TMT-based proteomics analysis identifies the interventional mechanisms of Qijia Rougan decoction in improving hepatic fibrosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Qijia Rougan decoction (QJ), consisting of eight herbs and two animal drugs, is an effective traditional Chinese medicine with hepatoprotective and antifibrotic effects. However, its underlying action mechanism remains unclear. AIM OF THE STUDY: To explore the mechanism underlying the treatment of liver fibrosis in rats by QJ. MATERIALS AND METHODS: Rats with fibrosis were constructed using carbon tetrachloride (CCl4). The QJ was orally administered to fibrotic rats. Hepatic pathological changes were evaluated using hematoxylin and eosin and Masson's trichrome staining. The differentially expressed proteins (DEPs) in QJ were analyzed using quantitative proteomics. Subsequently, the underlying mechanisms in liver fibrosis after QJ treatment were validated using Western blotting. RESULTS: The QJ markedly improved liver function and attenuated fibrotic progression. Based on the tandem mass-tag based (TMT) proteomics, we identified 818 common DEPs between QJ vs Model and Model vs Control, including 296 upregulated and 522 downregulated DEPs, which mostly participate in metabolic pathways, oxidation-reduction reactions, and collagen biosynthetic processes. In addition, we found that QJ reduced hepatocellular death by inhibiting the expression of caspase proteins, repressing pro-apoptotic proteins, and promoting anti-apoptotic proteins. We further demonstrated that QJ suppressed the Akt/mTOR pathway. CONCLUSION: QJ exerted hepatoprotective effects in CCl4-induced rats through multi-pathway regulation. This study provides protein information on liver fibrosis treated with QJ.

Qijia rougan formula ameliorates ECM deposition in hepatic fibrosis by regulating the JAK1/STAT6-microRNA-23a feedback loop in macrophage M2 polarization.

Hepatic fibrosis is the critical pathological stage in the progression of chronic liver disease to cirrhosis and hepatocellular carcinoma (HCC). However, no approved anti-hepatic fibrosis drugs are available currently. Qijia Rougan Formula (QRF) is a traditional Chinese medicine (TCM) with significant clinical efficacy on hepatic fibrosis. It was derived from Sanjiasan, a famous decoction documented in the Book of Treatise on the Pestilence in the Ming Dynasty of China. However, the underlying regulatory mechanisms remain elusive. This study further confirmed the therapeutic effects of QRF on hepatic fibrosis and dissected its underlying molecular mechanisms from the perspective of macrophage M2 polarization, one of the critical events in hepatic fibrosis. Experimentally, QRF significantly improved extracellular matrix (ECM) deposition and fibrosis in the liver of model rats. QRF diminished the proportion of M2 macrophages, decreased the levels of TGF-ß, PDGFB and IL-10, and regulated the expression of p-JAK1, p-STAT6, JAK1 and microRNA-23a both in vitro and in vivo. Collectively, it was confirmed that QRF effectively improves liver function and hepatocyte damage, and reduces ECM deposition. QRF ameliorates hepatic fibrosis by regulating JAK1/STAT6-microRNA-23a negative feedback loop to inhibit macrophage M2 polarization and thus reduce ECM deposition. Our study illustrates the potential of QRF for hepatic fibrosis therapy, suggesting that QRF is a promising anti-hepatic fibrosis drug candidate.

Proteomic investigation and biomarker identification of lung and spleen deficiency syndrome in HIV/AIDS immunological nonresponders.

Background: Human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) immunological nonresponders (HIV/AIDS-INRs) whose CD4+ cell counts do not rebound after highly active antiretroviral therapy (HAART) treatment usually experience severely impaired immune function and high mortality. Traditional Chinese medicine (TCM) has many advantages in the field of AIDS, especially its promotion of patients' immune reconstitution. Accurate differentiation of TCM syndromes is a prerequisite for guiding an effective TCM prescription. However, the objective and biological evidence for identification of the TCM syndromes in HIV/AIDS-INRs remains lacking. Lung and spleen deficiency (LSD) syndrome, a typical HIV/AIDS-INR syndrome, was examined on in this study. Methods: We first performed a proteomic study of LSD syndrome in INRs (INRs-LSD) using tandem mass tag combined with liquid chromatography-tandem mass spectrometry (TMT-LC-MS/MS) and screened them against the healthy and undocumented identifiable groups. The TCM syndrome-specific proteins were subsequently validated based on bioinformatics analysis and enzyme-linked immunosorbent assay (ELISA). Results: A total of 22 differentially expressed proteins (DEPs) were screened in INRs-LSD compared to the healthy group. Based on bioinformatic analysis, these DEPs were found to be mainly associated with the immunoglobin A (IgA)-generated intestinal immune network. In addition, we examined the TCM syndrome-specific proteins alpha-2-macroglobulin (A2M) and human selectin L (SELL) with ELISA and found that they were both upregulated, which was consistent with the proteomic screening results. Conclusions: A2M and SELL were finally identified as potential biomarkers for INRs-LSD, providing a scientific and biological basis for identifying typical TCM syndromes in HIV/AIDS-INRs and an opportunity to build a more effective TCM treatment system for HIV/AIDS-INRs.

Persistent Lipid Accumulation Leads to Persistent Exacerbation of Endoplasmic Reticulum Stress and Inflammation in Progressive NASH via the IRE1α/TRAF2 Complex.

Non-alcoholic steatohepatitis (NASH) is a metabolic disorder that often leads to other severe liver diseases, yet treatment options are limited. Endoplasmic reticulum (ER) stress is an important pathogenetic mechanism of NASH and plays a key role in tandem steatosis as well as liver inflammation. This study aims to develop a progressive NASH model through sustained lipid accumulation and to elucidate its molecular mechanism through IRE1α/TRAF2 complex. Male SD rats were fed a high-fat diet (HFD) for 4, 8, and 12 weeks to induce progressive NASH. MRNA sequencing and PPI analysis were used to screen core genes. Transmission electron microscopy, immunofluorescence staining, ELISA, qRT-PCR, and Western blotting were used at each time point to compare differences between each index of progressive NASH at 4, 8, and 12 weeks. Sustained lipid accumulation led to structural disruption of the ER, a reduction in ER number, and an increase of lipid droplet aggregation in hepatocytes. Persistent lipid accumulation led to a persistent increase in mRNA and protein expression of the IRE1α/TRAF2 complex, IKK/IκB/NF-κB signaling pathway and ASK1/JNK1 signaling pathway, and TNF-α, IL-1ß, and IL-6 also continued to increase. Persistent lipid accumulation led to a persistent exacerbation of ER stress and inflammation in progressive NASH via the IRE1α/TRAF2 complex.

Epidemiological investigation and proteomic profiling of typical TCM syndrome in HIV/AIDS immunological nonresponders.

HIV/AIDS pandemic remains the world's most severe public health challenge, especially for HIV/AIDS immunological nonresponders (HIV/AIDS-INRs), who tend to have higher mortality. Due to the advantages in promoting patients' immune reconstitution, Traditional Chinese medicine (TCM) has become one of the mainstays of complementary treatments for HIV/AIDS-INRs. Given that effective TCM treatments largely depend on precise syndrome differentiation, there is an increasing interest in exploring biological evidence for the classification of TCM syndromes in HIV/AIDS-INRs. In our study, to identify the typical HIV/AIDS-INRs syndrome, an epidemiological survey was first conducted in the Liangshan prefecture (China), a high HIV/AIDS prevalence region. The key TCM syndrome, Yang deficiency of spleen and kidney (YDSK), was evaluated by using a tandem mass tag combined with liquid chromatography-tandem mass spectrometry (TMT-LC-MS/MS). A total of 62 differentially expressed proteins (DEPs) of YDSK syndrome compared with healthy people were screened out. Comparative bioinformatics analyses showed that DEPs in YDSK syndrome were mainly associated with response to wounding and acute inflammatory response in the biological process. The pathway annotation is mainly enriched in complement and coagulation cascades. Finally, the YDSK syndrome-specific DEPs such as HP and S100A9 were verified by ELISA, and confirmed as potential biomarkers for YDSK syndrome. Our study may lay the biological and scientific basis for the specificity of TCM syndromes in HIV/AIDs-INRs, and may provide more opportunities for the deep understanding of TCM syndromes and the developing more effective and stable TCM treatment for HIV/AIDS-INRs.

Sorafenib Attenuates Fibrotic Hepatic Injury Through Mediating Lysine Crotonylation.

Background: Liver fibrosis is an independent contributor of chronic liver diseases, and regressing liver fibrosis is considered a potential therapeutic target for chronic liver diseases. We aimed to explore the effects and mechanism of sorafenib in liver fibrosis. Methods: Male Sprague Dawley (SD) rats were subjected to subcutaneous injection of carbon tetrachloride (CCl4) for 8 weeks to induce liver fibrosis and then treated with sorafenib. The degree of liver fibrosis was analyzed by hematoxylin-eosin (H&E) staining, Masson staining, and Picrosirius red (PSR) staining. Serum biochemical indexes were detected by fully automatic biochemical analyzer or enzyme-linked immunosorbent assay (ELISA). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of pro-fibrotic genes. Immunohistochemical staining and Western blotting were carried out to evaluate the levels of lysine crotonylation. Results: Liver index was reduced with oral sorafenib in CCl4-induced rats. Serum liver function (alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL)) and fibrosis indicators (type III procollagen (PC-III), hyaluronic acid (HA), and laminin (LN)) were attenuated with sorafenib treatment. Sorafenib improved the hepatic structure and fibrotic progression. The expression of fibrosis-related genes was remarkely reduced with sorafenib treatment. Meanwhile, sorafenib inhibited α-SMA and collagen I cumulation induced by CCl4 injection. Besides, protein lysine crotonylation especially the crotonylated H2BK12 (H2BK12cr) and crotonylated H3K18 (H3K18cr) were reversed by sorafenib, which were decreased in response to CCl4 treatment. Spearman correlation analysis shown lysine crotonylation expression was negatively correlated with serum fibrotic indicators. Conversely, crotonylation-regulated enzymes, which negatively regulate protein crotonylation, were increased in response to CCl4 treatment, while sorafenib reduced their expression. Conclusion: Sorafenib exerts significant anti-fibrotic effects through mediating crotonylation-regulated enzymes and protein crotonylation in fibrotic rats.

Active ingredients and molecular targets of Taraxacum mongolicum against hepatocellular carcinoma: network pharmacology, molecular docking, and molecular dynamics simulation analysis.

Background: Taraxacum mongolicum (TM) is a widely used herb. Studies have reported that TM exhibits growth-inhibitory and apoptosis-inducing on multiple tumors, including hepatocellular carcinoma (HCC). The active ingredients, targets, and molecular mechanisms of TM against HCC need to be further elucidated. Methods: We identified the active ingredients and targets of TM via HERB, PubChem, SwissADME, SwissTargetPrediction, and PharmMapper. We searched HCC targets from GeneCards, Comparative Toxicogenomics Database (CTD), and DisGeNET. Then, the intersection of drug targets and disease targets was uploaded to the STRING database to construct protein-protein interactions (PPI) networking whose topology parameters were analyzed in Cytoscape software to screen hub targets. Next, we used Metascape for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and we employed AutoDock vina, AMBER18 and PyMOL software along with several auxiliary tools for molecular docking and molecular dynamics (MD) simulation. Finally, based on the in silico findings, cellular experiments were conducted to investigate the effect of TM on HSP90AA1 gene expression. Results: A total of 228 targets and 35 active ingredients were identified. Twenty two hub targets were selected through PPI networking construction for further investigation. The enrichment analysis showed that protein kinase binding, mitogenactivated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways were mainly involved. Molecular docking and MD simulation results supported good interaction between HSP90 protein and Austricin/Quercetin. The in vitro assay showed that TM inhibited the proliferation of HepG2 cells and the expression of HSP90AA1 gene. Conclusions: This study is the first to use network pharmacology, molecular docking, MD simulation and cellular experiments to elucidate the active ingredients, molecular targets, and key biological pathways responsible for TM anti-HCC, providing a theoretical basis for further research.

Hepatoprotective Efficacy and Interventional Mechanism of Qijia Rougan Decoction in Liver Fibrosis.

Liver fibrosis is a leading contributor to chronic liver diseases such as cirrhosis and liver cancer, which pose a serious health threat worldwide, and there are no effective drugs to treat it. Qijia Rougan decoction was modified from Sanjiasan, a traditional Chinese medicine (TCM) described in the "Wenyilun" manuscript. Qijia Rougan decoction possesses hepatoprotective and antifibrotic effects for clinical applications. However, its underlying mechanisms remain largely unknown. In this study, fibrotic rats induced by carbon tetrachloride (CCl4) were treated with two doses of Qijia Rougan decoction. Histopathological and serum biochemical analyses were carried out to assess liver structure and function, respectively. High-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) was performed to identify bioactive compositions in Qijia Rougan decoction. Transcriptome analysis using mRNA-sequencing (mRNA-Seq) was used to explore the underlying mechanisms and verified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Qijia Rougan decoction significantly attenuated CCl4-induced hepatic fibrotic injury, supported by promoted liver function and improved liver fibrosis. Eight main representative components originating from raw materials in the Qijia Rougan decoction were found to possess an antifibrotic role. Mechanistically, Qijia Rougan decoction regulated biological processes such as oxidation-reduction, fatty acid metabolism, cell adhesion, and transforming growth factor beta (TGFß) signaling. We determined that Qijia Rougan decoction reversed the expression of inflammatory cytokines and inhibited the activation of fibrosis-related TGFß signaling. It also reversed the deterioration of liver structure and function in rats induced by CCl4. Overall, Qijia Rougan decoction significantly mediated metabolism-associated processes, inhibited inflammatory reactions, and repressed fibrosis-related TGFß signaling, which prevented liver fibrosis deterioration. Our study deepens our understanding of TCM in the diagnosis and treatment of liver fibrosis.