Development of a novel anti-liver fibrosis formula with luteolin, licochalcone A, aloe-emodin and acacetin by network pharmacology and transcriptomics analysis.

CONTEXT: Xiaoyaosan decoction (XYS), a classical Traditional Chinese Medicine (TCM) formula is used to treat liver fibrosis in clinics. OBJECTIVE: This study explores defined compound combinations from XYS decoction to treat liver fibrosis. MATERIALS AND METHODS: Network pharmacology combined with transcriptomics analysis was used to analyze the XYS decoction and liver depression and spleen deficiency syndrome liver fibrosis. From the constructed XYS-Syndrome-liver fibrosis network, the top 10 active formulas were developed by topological analysis according to network stability. The most active formula was determined by in vitro study. The anti-fibrosis effect was evaluated by in vitro and in vivo studies. RESULTS: According to the network XYS-Syndrome-liver fibrosis network, 8 key compounds and 255 combinations were predicted from in XYS. Luteolin, licochalcone A, aloe-emodin and acacetin formula (LLAAF) had a synergistic effect on the proliferation inhibition of hepatic stellate cells compared to individual compounds alone. The treatment of XYS and LLAAF showed a similar anti-liver fibrotic effect that reduced histopathological changes of liver fibrosis, Hyp content and levels of α-SMA and collagen I in CCl4-induced liver fibrosis in rats. Transcriptomics analysis revealed LLAAF regulated PI3K-Akt, AMPK, FoxO, Jak-STAT3, P53, cell cycle, focal adhesion, and PPAR signalling. Furthermore, LLAAF was confirmed to regulate Jak-STAT and PI3K-Akt-FoxO signalling in vitro and in vivo. CONCLUSIONS: This study developed a novel anti-liver formula LLAAF from XYS, and demonstrated its anti-liver fibrotic activity which may be involved in the regulation of Jak-STAT and PI3K-Akt-FoxO signalling.

Xiaoyaosan decoction alleviated rat liver fibrosis via the TGFß/Smad and Akt/FoxO3 signaling pathways based on network pharmacology analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver fibrosis is an outcome of many chronic liver diseases and often results in cirrhosis, liver failure, and even hepatocarcinoma. Xiaoyaosan decoction (XYS) as a classical Traditional Chinese Medicine (TCM) formula is used to liver fibrosis in clinical practice while its mechanism is unclear. AIM OF THE STUDY: The aim of this study was to investigate the anti-fibrosis effect of XYS and to explore the molecular mechanisms by combining network pharmacology and transcriptomic technologies. MATERIALS AND METHODS: The carbon tetrachloride (CCl4)-induced liver fibrosis rat were treated with three doses of XYS. The liver fibrosis and function were evaluated by histopathological examination and serum biochemical detection. The fibrosis related protein a-SMA and collagen I were assessed by Western blot. Different expressed genes (DEGs) between XYS-treated group and model group were analyzed. The herb-component-target network was constructed combined the network pharmacology. The predict targets and pathways were validated by in vitro and in vivo experiments. RESULTS: With XYS treatment, the liver function was significantly improved, and fibrotic changes were alleviated. The a-SMA and collagen I expression levels in the liver were also decreased in XYS-treated rats compared with CCl4 model rats. 108 active components and 42 targets from 8 herbs constituted herb-compound-target network by transcriptomics and network pharmacology analysis. The KEGG pathway and GO enrichment analyses showed that the FoxO, TGFß, AMPK, MAPK, PPAR, and hepatitis B and C pathways were involved in the anti-fibrosis effects of XYS. In the liver tissues, p-FoxO3a and p-Akt expression levels were significantly increased in the CCl4 model group but decreased in the XYS-treated group. The TGFß1/Smad pathway and Akt/FoxO3 pathway were verified in LX2 cells by inhibiting phosphorylation of Smad3 and Akt activity, respectively. CONCLUSIONS: Our findings suggested that XYS markedly alleviated CCl4-induced liver fibrosis in histopathological and serum liver function analyses, and this effect may occur via the TGFß1/Smad and Akt/FoxO signaling pathways.

Yinchenhao decoction suppresses rat liver fibrosis involved in an apoptosis regulation mechanism based on network pharmacology and transcriptomic analysis.

Yinchenhao decoction (YCHD) is a classical Traditional Chinese Medicine (TCM) formula that has been widely used in the treatment of liver fibrosis caused by chronic hepatitis B and jaundice for more than 1800 years. The purpose of this study was to investigate the apoptosis regulation mechanisms of YCHD and its active components suppresses liver fibrosis. The active components and putative targets of YCHD were predicted by network pharmacology approach. Functional and pathway enrichment analysis were presented in the present study by using clusterProfiler. Further, experimental validation was done by using terminal deoxynucleotidyl transferase (TDT) dUTP nick end labelling (TUNEL) assay and western blotting in dimethylnitrosamine (DMN)-induced liver fibrosis rats, and cell proliferation assay, apoptosis assay, and western blotting in human hepatic L02 cells and LX2 cells. 45 active compounds in YCHD formula, 592 potential target proteins and 1191 liver fibrosis-related human genes were identified. Functional and pathway enrichment analysis indicated that YCHD obviously influenced TNF, PI3K-Akt signaling pathways. Further, In vivo experiment indicated that YCHD treatment not only attenuated the symptoms of liver fibrosis, but also decrease the apoptosis of hepatic parenchyma cells. Moreover, in vitro experiments showed that rhein, kaempferol and quercetin treatments remarkably decreased the protein levels of cleaved caspase-3 and increased p-ERK1/2, PI3K and Bcl-XL protein expression in TNF-α-stimulated L02 cells. On the contrary, rhein, kaempferol, aloe-emodin and quercetin inhibited the proliferation of LX2 cells and up-regulated the protein levels of Bax and cleaved caspase-8. In conclusion, 45 active components and 296 potential targets of YCHD against liver fibrosis were identified by the analysis of network pharmacology and transcriptomics combination. The mechanisms of YCHD against liver fibrosis were involved in the regulation of multiple targets, especially affecting the apoptosis-related signaling pathways.

Metabolic mechanisms of Fuzheng-Huayu formula against liver fibrosis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Fuzheng-Huayu formula (FZHY) is traditionally used to treat liver fibrosis in clinic. The study was conducted to investigate the metabolic mechanisms of FZHY against liver fibrosis in rats. MATERIALS AND METHODS: Rats with CCl4 -induced liver fibrosis were treated with FZHY and its components, including amygdalin, cordyceps polysaccharide and gypenoside, respecitively. Liver fibrosis and function were assesed by histopathological examination, Western blot and serum biochemical detection. Metabolic profiling of liver tissue, serum and urine in each group were detected by gas chromatography-mass spectrometry (GC-MS) and transcriptomic changes were tested by gene chip. RT-qPCR was used to validate levels of different expressed genes (DEGs) with statistical significance. Metabolic network together with DEGs was constructed based on KEGG database. RESULTS: FZHY effectively improved liver fibrosis better than the mixture or single use of gypenoside, cordyceps sinensis mycelia and amygdalin. FZHY treatment widely modulated the metabolic profiles perturbed by liver fibrosis, involving several important metabolic pathways, including glycolysis/gluconeogenesis, glucose-alanine cycle, citrate cycle, galactose metabolism, tryptophan metabolism, urea cycle, etc. It also increased alanine and decreased glucose levels in liver tissue and decreased both of them in serum and urine, which were dysregulated by CCl4 treatment. Additionally, FZHY also upregulated expression of metabolic enzymes including Hk2, Adh1 and Gpt increased, and downregulated Gs and Acss2. CONCLUSION: FZHY improved liver fibrosis in rats via altering the metabolic pathways and regulating gene expression of involved metabolic enzymes.

Network Pharmacology-Based Study on the Mechanism of Bushen-Jianpi Decoction in Liver Cancer Treatment.

To investigate the mechanism of a Bushen-Jianpi decoction (BSJPD) in liver cancer (LC) treatment, we analyzed clinical therapy data, conducted network pharmacology analysis, and performed pharmacological experimental verification in vitro and in vivo. The univariate analysis of clinical therapy showed that the BSJPD was protective factor (p < 0.05). The network pharmacology analysis showed that 9 compounds were important nodes of BSJPD-LC therapy network. In experimental verification, the rate of apoptosis increased in the liver tumors of mice treated with the BSJPD (p < 0.05); drug serum with 20 % BSJPD inhibited cell viability (p < 0.05) and reduced the expression of PI3K, the Bcl-xL/BAD ratio, and the levels of p53 and p-Akt in HepG2 cells. Moreover, licochalcone A, alisol B, and hederagenin inhibited cell viability (p < 0.05), induced cell apoptosis (p < 0.01), reduced p-Akt levels, and increased cleaved-CASP3 (p < 0.05) and p53 expression levels in HepG2 cells. These data suggest that the BSJPD prolongs the survival of LC patients and induces apoptosis and that it may be associated with the regulation of PI3K, Akt, p53, CASP3, and Bcl-xL/BAD expression.

Active Compounds Derived from Fuzheng Huayu Formula Protect Hepatic Parenchymal Cells from Apoptosis Based on Network Pharmacology and Transcriptomic Analysis.

Fuzheng huayu formula (FZHY), an antifibrotic traditional Chinese medicine, is frequently used for the treatment of liver fibrosis. In this study, network analysis, transcriptomic analysis, assays of cell apoptosis, viability and protein expression were used for investigating the effects and mechanisms of compounds derived from FZHY on hepatic parenchymal cell (HPC) protection and hepatic stellate cell activation. Network pharmacology analysis found that 6 major compounds and 39 potential targets were important network nodes. Our analysis predicted that the active compounds of FZHY, including hederagenin, luteolin and tanshinone IIA inhibited cell apoptosis (p < 0.05), increased PI3K expression and reduced cleaved caspase 3 expression and the Bax/Bcl-w ratio (p < 0.05) in L02 cells that had apoptosis induced by TNF-α. Few significant changes caused by FZHY, hederagenin, luteolin and tanshinone IIA were observed in hepatic stellate Lx2 cells upon TGF-ß1 induction. These data suggest that FZHY is active against liver fibrosis, protects hepatic parenchymal cells from apoptosis, and recovers liver function, possibly through the effects of its active compounds hederagenin, luteolin and tanshinone IIA and is involved in the inhibition of apoptosis in HPCs, possibly through regulating the PI3K, ERK, cleaved caspase 3 and Bax/Bcl-w levels.

Yinchenhao Decoction Alleviates Liver Fibrosis by Regulating Bile Acid Metabolism and TGF-ß/Smad/ERK Signalling Pathway.

Yinchenhao decoction (YCHD), comprising Yinchenhao (Artemisiae Scopariae Herba), Zhizi (Gardeniae Fructus) and Dahuang (Radix Rhei et Rhizoma), is widely used for treating various diseases. We aimed to investigate the bile acid metabolic mechanism of YCHD in dimethylnitrosamine (DMN)-induced liver fibrosis model. Rats received DMN (10 mg/kg, intraperitoneally) for four successive weeks for liver fibrosis induction and were treated with YCHD for the last 2 weeks. Histopathological analysis showed that YCHD prevented DMN-induced histopathological changes in liver tissues. Serum liver function in YCHD group improved. Ultraperformance liquid chromatography-mass spectrometry analysis showed that YCHD significantly restored both free and conjugated bile acid levels increased by DMN, to normal levels. RT-qPCR results showed that YCHD treatment upregulated the expression of genes related to bile acid synthesis, reabsorption, and excretion. Western blotting analysis showed that YCHD downregulated α-SMA, TGF-ß1, p-Smad3, and p-ERK1/2 expression in chenodeoxycholic acid (CDCA)-activated hepatic stellate cells (HSCs). The viability of CDCA-activated HSCs significantly increased after treatment with YCHD and PD98059 (an ERK inhibitor) compared to YCHD treatment alone. Our findings suggest that YCHD alleviated DMN-induced liver fibrosis by regulating enzymes responsible for bile acid metabolism. Additionally, it inhibits CDCA-induced HSC proliferation and activation via TGF-ß1/Smad/ERK signalling pathway.

Systems biology approaches in the study of Chinese herbal formulae.

Systems biology is an academic field that attempts to integrate different levels of information to understand how biological systems function. It is the study of the composition of all components of a biological system and their interactions under specific conditions. The core of systems biology is holistic and systematic research, which is different from the manner of thinking and research of all other branches of biology to date. Chinese herbal formulae (CHF) are the main form of Chinese medicine and are composed of single Chinese herbal medicines (CHMs) with pharmacological and pharmacodynamic compatibility. When single CHMs are combined into CHF, the result is different from the original effect of a single drug and can be better adapted to more diseases with complex symptoms. CHF represent a complex system with multiple components, targets and effects. Therefore, the use of systems biology is conducive to revealing the complex characteristics of CHF. With the rapid development of omics technologies, systems biology has been widely and increasingly applied to the study of the basis of the pharmacological substances, action targets and mechanisms of CHF. To meet the challenges of multiomics synthesis-intensive studies and system dynamics research in CHF, this paper reviews the common techniques of genomics, transcriptomics, proteomics, metabolomics, and metagenomics and their applications in research on CHF.

A metabolic mechanism analysis of Fuzheng-Huayu formula for improving liver cirrhosis with traditional Chinese medicine syndromes.

Fuzheng-Huayu formula (FZHY), a Chinese herbal mixture prescription, has been proven effective in treating liver fibrosis and cirrhosis in both clinical trials and animal experiments. In this study we assessed the metabolic mechanisms of traditional Chinese medicine (TCM) syndrome-based FZHY treatment in liver cirrhosis (LC). A total of 113 participants, including 50 healthy controls and 63 LC patients, were recruited. According to the diagnosis and differentiation of the TCM syndromes, the LC patients were classified into 5 TCM syndrome groups including the liver stagnation syndrome (LSS), spleen deficiency and damp overabundance syndrome (SDDOS), damp-heat accumulation syndrome (DHAS), liver-kidney Yin deficiency syndrome (LKYDS), and blood stagnation syndrome (BSS), and administered FZHY for 6 months. FZHY treatment significantly decreased serum levels of hyaluronic acid (HA), a biochemical marker for LC, as well as TCM syndrome scores (the TCM syndrome scores were decreased in all the groups with significant decreases in the LSS and LKYDS groups). Furthermore, FZHY treatment gradually shifted the metabolic profiles of LC patients from a pathologic state to a healthy state, especially in LC patients with LSS and LKYDS. Twenty-two differently altered metabolites (DAMs) were identified, including carbohydrates, amino acids, fatty acids, etc with 9 DAMs in LSS patients, 9 in LKYDS patients, and 4 in other patients. The metabolic pathways involved in the conversion of amino acids and the body's detoxification process were regulated first, followed by the pathways involved in the body's energy supply process. In conclusion, the evaluation of the effect of TCM syndrome-based FZHY treatment show that FZHY has a better effect on LKYDS and LSS than on the other TCM syndromes, and the metabolic mechanisms might be involved in the increased detoxification function in LKYDS and the improvement of energy supply in LSS, which provides important evidence for the clinical application of TCM syndrome-based treatment.

Chinese herbal formula Fuzheng Huayu alleviates CCl4-induced liver fibrosis in rats: a transcriptomic and proteomic analysis.

Liver fibrosis is a consequence of chronic liver disease that can progress to liver cirrhosis or even hepatocarcinoma. Fuzheng Huayu (FZHY), a Chinese herbal formula, has been shown to exert anti-fibrotic effects. To better understand the molecular mechanisms underlying the anti-fibrotic effects of FZHY, we analyzed transcriptomic and proteomic combination profiles in CCl4-induced liver fibrosis in rats, which were treated with extracted FZHY powder (0.35 g·kg-1·d-1, ig) for 3 weeks. We showed that FZHY administration significantly improved liver function, alleviated hepatic inflammatory and fibrotic changes, and decreased the hydroxyproline content in the livers of CCl4-treated rats. When their liver tissues were examined using microarray and iTRAQ, we found 255 differentially expressed genes (fold change ≥1.5, P<0.05) and 499 differentially expressed proteins (fold change ≥1.2, P<0.05) in the FZHY and model groups. Functional annotation with DAVID (The Database for Annotation, Visualization and Integrated Discovery) showed that 15 enriched gene ontology terms, including drug metabolic process, response to extracellular stimulus, response to vitamins, arachidonic acid metabolic process, response to wounding, and oxidation reduction might be involved in the anti-fibrotic effects of FZHY; whereas KEGG pathway analysis revealed that eight enriched pathways, including arachidonic acid metabolism, retinol metabolism, metabolism of xenobiotics by cytochrome P450, and drug metabolism might also be involved. Moreover, the protein-protein interaction network demonstrated that 10 core genes/proteins overlapped, with Ugt2a3, Cyp2b1 and Cyp3a18 in retinol metabolism pathway overlapped to a higher degree. Compared to the model rats, the livers of FZHY-treated rats had significantly higher mRNA and protein expression levels of Ugt2a3, Cyp2b1 and Cyp3a18. Furthermore, the concentration of retinoic acid was significantly higher in the FZHY-treated rats compared with the model rats. The results suggest that the anti-fibrotic effects of FZHY emerge through multiple targets, multiple functions, and multiple pathways, including FZHY-regulated retinol metabolism, xenobiotic metabolism by cytochrome P450, and drug metabolism through up-regulated Ugt2a3, Cyp2b1, and Cyp3a18. These genes may play important anti-fibrotic roles in FZHY-treated rats.