,13(4):1126-1128.
[27] Runyan C E,Schnaper H W,Poncelet A C. Smad3 and PKCdelta mediate TGF-beta1-induced collagen I expression in human mesangial cells[J]. Am J Physiol Renal Physiol,2003,285(3):F413.
[28] Bottinger E P,Bitzer M. TGF-βsignaling in renal disease[J]. J Am Soc Nephrol,2002,13(10):2600-2610.
[29]冉龙娇,梁健,邓鑫. MicroRNA调控TGF-β/SMAD通路调节肝纤维化机制[J].世界华人消化杂志,2017,25(2):166-171.
[30]陈超,武德梅,孙建洁.转化生长因子β与肾脏纤维化的关系[J].医学综述,2014,20(11):1961-1962.
[31]周栋,傅海燕.细胞外基质的产生、重塑与肾脏纤维化[J].江苏医药,2015,41(8):936-938.
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