黄芪对高糖腹透液诱导大鼠腹膜间皮细胞EMT中TGF-β1/Smads信号通路的影响
Effect of Radix Astragali on TGF-β1/Smads Signaling Pathway in Epithelial-mesenchymal Transition of rat Peritoneal Mesothelial Cells Induced by High-glucose Peritoneal Dialysate
-
摘要: 目的 观察黄芪对高糖腹透液诱导大鼠腹膜间皮细胞EMT中TGF-β1/Smads信号蛋白的影响,探讨黄芪对腹膜纤维化的阻抑作用与机制。方法 实验分为5组,每组10只。阴性对照组:每日腹腔注射生理盐水20 mL/只,共35 d;模型组:每日腹腔注射4.25%葡萄糖腹透液20 mL/只,共35 d;黄芪低、中、高剂量组:于造模第16天开始每日腹腔注射黄芪腹透液(腹透液中加入黄芪注射液,分别含黄芪生药终浓度10、20、40 mg/mL)20 mL/只,共20 d。HE染色观察腹膜病理改变;快速免疫组化法检测腹膜组织TGF-β1、CollagenⅠ、Collagen Ⅲ、FN、E-cadherin、α-SMA的表达;Western blot检测腹膜组织TGF-β1、E-cadherin、α-SMA、Smad2/3、p-smad2/3、Smad7蛋白的表达;RT-PCR检测TGF-β1、E-cadherin、α-SMA、Smad7mRNA表达。结果 ①模型组PMCs呈圆形或柱形,有脱落,间皮下基质增生,大量成纤维样细胞及单核、巨噬细胞浸润,纤维素样物质沉积,腹膜组织TGF-β1、CollagenⅠ、Collagen Ⅲ、FN表达上调,黄芪各干预组上述变化有改善,以高剂量组作用较为明显;②模型组EMT标记蛋白α-SMA表达上调,E-cadherin表达下调,高、中剂量黄芪组能一定程度地逆转腹膜组织E-cadherin表达下降与α-SMA高表达;③模型组Samds信号蛋白p-smad2/3、Smad7表达上调,黄芪可不同程度地下调p-smad2/3高表达,增强Samd7的表达,高剂量组作用较为显著。结论 高糖腹透析液可促使PMCs EMT的发生,黄芪很可能通过抑制TGF-β1,并作用于Smads信号转导通路正、负反馈环路中的Smad2/3、Smad7关键信号蛋白,阻抑PMCs EMT的发生。
-
关键词:
- 黄芪注射液 /
- 腹膜纤维化 /
- PMCs EMT /
- TGF-β1/Smads信号通路
Abstract: OBJECTIVE To observe the effect of Radix Astragali on TGF-β1/Smads signaling pathway in epithelial-mesenchymal transition(EMT) of rat peritoneal mesothelial cells induced by high-glucose peritoneal dialysis and explore the inhibitory influence and mechanism of Radix Astragali on peritoneal fibrosis.METHODS They were divided into 2 groups, 10 rats in each. The contrast group's rats were intraperitoneal injected 35days normal saline 20 mL/rat. The model group's rats were intraperitoneal injected 35days Icodextrin 20 mL/rats. The low/middle and high doses astragalus mongholicus groups: After 16 days modeling, the rats were intraperitoneal injected 20 days astragalus mongholicus extraneal(put astragalus mongholicus injections into the externeal, and the concentation of crude drug is 10 mg/mL,20 mg/mL and 40 mg/mL) 20 mL/rats. The peritoneal pathological changes were observed through HE staining; the expressions of abdominal membrane TGF-β1, Collagen I, Collagen III, FN, E-cadherin, α-SMA were examined through rapid immunohistochemical method; the expressions of proteins of peritoneal tissues TGF-β1, E-cadherin, α-SMA, Smad2/3, p-smad2/3, of Smad7 were detected through Westernblot; the expressions of TGF-β1, E-cadherin, α-SMA, Smad7mRNA were detected through RT-PCR.RESULTS ①PMCs in model group were round or oval with cells falling off, subcutaneous matrix proliferating, a large number of fibroblast-like cells and monocytes and macrophage infiltrating, fibrinoid material depositing and the expression of peritoneal tissues TGF-β1, Collagen I, Collagen III and FN increasing. The astragalus intervention groups showed improvement in the above conditions with the high-dose group the most obvious. ②The expression of α -SMA in model group labeled by EMT showed an up-regulation while that of E-cadherin low-regulation. The middle and high doses of Radix Astragali groups could reverse the decrease in expression of E-cadherin in peritoneal tissues and high expression of α-SMA to some extent. ③In model group, the expression of signal proteins p-Smad2/3 and Smad7 showed an up-regulation. Radix Astragali could decease the high expression of p-Smad2/3 in different degrees and strengthen the expression of Samd7 taking the high-dose group as the more significant effect.CONCLUSION High-glucose abdominal dialysis fluid can prompt the occurrence of PMCsEMT while Radix Astragali may inhibit TGF-β1 and effect the key signal proteins Smad2/3 and Smad7 in the positive and negative feedback loop of Smads signal transduction pathways to prevent the occurrence of PMCsEMT. -
[1] 〖JP2〗Aroeira LS, Aguilera A, Sánchez-Tomero JA, et al. Epithelial to mesenchymal transition and peritoneal membrane failure in peritoneal dialysis patients: pathologic significance and potential therapeutic interventions[J]. J Am Soc Nephrol, 2007, 18(7): 2004-2013.〖JP〗 [2] Liu Y. Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention[J]. J Am Soc Nephrol, 2004, 15(1): 1-12. [3] 刁金囡,盛梅笑,朱萱萱,等.黄芪注射液对高通透性腹膜透析大鼠透析效能及腹膜结构的影响[J].南京中医药大学学报,2011, 27(1):58-62. [4] Diao JN, Sheng MX, Zhu XX, et al.The influence of Radix Astragali injection on dialysis efficiency and peritoneal structure of rat with high permeability of the peritoneal dialysis[J]. J Nanjing Univ Tradit Chin Med, 2011, 27(1): 58-62. [5] Gotloib L, Wajsbrot V, Cuperman Y, et al. Acute oxidative stress induces peritoneal hyperpermeability, mesothelial loss, and fibrosis[J]. J Lab Clin Med, 2004, 143(1): 31-40. [6] Witowski J, Wisniewska J, Korybalska K, et al. Prolonged exposure to glucose degradation products impairs viability and function of human peritoneal mesothelial cells[J]. J Am Soc Nephrol, 2001, 12(11): 2434-2441. [7] Jiménez-Heffernan JA, Aguilera A, Aroeira LS, et al. Immunohistochemical characterization of fibroblast subpopulations in normal peritoneal tissue and in peritoneal dialysis-induced fibrosis[J]. Virchows Arch, 2004, 444(3): 247-256. [8] 〖JP2〗邓英辉,林琼真,于洁,等.黄芪注射液改善肾间质纤维化的作用机制研究[J].中国中西医结合肾病杂志,2008, 9(5):393-396,474.〖JP〗 [9] Deng YH, Lin QZ, Yu J, et al.The study in the mechanism of Radix Astragali injection to improve the renal interstitial fibrosis[J]. Chin J Integr Tradit Western Med Kidney Dis, 2008, 9(5): 393-396,474. [10] 〖JP2〗Aroeira LS, Aguilera A, Selgas R, et al. Mesenchymal conversion of mesothelial cells as a mechanism responsible for high solute transport rate in peritoneal dialysis: role of vascular endothelial growth factor[J]. Am J Kidney Dis, 2005, 46(5): 938-948.〖JP〗 [11] Huang YJ, Wang ZH, Zhang JB, et al. Smad7 instead of Smad6 blocks epithelial-mesenchymal transition induced by TGF-beta in human renal proximal tubule epithelial cells[J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2008, 24(11): 1074-1078. [12] 〖JP2〗Li JH, Zhu HJ, Huang XR, et al. Smad7 inhibits fibrotic effect of TGF-Beta on renal tubular epithelial cells by blocking Smad2 activation[J]. J Am Soc Nephrol, 2002, 13(6): 1464-1472.〖JP〗 [13] 窦献蕊,余学清.TGF-β1及其信号蛋白在大鼠腹膜纤维化模型腹膜组织中的表达及可能作用[J].中华医学杂志,2005, 85(37):2613-2618. [14] Dou XR, Yu XQ.The expression and possible role of TGF-β1 and its signal protein in rat peritoneal tissues in peritoneal fibrosis model[J]. Chin Med J, 2005, 85(37): 2613-2618. [15] Zavadil J, Bttinger EP. TGF-beta and epithelial-to-mes-enchymal transitions[J]. Oncogene, 2005, 24(37): 5764-5774. [16] Schiffer M, Bitzer M, Roberts IS, et al. Apoptosis in podocytes induced by TGF-beta and Smad7[J]. J Clin Invest, 2001, 108: 807-816.
点击查看大图
计量
- 文章访问数: 982
- HTML全文浏览量: 2
- PDF下载量: 635
- 被引次数: 0