Effects of human umbilical cord mesenchymal stem cells-derived exosomes on fracture healing in rats through the Wnt signaling pathway.

OBJECTIVE: To investigate the role of human umbilical cord mesenchymal stem cell (hucMSC)-derived exosomes in the Wnt signaling pathway and their effects on fracture healing in rats. MATERIALS AND METHODS: A total of 24 healthy male Sprague-Dawley (SD) rats were randomly divided into 3 groups, of which the experimental groups were injected with Phosphate-Buffered Saline (PBS) and hUCMSC-derived exosomes, respectively, at the fracture site, and a blank control group was set. At 2 and 3 w after treatment, respectively, the healing condition at the fracture site in the rats was detected by micro-computed tomography (CT). The protein expressions of ß-catenin and Wnt3a of the Wnt signaling pathway in the bone tissue were measured via Western blotting (WB) assay. Quantitative Real Time-fluorescence Polymerase Chain Reaction (qRT-PCR) was performed to determine the expressions of osteogenic marker genes [collagen type I (COL-1), osteopontin (OPN) and runt-related transcription factor 2 (RUNX2)]. RESULTS: The results of the micro-CT scan showed that the rats treated with exosomes had better apposition of the fracture site, and the appearance of cortical bone was continuous. The fracture sites in the blank control group and PBS injection group were not healed, and the appearance of cortical bone was discontinuous, with significant fracture lines. According to the WB results, the protein expression levels of ß-catenin and Wnt3a in exosome treatment group were significantly higher than those in the blank control group and PBS injection group (p<0.01). The qRT-PCR results indicated that the expression levels of COL-1, OPN and RUNX2 in exosome treatment group were increased evidently compared with those in the other two groups (p<0.01). CONCLUSIONS: HucMSC-derived exosomes are probably involved in the repair of fracture in rats through the Wnt signaling pathway.

Characterization of the male-specific lethal 3 gene in the oriental river prawn, Macrobrachium nipponense.

In this study, male-specific lethal 3 homolog (Mnmsl3) was cloned and characterized from the freshwater prawn Macrobrachium nipponense (Crustacea: Decapoda: Palaemonidae) by rapid amplification of cDNA ends. The deduced amino acid sequences of Mnmsl3 showed high-sequence homology to the insect Msl3 and contained a conserved chromatin organization modifier domain and an MORF4-related gene domain. Real-time quantitative reverse transcription-polymerase chain reaction showed that the Mnmsl3 gene was expressed in all the investigated tissues, with the highest level of expression in the testis. The expression level of Mnmsl3 between males and females was different in the gonad (testis or ovary), abdominal ganglion, and heart. The results revealed that the Mnmsl3 gene might play roles in regulating chromatin and in dosage compensation of M. nipponense. Real-time quantitative reverse transcription-polymerase chain reaction also revealed that Mnmsl3 mRNA expression was significantly increased in both 5 and 20 days post-larvae after metamorphosis, suggesting that Mnmsl3 plays complex and important roles in the early embryonic development and sex differentiation of M. nipponense.

Absence of the adenosine A2A receptor confers pulmonary arterial hypertension and increased pulmonary vascular remodeling in mice.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by sustained elevation of pulmonary vascular resistance resulting from endothelial and smooth muscle cell dysfunction and collagen deposition in pulmonary vascular walls. In this study, we investigated the role of the adenosine A(2A) receptor (A(2A)R) in the development of PAH by determining the effect of genetic inactivation of A(2A)Rs on pulmonary vascular remodeling in mice. METHODS AND RESULTS: We characterized hemodynamic, histological and ultrastructural changes in pulmonary vascular remodeling in A(2A)R knockout (KO) mice compared with their wild-type (WT) littermates after exposure to normoxia and hypoxic conditions. After exposure to normoxia, compared to WT mice, A(2A)R KO mice displayed: (1) increased right ventricular systolic pressures and an elevated ratio of the right ventricle over left ventricle plus septum (Fulton index), (2) increased wall area and thickness as well as enhanced smooth muscle actin immunoreactivity in pulmonary resistance vessels, (3) increased proliferating cell nuclear antigen-positive cells in pulmonary resistance vessels and (4) increased smooth muscle cells hypertrophy and collagen deposition in the adventitia of pulmonary arteriole walls as revealed by electron microscope. By contrast, histological analysis revealed no features of hypertensive nephropathy in A(2A)R KO mice and there was no significant difference in systemic blood pressure, and left ventricular masses among the 3 genotypes. Furthermore, following chronic exposure to hypoxia, A(2A)R KO mice exhibited exacerbated elevation in right ventricular systolic pressure, hypertrophy of pulmonary resistance vessels and increased cell proliferation in pulmonary resistance vessels, compared to WT littermates. Thus, genetic inactivation of A(2A)Rs selectively produced PAH and associated increased smooth muscle proliferation and collagen deposition. CONCLUSIONS: Extracellular adenosine acting at A(2A)Rs represents an important regulatory mechanism to control the development of PAH and pulmonary vascular remodeling.

Transport stress in broilers. II. Superoxide production, adenosine phosphate concentrations, and mRNA levels of avian uncoupling protein, avian adenine nucleotide translocator, and avian peroxisome proliferator-activated receptor-gamma coactivator-1alpha in skeletal muscles.

The effect of transport stress on superoxide production and adenosine phosphate concentration in addition to avian uncoupling protein (avUCP), avian adenine nucleotide translocator, and avian peroxisome proliferator-activated receptor-gamma coactivator-1alpha mRNA levels of skeletal muscles in broilers was investigated. Arbor Acres chicks (n = 360, 46 d old, males) were randomly allotted to 1 of 5 treatments: unstressed control, 45-min (short-term) transport with 45-min (short-term) recovery, 45-min transport with 3-h (long-term) recovery, 3-h (long-term) transport with 45-min recovery, and 3-h transport with 3-h recovery. Each treatment consisted of 6 replicates with 12 birds each. All birds (except control group) were transported according to a designed protocol. Transport time affected reactive oxygen species production in the thigh muscle (P < 0.05), adenosine triphosphate (ATP) content and energy charge (EC) in both breast and thigh muscles (P < 0.05 for all 4 comparisons), ATP:adenosine diphosphate (ADP) ratio in the breast muscle (P < 0.05), and avUCP mRNA levels in the thigh muscle (P < 0.05). Long-term transport increased (P < 0.05) reactive oxygen species production, ATP content, ATP:ADP ratio, and EC in the thigh muscle, but it decreased ATP content, ATP:ADP ratio, and EC in the breast muscle. Long-term transport increased avUCP mRNA in the thigh muscle (P < 0.05). Long-term recovery increased the ATP (P < 0.05) and ADP (P < 0.05) concentrations, avian adenine nucleotide translocator mRNA (P < 0.05), and avian peroxisome proliferator-activated receptor-gamma coactivator-1alpha mRNA (P < 0.05) in the thigh muscle, whereas EC decreased (P < 0.05) in the breast muscle. There were interactions between transport and recovery time on ATP (P < 0.05), EC (P < 0.05), and avUCP mRNA level (P < 0.05) in the thigh muscle. This study suggests that long-term transport accelerates muscle energy metabolism and lipid peroxidation. A long-term recovery may help alleviate cellular damage and maintain meat quality by reducing the rate of energy metabolism and scavenging of free radicals formed.

Retroviral gene transfer of tissue-type plasminogen activator targets thrombolysis in vitro and in vivo.

Patients usually have serious complications of thrombosis and bleeding by eating anticoagulation medicine for their residual lives after mechanical valve replacement operation. Tissue-type plasminogen activator (tPA) could target thrombolysis by activating plasminogen to fibrinolysin. In this study, we recombined a retroviral vector pLEGFP-N1-tPA and cultured purified packaging cells PT67/pLEGFP-N1-tPA to produce high-titer retrovirus. In vitro, two target cells, endothelial cell of umbilical vein (ECUV) 304 and heart muscle cell (HMC) that consist of endocardium and heart muscle, were infected by pLEGFP-N1-tPA. The results demonstrated that exogenous tPA was successfully transferred into ECUV304 and HMC. tPA in the two cells shows significant thrombolysis in plasma plate and the activity and content of tPA were high. Furthermore, in vivo, no thrombus was seen on the surface of Dacron patches (the same material making up a ring of mechanical valve) by tPA locally transferring around Dacron patches that were transplanted in the inferior caval veins of rabbits. tPA was successfully transferred into the local inferior caval vein. Activity and content of tPA were high in local tissue and blood and thrombolysis was effectively demonstrated by tPA rapidly, efficiently and long expressing. This laid the foundation for study and appliance of the tPA gene valve.

Effect of 17beta-oestradiol and ginsenoside on osteoporosis in ovariectomised rats.

To study the anti-osteoporosis effects and mechanism of action of oestradiol (E2) and ginsenoside (tR), we measured the bone mineral densities (BMD) of lumbar vertebra and tibia and analysed the tibia histological morphological data, as well observed the activity and the number of osteoblasts and the activity of alkaline phosphatase (ALP) and the concentration of cAMP. Results showed that E2 (400 microg kg- 1 week- 1) and tR (10, 20, 30 mg kg- 1 day- 1) were able to countervail the decreasing in BMDs of lumbar vertebra and tibia induced by OVX in rats (P<0.05); E2 (0.1 micromol l- 1) and ginsenoside Rg1 (1 micromol l- 1 and 10 micromol l- 1) were able to increase the number of osteoblasts, the activity of ALP and the concentration of intercellular cAMP in cultured osteoblast cells. The present findings suggest that E2 and tR have an anti-osteoporosis effect in ovariectomised rats.

Effect of 17-beta-estradiol and ginsenoside Rg1 on reactive microglia induced by beta-amyloid peptides.

The reactive microglias induced by 25 micromol of beta-amyloid peptides (Abeta25-35) and/or IFN-gamma can initiate the microglial respiratory burst and release NO, respectively. Oxidative stress and inflammatory function have been implicated in Alzheimer's disease (AD). We showed that 10 micromol 17-beta-estradiol (E2) and 1-10 micromol ginsenoside Rg1 (Rg1) could prevent the toxicity of Abeta25-35 and/or IFN-gamma to microglias, inhibit the microglial respiratory burst activity and decrease the accumulation of NO. These results demonstrated the protectional effect of E2 or Rg1 on neuron from damaging by reactive microglias in AD.