肾衰泄浊汤及组方对UUO大鼠Wnt4/β-catenin信号通路作用的影响
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摘要
目的:基于Wnt/β-catenin信号通路探讨肾衰泄浊及组方抗肾间质纤维化的作用机制及肾间质纤维化中医本虚标实的病机。方法:将72只SD大鼠根据体质量随机分为模型组、假手术组、肾衰泄浊汤组、祛邪方组、补虚方组及贝那普利组,每组12只,采用单侧输尿管梗阻(UUO)动物模型。模型复制成功后,肾衰泄浊汤组、祛邪方组、补虚方组分别按照肾衰泄浊汤及其组方比例配制,浓缩剂灌胃给药,贝那普利组大鼠给贝那普利灌胃,模型组及空白组大鼠均予生理盐水灌胃,分别于术后7、14 d处死大鼠。检测24 h尿蛋白定量,血清肌酐、尿素氮水平,免疫组化检测Wnt4、β-catenin、E-cadherin含量,原位杂交检测Wnt4、β-catenin mRNA表达。结果:与假手术相比,同时期模型组大鼠24 h尿蛋白定量升高(P<0.05),与模型组相比,经各组治疗后24 h尿蛋白定量减少(P<0.05);与假手术相比,模型组血尿素氮、血清肌酐均升高(P<0.05),与模型组相比,经各组治疗后血尿素氮、清肌酐均有所下降(P<0.05);与假手术组相比,同时期模型组Wnt4、β-catenin表达升高(P<0.05),E-cadherin表达下降。与同时期模型组相比,各治疗组Wnt4、β-catenin表达下降(P<0.05),E-cadherin表达升高(P<0.05),肾衰泄浊汤组优于其他组(P<0.05)。与假手术组相比,同时期模型组及各治疗组Wnt4mRNA、β-cateninmRNA表达升高(均P<0.05),与模型组相比,各治疗组Wnt4mRNA、β-cateninmRNA表达下降(均P<0.05),尤以肾衰泄浊汤组效果最好。结论:肾衰泄浊汤及其组方可能是通过抑制Wnt4/β-catenin信号通路,达到改善肾功能,延缓肾间质纤维化的进展,且肾间质纤维化中医病机为本虚标实。
Objective:To explore the mechanism of Shenshuai Xiezhuo Decoction and its components and the mechanism of anti-renal interstitial fibrosis based on Wnt/β-catenin signal pathway and the pathogenesis of the deficiency of Chinese medicine in renal interstitial fibrosis. Methods:Seventy-two SD rats were randomly divided into model group, sham operation group, Shenshuai Xiezhuo Decoction and its components, Quxie Formula group, Buxu Formula group and benazepril group according to their body weights,each group 12, using unilateral ureteral obstruction(UUO) animal model. After the model replication was successful, the Shenshuai Xiezhuo Decoction group, the Formula group and the Buxu Formula group were respectively prepared in accordance with the proportion of Shenshuai Xiezhuo Decoction and its components, and the concentrated agent was given to the stomach by intragastric administration. The contents of Wnt4/β-catenin and E-cadherin were measured by immunohistochemistry, and the expressions of Wnt4 and β-catenin mRNA were detected by in situ hybridization. Results:Compared with the sham operation, 24 h urinary protein quantification was increased in the model group(P<0.05). Compared with the sham operation, both blood urea nitrogen and serum creatinine were increased in the model group(P<0.05). Compared with the model group, both blood urea nitrogen and serum creatinine were decreased after treatment in each group(P<0.05). Compared with the sham operation group, the expressions of Wnt4 and β-catenin in the same period were increased(P<0.05), while those of E-cadherin were decreased. Compared with the model group at the same period, the expressions of Wnt4 and β-catenin in the treatment groups were decreased(P<0.05), E-cadherin expression was increased(P<0.05) and Shenshuai Xiezhuo Decoction group's was superior to that of the other groups(P<0.05). Compared with the sham operation group, the expression levels of Wnt4 mRNA and β-cateninmrna in the model group and each treatment group at the same period were increased(P<0.05), and the expression levels of Wnt4 mRNA and β-cateninmrna in each treatment group were decreased(P<0.05) compared with those of the model group, especially in the Shenshuai Xiezhuo Decoction group. Conclusion: Shenshuai Xiezhuo Decoction and its components may improve renal function and delay the progress of renal interstitial fibrosis by inhibiting Wnt4/β-catenin signal pathway. In addition, the pathogenesis of renal interstitial fibrosis in traditional Chinese medicine is deficiency in origin and excess in superficiality.
Objective:To explore the mechanism of Shenshuai Xiezhuo Decoction and its components and the mechanism of anti-renal interstitial fibrosis based on Wnt/β-catenin signal pathway and the pathogenesis of the deficiency of Chinese medicine in renal interstitial fibrosis. Methods:Seventy-two SD rats were randomly divided into model group, sham operation group, Shenshuai Xiezhuo Decoction and its components, Quxie Formula group, Buxu Formula group and benazepril group according to their body weights,each group 12, using unilateral ureteral obstruction(UUO) animal model. After the model replication was successful, the Shenshuai Xiezhuo Decoction group, the Formula group and the Buxu Formula group were respectively prepared in accordance with the proportion of Shenshuai Xiezhuo Decoction and its components, and the concentrated agent was given to the stomach by intragastric administration. The contents of Wnt4/β-catenin and E-cadherin were measured by immunohistochemistry, and the expressions of Wnt4 and β-catenin mRNA were detected by in situ hybridization. Results:Compared with the sham operation, 24 h urinary protein quantification was increased in the model group(P<0.05). Compared with the sham operation, both blood urea nitrogen and serum creatinine were increased in the model group(P<0.05). Compared with the model group, both blood urea nitrogen and serum creatinine were decreased after treatment in each group(P<0.05). Compared with the sham operation group, the expressions of Wnt4 and β-catenin in the same period were increased(P<0.05), while those of E-cadherin were decreased. Compared with the model group at the same period, the expressions of Wnt4 and β-catenin in the treatment groups were decreased(P<0.05), E-cadherin expression was increased(P<0.05) and Shenshuai Xiezhuo Decoction group's was superior to that of the other groups(P<0.05). Compared with the sham operation group, the expression levels of Wnt4 mRNA and β-cateninmrna in the model group and each treatment group at the same period were increased(P<0.05), and the expression levels of Wnt4 mRNA and β-cateninmrna in each treatment group were decreased(P<0.05) compared with those of the model group, especially in the Shenshuai Xiezhuo Decoction group. Conclusion: Shenshuai Xiezhuo Decoction and its components may improve renal function and delay the progress of renal interstitial fibrosis by inhibiting Wnt4/β-catenin signal pathway. In addition, the pathogenesis of renal interstitial fibrosis in traditional Chinese medicine is deficiency in origin and excess in superficiality.
引文
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[13] 肖争,李瑛.β连环蛋白翻译后修饰与肾间质纤维化[J].肾脏病与透析肾移植杂志,2016,25(3):269-273.
[14] 任克军,王东,王亿平,等.清肾颗粒对单侧输尿管梗阻致肾间质纤维化大鼠肾组织Wnt/β-catenin信号通路的干预作用[J].安徽中医药大学学报,2017,36(2):60-63.
[15] Madan B,Patel M,Zhang J,et al.Experimental inhibition of porcupine mediated Wnt O-acylation attenuates kidney fibrosis[J].Kidney International,2016,89(5):1062-1074.
[16] Lin X,Zha Y,Zeng XZ,et al.Role of the Wnt/β-catenin signaling pathway in inducing apoptosis and renal fibrosis in 5/6-nephrectomized rats[J].Molecular Medicine Reports,2017,15(6):3575-3582.
[17] Xiao L,Zhou D,Tan RJ,et al.Sustained activation of Wnt/β-Catenin signaling drives AKI to CKD progression[J].J Am Soc Nephrol,2016,27:1724-1740.
[18] Wang L,Chi YF,Yuan ZT,et al.Astragaloside iv inhibits renal tubulointerstitial fibrosis by blocking tgf-beta/smad signaling pathway in vivo and in vitro[J].Exp Biol Med (Maywood),2014,239:1310-1324.
[19] Villa L,Boor P,Konieczny A,et al.Effects and mechanisms of angiotensin II receptor blockade with telmisartan in a normotensive model of mesangioproliferative nephritis[J].Nephrol Dial Transplant,2011,26(10):3131-3143.
[20] He W,Kang YS,Dai C,et al.Blockade of Wnt/beta-catenin signaling by paricalcitol ameliorates proteinuria and kidney injury[J].J Am Soc Nephrol,2011,22(1):90-103.
[2] 王身菊,朱美凤,郑宏香,等.保元排毒丸对单侧输尿管梗阻大鼠肾脏 Wnt/β-catenin信号通路的影响[J].世界科学技术—中医药现代化,2018,20(2):281-286.
[3] Ucero AC,Benito-Martin A,Izquierdo MC,et al.Unilateral ureteral obstruction:beyong obstruction[J].Int Urol Nephrol,2014,46(40):765-776.
[4] 李文歌,陈香美,师锁柱,等.漫型肾间质纤维化小鼠模型的快速制备及实验研究[J].军医进修学院学报,1996,17(3):188.
[5] Solez K,Axelsen RA,Benediktssen H,et al.International standardization of criteria for the histologic diagnosis of renal allograft rejection:the Banff working classification of kidney transplant pathology[J].Kidney Int,1993,44(2):411-422.
[6] Heungsoo K,Takashi O,Jesus LG,et al.TIMP-1 deficiency does not attenuate interstitial fibrosis in obstruetive nephropathy[J].J Am Soc Nephrol,2001,12(4):736-748.
[7] 晏子友,赵海.肾衰泄浊汤对肾间质纤维化小鼠HGF调节的作用[J].中国中西医结合肾病杂志,2007(4):223-225.
[8] 叶俊玲,李罗德,晏子友,等.肾衰泄浊汤对肾间质纤维化大鼠OPN、CD44的调节作用[J].中国医药科学,2013,3(2):25-27.
[9] YookⅡ,Li XY,Ota I,et al.Wnt—dependent regulation of the E-cadherin repressor snail[J].J Biol Chem,2008,280(12):11740-11748.
[10] 晏子友,申屠进军,熊芳,等.肾衰泄浊汤对BMP7/TGFβ1/Smads信号通路的调节作用[J].江西中医学院学报,2012,24(1):21-24.
[11] 晏子友,申屠进军,熊芳.肾衰泄浊汤对BMP_7、TGFβ_1调节作用的探讨[J].中医药信息,2010,27(3):34-37.
[12] 黄伟,沈金峰,谢娟,等.浅谈Wnt/β-catenin与肾间质纤维化的关系[J].江西医药,2018,53(1):90-93.
[13] 肖争,李瑛.β连环蛋白翻译后修饰与肾间质纤维化[J].肾脏病与透析肾移植杂志,2016,25(3):269-273.
[14] 任克军,王东,王亿平,等.清肾颗粒对单侧输尿管梗阻致肾间质纤维化大鼠肾组织Wnt/β-catenin信号通路的干预作用[J].安徽中医药大学学报,2017,36(2):60-63.
[15] Madan B,Patel M,Zhang J,et al.Experimental inhibition of porcupine mediated Wnt O-acylation attenuates kidney fibrosis[J].Kidney International,2016,89(5):1062-1074.
[16] Lin X,Zha Y,Zeng XZ,et al.Role of the Wnt/β-catenin signaling pathway in inducing apoptosis and renal fibrosis in 5/6-nephrectomized rats[J].Molecular Medicine Reports,2017,15(6):3575-3582.
[17] Xiao L,Zhou D,Tan RJ,et al.Sustained activation of Wnt/β-Catenin signaling drives AKI to CKD progression[J].J Am Soc Nephrol,2016,27:1724-1740.
[18] Wang L,Chi YF,Yuan ZT,et al.Astragaloside iv inhibits renal tubulointerstitial fibrosis by blocking tgf-beta/smad signaling pathway in vivo and in vitro[J].Exp Biol Med (Maywood),2014,239:1310-1324.
[19] Villa L,Boor P,Konieczny A,et al.Effects and mechanisms of angiotensin II receptor blockade with telmisartan in a normotensive model of mesangioproliferative nephritis[J].Nephrol Dial Transplant,2011,26(10):3131-3143.
[20] He W,Kang YS,Dai C,et al.Blockade of Wnt/beta-catenin signaling by paricalcitol ameliorates proteinuria and kidney injury[J].J Am Soc Nephrol,2011,22(1):90-103.
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