Resveratrol Enhances Inhibition Effects of Cisplatin on Cell Migration and Invasion and Tumor Growth in Breast Cancer MDA-MB-231 Cell Models In Vivo and In Vitro.

Triple-negative breast cancer (TNBC) is a refractory type of breast cancer that does not yet have clinically effective drugs. The aim of this study is to investigate the synergistic effects and mechanisms of resveratrol combined with cisplatin on human breast cancer MDA-MB-231 (MDA231) cell viability, migration, and invasion in vivo and in vitro. In vitro, MTS assays showed that resveratrol combined with cisplatin inhibits cell viability as a concentration-dependent manner, and produced synergistic effects (CI < 1). Transwell assay showed that the combined treatment inhibits TGF-ß1-induced cell migration and invasion. Immunofluorescence assays confirmed that resveratrol upregulated E-cadherin expression and downregulated vimentin expression. Western blot assay demonstrated that resveratrol combined with cisplatin significantly reduced the expression of fibronectin, vimentin, P-AKT, P-PI3K, P-JNK, P-ERK, Sma2, and Smad3 induced by TGF-ß1 (p < 0.05), and increased the expression of E-cadherin (p < 0.05), respectively. In vivo, resveratrol enhanced tumor growth inhibition and reduced body weight loss and kidney function impairment by cisplatin in MDA231 xenografts, and significantly reduced the expressions of P-AKT, P-PI3K, Smad2, Smad3, P-JNK, P-ERK, and NF-κB in tumor tissues (p < 0.05). These results indicated that resveratrol combined with cisplatin inhibits the viability of breast cancer MDA231 cells synergistically, and inhibits MDA231 cells invasion and migration through Epithelial-mesenchymal transition (EMT) approach, and resveratrol enhanced anti-tumor effect and reduced side of cisplatin in MDA231 xenografts. The mechanism may be involved in the regulations of PI3K/AKT, JNK, ERK and NF-κB expressions.

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.

Resveratrol Inhibits the Migration and Metastasis of MDA-MB-231 Human Breast Cancer by Reversing TGF-ß1-Induced Epithelial-Mesenchymal Transition.

Metastasis is a major cause of death in patients with breast cancer. In the process of cancer development, epithelial-mesenchymal transition (EMT) is crucial to promoting the invasion and migration of tumor cells. In a previous study, the role of resveratrol in migration and metastasis was investigated in MDA-MB-231 (MDA231) human breast cancer cells and a xenograft-bearing mouse model. Additionally, the related mechanism was explored. In the present study, in vitro Transwell assays showed that resveratrol can inhibit the migration of transforming growth factor (TGF)-ß1-induced MDA231 cells in a concentration-dependent manner. An enzyme-linked immunosorbent assay (ELISA) showed that resveratrol can reduce the secretion of matrix metalloproteinase (MMP)-2 and MMP-9. Immunofluorescence was performed to confirm the expression of EMT-related markers. Immunofluorescence assays confirmed that resveratrol changed the expression of the EMT-related markers E-cadherin and vimentin. Western blot analysis demonstrated that resveratrol decreased the expression levels of MMP-2, MMP-9, Fibronectin, α-SMA, P-PI3K, P-AKT, Smad2, Smad3, P-Smad2, P-Smad3, vimentin, Snail1, and Slug, as well as increased the expression levels of E-cadherin in MDA231 cells. In vivo, resveratrol inhibited lung metastasis in a mouse model bearing MDA231 human breast cancer xenografts without marked changes in body weight or liver and kidney function. These results indicate that resveratrol inhibits the migration of MDA231 cells by reversing TGF-ß1-induced EMT and inhibits the lung metastasis of MDA231 human breast cancer in a xenograft-bearing mouse model.

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.

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.

Curcumin reduces mitomycin C resistance in breast cancer stem cells by regulating Bcl-2 family-mediated apoptosis.

BACKGROUND: Curcumin, a natural compound derived from the turmeric rhizome Curcuma longa Linn, has anticancer and chemoresistance reduction biological activities. We evaluated the efficacy of curcumin in sensitizing chemotherapy drugs through regulation of Bcl-2-mediated apoptosis in breast cancer stem-like cells (BCSCs). METHODS: Cell survival was measured using MTT assay. Apoptosis-related proteins were observed using western blot analysis. Apoptosis was detected with flow cytometric analysis and by Hoechst 33258 staining. The mitochondrial membrane potential was observed with flow cytometric analysis. RESULTS: The ability of BCSCs to propagate decreased gradually along the passages and was completely lost at the fifth passage [0.1 µmol/L mitomycin C (MMC) with 5 µmol/L curcumin in MCF-7 and 0.5 µmol/L MMC with 5 µmol/L curcumin in MDA-MB-231 cells]. Curcumin combined with MMC treatment significantly decreased the levels of antiapoptotic Bcl-2 and Bcl-w expression, increased the levels of proapoptotic Bax, Bak, Bad, Bik, and Bim expression, and activated caspase-3 and caspase-9 in MCF-7 BCSCs. In the presence of Bcl-2 siRNA, the apoptosis rate increased by 15% in cells treated with curcumin and MMC. The mitochondrial membrane potential decreased by approximately 20% in MCF-7 BCSCs undergoing the combination treatment of curcumin and MMC. The combination-induced decrease in Bcl-2 was regulated by the presence of the Wnt-specific inhibitor PFK115-584 and PI3k inhibitor LY294002. CONCLUSIONS: Our study indicates that curcumin might represent a novel therapeutic agent for treating breast cancer chemoresistance induced by MMC.

Study of ZHENG differentiation in hepatitis B-caused cirrhosis: a transcriptional profiling analysis.

BACKGROUND: In traditional Chinese medicine (TCM) clinical practice, ZHENG (also known as TCM syndrome) helps to understand the human homeostasis and guide individualized treatment. However, the scientific basis of ZHENG remains unclear due to limitations of current reductionist approaches. METHODS: We collected the leukocyte samples of three hepatitis B-caused cirrhosis (HBC) patients with dampness-heat accumulation syndrome (DHAS) and three HBC patients with liver depression and spleen deficiency syndrome (LDSDS) for microarray analysis. We generated Gene-Regulatory-Networks (GeneRelNet) from the differentially expressed genes (DEGs) of microarray date. Core genes were validated using anther independent cohort of 40 HBC patients (20 DHAS, 20 LDSDS) with RT-PCR. RESULTS: There were 2457 mapped genes were differentially expressed between DHAS and LDSDS (Fold change ≥ 2.0, P < 0.05). There were markedly different genes co-expression patterns in DHAS and LDSDS. Furthermore, three differential co-expression genes including purine nucleoside phosphorylase (PNP); aquaporin 7 (AQP7) and proteasome 26S subunit, non-ATPase 2 (PSMD2) were screened by GeneRelNets, and their mRNA expressions were further validated by real time RT-PCR. The results were consistent with microarray. The PNP (P = 0.007), AQP7 (P = 0.038) and PSMD2 (P = 0.009) mRNA expression is significant difference between DHAS and LDSDS using the non-parametric test. Furthermore, we constructed an mRNA panel of PNP, AQP7 and PSMD2 (PAP panel) by logistic regression model, and evaluated the PAP panel to distinguish DHAS from LDSDS by area under the receiver operating characteristic curve (AUC) analysis, which showed a higher accuracy (AUC = 0.835). Gene ontology (GO) analysis indicated that the DHAS is most likely related to system process while the functions overrepresented by LDSDS most related to the response to stimulus. CONCLUSIONS: This study suggested that there are particular transcriptional profiles, genes co-expressions patterns and functional properties of DHAS and LDSDS, and PNP, AQP7, and PSMD2 may be involved in ZHENG differentiation of DHAS and LDSDS in HBC.

Synergistic effect of combinatorial treatment with curcumin and mitomycin C on the induction of apoptosis of breast cancer cells: a cDNA microarray analysis.

In order to explore the synergistic mechanisms of combinatorial treatment using curcumin and mitomycin C (MMC) for breast cancer, MCF-7 breast cancer xenografts were conducted to observe the synergistic effect of combinatorial treatment using curcumin and MMC at various dosages. The synergistic mechanisms of combinatorial treatment using curcumin and MMC on the inhibition of tumor growth were explored by differential gene expression profile, gene ontology (GO), ingenuity pathway analysis (IPA) and Signal-Net network analysis. The expression levels of selected genes identified by cDNA microarray expression profiling were validated by quantitative RT-PCR (qRT-PCR) and Western blot analysis. Effect of combinatorial treatment on the inhibition of cell growth was observed by MTT assay. Apoptosis was detected by flow cytometric analysis and Hoechst 33258 staining. The combinatorial treatment of 100 mg/kg curcumin and 1.5 mg/kg MMC revealed synergistic inhibition on tumor growth. Among 1501 differentially expressed genes, the expression of 25 genes exhibited an obvious change and a significant difference in 27 signal pathways was observed (p<0.05). In addition, Mapk1 (ERK) and Mapk14 (MAPK p38) had more cross-interactions with other genes and revealed an increase in expression by 8.14- and 11.84-fold, respectively during the combinatorial treatment by curcumin and MMC when compared with the control. Moreover, curcumin can synergistically improve tumoricidal effect of MMC in another human breast cancer MDA-MB-231 cells. Apoptosis was significantly induced by the combinatorial treatment (p<0.05) and significantly inhibited by ERK inhibitor (PD98059) in MCF-7 cells (p<0.05). The synergistic effect of combinatorial treatment by curcumin and MMC on the induction of apoptosis in breast cancer cells may be via the ERK pathway.

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.

Combining ZHENG Theory and High-Throughput Expression Data to Predict New Effects of Chinese Herbal Formulae.

ZHENG is the key theory in traditional Chinese medicine (TCM) and it is very important to find the molecular pharmacology of traditional Chinese herbal formulae. One ZHENG is related to many diseases and the herbal formulae are aiming to ZHENG. Therefore, many herbal formulae whose effects on a certain disease have been confirmed might also treat other diseases with the same ZHENG. In this study, the microarrays collected from patients with QiXuXueYu ZHENG (Qi-deficiency and Blood-stasis syndrome) before treatment and after being treated with Fuzheng Huayu Capsule were analyzed by a high-throughput gene microarrays-based drug similarity comparison method, which could find the small molecules which had similar effects with Fuzheng Huayu Capsule. Besides getting the results of anti-inflammatory and anti-fibrosis drugs which embody the known effect of Fuzheng Huayu Capsule, many other small molecules were screened out and could reflect other types of effects of this formula in treating QiXuXueYu ZHENG, including anti-hyperglycemic, anti-hyperlipidemic, hyposenstive effect. Then we integrated this information to display the effect of Fuzheng Huayu Capsule and its potential multiple-target molecular pharmacology. Moreover, through using clinical blood-tested data to verify our prediction, Fuzheng Huayu Capsule was proved to have effects on diabetes and dyslipidemia.

Molecular Mechanisms of Same TCM Syndrome for Different Diseases and Different TCM Syndrome for Same Disease in Chronic Hepatitis B and Liver Cirrhosis.

Traditional Chinese medicine (TCM) treatment is based on the traditional diagnose method to distinguish the TCM syndrome, not the disease. So there is a phenomenon in the relationship between TCM syndrome and disease, called Same TCM Syndrome for Different Diseases and Different TCM Syndrome for Same Disease. In this study, we demonstrated the molecular mechanisms of this phenomenon using the microarray samples of liver-gallbladder dampness-heat syndrome (LGDHS) and liver depression and spleen deficiency syndrome (LDSDS) in the chronic hepatitis B (CHB) and liver cirrhosis (LC). The results showed that the difference between CHB and LC was gene expression level and the difference between LGDHS and LDSDS was gene coexpression in the G-protein-coupled receptor protein-signaling pathway. Therein genes GPER, PTHR1, GPR173, and SSTR1 were coexpressed in LDSDS, but not in LGDHS. Either CHB or LC was divided into the alternative LGDHS and LDSDS by the gene correlation, which reveals the molecular feature of Different TCM Syndrome for Same Disease. The alternatives LGDHS and LDSDS were divided into either CHB or LC by the gene expression level, which reveals the molecular feature of Same TCM Syndrome for Different Diseases.

Classification of Traditional Chinese Medicine Syndromes in Patients with Chronic Hepatitis B by SELDI-Based ProteinChip Analysis.

Traditional Chinese medicine (TCM) syndrome, also called ZHENG, is the basis concept of TCM theory. It plays an important role in TCM practice. There are excess and deficiency syndromes in TCM syndrome. They are the common syndromes in chronic hepatitis B (CHB) patients. Here we aim to explore serum protein profiles and potential biomarkers for classification of TCM syndromes in CHB patients. 24 healthy controls and two cohorts of CHB patients of excess syndrome (n = 25) or deficiency syndrome (n = 19) were involved in this study. Protein profiles were obtained by surface-enhanced laser desorption ionization time-flight mass spectrometry (SELDI-TOF/MS) and multiple analyses were performed. Based on SELDI ProteinChip data, healthy controls and CHB patients or excess and deficiency syndromes in CHB patients were obviously differentiated by orthogonal partial least square (OPLS) analysis. Two significant serum proteins (m/z 4187 and m/z 5032) for classifying excess and deficiency syndromes were found. Moreover, the area under the receiver operating characteristic (ROC) curve was 0.887 for classifying excess and nonexcess syndrome, and 0.700 for classifying deficiency and nondeficiency syndrome, respectively. Therefore, the present study provided the possibility of TCM syndrome classification in CHB patients using a universally acceptable scientific approach.

Interleukin-10 genotype correlated to deficiency syndrome in hepatitis B cirrhosis.

Traditional Chinese medicine (TCM) syndrome is an important basis for TCM diagnosis and treatment. As Child-Pugh classification as well as compensation and decompensation phase in liver cirrhosis, it is also an underlying clinical classification. In this paper, we investigated the correlation between single nucleotide polymorphisms (SNPs) of Interleukin-10 (IL-10) and TCM syndromes in patients with hepatitis B cirrhosis (HBC). Samples were obtained from 343 HBC patients in China. Three SNPs of IL-10 (-592A/C, -819C/T, and -1082A/G) were detected with polymerase chain-reaction-ligase detection reaction (PCR-LDR). The result showed the SNP-819C/T was significantly correlated with Deficiency syndrome (P = 0.031), but none of the 3 loci showed correlation either with Child-Pugh classification and phase in HBC patients. The logistic regression analysis showed that the Excess syndrome was associated with dizzy and spider nevus, and the Deficiency syndrome was associated with dry eyes, aversion to cold, IL-10-819C/T loci, and IL-10-1082A/G loci. The odds ratio (OR) value at IL-10-819C/T was 4.022. The research results suggested that IL-10-819C/T locus (TC plus CC genotype) is probably a risk factor in the occurrence of Deficiency syndrome in HBC patients.

[Analysis of differential gene expression profile in peripheral blood of patients with chronic hepatitis B and syndromes of dual deficiency of liver and kidney yin and accumulation of dampness heat].

OBJECTIVE: To investigate the differential gene expression profile in two typical traditional Chinese medicine (TCM) syndromes of patients with chronic hepatitis B (CHB), and to find the relationship between TCM syndromes and gene expressions. METHODS: Patients with CHB were collected from Department of Liver Diseases of Longhua Hospital in Shanghai and diagnosed as dual deficiency of liver and kidney yin syndrome (n=3) or accumulation of dampness heat syndrome (n=3). Three healthy volunteers were used as a control. Blood samples were collected before treatment. After total RNA was isolated from leukocytes, the gene expression profiles were detected by microarray. For confirming the gene expressions, a further 10 patients with dual deficiency of liver and kidney yin syndrome or accumulation of dampness heat syndrome and 10 healthy volunteers were enrolled. Their peripheral blood was used to test the gene expression by real-time reverse transcription-polymerase chain reaction (RT-PCR) which was differently expressed by microarray. RESULTS: Microarray analysis identified that there were significant differences in gene expression between patients with dual deficiency of liver and kidney yin syndrome and patients with accumulation of dampness heat syndrome, as well as between CHB patients and healthy volunteers. Patients with dual deficiency of liver and kidney yin syndrome and accumulation of dampness heat syndrome were fully distinguished by clustering of 403 differentially expressed genes (P<0.05). A total of 239 genes were significantly differentially expressed (absolute value of foldchange≥2, P<0.05), among which 142 were up-regulated and 97 were down-regulated. Genes specially regulated by the dual deficiency of liver and kidney yin syndrome were mainly related to peroxide activity and stem cell maintenance; genes specially regulated by the accumulation of dampness heat syndrome were mainly related to cytokines, immune and inflammatory responses. ATP-binding cassette, sub-family C (CFTR/MRP), member 3, peroxisome proliferator-activated receptor α and phosphatidic acid phosphatase type 2A mRNA expressions detected by RT-PCR were similar to the results of microarray. CONCLUSION: Differential gene expressions are observed in CHB patients with dual deficiency of liver and kidney yin syndrome and accumulation of dampness heat syndrome, indicating that there is molecular foundation in the classification of TCM syndrome. The preliminary findings of this study suggest that TCM syndrome types are related to gene expression.

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.

Curcumin enhanced antiproliferative effect of mitomycin C in human breast cancer MCF-7 cells in vitro and in vivo.

AIM: To investigate the efficacy of mitomycin C (MMC) in combination with curcumin in suppressing human breast cancer in vitro and in vivo. METHODS: Human breast cancer MCF-7 cells were used. Cell viability was measured using MTT assay. The cell cycle phase was detected with flow cytometric analysis. Cell cycle-associated proteins were examined using Western blot analysis. MCF-7 breast cancer xenografts were established to monitor tumor growth and cell cycle-associated protein expression. RESULTS: Curcumin inhibited MCF-7 breast cancer cell viability in a concentration-dependent manner (IC(50) value=40 µmol/L). Similarly, MMC inhibited the cell viability with an IC(50) value of 5 µmol/L. Combined treatment of MMC and curcumin showed a synergistic antiproliferative effect. In the presence of curcumin (40 µmol/L), the IC(50) value of MMC was reduced to 5 µmol/L. In MCF-7 xenografts, combined administration of curcumin (100 mg/kg) and MMC (1-2 mg/kg) for 4 weeks produced significantly greater inhibition on tumor growth than either treatment alone. The combined treatment resulted in significantly greater G(1) arrest than MMC or curcumin alone. Moreover, the cell cycle arrest was associated with inhibition of cyclin D1, cyclin E, cyclin A, cyclin-dependent kinase 2 (CDK2) and CDK4, along with the induction of the cell cycle inhibitor p21 and p27 both in MCF-7 cells and in MCF-7 xenografts. These proteins were regulated through p38 MAPK pathway. CONCLUSION: The results suggest that the combination of MMC and curcumin inhibits MCF-7 cell proliferation and cell cycle progression in vitro and in vivo via the p38 MAPK pathway.

Aidi injection alters the expression profiles of microRNAs in human breast cancer cells.

OBJECTIVE: To investigate the effects of Aidi Injection on the MicroRNAs (miRNA) expression profiles in human breast cancer cells and explore the potential targets of the cancer treatment. METHODS: MCF-7 breast cancer cells were grown in RPMI 1640 medium supplemented with different concentrations of ADI. The inhibition of cell proliferation was measured by MTT assay. MCF-7 cells were treated by ADI with above 50% inhibiting concentration (IC50) for 48 h. The expression profiles of miRNA in ADI-treated and ADI-untreated MCF-7 cells were detected with miRNA microarray chips and the array data were verified by quantitative RT-PCR. MCF-7 cells were transiently transfected with miRNA mimics by liposome method. Potential mRNA targets were predicted by informatics analysis with TargetScan and PicTar software. RESULTS: ADI significantly inhibited the proliferation of MCF-7 cells in a dose-dependent manner. The IC50 of ADI was 55.71 mg/mL after treatment for 48 h. The 60 mg/mL ADI was used as the therapeutic drug concentration. Microarray analysis identified 45 miRNAs that were up-regulated and 55 miRNAs that were down-regulated in response to ADI treatment. Many ADI-induced miRNAs were related to breast cancers. The microarray data were validated by qRT-PCR. Ectopic expression of 100 nmol/L mir-126 mimics significantly inhibited the proliferation of MCF-7 cells. The 12 potential target genes of mir-126 were predicted by both TargetScan and PicTar software. CONCLUSIONS: The miRNA may serve as therapeutic targets, and the modulation of miRNA expression is an important mechanism of ADI inhibiting breast cancer cell growth.

[Research approach for biological basis of Chinese medical syndromes of chronic viral hepatitis B].

Chronic viral hepatitis B (CHB) is a major infectious disease greatly harmful to the health of Chinese people. Chinese medicine has its speciality and advantages in treating it depending syndrome-differentiation. The objectified researches regarding Chinese medical syndromes in CHB heretofore were reviewed in this article. Moreover, aiming at existing problems and taking the angle of "disease-syndrome combining" study, authors put forward research approach, and approaches for studying systemic biology based biological basis of Chinese medical syndrome in hepatitis B with reductionism and holism, cybernetics and system theories in combination.

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.