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.

Preliminary study of Yinhuapinggan granule against H1N1 influenza virus infection in mice through inhibition of apoptosis.

CONTEXT: Yinhuapinggan granule (YHPG) is frequently used for treating fever, cough, and viral pneumonia in traditional Chinese medicine. OBJECTIVE: This study investigated the antiviral effects of YHPG in H1N1 influenza virus (IFV)-infected mice and its possible mechanism. MATERIALS AND METHODS: ICR mice were intranasally infected with 10 LD50 viral dose of IFV and then oral administration of YHPG (6, 12, and 18 g/kg) or oseltamivir (positive control) once a day for 2 or 4 consecutive days, six mice in each group. The lung, spleen and thymus indexes of IFV-infected mice, the expression of viral loads and pathological changes in lung tissues were performed to evaluate the antiviral effects of YHPG. Real-time PCR, immunohistochemistry and western blot assays were used to determine the expression of Bax, Bcl-2 and caspase-3. RESULTS: LD50 in mice was 10-3.5/0.02 mL. YHPG (6, 12, and 18 g/kg) dose-dependently decreased the lung index and viral load; the inhibition ratio of lung index was 5.31, 18.22, and 34.06%, respectively. Further detection revealed that YHPG (12 and 18 g/kg) significantly attenuated lung pathological changes, and increased the spleen and thymus indexes. Moreover, YHPG significantly down-regulated the mRNA and protein expression of Bax and caspase-3 in lung tissues of mice infected with IFV, and up-regulated the expression of Bcl-2. CONCLUSIONS: YHPG has significant antiviral effects in IFV-infected mice, partially by inhibiting influenza virus replication and regulating the occurrence of apoptosis induced by influenza virus infection, suggesting that YHPG may be a promising antiviral agent with potential clinical application prospects.

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.

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.

Antiviral effects and mechanisms of Yinhuapinggan granule against H1N1 influenza virus infection in RAW264.7 cells.

Yinhuapinggan granule (YHPG), a modified prescription based on Ma-Huang-Tang (MHT), is used in traditional Chinese medicine (TCM) to treat influenza, cough, and viral pneumonia. In this study, we investigated the antiviral effects of YHPG by means of pre-, post-, and co-treatment, and its underlying mechanisms on regulating the levels of inflammatory-related cytokines, modulating the mRNA expressions of interferon-stimulated genes in influenza virus-infected murine macrophage cells (RAW264.7), and evaluating the protein expressions of key effectors in the Type I IFN and pattern recognition receptor (PRRs) signaling pathways. The results showed that YHPG markedly inhibited influenza virus (IFV) replication in pre-, post- and co-treatment assay, especially in post-treatment assay. Antiviral mechanisms studies revealed that YHPG (500 and 250 µg/mL) significantly up-regulated levels of IFN-ß, IFN-stimulated genes (Mx-1, ISG-15 and ISG-56) compared with the IFV control group, while the levels of IL-6 and TNF-α were significantly down-regulated. Furthermore, western blot analysis results revealed that the protein expressions of the phosphorylated forms of TBK1, IRF3, ERK1/2, P38 MAPK and NF-κB p65 were significantly down-regulated in RAW264.7 cells with the YHPG (500 and 250 µg/mL) treatment, while the expression of the phosphorylated form of STAT1 was significantly enhanced. Based on these results, YHPG had antiviral effects in IFV-infected RAW264.7 cells, which might be associated with regulation of the inflammatory cytokines production, evaluation of the levels of IFN-stimulated genes, and modulation of the protein expressions of key effectors in the Type I IFN and PRRs signaling pathways.

[Effect and mechanism of Mahuang Tang against influenza A/H1N1 virus in vitro].

To study the effect and underlying mechanism of Mahuang Tang against influenza A virus in vitro， the influenza virus-infected Madin-Darby canine kidney(MDCK) cells were used as the carrier in this study to detect the median tissue culture-infective dose(TCID50) of influenza A virus strains(A/PR8/34) on MDCK cells with cytopathic effect(CPE) assay. Blocking influenza virus invading host cells and anti-influenza virus biosynthesis were used as two different administration methods， and then the methyl thiazolyl tetrazolium(MTT) assay was utilized to determine the antiviral effective rate(ER)， median efficacious concentration(EC50) and therapeutic index(TI) of Mahuang Tang. The quantitative Real-time polymerase chain reaction(RT-PCR) was used to measure virus load and the mRNA expression levels of TLR4， TLR7， MyD88 and TRAF6 in MDCK cells at 24， 48 h after the treatment. The experiment results indicated that TCID50 of A/PR8/34 for MDCK cells was 1×10-4.32/mL. The EC50 values of two different treatment methods were 4.92，1.59 g·L⁻¹ respectively， the TI values were 12.53， 38.78 respectively， and when the concentration of Mahuang Tang was 5.00 g·L⁻¹ï¼Œ ER values were 50.21%， 98.41% respectively， showing that Mahuang Tang can block influenza virus into the host cells and significantly inhibit their biosynthesis. Meanwhile， as compared with the virus group， the virus load was significantly inhibited in Mahuang Tang groups， and Mahuang Tang high and middle doses had the significant effect on decreasing the mRNA expression of TLR4， TLR7，MyD88 and TRAF6 at 24， 48 h after the treatment. It can be demonstrated that the mechanisms of Mahuang Tang against influenza A virus are related to the inhibition of influenza virus replication and the mRNA expression of correlative genes in TLR4 and TLR7 signaling pathways.

[Immunologic mechanisms of Yinhua Pinggan granule and San-ao decoction against influenza virus in vivo].

This paper aimed to investigate the effect of Yinhua Pinggan granule and San-ao decoction on the immunologic mechanisms of influenza viral pneumonia mice in vivo, in order to study the activity of the combined administration of different formulas on influenza A/H1N1 virus. The model of pneumonia was established in mice through nasal dropping influenza virus, and then divided randomly into five groups: normal control group, influenza virus model group, oseltamivir control group, Yinhua Pinggan granule group, and San-ao decoction group. The animals were put to death at the 5th day after gavage administration with the corresponding drugs. The contents in mice serum of TNF-α, IL-6 and IFN-γ were respectively measured by ELISA. The mRNA expressions of TLR3/7, MyD88, JNK, p38MAPK and NF-κB p65 in lung tissues were respectively detected by RT-PCR. The protein expressions of JNK, p38MAPK and NF-κB p65 in lung tissues were determined by immunohistochemical analysis, respectively. According to the results, Yinhua Pinggan granule and San-ao decoction could significantly decrease the levels of TNF-α and IL-6, increase the level of IFN-γ in mice serum of lung tissues, significantly reduce the gene expressions of TLR3/7, MyD88, JNK, p38MAPK and NF-κB p65 in influenza virus-infected mice lung tissues, and significantly reduce the protein expressions of JNK, p38MAPK and NF-κB p65 in lung tissues. Furthermore, the regulatory effect of Yinhua Pinggan granule was superior to that of San-ao decoction. In conclusion, Yinhua Pingan granule and San-ao decoction have the therapeutic effect on pneumonia mice infected by H1N1 virus in vivo. The anti-influenza mechanisms of Yinhua Pinggan granule and San-ao decoction may be the results of interactions by regulating the immunologic function of influenza virus-infected mice and TLR3/7 signaling pathway with multiple links of the gene and protein expressions. Moreover, the combined administration of warm-natured and cold-natured Yinhua Pinggan granule with the effects of detoxification and exhalation has a better effect than the single administration of warm-natured San-ao decoction.

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.

[Effect of Yinghua Pinggan granule against influenza A/H1N1 virus in vivo].

To study the effect of Yinghua Pinggan granule (YHPG) against influenza A/H1N1 virus in vivo and on the immunologic function of infected mice. The intranasal influenza virus infection was adopted in ICR mouse to establish the influenza virus pneumonia model. At the 3rd and 7th day after the infection, the lung index and pathologic changes in lung tissues of mice were detected. Realtime PCR and flow cytometry were employed to observe the virus load in lung tissues and the levels of CD4+, CD8+, and CD4+/CD8+ in peripheral blood. The result showed that at the 3rd and 7th day after the infection, YHPG (15, 30 g x kg(-1)) can significant decrease in the lung index and virus load in lung tissues of mice infected with influenza virus, alleviate the pathologic changes in lung tissues, significantly increase the levels of CD4+ and CD4+/CD8+ ratio and reduce the levels of CD8+ in whole blood. This indicated that YHPG can inhibit the influenza virus replication, alleviate pulmonary damage and adjust the weak immunologic function of infected mice, with a certain therapeutic effect on mice infected by H1N1 virus in vivo.

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.

[Variations on hemagglutinin gene of Zhejiang measles virus strains and differences with measles strains circulated both at home and abroad].

OBJECTIVE: To investigate the variations on hemagglutinin (H) gene of measles virus (MV) in Zhejiang province, and to analyze the differences with strains circulated both at home and abroad. METHODS: In total, 33 MV strains isolated in Zhejiang province between 1999 and 2011 were collected.RNA of the isolated MV strains was extracted and the complete sequences on H gene were amplified using RT-PCR assay. The products were compared with the Chinese vaccine strain Shanghai-191, which was downloaded from GenBank, and other 95 different MV strains from all over the world. RESULTS: 33 MV strains, isolated from the throat swab specimens collected from MV patients in Zhejiang province during 1999 to 2001, were used to conduct phylogenetic analysis with MV strains circulated in other areas of China during 1993 to 2007. The phylogenetic tree based on H gene sequences showed that all the Zhejiang MV strains located in H1a cluster, and no apparent time series and geographic restrictions were observed. Compared to the referenced vaccine strain Shanghai-191, the average variation rate on nucleotides and amino acids, and the evolutionary rate of H1a viruses from China during 2003 to 2011 were separately 5.15%, 4.44% and 5.81%, which were higher than the rates of H1a viruses during 1965 to 1993 (4.75%, 3.86% and 5.30%), and the rates of viruses during 1994 to 2002 (4.80%, 4.08% and 5.37%).However, the dn/ds ratios of strains within the three time periods were 0.19,0.21 and 0.23 respectively, which indicated that no evidence of positive selection was found on H1a MV strains during 1993 to 2011. A 24 stable amino acid variation sites on H gene was found between H1a viruses during 2003 to 2011 and the vaccine strain Shanghai-191. The largest variation occurred between vaccine and H1a strains, with 0.053 of the p-distance and 26-28 of amino acid mutations.However, only 15 stable amino acid variations were found between vaccine strain and genotype B3 or D4 strains.In addition, significant differences were found between H1a viruses and genotype B or D viruses, with 0.074 and 0.071 of p-distance and 27-33 of amino acid differences. CONCLUSION: Significant differences were found on H gene between MV strains subtype H1a and vaccine strains and other genotype strains. The variations were enlarged with the time coursing; therefore, the surveillance on variation of Chinese MV strains should be taken into account.

[Analysis of genetic variation of hemagglutinin and three internal genes of influenza B virus isolated in Zhejiang province from 1999 to 2012].

OBJECTIVE: To explore the characteristics of the genetic variation of hemagglutinin( HA) and three internal genes coding for the nucleoprotein ( NP) , matrix protein ( M) and nonstructural protein ( NS) of influenza B virus. METHODS: A total of 31 strains of influenza B virus were isolated in Zhejiang province from 1999 to 2012, and then were amplified and sequenced the genes of HAl , NP, M and NS. The phylogenetic tree was constructed, the nucleotide substitution rate of the above individual gene was estimated and the variation sites of amino acids were analyzed. RESULTS: The 31 isolated strains of influenza B virus were divided into two distinct lineages Victoria and Yamagata in the phylogenetic tree of HAl gene,represented by B/Victoria/2/87 and B/Yamagata/16/88. Phylogenetic analysis of the NP gene showed that the NP gene of Victoria-like influenza B strains which were isolated after 2010 was highly homologous with Yamagata-like isolates, and thereby they were found to be on the same branch of the phylogenetic tree of the NP gene. Nucleotide substitution rates of HAl , NP, M and NS genes were estimated to be 2. 29 x 10 -3 ,1. 39 X 10-3 ,1. 78 X 10-3 ,1. 30 X 10-3 /site per year, respectively. Variations of amino acid of HAl domain of Victoria-like isolates mainly included K48E ,L58P ,N75K,K80R,K129N/S,N165K,S172P ,Sl97N/D and A202V; while those in Yamagata-like isolates were R48K, S1501, N166Y, N203S, G230D and D233N. Determined amino acid sequences of NP of Victoria-like influenza B isolates were similar to Yamagata-like isolates after 2010 and variations happened on four characteristic amino acid sites, naming A60D, I233V, N513S and V5341, compared with previous Victoria-like influenza B isolates. CONCLUSION: Significant variation was found among influenza B strains isolated in Zhejiang province from 1999 to 2012. The surface HAl gene evolved more rapidly than internal genes. Gene reassortment and gene mutation were the main evolutionary mechanism of influenza B virus.

Shenqi Fuzheng injection reverses M2 macrophage-mediated cisplatin resistance through the PI3K pathway in breast cancer.

BACKGROUND: Shenqi Fuzheng injection (SQFZ) combined with chemotherapy can sensitize tumour cells. However, the mechanisms underlying SQFZ's effects remain unknown. In human breast cancer cell lines and M2 macrophages, we showed that SQFZ was a significantly potent agent of sensitization. METHODS: The human breast cancer cell line, MDA-MB-231/DDP, and the human acute leukaemia mononuclear cell line, THP-1, were used. MDA-MB-231/DDP breast cancer xenografts were established to monitor tumour growth. Resistance-associated proteins were examined by western blotting. Levels of cytokines and chemokines were detected by ELISA. Cell viability was measured using the MTT assay. Apoptosis was detected by flow cytometric analysis. RESULTS: SQFZ significantly enhanced the capability of cisplatin to reduce tumour mass. SQFZ and cisplatin decreased the expression of CD206 by 1.89-fold and increased that of CD86 by 1.76-fold as compared to cisplatin alone. The levels of PGE2, IL-6, and CCL1 decreased significantly, and the activation of p-PI3K and the expressions of P-gp and ABCG2 were also inhibited by SQFZ in combination with cisplatin treatment in vivo. The survival following cisplatin administration of 60 µM and 120 µM reduced significantly in the presence of SQFZ in MDA-MB-231/DDP and M2 co-cultured cells. IGF-1, a PI3K activator, combined with SQFZ weakened the effects of SQFZ-induced apoptosis from 28.7% to 10.5%. The effects of IGF-1 on increasing the expressions of P-gp, ABCG2, and Bcl-2, and decreasing that of Bax were reversed by SQFZ. CONCLUSION: Our findings provide evidence that SQFZ is a potential therapeutic drug for cancer therapy.

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.

[Comparative analysis on the complete genome sequence of mumps epidemic strain and mumps vaccine strain S79 isolated in Zhejiang province, China between year 2005 and 2010].

OBJECTIVE: To compare the differences in the complete genome sequence between mumps epidemic strain and mumps vaccine strain S79 isolated in Zhejiang province. METHODS: A total of 4 mumps epidemic strains, which were separated from Zhejiang province during 2005 to 2010, named as ZJ05-1, ZJ06-3, ZJ08-1 and ZJ10-1 were selected in the study. The complete genome sequences were amplified using RT-PCR. The genetic differences between vaccine strain S79 and other genotype strains were compared; while the genetic-distance was calculated and the evolution was analyzed. RESULTS: The biggest difference between the 4 epidemic strains and the vaccine strain S79 was found on the membrane associated protein gene; whose average nucleotide differential number was 42.5 +/- 3.0 and the average variant ratio was 13.6%; while the mean amino acid differential number was 12.8 +/- 1.5 and the average variant ratio was 22.4%. The smallest difference among the 4 epidemic strains and the vaccine strain was found in stromatin genes, whose average nucleotide differential number was 73.8 +/- 2.5 and the average variant ratio was 5.9%; while the mean amino acid differential number was 3.0 +/- 0.8 and the average variant ratio was 0.8%. The dn/ds value of the stromatin genes of the 4 epidemic strains reached the highest, as 0.6526; but without any positive pressure (dn/ds < 1, chi2 = 0.87, P > 0.05). There were mutations happened on the known antigen epitope, as 8th amino acid of membrane associated protein genes and on the 336th and 356th amino acid of hemagglutinin/neuraminidase proteins. Compared with the vaccine strain, the glycosylation sites of ZJ05-1, ZJ06-3, ZJ08-1 and ZJ10-1 increased 1, 1, 2 and 2 respectively. The complete amino acid sequence of all strains showed that there were 17 characteristic sites found on the genotype-F mumps strain. Within the complete genome, the genetic-distance between epidemic strains and vaccine strains in Zhejiang province (0.071) was significantly larger than the genetic-distance between strains in Yunnan province (0.013); the difference showing statistical significance (t = 4.14, P < 0.05). Except nucleocapsid protein genes, all the genes shared similar evolution tree. CONCLUSION: There were significant differences found in the genes between mumps epidemic strain and mumps vaccine in Zhejiang province.

Elevated pretreatment neutrophil-to-lymphocyte ratio indicate low survival rate in apatinib-treated patients with non-small cell lung cancer: A STROBE-compliant article.

This study aimed to analyze the predictive value of the neutrophil-to-lymphocyte ratio (NLR) to better clarify which patients with advanced non-small cell lung cancer (NSCLC) would benefit most from apatinib after multiline treatment for drug resistance. This observational cohort study involved patients with advanced NSCLC who were treated with apatinib between May 2016 to May 2018. The participants in this study had previously been treated with at least two treatment regimens. Multivariate logistic regression and Cox proportional risk models were used to evaluate the overall survival (OS) and progression-free survival (PFS) of the pretreatment NLR. A total of 125 patients were reviewed. The median age was 64 years (range, 33-92); and 32.8% of the patients were female. Only 0.8% of the patients had an Eastern Cooperative Oncology Group Performance Status (ECOG-PS) score ≥ 2. In multivariate analysis, pretreatment NLR ≥ 5 had an independent correlation with inferior OS (median 2.07 vs 3.40 months; HR 1.493, 95% CI 1.022-2.182; P = .038) and inferior PFS (median 1.83 vs 2.76 months; HR 1.478, 95% CI 1.015-2.153; P = .042). Elevated pretreatment NLR is associated with shorter OS and PFS in patients with advanced NSCLC treated with apatinib after multiline treatment for drug resistance.