Identification of Q-markers for Schisandrae Sphenantherae Fructus in treating drug-induced liver injury based on network pharmacology, fingerprint and quantitative analysis / 中国中药杂志

This study aims to establish the ultra-performance liquid chromatography(UPLC) fingerprint and multi-indicator quantitative analysis method for Schisandrae Sphenantherae Fructus(SSF) and to screen out the potential quality markers(Q-markers) of hepatoprotection based on network pharmacology. The similarity analysis was performed using the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, which showed that the similarity of the fingerprints of 15 samples from different regions ranged from 0.981 to 0.998. Eighteen common components were identified, from which 3 differential components were selected by cluster analysis and principal component analysis. The "component-target-pathway" network was built to predict the core components related to the hepatoprotective effects. Fourteen core components were screened by network pharmacology. They acted on the targets such as AKT1, CCND1, CYP1A1, CYP3A4, MAPK1, MAPK3, NOS2, NQO1, and PTGS2 to regulate the signaling pathways of lipid metabolism and atherosclerosis, hepatitis B, interleukin-17, and tumor necrosis factor. Considering the chemical measurability, characteristics, and validity, schisantherin A, anwulignan, and schisandrin A were identified as the Q-markers. The content of schisantherin A, anwulignan, and schisandrin A in the test samples were 0.20%-0.57%, 0.13%-0.33%, and 0.42%-0.70%, respectively. Combining the fingerprint, network pharmacology, and content determination, this study predicted that schisantherin A, anwulignan, and schisandrin A were the Q-markers for the hepatoprotective effect of SSF. The results can provide reference for improving the quality evaluation standard and exploring the hepatoprotective mechanism of SSF.

Selection and Characterization of a Novel DNA Aptamer, Apt-07S Specific to Hepatocellular Carcinoma Cells.

BACKGROUND: The efficacy of traditional therapeutic methods for liver cancer is unsatisfying because of the poor targeting, and inefficient drug delivery system. A recent study has proven that aptamers, developed through cell-SELEX, could specifically recognize cancer cells and show great potential in the development of a delivery system for anticancer drugs. PURPOSE: To develop a hepatocellular carcinoma specific aptamer using two kinds of hepatocellular carcinoma cell lines, HepG2 and SMMC-7721, as double targets and a normal hepatocyte, L02, as a negative control cell. METHODS: Hepatocellular carcinoma specific aptamer was developed via cell-SELEX. The enrichment of the library was monitored by flow cytometric analysis. The specificity, affinity, and distribution of the candidate aptamer were explored. Further study was carried to assess its potential in drug delivery. RESULTS: The library was enriched after 14 rounds of screening. Candidate aptamer Apt-07S can recognize four kinds of hepatocellular carcinoma cells and show little cell-binding ability to normal cells and four cell lines of different cancer types, revealing a high specificity of Apt-07S. Confocal imaging showed that Apt-07S distributed both on the surface and in the cytoplasm of the two target cells. Moreover, an anti-sense nucleotide to gene Plk1 (ASO-Plk1) was connected at the 3' end of Apt-07S to form an integrated molecule (Apt-07S-ASO-Plk1); the functional analysis indicated that the structure of Apt-07S may help ASO-Plk1 enter the cancer cells. CONCLUSION: The study indicates that Apt-07S can specifically target HCC and may have potential in the delivery of anticancer drugs.

Antiviral effects of IFIT1 in human cytomegalovirus-infected fetal astrocytes.

The prominent feature of human cytomegalovirus (HCMV) is cell tropism specificity for human fetal nervous system, which leads to severe fetal nervous system damage especially in first-trimester gestation. In this study, human astrocytes isolated from fetal brain were infected with HCMV AD169 and whole genome transcriptome profile was performed. The results showed that the gene expression of interferon stimulated genes (ISGs), chemokine and chemokine receptors were significantly up-regulated (P < 0.01). The antiviral replication effects of IFIT1 (Interferon-induced protein with tetratricopeptide repeats 1, Fc = 148.17) was investigated. Lentivirus with IFIT1 overexpression or knockdown was transduced into astrocytes, respectively. The viral mRNA, protein expression and HCMV titers were determined. The results showed that IE1, IE2, pp65, and viral titers were significantly decreased in IFIT1 overexpression group and enhanced in the knockdown group compared with control one (P < 0.01). Taken together, this study revealed IFIT1 played an important antiviral role in HCMV infected fetal astrocytes. The prominent feature of human cytomegalovirus (HCMV) is cellular tropism specificity for human fetal brain nervous system leading to severe fetal nervous damage especially in first-trimester gestation. In this study, human astrocytes isolated from first-trimester fetal brain were infected with HCMV AD169 and IFIT1 was studied for its antiviral replication effects. The results provided insights into the function of IFIT1 as a key factor in antiviral defense contributing to development of targeted therapeutics to fetal brain with HCMV infection. J. Med. Virol. 89:672-684, 2017. © 2016 Wiley Periodicals, Inc.

RNAi-mediated gene silencing of vascular endothelial growth factor C suppresses growth and induces apoptosis in mouse breast cancer in vitro and in vivo.

Vascular endothelial cell growth factor (VEGF)-C promotes tumorigenesis by allowing lymph node metastasis and lymphangiogenesis, among other actions. RNA interference (RNAi) is a novel technique for suppressing target gene expression and may increase the effectiveness of cancer treatments. The present study assessed the influence of VEGF-C RNAi on the apoptosis and proliferation of mouse breast cancer cells in vitro and in vivo. A total of three pairs of small interfering RNA (siRNA) targeting mouse VEGF-C were designed and synthesized prior to transfection into 4T1 cells via a liposomal approach. Reverse transcription polymerase chain reaction, western blot analysis, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Hoechst 33258 staining and flow cytometry were performed in vitro to analyze VEGF-C expression, cleaved caspase-3 protein expression and 4T1 cell proliferation and apoptosis. Experiments were also conducted in vivo on BALB/c mice with breast cancer. Tumor weight and volume were measured and the number of apoptotic cells in tumor tissues was assessed by a TUNEL assay. Immunohistochemical assays and an enzyme-linked immunosorbent assay were used to measure the expression of VEGF-C in tumor tissues. The results demonstrated that the three pairs of siRNA, particularly siV2, significantly reduced VEGF-C mRNA and protein levels in 4T1 cells. siV2 was deemed to be the most efficient siRNA and therefore was selected to be used in subsequent experiments. Furthermore, in vitro studies indicated that VEGF-C RNAi significantly decreased cell growth, induced apoptosis and upregulated the expression of cleaved caspase-3 protein. Tumor weight and volume in breast cancer in vivo models was reduced by the intratumoral injection of siV2. Antitumor efficacy was associated with decreased VEGF-C expression and increased induction of apoptosis. The present study therefore indicated that VEGF-C RNAi inhibited mouse breast cancer growth in vitro and in vivo and that it may be a novel targeted therapy for breast cancer.

Downregulation of survivin expression exerts antitumoral effects on mouse breast cancer cells in vitro and in vivo.

Metastasis constantly occurs in the majority of cases of primary breast cancer at late stage or following surgical treatment. Survivin, a member of the inhibitor of apoptosis protein family, has long been recognized as a promising anticancer target, but its antitumor effects remain largely unexplored. In order to elucidate the role of survivin in breast cancer metastasis, short interfering RNA (siRNA) was used in the present study to specifically downregulate survivin expression in the murine breast cancer cell line 4T1. The results demonstrated that blocking the expression of survivin by siRNA inhibited the proliferation, migration and invasion abilities of murine breast cancer cells in vitro. Vascular endothelial growth factor (VEGF)-C is a lymphatic endothelial cell-stimulating factor that may lead to the formation of lymphatic vessels in lymph nodes. In the present study, the inhibition of survivin by siRNA was able to reduce the overexpression of VEGF-C in 4T1 cells. Furthermore, intratumoral injections of the survivin-siRNA significantly inhibited the growth of orthotopically transplanted 4T1 tumors in vivo. In addition, the number of pulmonary metastases and the microlymphatic vessel density were significantly reduced in vivo, following transfection with survivin-siRNA. The results of the present study suggested that the Akt/hypoxia-inducible factor-1α signaling pathway participates in the survivin-mediated downregulation of VEGF-C expression observed in breast cancer cells treated with survivin-siRNA. Therefore, the use of siRNA specifically targeting survivin may be a potential anticancer method in the future.

Form-deprivation myopia induces decreased expression of bone morphogenetic protein-2, 5 in guinea pig sclera.

AIM: To identify the presence of various bone morphogenetic proteins (BMPs) and their receptors in normal sclera of human, rat and guinea pigs, and to determine whether their expression changed with form-deprivation myopia (FDM) in guinea pig sclera. METHODS: The expression of BMPs and BMP receptors were detected using reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence. Two-week-old guinea pigs were monocularly form-deprived with a translucent lens. After fourteen days induction of FDM, total RNA was isolated and subjected to RT-PCR to examine the changes of BMPs and BMP receptors in tissues from the posterior sclera. Western blotting analysis was used to investigate their changes in protein levels. RESULTS: Human sclera expressed mRNAs for BMP-2, -4, -5, -7, -RIA, -RIB and BMP-RII. Conversely, rat sclera only expressed mRNA for BMP-7 and BMP-RIB, while the expression of BMPs and BMP receptors in guinea pigs were similar to that of humans. Human sclera also expresses BMP-2, -4, -5,-7 in protein level. Fourteen days after the induction of myopia, significant decreased expressions for BMP-2 and BMP-5 in the posterior sclera of FDM-affected eyes (P<0.05 vs internal control eyes). CONCLUSION: Various BMPs were expressed in human and guinea pig sclera. In the posterior sclera, expressions of BMP-2 and BMP-5 significantly decreased in FDM eyes. This finding indicates that various BMPs as components of the scleral cytokines regulating tissue homeostasis and provide evidence that alterations in the expression of BMP-2 and BMP-5 are associated with sclera remodeling during myopia induction.

Chemically modified siRNA directed against the KDR gene inhibits the proliferation of breast cancer cells.

Vascular endothelial growth factor receptor-2 or kinase insert domain-containing receptor (VEGFR2/KDR) is secreted by most solid tumors, including breast cancer, and is an important mediator of angiogenesis. To observe the effects of KDR gene expression on cell proliferation and the cell cycle in MCF-7 cells in vitro and in vivo, we used chemically modified siRNA directed against KDR. The results revealed that chemically modified siRNA transfection of the KDR gene effectively inhibited the proliferation of MCF-7 cells, arrested cells in the G1 phase and down-regulated the expression of KDR. In addition, in the progression of cell cycle arrest induced by siRNA, phosphorylated ERK and CDK1 expression was down-regulated (P<0.01). In vivo, the growth of tumors was visibly suppressed. RT-PCR and the results of immunohistochemistry indicated that KDR mRNA and protein expression was reduced in the excised tumors. In contrast, there were no obvious changes in the control groups. This implies that chemically modified KDR siRNA markedly decreases KDR gene expression and inhibits cellular proliferation in vitro, as well as suppressing tumor growth in a xenograft model. KDR may be a new target for breast cancer treatment.

[Study on influence of different dosage of vitamin E on peripheral blood cell activities in rats].

OBJECTIVE: To investigate the influences of vitamin E (VE) at different dosage on peripheral blood lymphocyte (PBL) proliferation and anti-DNA oxidative damage activities and erythrocyte membrane fluidity. METHODS: 48 Wistar rats were randomly divided into four groups including control group, VE1, VE2, and VE3 groups supplemented with 7.5, 50, 200, 750 IU/kg bw x d VE, respectively. The trial lasted 8 weeks and the blood samples were collected at the end of the trial. The level of plasma VE was analyzed by fluorescent spectrometry. Plasma MDA and membrane GSH-Px were analyzed by kits. The blood erythrocyte membrane fluidity was detected by fluorescence polarization method, lymphocyte transformation rate by MTT method and DNA oxidative damage by comet assay. RESULTS: The results showed that plasma VE levels significantly increased in VE1, VE2, and VE3 groups. Plasma MDA and erythocyte membrane GSH-Px activity in the rats in 50 IU/kg bw x d (VE1) group were (2.29 +/- 0.55) nmol/ml and (367.17 +/- 129.86) U/mg prot, respectively. P (fluorescence polarization) and eta(microviscosity), which were inversely related with membrane fluidity, in VE1 group were significantly lower. Lymphocyte transformation rate was significantly increased by 261.86%, 199.23% and 412.97% and H2O2 induced DNA damage significantly decreased compared with the control, VE2, and VE3 groups. CONCLUSION: It is indicated that an effective intake of VE for enhancing erythrocyte membrane fluidity, lymphocyte proliferation and DNA stability was 50 IU/kg bw x d, while too excessive intake of VE could not be found to be beneficial.