Alfalfa as a vegetable source of ß-carotene: The change mechanism of ß-carotene during fermentation.

The objectives of this study were to explore the ß-carotene-producing bacteria and ascertain the main factors affecting ß-carotene content via investigating the effects of various additives on ß-carotene content, bacterial community succession, and quality of fermented alfalfa, using single-molecule real-time (SMRT) sequencing technology. Fresh alfalfa was fermented without (CON) or with squalene (SQ), the combination of Lactobacillus plantarum and cellulase (LPEN), and the combination of SQ and LPEN (SQLPEN) for 3, 45, and 90 d. The results showed that relative to the fresh alfalfa, extensive ß-carotene loss in all groups occurred in the early fermentation phase (3 d) since epiphytic Pantoea agglomerans with the ability to produce ß-carotene disappeared and ß-carotene was oxidized by lipoxygenase and peroxidase. With the prolonged fermentation days, ß-carotene content in all groups increased due to bacterial community succession in the middle and late phases of fermentation (45 and 90 d). The species L. parabuchneri, L. kunkeei, and L. kullabergensis (r = 0.591, 0.366, 0.341, orderly) had positive correlations with ß-carotene content (P < 0.05). Bacterial functional potential prediction showed that species L. kunkeei, L. helsingborgensis, and L. kullabergensis had positive (r = 0.478, 0.765, 0.601) correlations with C10-C20 isoprenoid biosynthesis (P < 0.01), and L. helsingborgensis and L. kullabergensis had positive (r = 0.805, 0.522) correlations with ß-carotene biosynthesis (P < 0.01). Additionally, the pH and propionic acid (r = -0.567, -0.504) had negative correlations with ß-carotene content (P < 0.01). The CON group was preserved well after 90 d, LPEN and SQLPEN further improved fermentation quality. In conclusion, certain Lactobacillus had the potential for ß-carotene biosynthesis, and high pH and propionic acid content were the unbenefited factors for ß-carotene retention in fermented alfalfa.

Recycling deteriorated silage to remove hazardous mycotoxins and produce a value-added product.

Silage, an important forage feed, contains hazardous mycotoxins due to spoilage caused by unreasonable management. Deteriorated silage becomes a mycotoxin source and threatens human health and the eco-environment. Recycling deteriorated silage and exploiting beneficial substances would be profitable and environmentally friendly. Squalene [60.3-73.9 mg/kg fresh matter (FM)] and 6 types of mycotoxins (4.56-10,080 ug/kg FM) were found in deteriorated silages. To clarify the source and synthesis mechanism of squalene, alfalfa was ensiled at low temperature (LT, 3-20 â„ƒ), 25 â„ƒ (T25), 30 â„ƒ (T30) or 35 â„ƒ (T35) for 10, 40 and 70 d. The highest squalene was detected when alfalfa ensiled for 40 d (P = 0.033) or ensiled at LT and T30 (P < 0.001). Squalene source was traced as lactic acid bacteria (LAB) using next-generation sequencing. Multiple linear regression models inferred that squalene synthase of LAB positively contributed to the squalene synthesis but was negatively adjusted by ammonia-N during ensiling. Two promising squalene-producing LAB strains were screened from alfalfa silage, which fermented deteriorated silage to enhanced squalene yield (190~279 mg/L) with low cost and high mycotoxin removal ratios (up to 85.5%). Therefore, the environmentally friendly strategy of recycling deteriorated silage to produce beneficial squalene was created.

Glutamine protects cardiomyocytes from hypoxia/reoxygenation injury under high glucose conditions through inhibition of the transforming growth factor-ß1-Smad3 pathway.

Activation of transforming growth factor-ß1 (TGF-ß1)-Smad3 pathway aggravates myocardial ischemia/reperfusion injury (IRI). We previously showed that glutamine (Gln) protects cardiomyocytes from hypoxia/reoxygenation (H/R) injury under high glucose (HG) conditions. The aim of this study was to investigate whether Gln exerts its protective effect in H/R via inhibiting TGF-ß1-Smad3 pathway. In vitro, H9c2 rat cardiomyocytes were treated with Gln with HG (33 mM) and/or H/R. We also performed in vivo experiments in which we treated normal and diabetic rats with Gln or solvent control following IRI. We assessed protein levels of TGF-ß1, total Smad3, phosphorylated (p)-Smad3 and cleaved caspase-3 in H9c2 cells and rat myocardium by Western blotting. H9c2 cells treated with HG + H/R exhibited high apoptosis rates, as well as a highly activated TGF-ß1-Smad3 pathway. TGF-ß1 receptor inhibitor (SB431542) or Smad3 inhibitor (SIS3) reduced HG + H/R induced apoptosis. Similarly, Gln supplementation alleviated apoptosis and decreased p-Smad3 levels. However, Gln's protective effect was significantly weakened by TGF-ß1. Diabetic rats treated with Gln had improved hemodynamics, smaller infarct size after IRI, and a significant decrease in TGF-ß1-Smad3 pathway activation. We conclude that Gln inhibits HG + H/R induced activation of the TGF-ß1-Smad3 pathway and decreases cell apoptosis in cardiomyocytes.

A novel model for evaluating thrombolytic therapy in dogs with ST-elevation myocardial infarction.

BACKGROUND: There is still no standard large animal model for evaluating the effectiveness of potential thrombolytic therapies. Here, we aimed to develop a new beagle model with ST-elevation myocardial infarction (STEMI) by injecting autologous emboli with similar components of coronary thrombus. METHODS: 18 male beagles were included and divided into three groups: red embolus group (n = 6), white embolus group (n = 6) or white embolus + rt-PA group (n = 6). Autologous emboli were infused into the mid-distal region of the left anterior descending coronary artery. The composition of embolus was examined by scanning electron microscope (SEM). Coronary angiography was performed to verify the status of embolism. Myocardial infarct size was measured by 2, 3, 5- triphenyltetrazolium chloride (TTC) staining. RESULTS: Red thrombus was characteristic of loose reticular structure of erythrocytes under SEM, while the white embolus had compacted structure that mainly consisted of a dense mass of fibrin. Coronary angiography showed the recanalization rate was 2/6 in the red embolus group versus 0/6 in the white embolus group in three hours after occlusion. Arrhythmia, resolution of ST-segment elevation and lower T wave on the electrocardiogram appeared in the red embolus group but not in the white embolus group. Another six dogs with white thrombi were treated with rt-PA. Five out of six dogs exhibited coronary recanalization after two hours of therapy, compared to zero dogs without rt-PA treatment. The size of myocardial infarction in rt-PA group reduced significantly compared with white embolus group using TTC staining method. CONCLUSIONS: The white embolism model was more convenient experimentally and had a higher uniformity, stability and success rate. The major innovation of our study is that we applied fibrin-rich white thrombi to establish beagle model possessing features of clinically observed coronary thrombi in time window of intravenous thrombolysis of STEMI. This model can be used to evaluate new thrombolytic drugs for the treatment of STEMI.

Glutamine Reduces the Apoptosis of H9C2 Cells Treated with High-Glucose and Reperfusion through an Oxidation-Related Mechanism.

Mitochondrial overproduction of reactive oxygen species (ROS) in diabetic hearts during ischemia/reperfusion injury and the anti-oxidative role of glutamine have been demonstrated. However, in diabetes mellitus the role of glutamine in cardiomyocytes during ischemia/reperfusion injury has not been explored. To examine the effects of glutamine and potential mechanisms, in the present study, rat cardiomyoblast H9C2 cells were exposed to high glucose (33 mM) and hypoxia-reoxygenation. Cell viability, apoptosis, intracellular glutamine, and mitochondrial and intracellular glutathione were determined. Moreover, ROS formation, complex I activity, membrane potential and adenosine triphosphate (ATP) content were also investigated. The levels of S-glutathionylated complex I and mitochondrial apoptosis-related proteins, including cytochrome c and caspase-3, were analyzed by western blot. Data indicated that high glucose and hypoxia-reoxygenation were associated with a dramatic decline of intercellular glutamine and increase in apoptosis. Glutamine supplementation correlated with a reduction in apoptosis and increase of glutathione and glutathione reduced/oxidized ratio in both cytoplasm and mitochondria, but a reduction of intracellular ROS. Glutamine supplementation was also associated with less S-glutathionylation and increased the activity of complex I, leading to less mitochondrial ROS formation. Furthermore, glutamine supplementation prevented from mitochondrial dysfunction presented as mitochondrial membrane potential and ATP levels and attenuated cytochrome c release into the cytosol and caspase-3 activation. We conclude that apoptosis induced by high glucose and hypoxia-reoxygenation was reduced by glutamine supplementation, via decreased oxidative stress and inactivation of the intrinsic apoptotic pathway.

A pig model of ischemic mitral regurgitation induced by mitral chordae tendinae rupture and implantation of an ameroid constrictor.

A miniature pig model of ischemic mitral regurgitation (IMR) was developed by posterior mitral chordae tendinae rupture and implantation of an ameroid constrictor. A 2.5-mm ameroid constrictor was placed around the left circumflex coronary artery (LCX) of male Tibetan miniature pigs to induce ischemia, while the posterior mitral chordae tendinae was also ruptured. X-ray coronary angiography, ECG analysis, echocardiography, and magnetic resonance imaging (MRI) were used to evaluate heart structure and function in pigs at baseline and one, two, four and eight weeks after the operation. Blood velocity of the mitral regurgitation was found to be between medium and high levels. Angiographic analyses revealed that the LCX closure was 10-20% at one week, 30-40% at two weeks and 90-100% at four weeks subsequent ameroid constrictor implantation. ECG analysis highlighted an increase in the diameter of the left atria (LA) at two weeks post-operation as well as ischemic changes in the left ventricle (LV) and LA wall at four weeks post-operation. Echocardiography and MRI further detected a gradual increase in LA and LV volumes from two weeks post-operation. LV end diastolic and systolic volumes as well as LA end diastolic and systolic volume were also significantly higher in pig hearts post-operation when compared to baseline. Pathological changes were observed in the heart, which included scar tissue in the ischemic central area of the LV. Transmission electron microscopy highlighted the presence of contraction bands and edema surrounding the ischemia area, including inflammatory cell infiltration within the ischemic area. We have developed a pig model of IMR using the posterior mitral chordae tendineae rupture technique and implantation of an ameroid constrictor. The pathological features of this pig IMR model were found to mimic the natural history and progression of IMR in patients.

Application of sodium alginate microspheres in ischemic stroke modeling in miniature pigs.

The miniature pig is an optimal animal model for studying nervous system disease because of its physiologic and pathologic features. However, the rete mirabile composed of arteries and veins at the skull base limits their application as a model of ischemic stroke by middle cerebral artery occlusion. The present study investigated the possibility of establishing an ischemic stroke model in the miniature pig by blocking the skull base retia with sodium alginate microspheres. Three Bama miniature pigs were used. Using the monitor of C-arm X-ray machine, sodium alginate microspheres (100-300 µm), a novel embolic material, were injected through the femoral artery, aortic arch, common carotid artery, ascending pharyngeal artery and the retia. Results were evaluated using carotid arteriography, MRI, behavior observation and histology. The unilateral rete mirabile was completely blocked, resulting in disturbance in blood supply to the basal ganglia, astasia of the right hind limb and salivation. MRI and hematoxylin-eosin staining showed an evident infarction focus in the basal ganglia. These findings indicate that sodium alginate microspheres are a suitable embolic material for blocking the skull base retia in miniature pigs to establish an ischemic stroke models.

Neural progenitor cells derived from adult bone marrow mesenchymal stem cells promote neuronal regeneration.

AIM: It is well known that neural stem/progenitor cells (NS/PC) are an ideal cell type for the treatment of central nervous system (CNS) disease. However, ethical problems have severely hampered fetal NS/PC from being widely used as a source for stem cell therapy. Recently, it has been demonstrated that autologous bone marrow mesenchymal stem cells (BMSC) can transdifferentiate into neural progenitor cells (NPC). The biological function of BMSC derived NPC (MDNPC) in neuronal systems remains unknown. In the present study, we aimed to investigate whether MDNPC can promote in vitro neural regeneration, a process comprising mainly the generation of neurons and neurotransmitters. MAIN METHODS: We co-cultured BMSC, MDNPC or fetal NS/PC with PC12 cells and studied their roles on proliferation, neurite formation and dopamine release from PC12 cells. Furthermore, we also explored the mechanisms by which MDNPC regulate dopamine secretion from PC12 derived neural cells using Western blot. KEY FINDINGS: We found that both MDNPC and NS/PC had similar morphologies and there were no significant differences between MDNPC and NS/PC in promoting PC12 cell proliferation, neurite outgrowth, and dopamine release. We also demonstrated that NS/PC induced dopamine secretion was associated with an upregulation of dopamine transporter (DAT) levels. SIGNIFICANCE: In summary, MDNPC were comparable to NS/PC in promoting neural regeneration, indicating that MDNPC are a promising candidate source of neural stem cells for the treatment of neurological diseases.

The optimal flow rate for antegrade cerebral perfusion during deep hypothermic circulatory arrest.

The aim of this study is to compare cerebral protection using antegrade cerebral perfusion (ACP) with various flow rates during deep hypothermic circulatory arrest (DHCA) in a piglet model. Twenty-three piglets were randomized to five groups: the control group (n = 3), DHCA group (n = 5), ACP25 group (n = 5), ACP50 group (n = 5), and ACP80 group (n = 5). Three control piglets did not undergo operations. Twenty piglets underwent cardiopulmonary bypass (CPB) and DHCA for 60 min at 20°C. ACP was conducted at 0, 25, 50, and 80 mL/kg/min in the DHCA, ACP25, ACP50, and ACP80 group, respectively. Serum S-100B protein and neuron-specific enolase were monitored, and brain tissues were assayed for the activities of caspase-3 and stained for the evidence of apoptotic cellular injury. Rise in serum S-100B level (post-CPB-pre-CPB) in the ACP50 group was significantly lower than that in the ACP80 group (P = 0.001). Caspase-3 levels were significantly elevated in the ACP80 group compared with the ACP25 (P = 0.041) and ACP50 group (P = 0.01), while positive terminal deoxyneucleotidyl transferase-mediated biotin-dUTP nick end labeling reaction scores in the ACP80 group were significantly higher than those in the ACP25 (P = 0.043) and ACP50 group (P = 0.023). Cerebral protection effects of ACP at 25 and 50 mL/kg/min were superior to that of ACP at 80 mL/kg/min as determined by cerebral markers, immunology, and histology.

Cell viability and dopamine secretion of 6-hydroxydopamine-treated PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells.

In the present study, PC12 cells induced by 6-hydroxydopamine as a model of Parkinson's Disease, were used to investigate the protective effects of bone marrow-derived mesenchymal stem cells bone marrow-derived mesenchymal stem cells against 6-hydroxydopamine-induced neurotoxicity and to verify whether the mechanism of action relates to abnormal α-synuclein accumulation in cells. Results showed that co-culture with bone marrow-derived mesenchymal stem cells enhanced PC12 cell viability and dopamine secretion in a cell dose-dependent manner. MitoLight staining was used to confirm that PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells demonstrate reduced levels of cell apoptosis. Immunocytochemistry and western blot analysis found the quantity of α-synuclein accumulation was significantly reduced in PC12 cell and bone marrow-derived mesenchymal stem cell co-cultures. These results indicate that bone marrow-derived mesenchymal stem cells can attenuate 6-hydroxydopamine-induced cytotoxicity by reducing abnormal α-synuclein accumulation in PC12 cells.

Effects of ligustilide on lipopolysaccharide-induced endotoxic shock in rabbits.

In this study, we investigated the protective effects of ligustilide against lipopolysaccharide (LPS)-induced endotoxic shock in Japanese white rabbits and attempted to elucidate the possible mechanism underlying these effects. Forty-two rabbits were randomized into 6 groups: normal group, LPS group, dexamethasone group (5 mg/kg), and 3 ligustilide groups (20, 40, and 80 mg/kg). After the rabbits had received a LPS infusion (0.3 mg/kg), dexamethasone and ligustilide were intravenously injected at the above-mentioned dosages. Heart rate (HR), mean arterial pressure (MAP), and rectal temperature (RT) were recorded throughout the experiment. Tumor necrosis factor- α (TNF- α), interleukin-1 ß (IL-1 ß), and nitric oxide (NO) levels were measured by radioimmunoassay every 30 minutes for the first hour and every 60 minutes thereafter until the end of the experiment. The serum levels of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), creatinine kinase (CK), lactate dehydrogenase (LDH), total protein (TP), creatinine (Scr), blood urea nitrogen (BUN), total bilirubin (T. BIL), and counts of formed elements of blood were measured at 0, 120, and 300 minutes after the administration of LPS. Hemorheology was assayed 300 minutes after the LPS injection. The vital organs were collected and weighed before histopathologic examination. A comparison between the LPS group and the ligustilide groups showed that ligustilide significantly inhibited the decline in MAP and RT and decreased the levels of TNF- α, IL-1 ß, and NO, but had no apparent effect on HR. Ligustilide also inhibited the increase in the levels of biochemical markers, such as ALT, AST, ALP, GGT, LDH, CK, BUN, and Scr, but showed no apparent effect on T. BIL and TP. Furthermore, ligustilide partly restored the function of injured vital organs, including the heart, liver, lungs, and kidneys. These results suggest that ligustilide protected the rabbits against LPS-induced endotoxic shock.

Neuroprotective effect of liquiritin against focal cerebral ischemia/reperfusion in mice via its antioxidant and antiapoptosis properties.

Our present study was conducted to investigate whether liquiritin (7-hydroxy-2-[4-[3,4,5-trihydroxy-6-(hydroxymethyl) oxan-2-yl] oxyphenyl]-chroman-4-one, 1), an active component of Glycyrrhiza uralensis Fisch., exerts a neuroprotective effect against focal cerebral ischemia/reperfusion (I/R) in male Institute of Cancer Research (ICR) mice. On the establishment of mice with middle cerebral artery occlusion (MCAO) for 2 h and reperfusion for 22 h, liquiritin at the doses of 40, 20, and 10 mg/kg was administered before MCAO once a day intragastrically for a subsequent 3 days. Neurological deficits and infarct volume were measured, respectively. The levels of malondialdehyde (MDA) and carbonyl, activities of superoxide anion (SOD), catalase (CAT) and glutathion peroxidase (GSH-Px) and reduced glutathione/oxidized disulfide (GSH/GSSG) ratio in brain were estimated spectrophotometrically. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) and terminal deoxynucleotidyl transferase-mediated DuTP-biotin nick end labeling (TUNEL)-positive cells were detected by immunohistochemical analysis. Our results showed that the neurological deficits, infarct volume, and the levels of MDA and carbonyl decreased, the ratio of GSH/GSSG and the activities of SOD, CAT, and GSH-Px were compensatorily up-regulated, and 8-OHdG and TUNEL-positive cells decreased after 22 h of reperfusion in liquiritin-treated groups. These findings suggest that liquiritin might be a potential agent against cerebral I/R injury in mice by its antioxidant and antiapoptosis properties.

Salvianolic acid A displays cardioprotective effects in in vitro models of heart hypoxia/reoxygenation injury.

Oxidative stress induced by overproduction of reactive oxygen species (ROS) plays an important role in hypoxia/reoxygenation (H/R) injury. In the present study, effects of salvianolic acid A (1) on heart H/R injury through its antioxidant activity were examined, using a molecule-based ROS scavenging system and cardiomyocyte model of H/R injury, as well as isolated rat heart model. As a result, 1 showed a potent antioxidant activity, scavenging all of the tested ROS and DPPH (2,2-diphenyl-1-picrylhydrazyl). The antioxidant effect of 1 was also observed in cardiomyocytes exposed to H/R. Compound 1 remarkably decreased dihydroethidium and dichlorofluorescein fluorescence and increased cell viability and mitochondrial membrane potential, ΔΨ(m), when compared to the H/R group. In isolated rat hearts exposed to H/R, 1 markedly increased the coronary flow, the peak of pressure development and the valley of pressure development, and significantly reduced the left ventricular end diastolic pressure when compared to the H/R group. These results suggested that 1 had significant protective effects against H/R-induced myocardial injury through its antioxidant activity.