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目的：采用网络药理学方法结合体内外实验研究苓桂术甘汤（LGZGD）干预高脂诱导心肌纤维化的作用及机制。方法：运用网络药理学方法筛选LGZGD的有效成分及靶点，获取心肌纤维化和代谢性心肌病的靶点，对共同靶点进行GO功能及KEGG通路富集分析，并建立交集靶点的PPI网络和有效成分-核心靶点关联网络。构建高脂喂养诱导的高脂血症大鼠模型，给予LGZGD干预后，采用H&E染色和Sirius red染色观察心肌损伤和胶原纤维沉积，RT-qPCR检测观察心肌组织MMP9、TGF-β、SMAD3、SMAD7mRNA表达水平，并进行免疫组化染色检测心肌组织的CollagenⅠ、CollagenⅢ表达水平；构建棕榈酸（Palmitic acid,PA）诱导的大鼠H9C2心肌损伤细胞模型，采用流式细胞术、RT-qPCR检测，探究LGZGD含药血清对凋亡水平以及MMP9、TGF-β、SMAD3、SMAD7 mRNA表达水平的影响。结果：网络药理学分析得出共筛选出LGZGD 103个活性成分和886个核心靶点，与疾病相关的核心靶点有12个，与代谢性心肌病引起的心肌纤维化密切相关的富集通路有245条等。动物实验显示，与正常组比较，模型组大鼠心肌组织出现明显的心肌细胞排列紊乱，心肌纤维部分断裂及间质纤维化等病理变化，心肌组织CollagenⅠ、CollagenⅢ含量及CollagenⅠ/CollagenⅢ比值显著升高，TGF-β、SMAD3、MMP9 mRNA表达显著升高，而SMAD7mRNA表达显著降低（P<0.05）。LGZGD干预8周后，心脏损伤有明显改善，使心肌组织CollagenⅠ、CollagenⅢ含量及CollagenⅠ/CollagenⅢ比值，TGF-β、SMAD3、MMP9 mRNA表达下调，SMAD7 mRNA表达上调（P<0.05）。细胞实验显示，与正常组比较，PA组细胞凋亡水平显著升高，且H9C2心肌细胞TGF-β、SMAD3、MMP9 mRNA表达显著升高，而SMAD7 mRNA表达显著降低（P<0.01）；与PA组比较，LGZGD干预后，H9C2心肌细胞TGF-β、SMAD3、MMP9 mRNA表达下调，SMAD7 mRNA表达上调以及细胞凋亡水平均显著改善（P<0.01）。结论：LGZGD可通过干预心肌组织TGF-β/SMAD通路，从而抑制高脂模型大鼠心肌纤维化。
Abstract：Objective: To study the effect and mechanism of Linggui Zhugan Decoction(LGZGD) in the intervention of highlipid-induced myocardial fibrosis by using network pharmacology combined with in vitro and in vivo experiments. Methods: The network pharmacology method was used to screen the active ingredients and targets of LGZGD, the targets of myocardial fibrosis and metabolic cardiomyopathy were obtained, the GO function and KEGG pathway enrichment analysis of the common targets were carried out, and the PPI network and the active ingredient-core target association network of the intersecting targets were established. A rat model of hyperlipidemia induced by high-fat feeding was constructed, and after the intervention of LGZGD, myocardial injury and collagen fiber deposition were observed by H&E staining and Sirius red staining, the mRNA expression levels of MMP9, TGF-β, SMAD3, and SMAD7 in myocardial tissues were detected by RT-qPCR, and the expression levels of CollagenⅠ and CollagenⅢ in myocardial tissues were detected by immunohistochemical staining. A model of H9C2 cardiomyocyte injury induced by palmitic acid(PA) in rats was constructed, and the effects of LGZGD on the level of apoptosis and the mRNA expression levels of MMP9, TGF-β, SMAD3, and SMAD7 were investigated by flow cytometry and RT-qPCR. Results: The network pharmacology analysis showed that a total of 103 active ingredients and 886 core targets of LGZGD were screened, including 12 core targets related to the diseases, and 245 enrichment pathways closely related to myocardial fibrosis caused by metabolic cardiomyopathy. Animal experiments showed that compared to the normal group, the myocardial tissue of the model group showed obvious myocardial cell arrangement disorder, partial breakage of myocardial fibers, and interstitial fibrosis, the content of CollagenⅠ, CollagenⅢ and the ratio of CollagenⅠ/CollagenⅢ in myocardial tissue were significantly increased, the mRNA expressions of TGF-β, SMAD3, and MMP9 were significantly increased, and the mRNA expression of SMAD7 expression was significantly reduced(P<0.05). LGZGD significantly improved the cardiac injury after 8 weeks of intervention, and the content of CollagenⅠ, CollagenⅢ and the ratio of CollagenⅠ/CollagenⅢ in myocardial tissue, the mRNA expression of TGF-β, SMAD3, and MMP9 was down-regulated, and the mRNA expression of SMAD7 was up-regulated(P<0.05). Cell experiments showed that compared to the normal group, the apoptosis level of the PA group was significantly increased, and the mRNA expression of TGF-β, SMAD3, and MMP9 of H9C2 cardiomyocytes was significantly increased, while the mRNA expression of SMAD7 was significantly reduced(P<0.01); and compared to the PA group, LGZGD could down-regulate the mRNA expression of TGF-β, SMAD3, and MMP9 in H9C2 cardiomyocytes, up-regulate the mRNA expression of SMAD7, and significantly improve the level of apoptosis(P<0.01). Conclusion: LGZGD can inhibit myocardial fibrosis in high-fat rats by interfering with the TGF-β/SMAD pathway in myocardial tissue.
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基本信息:
DOI：10.13210/j.cnki.jhmu.20241023.002
中图分类号:R285.5
引用信息:
[1]彭红艳,张文龙,郭露琴,等.苓桂术甘汤干预高脂诱导大鼠心肌纤维化的作用机制研究[J].海南医科大学学报,2025,31(16):1240-1253.DOI:10.13210/j.cnki.jhmu.20241023.002.
基金信息:
安徽自然科学基金面上项目(2208085MH277)~~
2024-10-24
2024-10-24
2024-10-24