An Integration of RNA Sequencing and Network Pharmacology Approaches Predicts the Molecular Mechanisms of the Huo-Xue-Shen Formula in the Treatment of Liver Fibrosis

<b>Background</b>: Liver fibrosis is a prevalent, chronic inflammatory condition characterized by the excessive accumulation of extracellular matrix components and, primarily, collagen in the liver. Huo-xue-shen (HXS) has proven effective for the treatment of liver fibrosis. However, the mechanism is yet to be deciphered. <b>Methods</b>: Network pharmacology, machine learning algorithms and RNA-seq were used to predict the immune-treated targets and mechanisms associated with HXS in liver fibrosis. Molecular docking was employed to screen for effective agents based on the drug-compound-hub gene network in HXS, aiming to identify the most critical bioactive compound in HXS for the treatment of liver fibrosis. <b>Results</b>: A total of 100 immune-treated targets (ITTs) of HXS were found to significantly regulate the PI3K-Akt signaling pathway and the MAPK signaling pathway. Among these, CDKN1A, NR1I3, and TUBB1, which can concurrently interact with quercetin, were associated with the prognosis of liver fibrosis, indicating that HXS may inhibit or reverse HSC activation primarily by suppressing neutrophil extracellular trap formation, stimulating oxidative phosphorylation and promoting thyroid hormone synthesis in the regulation of the liver microenvironment. <b>Conclusions</b>: Our study suggests that HXS may delay the progression of liver fibrosis by targeting multiple pathways, as shown by the network pharmacology and transcriptome profiling used to examine the liver immune environment. Quercetin, its key ingredient, likely plays an important role by mediating the CDKN1A, NR1I3, and TUBB1 signaling pathways. Overall, our findings provide a new perspective on the potential biological mechanisms of this traditional Chinese medicine formula.