Echinococcus granulosus promotes bone resorption by increasing osteoclasts differentiation.

Osseous cystic echinococcosis (CE) is a rare disease caused by Echinococcus granulosus, which is characterized by high morbidity, disability, and mortality. However, it is severely neglected due to its mainly regional epidemic. The development of osseous CE is usually accompanied by severe bone erosion and destruction at the site of infection; however, there is a gap in research on the mechanism of this phenomenon. The current treatment for this disease is single-sided, ineffective, and has a high rate of disability and recurrence. Our study investigated the mechanism of bone destruction caused by osseous CE and provided a theoretical basis for basic research and innovative ideas for treating clinical disease. A co-culture system of osteoclast progenitor cells and protoscoleces (PSCs) was established to test the effects of PSCs on osteoclast differentiation. We also created two disease models of spinal and femoral CE, with the highest incidence of osseous CE. We verified the effect of E. granulosus on osteoclasts at the infection site in vivo. The stimulatory effect of E. granulosus on osteoclast formation was confirmed by in vivo and in vitro experiments. This study elucidates the elementary mechanism of bone destruction in osseous CE and fills a gap in the field of basic osseous CE research, which is conducive to treating the disease.

An evaluation of COVID-19 transmission control in Wenzhou using a modified SEIR model.

In December 2019, the first confirmed case of pneumonia caused by a novel coronavirus was reported. Coronavirus disease 2019 (COVID-19) is currently spreading around the world. The relationships among the pandemic and its associated travel restrictions, social distancing measures, contact tracing, mask-wearing habits and medical consultation efficiency have not yet been extensively assessed. Based on the epidemic data reported by the Health Commission of Wenzhou, we analysed the developmental characteristics of the epidemic and modified the Susceptible-Exposed-Infectious-Removed (SEIR) model in three discrete ways. (1) According to the implemented preventive measures, the epidemic was divided into three stages: initial, outbreak and controlled. (2) We added many factors, such as health protections, travel restrictions and social distancing, close-contact tracing and the time from symptom onset to hospitalisation (TSOH), to the model. (3) Exposed and infected people were subdivided into isolated and free-moving populations. For the parameter estimation of the model, the average TSOH and daily cured cases, deaths and imported cases can be obtained through individual data from epidemiological investigations. The changes in daily contacts are simulated using the intracity travel intensity (ICTI) from the Baidu Migration Big Data platform. The optimal values of the remaining parameters are calculated by the grid search method. With this model, we calculated the sensitivity of the control measures with regard to the prevention of the spread of the epidemic by simulating the number of infected people in various hypothetical situations. Simultaneously, through a simulation of a second epidemic, the challenges from the rebound of the epidemic were analysed, and prevention and control recommendations were made. The results show that the modified SEIR model can effectively simulate the spread of COVID-19 in Wenzhou. The policy of the lockdown of Wuhan, the launch of the first-level Public Health Emergency Preparedness measures on 23 January 2020 and the implementation of resident travel control measures on 31 January 2020 were crucial to COVID-19 control.

Crocetin Attenuates Sepsis-Induced Cardiac Dysfunction via Regulation of Inflammatory Response and Mitochondrial Function.

Sepsis-induced systemic inflammation can induce cardiac dysfunction, which can result in heart failure and death. Recently, natural drugs/compounds have received increased attention as therapeutic agents to prevent sepsis-induced cardiac dysfunction. Crocetin (CRO) is a natural compound that has been shown to reduce inflammation and cytotoxicity in cardiac ischemia/reperfusion injury. However, the effects of CRO on sepsis-induced cardiac dysfunction have not been evaluated. In this study, we used lipopolysaccharide (LPS)-induced H9c2 cells as an in vitro model to mimic cardiac sepsis. Crocetin significantly alleviated LPS-induced cytotoxicity, cellular apoptosis, and oxidative stress through increased Bcl-2 activity and PI3K-Akt signaling and suppression of caspase 3 and caspase 9 activities. Furthermore, CRO dramatically decreased the mRNA levels of TNF-α, IL-1, IL-6, and IL-8 via suppression of p65/Keap1 signaling and activation of Nrf2/HO-1/NQO1 signaling. In addition, CRO protected mitochondrial respiration, free fatty acid ß-oxidation, and mitochondrial morphology in LPS-induced H9c2 cells. This study showed that CRO attenuated LPS-induced cardiac dysfunction via regulation of the inflammatory response and mitochondrial function and potentially had an effect on sepsis-induced cardiac dysfunction.

Crocetin alleviates myocardial ischemia/reperfusion injury by regulating inflammation and the unfolded protein response.

Crocetin, a natural compound, has been demonstrated to exhibit beneficial effects in cardiovascular diseases. Previous studies demonstrated that crocetin reduced ischemia/reperfusion (I/R) injury by attenuating cytotoxicity and cellular apoptosis. However, the previous mechanistic studies did not fully elucidate its pharmacological effects on cardiac damage, especially I/R injury. The present study verified its cardioprotective effects in a Langendorff perfusion system, an ex vivo model of I/R. It was demonstrated that crocetin significantly attenuated the activities of pro­inflammatory cytokines and nuclear factor erythroid­2 related factor 2 (Nrf2)/heme oxygenase­1 signaling. The present study provided novel insight that crocetin regulated the unfolded protein response (UPR) and decreased associated protein levels to protect the heart. Furthermore, it was identified that Nrf2 played a key role in the cardioprotective effect of crocetin by attenuating inflammation and the UPR.

Different protein expression patterns in rat spinal nerves during Wallerian degeneration assessed using isobaric tags for relative and absolute quantitation proteomics profiling.

Sensory and motor nerve fibers of peripheral nerves have different anatomies and regeneration functions after injury. To gain a clear understanding of the biological processes behind these differences, we used a labeling technique termed isobaric tags for relative and absolute quantitation to investigate the protein profiles of spinal nerve tissues from Sprague-Dawley rats. In response to Wallerian degeneration, a total of 626 proteins were screened in sensory nerves, of which 368 were upregulated and 258 were downregulated. In addition, 637 proteins were screened in motor nerves, of which 372 were upregulated and 265 were downregulated. All identified proteins were analyzed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of bioinformatics, and the presence of several key proteins closely related to Wallerian degeneration were tested and verified using quantitative real-time polymerase chain reaction analyses. The differentially expressed proteins only identified in the sensory nerves were mainly relevant to various biological processes that included cell-cell adhesion, carbohydrate metabolic processes and cell adhesion, whereas differentially expressed proteins only identified in the motor nerves were mainly relevant to biological processes associated with the glycolytic process, cell redox homeostasis, and protein folding. In the aspect of the cellular component, the differentially expressed proteins in the sensory and motor nerves were commonly related to extracellular exosomes, the myelin sheath, and focal adhesion. According to the Kyoto Encyclopedia of Genes and Genomes, the differentially expressed proteins identified are primarily related to various types of metabolic pathways. In conclusion, the present study screened differentially expressed proteins to reveal more about the di?erences and similarities between sensory and motor nerves during Wallerian degeneration. The present findings could provide a reference point for a future investigation into the differences between sensory and motor nerves in Wallerian degeneration and the characteristics of peripheral nerve regeneration. The study was approved by the Ethics Committee of the Chinese PLA General Hospital, China (approval No. 2016-x9-07) in September 2016.

Association of vitamin D receptor ApaI gene polymorphism with osteoporosis susceptibility in postmenopausal Han Chinese women in Xinjiang.

Osteoporosis is a polygenic disorder and has been demonstrated to be associated with ~30 candidate genes, the majority of which have also been implicated in the regulation of bone mineral density (BMD). Vitamin D receptor (VDR) is the candidate gene that has been most extensively studied. Certain studies have reported that the VDR single nucleotide polymorphism ApaI is associated with the risk of osteoporosis in Caucasian and African women. However, this association has not yet been studied in postmenopausal Han Chinese women in the Xinjiang area. In the present study, ApaI polymorphisms of VDR were defined by polymerase chain reaction-restriction fragment length polymorphism, in order to analyze the distribution of ApaI polymorphisms in postmenopausal Han Chinese women from Xinjiang. BMD was measured by dual energy X-ray absorptiometry at the lumbar spine (L2-4), Ward's triangle, great trochanter and femoral shaft. A total of 336 women were included in this study. The genotype distribution of ApaI was consistent with the Hardy-Weinberg equilibrium (all P>0.05). There were no significant differences in ApaI genotype frequencies between the 90 cases in the osteoporosis group and 246 cases in the non-osteoporosis group (P=0.946). Meanwhile, it was identified that BMD values of the tested locations were negatively correlated with age (P<0.05) and positively correlated with body mass index (BMI; P<0.05). On further attribution risk analysis, BMD was identified as a risk factor [odds ratio (OR): 0.464, 95% confidence interval (CI): 0.372-0.580, P=0.001] and BMI a protective factor (OR: 1.502, 95% CI: 1.008-2.240, P=0.032) in osteoporosis. When BMD was adjusted for confounding factors including age and BMI, it was observed that the ApaI polymorphism was not associated with BMD at the sites tested (P>0.05). In conclusion, the present study identified no significant association of the common VDR polymorphism ApaI with BMD at several skeletal sites in postmenopausal Han Chinese women in the Xinjiang area. Age was negatively correlated with BMD at different sites and identified as a risk factor; while BMI was positively correlated with BMD and identified as a protective factor.

Efficacy of mesenchymal stem cells in treating patients with osteoarthritis of the knee: A meta-analysis.

To assess the clinical efficacy and safety of mesenchymal stem cell (MSC) treatment for osteoarthritis of the knee (KOA), a systematic electronic literature search was performed on PubMed, EMBASE and Web of Science. Studies published in English from the earliest record to December 2014 were searched using the following keywords: Cartilage defect, cartilage repair, osteoarthritis, KOA, stem cells, MSCs, bone marrow concentrate (BMC), adipose-derived mesenchymal stem cells, synovial-derived mesenchymal stem cells and peripheral blood-derived mesenchymal stem cells. The effect sizes of selected studies were determined by extracting pain scores from the visual analog scale and functional changes from International Knee Documentation Committee and Lysholm and Western Ontario and McMaster Universities Osteoarthritis Index before and after MSCs or reference treatments at 3, 6, 12, and 24 months. The factors were analyzed and the outcomes were modified after comparing the MSC group pooled values with the pretreatment baseline or between different treatment arms. A systematic search identified 18 clinical trials on this topic, including 10 single-arm prospective studies, four quasi-experimental studies and four randomized controlled trials that used BMCs to treat 565 patients with KOA in total. MSC treatment in patients with KOA showed continual efficacy for 24 months compared with their pretreatment condition. Effectiveness of MSCs was improved at 12 and 24 months post-treatment, compared with at 3 and 6 months. No dose-responsive association in the MSCs numbers was demonstrated. However, patients with arthroscopic debridement, activation agent or lower degrees of Kellgren-Lawrence grade achieved improved outcomes. MSC application ameliorated the overall outcomes of patients with KOA, including pain relief and functional improvement from basal evaluations, particularly at 12 and 24 months after follow-up.

Effect of lentivirus-mediated uPA silencing on the proliferation and apoptosis of chondrocytes and the expression of MMPs.

The lentivirus-mediated uPA interference in the proliferation, apoptosis, and secretion of osteoarthritic chondrocytes was examined in this study. Cells were obtained from the cartilage tissues of New Zealand white rabbits. They were cultured with interleukin (IL)-1ß (10 ng/mL) for 24 h and then divided into three groups: uPA-siRNA group (cells transfected with uPA-siRNA lentiviruses), blank control group (untreated cells), and negative control group (cells transfected with empty vectors). Western blotting and real-time quantitative reverse transcription-PCR (RT-QPCR) were performed to detect the protein and mRNA expression levels of uPA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13 and MMP-14 in osteoarthritic chondrocytes. Cell Counting Kit-8, flow cytometry, and colony formation assay were used to examine the proliferation and apoptosis of chondrocytes. The results showed that after uPA-siRNA transfection, the protein and mRNA expression levels of uPA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13, and MMP-14 were significantly decreased (P<0.05 for MMP-1, MMP-9, MMP-10 and MMP-14, P<0.01 for uPA, MMP-3 and MMP-13). Cell proliferation and colony formation rate were significantly higher and the cell apoptosis rate was significantly lower in uPA-siRNA group than in control groups (P<0.01). The proportion of cells in G0/G1 phase was markedly increased and that in the S phase decreased, and the cell cycle was arrested at the G1/S phase in the control group. In the uPA-siRNA group, the proportion of cells in the S phase was significantly increased, resulting in a different proportion of cells in cell cycle phase (P<0.01). It was suggested that the down-regulation of uPA gene could inhibit the expression of MMPs protein and cell apoptosis, increase the proliferation and colony formation of osteoarthritic chondrocytes.

Construction and verification of the targeted uPA-shRNA lentiviral vector and evaluation of the transfection and silencing rate.

Urokinase-type plasminogen activator (uPA) receptors, which are released by the synovial tissue, are responsible for the activation of cartilage-breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). RNA interference (RNAi) technology has emerged as a potent tool to generate cellular knockdown phenotypes of a desired gene. The aims of the present study were to investigate the effect of siRNA specific to the uPA gene on chondrocytes and to investigate the possible mechanisms of OA. Firstly, four types of small hairpin RNA (shRNA) sequence (P1, P2, P3 and P4) were obtained from the targeted uPA gene of the New Zealand rabbit, based on siRNA theory. The sequences were designed, constructed and subjected to restriction enzyme digestion, transformation, polymerase chain reaction (PCR) identification, positive clone sequencing and lentivirus packaging. Secondly, primary culturing cartilage cells from the New Zealand rabbit were transfected with P1, P2, P3 or P4 to observe the transfection rate under a fluorescence microscope. The mRNA expression levels of uPA were analyzed in cartilage cells using quantitative PCR, while protein expression levels were analyzed in the cartilage cells using western blot technology. Four types of uPA-shRNA lentiviral vectors were constructed successfully, which were all able to be transfected into the primary culturing cartilage cells. The transfection rate was as high as 85% when the multiplicity of infection was 100, which demonstrated that P1, P2, P3 and P4 were all capable of inhibiting the mRNA and protein expression of uPA in cartilage cells. In addition, among the four sequences, the P2 sequence exhibited the highest silencing rate of 70%. Statistical significance (P<0.05) was observed when analyzing the silencing rate of P2 compared to the other three groups. The most efficient targeted uPA-shRNA sequence was identified following screening. The results strongly verified that siRNA lentiviral vectors can be transfected into cartilage cells to further inhibit the expression of the uPA gene efficiently and steadily. Thus, the results provide the foundation for further research on the role of uPA in the pathogenesis of OA.

Mesenchymal stem cells recruited by active TGFß contribute to osteogenic vascular calcification.

Vascular calcification is an actively regulated process that culminates in organized extracellular matrix mineral deposition by osteoblast-like cells. The origins of the osteoblastic cells involved in this process and the underlying mechanisms remain to be defined. We previously revealed that active transforming growth factor (TGFß) released from the injured arteries mobilizes mesenchymal stem cells (MSCs) to the blood stream and recruits the cells to the injured vessels for neointima formation. In this study, we used a low-density lipoprotein receptor (LDLR)-deficient mouse model (ldlr(-/-)), which develop progressive arterial calcification after having fed high-fat western diets (HFD), to examine whether TGFß is involved in the mobilization of MSCs during vascular calcification. Nestin(+)/Sca1(+) cells were recruited to the diseased aorta at earlier time points, and osteocalcin(+) osteoblasts and the aortic calcification were seen at later time point in these mice. Importantly, we generated parabiotic pairs with shared blood circulation by crossing ldlr(-/-)mice fed HFD with transgenic mice, in which all the MSC-derived cells were fluorescently labeled. The labeled cells were detected not only in the peripheral blood but also in the arterial lesions in ldlr(-/-) mouse partners, and these blood circulation-originated cells gave rise to Ocn(+) osteoblastic cells at the arterial lesions. Both active TGFß1 levels and MSCs in circulating blood were upregulated at the same time points when these cells appeared at the aortic tissue. Further, conditioned medium prepared by incubating the aortae from ldlr(-/-)mice fed HFD stimulated the migration of MSCs in the ex vivo transwell assays, and either TGFß neutralizing antibody or the inhibitor of TGFß Receptor I kinase (TßRI) antagonized this effect. Importantly, treatment of the mice with TßRI inhibitor blocked elevated blood MSC numbers and their recruitment to the arterial lesions. These findings suggest that TGFß-recruited MSCs to the diseased vasculature contribute to the development of osteogenic vascular calcification.

Disruption of LRP6 in osteoblasts blunts the bone anabolic activity of PTH.

Mutations in low-density lipoprotein receptor-related protein 6 (LRP6) are associated with human skeletal disorders. LRP6 is required for parathyroid hormone (PTH)-stimulated signaling pathways in osteoblasts. We investigated whether LRP6 in osteoblasts directly regulates bone remodeling and mediates the bone anabolic effects of PTH by specifically deleting LRP6 in mature osteoblasts in mice (LRP6 KO). Three-month-old LRP6 KO mice had a significant reduction in bone mass in the femora secondary spongiosa relative to their wild-type littermates, whereas marginal changes were found in femoral tissue of 1-month-old LRP6 KO mice. The remodeling area of the 3-month-old LRP6 KO mice showed a decreased bone formation rate as detected by Goldner's Trichrome staining and calcein double labeling. Bone histomorphometric and immumohistochemical analysis revealed a reduction in osteoblasts but little change in the numbers of osteoclasts and osteoprogenitors/osteoblast precursors in LRP6 KO mice compared with wild-type littermates. In addition, the percentage of the apoptotic osteoblasts on the bone surface was higher in LRP6 KO mice compared with wild-type littermates. Intermittent injection of PTH had no effect on bone mass or osteoblastic bone formation in either trabecular and cortical bone in LRP6 KO mice, whereas all were enhanced in wild-type littermates. Additionally, the anti-apoptotic effect of PTH on osteoblasts in LRP6 KO mice was less significant compared with wild-type mice. Therefore, our findings demonstrate that LRP6 in osteoblasts is essential for osteoblastic differentiation during bone remodeling and the anabolic effects of PTH.

Injury-activated transforming growth factor ß controls mobilization of mesenchymal stem cells for tissue remodeling.

Upon secretion, transforming growth factor ß (TGFß) is maintained in a sequestered state in extracellular matrix as a latent form. The latent TGFß is considered as a molecular sensor that releases active TGFß in response to the perturbations of the extracellular matrix at the situations of mechanical stress, wound repair, tissue injury, and inflammation. The biological implication of the temporal discontinuity of TGFß storage in the matrix and its activation is obscure. Here, using several animal models in which latent TGFß is activated in vascular matrix in response to injury of arteries, we show that active TGFß controls the mobilization and recruitment of mesenchymal stem cells (MSCs) to participate in tissue repair and remodeling. MSCs were mobilized into the peripheral blood in response to vascular injury and recruited to the injured sites where they gave rise to both endothelial cells for re-endothelialization and myofibroblastic cells to form thick neointima. TGFßs were activated in the vascular matrix in both rat and mouse models of mechanical injury of arteries. Importantly, the active TGFß released from the injured vessels is essential to induce the migration of MSCs, and cascade expression of monocyte chemotactic protein-1 stimulated by TGFß amplifies the signal for migration. Moreover, sustained high levels of active TGFß were observed in peripheral blood, and at the same time points following injury, Sca1+ CD29+ CD11b- CD45- MSCs, in which 91% are nestin+ cells, were mobilized to peripheral blood and recruited to the remodeling arteries. Intravenously injection of recombinant active TGFß1 in uninjured mice rapidly mobilized MSCs into circulation. Furthermore, inhibitor of TGFß type I receptor blocked the mobilization and recruitment of MSCs to the injured arteries. Thus, TGFß is an injury-activated messenger essential for the mobilization and recruitment of MSCs to participate in tissue repair/remodeling.

Antagonists of LRP6 regulate PTH-induced cAMP generation.

LRP6 is a common coreceoptor for different G protein-coupled seven-transmembrane receptors in production of cAMP. Extracelluar proteins sclerostin and DKK1, initially identified as antagonists for Wnt signaling by binding to LRP6, are negative regulators for bone formation. Here, we show that both sclerostin and DKK1 inhibit PTH-stimulated cAMP production. In addition, PTH suppresses expression of sclerostin in osteocytes in mice. We also found that sclerostin and DKK1 binds to LRP6 as antagonists to increase the availability of LRP6 to facilitate PTH signaling in a positive-feedback fashion. These studies reveal a previously unrecognized function of sclerostin and DKK1, which provides an alternative explanation for the application of sclerostin and DKK1 neutralization on enhancing bone formation as a potential therapy for skeletal diseases.

Determination of oxadiargyl residues in environmental samples and rice samples.

Oxadiargyl is a commonly used herbicide in China. We developed a simple, fast, and high-throughput method employing gas chromatography with electron capture detector to determine oxadiargyl residues in food samples (rice, straws) and environmental samples (soil, water). Samples were prepared by a modified QuEChERS procedure. In this method, acetonitrile was used as the extracting solvent. The purifying step was omitted when the chromatographic conditions were optimized. Recoveries ranged from 82.9 to 112.0% for oxadiargyl in all samples, with relative standard deviation values lower than 6.2% at 0.01 mg/kg fortified concentration level.