Lipoma preferred partner (LPP) has been identified as a protein which is highly selective for smooth muscle progenitor cells (SMPCs) and regulates differentiation and migration of SMPCs, but mechanisms of LPP expression are not elucidated clearly. The aim of the present study was to discuss the mechanisms by which LPP expression is regulated in the differentiation and migration of SMPCs induced by TGF-β1. It was found that TGF-β1 could significantly increase the expression of LPP, smooth muscle α-actin, smooth muscle myosin heavy chain (SM-MHC), and smoothelin in SMPCs. Moreover, inactivation of Rho kinase (ROK) with ROK inhibitors significantly inhibited LPP mRNA expression in TGF-β1-treated SMPCs and mouse aortic smooth muscle cells (MAoSMCs). At the same time, LPP silencing with short interfering RNA significantly decreased SMPCs migration. In conclusion, LPP appears to be a ROK-dependant SMPCs differentiation marker that plays a role in regulating SMPCs migration.
Mitochondrial DNA (mtDNA) common deletion (CD) plays a significant role in aging and age-related diseases. In this study, we used D-galactose (D-gal) to generate an animal model of aging and the involvement and causative mechanisms of mitochondrial damage in such a model were investigated. Twenty 5-week-old male Sprague-Dawley rats were randomly divided into two groups: D-gal group (n=10) and control group (n=10). The quantity of the mtDNA CD in the hippocampus was determined using a TaqMan real-time PCR assay. Transmission electron microscopy was used to observe the mitochondrial ultrastructure in the hippocampus. Western blot was used to detect the protein levels of NADPH oxidase (NOX) and uncoupling protein 2 (UCP2). We found that the level of mtDNA CD was significantly higher in the hippocampus of D-gal-induced aging rats than in control rats. In comparison with the control group, the mitochondrial ultrastructure in the hippocampus of D-gal-treated rats was damaged, and the protein levels of NOX and UCP2 were significantly increased in the hippocampus of D-gal-induced aging rats. This study demonstrated that the levels of mtDNA CD and NOX protein expression were significantly increased in the hippocampus of D-gal-induced aging rats. These findings indicate that NOX-dependent reactive oxygen species generation may contribute to D-gal-induced mitochondrial damage.
In order to confirm the existence of indoleamine 2, 3-dioxygenase (IDO) gene in swine, and to clone the novel gene followed by the molecule structure properties and expression pattern analysis, the porcine mRNA sequences homologous to human IDO were obtained from GenBank database by bioinformatics method. By using RT-PCR, the IDO gene was cloned from porcine endothelial cell line and the accuracy of the nucleic acid sequence was confirmed, and the expression pattern of the gene was detected. The three-dimensional structure model of porcine IDO was built referring to the tertiary structure of human IDO using biological sequence analysis software and database. The results showed that the porcine IDO was identified by sequencing. The nucleotide sequences were confirmed as a novel gene after submitted to Genbank. Porcine IDO was expressed in the lung, thymus, epididymis and anterior chamber with a basic level, however in peripheral blood mononuclear cells (PBMCs) the IDO gene was highly expressed. The three-dimensional structure model of porcine IDO was similar to that of human IDO. It was suggested that identification of the structure information of porcine IDO is essential to further investigate the immunologic function of the gene. Study of IDO on NK cells-mediated xenograft rejection will be a novel therapeutic target for the development of xenotransplantation.
The effects of ATP-sensitive mitochondrial K+ channel (mitoKATP) on mitochondrial membrane potential (Δψm), cell proliferation and protein kinase C alpha (PKCα) expression in airway smooth muscle cells (ASMCs) were investigated. Thirty-six Sprague-Dawley (SD) rats were immunized with saline (controls) or ovalbumin (OVA) with alum (asthma models). ASMCs were cultured from the lung of control and asthma rats. ASMCs were treated with diazoxide (the potent activator of mitoKATP) or 5-hydroxydencanote (5-HD, the inhibitor of mitoKATP). Rhodamine-123 (R-123) was used to detect Δψm. The expression of PKCα protein was examined by using Western blotting, while PKCα mRNA expression was detected by using real-time PCR. The proliferation of ASMCs was measured by MTT assay and cell cycle analysis. In diazoxide-treated normal ASMCs, the R-123 fluorescence intensity, protein and mRNA levels of PKCα, MTT A values and percentage of cells in S phase were markedly increased as compared with untreated controls. The ratio of G0/G1 cells was decreased (P<0.05) in diazoxide-treated ASMCs from normal rats. However, there were no significant differences between the ASMCs from healthy rats treated with 5-HD and the normal control group. In untreated and diazoxide-treated ASMCs of asthmatic rats, the R-123 fluorescence intensity, protein and mRNA levels of PKCα, MTT A values and the percentage of cells in S phase were increased in comparison to the normal control group. Furthermore, in comparison to ASMCs from asthmatic rats, these values were considerably increased in asthmatic group treated with diazoxide (P<0.05). After exposure to 5-HD for 24 h, these values were decreased as compared with asthma control group (P<0.05). In ASMCs of asthma, the signal transduction pathway of PKCα may be involved in cell proliferation, which is induced by the opening of mitoKATP and the depolarization of Δψm.
Cardiac hypertrophy is an independent risk factor for sudden cardiac death in clinical settings and the incidence of sudden cardiac death and ventricular arrhythmias are closely related. The aim of this study was to determine the effects of the calmodulin-dependent protein kinase (CaMK) II inhibitor, KN-93, on L-type calcium current (ICa, L) and early after-depolarizations (EADs) in hypertrophic cardiomyocytes. A rabbit model of myocardial hypertrophy was constructed through abdominal aortic coarctation (LVH group). The control group (sham group) received a sham operation, in which the abdominal aortic was dissected but not coarcted. Eight weeks later, the degree of left ventricular hypertrophy (LVH) was evaluated using echocardiography. Individual cardiomyocyte was isolated through collagenase digestion. Action potentials (APs) and ICa, L were recorded using the perforated patch clamp technique. APs were recorded under current clamp conditions and ICa, L was recorded under voltage clamp conditions. The incidence of EADs and Ica, L in the hypertrophic cardiomyocytes were observed under the conditions of low potassium (2 mmol/L), low magnesium (0.25 mmol/L) Tyrode’s solution perfusion, and slow frequency (0.25–0.5 Hz) electrical stimulation. The incidence of EADs and Ica, L in the hypertrophic cardiomyocytes were also evaluated after treatment with different concentrations of KN-92 (KN-92 group) and KN-93 (KN-93 group). Eight weeks later, the model was successfully established. Under the conditions of low potassium, low magnesium Tyrode’s solution perfusion, and slow frequency electrical stimulation, the incidence of EADs was 0/12, 11/12, 10/12, and 5/12 in sham group, LVH group, KN-92 group (0.5 μmol/L), and KN-93 group (0.5 μmol/L), respectively. When the drug concentration was increased to 1 μmol/L in KN-92 group and KN-93 group, the incidence of EADs was 10/12 and 2/12, respectively. At 0 mV, the current density was 6.7±1.0 and 6.3±0.7 PA·PF−1 in LVH group and sham group, respectively (P>0.05, n=12). When the drug concentration was 0.5 μmol/L in KN-92 and KN-93 groups, the peak ICa, L at 0 mV was decreased by (9.4±2.8)% and (10.5±3.0)% in the hypertrophic cardiomyocytes of the two groups, respectively (P>0.05, n=12). When the drug concentration was increased to 1 μmol/L, the peak ICa, L values were lowered by (13.4±3.7)% and (40±4.9)%, respectively (P<0.01, n=12). KN-93, a specific inhibitor of CaMKII, can effectively inhibit the occurrence of EADs in hypertrophic cardiomyocytes partially by suppressing ICa, L, which may be the main action mechanism of KN-93 antagonizing the occurrence of ventricular arrhythmias in hypertrophic myocardium.
The antibody against AT1-EC2 plays a role in some kinds of inflammatory vascular diseases including malignant hypertension, preeclampsia, and renal-allograft rejection, but the detailed mechanisms remain unclear. In order to investigate the changes of NADPH oxidase and reactive oxygen species in the aorta in a mouse model which can produce AT1-EC2 antibody by active immunization with AT1-EC2 peptide, 15 mice were divided into three groups: control group, AT1-EC2-immunized group, and AT1-EC2-immunized and valsartan-treated group. In AT1-EC2-immunized group and AT1-EC2-immunized and valsartan-treated group, the mice were immunized by 50 μg peptide subcutaneously at multiple points for 4 times: 0, 5, 10, and 15 days after the experiment. In AT1-EC2-immunized and valsartan-treated group, valsartan was given at a dose of 100 mg/kg every day for 20 days. After the experiment, the mice were sacrificed under anesthesia and the aortas were obtained and frozen in liquid nitrogen for the preparation of frozen section slides and other experiments. The titer of AT1-EC2 was assayed by using ELISA. The level of NOX1 mRNA in the aorta was determined by using RT-PCR. The expression of NOX1 was detected by using Western blotting. Confocal scanning microscopy was used to assay the α-actin and NOX1 expression in the aortic tissue. The O2∸ production was detected in situ after DHE staining. The mice produced high level antibody against AT1-EC2 in AT1-EC2-immunized group and AT1-EC2-immunized and valsartan-treated group, and the level of NOX1 mRNA in the aortic tissues was 1.6±0.4 times higher and the NOX1 protein expression was higher in AT1-EC2-immunized group than in control group. There were no significant differences in the level of NOX1 mRNA and protein expression between control group and AT1-EC2-immunized and valsartan-treated group. The expression and co-localization of α-actin and NOX1 in AT1-EC2-immunized group increased significantly as compared with those in control group, and the O2∸ production increased about 2.7 times as compared with control group. There were no significant differences between control group and AT1-EC2-immunized and valsartan-treated group. It is concluded that active immunization with AT1-EC2 can activate NOX1-ROS, and increase vascular inflammation, which can be inhibited by AT1 receptor blocker valsartan. This may partially explain the mechanism of the pathogenesis of inflammatory vascular diseases related to antibody against AT1-EC2.
In this study, we administered a modified schedule of weekly intravenous Bortezomib at 1.6 mg/m2 with dexamethasone (BD) and compared it to the standard 1.3 mg/m2 twice-weekly BD regimen in Chinese patients with newly diagnosed multiple myeloma (MM). We assessed the difference in efficacy, safety profile and survival between the once-weekly and twice-weekly cohorts (13 vs. 24 patients). The over response rate was similar with both arms of the study, being 77% in the once-weekly schedule and 74.9% in the twice-weekly schedule (P=0.690). The median overall survival was not reached in either schedule. Also, the median progression-free survival and duration of response of the once-weekly schedule did not significantly differ from those of the twice-weekly schedule (8 months vs.10 months, P=0.545 and 6 months vs.7 months, P=0.467 respectively). Peripheral sensory neuropathy and grade 3/4 hematologic toxic effects were more frequently reported in the twice-weekly schedule than the once-weekly schedule, but there was no statistically significant difference. This preliminary experience in Chinese patients with newly diagnosed MM indicated that once-weekly infusion of Bortezomib plus dexamethasone may improve safety without affecting outcome.
Nowadays the role of genetic findings in determining the diagnosis, therapy and prognosis of acute myeloid leukemia (AML) has become more valuable. To improve and validate the detection of clonal chromosomal aberrations in leukemia, we designed a combined application of karyotyping with multiplex reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH), and addressed the expression and distribution of fusion genes among the subtypes of Chinese adult patients with de novo AML. Multiplex RT-PCR assays were performed on 477 samples from newly diagnosed AML patients, and cytogenetic data were obtained from 373 of them by R or G banding techniques and those in some cases were confirmed by FISH. The PCR products in some suspected cases were tested by two-directional sequencing. The results showed that except unqualified samples, fusion genes were detected by multiplex RT-PCR in 211 of 474 patients (44.51%), including AML1-ETO, CBFβ-MYH11, PML-RARα, PLZF-RARα, NPM-RARα, MLL rearrangements, BCR-ABL, DEK-CAN, SET-CAN, TEL-PDGFR, TLS-ERG, AML1-MDS1 (EVI-1). In 373 patients, who took both multiplex RT-PCR and karyotype analysis, the detection rate of chromosomal aberrations by using multiplex RT-PCR and karyotyping was 160/373 (42.89%) and 179/373 (47.98%) respectively, and the combination could optimize the detection rate of clonal genetic abnormalities to 216/373 (57.90%). The PCR results from 11 cases “normal” in karyotyping but abnormal in RT-PCR for MLL rearrangements were confirmed by two-directional sequencing. It is concluded that karyotype studies remain the cornerstone for genetic testing; conventional cytogenetics and molecular-based methods are complementary tests for the detection of clonal genetic aberrations in AML, especially for the cryptic or submicroscopic aberrations. Once a genetic marker has been identified by combined analysis, it could be used to monitor residual disease during/after chemotherapy, by quantitative RT-PCR and/or FISH.
T cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia. However the poor prognosis and low morbidity restrict further analysis of the disease. Therefore there is an increasing demand to develop animal models for identifying novel therapeutic approaches. In this study, we inoculated the anti-mouse CD122 monoclonal antibody conditioned NOD/SCID mice with the leukemia cells from 9 T-ALL patients and 1 cell line via the tail vein. Four of the 9 patients and the cell line were successfully engrafted. Flow cytometry detected high percentage of human CD45+ cells in recipient mice. Immunohistochemistry showed infiltration of human CD45+ cells in different organs. Serial transplantation was also achieved. In vivo drug treatment showed that dexamethasone could extend survival, which was consistent with clinical observation. These results demonstrated that we successfully established 5 xenotransplantation models of T-ALL in anti-mCD122 mAb conditioned NOD/SCID mice, which recapitulated the characteristics of original disease.
The aim of this study was to evaluate the safety and efficiency of a novel, oncolytic adenovirus mutant M1 administered in conjunction with immunosuppressive agents. Animal models were established by administering purified M1 either intravenously or retroperitoneally. At different time points, blood samples were taken from the mice for testing of liver and renal function. Microscopic examination of the liver was performed to observe pathological changes. Immunohistochemical analyses were used to evaluate the expression of the adenovirus in the liver. Lymphocyte recruitment to the liver and the activation of adenovirus specific T cells were also analyzed. No signs of general toxicity were observed, but transient increases in ALT and Scr were observed following the administration of M1. Microscopic examination revealed a mild inflammatory response in the liver. Compared to intravenous injection, higher expression levels of adenoviral proteins were observed after retroperitoneal injection. Combined treatment with cyclosporine A resolved the liver and kidney dysfunction and increased the concentration of the adenovirus in the liver. The use of the novel oncolytic adenovirus mutant M1 in vivo is safe, and the combined administration of M1 with immunosuppressive agents was able to enhance the effectiveness and safety profile of M1.
This study examined the protective effect of ischemic postconditioning (IPoC) and minocycline postconditioning (MT) on myocardial ischemia-reperfusion (I/R) injury in atherosclerosis (AS) animals and the possible mechanism. Forty male healthy rabbits were injected with bovine serum albumin following feeding on a high fat diet for 6 weeks to establish AS model. AS rabbits were randomly divided into 3 groups: (1) I/R group, the rabbits were subjected to myocardial ischemia for 35 min and then reperfusion for 12 h; (2) IPoC group, the myocardial ischemia lasted for 35 min, and then reperfusion for 20 s and ischemia for 20 s [a total of 3 cycles (R20s/I20s×3)], and then reperfusion was sustained for 12 h; (3) MT group, minocycline was intravenously injected 10 min before reperfusion. The blood lipids, malondialdehyde (MDA), superoxide dismutase (SOD), soluble cell adhesion molecule (sICAM), myeloperoxidase (MPO), and cardiac troponin T (cTnT) were biochemically determined. The myocardial infarction size (IS) and apoptosis index (AI) were measured by pathological examination. The expression of bcl-2 and caspase-3 was detected in the myocardial tissue by using reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the AS models were successfully established. The myocardial IS, the plasma levels of MDA, sICAM, MPO and cTnT, and the enzymatic activity of MPO were significantly decreased, and the plasma SOD activity was significantly increased in IPoC group and MT group as compared with I/R group (P<0.05 for all). The myocardial AI and the caspase-3 mRNA expression were lower and the bcl-2 mRNA expression was higher in IPoC and MT groups than those in I/R group (all P<0.05). It is concluded that the IPoC and MT can effectively reduce the I/R injury in the AS rabbits, and the mechanisms involved anti-oxidation, anti-inflammation, up-regulation of bcl-2 expression and down-regulation of caspase-3 expression. Minocycline can be used as an effective pharmacologic postconditioning drug to protect myocardia from I/R injury.
We aimed to examine the effect of pioglitazone on transdifferentiation of preosteoblasts from rat bone marrow mesenchymal stem cells (BMSCs) into adipocytes and investigate its effect on bone metabolism. BMSCs were harvested from the femurs and tibias of a rat, then separated, purified, proliferated for 3 generations and differentiated into preosteoblasts for 5 days and 14 days respectively in the presence of osteogenic medium. Thereafter, the preosteoblasts were cultured for 21 days in the presence of adipogenic medium with and without pioglitazone (1 μg/mL). Partially-differentiated osteoblasts were identified by mineralized nodules with Alizarin red S staining. Transdifferentiated adipocytes were identified by Oil Red O staining. Reverse transcription PCR (RT-PCR) was performed to assay the expression levels of osteogenic markers Runx2 and ALP, and an adipogenic marker PPARγ. Those cells cultured for 5 days did not show mineralized nodules as detected by staining of Alizarin red S, while those cultured for 14 days showed dispersed mineralized centers in the form of brown spots, although without obvious red mineralized nodules. After adipogenic transdifferentiation for 21 days, adipose-drops were found in cells of 5CG and 5EG earlier than those of 14CG and 14EG, and the former showed much more adipocytes separately as detected by Oil Red O staining. Whatever the time was 5 days or 14 days of BMSCs osteogenic differentiation, the cells cultured with pioglitazone showed much more adipocytes than those without pioglitazone. Our experiment showed that the less time it took for BMSCs osteogenic differentiation, a stronger ability remained for BMSCs to transdifferentiate into adipocytes. The mRNA expression levels of Runx2 and ALP were decreased by 1.79 and 1.90 times respectively in 5EG (P< 0.05) as compared with 5CG, and that of PPARγ was increased by 1.31 times in 5EG (P<0.05) as compared with 5CG. The mRNA expression levels of Runx2 and ALP were decreased by 1.45 and 1.54 times respectively in 14EG (P<0.05) as compared with 14CG, and that of PPARγ was increased by 1.39 times in 14EG (P<0.05) as compared with 14CG. It was concluded that pioglitazone stimulated the transdifferentiation of BMSCs into adipocytes. These observations provided a potential mechanism of imbalance in thiazolidinedione induced bone metabolism.
The changes of plasma myostatin levels in patients with type 2 diabetes mellitus (T2D) and their clinical correlation were investigated. We recruited 43 T2D patients and 20 age-matched healthy subjects. Plasma myostatin, lipid and glucose, and serum insulin were determined. T2D patients showed significantly higher fasting plasma glucose (FPG), serum insulin and triglyceride levels, and lower high-density lipoprotein levels than normal control subjects (P<0.01). Mean plasma myostatin level in T2D patients and health controls was (66.5±17.8) and (46.2±13.8) ng/mL, respectively. An unpaired t test showed that the increase of myostatin in the T2D patients was significant (P<0.001). In both healthy control and T2D groups, the female subjects showed higher myostatin levels than the male subjects. In the T2D patients, plasma level of myostatin was negatively correlated with body mass index (BMI, r=−0.42, P<0.01) and FPG (r=−0.51, P〈0.01), but positively correlated with insulin resistance index (HOMA-IR, r=0.48, P<0.01). Up-regulation of plasma myostatin in the T2D patients and its correlation with BMI, FPG and blood insulin sensitivity suggests that plasma myostatin may be implicated in the pathogenesis of T2D and thus presented as a therapeutic target for treating the disease. Furthermore, circulating myostatin levels may be used as a biomarker for the disease.
CD4+CD25+CD127dim/− regulatory T cells (Tregs) have been implicated in suppressing T cell immune responses to hepatitis B virus (HBV), but the inhibition mechanism has not being clear yet. This study investigated the effects of soluble FGL2 (sFGL2) secreted by Tregs on immune suppression in chronic HBV-infected patients. We verified that sFGL2 protein and mRNA were highly expressed in Tregs. The separated Tregs by using magnetic beads from peripheral blood mononuclear cells (PBMCs) in 20 patients with chronic hepatitis B were co-cultured with PBMCs at a ratio of 1:3 with anti-CD3 stimulating antibody or FGL2 blocking antibody. The proliferation index of CD8+T cells after blocking FGL2 was higher than that in blank group (3.58±0.18 vs. 3.28±0.17, P=0.034) in 18 of 20 samples, and lower than that in CD3 stimulation group (3.82±0.19, P=0.026) in 16 of 20 samples. The IFN-γ secreted in the mixed culture in the absence of Tregs was higher than that in the culture in the presence of Tregs, but it could be abolished by FGL2 blocking antibody. These results suggest that sFGL2 protein secreted by Tregs suppresses the proliferation and function of CD8+ T cells in chronic hepatitis B.
In order to investigate the role of the MexA-MexB-OprM efflux pump system in the pathogenesis of Pseudomonas aeruginosa (PA)-induced pulmonary infection, pulmonary infection models were established by intratracheal injection of K767 (wild type), nalB (MexA-MexB-OprM up-regulated mutant), and ΔmexB (knockout) strains, separately. All mice were treated with Meropenem (intraper Δ itoneal injection, 100 mg/kg body weight, twice every day), and strain-related pathology, bacteria count, cytokine level, myeloperoxidase (MPO, indicator of neutrophil recruitment) activity, and macrophage inflammatory protein-2 (MIP-2) expression were evaluated at early (3rd day post-infection) and late (7th and 14th day post-infection) stages of infection. E-test showed that ΔmexB was more significantly Δ sensitive to panipenan (ETP), meropenem (MP) and imipenem (IP) than K767 and nalB strains. There was no significant difference in sensitivity to cefepime (TM) among the three stains. In contrast to the K767 and nalB groups, the ΔmexB group showed decreased bacteria burden over time and less exte Δ nsive pathological change. Additionally, MPO activity and levels of inflammatory cytokines (IL-1b, IL-12, and TNF-α) were increased at the early stage (day 3) and decreased at the later stage (day 14). Serum MIP-2 expression level was steadily increased in all three groups from early to late stages, but significantly higher in ΔmexB group than in K767 and nalB groups ( Δ P<0.05). In conclusion, the MexA-MexB-OprM efflux pump system might play an important role in PA-induced chronic pulmonary infection. High expression of the MexA-MexB-OprM efflux pump could increase antibacterial resistance and promote infection.
Recently, the Th17 cells and IL-17 have been shown to play a critical role in the immune-mediated liver injury in hepatitis B, while their functions in acute liver failure have not been well elucidated yet. In this study, we primarily investigated the role of IL-17 in the development of mouse hepatitis virus strain 3 (MHV-3)-induced acute liver failure. IL-17 mRNA levels in liver tissue were quantified by using quantitative real-time polymerase chain reaction, and cytokine IL-17 levels in liver tissue and serum were determined by using ELISA in MHV-3-induced murine fulminant hepatitis model. The IL-17 expression levels on CD4+T and CD8+T cells were determined by using flow cytometry. The correlation between IL-17 level and liver injury was studied. Th17 associated cytokines were also investigated by intracellular staining. Our results showed that the IL-17 expression was significantly elevated in the liver and serum of BALB/cJ mice infected with MHV-3. Moreover, a time course study showed that the percentage of both IL-17-producing CD4+T cells and IL-17-producing CD8+T cells was increased remarkably in the liver starting from 48 h and peaked at 72 h post-infection. There was a close correlation between hepatic or serum IL-17 concentration and the severity of liver injury defined by ALT level, respectively. Th17 associated cytokines, IL-6, IL-21 and IL-22, were also increased significantly at 72 h post-infection. It was concluded that IL-17 may contribute to the pathogenesis of MHV-3-induced acute liver failure.
Different antiepileptic drugs (AEDs) may cause similar adverse effects, one of which is diplopia. However, the AEDs causing diplopia and the dose-response effect of each drug remains uncertain. In this study, we compared several second-generation AEDs to find out whether they would contribute to the risk of diplopia and their effect-causing dose. A meta-analysis was performed on 19 studies in agreement with our inclusion criteria. The results showed that eight commonly used second-generation AEDs (gabapentin, levetiracetam, oxcarbazepine, lamotrigine, pregabalin, topiramate, vigabatrin and zonisamide) could cause diplopia. The reported odds ratios (ORs) ranged from 1.406 to 7.996. Ranking risks from the highest to the lowest ORs of the eight AEDs of any dose resulted in the following order: use of oxcarbazepine (7.996), levetiracetam (7.472), lamotrigine (5.258), vigabatrin (3.562), pregabalin (3.048), topiramate (2.660), gabapentin (1.966), zonisamide (1.406). Taking into account the ORs above, we can conclude that second-generation AEDs of any dose may cause diplopia. However, the levetiracetam-caused diplopia needs to be further studied according to the data (OR, 7.472; 95% confidence interval, 0.375–148.772). These findings ask for better concerns about patients’ quality of life when giving antiepileptic treatments.
A novel unsaturated polyphosphoester (UPPE) was devised in our previous research, which is a kind of promising scaffold for improving bone regeneration. However, the polymerization process of UPPE scaffolds was unfavorable, which may adversely affect the bioactivity of osteoinductive molecules added if necessary, such as recombinant human bone morphogenetic protein-2 (rhBMP2). The purpose of this study was to build a kind of optimal scaffold named UPPE-PLGA-rhBMP2 (UPB) and to investigate the bioactivity of rhBMP2 in this scaffold. Furthermore, the cytotoxicity and biocompatibility of UPB scaffold was assessed in vitro. A W1/O/W2 method was used to fabricate PLGA-rhBMP2 microspheres, and then the microspheres were added to UPPE for synthesizing UPB scaffold. The morphological characters of PLGA-rhBMP2 microspheres and UPB scaffolds were observed under the scanning electron microscopy and laser scanning confocal microscopy. The cumulative release of UPB scaffolds was detected by using ELISA. The cytotoxicity and biocompatibility of UPB scaffolds were evaluated through examining the adsorption and apoptosis of bone marrow stromal cells (bMSCs) seeded on the surface of UPB scaffolds. The bioactivity of rhBMP2 in UPB scaffolds was assessed through measuring the alkaline phosphates (ALP) activity in bMSCs seeded. The results showed that UPB scaffolds sequentially exhibited burst and sustained release of rhBMP2. The cytotoxicity was greatly reduced when the scaffolds were immersed in buffer solution for 2 h. bMSCs attached and grew on the surface of soaked UPB scaffolds, exerting well biocompatibility. The ALP activity of bMSCs seeded was significantly enhanced, indicating that the bioactivity of rhBMP2 remained and still took effect after the unfavorable polymerization process of scaffolds. It was concluded that UPB scaffolds have low cytotoxicity, good biocompatibility and preserve bioactivity of rhBMP2. UPB scaffolds are promising in improving bone regeneration.
The purpose of this study was to compare the biomechanical stability obtained by using our technique featured an anatomical plate and compression bolts versus that of the conventional anatomic plate and cancellous screws in the fixation of intraarticular calcaneal fractures. Eighteen fresh frozen lower limbs of cadavers were used to create a reproductive Sanders type-III calcaneal fracture model by using osteotomy. The calcaneus fractures were randomly selected to be fixed either using our anatomical plate and compression bolts or conventional anatomic plate and cancellous screws. Reduction of fracture was evaluated through X radiographs. Each calcaneus was successively loaded at a frequency of 1 Hz for 1000 cycles through the talus using an increasing axial force 20 N to 200 N and 20 N to 700 N, representing the partial weight bearing and full weight bearing, respectively, and then the specimens were loaded to failure. Data extracted from the mechanical testing machine were recorded and used to test for difference in the results with the Wilcoxon signed rank test. No significant difference was found between our fixation technique and conventional technique in displacement during 20–200 N cyclic loading (P=0.06), while the anatomical plate and compression bolts showed a great lower irreversible deformation during 20–700 N cyclic loading (P=0.008). The load achieved at loss of fixation of the constructs for the two groups had significant difference: anatomic plate and compression bolts at 3839.6±152.4 N and anatomic plate and cancellous screws at 3087.3±58.9 N (P=0.008). There was no significant difference between the ultimate displacements. Our technique featured anatomical plate and compression bolts for calcaneus fracture fixation was demonstrated to provide biomechanical stability as good as or better than the conventional anatomic plate and cancellous screws under the axial loading. The study supports the mechanical viability of using our plate and compression bolts for the fixation of calcaneal fracture.
This study aimed to investigate infiltration related microRNAs (miRNAs) in bladder urothelial carcinoma (BUC). Twenty patients with BUC were enrolled and divided into 2 groups according to infiltration or not: infiltrating BUC group (n=12) and non-infiltrating BUC group (n=8). Gene chip was used to detect infiltration related miRNAs in the BUC samples. In other recruited 17 patients with BUC who were divided into infiltrating BUC samples (n=14) and non-infiltrating BUC samples (n=3), and in 4 BUC cell lines (EJ, 5637, T24 and BIU-87), the expression of miRNAs was assayed by using reverse transcription-polymerase chain reaction (RT-PCR). In infiltrating BUC group, as compared with non-infiltrating BUC group, there were 7 differentially expressed miRNAs: hsa-miR-29c, hsa-miR-200a, hsa-miR-378, hsa-miR-429, hsa-miR-200c and hsa-miR-141 were up-regulated, while hsa-miR-451 was down-regulated. In the BUC samples, the results of RT-PCR were consistent with those by the miRNA array. In the cancer cell lines, RT-PCR in T24 only revealed the similar expression pattern of miRNAs to that by the miRNA array. It is suggested that infiltration of BUC is related with different expression of miRNAs, which may provide a novel platform for further study on function and action mechanism of miRNAs.
The inhibitory effects of diallyl sulfide (DAS) derived from allicin on in vitro and in vivo proliferation of human osteosarcoma MG-63 cells and the action mechanism, and the influence of DAS on invasive capability of MG-63 cells were investigated in order to search for the novel medicines for osteosarcoma. In the in vitro experiment, MG-63 cells were treated with different concentrations of DSA, and the morphological changes of MG-63 cells were observed under an inverted phase microscope. MTT method was used to assay the proliferation of MG-63 cells. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect the VEGF mRNA expression level in MG-63 cells. By using Transwell invasion assay, the influence of DAS on invasive ability of MG-63 cells was tested. In the in vivo experiment, the nude mice MG-63 cells tumor-bearing model was established, and different concentrations of DAS were injected beside the tumor. Twenty-one days after treatment, the mice were killed, the tumor size and tumor inhibition rate were calculated. The microvessel density (MVD) was determined by using immunohistochemistry. In the in vitro experiment, different concentrations of DAS could obviously inhibit proliferation of MG-63 cells in a time- and concentration-dependent manner. RT-PCR revealed that the expression levels of VEGF mRNA in DSA groups (different concentrations) were significant reduced as compared with those in control group (all P<0.05). Transwell invasion assay indicated that in 20 and 40 μg/mL DAS groups, the number of migratory cells was 91.4±8.3 and 81.8±7.4 respectively, which was significantly declined as compared with that in control group (150.4±14.7, both P<0.05). In the in vivo experiment, DAS could significantly suppress the growth of MG-63 tumor-bearing tissue. Immunohistochemistry demonstrated that different concentrations (20 and 40 μg/mL) of DAS could significantly decrease MVD of MG-63 tumor-bearing tissue (all P<0.05). It was suggested that DAS could inhibit the growth of MG-63 cells probably by suppressing the expression of VEGF mRNA.
Chemotherapy-induced neuropathy is a serious clinical problem for patients receiving cancer treatment. The aim of this study was to investigate the potential efficacy of pregabalin in chemotherapy-induced neuropathy in rats. A total of 35 male Sprague-Dawley rats were randomly divided into 5 groups: group 1, naive control; group 2, treated with pregabalin (30 mg/kg p.o., for 8 days); group 3, docetaxel was given by single intravenous infusion at 10 mg/kg; groups 4 and 5, pregabalin at 10 mg/kg and 30 mg/kg respectively was orally administered for 8 days after the docetaxel treatment. On day 8, behavioral test was performed, and substance P and CGRP release in dorsal root ganglion (DRG) and sciatic nerve were analyzed by electron microscope. Our results showed that docetaxel induced mechanical allodynia, mechanical hyperalgesia, heat hypoalgesia, cold allodynia, and sciatic nerve impairment and substance P and CGRP release in DRG. However, oral administration of pregabalin (10 mg/kg and 30 mg/kg) for 8 consecutive days significantly attenuated docetaxel-induced neuropathy by ameliorating heat hypoalgesia, cold allodynia, impairment of sciatic nerve and reducing the release of substance P and CGRP. The findings in the present study reveal that pregabalin may be a potential treatment agent against chemotherapy-induced neuropathy.
Listeria monocytogenes (LM), a Gram-positive facultative intracellular bacterium, can be used as an effective exogenous antigen expression vector in tumor-target therapy. But for successful clinical application, it is necessary to construct attenuated LM stain that is safe yet retains the potency of LM based on the full virulent pathogen. In this study, attenuated LM and recombinants of LM expressing melanoma inhibitory activity (MIA) were constructed successfully. The median lethal dose (LD50) and invasion efficiency of attenuated LM strains were detected. The recombinants were utilized for immunotherapy of animal model of B16F10 melanoma. The level of MIA mRNA expression in tumor tissue was detected by using real-time polymerase chain reaction (PCR) with specific sequence, meanwhile the anti-tumor immune response was assayed by flow cytometric analysis and enzyme-linked immunosorbent spot (ELISPOT) assay. The results showed the toxicity and invasiveness of attenuated LM were decreased as compared with LM, and attenuated LM expressing MIA, especially the double-genes attenuated LM recombinant, could significantly induce anti-tumor immune response and inhibit tumor growth. This study implicates attenuated LM may be a safer and more effective vector for immunotherapy of melanoma.
The effectiveness of liver autotransplantation for patients with partial hepatic alveolar echinococcosis was analyzed. We retrospectively studied 6 patients with hepatic alveolar echinococcosis who underwent liver autotransplantation in our hospital from 2008 to 2010. We also summarized the surgical indications of liver autotransplantation for hepatic alveolar echinococcosis and our experience in the management of postoperative complications of liver autotransplantation. Of 6 patients, 5 achieved good curative results, and one died of multiple organ failure caused by portal vein thrombosis. Main complications included postoperative bleeding, bile leak and small-for-size liver graft syndrome. Liver autotransplantation offers a new approach to cure hepatic alveolar echinococcosis with non-resectable lesions. It could be the most effective method to cure intractable hepatic alveolar echinococcosis if correct handling in operation and proper prevention of complications are performed. But the long-term outcomes are still needed to be confirmed in longer follow-up.
This study investigated the effect of etoposide, an anticancer chemotherapy drug, on B7-H1 expression in retinoblastoma (Rb) cells and the role of miR-513a-5p in the process. Rb cells were divided into control and etoposide groups. In the etoposide group, cells were treated with etoposide at different concentrations (2.5, 5, 10, 20 and 40 μg/mL) for 24 h. Those given no treatment of etopside served as controls. Reverse transcription polymerase chain reaction (RT-PCR), fluorescence quantitative PCR and flow cytometry were performed to measure the mRNA and protein expression of B7-H1 in Rb cells. The mRNA expression of miR-513a-5p in Rb cells before and after etoposide treatment was also detected by fluorescence quantitative PCR. The miR-513a-5p mimics and the miR-513a-5p inhibitor were transfected into Rb cells separately, and fluorescence quantitative PCR and flow cytometry were used to detect the effect of the miR-513a-5p mimics or inhibitor on B7-H1 expression. TargetScan5.2 was employed to predict the miR-513a-5p binding sites in the 3′-untranslated region of B7-H1 mRNA. Luciferase reporter plasmids carrying this site were prepared and transfected into Rb cells and luciferase activity analyzed. The results showed that etoposide stimulated the mRNA and protein expression of B7-H1 in Rb cells, which reached a maximal level after treatment with 5 μg/mL etoposide (P<0.05). However, miR-513a-5p expression was decreased in Rb cells after etoposide treatment. When the miR-513a-5p inhibitor was added, B7-H1 expression was increased with the concentration of the miR-513a-5p inhibitor (P<0.05). Moreover, B7-H1 expression was decreased gradually with the concentration of the miR-513a-5p mimics increased (P<0.01). Additionally, the miR-513a-5p mimics were found to inhibit the luciferase activity. It was concluded that etoposide can promote B7-H1 expression in Rb cells, which may be associated with chemoresistance. The promoting effect of etoposide on B7-H1 expression can be reversed by miR-513a-5p mimics. MiR-513a-5p inhibits the mRNA and protein expression of B7-H1 via binding to the 3′-UTR of B7-H1 mRNA.
The aim of this study was to investigate the effects of Avastin on aquaporin4 (AQP4) expression in human retinal Müller cells in vitro under hypoxia, so as to explore the mechanism of Avastin treating retinal edema. The human Müller cells were cultured using the enzymatic digestion method. Müller cells were identified under the transmission electron microscopy and by using immunofluorescence staining. By using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), the expression of AQP4 mRNA and VEGF mRNA in Müller cells cultured with 500 μmol/L CoCl2 for 0, 3, 6, 12 and 24 h, and with 0, 100, 300, 500 and 700 μmol/L CoCl2 for 24 h was detected. The expression of AQP4 mRNA in Müller cells cultured with 50 ng/mL exogenous vascular endothelial growth factor (VEGF) for 0, 0.5, 1, 2 and 4 h, and with 0, 25, 50 and 75 ng/mL VEGF for 24 h was detected. Amplified cDNA products of AQP4 mRNA in Müller cells cultured with 500 μmol/L CoCl2 and 200 μg/mL Avastin for 24 h were detected. The results showed that more than 95% cells displayed positive immunofluorescence reaction. Characteristic 8–10 nm intracellular filaments could be seen in the cytoplasm under the transmission electron microscopy. In the CoCl2 experimental groups, the expression of AQP4 mRNA and VEGF mRNA in Müller cells was increased as compared with the control group. Alteration of AQP4 mRNA and VEGF mRNA levels showed a significantly positive correlation (r2=0.822, P<0.05). The expression of AQP4 mRNA in Müller cells was increased by VEGF. The expression of AQP4 mRNA was significantly decreased by Avastin as compared with the control group. It is suggested that Avastin can decrease the expression of AQP4 mRNA in human Müller cells under chemical hypoxic conditions partially via VEGF path, which may be one of the mechanisms of Avastin treating retinal edema.
This study investigated the inhibitory effect of grape seed proanthocyanidin extract (GSPE) on selenite-induced cataract formation in rats and the possible mechanism. Eighty 8-day-old Sprague-Dawley rats were divided randomly into 5 groups: control group, model group, three GSPE groups (low dose, medium dose and high dose). Control group received subcutaneous injection of physiological saline. Model group was given subcutaneous injection of sodium selenite (20 μmol/kg body weight) on the postpartum day 10, and once every other day for consecutive three times thereafter. GSPE treated groups were respectively administered GSPE at doses of 50, 100, and 200 mg/kg body weight intragastrically 2 days prior to the selenite injection (that was, on the postpartum day 8), and once daily for fourteen consecutive days thereafter. The opacity of lenses was observed, graded and photographed under the slit lamp microscopy and the maximal diameter of the nuclear cataract plaques was measured. The lenses were analyzed for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), malondialdehyde (MDA), calcium (Ca2+), nitric oxide (NO) and anti-hydroxyl radical ability (anti-OH−). The histomorphology of lenses was observed with HE staining under a light microscope. The levels of calpainII, and iNOS protein and mRNA expression in lenses were detected by using immunohistochemistry and real-time quantitative RT-PCR. The results showed subcutaneous injection of sodium selenite led to severe nuclear cataract in model group, and the achievement ratio of model group was 100%. As compared with model group, the degree of lenses opacity and the maximal diameter of nuclear cataract plaques were significantly reduced in GSPE-treated groups. Moreover, we observed selenite treatment caused a significant decrease in the activities of antioxidative enzymes (SOD, CAT, GSH-PX) and anti-OH− ability, accompanied by a significant increase in the levels of MDA, NO, Ca2+ as well as iNOS, and calpainII protein and mRNA expression. Administration of GSPE could dose-dependently preserve the activities of these antioxidative enzymes and anti-OH− ability, accompanied by a significant reduction in the levels of MDA, NO, Ca2+ as well as iNOS, and calpainII protein and mRNA expression. These results suggested that GSPE markedly prevented selenite-induced cataract formation probably by suppressing the generation of lipid peroxidation and free radicals as well as the activation of iNOS, and calpainII in the lenses.
The presence of insufficient bone volume affects the implant healing and success. The aim of this study was to evaluate osteogenic capacity of dental pulp stem cells (DPSCs) on micro-arc oxidation (MAO) titanium surface. DPSCs were challenged at MAO and smooth titanium surface separately for different durations, and the bone marrow mesenchymal stem cells (BMSCs) served as the positive controls. The osteogenic capacity of DPSCs on MAO titanium surface was assessed by using scanning electron microscopy, energy dispersive spectroscopy, biochemical tests and real-time quantitative PCR. Data showed that DPSCs differentiated into osteoblasts and expressed bone morphogenetic genes on MAO titanium surface. The results of this study revealed that DPSCs had good potential to generate mineralized tissue on MAO titanium plates. The differential potential of DPSCs may be regulated by MAO titanium surface. The osteogenesis potential of DPSCs on the MAO titanium was similar with BMSCs.
The overuse of injection exists more than 20 years since economic reform in China. It is a persistent problem and seems becoming a new challenge in the new health reform period. This study was designed to assess the effect of national essential medicines policy (NEMP) on injection use at primary health facilities in China by investigating their prescription information. Questionnaires were designed and disseminated to collect empirical data on injection use at 120 primary health facilities in 6 provinces from January to September in 2010 and 2011. The injection use was measured as the indicator as the percentage of prescriptions with one or more injections. The results showed that the percentage of prescriptions with one or more injections was decreased from 38.91% to 36.82% (χ2=11.158, P=0.001) in the all survey areas during the NEMP reform. The difference in level of the injection use in 2011 was significant among the eastern, central and western regions (χ2=223.584, P=0.000); level of the injection use in western region was the lowest (27.73%), while that in the central region was the highest (43.10%). The level of the injection use in 2011 among different provinces was also of great difference (26.00%–58.25%, range: 32.25%). The level of the injection use in 2011 was still much higher than the standard suggested by WHO for developing countries (13.4%–24.1%). It was concluded that NEMP has improved injection use in China, but the injection abuse situation remains serious, indicating that one of the priorities to the next stage of NEMP is to promote the rational use of drugs, especially the injection use.
There are actually no sufficient data for lifting technique assessment. A laboratory study was undertaken to determine the effect of 3 regular lifting techniques on erector muscle activation, cardiovascular strain and subjective response. Thirty student volunteers participated in the study, and were required to lift a weight with different techniques. Stoop-, squat-, semi-squat-lifting resulted in 3, 2 and 1 time of the highest percentage of the maximum voluntary electrical activation (MVE%) respectively. In the same order, the lowest median frequency (MF) existed 1, 1 and 2 times. Muscle fatigue was 4 times in squat, 36 in semi-squat and 43 in stoop lifting. Heart rate was the highest in squat and lowest in stoop respectively, with a middle level in semi-squat lifting. It may be recommended to adopt mainly the semi-squat technique for daily lifting works. For heavy lifting, it should use the squat technique. Stoop lifting may also be used alternatively but for light things.