PDF下载 ( 5010 KB)
基于网络药理学探讨消癥活络方通过ERK5通路抗大鼠肝纤维化的机制
DOI: 10.3969/j.issn.1001-5256.2021.03.016
利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突，特此声明。
作者贡献声明：田乐负责课题设计，资料分析，撰写论文；王颖航参与收集数据，修改论文；潘志负责拟定写作思路，指导撰写文章并最后定稿。
Mechanism of Xiaozheng Huoluo prescription in preventing liver fibrosis in rats via the ERK5 pathway: An analysis based on network pharmacology
-
摘要:
目的 基于网络药理学方法探索消癥活络方(XZHLF)防治肝纤维化的作用机制。 方法 通过TCMSP数据库、化学专业数据库、ETCM数据库、化源网数据库、PubChem数据库以及文献查阅，收集XZHLF各中药化学成分，利用Swiss ADME数据库筛选出XZHLF各中药活性成分，利用Swiss Target Prediction数据库预测XZHLF各中药活性成分靶点；通过GeneCards、OMIN数据库收集肝纤维化疾病靶点，利用韦恩图获得XZHLF防治肝纤维化的潜在作用靶点。使用Cytoscape 3.7.1软件建立XZHLF“药物-活性成分”网络和XZHLF防治肝纤维化的“活性成分-潜在作用靶点”网络。在Metascape数据库对潜在作用靶点进行GO和KEGG富集分析，选取富集基因数最多的前20条绘制气泡图。对前20条KEGG通路中的MAPK信号通路进行分析，绘制“活性成分-潜在作用靶点-通路”网络图。将健康的SD大鼠随机分为6组，分别为空白对照组(K组)、模型组(M组)、秋水仙碱阳性对照组(Y组)、XZHLF高(G组)、中(Z组)、低(D组)剂量组，利用CCl4诱导大鼠肝纤维化进行造模，造模同时给药，共8周。通过Western Blot法检测各组大鼠肝组织内ERK5、p-ERK5、MEK5、MEKK3蛋白的相对含量和表达情况。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。 结果 从XZHLF中筛选出110个活性成分，作用于923个活性成分靶点，与6823个肝纤维化疾病靶点相映射得到潜在作用靶点647个。XZHLF可能通过MAPK信号通路等通路，通过MAPK级联反应的调控等生物过程，作用于EGFR、AKT1、IKBKB、MAPK8、PDGFRB等多个蛋白靶点，从而发挥防治肝纤维化的作用。M组的大鼠肝组织内ERK5、p-ERK5、MEK5和MEKK3蛋白水平较K、Y、G、Z、D组明显增加，差异均有统计学意(P值均＜0.05)；K组的大鼠肝组织内ERK5、p-ERK5、MEK5和MEKK3蛋白水平较Y、G、Z、D组明显减少，差异均有统计学意义(P值均＜0.05)。 结论 基于网络药理学的方法预测出XZHLF可能通过MAPK信号通路防治肝纤维化，通过实验验证XZHLF是通过MAPK信号通路家族中ERK5通路抗肝纤维化。XZHLF高剂量组的抗肝纤维化作用效果最为明显。 -
关键词:
- 网络药理学 /
- 消癥活络方 /
- 肝硬化 /
- 细胞外信号调节激酶5
Abstract:Objective To investigate the mechanism of Xiaozheng Huoluo prescription (XZHLF) in the prevention and treatment of liver fibrosis based on network pharmacology. Methods TCMSP database, Chemistry Database, ETCM database, chemical source network database, ChemSrc database, and PubChem database were searched and a literature review was performed to collect the chemical components of each traditional Chinese medicine of XZHLF, Swiss ADME database was used to screen out the active components of each traditional Chinese medicine of XZHLF, and Swiss Target Prediction database was used to predict the targets of these active components; GeneCards and OMIN databases were used to collect the disease targets of liver fibrosis, and Venn diagrams were used to obtain the potential targets of XZHLF in the prevention and treatment of liver fibrosis. Cytoscape 3.7.1 software was used to establish a "drug-active component" network for XZHLF and an "active component-potential target" network for XZHLF in the prevention and treatment of liver fibrosis. Metascape database was used to perform GO and KEGG enrichment analysis of potential targets, and bubble charts were plotted for the top 20 pathways with the highest number of enriched genes. The MAPK signaling pathway among the top 20 KEGG pathways was analyzed to plot an "active component-potential target-pathway" network. Healthy Sprague-Dawley rats were randomly divided into blank control group (K group), model group (M group), colchicine-positive control group (Y group), high-dose XZHLF group (G group), middle-dose XZHLF group (Z Group), and low-dose XZHLF group (D group), and a rat model of liver fibrosis was established by CCl4; the drug was administered simultaneously for 8 weeks. Western blot was used to measure the protein expression of ERK5, p-ERK5, MEK5, and MEKK3 in liver tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results A total of 110 active components were screened out from XZHLF and they acted on 923 targets, which were mapped to 6823 disease targets of liver fibrosis to obtain 647 potential targets. XZHLF might act on multiple protein targets such as EGFR, AKT1, IKBKB, MAPK8, and PDGFRB through the pathways including the MAPK signaling pathway and the biological processes including the regulation of MAPK cascade, thereby playing a role in the prevention and treatment of liver fibrosis. The M group had significant increases in the protein levels of ERK5, p-ERK5, MEK5, and MEKK3 in liver tissue compared with the K, Y, G, Z, and D groups (all P < 0.05), and the K group had significant reductions in the protein levels of ERK5, p-ERK5, MEK5, and MEKK3 in liver tissue compared with the Y, G, Z, and D groups (all P < 0.05). Conclusion Based on the method of network pharmacology, it is predicted that XZHLF may prevent and treat liver fibrosis via the MAPK signaling pathway, and it is verified through experiments that XZHLF prevents and treats liver fibrosis via the ERK5 pathway in the MAPK signaling pathway family. The high-dose XZHLF group shows the most obvious anti-liver fibrosis effect. -
图 1 XZHLF“药物-活性成分”网络图
注：类三角形代表XZHLF各中药，圆形代表XZHLF各中药活性成分并用不同的颜色标记，其中粉色代表各中药共有的活性成分，圆形面积越大代表XZHLF各中药活性成分连接的XZHLF各中药活性成分靶点越多。
图 4 “活性成分-潜在作用靶点”网络图
注：图中粉色圆形代表XZHLF各中药活性成分，圆形面积越大代表XZHLF各中药活性成连接的XZHLF防治肝纤维化潜在作用靶点越多；蓝色菱形代表XZHLF防治肝纤维化潜在作用靶点，菱形面积越大代表XZHLF防治肝纤维化潜在作用靶点连接的XZHLF各中药活性成分越多。
-
[1] WEISKIRCHEN R. Hepatoprotective and anti-fibrotic agents: It's time to take the next step[J]. Front Pharmacol, 2015, 6: 303. http://pubmedcentralcanada.ca/pmcc/articles/PMC4703795/ [2] WYNN TA, RAMALINGAM TR. Mechanisms of fibrosis: Therapeutic translation for fibrotic disease[J]. Nat Med, 2012, 18(7): 1028-1040. DOI: 10.1038/nm.2807 [3] SEKI E, BRENNER DA. Recent advancement of molecular mechanisms of liver fibrosis[J]. J Hepatobiliary Pancreat Sci, 2015, 22(7): 512-518. DOI: 10.1002/jhbp.245 [4] TANG CF. Experimental and clinical study on Xiaozheng Huoluo Fang in prevention and treatment of liver arthralgia[D]. Changchun: Changchun University of Chinese Medicine, 2016. (in Chinese)唐成芳. 消癥活络方防治肝痹的实验与临床研究[D]. 长春: 长春中医药大学, 2016. [5] DAI DX, PAN Z, TANG CF, et al. Effect of Xiaozheng Huoluo Fang on the expression of α-SMA and TGF-β1 in liver tissue of rats with hepatic fibrosis[J]. Jilin J Chin Med, 2017, 37(2): 162-166. (in Chinese) https://www.cnki.com.cn/Article/CJFDTOTAL-ZYJL201702016.htm代东雪, 潘志, 唐成芳, 等. 消癥活络方对肝纤维化大鼠肝组织α-SMA和TGF-β1表达的影响[J]. 吉林中医药, 2017, 37(2): 162-166. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYJL201702016.htm [6] LI S, ZHANG B. Traditional Chinese medicine network pharmacology: Theory, methodology and application[J]. Chin J Nat Med, 2013, 11(2): 110-120. http://europepmc.org/abstract/MED/23787177 [7] JIN JJ, ZHONG M, YU SM, et al. Therapeutic effect of water extract of chuanposhi on hepatic fibrosis induced by carbon tetrachloride in rats[J]. Chin J Experi Tradi Med Form, 2012, 18(22): 258-262. (in Chinese) https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX201222075.htm金俊杰, 钟鸣, 余胜民, 等. 穿破石水提取物对四氯化碳致大鼠肝纤维化的治疗作用[J]. 中国实验方剂学杂志, 2012, 18(22): 258-262. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX201222075.htm [8] XU SY, BIAN RL, CHEN X. Methodology of pharmacological experiment[M]. 3rd Ed. Beijing: People's Medical Publishing House, 2002: 1350-1351. (in Chinese)徐淑云, 卞如濂, 陈修. 药理实验方法学[M]. 第3版. 北京: 人民卫生出版社, 2002: 1350-1351. [9] LI ZY, TIAN YZ. Tibetan medicine Langqing atta treats liver fibrosis from stasis and heat[J]. J Changchun Univ Tradit Chin Med, 2019, 35(6): 1223-1225. (in Chinese) https://www.cnki.com.cn/Article/CJFDTOTAL-CZXX201906062.htm李忠意, 田耀洲. 藏药郎庆阿塔从瘀热论治肝纤维化[J]. 长春中医药大学学报, 2019, 35(6): 1223-1225. https://www.cnki.com.cn/Article/CJFDTOTAL-CZXX201906062.htm [10] YI L, YANG JS. Discussion on TCM pathogenesis of liver fibrosis[J]. J Tradit Chin Med, 2005, 46(11): 806-808. (in Chinese) https://www.cnki.com.cn/Article/CJFDTOTAL-ZZYZ200511001.htm衣蕾, 杨俊生. 肝纤维化中医病机浅探[J]. 中医杂志, 2005, 46(11): 806-808. https://www.cnki.com.cn/Article/CJFDTOTAL-ZZYZ200511001.htm [11] HAN B. Treatment of liver fibrosis with traditional Chinese Medicine[J]. Nei Mongol J Tradit Chin Med, 2014, 33(19): 39-40. (in Chinese) https://www.cnki.com.cn/Article/CJFDTOTAL-NZYY201419039.htm韩斌. 浅谈肝纤维化的中医治疗[J]. 内蒙古中医药, 2014, 33(19): 39-40. https://www.cnki.com.cn/Article/CJFDTOTAL-NZYY201419039.htm [12] NISHIMOTO S, NISHIDA E. MAPK signalling: ERK5 versus ERK1/2[J]. EMBO Rep, 2006, 7(8): 782-786. DOI: 10.1038/sj.embor.7400755 [13] CHAO TH, HAYASHI M, TAPPING RI, et al. MEKK3 directly regulates MEK5 activity as part of the big mitogen-activated protein kinase 1 (BMK1) signaling pathway[J]. J Biol Chem, 1999, 274(51): 36035-36038. DOI: 10.1074/jbc.274.51.36035 [14] SUN W, KESAVAN K, SCHAEFER BC, et al. MEKK2 associates with the adapter protein Lad/RIBP and regulates the MEK5-BMK1/ERK5 pathway[J]. J Biol Chem, 2001, 276(7): 5093-5100. DOI: 10.1074/jbc.M003719200 [15] ENGLISH JM, VANDERBILT CA, XU S, et al. Isolation of MEK5 and differential expression of alternatively spliced forms[J]. J Biol Chem, 1995, 270(48): 28897-28902. DOI: 10.1074/jbc.270.48.28897 [16] FANG DJ, LIANG WG, SHEN Y. Recent progress of extracellular signal regulated kinase 5 signal transduction pathway[J]. Guangdong Med J, 2012, 33(1): 140-143. (in Chinese) https://www.cnki.com.cn/Article/CJFDTOTAL-GAYX201201065.htm方德健, 梁伟国, 沈雁. 细胞外信号调节激酶5信号转导通道研究的最新进展[J]. 广东医学, 2012, 33(1): 140-143. https://www.cnki.com.cn/Article/CJFDTOTAL-GAYX201201065.htm [17] WANG Y, SU B, XIA Z. Brain-derived neurotrophic factor activates ERK5 in cortical neurons via a Rap1-MEKK2 signaling cascade[J]. J Biol Chem, 2006, 281(47): 35965-35974. DOI: 10.1074/jbc.M605503200 [18] DORADO F, VELASCO S, ESPARÍS-OGANDO A, et al. The mitogen-activated protein kinase Erk5 mediates human mesangial cell activation[J]. Nephrol Dial Transplant, 2008, 23(11): 3403-3411. DOI: 10.1093/ndt/gfn333 [19] RAMSAY AK, MCCRACKEN SR, SOOFI M, et al. ERK5 signalling in prostate cancer promotes an invasive phenotype[J]. Br J Cancer, 2011, 104(4): 664-672. DOI: 10.1038/sj.bjc.6606062 [20] URUSHIHARA M, TAKAMATSU M, SHIMIZU M, et al. ERK5 activation enhances mesangial cell viability and collagen matrix accumulation in rat progressive glomerulonephritis[J]. Am J Physiol Renal Physiol, 2010, 298(1): F167-F176. DOI: 10.1152/ajprenal.00124.2009 -
下载:
本文二维码
图(8)
计量
- 文章访问数: 479
- HTML全文浏览量: 190
- PDF下载量: 26
- 被引次数: 0
