基于生物信息学、机器学习及动物实验揭示天香丹调控胆汁酸代谢治疗动脉粥样硬化的作用

Research on the mechanism of Tianxiangdan regulating mitochondrial energy metabolism in treating myocardial ischemic injury

目的:通过生物信息学及机器学习,筛选与动脉粥样硬化(atherosclerosis,AS)相关的胆汁酸代谢差异基因,并在动物实验中验证天香丹治疗AS的作用机制是否与调控胆汁酸代谢相关。方法:通过生物信息学得到“天香丹-AS-胆汁酸代谢”差异表达基因(DEGs)并分析其相关性,进一步通过富集分析和蛋白互作网络筛选目的基因以进行生物信息验证。通过对目的基因免疫浸润分析和数据集样本的聚类分型,解析各分型间的免疫细胞差异。通过机器学习模型的构建,预测目的基因与AS疾病间的关联性,进一步通过外部数据集构建风险预测列线图模型验证其临床诊断效能。通过对目的基因的临床相关性分析,评价其临床应用价值。最后,采用动物实验再次验证目的基因FXR、LXRa、CYP7A1是否为天香丹调节胆汁酸代谢治疗AS的关键分子靶点。结果:共获得“天香丹-AS-胆汁酸代谢”核心基因CYP7A1、CYP19A1、FXR、LXRa,通过基因富集和关键基因分析显示富集通路最多、排名前三的是CYP7A1、LXRa、FXR,并对其进行进一步的机器学习分析、外部数据集和动物实验双重验证。免疫浸润结果显示CYP7A1、LXRa、FXR与22种免疫细胞中的1个或多个显著相关。通过风险预测列线图模型验证FXR对AS患病风险较明显。其中CYP7A1、FXR的表达与AS患者年龄负相关。通过动物实验发现:与空白对照组比较,模型组小鼠主动脉管腔较大粥样斑块,管壁见大量脂质沉积,天香丹治疗后小鼠主动脉管腔粥样斑块及脂质沉积情况均出现不同程度的缓解;模型组小鼠主动脉FXR蛋白水平升高,LXRa,CYP7A1蛋白水平降低,天香丹治疗后小鼠主动脉组织中FXR蛋白水平显著降低,LXRa,CYP7A1蛋白水平显著升高。结论:FXR、LXRa、CYP7A1是与AS发病密切相关的胆汁酸代谢关键基因,天香丹可通过下调FXR,上调LXRa、CYP7A1的水平,调节胆汁酸代谢平衡,从而防治AS。

Objective:To screen the differential genes of bile acid metabolism related to atherosclerosis(AS)through bioinformatics and machine learning,and to verify whether the mechanism of Tianxiangdan in treating AS is related to regulating bile acid metabolism in animal experiments.Method:Differential expression genes(DEGs)of"Tianxiangdan-AS-bile acid metabolism"were obtained through bioinformatics and their correlation was analyzed.Further enrichment analysis and protein interaction network screening were used to screen target genes for further bioinformatics validation.The differences in immune cells between different subtypes were analyzed through immune infiltration analysis of target genes and clustering typing of dataset samples.By constructing machine learning models,we can predict the association between key genes and AS diseases,and further validate its clinical diagnostic efficacy by constructing a risk prediction column chart model using external datasets.The clinical application value of the target gene was evaluated through clinical correlation analysis.Finally,animal experiments were conducted to validate whether the target genes FXR,LXRa,and CYP7A1 are key molecular targets for Tianxiangdan in regulating bile acid metabolism and treating AS.Result:Core genes CYP7A1 CYP19A1,FXR,LXRa for"Tianxiangdan-AS-bile acid metabolism"were obtained,Through gene enrichment and key gene analysis,CYP7A1,LXRa,and FXR were found to have the most enriched pathways and ranked in the top three.Further machine learning analysis,external datasets,and animal experiments were conducted to verify these pathways.The immune infiltration results showed a significant correlation between CYP7A1,LXRa,FXR and one or more of the 22 types of immune cells.Verify through the risk prediction column chart model that FXR has a significant impact on the risk of AS.The expression of CYP7A1 and FXR is negatively correlated with the age of AS patients.Through animal experiments,it was found that compared with the blank control group,the model group mice had larger atherosclerotic plaque in the aortic lumen,and a large amount of lipid deposition was seen in the wall of the aorta.After the treatment of Tianxiangdan,the atherosclerotic plaque and lipid deposition in the aortic lumen of mice were alleviated to varying degrees;The FXR protein level in the aorta of the model group mice increased,while the LXRa and CYP7A1 protein levels decreased.After treatment with Tianxiangdan,the FXR protein level in the aortic tissue of mice significantly decreased,while the LXRa and CYP7A1 protein levels significantly increased.Conclusion:FXR,LXRa,and CYP7A1 are key genes closely related to bile acid metabolism in the pathogenesis of AS.Tianxiangdan can regulate bile acid metabolism balance by downregulating FXR and upregulating the levels of LXRa and CYP7A1,thereby preventing and treating AS.