基于网络药理学和试验验证探讨茵陈蒿汤治疗糖尿病的作用机制

doi:10.16431/j.cnki.1671-7236.2024.09.038

摘要/Abstract

摘要： 【目的】通过网络药理学、分子对接和试验验证的方法探讨茵陈蒿汤(YCHD)治疗糖尿病(DM)的作用机制。【方法】利用TCMSP、ETCM、UniProt、SwissTargetPrediction数据库及文献查阅等查找YCHD的活性成分和相关作用靶点；利用OMIM、PharmGkb、TTD、DrugBank数据库获取DM的疾病靶点。构建YCHD和DM的蛋白互作(PPI)网络图，对关键作用靶点进行GO功能和KEGG通路富集分析。采用AutoDockTool 1.5.7软件进行分子对接，预测活性成分和核心靶点的结合能。将细胞分为对照组(Control)、模型组(Model)及YCHD含药血清低(YCHD-L)、中(YCHD-M)、高(YCHD-H)剂量组，通过体外细胞试验检测诱导型一氧化氮合酶(iNOS)、环氧化酶2(COX2)、Toll样受体4(TLR4)、核因子κB(NF-κB)p65、白细胞介素-1β(IL1β)、IL6的mRNA表达水平，以及iNOS、COX2、髓样分化因子88(MyD88)、NF-κB p65蛋白表达水平，并对关键通路进行验证。【结果】共获得YCHD活性成分147个，DM疾病靶点486个，YCHD作用于DM的相关靶点基因57个，其中胰岛素(INS)、IL1β、肿瘤坏死因子(TNF)、B细胞中κ轻型多肽基因增强子的核因子1(NFKB1)、信号传导子和转录活化子1(STAT1)、白蛋白(ALB)、TLR4、IL1A、IL10、IL8等为关键靶基因。根据Counts值筛选出GO前十的条目中包含生物过程1条、细胞组分7条、分子功能2条，主要包括RNA聚合酶Ⅱ启动子转录的正调控、内质网膜、质膜、胞外区、核质、胞质溶胶、细胞质、膜的组成部分、相同蛋白质结合和蛋白质结合。KEGG通路富集分析显示，与NOD样受体、NF-κB、Toll样受体等炎症信号通路密切相关。分子对接结果显示，YCHD的活性有效成分与主要靶点具有良好的结合能力。体外试验结果表明，与对照组相比，模型组细胞TLR4、iNOS、COX2、NF-κB p65、IL1β和IL6 mRNA表达水平及iNOS、COX2、MyD88、NF-κB p65蛋白表达水平均显著升高(P＜0.05)；与模型组相比，YCHD-L、YCHD-M、YCHD-H组细胞iNOS、COX2、IL1β和IL6 mRNA表达水平及iNOS、COX2蛋白表达水平均显著降低(P＜0.05)，YCHD-M、YCHD-H组细胞TLR4、NF-κB p65 mRNA水平显著降低(P＜0.05)，YCHD-H组细胞MyD88、NF-κB p65蛋白表达水平显著降低(P＜0.05)。【结论】 YCHD可能通过多成分、多靶点，调控TLR4、NF-κB等多条炎症信号通路，发挥抗DM的作用。以YCHD-H组对炎症信号通路相关指标的抑制最明显。

关键词: 糖尿病; 茵陈蒿汤; 网络药理学; 分子对接; 试验验证

Abstract: 【Objective】 This study was aimed to explore the action mechanism of Yinchenhao decoction (YCHD) in the treatment of diabetes mellitus (DM) by network pharmacology, molecular docking and experimental validation. 【Method】 TCMSP, ETCM, UniProt, SwissTargetPrediction databases and literature reviews were utilized to find the active ingredients and their related targets.OMIM, PharmGkb, TTD and DrugBank databases were utilized to obtain the disease targets of DM.The protein-protein interaction (PPI) network diagrams of YCHD and DM were constructed, and the key targets were analyzed by GO function and KEGG signal pathway enrichment.Molecular docking was performed using AutoDockTool 1.5.7 software to predict the binding energy of active ingredients and core targets.The cells were divided into control group (Control), model group (Model), and YCHD-containing serum low (YCHD-L), medium (YCHD-M) and high (YCHD-H) dose groups.The levels of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (Cox2), Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB) p65, interleukin-1β (IL1β), IL6 mRNA and iNOS, COX2, Myd88, NF-κB P65 protein were examined by in vitro cell assay to verify the key pathways. 【Result】 A total of 147 YCHD active ingredients were obtained, 486 DM disease targets were identified, and 57 genes related to the action of YCHD on DM were identified, including insulin (INS) and IL1β, tumor necrosis factor (TNF), nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NFKB1), signal transducer and transcriptional activator 1 (STAT1), albumin (ALB), TLR4, IL1A, IL10, and IL8 were key target genes.According to the counts value, the top ten entries in GO included 1 biological process, 7 cellular components, and 2 molecular functions, which were the positive regulation of RNA polymerase Ⅱ promoter transcription, endoplasmic reticulum membrane, plasma membrane, extracellular region, nucleoplasm, cytosol, cytoplasm, integral component of membrane, identical protein binding, and protein binding.KEGG pathway analysis showed that it was closely related with NOD-like receptors and NF-κB, Toll-like receptors and other inflammatory signaling pathways.The molecular docking results showed that the active ingredients of YCHD had good binding ability with the main targets.The experimental results showed that compared with control group, the mRNA levels of TLR4, iNOS, COX2, NF-κB p65, IL1β, IL6 and the protein expression levels of INOS, COX2, MyD88, NF-κB p65 of cells were significantly increased (P＜0.05) in model group.Compared with model group, the mRNA levels of iNOS, COX2, IL1β, IL6 and the protein expression levels of iNOS and COX2 of cells were significantly reduced (P＜0.05) in YCHD-L, YCHD-M and YCHD-H groups.The mRNA levels of TLR4 and NF-κB p65 of cells in YCHD-M and YCHD-H groups were significantly decreased (P＜0.05), and the protein levels of Myd88 and NF-κB p65 of cells in YCHD-H group were significantly decreased (P＜0.05). 【Conclusion】 YCHD might regulate TLR4, NF-κB and other inflammatory signaling pathways through multiple components and multiple targets to treat the DM.The YCHD-H group showed the most significant inhibition of inflammatory signaling pathway related indicators.

Key words: diabetes mellitus; Yinchenhao decoction; network pharmacology; molecular docking; experimental verification

中图分类号:

S853.74

宋雨, 孙彦刚, 张艺媛, 周思奇, 张璐, 李伟, 张明昊, 张紫娟, 苗晋鑫, 陈芳, 曹珊, 张小莉, 谢莎. 基于网络药理学和试验验证探讨茵陈蒿汤治疗糖尿病的作用机制[J]. 中国畜牧兽医, 2024, 51(9): 4106-4119.

SONG Yu, SUN Yangang, ZHANG Yiyuan, ZHOU Siqi, ZHANG Lu, LI Wei, ZHANG Minghao, ZHANG Zijuan, MIAO Jinxin, CHEN Fang, CAO Shan, ZHANG Xiaoli, XIE Sha. Study on the Mechanism of Yinchenhao Decoction in Treating Diabetes Mellitus Based on Network Pharmacology and Experimental Verification[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(9): 4106-4119.

参考文献

[1] 李宝铜,夏星,钟斯然,等.罗汉果总皂苷对高糖高脂饲料联合链脲佐菌素致2型糖尿病大鼠的作用[J].中国畜牧兽医,2020,47(12):4148-4155. LI B T,XIA X,ZHONG S R,et al.Effects of Momordica grosvenori saponins on type 2 diabetes rats induced by high-sucrose/high-fat diet and streptozotocin[J] China Animal Husbandry & Veterinary Medicine,2020,47(12):4148-4155.(in Chinese)
[2] RAMI-MERHAR B,FRÖHLICH-REITERER E,HOFER S E.Diabetes mellitus in childhood and adolescence (Update 2019)[J].Wien Klin Wochenschr,2019,131(Suppl 1):85-90.
[3] GENAZZANI A D,BATTIPAGLIA C,PETRILLO T,et al.Familial diabetes predisposes PCOS patients to insulin resistance (IR),reproductive impairment and hepatic dysfunction:Effects of d-chiro inositol (DCI) and alpha lipoic acid (ALA) administration on hepatic insulin extraction (HIE) index[J].Gynecological Endocrinology,2022,38(8):681-688.
[4] TAKITA M.Multicenter trial of closed-loop control in type 1 diabetes[J].New England Journal of Medicine,2020,382(6):577.
[5] LECUMBERRI E,ORTEGA M,ITURREGUI M,et al.Quality-of-life and treatment satisfaction in actual clinical practice of patients with type 1 diabetes mellitus (T1DM) and hypoglycemia treated with insulin degludec[J].Current Medical Research and Opinion,2018,34(6):1053-1059.
[6] EL-SHABRAWY R M,AHMED A M,SELIM F O,et al.Association between CD4⁺,CD25⁺,FOXP3⁺ regulatory T-cells and cardiovascular complications in diabetic patients type 1[J].The Egyptian Journal of Immunology,2019,26(1):129-139.
[7] ESPONA-NOGUERA A,CIRIZA J,CAÑIBANO-HERNÁNDEZ A,et al.Review of advanced hydrogel-based cell encapsulation systems for insulin delivery in type 1 diabetes mellitus[J].Pharmaceutics,2019,11(11):597.
[8] XIN G L L,KHEE Y P,YING T Y,et al.Current status on immunological therapies for type 1 diabetes mellitus[J].Current Diabetes Reports,2019,19(5):22.
[9] 樊亚男,关伟军,刘学东.干细胞治疗1型糖尿病研究进展[J].中国畜牧兽医,2015,42(11):3099-3104. FAN Y N,GUAN W J,LIU X D.Research progress on stem cell therapy for type 1 diabetes mellitus[J]. China Animal Husbandry & Veterinary Medicine,2015,42(11):3099-3104.(in Chinese)
[10] 于蕾妍,隋君霞,李华涛,等.螺旋藻多糖与银杏叶提取物复方对四氧嘧啶模型小鼠的降血糖作用[J].中国畜牧兽医,2019,46(7):2176-2182. YU L Y,SUI J X,LI H T,et al.Hypoglycemic effect of polysaccharide from Soirulina platensis and Ginkgo biloba extract compound on alloxan-induced model mice[J].China Animal Husbandry & Veterinary Medicine,2019,46(7):2176-2182.(in Chinese)
[11] ZHOU H B,CHEN J M,SHAO L M,et al.Apoptosis of human pancreatic carcinoma cell-1 cells induced by Yin Chen Hao decoction[J].World Journal of Gastroenterology,2015,21(27):8352-8357.
[12] CHEN S D,FAN Y,XU W J.Effects of Yinchenhao decoction (see text) for non-alcoholic steatohepatitis in rats and study of the mechanism[J].Journal of Traditional Chinese Medicine,2011,31(3):220-223.
[13] 程欢欢.应用茵陈蒿汤治疗Ⅱ型糖尿病的临床效果分析[J].当代医药论丛,2015,13(19):30-31. CHENG H H.Analysis of clinical effect of Yinchenhao decoction on type diabetes[J].Contemporary Medical Symposium,2015,13(19):30-31.(in Chinese)
[14] 潘竞锵,韩超,刘惠纯,等.茵陈蒿汤对正常和多种糖尿病模型动物血糖的影响[J].中药材,2001,24(2):128-131. PAN J Q,HAN C,LIU H C,et al.Effects of Yinchenhao decoction on normal animals and animal models of diabetes mellitus[J]. Journal of Chinese Medicinal Materials,2001,24(2):128-131.(in Chinese)
[15] DARENSKAYA M A,KOLESNIKOVA L I,KOLESNIKOV S I.Oxidative stress:Pathogenetic role in diabetes mellitus and its complications and therapeutic approaches to correction[J].Bulletin of Experimental Biology and Medicine,2021,171(2):179-189.
[16] FRANCE N L,SYED Y Y.Tirzepatide:A review in type 2 diabetes[J]. Drugs,2024，84(2)：227-238.
[17] SHREYA REDDY C S,RAMALINGAM G D,SELVARAJ J,et al.In vitro antioxidant and anti-diabetic analysis of Andrographis echioides and Andrographis paniculata ethanol extract[J].Bioinformation,2022,18(4):337-342.
[18] NASROLLAHI Z,SHAHANIPOUR K,MONAJEMI R,et al.Effect of quercetin and Abelmoschus esculentus (L.) Moench on lipids metabolism and blood glucose through AMPK-α in diabetic rats (HFD/STZ)[J].Journal of Food Biochemistry,2022,46(12):e14506.
[19] AL-ABBASI F A,KAZMI I.Therapeutic role of kaempferol and myricetin in streptozotocin-induced diabetes synergistically via modulation in pancreatic amylase,glycogen storage and insulin secretion[J].Molecular and Cellular Biochemistry,2023,478(9):1927-1937.
[20] AL-NUMAIR K S,VEERAMANI C,ALSAIF M A,et al.Influence of kaempferol,a flavonoid compound,on membrane-bound ATPases in streptozotocin-induced diabetic rats[J].Pharmaceutical Biology,2015,53(9):1372-1378.
[21] XIONG D,HU W,HAN X,et al.Rhein inhibited ferroptosis and EMT to attenuate diabetic nephropathy by regulating the Rac1/NOX1/β-catenin axis[J].Frontiers in Bioscience-Landmark,2023,28(5):100.
[22] XU K,WANG H,WU Z.Genkwanin suppresses mitochondrial dysfunction to alleviate IL-1β-elicited inflammation,apoptosis,and degradation of extracellular matrix in chondrocytes through upregulating DUSP1[J].Chinese Journal of Physiology,2023,66(4):284-293.
[23] 孙伟翔,宗莹莹,刘博,等.梓醇抑制Galectin-3和CD146互作改善AGEs致肝窦内皮细胞的损伤作用[J].中国畜牧兽医,2023,50(7):2820-2831. SUN W X,ZONG Y Y,LIU B,et al.Catalpol alleviates the damaging effect of AGEs on the endothelial cells in the hepatic sinus by disrupting the interaction between Galectin-3 and CD146[J]. China Animal Husbandry & Veterinary Medicine,2023,50(7):2820-2831.(in Chinese)
[24] ALIBASHE-AHMED M,BRIOUDES E,REITH W,et al.Toll-like receptor 4 inhibition prevents autoimmune diabetes in NOD mice[J].Scientific Reports,2019,9(1):19350.
[25] DEVARAJ S,TOBIAS P,JIALAL I.Knockout of Toll-like receptor-4 attenuates the pro-inflammatory state of diabetes[J].Cytokine,2011,55(3):441-445.
[26] 方颖,王亚东,周雯,等.黄芪桂枝五物汤对糖尿病周围神经病变大鼠模型AGEs/RAGE/NF-κB信号通路的影响[J].中国实验方剂学杂志,2020,26(13):52-58. FANG Y,WANG Y D,ZHOU W,et al.Effect of Huangqi Guizhi Wuwutang on AGEs/RAGE/NF-κB signaling pathway in diabetic peripheral neuropathy rats[J]. Chinese Journal of Experimental Traditional Medical Formulae,2020,26(13):52-58.(in Chinese)
[27] CHEN Y,ZHAO T,HAN M,et al.Gigantol protects retinal pigment epithelial cells against high glucose-induced apoptosis,oxidative stress and inflammation by inhibiting MTDH-mediated NF-κB signaling pathway[J].Immunopharmacology and Immunotoxicology,2024,46(1):33-39.
[28] CHOI Y H.Reduction of high glucose-induced oxidative injury in human retinal pigment epithelial cells by sarsasapogenin through inhibition of ROS generation and inactivation of NF-κB/NLRP3 inflammasome pathway[J].Genes Genomics,2023,45(9):1153-1163.