温阳泄浊通络法对慢性肾衰竭大鼠肾间质纤维化的干预作用及其机制研究
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摘要
目的观察温阳泄浊通络法对5/6肾切除所致慢性肾衰竭大鼠肾间质纤维化的干预作用,并探讨其机制。方法 32只SD大鼠随机选取8只为假手术组,其余24只行5/6肾切除术制备肾纤维化模型。造模完成后随机分为模型组、温阳泄浊通络法组、尿毒清组,并ig予以相应药物。干预12周后,取肾组织,行HE、PAS染色检测病理损伤程度,行Masson、天狼星红染色检测纤维化程度,并应用免疫荧光、Westernblotting法检测I型胶原蛋白及m RNA的表达,应用免疫组化、Western blotting法检测肾小管上皮细胞EMT相关蛋白(α-SMA、Vimentin、E-cadherin)的表达。结果与模型组比较,温阳泄浊通络法可减轻肾脏病理损伤及肾纤维化程度,并显著降低I型胶原蛋白及m RNA的表达;下调α-SMA、Vimentin的高表达,上调E-cadherin的低表达。结论温阳泄浊通络法可有效减轻慢性肾衰竭大鼠肾损伤、改善肾纤维化,其机制可能与抑制肾小管上皮细胞EMT及I型胶原的表达相关。
Objective To investigate the effects of warming yang-dispelling turbidity-dredging collaterals method on renal interstitial fibrosis in rats with chronic renal failure caused by 5/6 nephrectomy and explore its mechanisms. Methods Eight rats were randomly chosen as sham operation group(Sham), while the remaining 24 rats underwent 5/6 nephrectomy were divided into model group(5/6 Nx), warming yang-dispelling turbidity-dredging collaterals method group(WDD) and Niaoduqing group(NDQ), and the rats in each group were administrated with corresponding drugs. After the intervention of 12 weeks, all the rats were sacrificed and the kidneys were then removed. HE, PAS Masson, Picrosirius red staining were conducted to assess kidney pathological changes and renal fibrosis.The protein expression of Collagen Ⅰ was tested by both immunofluorescence and Western blotting, and the m RNA expression was evaluated using qRT-PCR. The expression of protein related with the process of EMT, including α-SMA, E-Cadherin and Vimentin, were tested with immunohistochemistry and Western blotting. Results Compared with the model group, warming yang-dispelling method alleviated kidney pathological injury and renal fibrosis, and significantly down-regulated the protein and mRNA expression of CollagenⅠ. Moreover, warming yang-dispelling turbidity-dredging collaterals method regulated the process of EMT by decreasing the high expression of α-SMA and Vimentin, and increasing the low expression of E-Cadherin. Conclusion Warming yang-dispelling turbidity-dredging collaterals method can effectively attenuate renal fibrosis and injury and its underlying mechanisms may be related with its inhibitory effects on Collagen Ⅰ expression and regulation of EMT.
Objective To investigate the effects of warming yang-dispelling turbidity-dredging collaterals method on renal interstitial fibrosis in rats with chronic renal failure caused by 5/6 nephrectomy and explore its mechanisms. Methods Eight rats were randomly chosen as sham operation group(Sham), while the remaining 24 rats underwent 5/6 nephrectomy were divided into model group(5/6 Nx), warming yang-dispelling turbidity-dredging collaterals method group(WDD) and Niaoduqing group(NDQ), and the rats in each group were administrated with corresponding drugs. After the intervention of 12 weeks, all the rats were sacrificed and the kidneys were then removed. HE, PAS Masson, Picrosirius red staining were conducted to assess kidney pathological changes and renal fibrosis.The protein expression of Collagen Ⅰ was tested by both immunofluorescence and Western blotting, and the m RNA expression was evaluated using qRT-PCR. The expression of protein related with the process of EMT, including α-SMA, E-Cadherin and Vimentin, were tested with immunohistochemistry and Western blotting. Results Compared with the model group, warming yang-dispelling method alleviated kidney pathological injury and renal fibrosis, and significantly down-regulated the protein and mRNA expression of CollagenⅠ. Moreover, warming yang-dispelling turbidity-dredging collaterals method regulated the process of EMT by decreasing the high expression of α-SMA and Vimentin, and increasing the low expression of E-Cadherin. Conclusion Warming yang-dispelling turbidity-dredging collaterals method can effectively attenuate renal fibrosis and injury and its underlying mechanisms may be related with its inhibitory effects on Collagen Ⅰ expression and regulation of EMT.
引文
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[2]Farris A B,Colvin R B.Renal interstitial fibrosis:Mechanisms and evaluation[J].Curr Opin Nephrol Hypertens,2012,21(3):289-300.
[3]Zeisberg M,Neilson E G.Mechanisms of tubulointerstitial fibrosis[J].J Am Soc Nephrol,2010,21(11):1819-1834.
[4]Inoue T,Umezawa A,Takenaka T,et al.The contribution of epithelial-mesenchymal transition to renal fibrosis differs among kidney disease models[J].Kidney Int,2015,87(1):233-238.
[5]Liu Y H.New insights into epithelial-mesenchymal transition in kidney fibrosis[J].J Am Soc Nephrol,2010,21(2):212-222.
[6]Wing M R,Ramezani A,Gill H S,et al.Epigenetics of progression of chronic kidney disease:Fact or fantasy?[J].Semin Nephrol,2013,33(4):363-374.
[7]潘永梅,郭倩,贾蕊,等.陈志强治疗慢性肾功能衰竭的临床经验[J].辽宁中医杂志,2015,42(6):1207-1208.
[8]徐晶,周旭,郭倩,等.数据挖掘陈志强教授对早中期慢性肾衰竭辨证思路[J].天津中医药,2015,32(12):712-715.
[9]徐晶,周旭,郭倩,等.数据挖掘陈志强教授治疗早中期慢性肾衰竭用药规律[J].中国中西医结合肾病杂志,2015,16(12):1088-1090.
[10]郭立芳,王凤丽,王月华,等.益气通络解毒降浊方结合西医基础疗法治疗早、中期慢性肾功能衰竭疗效观察[J].中国中西医结合杂志,2012,32(8):1042-1045.
[11]Genovese F,Manresa A A,Leeming D J,et al.The extracellular matrix in the kidney:A source of novel non-invasive biomarkers of kidney fibrosis?[J].Fibrog Tissue Repair,2014,doi:10.1186/1755-1536-7-4.
[12]Stefanovic B.RNA protein interactions governing expression of the most abundant protein in human body,type I collagen[J].Wiley Interdiscip Rev RNA,2013,4(5):535-545.
[13]Lovisa S,LeBleu V S,Tampe B,et al.Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis[J].Nat Med,2015,21(9):998-1009.
[14]Grande M T,Sánchez-Laorden B,López-Blau C,et al.Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease[J].Nat Med,2015,21(9):989-997.
[15]Lamouille S,Xu J,Derynck R.Molecular mechanisms of epithelial-mesenchymal transition[J].Nat Rev Mol Cell Biol,2014,15(3):178-196.
[16]Saito A.EMT and End MT:Regulated in similar ways?[J].J Biochem,2013,153(6):493-495.