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郭心仪, 黄栊瑢, 郭永红, 陈向东, 付美林.利用LC-MS/MS和网络药理学探讨丹黄明目汤对糖尿病视网膜病变的潜在治疗作用[J].湖南中医药大学学报,2024,44(12):2257-2266[点击复制] |
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| 利用LC-MS/MS和网络药理学探讨丹黄明目汤对糖尿病视网膜病变的潜在治疗作用 |
| 郭心仪,黄栊瑢,郭永红,陈向东,付美林 |
| (湖南中医药大学第一中医临床学院, 湖南 长沙 410007;湖南中医药大学, 湖南 长沙 410208;衡阳市中医医院, 湖南 衡阳 421000) |
| 摘要: |
| 目的 探讨丹黄明目汤对糖尿病视网膜病变（diabetic retinopathy,DR）的潜在治疗作用。方法 采用液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry,LC-MS/MS）技术检测丹黄明目汤入血成分。采用网络药理学分析丹黄明目汤的药效成分及其治疗DR的关键通路及潜在靶点。将60只大鼠随机分为未造模组（24只）和造模组（36只）。将24只未造模组大鼠分为空白组（等体积生理盐水）、空白给药组（等体积生理盐水+1.25 g/kg丹黄明目汤浸膏），每组12只。造模组腹腔一次性注射40 mg/kg链脲佐菌素（streptozocin,STZ）联合高脂饲养建立DR模型后，分为模型组（40 mg/kg STZ）、阳性对照组（40 mg/kg STZ+羟苯磺酸钙胶囊0.09 g/kg）、丹黄明目汤样本组（40 mg/kg STZ+1.25 g/kg丹黄明目汤浸膏），每组12只，连续干预4周。生化试剂盒检测超氧化物歧化酶（superoxidedismutase,SOD）和过氧化氢酶（catalase,CAT）的活性；ELISA检测大鼠血清肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）、白细胞介素（interleukin,IL）-6和血清炎症因子IL-1β的水平；Western blot检测大鼠视网膜组织p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase,p38 MAPK）和细胞外调节蛋白激酶1/2（extracellular regulated protein kinases 1/2,ERK1/2）的蛋白表达水平。结果 LC-MS/MS结果表明，丹黄明目汤中共检测到347个入血化合物，其中前7个含量较高的入血代谢物分别为1,7-bis（4-hydroxyphenyl）heptan-3-one、5-hydroxy-2,2-dimethyl-10-（2-methylbut-3-en-2-yl）pyrano[3,2-g]chromen-8-one、5,7，4'-Trimethoxyflavone、Cillin、Deacetylto mentosideI、canescinA CanescinA、VentiloquinoneI。网络药理学结果显示，丹黄明目汤可能通过调节丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）信号通路、紧密连接和趋化因子信号通路等关键生物通路的多靶点干预DR。与空白组相比，模型组SOD、CAT水平及p38 MAPK、ERK1/2总蛋白表达降低（P＜0.05，P<0.01），TNF-α、IL-6、IL-1β水平及p38 MAPK、ERK1/2磷酸化水平升高（P<0.05，P<0.01）。与模型组相比，阳性对照组、空白给药组和丹黄明目汤样本组的SOD、CAT、TNF-α、IL-6、IL-1β水平及p38 MAPK、ERK1/2磷酸化水平降低（P<0.05，P<0.01）。结论 丹黄明目汤可通过调节MAPK信号通路抑制p38 MAPK等因子的表达和活性，促进SOD和CAT的表达，从而减轻DR的炎症反应、氧化应激、血管损伤，最终起到治疗DR的作用。 |
| 关键词: 糖尿病视网膜病变 丹黄明目汤 液相色谱-串联质谱 抗炎 抗氧化 p38丝裂原活化蛋白激酶 超氧化物歧化酶 过氧化氢酶 |
| DOI：10.3969/j.issn.1674-070X.2024.12.018 |
| 投稿时间：2024-09-18 |
| 基金项目:国家自然科学基金项目（82374525）;湖南省中医药科研计划项目（C2022003）;湖南省眼科疾病（中医）临床医学研究中心（2023SK4038）;湖南省自然科学基金项目（2021JJ30520）;湖南中医药大学校级科研项目（2023CX39）。 |
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| Exploring the potential therapeutic effects of Danhuang Mingmu Decoction on diabetic retinopathy through LC-MS/MS and network pharmacology |
| GUO Xinyi, HUANG Longrong, GUO Yonghong, CHEN Xiangdong, FU Meilin |
| (The First Clinical School of Chinese Medicine, Hunan University of Chinese Medicine, Changsha, Hunan 410007, China;Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;Hengyang Municipal Hospital of Chinese Medicine, Hengyang, Hunan 421000, China) |
| Abstract: |
| Objective To explore the potential therapeutic effects of Danhuang Mingmu Decoction (DHMMD) on diabetic retinopathy (DR). Methods Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to identify the absorbed components of DHMMD in the blood. The pharmacological components of DHMMD and their key pathways and potential targets for treating DR were analyzed using network pharmacology. Sixty rats were randomized into a non-model group (n=24) and a model induction group (n=36). The 24 rats in the non-model group were subdivided into blank group (equal volume of normal saline) and blank treatment group (equal volume of normal saline+DHMMD extract 1.25 g/kg), with 12 rats in each group. After establishing the model by a single intraperitoneal injection of streptozotocin (STZ) 40 mg/kg combined with a high-fat diet, the 36 rats in the model induction group were subdivided into model group (STZ 40 mg/kg), positive control group (STZ 40 mg/kg+calcium hydroxyl sulfonate capsule 0.09 g/kg), and DHMMD sample group (STZ 40 mg/kg+DHMMD extract 1.25 g/kg), with 12 rats in each group. The intervention lasted for four weeks. The activity of superoxide dismutase (SOD) and catalase (CAT) was determined using biochemical kits. The rat serum levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and the inflammatory cytokine IL-1β were measured by ELISA. The protein expression levels of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular regulated protein kinases 1/2 (ERK1/2) in rat retinal tissues were examined by Western blot. Results The LC-MS/MS revealed that a total of 347 compounds of DHMMD were examined in the blood after administration, with the first seven metabolites with higher content checked in the blood being 1,7-bis (4-hydroxyphenyl) heptan-3-one, 5-hydroxy-2,2-dimethyl-10-(2-methylbut-3-en-2-yl) pyrano[3,2-g] chromen-8-one, 5,7,4'-Trimethoxyflavone, Cillin, Deacetylto mentosideI, canescinA CanescinA, and VentiloquinoneI. The network pharmacology revealed that DHMMD might intervene in DR through multi-target regulation of key biological pathways such as the mitogen-activated protein kinase (MAPK) signaling pathway, tight junctions, and chemotaxis factor signaling pathway. Compared with the blank group, the levels of SOD and CAT, as well as the total protein expressions of p38 MAPK and ERK1/2 in the model group decreased (P<0.05, P<0.01), while the levels of TNF-α, IL-6, and IL-1β, as well as the phosphorylation levels of p38 MAPK and ERK1/2 increased (P<0.05, P<0.01). Compared with the model group, the levels of SOD, CAT, TNF-α, IL-6, and IL-1β, as well as the phosphorylation levels of p38 MAPK and ERK1/2 decreased in the positive control, blank treatment, and DHMMD sample groups (P<0.05, P<0.01). Conclusion DHMMD can inhibit the expression and activity of factors such as p38 MAPK by regulating the MAPK signaling pathway, promote the expression of SOD and CAT, thereby reducing the inflammatory response, oxidative stress, and vascular damage in DR, and ultimately play a therapeutic role in DR. |
| Key words: diabetic retinopathy Danhuang Mingmu Decoction liquid chromatography-tandem mass spectrometry anti-inflammation anti-oxidation p38 mitogen-activated protein kinase superoxide dismutase catalase |
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