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苦参碱调控Snail2表达水平抑制转化生长因子β1诱导的人腹膜间皮细胞上皮间充质转分化
李福记, 霍冬梅, 梁靖梅, 段秀萍, 廖蕴华
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苦参碱调控Snail2表达水平抑制转化生长因子β1诱导的人腹膜间皮细胞上皮间充质转分化
Matrine inhibits transforming growth factor-β1-induced epithelial mesenchymal transdifferentiation in human peritoneal mesothelial cells by regulating the expression of Snail2
目的 研究苦参碱对转化生长因子β1（TGF-β1）诱导腹膜间皮细胞上皮间充质转分化（EMT）后转录因子Snail2的影响。 方法 采用TGF-β1刺激人腹膜间皮细胞并同时予不同浓度苦参碱干预处理，实验分为空白对照组、TGF-β1（5 ng/ml）诱导组、TGF-β1+0.4 mg/ml苦参碱干预组、TGF-β1+0.6 mg/ml苦参碱干预组、TGF-β1+0.8 mg/ml苦参碱干预组和 TGF-β1+1.0 mg/ml苦参碱干预组。实时荧光定量PCR和Western印迹检测Snail2、上皮标志分子E钙黏蛋白（E-cadherin）和间质标志分子α平滑肌肌动蛋白（α-SMA）、纤维连接蛋白（FN）、胶原Ⅲ（ColⅢ）的表达，Western印迹检测Smad2、Smad3和细胞外调节蛋白激酶1/2（ERK1/2）的蛋白磷酸化水平。 结果 TGF-β1（5 ng/ml）刺激能上调人腹膜间皮细胞Snail2、 α-SMA、FN和ColⅢ mRNA和蛋白的表达水平，上调Smad2、Smad3和ERK1/2的蛋白磷酸化水平，下调E-cadherin mRNA和蛋白的表达水平;苦参碱（0.4、0.6、0.8、1.0 mg/ml）干预处理后能下调Snail2和α-SMA、FN和ColⅢ mRNA和蛋白的表达水平以及ERK1/2的蛋白磷酸化水平，上调 E-cadherin mRNA和蛋白的表达水平。 结论 TGF-β1能诱导人腹膜间皮细胞EMT，苦参碱可能通过ERK1/2信号通路下调Snail2的表达水平来抑制TGF-β1诱导的人腹膜间皮细胞EMT。
Objective To study the effect of matrine on the expression of transcription factor Snail2 in peritoneal mesothelial cells epithelial-mesenchymal transition (EMT) induced by transforming growth factor-β1 (TGF-β1). Methods Human peritoneal mesothelial cells were stimulated by TGF-β1 and treated with matrine. The experiment was divided into six groups (control group, TGF-β1-induced group (5 ng/ml), TGF-β1+0.4 mg/ml matrine intervention group, TGF-β1+0.6 mg/ml matrine intervention group, TGF-β1+0.8 mg/ml matrine intervention group and TGF-β1+1.0 mg/ml matrine intervention group). The expressions of Snail2, E-cadherin, α-smooth muscle actin (α-SMA), Fibronectin and collagen (Col)Ⅲ were detected by real-time fluorescence quantitative PCR and Western blotting. The protein phosphorylation levels of Smad2, Smad3 and extracellular signal-regulated kinase (ERK)1/2 were detected by Western blotting. Results The mRNA and protein expressions of Snail2, α-SMA, Fibronectin and ColⅢ were up-regulated after being stimulated by TGF-β1 (5 ng/ml) in human peritoneal mesothelial cells, while the mRNA and protein expression of E-cadherin was down-regulated. TGF-β1 (5 ng/ml) could up-regulate the protein phosphorylation levels of Smad2, Smad3 and ERK1/2. Matrine (0.4, 0.6, 0.8, 1.0 mg/ml) could down-regulate the mRNA and protein expression levels of Snail2, α-SMA, Fibronectin and ColⅢ after being stimulated by TGF-β1 in human peritoneal mesothelial cells. Matrine could down-regulate the protein phosphorylation of ERK1/2, and up-regulate the mRNA and protein expression levels of E-cadherin with treatment of TGF-β1 in human peritoneal mesothelial cells. Conclusions TGF-β1 can induce EMT of human peritoneal mesothelial cells. Matrine may inhibit TGF-β1-induced EMT of human peritoneal mesothelial cells by down-regulating the expression of Snail2 through the ERK1/2 signaling pathway.
苦参碱 / 转化生长因子β1 / 腹膜 / Snail2 / 上皮细胞间充质转分化 {{custom_keyword}} /
Matrine / Transforming growth factor beta 1 / Peritoneum / Snail2 / Epithelial-mesenchymal transition {{custom_keyword}} /
彭苗、杨克魁 , {{custom_editor}}
表1 实时荧光定量PCR扩增引物序列 |
| 引物名称 | 引物序列 |
|---|---|
| β-actin（内参） | 上游引物AGGATTCCTATGTGGGCGAC |
| 下游引物CGTACAGGGATAGCACAGCCT | |
| Snail2 | 上游引物GCCAAACTACAGCGAACTGG |
| 下游引物GAGAGAGGCCATTGGGTAGC | |
| E钙黏蛋白 | 上游引物TGAGGTCTCTCTCACCACCT |
| 下游引物TAGGGCTGTGTACGTGCTGT | |
| α平滑肌肌动蛋白 | 上游引物GGCAAGTGATCACCATCGGA |
| 下游引物GTGGTTTCATGGATGCCAGC | |
| 纤维连接蛋白 | 上游引物CACCTCTGTGCAGACCACAT |
| 下游引物AGTTGCCACCGTAAGTCTGG | |
| 胶原Ⅲ | 上游引物GCTGGCATCAAAGGACATCG |
| 下游引物TCTCTGCAGTTTCTAGCGGG |
图1 不同浓度苦参碱对E-cadherin、α-SMA、FN和ColⅢ mRNA表达水平的影响（实时荧光定量PCR）注：1：空白对照组;2：TGF-β1（5 ng/ml）诱导组;3：TGF-β1+0.4 mg/ml苦参碱干预组;4：TGF-β1+0.6 mg/ml苦参碱干预组;5：TGF-β1+0.8 mg/ml苦参碱干预组;6：TGF-β1+1.0 mg/ml苦参碱干预组;E-cadherin：E钙黏蛋白;α-SMA：α平滑肌肌动蛋白;FN：纤维连接蛋白;ColⅢ：胶原Ⅲ;与空白对照组比较，aP<0.05;与TGF-β1诱导组比较，bP<0.05，cP<0.01 |
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The annual payer costs for patients treated with peritoneal dialysis (PD) are lower than with hemodialysis (HD), but in 2007, only 7% of dialysis patients in the United States were treated with PD. Since 1996, there has been no change in the first-year mortality of HD patients, but both short- and long-term outcomes of PD patients have improved.Data from the US Renal Data System were examined for secular trends in survival among patients treated with HD and PD on day 90 of end-stage renal disease (HD, 620 020 patients; PD, 64 406 patients) in three 3-year cohorts (1996-1998, 1999-2001, and 2002-2004) for up to 5 years of follow-up using a nonproportional hazards marginal structural model with inverse probability of treatment and censoring weighting.There was a progressive attenuation in the higher risk for death seen in patients treated with PD in earlier cohorts; for the 2002-2004 cohort, there was no significant difference in the risk of death for HD and PD patients through 5 years of follow-up. The median life expectancy of HD and PD patients was 38.4 and 36.6 months, respectively. Analyses in 8 subgroups based on age (<65 and ≥65 years), diabetic status, and baseline comorbidity (none and ≥1) showed greater improvement in survival among patients treated with PD relative to HD at all follow-up periods.In the most recent cohorts, patients who began treatment with HD or PD have similar outcomes.
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达静静, 董蓉, 沈燕, 等.1, 25-二羟基维生素D3调控腹膜纤维化大鼠结缔组织生长因子和热休克蛋白47的表达[J]. 中华肾脏病杂志, 2018, 34(7): 531-538. DOI:10.3760/cma.j.issn.1001-7097.2018.07.009.
目的 观察腹膜纤维化大鼠模型中结缔组织生长因子（CTGF）和热休克蛋白47（HSP47）在腹膜中的表达，探讨1,25-二羟基维生素D3[1,25-(OH)2-VitD3]抑制腹膜纤维化的可能机制。 方法 按随机数字表法将6周龄雄性Sprague-Dawley (SD)大鼠分为3组：对照组(n=8)、模型组(n=8)和1,25-(OH)2-VitD3（VitD3）组(n=8)。腹腔注射含有0.1%葡萄糖和15%乙醇的氯己定（CHX）溶液0.2 ml/d构建腹膜纤维化（PF）模型。VitD3组于造模当日开始腹腔注射1,25-(OH)2-VitD3[6 ng?(100 g)-1?d-1]。4周后，观察各组大鼠腹膜转运功能、肾功能、腹膜厚度及血清25羟基维生素D3水平。原代培养SD大鼠腹膜间皮细胞分组为：对照组、高糖诱导组（HG，2.5%）、CTGF siRNA干预组（CTGF siRNA+HG）、VitD3干预组（VitD3+HG）和联合干预组（CTGF siRNA+VitD3+HG）。采用荧光定量PCR、Western印迹和免疫荧光检测CTGF和HSP47，ELISA检测纤维连接蛋白（FN）在腹膜组织和腹膜间皮细胞中的表达。 结果 与对照组相比，模型组大鼠腹膜超滤量明显降低（P＜0.05），腹膜厚度和腹透液尿素氮/血尿素氮（DUN/BUN）比值增加（均P＜0.05），腹膜组织CTGF及HSP47的表达均明显增加（均P＜0.05）。应用1,25-(OH)2-VitD3干预后，大鼠腹膜纤维化程度明显改善（P＜0.05），腹膜厚度下降（P＜0.05），CTG及HSP47的表达明显减少（均P＜0.05）。体外实验，经CTGF siRNA、1,25-(OH)2-VitD3和二者联合分别干预2.5%高糖诱导的腹膜间皮细胞，FN、CTGF及HSP47的表达量较高糖组均明显减少（均P＜0.05）。 结论 腹膜纤维化模型大鼠腹膜CTGF和HSP47的表达明显增加，1,25-(OH)2-VitD3可能通过下调CTGF的表达，降低HSP47和FN的表达，从而改善腹膜纤维化。
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Long-term peritoneal dialysis is accompanied by functional and histopathological alterations in the peritoneal membrane. In the long process of peritoneal dialysis, high-glucose peritoneal dialysis solution (HGPDS) will aggravate the peritoneal fibrosis, leading to decreased effectiveness of peritoneal dialysis and ultrafiltration failure. In this study, we found that the coincidence of elevated TGF-β1 expression, autophagy, apoptosis and fibrosis in peritoneal membrane from patients with peritoneal dialysis. The peritoneal membranes from patients were performed with immunocytochemistry and transmission electron microscopy. Human peritoneal mesothelial cells were treated with 1.5%, 2.5% and 4.25% HGPDS for 24 hrs; Human peritoneal mesothelial cells pre-treated with TGF-β1 (10 ng/ml) or transfected with siRNA Beclin1 were treated with 4.25% HGPDS or vehicle for 24 hrs. We further detected the production of TGF-β1, activation of TGF-β1/Smad2/3 signalling, induction of autophagy, EMT, fibrosis and apoptosis. We also explored whether autophagy inhibition by siRNA targeting Beclin 1 reduces EMT, fibrosis and apoptosis in human peritoneal mesothelial cells. HGPDS increased TGF-β1 production, activated TGF-β1/Smad2/3 signalling and induced autophagy, fibrosis and apoptosis hallmarks in human peritoneal mesothelial cells; HGPDS-induced Beclin 1-dependent autophagy in human peritoneal mesothelial cells; Autophagy inhibition by siRNA Beclin 1 reduced EMT, fibrosis and apoptosis in human peritoneal mesothelial cells. Taken all together, these studies are expected to open a new avenue in the understanding of peritoneal fibrosis, which may guide us to explore the compounds targeting autophagy and achieve the therapeutic improvement of PD.© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
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Peritoneal solute transport increases with time on treatment in a proportion of peritoneal dialysis (PD) patients, contributing to ultrafiltration failure. Continuous exposure of the peritoneum to hypertonic glucose solutions results in morphologic damage that may have a causative role in changes in peritoneal function. The purpose of this analysis was to establish whether increased exposure to glucose preceded changes in solute transport in a selected group of long-term PD patients. Peritoneal solute transport, residual renal function, peritonitis rate, and peritoneal exposure to glucose were recorded prospectively in a cohort of 303 patients at a single dialysis center. A subgroup of individuals, treated continuously for 5 yr, were identified and defined retrospectively as having either stable or increasing transport status. Of the 22 patients who were treated continuously for 5 yr, 13 had stable solute transport (solute transport at start, 0.67 [+/-0.1]; at 5 yr, 0.67 [+/-0.1]), whereas 9 had a sustained increase (solute transport at start, 0.56 [+/-0.08]; at 5 yr, 0.77 [+/-0.09]). Compared with the stable patients, those with increasing transport had earlier loss in residual renal function and were exposed to significantly more hypertonic glucose during the first 2 yr of treatment that preceded the increase in solute transport. This was associated with greater achieved ultrafiltration compensating for the reduced urinary volumes in these patients. Further increases in glucose exposure were observed as solute transport continued to rise. Peritonitis, including severity of infection and causative organism, was similar in both groups. In this selected group of long-term survivors on PD, an increase in solute transport with time was preceded by increased peritoneal exposure to hypertonic glucose. This is supportive evidence that hypertonic glucose may play a causative role in alterations in peritoneal membrane function.
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肖静, 王聪, 宫亚楠, 等. 白细胞介素6通过STAT3诱导腹膜间皮细胞上皮间质转分化[J]. 中华肾脏病杂志, 2017, 33(9): 711-717. DOI: 10.3760/cma.j.issn.1001-7097.2017.09.011.
目的 探讨信号传导与转录激活基因3（STAT3）对白细胞介素6（IL-6）诱导人腹膜间皮细胞（HPMC）上皮间质转分化（EMT）的作用。 方法 体外培养HPMC并分组：（1）50 μg/L IL-6刺激HPMC不同时间，按刺激时间24、48、72 h分组；（2）不同浓度IL-6刺激HPMC 24 h，按IL-6浓度50、100 μg/L分组；（3）应用慢病毒介导的RNA干扰技术建立稳定沉默STAT3基因的HPMC，分成空白对照组、IL-6组、空载体组、空载体+IL-6组、慢病毒感染组、慢病毒感染+IL-6组，其中IL-6刺激均以50 μg/L终浓度刺激24 h。实时荧光定量PCR检测上皮钙黏素（E-cadherin）、α平滑肌肌动蛋白（α-SMA）及血管内皮生长因子（VEGF）mRNA的表达；Western印迹检测上述分子的蛋白表达及通路蛋白Janus激酶2（JAK2）、STAT3磷酸化水平；细胞免疫荧光观察E-cadherin、α-SMA的表达和分布。 结果 与对照组比较，不同IL-6浓度组和不同作用时间组细胞E-cadherin蛋白和mRNA表达均下降（均P＜0.05），α-SMA、VEGF的蛋白和mRNA表达均升高（均P＜0.05），通路蛋白磷酸化（p）-JAK2/JAK2比值和p-STAT3/STAT3比值均升高（均P＜0.05），且均呈剂量和时间依赖性。沉默STAT3基因后，与空载体+IL-6组比较，慢病毒感染+IL-6组α-SMA、VEGF蛋白表达均下降（均P＜0.05），E-cadherin蛋白表达上升（P＜0.05）。 结论 IL-6通过激活STAT3通路而刺激HPMC分泌VEGF并诱导EMT的发生。沉默STAT3基因可抑制人腹膜间皮细胞EMT的发生。
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Transition of peritoneal mesothelial cells to a mesenchymal phenotype plays an integral role in the angiogenic and fibrotic changes seen in the peritoneum of patients receiving long-term peritoneal dialysis. While signaling by transforming growth factor (TGF)-beta through Smad proteins likely causes these changes, it is possible that non-Smad pathways may also play a role. Here, we found that Smad3-deficient mice were protected from peritoneal fibrosis and angiogenesis caused by adenovirus-mediated gene transfer of active TGF-beta1 to mesothelial cells; however, mesothelial transition occurred in this setting, suggesting involvement of non-Smad mechanisms. The phosphatidyl inositol 3 kinase (PI3K) target, Akt, was upregulated in both Smad-deficient and wild-type mice after exposure to TGF-beta1. In vivo inhibition of the mammalian target of rapamycin (mTOR) by rapamycin completely abrogated the transition response in Smad3-deficient but not in wild-type mice. Rapamycin blocked nuclear localization of beta-catenin independent of glycogen synthase kinase 3beta activity. Further, in Smad3-deficient mice rapamycin reduced the expression of alpha-smooth muscle actin, which is an epithelial-to-mesenchymal transition-associated gene. Hence, we conclude that TGF-beta1 causes peritoneal injury through Smad-dependent and Smad-independent pathways; the latter involves redundant mechanisms inhibited by rapamycin, suggesting that suppression of both pathways may be necessary to abrogate mesothelial transition.
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Epithelial mesenchymal transition (EMT), a process involved in many growth and repair functions, has been identified in the peritoneal tissues of patients who undergo peritoneal dialysis. The sequence of changes in gene regulation and cellular events associated with EMT after TGF-beta1-induced peritoneal fibrosis is reported. Sprague-Dawley rats received an intraperitoneal injection of an adenovirus vector that transfers active TGF-beta1 (AdTGF-beta1) or control adenovirus, AdDL. Animals were killed 0 to 21 days after infection. Peritoneal effluent and tissue were analyzed for markers of EMT. In the animals that were treated with AdTGF-beta1, an increase in expression of genes associated with EMT and fibrosis, such as type I collagen A2, alpha-smooth muscle actin, and the zinc finger regulatory protein Snail, was identified. Transition of mesothelial cells 4 to 7 d after infection, with appearance of epithelial cells in the submesothelial zone 7 to 14 d after exposure to AdTGF-beta1, was demonstrated. This phase was associated with disruption of the basement membrane and increased expression of matrix metalloproteinase 2. By 14 to 21 d after infection, there was evidence of restoration of normal submesothelial architecture. These findings suggest that EMT occurs in vivo after TGF-beta1 overexpression in the peritoneum. Cellular changes and gene regulation associated with EMT are evident throughout the fibrogenic process and are not limited to early time points. This further supports the central role of TGF-beta1 in peritoneal fibrosis and provides an important model to study the sequence of events involved in TGF-beta1-induced EMT.
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Cardiac function and compliance impairments are the features of cardiac fibrosis. Matrine shows therapeutic effects on cardiovascular diseases and organ fibrosis. In this study, we examined the therapeutic effects and mechanisms of matrine on cardiac fibrosis of DbCM. Matrine was administrated orally to rats with DbCM. Cardiac functions and compliance were evaluated. The collagen deposition was visualized by sirius red staining. Real-time PCR was used to determine the expression level of miRNA. Western blotting was performed to assess the protein expression. NFAT nuclear translocation was evaluated by fluorescent immunochemistry staining and Western blotting. Intracellular calcium level was assessed by fura-2/AM staining. A colorimetric method was used to determine calcineurin enzymatic activity. Impaired cardiac function and compliance were observed in rats with DbCM. Increased collagen deposition in cardiac tissue was found. Furthermore, ATF6 signaling was activated, leading to intracellular calcium accumulation and NFAT activation which further initiated ECM gene expressions. Matrine administration recovered cardiac function and improved compliance by exerting inhibitory effects against ATF6 signaling- induced fibrosis. The high- glucose incubation induced ATF6 signaling activation in cultured CFs to increase the synthesis of ECM. Matrine blocked the ATF6 signaling in CFs to inhibit ECM synthesis within non- cytotoxic concentrations. ATF6 signaling induced cardiac fibrosis was one of the mechanisms involved in DbCM, which was characterized by loss of cardiac compliance and functions. Matrine attenuated cardiac compliance and improved left ventricular functions by exerting therapeutic effects against cardiac fibrosis via affecting ATF6 signaling pathway.Copyright © 2017 Elsevier B.V. All rights reserved.
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姜燕, 龚燕梅, 曾红兵, 等. 苦参碱对实验性大鼠腹膜纤维化的影响[J]. 中国医院药学杂志, 2008, 28(24): 2110-2112. DOI: 10.3321/j.issn:1001-5213.2008.24.012.
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Idiopathic pulmonary fibrosis (IPF), the prototypic progressive fibrotic interstitial lung disease, is thought to be a consequence of repetitive micro-injuries to an ageing, susceptible alveolar epithelium. Ageing is a risk factor for IPF and incidence has been demonstrated to increase with age. Decreased (macro)autophagy with age has been reported extensively in a variety of systems and diseases, including IPF. However, it is undetermined whether the role of faulty autophagy is causal or coincidental in the context of IPF. Here, we report that in alveolar epithelial cells inhibition of autophagy promotes epithelial-mesenchymal transition (EMT), a process implicated in embryonic development, wound healing, cancer metastasis and fibrosis. We further demonstrate that this is attained, at least in part, by increased p62/SQSTM1 expression that promotes p65/RELA mediated-transactivation of an EMT transcription factor, Snail2 (SNAI2), which not only controls EMT but also regulates the production of locally acting profibrogenic mediators. Our data suggest that reduced autophagy induces EMT of alveolar epithelial cells and can contribute to fibrosis via aberrant epithelial-fibroblast crosstalk.
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The Snail family of transcription factors has previously been implicated in the differentiation of epithelial cells into mesenchymal cells (epithelial-mesenchymal transitions) during embryonic development. Epithelial-mesenchymal transitions are also determinants of the progression of carcinomas, occurring concomitantly with the cellular acquisition of migratory properties following downregulation of expression of the adhesion protein E-cadherin. Here we show that mouse Snail is a strong repressor of transcription of the E-cadherin gene. Epithelial cells that ectopically express Snail adopt a fibroblastoid phenotype and acquire tumorigenic and invasive properties. Endogenous Snail protein is present in invasive mouse and human carcinoma cell lines and tumours in which E-cadherin expression has been lost. Therefore, the same molecules are used to trigger epithelial-mesenchymal transitions during embryonic development and in tumour progression. Snail may thus be considered as a marker for malignancy, opening up new avenues for the design of specific anti-invasive drugs.
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The molecular mechanisms that underlie tumour progression are still poorly understood, but recently our knowledge of particular aspects of some of these processes has increased. Specifically, the identification of Snail, ZEB and some basic helix-loop-helix (bHLH) factors as inducers of epithelial-mesenchymal transition (EMT) and potent repressors of E-cadherin expression has opened new avenues of research with potential clinical implications.
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朱士彦, 甘平, 曹院国, 等. 苦参碱对高糖致大鼠腹膜间皮细胞NF-κB p65表达的影响[J]. 临床军医杂志, 2010, 38(5): 689-692. DOI: 10.3969/j.issn.1671-3826.2010.05.003.
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所有作者均声明不存在利益冲突
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表1 实时荧光定量PCR扩增引物序列
图1 不同浓度苦参碱对E-cadherin、α-SMA、FN和ColⅢ mRNA表达水平的影响（实时荧光定量PCR）图2 苦参碱对E-cadherin、α-SMA、FN和Col Ⅲ的蛋白表达水平的影响（Western印迹）图3 苦参碱对转化生长因子β1（TGF-β1）诱导的人腹膜间皮细胞上皮间充质转分化形态学的影响（光镜 ×100）图4 苦参碱（0.8 mg/ml）对Smad2、Smad3和ERK1/2蛋白磷酸化水平的影响（Western印迹）图5 苦参碱（0.8 mg/ml）对Snail2 mRNA和蛋白表达水平的影响/
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