桑黄多糖通过P311/TGF-β1/Snail1通路抗糖尿病小鼠肾间质纤维化的研究
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摘要
目的:观察桑黄多糖对糖尿病肾病(DN)模型小鼠肾间质纤维化的影响,并探讨其可能的作用机制。方法:一次性尾静脉注射链脲佐菌素(STZ)溶液120 mg/kg构建糖尿病小鼠模型,并将成模小鼠随机分为正常组、模型组、桑黄多糖40、80 mg/kg组、罗格列酮4 mg/kg组。小鼠连续灌胃给药12周后采血、留尿、留取肾脏组织。采用血糖仪检测空腹血糖(FBG)水平;采用磺基水杨酸-硫酸钠比浊法检测24 h尿蛋白(UP 24 h)水平;采用全自动生化分析仪测定血清肌酐(Scr)和尿素氮(BUN)水平;Masson染色观察肾脏组织的病理变化;免疫组织化学法检测肾皮质中E-钙黏蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-SMA)蛋白表达;酶联免疫吸附法(ELISA)检测肾皮质中基质金属蛋白酶-2(MMP-2)水平;Western blot检测肾皮质中组织金属蛋白酶抑制因子-2(TIMP-2)蛋白的表达及信号通路P311/TGF-β1/Snail1相关蛋白的表达。结果:与模型组比较,除桑黄多糖40 mg/kg组小鼠肾皮质中TIMP-2及P311蛋白表达无显著性变化外,其余剂量组FBG、UP 24 h、Scr和BUN水平、肾组织中胶原纤维沉积的比例、α-SMA蛋白表达及TIMP-2、P311、TGF-β1、Snail1的蛋白表达较模型组均明显降低,而E-cadherin蛋白表达和MMP-2水平均明显升高。结论:桑黄多糖可调节MMP-2/TIMP-2平衡,减轻糖尿病肾病小鼠肾间质纤维化,可能与抑制P311/TGF-β1/Snail1信号通路的激活有关。
Objective: To investigate the effect of Phellinus igniarius polysaccharide( PIP) on renal fibrosis in mice with diabetic nephropathy( DN) and its possible mechanism. Methods: C57BL/6 mice were intraperitoneally injected with 120 mg/kg streptozotocin( STZ) to establish DN model. Mice were randomly divided into the model group,PIP low-dose( 40 mg/kg) group,PIP high-dose( 80 mg/kg) group,rosiglitazone( 4 mg/kg)( positive control) group and blank control group,10 mice in each group. All mice were given the corresponding medicines by gavage for 12 weeks. After the last administration,blood,urine and the kidney tissues were taken. The levels of fasting blood-glucose( FBG),24 h urinary protein( UP 24 h),serum creatinine( Scr) and blood urea nitrogen( BUN) were measured. Renal pathological changes were observed by Masson trichrome staining. The distributions and expressions of E-cadherin and α-Smooth muscle actin( α-SMA)protein were evaluated by immunohistochemistry. Matrix metalloproteinase-2( MMP-2) level in the renal cortex was determined by enzyme linked immunosorbent assay( ELISA). The protein expressions related to P311/TGF-β1/Snail1 signaling pathway as well as the tissue inhibitor of metalloproteinase-2( TIMP-2) in the renal cortex were detected by Western blot. Results: Compared with the model group,FBG,mA lb,Scr and BUN contents,collagen fiber area ratio,α-SMA,TGF-β1,Snail1 protein expression levels in 40 mg/kg and 80 mg/kg PIP,TIMP-2 and P311 in 80 mg/kg PIP were significantly decreased,but E-cadherin protein expression and MMP-2 level were significantly increased. Conclusion: PIP may regulate the balance between MMP-2 and TIMP-2,and has the inhibitory effect on the renal fibrosis in DN mice model by down-regulating the expression of P311/TGF-β1/Snail1 pathway.
Objective: To investigate the effect of Phellinus igniarius polysaccharide( PIP) on renal fibrosis in mice with diabetic nephropathy( DN) and its possible mechanism. Methods: C57BL/6 mice were intraperitoneally injected with 120 mg/kg streptozotocin( STZ) to establish DN model. Mice were randomly divided into the model group,PIP low-dose( 40 mg/kg) group,PIP high-dose( 80 mg/kg) group,rosiglitazone( 4 mg/kg)( positive control) group and blank control group,10 mice in each group. All mice were given the corresponding medicines by gavage for 12 weeks. After the last administration,blood,urine and the kidney tissues were taken. The levels of fasting blood-glucose( FBG),24 h urinary protein( UP 24 h),serum creatinine( Scr) and blood urea nitrogen( BUN) were measured. Renal pathological changes were observed by Masson trichrome staining. The distributions and expressions of E-cadherin and α-Smooth muscle actin( α-SMA)protein were evaluated by immunohistochemistry. Matrix metalloproteinase-2( MMP-2) level in the renal cortex was determined by enzyme linked immunosorbent assay( ELISA). The protein expressions related to P311/TGF-β1/Snail1 signaling pathway as well as the tissue inhibitor of metalloproteinase-2( TIMP-2) in the renal cortex were detected by Western blot. Results: Compared with the model group,FBG,mA lb,Scr and BUN contents,collagen fiber area ratio,α-SMA,TGF-β1,Snail1 protein expression levels in 40 mg/kg and 80 mg/kg PIP,TIMP-2 and P311 in 80 mg/kg PIP were significantly decreased,but E-cadherin protein expression and MMP-2 level were significantly increased. Conclusion: PIP may regulate the balance between MMP-2 and TIMP-2,and has the inhibitory effect on the renal fibrosis in DN mice model by down-regulating the expression of P311/TGF-β1/Snail1 pathway.
引文
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[2]Tang F, Hao Y, Zhang X, et al. Effect of echinacoside on kidney fibrosis by inhibition of TGF-β1/Smads signaling pathway in the db/db mice model of diabetic nephropathy. Drug Design Development and Therapy, 2017,11∶2813～2826.
[3]方开云, 石明隽, 肖瑛, 等. p38 MAPK介导高糖诱导的肾小管上皮细胞向间充质细胞转变.生理学报, 2008,60(6)∶759～766.
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[6]Wang H, Wu G, Park H J, et al. Protective effect of Phellinus linteus polysaccharide extracts against thioacetamide-induced liver fibrosis in rats:a proteomics analysis. Chinese Medical Journal, 2012,7(1)∶23～33.
[7]黄倩, 张素萍, 施子禄. 人参多糖通过cAMP/PKA/CREB信号通路抗糖尿病肾病肾纤维化作用机制研究. 中国药理学通报, 2018,34(5)∶695～670.
[8]史得君, 崔清美, 齐欣, 等. 桑黄多糖对I型糖尿病小鼠模型的影响. 延边大学农学学报, 2017,39(3)∶33～38.
[9]Kramann R, Fleig S V, Schneider R K, et al. Pharmacological GLI2 inhibition prevents myofibroblast cell-cycle progression and reduces kidney fibrosis. Journal of Clinical Investigation, 2015,125(8)∶2935～2951.
[10]Geng Z M, Zheng J B, Zhang X X, et al. Role of transforming growth factor-beta signaling pathway in pathogenesis of benign biliary stricture. World Journal of Gastroenterology, 2005,11(2)∶293～295.
[11]边晓慧. MMPs表达与调控的研究进展. 国外医学.泌系统分册, 2004,24(1)∶136～139.
[12]蔡朕, 郑桂敏, 刘宝利, 等. 改良保肾方II号对糖尿病肾病大鼠肾脏病理和肾组织MMP-2/TIMP-2表达的影响. 北京中医药, 2010,29(6)∶452～455.
[13]李承德, 王煜, 曲敬蓉, 等. 黄芪多糖对糖尿病大鼠肾脏TGF-β1/Smads信号通路的影响.中国药理学通报, 2018,34(4)∶512～516.
[14]Park M J, Lee D E, Shim M K, et al. Piperlongumine inhibits TGF-β-induced epithelial-to-mesenchymal transition by modulating the expression of E-cadherin,Snail1, and Twist1. European Journal of Pharmacology, 2017,812∶243～249.
[15]方开云, 娄晶, 肖瑛, 等. 转化生长因子β1和Snail1参与糖尿病大鼠肾小管上皮细胞向间充质细胞转化. 生理学报, 2008,60(1)∶125～134.
[16]陈结慧, 姚文萃, 祝胜郎, 等. 虫草制剂通过TGF-β/Snail信号通路拮抗糖尿病小鼠肾小管上皮细胞间充质转分化. 临床军医杂志, 2017,45(2)∶140～144.
[17]Li H, Yao Z, He W, Gao H, et al. P311 induces the transdifferentiation of epidermal stem cells to myofibroblast-like cells by stimulating transforming growth factor β1 expression. Stem Cell Research Therapy, 2016,7(1)∶175～191.
[18]Pan D, Zhe X, Jakkaraju S, et al. P311 induces a TGF-beta1-independent, nonfibrogenic myofibroblast phenotype. Journal of Clinical Investigation, 2002,110(9)∶1349～1358.
[2]Tang F, Hao Y, Zhang X, et al. Effect of echinacoside on kidney fibrosis by inhibition of TGF-β1/Smads signaling pathway in the db/db mice model of diabetic nephropathy. Drug Design Development and Therapy, 2017,11∶2813～2826.
[3]方开云, 石明隽, 肖瑛, 等. p38 MAPK介导高糖诱导的肾小管上皮细胞向间充质细胞转变.生理学报, 2008,60(6)∶759～766.
[4]Yao Z, Yang S, He W, et al. P311 promotes renal fibrosis via TGF-β1/Smad signaling. Scientific Reports, 2015,5∶17032～17044.
[5]郎明紫, 曾鹏, 刘明明, 等. 桑黄多糖的研究进展. 桑蚕通报, 2017,(2)∶14～18.
[6]Wang H, Wu G, Park H J, et al. Protective effect of Phellinus linteus polysaccharide extracts against thioacetamide-induced liver fibrosis in rats:a proteomics analysis. Chinese Medical Journal, 2012,7(1)∶23～33.
[7]黄倩, 张素萍, 施子禄. 人参多糖通过cAMP/PKA/CREB信号通路抗糖尿病肾病肾纤维化作用机制研究. 中国药理学通报, 2018,34(5)∶695～670.
[8]史得君, 崔清美, 齐欣, 等. 桑黄多糖对I型糖尿病小鼠模型的影响. 延边大学农学学报, 2017,39(3)∶33～38.
[9]Kramann R, Fleig S V, Schneider R K, et al. Pharmacological GLI2 inhibition prevents myofibroblast cell-cycle progression and reduces kidney fibrosis. Journal of Clinical Investigation, 2015,125(8)∶2935～2951.
[10]Geng Z M, Zheng J B, Zhang X X, et al. Role of transforming growth factor-beta signaling pathway in pathogenesis of benign biliary stricture. World Journal of Gastroenterology, 2005,11(2)∶293～295.
[11]边晓慧. MMPs表达与调控的研究进展. 国外医学.泌系统分册, 2004,24(1)∶136～139.
[12]蔡朕, 郑桂敏, 刘宝利, 等. 改良保肾方II号对糖尿病肾病大鼠肾脏病理和肾组织MMP-2/TIMP-2表达的影响. 北京中医药, 2010,29(6)∶452～455.
[13]李承德, 王煜, 曲敬蓉, 等. 黄芪多糖对糖尿病大鼠肾脏TGF-β1/Smads信号通路的影响.中国药理学通报, 2018,34(4)∶512～516.
[14]Park M J, Lee D E, Shim M K, et al. Piperlongumine inhibits TGF-β-induced epithelial-to-mesenchymal transition by modulating the expression of E-cadherin,Snail1, and Twist1. European Journal of Pharmacology, 2017,812∶243～249.
[15]方开云, 娄晶, 肖瑛, 等. 转化生长因子β1和Snail1参与糖尿病大鼠肾小管上皮细胞向间充质细胞转化. 生理学报, 2008,60(1)∶125～134.
[16]陈结慧, 姚文萃, 祝胜郎, 等. 虫草制剂通过TGF-β/Snail信号通路拮抗糖尿病小鼠肾小管上皮细胞间充质转分化. 临床军医杂志, 2017,45(2)∶140～144.
[17]Li H, Yao Z, He W, Gao H, et al. P311 induces the transdifferentiation of epidermal stem cells to myofibroblast-like cells by stimulating transforming growth factor β1 expression. Stem Cell Research Therapy, 2016,7(1)∶175～191.
[18]Pan D, Zhe X, Jakkaraju S, et al. P311 induces a TGF-beta1-independent, nonfibrogenic myofibroblast phenotype. Journal of Clinical Investigation, 2002,110(9)∶1349～1358.