基于转录组学分析不同肝损伤诱导因素对肝星状细胞的影响

Transcriptomics-based analysis of the effects of different liver injury-inducing factors on hepatic stellate cells

关键词:  肝损伤  肝纤维化  炎症  肝星状细胞  转录组学

Key words:  Liver injury  liver fibrosis  inflammation  hepatic stellate cells  transcriptomics

摘要:

目的 探讨不同的肝纤维化刺激因子包括转化生长因子β1（transforming growth factor-β1, TGF- β1）、药物损伤诱导剂对乙酰氨基酚（acetaminophen, APAP）和脂多糖（lipopolysaccharide, LPS）对肝星状细胞（hepatic stellate cells, HSCs）的影响及潜在作用机制。方法 实验分为未做任何处理的对照组LX-2细胞和实验组TGF- β1组（50ng/mL）、APAP组（20mM）和LPS组（100ng/mL），分别处理LX-2细胞24 h，收集各组细胞组织样本。利用转录组测序从HSCs的增殖、凋亡、炎症、纤维化等生物标志物的表达情况进行分析。RT-PCR检测平滑肌激动蛋白（actin alpha 2, ACTA2）、Ⅰ型胶原（recombinant collagen type I alpha 1, COL1A1）和粘连蛋白（fibronectin, FN1）的表达。采用功能富集分析和蛋白互作网络探讨潜在作用机制。数据统计分析多组间比较采用单因素方差分析（One-way ANOVY），两两比较采用LSD-t检验。结果 TGF- β1可以有效地启动HSCs的活化过程，表达经典的纤维化标记基因ACTA2、COL1A1和FN1；LPS可以激活ACTA2的表达，但是COL1A1和FN1这些肌成纤维细胞(myofibroblast, MFB)的经典细胞外基质(extracellular matrixc, ECM)沉积相关基因反而是下降；APAP不能引起ACTA2和COL1A1表达的激活，只能影响FN1表达的增加(P值均＜0.001)。通过RT-PCR验证了测序数据的可靠性(P值均＜0.05)。通过功能富集分析和蛋白互作网络，发现TGF- β1和LPS可能通过与其密切相关的PI3K-Akt信号通路、细胞因子-细胞因子受体相互作用信号通路参与炎症和肝纤维化的调控，同时，TGF- β1还可能通过ECM受体相互作用和糖尿病并发症中的AGE-RAGE信号通路参与纤维化和糖代谢的过程，LPS可能通过肿瘤坏死因子（tumor necrosis factor, TNF）信号通路、补体和凝血级联信号通路参与炎症反应的发生。而APAP则主要可能通过调控细胞的衰老、凋亡、癌症中的蛋白聚糖以及MAPK信号通路参与细胞增殖凋亡等代谢过程。其中ITGB3、ITGAV、FN1、ITGA2、COL1A2、MMP2、FOS、THBS1和EGF可以作为TGF-β1刺激的关键调控基因；PIK3R1、FOS、CDC42、CASP3、FOXO1和ACTB可以作为APAP刺激的关键调控基因；ITGAM、CXCL8、CCL2、ITGAV、DDX58、IFI44L、IFIH1、IFIT3、IFIT2、OAS2、MMP9和XAF1可以作为LPS刺激的关键调控基因，以上可能是肝纤维化不同刺激条件的关键调控基因。结论 TGF- β1和LPS会引发一定程度的炎症反应，其中LPS炎症现象更为明显，TGF- β1会伴随肝纤维化发生，而LPS则可能引起纤维化胶原降解，APAP可能导致HSCs的凋亡和坏死。基于不同刺激因子的模型选择对于疾病的机制研究、靶点发现和药物研发具有重要作用。

Abstract:

OBJECTIVE To investigate the effects of different liver fibrosis stimulating factors including transforming growth factor β 1 (transforming growth factor-β1, TGF- β1), drug injury inducer acetaminophen (acetaminophen, APAP) and lipopolysaccharide (lipopolysaccharide, LPS) on hepatic stellate cells (hepatic stellate cells, HSCs) and their potential mechanisms of action. METHODS The experiment was divided into control LX-2 cells without any treatment and experimental TGF- β1 group (50 ng/mL), APAP group (20 mM) and LPS group (100 ng/mL), and LX-2 cells were treated for 24 h. Tissue samples were collected from each group. The expression of biomarkers from HSCs such as proliferation, apoptosis, inflammation and fibrosis were analyzed using transcriptome sequencing. RT-PCR was performed to detect the expression of smooth muscle agonist protein (actin alpha 2, ACTA2), type I collagen (recombinant collagen type I alpha 1, COL1A1) and adhesion protein (fibronectin, FN1). Functional enrichment analysis and protein interaction networks were used to explore potential mechanisms of action. One-way ANOVY was used for comparison between multiple groups for measurement data, and LSD-t test was used for two-way comparison. RESULTS TGF- β1 could effectively initiate the activation process of HSCs to express the classical fibrosis marker genes ACTA2, COL1A1 and FN1; LPS could activate the expression of ACTA2, but COL1A1 and FN1, the classical extracellular matrix (extracellular matrixc, ECM) deposition-related genes of myofibroblasts (myofibroblast, MFB), were instead decreased; APAP could not cause the activation of ACTA2 and COL1A1 expression, but only affected the increase of FN1 expression(P＜0.001). The reliability of the sequencing data was verified by RT-PCR(P＜0.05). Functional enrichment analysis and protein interaction network revealed that TGF- β1 and LPS may be involved in the regulation of inflammation and liver fibrosis through their closely related PI3K-Akt signaling pathway, cytokine-cytokine receptor interaction signaling pathway, while TGF-β1 may also be involved in the regulation of liver fibrosis through ECM receptor interaction and AGE-RAGE signaling pathway in diabetic complications are involved in the process of fibrosis and glucose metabolism, and LPS may be involved in the development of inflammatory responses through tumor necrosis factor (tumor necrosis factor, TNF) signaling pathway, complement and coagulation cascade signaling pathways. In contrast, APAP may be involved in metabolic processes such as cell proliferation and apoptosis mainly through regulation of cell senescence, apoptosis, proteoglycans in cancer and MAPK signaling pathway. Among them, ITGB3, ITGAV, FN1, ITGA2, COL1A2, MMP2, FOS, THBS1 and EGF could be the key regulatory genes for TGF-β1 stimulation; PIK3R1, FOS, CDC42, CASP3, FOXO1 and ACTB could be the key regulatory ITGAM, CXCL8, CCL2, ITGAV, DDX58, IFI44L, IFIH1, IFIT3, IFIT2, OAS2, MMP9 and XAF1 could be key regulatory genes for LPS stimulation, and the above could be key regulatory genes for different stimulation conditions of liver fibrosis. CONCLUSION TGF- β1 and LPS trigger a certain degree of inflammatory response, with LPS inflammatory phenomenon being more pronounced, TGF- β1 accompanies the occurrence of liver fibrosis, while LPS may cause fibrotic collagen degradation and APAP may lead to apoptosis and necrosis of HSCs. The selection of models based on different stimuli plays an important role in the study of disease mechanisms, target discovery and drug development.