Mechanisms of Ganweikang Tablets against Chronic Hepatitis B: A Comprehensive Study of Network Analysis, Molecular Docking, and Chemical Profiling.

The hepatitis B virus (HBV) is one of the major viral infection problems worldwide in public health. The exclusive proprietary Chinese medicine Ganweikang (GWK) tablet has been marketed for years in the treatment of chronic hepatitis B (CHB). However, the pharmacodynamic material basis and underlying mechanism of GWK are not completely clear. This study is aimed at investigating the pharmacological mechanism of the GWK tablet in the treatment of CHB. The chemical ingredient information was obtained from the Traditional Chinese Medicine Database and Analysis Platform (TCMSP), Traditional Chinese Medicines Integrated Database (TCMID), and Shanghai Institute of Organic Chemistry of CAS. Ingredients and disease-related targets were defined by a combination of differentially expressed genes from CHB transcriptome data and open-source databases. Target-pathway-target (TPT) network analysis, molecular docking, and chemical composition analysis were adopted to further verify the key targets and corresponding active ingredients of GWK. Eight herbs of GWK were correlated to 330 compounds with positive oral bioavailability, and 199 correlated targets were identified. The TPT network was constructed based on the 146 enriched targets by KEGG pathway analysis, significantly associated with 95 pathways. Twenty-five nonvolatile components and 25 volatile components in GWK were identified in UPLC-QTOF/MS and GC-MS chromatograms. The key active ingredients of GWK include ferulic acid, oleanolic acid, ursolic acid, tormentic acid, 11-deoxyglycyrrhetic acid, dibenzoyl methane, anisaldehyde, wogonin, protocatechuic acid, psoralen, caffeate, dimethylcaffeic acid, vanillin, ß-amyrenyl acetate, formonentin, aristololactam IIIa, and 7-methoxy-2-methyl isoflavone, associated with targets CA2, NFKB1, RELA, AKT1, JUN, CA1, CA6, IKBKG, FOS, EP300, CREB1, STAT1, MMP9, CDK2, ABCB1, and ABCG2.

DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B.

OBJECTIVE: To explore the molecular bases of Chinese medicine (CM) syndrome classification in chronic hepatitis B (CHB) patients in terms of DNA methylation, transcription and cytokines. METHODS: Genome-wide DNA methylation and 48 serum cytokines were detected in CHB patients (DNA methylation: 15 cases; serum cytokines: 62 cases) with different CM syndromes, including dampness and heat of Gan (Liver) and gallbladder (CHB1, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan stagnation and Pi (Spleen) deficiency (CHB2, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan and Shen (Kidney) yin deficiency (CHB3, DNA methylation: 5 cases, serum cytokines: 16 cases), CHB with hidden symptoms (HS, serum cytokines:16 cases) and healthy controls (DNA methylation: 6 cases). DNA methylation of a critical gene was further validated and its mRNA expression was detected on enlarged samples. Genome-wide DNA methylation was detected using Human Methylation 450K Assay and furthered verified using pyrosequencing. Cytokines and mRNA expression of gene were evaluated using multiplex biometric enzyme-linked immunosorbent assay (ELISA)-based immunoassay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively. RESULTS: Totally 28,667 loci, covering 18,403 genes were differently methylated among CHB1, CHB2 and CHB3 (P<0.05 and |Δß value| > 0.17). Further validation showed that compared with HS, the hg19 CHR6: 29691140 and its closely surrounded 2 CpG loci were demethylated and its mRNA expressions were significantly up-regulated in CHB1 (P<0.05). However, they remained unaltered in CHB2 (P>0.05). Levels of Interleukin (IL)-12 were higher in CHB3 and HS than that in CHB1 and CHB2 groups (P<0.05). Levels of macrophage inflammatory protein (MIP)-1α and MIP-1ß were higher in CHB3 than other groups and leukemia inhibitory factor level was higher in CHB1 and HS than CHB2 and CHB3 groups (P<0.05). IL-12, MIP-1α and MIP-1ß concentrations were positively correlated with human leukocyte antigen F (HLA-F) mRNA expression (R2=0.238, P<0.05; R2=0.224, P<0.05; R=0.447, P<0.01; respectively). Furthermore, combination of HLA-F mRNA and differential cytokines greatly improved the differentiating accuracy among CHB1, CHB2 and HS. CONCLUSIONS: Demethylation of CpG loci in 5' UTR of HLA-F may up-regulate its mRNA expression and HLA-F expression was associated with IL-12, MIP-1α and MIP-1ß levels, indicating that HLA-F and the differential cytokines might jointly involve in the classification of CM syndromes in CHB. REGISTRATION NO: ChiCTR-RCS-13004001.

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.

Glycyrrhetinic Acid-Induced MiR-663a Alleviates Hepatic Stellate Cell Activation by Attenuating the TGF-ß/Smad Signaling Pathway.

Glycyrrhetinic acid (GA), a hydrolysate of glycyrrhizic acid from licorice root extract, has been used to treat liver fibrotic diseases. However, the molecular mechanism involved in the antifibrotic effects of GA remains unclear. The involvement of miR-663a and its roles in TGF-ß-1-induced hepatic stellate cell (HSC) activation remains unclear. In this study, we investigated the roles of miR-663a in the activation of HSCs and the antifibrosis mechanism of GA. MiR-663a expression was downregulated in TGF-ß-treated HSCs. The overexpression of miR-663a inhibited HSC proliferation. TGF-ß-1was confirmed as a direct target gene of miR-663a. MiR-663a alleviated HSC activation, concomitant with decreased expression of α-smooth muscle actin (α-SMA), human α2 (I) collagen (COL1A2), TGF-ß1, TGF-ßRI, Smad4, p-Smad2, and p-Smad3. GA upregulated miR-663a expression and inhibited the TGF-ß/Smad pathway in HSCs. Further studies showed that miR-663a inhibitor treatment reversed GA-mediated downregulation of TGF-ß1, TGF-ßRI, Smad4, p-Smad2, p-Smad3, α-SMA, and CoL1A2 in TGF-ß1-treated HSCs. These results show that miR-663a suppresses HSC proliferation and activation and the TGF-ß/Smad signaling pathway, highlighting that miR-663a can be utilized as a therapeutic target for hepatic fibrosis. GA inhibits, at least in part, HSC proliferation and activation via targeting the miR-663a/TGF-ß/Smad signaling pathway.

A network pharmacology approach to investigating the mechanism of Tanshinone IIA for the treatment of liver fibrosis.

ETHNOPHARMACOLOGICAL REVELVANCE: Tanshinone IIA (TIIA) is a major component extracted from the traditional herbal medicine salvia miltiorrhiza (Danshen), which activates blood circulation and treats chronic hepatitis and liver fibrosis. However, the underlying molecular mechanism of TIIA against hepatic fibrosis is still largely unknown. AIM OF THE STUDY: The present study aimed to evaluate the antifibrotic effects of TIIA in liver fibrosis and investigate its underlying mechanism through network pharmacology-based prediction and experimental verification. MATERIALS AND METHODS: In this study, a "TIIA-targets-liver fibrosis" network was constructed by combining the TIIA-specific and hepatic fibrosis-specific targets with protein-protein interactions (PPIS), and network pharmacology was applied to identify the potential targets and mechanisms of TIIA in the treatment of hepatic fibrosis. The antifibrotic effect of TIIA was investigated in CCl4-induced liver fibrosis in rats in vivo and in the human HSC line LX2 in vitro. RESULTS: We identified 75 potential targets of TIIA and 1382 targets of liver fibrosis. Subsequently, the 29 target proteins that overlapped between the potential TIIA targets and the liver fibrosis targets indicated that TIIA has potential antifibrotic effects through regulating multiple targets, including c-Jun, c-Myc, CCND1, MMP9 and P65. Pathway and functional enrichment analysis of these putative targets showed that TIIA could regulate the MAPK, PI3K/Akt and Wnt signaling pathways. Consistently, in vivo and in vitro experiments indicated that TIIA attenuated CCl4-induced liver injury and fibrosis and inhibited hepatic stellate cell (HSC) proliferation and activation; these findings were concomitant with the decreased expression of α-smooth muscle actin (α-SMA) and human α2 (I) collagen (COL1A2). Moreover, TIIA remarkably downregulated the expression of c-Jun, c-Myc, MMP9, PI3K and P38 proteins, which were upregulated in CCl4-induced hepatic fibrosis in vivo. TIIA significantly downregulated the expression of c-Jun, p-c-Jun, c-Myc, CCND1, MMP9, P65, P-P65, PI3K and P38 proteins, which were upregulated during HSC activation in vitro. CONCLUSION: Our study demonstrated that TIIA could significantly improve liver function, decrease liver injury, alleviate ECM accumulation, and attenuate HSC proliferation and activation, thus exerting an antifibrotic effect. The possible molecular mechanism involved MAPK, Wnt and PI3K/Akt signaling pathways via inhibiting c-Jun, p-c-Jun, c-Myc, CCND1, MMP9, P65, P-P65, PI3K and P38. Overall, our results suggest that TIIA could alleviate liver fibrosis through multiple targets and multiple signaling pathways and provide deep insight into the pharmacological mechanisms of TIIA in the treatment of hepatic fibrosis.

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.

Preventive and therapeutic role of Tanshinone â ¡A in hepatology.

Liver diseases cause serious public health problems because of their high global prevalence and poor long-term clinical outcomes. With its mild and broad therapeutic efficacy, traditional Chinese medicine (TCM) has played an important role in the prevention and treatment of liver diseases in China for hundreds of years. Tanshinone IIA (TIIA), a major component extracted from the traditional herbal medicine Salvia miltiorrhiza Bunge, has been broadly studied for its multiple biological activities, including antiangiogenic, antioxidant, anti-inflammatory and anticancer properties. In this article, we focus on the effects of TIIA on the development of liver diseases, including hepatic injury, fatty liver, hepatic fibrosis and hepatocellular carcinoma (HCC), as well as the mechanism underlying TIIA and its new formulations and carriers in order to provide a reference for its further study and clinical therapeutic use in liver diseases.

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.

Suppression of lung cancer progression by isoliquiritigenin through its metabolite 2, 4, 2', 4'-Tetrahydroxychalcone.

BACKGROUND: Licorice is an herb extensively used for both culinary and medicinal purposes. Various constituents of licorice have been shown to exhibit anti-tumorigenic effect in diverse cancer types. However, majority of these studies focus on the aspect of their growth-suppressive role. In this study, we systematically analyzed known licorice's constituents on the goal of identifying component(s) that can effectively suppress both cell migration and growth. METHODS: Effect of licorice's constituents on cell growth was evaluated by MTT assay while cell migration was assessed by both wound-healing and Transwell assays. Cytoskeleton reorganization and focal adhesion assembly were visualized by immunofluorescence staining with labeled phalloidin and anti-paxillin antibody. Activity of Src in cells was judged by western blot using phosphor-Src416 antibody while Src kinase activity was measured using Promega Src kinase assay system. Anti-tumorigenic capabilities of isoliquiritigenin (ISL) and 2, 4, 2', 4'-Tetrahydroxychalcone (THC) were investigated using lung cancer xenograft model. RESULTS: Using a panel of lung cancer cell lines, ISL was identified as the only licorice's constituent capable of inhibiting both cell migration and growth. ISL-led inhibition in cell migration resulted from impaired cytoskeleton reorganization and focal adhesion assembly. Assessing the phosphorylation of 141 cytoskeleton dynamics-associated proteins revealed that ISL reduced the abundance of Tyr421-phosphorylation of cortactin, Tyr925- and Tyr861-phosphorylation of FAK, indicating the involvement of Src because these sites are known to be phosphorylated by Src. Enigmatically, ISL inhibited Src in cells while displayed no effect on Src activity in cell-free system. The discrepancy was explained by the observation that THC, one of the major ISL metabolite identified in lung cancer cells abrogated Src activity both in cells and cell-free system. Similar to ISL, THC deterred cell migration and abolished cytoskeleton reorganization/focal adhesion assembly. Furthermore, we showed both ISL and THC suppressed in vitro lung cancer cell invasion and in vivo tumor progression. CONCLUSION: Our study suggests that ISL inhibits lung cancer cell migration and tumorigenesis by interfering with Src through its metabolite THC. As licorice is safely used for culinary purposes, our study suggests that ISL or THC may be safely used as a Src inhibitor.

Plasma metabolic profiling on postoperative colorectal cancer patients with different traditional Chinese medicine syndromes.

OBJECTIVES: This study aims to investigate the metabolic profiles of postoperative colorectal cancer (PCRC) patients with different traditional Chinese medicine (TCM) syndromes and to discuss the metabolic mechanism under PCRC progression and TCM syndrome classification. METHODS: Fifty healthy controls (HC) and 70 PCRC patients, including 10 Dampness and heat syndrome (DHS), 33 Spleen deficiency syndrome (SDS), 19 Liver and kidney Yin deficiency syndrome (LKYDS) and 8 with non-TCM syndrome (NS) were enrolled. Plasma metabolic profiles were detected by Gas chromatography-mass spectrometry (GC-MS) and analyzed by principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA). Furthermore, pathway enrichment was analyzed based on KEGG and DAVID databases and metabolic network was constructed via metaboanalyst and cytoscape. RESULTS: The top-3 metabolites with higher abundance in PCRC compared with HC were terephthalic acid (165.417-fold), ornithine (24.484-fold) and aminomalonic acid (21.346-fold). And the cholesterol (0.588-fold) level was decreased in PCRC. l-Alanine, 1, 2-ethanediamine, urea, glycerol, glycine, aminomalonic acid, creatinine and palmitic acid were specifically altered in the DHS, while d-tryptophan was exclusively changed in SDS, and l-proline, 1, 2, 3-propanetricarboxylic acid, d-galactose and 2-indolecarboxylic acids in LKYDS. CONCLUSIONS: The plasma metabolic profiles were perturbed in PCRC patients. Increased levels of terephthalic acid might indicate high risk of relapse and elevated ornithine may contribute to the post-operational recovery or may raise the susceptibility to PCRC recurrence. The metabolic profiles of DHS, SDS, LKYDS and NS were almost separately clustered, indicating the possibility of explaining TCM syndromes classification using metabolomics. Furthermore, creatinine and aminomalonic acid alternation might correlate with the formation of DHS, while d-tryptophan may associate with SDS and d-galactose and 1, 2, 3-propanetricarboxylic acid may relate to LKYDS. As numbers of patients in each TCM syndrome are small, further study is needed to verify those results.

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.

Berberine and Evodiamine Act Synergistically Against Human Breast Cancer MCF-7 Cells by Inducing Cell Cycle Arrest and Apoptosis.

BACKGROUND/AIM: The synergistic combinations of natural products have long been the basis of Traditional Chinese herbal Medicine formulas. In this study, we investigated the synergistic effects of a combination of berberine and evodiamine against human breast cancer MCF-7 cells in vitro and in vivo, and explored its mechanism. MATERIALS AND METHODS: Cell survival was measured using the MTT assay. Apoptosis-related proteins were observed using western blot analysis. Apoptosis was detected with flow cytometric analysis and by Hoechst 33258 staining. Tumor xenografts were used in vivo. RESULTS: Compared to berberine or evodiamine treatments alone, the combination treatment of berberine (25 µM) and evodiamine (15 µM) synergistically inhibited the proliferation of MCF-7 cells in a time-dependent manner and resulted in the G0/G1 phase accumulation of cells that exhibited increased expression levels of the CDK inhibitors p21 and p27 with a concomitant reduction in the expression levels of cell-cycle checkpoint proteins cyclin D1, cyclin E, CDK4, and CDK6. Furthermore, the combination treatment induced apoptosis that was accompanied by increased expression levels of p53 and Bax, reduced expression levels of Bcl-2, activation of caspase-7, and caspase-9, and the cleavage of PARP. The combination of berberine and evodiamine synergistically inhibited tumor growth in vivo in MCF-7 human breast cancer xenografts. CONCLUSION: Combination of berberine and evodiamine acts synergistically to suppress the proliferation of MCF-7 cells by inducing cell cycle arrest and apoptosis, illustrating the potential synergistic and combinatorial application of bioactive natural products.

Metabolomic mechanisms of gypenoside against liver fibrosis in rats: An integrative analysis of proteomics and metabolomics data.

AIMS: To investigate mechanisms and altered pathways of gypenoside against carbon tetrachloride (CCl4)-induced liver fibrosis based on integrative analysis of proteomics and metabolomics data. METHODS: CCl4-induced liver fibrosis rats were administrated gypenoside. The anti-fibrosis effects were evaluated by histomorphology and liver hydroxyproline (Hyp) content. Protein profiling and metabolite profiling of rats liver tissues were examined by isobaric tags for relative and absolute quantitation (iTRAQ) approach and gas chromatography-mass spectrometer (GC-MS) technology. Altered pathways and pivotal proteins and metabolites were searched by integrative analysis of proteomics and metabolomics data. The levels of some key proteins in altered pathways were determined by western blot. RESULTS: Histopathological changes and Hyp content in gypenoside group had significant improvements (P<0.05). Compared to liver fibrosis model group, we found 301 up-regulated and 296 down-regulated proteins, and 9 up-regulated and 8 down-regulated metabolites in gypenoside group. According to integrative analysis, some important pathways were found, including glycolysis or gluconeogenesis, fructose and mannose metabolism, glycine, serine and threonine metabolism, lysine degradation, arginine and proline metabolism, glutathione metabolism, and sulfur metabolism. Furthermore, the levels of ALDH1B1, ALDH2 and ALDH7A1 were found increased and restored to normal levels after gypenoside treated (P<0.05). CONCLUSIONS: Gypenoside inhibited CCl4-induced liver fibrosis, which may be involved in the alteration of glycolysis metabolism and the protection against the damage of aldehydes and lipid peroxidation by up-regulating ALDH.

An overview of epigenetics in Chinese medicine researches.

Chinese medicine (CM) has contributed to human health care for several thousand years and is still popular in Asian countries. But the mechanisms underlying CM remain obscure, and need to be clarifified by modern biological subjects and methods. Recently, CM researches on epigenetics have gained obvious improvement following rapidly developed molecular technologies. In this review, we revealed mutual characteristics of epigenetics and CM, such as holism, yin-yang dynamic nature, reversibility and balance, and visceral manifestation. Moreover, epigenetic modifications, including DNA methylation, histone post-transcriptional modifications, micro RNA interference, etc. may help explore the molecular basis of CM syndrome classifification, and mechanisms of Chinese herbal medicine (CHM), CHM compounds and Chinese herbal formulae activities. Meanwhile, CM and epigenetics might promote each other and jointly develop following the continuous progress of epigenetics in CM researches.