干预Wnt/β-catenin信号通路对硬皮病模型小鼠皮肤组织纤维化及上皮间质转化的影响
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摘要
背景:研究证实,Wnt/β-catenin信号通路在系统性硬化症的发生发展中起着重要作用,然而有关抑制Wnt/β-catenin抑制患者皮损信号是否改善硬皮病的报道较少。目的:探究抑制Wnt/β-catenin信号通路对硬皮病小鼠皮肤组织纤维化及上皮间质转化的影响。方法:选取雌性BALB/c小鼠24只,购自长沙市天勤生物技术有限公司。实验方案经南华大学动物实验伦理委员会批准。随机将24只BALB/c小鼠均分为3组:对照组背部皮下注射生理盐水;模型组和干预组采用背部注射博来霉素500mg/L建立硬皮病小鼠模型;干预组同时腹腔注射5mg/kg的IGC-001(Wnt/β-catenin信号通路抑制剂),1次/d,连续注射4周。麻醉后取各组小鼠背部注射区皮肤,采用苏木精-伊红和Masson染色,观察皮损组织病理学变化、胶原纤维分布和真皮厚度;采用水解法检测皮损组织羟脯氨酸含量;Westernblot检测皮损组织中β-链蛋白(β-catenin)、Ⅰ型胶原蛋白、纤维链接蛋白、α-平滑肌肌动蛋白、上皮性钙黏附蛋白、波形蛋白和神经性钙黏附蛋白的表达。结果与结论:①博来霉素连续皮下注射4周后,模型组大鼠注射区域真皮层明显增厚、胶原纤维明显增多;干预组真皮层较模型组明显变薄,胶原纤维明显减少;②与对照组比较,模型组皮损组织中羟脯氨酸含量及Ⅰ型胶原蛋白、纤维链接蛋白、α-平滑肌肌动蛋白、波形蛋白、神经性钙黏蛋白和β-链蛋白表达量显著增加(均P <0.05),上皮性钙黏附蛋白表达显著降低(P <0.05);与模型组比较,干预组皮损组织中羟脯氨酸含量及Ⅰ型胶原蛋白、纤维链接蛋白、α-平滑肌肌动蛋白、波形蛋白和神经性钙黏蛋白表达量显著降低(均P <0.05),上皮性钙黏附蛋白表达显著增加(P <0.05),β-链蛋白表达无显著性变化(P> 0.05);③结果说明,抑制Wnt/β-catenin信号通路可抑制硬皮病小鼠皮肤组织纤维化,其机制可能与抑制皮肤组织上皮间质转化有关。
BACKGROUND: Wnt/β-catenin signaling pathway has been shown to play an important role in the occurrence and development of systemic sclerosis, but the whether inhibiting Wnt/β-catenin can improve sclerosis is little reported.OBJECTIVE: To explore the effect of inhibition of Wnt/β-catenin signaling pathway on skin tissue fibrosis and epithelial-mesenchymal transition in mice with scleroderma.METHODS: Twenty-four BALB/c female mice were provided by Changsha Tianqin Biotechnology Co., Ltd., and the study was approved by the Experimental Animal Ethics Committee of University of South China. The mice were randomly divided into three groups: control group,model group and intervention group. Bleomycin was injected into the dorsal area of mice to establish the model of scleroderma. The control mice were subcutaneously injected with normal saline; the model mice were subcutaneously injected with 500 mg/L bleomycin, Mice of the intervention group were subcutaneously injected with 500 mg/L bleomycin and intraperitoneally injected with 5 mg/kg IGC-001(Wnt/β-catenin signaling pathway inhibitor) at the same time, once daily, for 4 consecutive weeks. All mice were decapitated and the dorsal area skin of injection region was collected. Hematoxylin-eosin staining and Masson staining were used to observe the pathological changes of skin lesions,collagen fiber distribution and dermal thickness. The content of hydroxyproline in skin tissues was detected by hydrolysis method. The protein expression levels of β-catenin, type I collagen, fibronectin, α-smooth muscle actin, E-cadherin, vimentin and N-cadherin in skin tissues were detected by western blot assay.RESULTS AND CONCLUSION:(1) After 4 weeks of continuous subcutaneous injection of bleomycin, the dermal thickness and collagen fibers in the skin tissues of model mice were significantly increased, while that in the intervention group were significantly decreased.(2)Compared with the control group, the hydroxyproline content and the protein expression levels of type I collagen, fibronectin, α-smooth muscle actin, vimentin, N-cadherin and β-catenin were significantly increased in the skin tissues of model group(all P < 0.05), and the protein expression of E-cadherin protein was significantly decreased(P < 0.05). Compared with the model group, the hydroxyproline content and the protein expression levels of type I collagen, fibronectin, α-smooth muscle actin, vimentin, N-cadherin were significantly decreased(P < 0.05),the protein expression level of E-cadherin protein was significantly increased(P < 0.05), while there was no significant change in the expression of β-catenin(P > 0.05).(3) To conclude, inhibition of Wnt/β-catenin signaling pathway may improve skin fibrosis in mice with scleroderma, and its mechanism may be related to inhibition of epithelial-mesenchymal transition in skin tissue.
BACKGROUND: Wnt/β-catenin signaling pathway has been shown to play an important role in the occurrence and development of systemic sclerosis, but the whether inhibiting Wnt/β-catenin can improve sclerosis is little reported.OBJECTIVE: To explore the effect of inhibition of Wnt/β-catenin signaling pathway on skin tissue fibrosis and epithelial-mesenchymal transition in mice with scleroderma.METHODS: Twenty-four BALB/c female mice were provided by Changsha Tianqin Biotechnology Co., Ltd., and the study was approved by the Experimental Animal Ethics Committee of University of South China. The mice were randomly divided into three groups: control group,model group and intervention group. Bleomycin was injected into the dorsal area of mice to establish the model of scleroderma. The control mice were subcutaneously injected with normal saline; the model mice were subcutaneously injected with 500 mg/L bleomycin, Mice of the intervention group were subcutaneously injected with 500 mg/L bleomycin and intraperitoneally injected with 5 mg/kg IGC-001(Wnt/β-catenin signaling pathway inhibitor) at the same time, once daily, for 4 consecutive weeks. All mice were decapitated and the dorsal area skin of injection region was collected. Hematoxylin-eosin staining and Masson staining were used to observe the pathological changes of skin lesions,collagen fiber distribution and dermal thickness. The content of hydroxyproline in skin tissues was detected by hydrolysis method. The protein expression levels of β-catenin, type I collagen, fibronectin, α-smooth muscle actin, E-cadherin, vimentin and N-cadherin in skin tissues were detected by western blot assay.RESULTS AND CONCLUSION:(1) After 4 weeks of continuous subcutaneous injection of bleomycin, the dermal thickness and collagen fibers in the skin tissues of model mice were significantly increased, while that in the intervention group were significantly decreased.(2)Compared with the control group, the hydroxyproline content and the protein expression levels of type I collagen, fibronectin, α-smooth muscle actin, vimentin, N-cadherin and β-catenin were significantly increased in the skin tissues of model group(all P < 0.05), and the protein expression of E-cadherin protein was significantly decreased(P < 0.05). Compared with the model group, the hydroxyproline content and the protein expression levels of type I collagen, fibronectin, α-smooth muscle actin, vimentin, N-cadherin were significantly decreased(P < 0.05),the protein expression level of E-cadherin protein was significantly increased(P < 0.05), while there was no significant change in the expression of β-catenin(P > 0.05).(3) To conclude, inhibition of Wnt/β-catenin signaling pathway may improve skin fibrosis in mice with scleroderma, and its mechanism may be related to inhibition of epithelial-mesenchymal transition in skin tissue.
引文
[1]Li SC. Scleroderma in children and adolescents:localized scleroderma and systemic sclerosis. Pediatr Clin North Am.2018;65(4):757-781.
[2]Bazan IS,Mensah KA,Rudkovskaia AA,et al. Pulmonary arterial hypertension in the setting of scleroderma is different than in the setting of lupus:A review. Respir Med.2018;134:42-46.
[3]贺欢,孙丹,闫小宁.中医治疗硬皮病研究进展[J].亚太传统医药,2017,13(20):63-65.
[4]Mo C,Zeng Z,Deng Q,et al. Imbalance between T helper 17and regulatory T cell subsets plays a significant role in the pathogenesis of systemic sclerosis. Biomed Pharmacother.2018;108:177-183.
[5]Vehe RK,Riskalla MM. Collagen vascular diseases:SLE,dermatomyositis, scleroderma, and MCTD. Pediatr Rev.2018;39(10):501-515.
[6]Cantatore FP,Maruotti N,Corrado A,et al. Angiogenesis dysregulation in the pathogenesis of systemic sclerosis.Biomed Res Int,2017,2017:5345673.
[7]Manetti M,Romano E,Rosa I,et al. Endothelial-tomesenchymal transition contributes to endothelial dysfunction and dermal fibrosis in systemic sclerosis. Ann Rheum Dis.2017;76(5):924-934.
[8]Piera-Velazquez S,Mendoza FA,Jimenez SA.Endothelial to mesenchymal transition(EndoMT)in the pathogenesis of human fibrotic diseases. J Clin Med.2016;5(4):45-67.
[9]Nakamura M,Tokura Y. Expression of SNAI1 and TWIST1 in the eccrine glands of patients with systemic sclerosis:possible involvement of epithelial-mesenchymal transition in the pathogenesis. Br J Dermatol.2011;164(1):204-205.
[10]徐文华,许春姣,周美璐,等.上皮间质转化相关蛋白在口腔黏膜下纤维化及其癌变组织中的表达[J].临床口腔医学杂志, 2016,32(4):203-207.
[11]Yu K,Li Q,Shi G,et al.Involvement of epithelial-mesenchymal transition in liver fibrosis.Saudi J Gastroenterol. 2018;24(1):5-11.
[12]刘金娟,刘彤. Wnt2、Wnt3a和β-catenin在硬皮病患者皮损中的作用[J].南方医科大学学报,2012,32(12):1781-1786.
[13]白炜,王婷,徐东,等.白藜芦醇对博来霉素诱导的硬皮病小鼠肺及皮肤病变的作用[J].中华风湿病学杂志,19(11):724-729.
[14]黄健,刘畅,陈松,等. Wnt/β-catenin信号通路在肾缺血再灌注大鼠中的表达及ICG-001阻断对慢性肾间质纤维化的作用[J].实用医学杂志,2018,34(7):1085-1088.
[15]Klaus A,Birchmeier W. Wnt signaling and its impact on development and cancer. Nature Reviews Cancer. 2008,8(5):387-398.
[16]Garcia A L,Udeh A,Kalahasty K,et al. A growing field:The regulation of axonal regeneration by Wnt signaling. Neural Regen Res.2018,13(1):43-52.
[17]Burgy O,Konigshoff M. The WNT signaling pathways in wound healing and fibrosis. Matrix Biol.2018;69:67-80.
[18]黄晶,孙兆瑞,杨志洲,等.调控Wnt信号通路对百草枯中毒大鼠肺纤维化的影响[J].医学研究生学报,2017,30(3):228-232.
[19]Huang GR,Wei SJ,Huang YQ,et al. Mechanism of combined use of vitamin D and puerarin in anti-hepatic fibrosis by regulating the Wnt/beta-catenin signalling pathway. World J Gastroenterol.2018;24(36):4178-4185.
[20]Wei J,Melichian D,Komura K,et al. Canonical Wnt signaling induces skin fibrosis and subcutaneous lipoatrophy:a novel mouse model for scleroderma?. Arthritis Rheum.2011;63(6):1707-1717.
[21]商安全,吉萍,李冬,等. MyD88基因缺失型与野生型小鼠在博莱霉素诱导肺纤维化过程中的病理学比较[J].临床与实验病理学杂志,2018,34(8):871-874.
[22]李清兰,杨拉维,刘刚. PM2.5对博莱霉素诱导小鼠肺纤维化的影响[J].广东医科大学学报,2017,35(3):237-240.
[23]Cao H,Wang C,Chen X,et al. Inhibition of Wnt/β-catenin signaling suppresses myofibroblast differentiation of lung resident mesenchymal stem cells and pulmonary fibrosis. Sci Rep.2018;8(1):13644.
[24]黄健,刘畅,陈松,等. Wnt/β-catenin信号通路在肾缺血再灌注大鼠中的表达及ICG-001阻断对慢性肾间质纤维化的作用[J].实用医学杂志,2018,34(7):1085-1088.
[25]郭鱼波,王春晖,裴晓华.乳腺癌细胞上皮间质转化的分子机制及其防治的研究进展[J].中国肿瘤,2018,27(12):933-943.
[26]杜晓欣,赵素芬.与肿瘤转移相关的上皮间质转化信号通路研究进展[J].中国癌症防治杂志,2017,9(05):412-414.
[27]Chen L,Munoz-Antonia T,Cress WD1. Trim28 contributes to EMT via regulation of E-cadherin and N-cadherin in lung cancer cell lines. PLoS One.2014;9(7):e101040.
[28]Emami KH,Nguyen C,Ma H,et al. A small molecule inhibitor ofβ-catenin/cyclic AMP response element-binding protein transcription. Proc Natl Acad Sci U S A.2004;101(34):12682-12687.
[2]Bazan IS,Mensah KA,Rudkovskaia AA,et al. Pulmonary arterial hypertension in the setting of scleroderma is different than in the setting of lupus:A review. Respir Med.2018;134:42-46.
[3]贺欢,孙丹,闫小宁.中医治疗硬皮病研究进展[J].亚太传统医药,2017,13(20):63-65.
[4]Mo C,Zeng Z,Deng Q,et al. Imbalance between T helper 17and regulatory T cell subsets plays a significant role in the pathogenesis of systemic sclerosis. Biomed Pharmacother.2018;108:177-183.
[5]Vehe RK,Riskalla MM. Collagen vascular diseases:SLE,dermatomyositis, scleroderma, and MCTD. Pediatr Rev.2018;39(10):501-515.
[6]Cantatore FP,Maruotti N,Corrado A,et al. Angiogenesis dysregulation in the pathogenesis of systemic sclerosis.Biomed Res Int,2017,2017:5345673.
[7]Manetti M,Romano E,Rosa I,et al. Endothelial-tomesenchymal transition contributes to endothelial dysfunction and dermal fibrosis in systemic sclerosis. Ann Rheum Dis.2017;76(5):924-934.
[8]Piera-Velazquez S,Mendoza FA,Jimenez SA.Endothelial to mesenchymal transition(EndoMT)in the pathogenesis of human fibrotic diseases. J Clin Med.2016;5(4):45-67.
[9]Nakamura M,Tokura Y. Expression of SNAI1 and TWIST1 in the eccrine glands of patients with systemic sclerosis:possible involvement of epithelial-mesenchymal transition in the pathogenesis. Br J Dermatol.2011;164(1):204-205.
[10]徐文华,许春姣,周美璐,等.上皮间质转化相关蛋白在口腔黏膜下纤维化及其癌变组织中的表达[J].临床口腔医学杂志, 2016,32(4):203-207.
[11]Yu K,Li Q,Shi G,et al.Involvement of epithelial-mesenchymal transition in liver fibrosis.Saudi J Gastroenterol. 2018;24(1):5-11.
[12]刘金娟,刘彤. Wnt2、Wnt3a和β-catenin在硬皮病患者皮损中的作用[J].南方医科大学学报,2012,32(12):1781-1786.
[13]白炜,王婷,徐东,等.白藜芦醇对博来霉素诱导的硬皮病小鼠肺及皮肤病变的作用[J].中华风湿病学杂志,19(11):724-729.
[14]黄健,刘畅,陈松,等. Wnt/β-catenin信号通路在肾缺血再灌注大鼠中的表达及ICG-001阻断对慢性肾间质纤维化的作用[J].实用医学杂志,2018,34(7):1085-1088.
[15]Klaus A,Birchmeier W. Wnt signaling and its impact on development and cancer. Nature Reviews Cancer. 2008,8(5):387-398.
[16]Garcia A L,Udeh A,Kalahasty K,et al. A growing field:The regulation of axonal regeneration by Wnt signaling. Neural Regen Res.2018,13(1):43-52.
[17]Burgy O,Konigshoff M. The WNT signaling pathways in wound healing and fibrosis. Matrix Biol.2018;69:67-80.
[18]黄晶,孙兆瑞,杨志洲,等.调控Wnt信号通路对百草枯中毒大鼠肺纤维化的影响[J].医学研究生学报,2017,30(3):228-232.
[19]Huang GR,Wei SJ,Huang YQ,et al. Mechanism of combined use of vitamin D and puerarin in anti-hepatic fibrosis by regulating the Wnt/beta-catenin signalling pathway. World J Gastroenterol.2018;24(36):4178-4185.
[20]Wei J,Melichian D,Komura K,et al. Canonical Wnt signaling induces skin fibrosis and subcutaneous lipoatrophy:a novel mouse model for scleroderma?. Arthritis Rheum.2011;63(6):1707-1717.
[21]商安全,吉萍,李冬,等. MyD88基因缺失型与野生型小鼠在博莱霉素诱导肺纤维化过程中的病理学比较[J].临床与实验病理学杂志,2018,34(8):871-874.
[22]李清兰,杨拉维,刘刚. PM2.5对博莱霉素诱导小鼠肺纤维化的影响[J].广东医科大学学报,2017,35(3):237-240.
[23]Cao H,Wang C,Chen X,et al. Inhibition of Wnt/β-catenin signaling suppresses myofibroblast differentiation of lung resident mesenchymal stem cells and pulmonary fibrosis. Sci Rep.2018;8(1):13644.
[24]黄健,刘畅,陈松,等. Wnt/β-catenin信号通路在肾缺血再灌注大鼠中的表达及ICG-001阻断对慢性肾间质纤维化的作用[J].实用医学杂志,2018,34(7):1085-1088.
[25]郭鱼波,王春晖,裴晓华.乳腺癌细胞上皮间质转化的分子机制及其防治的研究进展[J].中国肿瘤,2018,27(12):933-943.
[26]杜晓欣,赵素芬.与肿瘤转移相关的上皮间质转化信号通路研究进展[J].中国癌症防治杂志,2017,9(05):412-414.
[27]Chen L,Munoz-Antonia T,Cress WD1. Trim28 contributes to EMT via regulation of E-cadherin and N-cadherin in lung cancer cell lines. PLoS One.2014;9(7):e101040.
[28]Emami KH,Nguyen C,Ma H,et al. A small molecule inhibitor ofβ-catenin/cyclic AMP response element-binding protein transcription. Proc Natl Acad Sci U S A.2004;101(34):12682-12687.