[Exploration on mechanism of Polygalae Radix and Acori Tatarinowii Rhizoma in treating Alzheimer's disease based on network pharmacology and experimental verification].

This study aimed to explore the action targets and mechanisms of Polygala tenuifolia and Acorus tatarinowii in treating Alzheimer's disease(AD) based on network pharmacology, molecular docking, and animal tests. The AD-related targets were collec-ted from GeneCard and the main active ingredients and targets of P. tenuifolia and A. tatarinowii from the TCMSP. Cytoscape was applied to construct the "Chinese herb-active ingredient-target-disease" network, followed by the construction of protein-protein interaction(PPI) network using STRING. GO biological function and KEGG pathway enrichment analysis was performed by DAVID and Metascape. The main active components of P. tenuifolia and A. tatarinowii and their potential core targets were docking using AutoDock Vina. The effects of P. tenuifolia and A. tatarinowii on the cognitive function were verified in mice with scopolamine(SCOP)-induced cognitive impairment. A total of seven active ingredients including kaempferol, onjixanthone Ⅰ, and marmesin and 56 potential targets of P. tenuifolia and A. tatarinowii were screened out, with the core targets covering AKT1, PTGS2, TNF, and NF-κB inflammation pathway mainly involved. The results of molecular docking also showed that the main active components of P. tenuifolia and A. tatarinowii stably bond to the core targets predicted by network pharmacology. The new object recognition experiment suggested that P. tenuifolia and A. tatarinowii improved the learning and memory abilities of mice after SCOP induction. As revealed by pathological section observation and relevant kit assay, P. tenuifolia and A. tatarinowii reduced the damage of central cholinergic neurons and enhanced the antioxidant ability of SCOP-induced mice. Western blot confirmed that P. tenuifolia and A. tatarinowii down-regulated the protein expression levels of TLR4, NF-κB, and related inflammatory factors(TNF-α, IL-1ß, and IL-6). All these have suggested that P. tenuifolia and A. tatarinowii inhibits AD via multiple components, multiple targets, and multiple pathways, which has provided an experimental basis for the clinical application of P. tenuifolia and A. tatarinowii for the treatment of AD.

Mitochondrial targeted doxorubicin derivatives delivered by ROS-responsive nanocarriers to breast tumor for overcoming of multidrug resistance.

Multidrug resistance (MDR) is a serious challenge in chemotherapy and also a major threat to breast cancer treatment. As an intracellular energy factory, mitochondria provide energy for drug efflux and are deeply involved in multidrug resistance. Mitochondrial targeted delivery of doxorubicin can overcome multidrug resistance by disrupting mitochondrial function. By incorporating a reactive oxygen species (ROS)-responsive hydrophobic group into the backbone structure of hyaluronic acid - a natural ligand for the highly expressed CD44 receptor on tumor surfaces, a novel ROS-responsive and CD44-targeting nano-carriers was constructed. In this study, mitochondria-targeted triphenylphosphine modified-doxorubicin (TPP-DOX) and amphipathic ROS-responsive hyaluronic acid derivatives (HA-PBPE) were synthesized and confirmed by 1H NMR. The nanocarriers TPP-DOX @ HA-PBPE was prepared in a regular shape and particle size of approximately 200 nm. Compared to free DOX, its antitumor activity in vitro and tumor passive targeting in vivo has been enhanced. The ROS-responsive TPP-DOX@HA-PBPE nanocarriers system provide a promising strategy for the reverse of MDR and efficient delivery of doxorubicin derivatives into drug-resistant cancer cells.

Analysis of Coding RNA and LncRNA Expression Profile of Stem Cells from the Apical Papilla After Depletion of Sirtuin 7.

OBJECTIVE: To explore the effects of Sirtuin 7 (SIRT7) on the gene expression profile of stem cells from the apical papilla (SCAPs). METHODS: SCAPs were isolated and cultured. SIRT7 short hairpin ribonucleic acid (shRNA) was used to knock down the expression of SIRT7 in SCAPs. After library construction and RNA sequencing (RNA-seq), differentially expressed genes were identified using Cuffdiff with a false discovery rate (FDR) ≤ 0.05 and fold change ≥ 2. Pathway and Gene Ontology (GO) analyses were conducted to elucidate the changes in important functions and pathways after SIRT7 gene knockdown. Gene set enrichment analysis (GSEA) was performed and enrichment of a gene set with an FDR lower than 0.25 was considered significant. RESULTS: The most striking GO terms related to SIRT7sh SCAPs and Consh SCAPs were response to nucleus, nucleolus, cytoplasm, protein binding and intrinsic apoptotic signalling pathway. Signalling pathway analysis revealed the top five pathways to be metabolic, pyrimidine metabolism, protein processing in endoplasmic reticulum, phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling and p53 signalling. The results of GSEA showed that genes were mainly enriched in cell cycle, cell proliferation, transforming growth factor beta (TGF-ß) signalling and cytokine-cytokine receptor interaction pathways. CONCLUSION: SIRT7 may affect the functions of SCAPs through cell cycle, cell proliferation and apoptosis pathways.

Delivery of mitochondriotropic doxorubicin derivatives using self-assembling hyaluronic acid nanocarriers in doxorubicin-resistant breast cancer.

Breast cancer is the leading cause of cancer-related death for women, and multidrug resistance (MDR) is the major obstacle faced by chemotherapy for breast cancer. We have previously synthesized a doxorubicin (DOX) derivative by conjugating DOX with triphenylphosphonium (TPP) to achieve mitochondrial delivery, which induced higher cytotoxicity in drug-resistant breast cancer cells than DOX itself. Due to its amphiphilicity, TPP-DOX is difficult to physically entrap in nanocarriers. Thus, we linked it to hyaluronic acid (HA) by a novel ionic bond utilizing the specific bromide ion of TPP to form supra-molecular self-assembled structures (HA-ionic-TPP-DOX). The product was analyzed uisng 1H-NMR, 13C-NMR and mass spectrometry. The HA nanocarriers (HA-ionic-TPP-DOX) were shown to self-assemble into spherical nanoparticles, and sensitive to acidic pH in terms of morphology and drug release. Compared with free DOX, HA-ionic-TPP-DOX produced much greater intracellular DOX accumulation and mitochondrial localization, leading to increased ROS production, slightly decreased mitochondrial membrane potential, increased cytotoxicity in MCF-7/ADR cells and enhanced tumor targeting in vivo. In xenotransplant zebrafish model with the MCF-7/ADR cell line, both TPP-DOX and HA-ionic-TPP-DOX inhibited tumor cell proliferation without inducing significant side effects compared with free DOX. In addition, we observed a better anti-tumor effect of HA-ionic-TPP-DOX on MCF-7/ADR cells in zebrafish than that of TPP-DOX treatment. Furthermore, HA-ionic-DOX-TPP exhibited favorable biocompatibility and anti-tumor effects in MCF-7/ADR tumor-bearing nude mice in comparison with the effects of TPP-DOX and DOX, suggesting the potential of HA-ionic-TPP-DOX for the targeted delivery and controlled release of TPP-DOX, which can lead to the sensitization of resistant breast tumors.

Mitochondrial Targeted Doxorubicin-Triphenylphosphonium Delivered by Hyaluronic Acid Modified and pH Responsive Nanocarriers to Breast Tumor: in Vitro and in Vivo Studies.

Multidrug resistance (MDR) is the major obstacle for chemotherapy. In a previous study, we have successfully synthesized a novel doxorubicin (DOX) derivative modified by triphenylphosphonium (TPP) to realize mitochondrial delivery of DOX and showed the potential of this compound to overcome DOX resistance in MDA-MB-435/DOX cells. (1) To introduce specificity for DOX-TPP to cancer cells, here we report on the conjugation of DOX-TPP to hyaluronic acid (HA) by hydrazone bond with adipic acid dihydrazide (ADH) as the acid-responsive linker, producing HA- hydra-DOX-TPP nanoparticles. Hyaluronic acid (HA) is a natural water-soluble linear glycosaminoglycan, which was hypothesized to increase the accumulation of nanoparticles containing DOX-TPP in the mitochondria of tumor cells upon systemic administration, overcoming DOX resistance, in vivo. Our results showed HA- hydra-DOX-TPP to self-assemble to core/shell nanoparticles of good dispersibility and effective release of DOX-TPP from the HA- hydra-DOX-TPP conjugate in cancer cells, which was followed by enhanced DOX mitochondria accumulation. The HA- hydra-DOX-TPP nanoparticles also showed improved anticancer effects, better tumor cell apoptosis, and better safety profile compared to free DOX in MCF-7/ADR bearing mice.

MMP-2-Sensitive HA End-Conjugated Poly(amidoamine) Dendrimers via Click Reaction To Enhance Drug Penetration into Solid Tumor.

Currently, the limited penetration of nanoparticles remains a major challenge for antitumor nanomedicine to penetrate into the tumor tissues. Herein, we propose a size-shrinkable drug delivery system based on a polysaccharide-modified dendrimer with tumor microenvironment responsiveness for the first time to our knowledge, which was formed by conjugating the terminal glucose of hyaluronic acid (HA) to the superficial amidogen of poly(amidoamine) (PAMAM), using a matrix metalloproteinase-2 (MMP-2)-cleavable peptide (PLGLAG) via click reaction. These nanoparticles had an initial size of ∼200 nm, but once deposited in the presence of MMP-2, they experienced a dramatic and fast size change and dissociated into their dendrimer building blocks (∼10 nm in diameter) because of cleavage of PLGLAG. This rapid size-shrinking characteristic not only promoted nanoparticle extravasation and accumulation in tumors benefited from the enhanced permeability and retention effect but also achieved faster nanoparticle diffusion and penetration. We have further conducted comparative studies of MMP-2-sensitive macromolecules (HA-pep-PAMAM) and MMP-2-insensitive macromolecules (HA-PAMAM) synthesized with a similar particle size, surface charge, and chemical composition and evaluated in both monolayer cells and multicellular spheroids. The results confirmed that the enzyme-responsive size shrink is an implementable strategy to enhance drug penetration and to improve therapeutic efficacy. Meanwhile, macromolecule-based nanoparticles with size-variable characteristics not only promote drug penetration, but they can also be used as gene delivery systems, suggesting great potential as nano-delivery systems.

Fabrication and characterization of drug-loaded nano-hydroxyapatite/polyamide 66 scaffolds modified with carbon nanotubes and silk fibroin.

Nano-hydroxyapatite/polyamide 66 (nHA/PA66) porous scaffolds were fabricated by a phase inversion method. Carbon nanotubes (CNTs) and silk fibroin (SF) were used to modify the surface of the nHA/PA66 scaffolds by freeze-drying and cross-linking. Dexamethasone was absorbed to the CNTs to promote the osteogenic differentiation of bone mesenchymal stem cells (BMSCs). The cell viability of BMSCs was investigated by changing the concentration of the CNT dispersion, and the most biocompatible scaffold was selected. In addition, the morphology and mechanical property of the scaffolds were investigated. The results showed that the nHA/PA66 scaffolds modified with CNTs and SF met the requirements of bone tissue engineering scaffolds. The dexamethasone-loaded CNT/SF-nHA/PA66 composite scaffold promoted the osteogenic differentiation of BMSCs, and the drug-loaded scaffolds are expected to function as effective bone tissue engineering scaffolds.

[Mitochondrial drug delivery for cancer therapy].

Mitochondrion is one of the most vital organelles in cells of human body, and it is involved in many metabolic processes. Mitochondrion dysfunction is closely related to many diseases such as cancers, neurodegenerative diseases, obesity and ischemia reperfusion injury. As a result, mitochondrial drug delivery has gained more and more attention in the drug discovery against these diseases. This review gives a brief introduction to the relationship between mitochondria and human diseases(e.g., cancer), and summarizes the latest trend of mitochondrial targeting drug delivery system(MTDDS).

ScreenFect A: an efficient and low toxic liposome for gene delivery to mesenchymal stem cells.

Mesenchymal stem cells (MSCs) hold great promise in variety of therapeutic applications including tissue engineering and cancer therapy. Genetic modification of MSCs can be used to enhance the therapeutic effect of MSCs by facilitating a specific function or by transforming MSCs into more effective gene therapy tools. However, the successful generation of genetically modified MSCs is often limited by the poor transfection efficiency or high toxicity of available transfection reagents. In our previous study, we used thiol-yne click chemistry to develop new liposomal vectors, including ScreenFect(®) A (SF) (Li et al., 2012). In this study, we investigated the transfection performance of SF on MSCs. A comparative evaluation of transfection efficiency, cell viability and cellular DNA uptake was performed using the Lipofectamine™ 2000 (L2K) as a control, and the results show that SF is superior to L2K for MSC transfection. The presence of serum did not significantly influence the transfection efficiency of either SF or L2K but greatly reduced the viability of MSC transfected by L2K. The higher efficiency of SF-mediated transfection compared to L2K was also correlated with better proliferation of cells. These results were supported by monitoring the intracellular fate of DNA, which confirmed stable transportation of DNA from lysosomes and efficient nuclear localization. TGF-ß1 gene delivery by SF promoted MSC osteogenic differentiation in an osteogenic induction condition. As the first study of SF lipofection on stem cells, this study highlights a promising role of SF in gene delivery to MSCs as well as other stem cells to facilitate tissue engineering and other therapeutic effects based on genetically modified stem cells.

The TLR3, PI3K, survivin, FasL, and Fas genes as major risk factors of occurrence and development of cervical cancer disease.

To investigate the role of TLR3/PI3K signals in the occurrence and development of cervical cancer disease, TLR3-siRNA was used to block key signaling pathways involved in cervical cancer metastasis that are pivotal to metastatic tumor cells but not to normal cells under ordinary physiologic conditions. Results show that tumor U14 cell growth, migration and invasion in TLR3-siRNA treatment group were significantly decreased. Through LY294002 suppressing targeted gene, the LY294002 treatment specifically and significantly knocked down the expressions of tumor TLR3 and PI3K proteins in cervical cancer mice. Furthermore, expressions of tumor Survivin and FasL proteins were markedly suppressed, whereas expressions of Fas protein were upregulated in LY294002 treatment group mice. LY294002 treatment suppressed tumor growth and increased the thymus and spleen indeces and survival days of cervical cancer mice. This study demonstrates that TLR3-siRNA and LY294002 treatments can markedly suppress cervical cancer cell invasion and tumor growth and increase survival life by silencing targeted genes.

[Correction of secondary lip whistle deformities and nasal base depression after bilateral cleft lip repair with lip subdermal soft tissue flap].

OBJECTIVE: To explore a new method to correct secondary lip whistle deformities and nasal base depression after bilateral complete cleft lip (BCCL) repair with lip subdermal soft tissue flap. METHODS: Bilateral subdermal soft tissue "C" flaps and "lambda" flap were designed to repair secondary deformities of nasal base and reconstruct vermilion tubercle in patients after BCCL repair. RESULTS: Good results were achieved in all the patients with primary healing. No flap necrosis happened. The result was satisfactory. CONCLUSIONS: With bilateral subdermal soft tissue "C" flaps and " lambda" flap, nasal base depression deformities and lip whistle deformities can be corrected. It is an ideal method for correction of deformities after BCCL repair.

[Expressions of MACC1, HGF, and C-met protein in epithelial ovarian cancer and their significance].

OBJECTIVE: To investigate the expressions of metastasis-associated in colon cancer-1 (MACC1), hepatocyte growth factor (HGF), and C-met proteins in epithelial ovarian cancer and their significance. METHODS: The expressions of MACC1, HGF and C-met in 20 specimens of normal ovarian tissues, 19 specimens of benign epithelial ovarian tumor and 52 specimens of epithelial ovarian cancer were measured by immunohistochemistry and Western blotting. The correlations of the expressions of MACC1, HGF and C-met protein to the clinicopathologic characteristics of epithelial ovarian cancer were analyzed, and the correlations between the expressions of the 3 proteins were also evaluated. RESULTS: The positivity rates of MACC1, HGF and C-met proteins were 73.1%, 63.5% and 78.8% in epithelial ovarian cancer with relative expressions of 0.72∓0.05, 0.64∓0.04 and 0.79∓0.04, respectively, showing significant differences from those in normal ovarian tissues and benign ovarian tumors (P<0.05). In epithelial ovarian cancer, the up-regulation of MACC1, HGF and C-met expressions were associated with advanced FIGO stage, poor differentiation and lymph node metastasis (P<0.05). MACC1 expression was positively correlated to HGF (r=0.350, P=0.011) and C-met expressions (r=0.429, P=0.002), and the latter two was also positively correlated (r=0.487, P=0.000). CONCLUSIONS: MACC1 may serve as a potential biomarker for advanced ovarian cancer. Deregulation of MACC1, HGF and C-met proteins may synergistically participate in the malignant progression of epithelial ovarian cancer.

[Expression of TGF-beta1 and E-cadherin in primary and metastatic ovarian carcinoma].

OBJECTIVE: To detect the expression of the protein of TGF-beta1 and E-cadherin in the primary and metastatic lesions of ovarian carcinoma and explore the mechanism of the metastasis of ovarian carcinoma. METHODS: Immunohistochemistry (IHC) was performed to detect the expression of TGF-beta1 and E-cadherin proteins in primary and metastatic ovarian carcinoma, benign epithelial ovarian tumor and normal ovarian tissue. RESULTS: The expression of TGF-beta1 was significantly higher in ovarian carcinoma (67.2%) than in benign tumors (28.6%) and normal ovarian tissue (18.9%) (Chi2=26.94, P<0.001), but E-cadherin expression showed a reverse pattern. TGF-beta1 expression in the primary ovarian carcinoma carcinoma was associated with the FIGO stage, lymph metastasis and ascites of the tumor (P=0.01, P=0.01, and P=0.04, respectively). E-cadherin expression in the tumor was associated with the differentiation (P=0.02) and lymph metastasis of ovarian carcinoma (P=0.04). The expressions of TGF-beta1 and E-cadherin were all significantly lower in the primary tumors than in the metastatic tumor (Chi2=4.70, P=0.03; Chi2=5.91, P=0.015). A significant correlation was found between the expressions of the TGF-beta1 and E-cadherin in the primary carcinoma (Kappa value of -0.32, P=0.01). CONCLUSION: TGF-beta1 and E-cadherin are closely associated with the metastasis of ovarian carcinoma and might be potential targets for controlling the metastasis of ovarian carcinoma.

[Comparison of microbial community structure in MBRs treating different wastewater].

In order to investigate the microbial community structures in different membrane bioreactors, total bacterial genomic DNA was extracted from biomass in four MBRs treating different wastewater. The microbial community structures were studied by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and cloning-sequencing. Furthermore, and the sequences were used for homology analysis and then two phylogenetic trees were constructed. Results indicate that each MBR owns its specific ecological community after a long-term of operation. Influent wastewater composition has a significant impact on total bacterial community structures. Population diversity, which is in the bioreactor fed with more complex components wastewater, is higher than those of other MBRs. The Shannon Index are 0.77 and 0.78. Proteobacteria and Bacillus, which contain 8 OUTs and 2 OUTs, are the climax communities in the investigable MBRs. The profiles of ammonia-oxidizing bacterial community in the four reactors are very similar. There may exist several modes of nitrification and denitrification in MBR because sequences revealed that there exist multitude nitrifying organisms, in which Nitrosomonas are the most predominant, and two denitrifying bacterium (uncultured Achromobacter sp. and uncultured denitrifying bacterium)in reactors.