Clinical features and prognosis of advanced intra- and extra-pulmonary neuroendocrine carcinomas.

Objective: We examined the clinical features and prognosis of advanced intra- and extra-pulmonary neuroendocrine carcinomas (NECs) to offer additional guidance for the clinical treatment of small-cell lung cancer (SCLC), which is a type of advanced intrapulmonary NEC (IPNECs). Materials and Methods: The clinical data and survival of 123 patients with advanced IPNECs and extrapulmonary NECs (EPNECs) were obtained. We retrospectively examined the corresponding clinical diagnosis and treatment and investigated the significant factors influencing the survival prognosis of patients with NECs. Results: There were 90 cases of IPNECs (including 81 cases of SCLC), and 33 cases of EPNECs. The median overall survival (OS) of IPNECs was significantly longer than that of the EPNECs in the gastrointestinal tract and in the other regions (P < 0.05). The median OS of patients with other IPNECs was longer than that of patients with SCLC (P > 0.05). Multivariate analysis demonstrated that age, liver metastasis, number of cycles of first-line chemotherapy, and chest radiotherapy were risk factors influencing OS in patients with NECs (P < 0.05). Conclusions: The survival of IPNECs was significantly longer than that of EPNECs in the gastrointestinal tract and other regions. Nevertheless, patients with advanced NECs who were older and had liver metastases had a poorer prognosis. Multidisciplinary treatments including multicycle chemotherapy and a combination of chemotherapy and radiotherapy should function significantly in extending the survival of NECs.

Iridoid from Eucommia ulmoides Oliv. Exerts Antiarthritis Effects by Inhibiting the JAK2/STAT3 Signaling Pathway In Vivo and In Vitro.

The purpose of this study was to investigate the anti-inflammatory effects of EU-Idd both in vivo and in vitro. In vivo, we used the collagen-induced arthritis (CIA) rat model to investigate the efficacy of EU-Idd on rheumatoid arthritis. Hematoxylin-eosin staining and Safranin O-fast green staining were used to evaluate the pathological status of the ankle joints in CIA rats. Micro-CT scanning was used to investigate bone erosion of the ankle joints. In vitro, the effect of EU-Idd on Th17 cell differentiation was identified by flow cytometry. TRAP staining was used to detect osteoclast cells. HFLS-RA model cells, induced by tumor necrosis factor-α(TNF-α), were used to evaluate the anti-inflammatory effects of EU-Idd while the levels of related inflammatory cytokines and JAK2/STAT3 proteins were detected by RT-qPCR and western blotting. EU-Idd alleviated joint inflammation in CIA rats and exerted protective effects on the ankle joints. EU-Idd also prevented the differentiation of CD4+ T cells into Th17 cells, reduced the number of osteoclasts, and improved the expression levels of bone metabolism-related proteins including OPG and RANKL. Moreover, EU-Idd inhibited the invasion and migration of HFLS-RA cells and downregulated the expression of related inflammatory cytokine genes and the protein expression levels of p-JAK2 and p-STAT3, both in vivo and in vitro. EU-Idd exerts anti-inflammatory and osteoprotective effects by regulating the JAK2/STAT3 pathway in rheumatoid arthritis. These results are beneficial to excavate new pharmaceutical ingredients for rheumatoid arthritis from iridoid.

Follow-Up of Adefovir Dipivoxil Induced Osteomalacia: Clinical Characteristics and Genetic Predictors.

Adefovir dipivoxil (ADV) is widely used for chronic hepatitis B therapy in China. To explore the clinical features and prognosis of ADV-induced osteomalacia and to analyze the association between osteomalacia and genetic variants in 51 drug transporters genes. Clinical and follow-up data of the ADV-treated patients were collected. Target capture sequencing was used to identify genetic variations of 51 drug transporter genes. A total of 193 hepatitis B patients treated with ADV were enrolled, of whom 140 had osteomalacia. The other 53 without osteomalacia were included in the control group. The median duration of ADV treatment before the onset of osteomalacia was 6.5 years (range:1.5-7 years). We found that most patients with osteomalacia had hypophosphatemia, high serum alkaline phosphatase levels, hypouricemia, nondiabetic glycosuria, proteinuria. Stopping ADV administration, supplementing calcitriol and calcium were effective treatments. During 3-6 months of follow-up, the clinical symptoms and biochemical indicators of patients with osteomalacia have been significantly improved. There was no significant difference in duration of adefovir treatment in patients with or without osteomalacia (p = 0.791). Through regression analysis, we found that age was a risk factor for osteomalacia [per 1 year, odds ratio (OR), 1.053; 95% confidence interval (95% CI), 1.020-1.087; p = 0.015]. 1992 single nucleotide variants were found using target capture sequencing. However, the associations of genetic variants of 51 drug transporter genes and the risk of osteomalacia were negligible. Osteomalacia is prone to occur in patients with chronic hepatitis B treated with long-term ADV at a therapeutic dose. After standard treatment, the prognosis is mostly good. We failed to find genetic variants that can predict the risk of ADV-induced osteomalacia.

Inhibition of Rheumatoid Arthritis Using Bark, Leaf, and Male Flower Extracts of Eucommia ulmoides.

Eucommia ulmoides Oliv., a native Chinese plant species, has been used as a traditional Chinese medicine formulation to treat rheumatoid arthritis (RA), strengthen bones and muscles, and lower blood pressure. Various parts of this plant such as the bark, leaves, and flowers have been found to have anti-inflammatory properties. E. ulmoides has potential applications as a therapeutic agent against bone disorders, which were investigated in this study. In vitro, RA joint fibroblast-like synoviocytes (RA-FLS) were treated with different concentrations (0, 25, 50, 100, 200, 400, 800, and 1000 µg/mL) of E. ulmoides bark, leaf, and male flower alcoholic extracts (EB, EL, and EF, respectively) to determine their potential cytotoxicity. Tumor necrosis factor- (TNF-) α and nitric oxide (NO) levels in RA-FLS were quantified using enzyme-linked immunosorbent assay (ELISA). Furthermore, collagen-induced arthritis (CIA) rats were treated with EB, EL, EF, Tripterygium wilfordii polyglycoside (TG) or the normal control (Nor), and then ankle joint pathology, bone morphology, and serum and spleen inflammatory cytokine levels were evaluated. The results showed that, in RA-FLS, EB, EL, and EF were not cytotoxic; EB and EF reduced TNF-α supernatant levels; and EB, EL, and EF reduced NO levels. The results of in vivo experiments showed that EB, EL, and EF alleviated ankle swelling and joint inflammation, while all extracts diminished inflammatory cell infiltration, pannus and bone destruction, and bone erosion. All tested extracts inhibited interleukin- (IL-) 6, IL-17, and TNF-α mRNA in the spleen of CIA rats, while EB most effectively reduced osteoclasts and inhibited bone erosion. EF showed the most obvious inhibition of inflammatory factors and pannus. Thus, EB, EL, and EF may alleviate bone destruction by inhibiting inflammation.

Effects of betulinic acid on synovial inflammation in rats with collagen-induced arthritis.

Betulinic acid (BA) inhibits the migration, invasion, and cytoskeletal reorganization of fibroblast-like synoviocytes (RA-FLS) in patients with rheumatoid arthritis. Here, to further explore the mechanism of action of BA in collagen-induced arthritis (CIA) rats, we investigated the pharmacodynamic effects of BA on synovial inflammation in a rat model of type II CIA. After inducing hind paw swelling, the rats were divided into four groups: healthy controls (normal), and rats that underwent CIA and received methotrexate treatment (MTX), BA treatment (BA), or no treatment (CIA). Body weight and hind paw swelling were determined regularly, and arthritis scores were calculated weekly. On day 35, rats were sacrificed and their hind ankle joints sectioned and stained with hematoxylin and eosin for histopathological evaluation. BA significantly reduced CIA-induced hind paw swelling, synovial tissue proliferation, cartilage destruction, and vasospasm. BA treatment also decreased serum interleukin (IL)-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α) levels in rats with CIA. The CCK-8 assay was used to detect the proliferation of isolated vimentin+CD68- RA-FLS; RA-FLS were stimulated with TNF-α in vitro. BA significantly inhibited TNF-α-stimulated RA-FLS proliferation, as well as IL-1ß and IL-6 secretion. BA also downregulated the transcription of vascular endothelial growth factor (VEGF) and transforming growth factor ß (TGF-ß) and decreased the expression of the NF-кB pathway proteins (NF-kB-P65, IkBα, and IKKα/ß) in the TNF-α-stimulated RA-FLS. These results indicate that BA alleviated the symptoms of CIA by inhibiting synoviocyte proliferation, modifying TNF-α- and NF-кB-related inflammatory pathways, and downregulating inflammatory mediators and growth factors including IL-1ß, IL-6, VEGF, and TGF-ß.

MicroRNA-153-3p enhances the sensitivity of chronic myeloid leukemia cells to imatinib by inhibiting B-cell lymphoma-2-mediated autophagy.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease caused by abnormal DNA replication of bone marrow stem cells and chemotherapy resistance is a major obstacle to the effective treatment of patients with CML. Imatinib (IM), a tyrosine kinase inhibitor (TKI), is a first-line drug clinically used for CML. Mounting evidence has indicated that the dysregulation of microRNAs (miRNAs) is associated with the chemoresistance of CML. In this study, miR-153-3p, which had been implicated with numerous types of tumors, was identified to be downregulated in IM-resistant CML cells. Upregulation of miR-153-3p significantly increased IM sensitivity and decreased the survival rate of IM-resistant CML cells, whereas downregulation of miR-153-3p attenuated these effects in IM-resistant CML cells. Upregulated miR-153-3p could decrease the autophagy caused by IM in IM-resistant CML cells. Dual-luciferase reporter assays confirmed that Bcl-2 is a direct target of miR-153-3p. Bcl-2 restoration reversed the increased sensitivity to IM induced by miR-153-3p-mimic transfection in IM-resistant CML cells. The results of the present study showed that dysregulated miR-153-3p may target Bcl-2 to promote the development of IM resistance and attenuate IM-induced apoptosis in CML. Therefore, miR-153-3p upregulation combined with IM treatment may serve as a promising therapeutic strategy for patients with low sensitivity.

MicroRNA-21 attenuates BDE-209-induced lipid accumulation in THP-1 macrophages by downregulating Toll-like receptor 4 expression.

Growing evidence demonstrates a possible response of specific microRNA (miRNA) to environmental pollutant stimuli in multiple biological processes. We previously reported that a persistent organic pollutant, decabromodiphenyl ether (BDE-209), can enhance Toll-like receptor 4 (TLR4)-dependent lipid uptake in THP-1 macrophages; whether miRNAs are involved in this process remains unclear. In the present study, we investigated the levels of several miRNAs related to TLR4 signaling, including miRs-9, -21, -27b, -125b, -132, -146a, -147, -155, and -let-7e, in THP-1 macrophages after stimulation by BDE-209 and oxidized low-density lipoprotein. The results showed that the levels of miR-21 were significantly suppressed by BDE-209 at concentrations of 6.25, 12.5 and 25 µM, in a dose-dependent manner; whereas there was no significant changes for the other miRNAs investigated. Moreover, the suppression of miR-21 was accompanied by an upregulated TLR4 expression, at both mRNA and protein levels. Further analysis showed that the up-regulated TLR4 induced by BDE-209 was inhibited in macrophages transfected with miR-21 mimic; meanwhile opposite results were exhibited when an anti-miR-21 inhibitor was transfected to the macrophages. Additionally, transfection with miR-21 mimic effectively attenuated BDE-209-induced lipid accumulation in macrophages. Together, these data illustrate that miR-21 inhibits BDE-209-triggered lipid accumulation in macrophages through down-regulating TLR4 expression.

Decarbromodiphenyl ether (BDE-209) promotes monocyte-endothelial adhesion in cultured human aortic endothelial cells through upregulating intercellular adhesion molecule-1.

There is growing evidence that exposure to persistent organic pollutants (POPs) is statistically associated with incidence of cardiovascular disease (CVD) or its risk factors. Decarbromodiphenyl ether (BDE-209) is a new POP which exists extensively in human tissues, but its potential effects on CVD have so far received less focus. The adhesion of circulating monocytes to endothelial cells is one of the critical underlying steps in the initiation and development of CVD. In the present study, we investigated the effect of BDE-209 on the adhesion of THP-1 monocytes to human aortic endothelial cells (HAECs) and identified the molecular mechanisms involved. Our results showed that 6.25, 12.5 and 25 µM of BDE-209 exposures caused significant increases in monocyte-endothelial cell adhesion, in a dose-dependent manner. Mechanistically, BDE-209 exposure increased the expression of intercellular adhesion molecule-1 (ICAM-1). Moreover, the up-regulation of ICAM-1 was accompanied by a decrease in the expression of microRNA-141 (miR-141). Furthermore, the up-regulation of ICAM-1 and the increased adhesion induced by BDE-209 could be reversed by miR-141 supplement. Taken together, our results show that BDE-209 potentiates monocyte-endothelial cell interaction via miR-141/ICAM-1 pathway in HAECs.

Decabromodiphenyl ether (BDE-209) enhances foam cell formation in human macrophages via augmenting Toll-like receptor 4-dependent lipid uptake.

Growing epidemiological evidence is substantiating an association between exposure to persistent organic pollutants (POPs) and incidence of atherosclerosis. Decabromodiphenyl ether (BDE-209) is a new POP which presents extensively in human populations; whether this contaminant is potentially arteriosclerotic remains unclear. In this study, we investigated the effects of BDE-209 on macrophage-derived foam cell formation, a hallmark of early atherosclerosis, using THP-1-derived macrophages incubated with oxidized low-density lipoprotein (oxLDL) as a foam cell model. The results showed that 6.25, 12.5 and 25.0 µM of BDE-209 significantly enhanced lipid accumulation inside the foam cells, in a dose-dependent manner. Further mechanism assays suggested that BDE-209 significantly increased the expression of Toll-like receptor 4 (TLR4), a signal transducing integral membrane protein mediating lipid uptake in macrophages, at both the mRNA and protein levels. In contrast, there was no significant changes for several key regulators involving in lipid efflux, lipogenesis, and lipid oxidation in macrophages. Furthermore, the augmented lipid accumulation was almost completely abrogated by treatment with an anti-TLR4 antibody. Together, these data illustrate that BDE-209 enhances oxLDL-induced macrophage foam cell formation via augmenting TLR4-dependent lipid uptake in the cells.

ILK enhances migration and invasion abilities of human endometrial stromal cells by facilitating the epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) plays a significant part in the pathogenesis of endometriosis by facilitating the migration and invasion abilities of cells. Integrin-linked kinase (ILK) increases the cell migration and invasion abilities by inducing the EMT. Eutopic and control endometrial stromal cells (EuSCs and CSCs) were isolated and cultured. Cell migration and invasion abilities were detected by transwell assays. Levels of proteins were detected by Western blot. EuSCs showed higher levels of ILK, N-cadherin, vimentin and stronger migration and invasion abilities. After transfection of siRNA-ILK, E-cadherin and keratin levels were increased while N-cadherin and vimentin levels were decreased in EuSCs. Besides that, the migration and invasion abilities of EuSCs were significantly decreased after transfection of siRNA-ILK. On the contrary, levels of ILK, N-cadherin and vimentin were increased while levels of E-cadherin and keratin were decreased simultaneously after transfecting CSCs with pEGFP-C1-ILK. Simultaneously, the migration and invasion abilities of CSCs were increased after transfection of pEGFP-C1-ILK. Our study verified that high expression of ILK enhanced the migration and invasion abilities of ESCs by facilitating the EMT. Given that ILK played crucial roles in the pathogenesis of endometriosis, it may be considered as a promising targeted therapy for endometriosis.

Anti-glioma Activity of Dapsone and Its Enhancement by Synthetic Chemical Modification.

The sulfone dapsone is an old antibiotic used for the treatment of mycobacterial and protozoal infections. We postulated before that dapsone might possess biological activity exceeding its anti-infectious properties and that it could potentially be repurposed for the treatment of glioma. To test this hypothesis, we treated established and primary cultured glioma cells with dapsone or several dapsone analogues which we previously synthesized (D2-D5) and determined effects on proliferation, anchorage-independent growth and migration. While dapsone and its synthetic analogues D2-D5 displayed only modest anti-proliferative activity, important neoplastic features such as anchorage-independent growth, clonogenic survival and directed migration were significantly inhibited by dapsone treatment. Moreover, dapsone analogues D3, D4 and D5 yielded even enhanced anti-glioma activity against different pro-neoplastic features. Overall these data suggest that dapsone provides activity against glioma which can be further enhanced by molecular modifications. These compounds could potentially serve as a therapeutic adjunct to the treatment of gliomas in a repurposing approach.

Immunogenicity of a recombinant Sendai virus expressing the capsid precursor polypeptide of foot-and-mouth disease virus.

In this study, SeV was used as a vector to express capsid precursor polypeptide (P1) of Foot-and-mouth disease virus (FMDV) by using reverse genetics. The rescue recombinant SeV (rSeV-P1) can efficiently propagate and express P1 protein by Western blot and IFA analysis. To evaluate the immunogenicity of rSeV-P1, BALB/c mice were divided into several groups and immunized intramuscularly with various doses of rSeV-P1, rSeV-eGFP, PBS and commercial FMD vaccine, respectively, and then challenged with an intraperitoneal injection of 1 × 10(6) TCID50 of virulent serotype O FMDV O/ES/2001 strain 4 weeks after booster immunization. Mice vaccinated with rSeV-P1 acquired FMDV-specific ELISA antibodies, neutralizing antibodies as well as cellular immune response. Meantime, mice immunized with rSeV-P1 (dose-dependent) had the ability to inhibit the replication of FMDV in the sera after FMDV challenge. Our results indicated that the recombinant SeV-P1 virus could be utilized as an alternative strategy to develop a new generation of safety and efficacious vaccine against FMDV infection.

A natural product, resveratrol, protects against high-glucose-induced developmental damage in chicken embryo.

Resveratrol, a famous plant-derived polyphenolic phytoalexin, has been considered to play physiological roles such as antioxidative, neuroprotective, and anticancer effects in adults. However, its antioxidative activity and neuroprotective effect were seldom discussed in the embryonic system. In this study, the effect of resveratrol on chicken embryo development under high glucose and its underlying mechanism of resveratrol were investigated. High glucose administrated to chicken embryo at embryonic Day 1 induced stillbirth, growth retardation, and impaired blood vessel development on yolk sac. However, resveratrol supplementation before glucose exposure showed significant effect on decreasing the death rate, developmental damage, and vessel injury. In addition, oxidative stress was caused by high-glucose exposure, and resveratrol could rescue this high-glucose-induced oxidative stress. Moreover, the neural developmental marker paired box 3 was significantly decreased by high glucose and recovered by resveratrol. Cell cycle-regulated gene expression was also intervened by resveratrol. This study had found an association between resveratrol and hyperglycemia-induced embryonic damage, which suggested a potential protective effect of resveratrol on gestational diabetes.

[Evaluation on contribution rate of each component total salvianolic acids and characterization of apparent oil/water partition coefficient].

The difference between three representative components of total salvianolic acids in pharmacodynamic activity were compared by three different pharmacological experiments: HUVECs oxidative damage experiment, 4 items of blood coagulation in vitro experiment in rabbits and experimental myocardial ischemia in rats. And the effects of contribution rate of each component were calculated by multi index comprehensive evaluation method based on CRITIC weights. The contribution rates of salvianolic acid B, rosmarinic acid and Danshensu were 28.85%, 30.11%, 41.04%. Apparent oil/water partition coefficient of each representative components of total salvianolic acids in n-octyl alcohol-buffer was tested and the total salvianolic acid components were characterized based on a combination of the approach of self-defined weighting coefficient with effects of contribution rate. Apparent oil/water partition coefficient of total salvianolic acids was 0.32, 1.06, 0.89, 0.98, 0.90, 0.13, 0.02, 0.20, 0.56 when in octanol-water/pH 1.2 dilute hydrochloric acid solution/ pH 2.0, 2.5, 5.0, 5.8, 6.8, 7.4, 7.8 phosphate buffer solution. It provides a certain reference for the characterization of components.

[Clinical study of 32 patients with adult Philadelphia chromosome-positive acute lymphoblastic leukemia].

This study was aimed to evaluate the efficacy and safety of imatinib in the treatment of patients with adult Ph chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL). A total of 32 diagnosed adult Ph(+)ALL patients from July 2007 to February 2014 in our hospital were retrospectively analyzed and were divided into two groups: imatinib plus chemotherapy group and traditional chemotherapy group. The differences between two groups were analysed in disease-free survival time (DFS), overall survival time (OS) and toxicity. The G banding technigue was used to analyse the karyotype, and the flow cytometry was applyed to detect the immune markers on surface of cells. The results showed that all patients expressed B cell and hematopietic stem/progenitor cell immune markers, out of them 21 patients (65.6%) were with myeloid antigens, 27 patients with simple Ph (+) phenotype and 5 patients with additional chromosome abnormality. The DFS and OS of the imatinib group were statistically longer than those of the traditional chemotherapy group (14.3 ± 4.7 months vs 10.7 ± 3.8 months) (P < 0.05) and 22.6 ± 6.8 months vs 10.7 ± 3.8 months) (P < 0.05)). There was no significant difference in toxic effects between two groups (P > 0.05)). It is concluded that the all cases of adult Ph(+)ALL are with B cell phenotype and express hematopietic stem/progenitor cell antigen. They often accompanied by expression of myeloid antigens and additonal chromosome abnormality in genetics. The combination of imatinib with chemotherapy can prolong remission time and survival time for patients of non-hematopietic stem cell transplantation on the basis of no notably increasing the toxic effects.

[Studies on hydroxyapatite applicatied in coprecipitate of total salvianolic acids phospholipid complex].

The purpose of this research was to prepare total salvianolic acids-phytosome-HA coprecipitate to improve drug dissolution and its micromeritic properties. Firstly, the coprecipitate was prepared by solvent method and in vitro dissolution of tripterine was performed with the salvianolic acid B and danshensu as criteria. At the same time, the micromeritic properties was characterizated, the structure of samples was characterized by TEM, DSC, XRD and FTIR. Results showed that when the ratio of drug to HA was 1:2, it had a better dissolution, the accumulative drug-release percent in vitro at 60 min was over 90%. At the same time, it has good liquidity and low moisture absorption. Its micromeritic properties have improved. It is proved that the drug still existed amorphously by microstructure analysis. The preparation process is simple and feasible, it has practical value.

[Study on preparation of salvianolic acid phospholipid compound].

To prepare salvianolic acid phospholipid compound. With the compound of salvianolic acids and soybean phospholipid as the index, mono-factor experiment and orthogonal design experiment were conducted to screen its technical parameters. According to the results, the optimal preparation conditions of salvianolic acid phospholipid compound were that THF were taken as the reaction solvent, the concentration time was 3 h, the reactant concentration was 5 g x L(-1), the mass ratio of salvianolic acids and phospholipid was 1: 1.5, and the reaction temperature was 40 degrees C. The oil/water partition coefficient of the prepared salvianolic acid phospholipid compound significant increased in water and buffers with different pH values. The results of phase analysis such as DSC, XRD and FTIR indicated that salvianolic acids existed in phospholipid in an amorphous state.

[Influence of recombinant lentiviral vector encoding miR-15a/16-1 in biological features of human nasopharyngeal carcinoma CNE-2Z cells].

OBJECTIVE: To study the influence of recombinant lentiviral vector encoding miR-15a/16-1 on biological features of human nasopharyngeal carcinoma CNE-2Z cells and underlying mechanisms. METHODS: GFP-positive CNE-2Z cells transfected with recombinant lentiviral vector were selected. The experiment was divided into control group, transfected group, radiotherapy group, transfected-radiotherapy group. Cell proliferation was analyzed by MTT. Apoptosis was detected by flow cytometry. Radiotherapy sensitivity of the cells in control group and transfected group was evaluated by colony forming experiment. The expressions of miR-15a, miR-16-1 and bcl-2 mRNA were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The expression of bcl-2 protein was detected by Western blot. The activation of Caspase-2 and Caspase-3 was evaluated by spectrophotometry. RESULTS: Relative expression quantities of miR-15a and miR-16-1 in infected group were 524.80 ± 40.79 (t = 494.611, P = 0.000) and 466.11 ± 40.96 (t = 386.8, P = 0.000), respectively. The proliferation of the cells in transfected-radiotherapy group was the most obvious, followed by the cells in radiotherapy group and transfected group (F = 424.3, P = 0.000). The apoptosis rates of control group, transfected group, radiotherapy group and transfected-radiotherapy group were (2.2 ± 1.4)%, (9.6 ± 0.8)%, (2.9 ± 1.1)%, and (18.6 ± 0.7)% respectively(F = 158.5, P = 0.000). Clonogenic assay showed that the values of SF2, Do (1.473) and Dq (1.581) in transfected group were lower than those in control group. The relative expression levels of bcl-2 mRNA in transfected group, radiotherapy group, and transfected-radiotherapy group had no significant difference (P > 0.05). Decrease in the expression of bcl-2 protein in transfected-radiotherapy group was most significantly, followed by that in transfected group. The percentages of activated Caspase-2 in control group, radiotherapy group, transfected group and transfected -radiotherapy group were 0.12 ± 0.01, 0.24 ± 0.04, 0.35 ± 0.02, and 0.44 ± 0.04, respectively (F = 115.500, P = 0.000). The percentages of activated Caspase-3 in the groups were 0.13 ± 0.01, 0.27 ± 0.01, 0.43 ± 0.02, and 0.83 ± 0.06, respectively (F = 439.921, P = 0.000). CONCLUSIONS: Recombinant lentiviral vector LV-miR15a/16-1 could improve the expression of miR-15a and miR-16-1 in CNE-2Z cells, inhibit the proliferation of CNE-2Z cells, promote apoptosis and enhance the sensitivity of the cells to radiotherapy probably by inhibiting bcl-2 expression, activating Caspase-2 and Caspase-3.

A novel drug-phospholipid complex enriched with micelles: preparation and evaluation in vitro and in vivo.

Mixed micelles are widely used to increase solubility and bioavailability of poorly soluble drugs. One promising antitumor drug candidate is 20(S)-protopanaxadiol (PPD), although its clinical application is limited by low water solubility and poor bioavailability after oral administration. In this study, we developed mixed micelles consisting of PPD-phospholipid complexes and Labrasol(®) and evaluated their potential for oral PPD absorption. Micelles were prepared using a solvent-evaporation method, and their physicochemical properties, including particle size, zeta potential, morphology, crystal type, drug loading, drug entrapment efficiency, and solubility, were characterized. Furthermore, in vitro release was investigated using the dialysis method, and transport and bioavailability of the mixed micelles were investigated through a Caco-2 cell monolayer and in vivo absorption studies performed in rats. Compared with the solubility of free PPD (3 µg/mL), the solubility of PPD in the prepared mixed micelles was 192.41 ± 1.13 µg/mL in water at room temperature. The in vitro release profiles showed a significant difference between the more rapid release of free PPD and the slower and more sustained release of the mixed micelles. At the end of a 4-hour transport study using Caco-2 cells, the apical-to-basolateral apparent permeability coefficients (P(app)) increased from (1.12 ± 0.21) × 10(6) cm/s to (1.78 ± 0.16) × 10(6) cm/s, while the basolateral-to-apical P(app) decreased from (2.42 ± 0.16) × 10(6) cm/s to (2.12 ± 0.32) × 10(6). In this pharmacokinetic study, compared with the bioavailability of free PPD (area under the curve [AUC](0-∞)), the bioavailability of PPD from the micelles (AUC(0-∞)) increased by approximately 216.36%. These results suggest that novel mixed micelles can significantly increase solubility, enhance absorption, and improve bioavailability. Thus, these prepared micelles might be potential carriers for oral PPD delivery in antitumor therapies.

[Construction of recombinant adenovirus expressing shRNA targeting human leukocyte-derived arginine aminopeptidase gene].

AIM: To construct the recombinant adenovirus expressing small hairpin RNA (shRNA) targeting human leukocyte-derived arginine aminopeptidase (LRAP) gene and silence the expression of LRAP in human retinal microvascular endothelial cells (HRMECs). METHODS: Three pairs of oligonucleotides coding for shRNAs targeting human LRAP gene were designed and synthesized, and were cloned into the shuttle vector pRNAT-H1.1/Adeno after annealing. The three constructed shuttle plasmids, through homologous recombination with adenoviral backbone vector pAdEasy-1, were transformed into E.coli BJ5183-AD- to produce recombinant adenoviral plasmids. And then the recombinants linearized with Pac I were transfected into 293a cells to package adenoviruses. After amplification and titter determination, the recombinant adenoviruses were used to infect the primary HRMECs. Quantitative real-time PCR was taken to determine the relative amount of LRAP mRNA to screen the adenovirus with the highest silencing efficiency. The level of LRAP protein after RNA interference in HRMECs was further determined by Western blotting. RESULTS: PCR, restriction digestion and DNA sequencing confirmed that the purpose shRNA-coding sequences were correctly inserted into the shuttle vectors and adenoviral plasmids, and the recombinant adenoviruses were packaged successfully in 293a cells. The most effective adenovirus with the silencing efficiency up to 79% was selected by quantitative real-time PCR, which significantly lowered the expression of LRAP in HRMECs. CONCLUSION: The recombinant adenovirus expressing shRNA effectively silencing LRAP gene was constructed successfully, which would facilitate further study of the role that LRAP plays in the development of diabetic retinopathy.