Unveiling the mechanism of source-sink rebalancing in cucumber-pumpkin heterografts: the buffering roles of rootstock cotyledon.

Grafting onto pumpkin rootstock is widely applied in cucumber production to improve growth and yield, as well as to overcome soil-borne diseases and enhance resistance to abiotic stresses. In this study, we constructed the cucumber-pumpkin heterografts with the one-cotyledon grafting method, and examined the effects of heterografting on biomass allocation and sugar partitioning, with cucumber and pumpkin self-grafts used as control. Compared with cucumber self-grafts, heterografting onto pumpkin rootstock promoted photosynthesis in cucumber scion, and led to higher sucrose contents in the 1st true leaf (source) and newly emerged leaf (sink). Thereby, the scion part of heterografts accumulated more biomass than cucumber self-grafts. In contrast, when compared to pumpkin self-grafts, grafting with cucumber scion reduced root vigor and biomass but promoted cotyledon growth in pumpkin rootstock. The roots (sink) of heterografts contained less sucrose and hexoses, and showed reduced sucrose synthase (SuSy) and hexokinase (HXK) activities. However, the rootstock cotyledon (source) contained more sucrose and starch, and showed higher activities of HXK, cell-wall invertase (CWIN), and enzymes for starch synthesis and degradation. Furthermore, removal or shade of rootstock cotyledon led to reduced growth of root and scion. Silencing of CmoMEX1a gene in rootstock cotyledon inhibited maltose export and reduced root growth of heterografts. These results indicated that rootstock cotyledon, especially its starch content, played a buffering role in the growth regulation of cucumber-pumpkin heterografts. Taken together, our results provided a major contribution to our understanding of source-sink sugar partitioning and scion-rootstock growth balancing in cucumber-pumpkin heterografts.

[EGF and SCF promote the proliferation and differentiation of mouse spermatogenic cells in vitro].

OBJECTIVE: To study the promoting effects of the epidermal growth factor (EGF) and stem cell factor (SCF) on the proliferation and differentiation of spermatogenic cells in mice. METHODS: We cocultured in vitro spermatogenic cells of male mice aged 7 - 8 days in the medium with EGF and/or SCF at the concentrations of 5, 10, 20, 40 and 100 ng/ml, respectively. Then we observed the survival rate and morphological changes of the spermatogenic cells, detected the expressions of the pachytene-specific phosphoprotein gene (P19) and haploid sperm cell-specific transition protein gene (TP1), and analyzed the ploidy of the cells. RESULTS: After cocultured with EGF or SCF for 2 - 4 days, the spermatogenic cells began to proliferate in masses or chains in all concentration groups, most obviously in the 20 ng/ml EGF and 40 ng/ml SCF groups. At 7 days, both the number and survival rate of spermatogenic cells were significantly higher in the 20 ng/ml EGF and 40 ng/ml SCF groups than in the others (P < 0.05), and meanwhile, the P19/TP1 ratio was obviously decreased and the rate of haploid sperm markedly increased in the 40 ng/ml SCF group (P < 0.05). Combination of EGF and SCF remarkably promoted the proliferation of the spermatogenic cells (P < 0.05). CONCLUSION: Both EGF and SCF could increase the number and survival rate of spermatogenic cells. SCF could promote the formation of haploid sperm, and the combination of the two cytokines could enhance the effect on the proliferation of spermatogenic cells.

Phytosterols Alleviate Hyperlipidemia by Regulating Gut Microbiota and Cholesterol Metabolism in Mice.

Phytosterols (PS) have been shown to regulate cholesterol metabolism and alleviate hyperlipidemia (HLP), but the mechanism is still unclear. In this study, we investigated the mechanism by which PS regulates cholesterol metabolism in high-fat diet (HFD) mice. The results showed that PS treatment reduced the accumulation of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in the serum of HFD mice, while increasing the serum levels of high-density lipoprotein cholesterol (HDL-C). Compared with HFD mice, PS not only increased the antioxidant activity of the liver but also regulated the mRNA expression levels of enzymes and receptors related to cholesterol metabolism. The hypolipidemic effect of PS was abolished by antibiotic (Abx) intervention and reproduced by fecal transplantation (FMT) intervention. The results of 16S rRNA sequencing analysis showed that PS modulated the gut microbiota of mice. PS reduced the relative abundance of Lactobacillus and other bile salt hydrolase- (BSH-) producing gut microbiota in HFD mice, which are potentially related to cholesterol metabolism. These findings partially explain the mechanisms by which PS regulates cholesterol metabolism. This implies that regulation of the gut microbiota would be a potential target for the treatment of HLP.

Portulaca oleracea L. extracts alleviate 2,4-dinitrochlorobenzene-induced atopic dermatitis in mice.

Atopic dermatitis (AD) is a common chronic allergic skin disease characterized clinically by severe skin lesions and pruritus. Portulaca oleracea L. (PO) is a resourceful plant with homologous properties in medicine and food. In this study, we used two different methods to extract PO, and compared the therapeutic effects of PO aqueous extract (POAE) and PO ultrasound-assisted ethanol extract (POEE) on 2,4-dinitrochlorobenzene (DNCB)-induced AD mice. The results showed that in POAE and POEE, the extraction rates of polysaccharides were 16.95% and 9.85%, while the extraction rates of total flavonoids were 3.15% and 3.25%, respectively. Compared with AD mice, clinical symptoms such as erythema, edema, dryness and ulceration in the back and left ear were alleviated, and pruritus behavior was reduced after POAE and POEE treatments. The thickness of the skin epidermis was thinned, the density of skin nerve fibers labeled with protein gene product 9.5 (PGP9.5) was decreased, and mast cell infiltration was reduced. There was a decrease in blood lymphocytes, eosinophils and basophils, a significant decrease in spleen index and a noticeable decrease in serum immunoglobulin E (Ig E). POEE significantly reduced the concentration of the skin pruritic factor interleukin (Il)-31. POAE and POEE reduced the concentration of skin histamine (His), down-regulated mRNA expression levels of interferon-γ (Ifnγ), tumor necrosis factor-α (Tnf-α), thymic stromal lymphopoietin (Tslp) and Il-4, with an increase of Filaggrin (Flg) and Loricrin (Lor) in skin lesions. These results suggested that POAE and POEE may inhibit atopic response and alleviate the clinical symptoms of AD by inhibiting the expression of immune cells, inflammatory mediators and cytokines. PO may be a potential effective drug for AD-like diseases.

A rat model for studying neural stem cell transplantation.

AIM: The goal of this project was to develop a rat model for neural stem cell (NSC) transplantation studies in which NSCs were modified with brain-derived neurotrophic factor (BDNF) genes that may permit extensive and reliable analysis of the transplants. METHODS: NSCs were cultured and purified by limiting dilution assay in vitro and infected with recombinant retrovirus pLXSN-BDNF (BDNF-NSCs) and retrovirus pLXSN (p-NSCs). The expression of BDNF genes in transgenic and control NSC groups was measured by FQ-PCR and ELISA assays. NSCs were then transplanted into the subretinal space of normal rat retinas in four groups, which included NSCs alone, BDNF-NSCs, phosphate buffered saline (PBS) control, and normal control. Survival, migration, and differentiation of donor cells in host retinas were observed with optical coherence tomography (OCT), Heidelberg retina angiograph (HRA), and immunohistochemistry, respectively. RESULTS: The results obtained by FQ-PCR demonstrated that the copy numbers of BDNF gene templates from BDNF-NSCs were the highest among the four groups (P<0.05). Consistent with the results of FQ-PCR, BDNF protein level from the supernatant of the BDNF-NSCs group was much higher than that of the other two groups (P<0.05) as suggested by the ELISA assays. HRA and OCT showed that graft cells could successfully survive. Immunohistochemical analysis revealed that transplanted BDNF-NSCs could migrate in the host retinas and differentiate into glial cells and neurons three months after transplantation. CONCLUSION: BDNF promotes NSCs to migrate and differentiate into neural cells in the normal host retinas.

[Effect of Radix Polygoni Multifori praeparata by different processing on model of blood deficiency rats].

OBJECTIVE: To evaluate a proper processed method of Radix Polygoni Multiflori by pharmacodynamic action. METHODS: Model of blood deficiency rats were used in this experiment. The reagents were alcohol extracts of radix polygoni multiflori and its dreferent processing drugs made without any adjuvants in the first experiment. The reagents were alcohol extracts of te processing drugs made without any adjuvants and that made with decoction of black soybean in the second experiment. Their effecacy on model blood deficiency rats was observed. RESULTS: It was showed that some of radix polygoni multiflori praeparata could nourish the blood. The effecacy was different for alcohol extracts of variously processing drugs. Decoction of black soybean could reinforce effectiveness of radix polygoni multiflori praeparata. But it was related to the processed method. CONCLUSION: The processed method directly influences the pharmacodynamic action of radix polygoni multiflori praeparata.

Scutellarin protects oxygen/glucose-deprived astrocytes and reduces focal cerebral ischemic injury.

Scutellarin, a bioactive flavone isolated from Scutellaria baicalensis, has anti-inflammatory, anti-neurotoxic, anti-apoptotic and anti-oxidative effects and has been used to treat cardiovascular and cerebrovascular diseases in China. However, the mechanisms by which scutellarin mediates neuroprotection in cerebral ischemia remain unclear. The interaction between scutellarin and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) was assessed by molecular docking study, which showed that scutellarin selectively binds to NOX2 with high affinity. Cultures of primary astrocytes isolated from the cerebral cortex of neonatal Sprague-Dawley rats were pretreated with 2, 10 or 50 µM scutellarin for 30 minutes. The astrocytes were then subjected to oxygen/glucose deprivation by incubation for 2 hours in glucose-free Dulbecco's modified Eagle's medium in a 95% N2/5% CO2 incubator, followed by simulated reperfusion for 22 hours. Cell viability was assessed by cell counting kit-8 assay. Expression levels of NOX2, connexin 43 and caspase-3 were assessed by western blot assay. Reactive oxygen species were measured spectrophotometrically. Pretreatment with 10 or 50 µM scutellarin substantially increased viability, reduced the expression of NOX2 and caspase-3, increased the expression of connexin 43, and diminished the levels of reactive oxygen species in astrocytes subjected to ischemia-reperfusion. We also assessed the effects of scutellarin in vivo in the rat transient middle cerebral artery occlusion model of cerebral ischemia-reperfusion injury. Rats were given intraperitoneal injection of 100 mg/kg scutellarin 2 hours before surgery. The Bederson scale was used to assess neurological deficit, and 2,3,5-triphenyltetrazolium chloride staining was used to measure infarct size. Western blot assay was used to assess expression of NOX2 and connexin 43 in brain tissue. Enzyme-linked immunosorbent assay was used to detect 8-hydroxydeoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE) and 3-nitrotyrosin (3-NT) in brain tissue. Immunofluorescence double staining was used to determine the co-expression of caspase-3 and NeuN. Pretreatment with scutellarin improved the neurological function of rats with focal cerebral ischemia, reduced infarct size, diminished the expression of NOX2, reduced levels of 8-OHdG, 4-HNE and 3-NT, and reduced the number of cells co-expressing caspase-3 and NeuN in the injured brain tissue. Furthermore, we examined the effect of the NOX2 inhibitor apocynin. Apocynin substantially increased connexin 43 expression in vivo and in vitro. Collectively, our findings suggest that scutellarin protects against ischemic injury in vitro and in vivo by downregulating NOX2, upregulating connexin 43, decreasing oxidative damage, and reducing apoptotic cell death.

A new cytotoxic cholesterol sulfate from marine sponge Halichondria rugosa.

A new steroid, 24xi,25-dimethyl-3alpha-hydroxyl-cholest-5-ene-2beta-ol sodium sulfate (1), together with a known steroid, 24xi,25-dimethyl-cholest-5-ene-2beta,3alpha-diol disodium sulfate (2), was isolated from the ethanol extract of marine sponge Halichondria rugosa. Their structures were elucidated on the base of spectroscopic analysis. Both compounds showed cytotoxicity to four human cancer cell lines (BEL-7402, HT-29, SPC-A1 and U-251) with IC(50) values between 6.5 and 23.1 microM.

Effect of gastrin on differentiation of rat intestinal epithelial cells in vitro.

AIM: To investigate the effect of gastrin on differentiation of IEC-6 cell line in vitro. METHODS: IEC-6 cells were incubated with gastrin. On day 7 after treatment, cell morphology was examined by light microscope, and on day 20, the cellular ultrastructures were examined by electron microscope. After exposure to gastrin for 6 hours, villin mRNA was analyzed by reverse transcription-polymerase chain reaction, and on day 7, the expression of villin was examined by immunocytochemical analysis with laser confocal microscope. RESULTS: After exposure to gastrin, IEC-6 cells showed differentiated phenotypes as villas enterocytes and contained an abundance of plasma, small nuclei with nucleoli, and were arranged regularly. There were numerous microvilli around edge of the cells, and several cells showed columnar structures. Villin mRNA expression in cytoplasm was increased in comparison with control. CONCLUSION: Differentiated characteristics of villus enterocytes and phenotypic changes of rat intestinal epithelial cells (IEC-6) are induced by gastrin, and the effects of gastrin are correlated to increased villin expression.

[Effect of yirong oral liquid on the reperfusion injury in rats with cerebral infarction undergoing thrombolysis].

OBJECTIVE: To observe the effect of Yirong Oral Liquid (YROL) on reperfusion injury in rats with cerebral infarction undergoing thrombolysis. METHODS: Clinical reperfusion under thrombolysis was simulated by applying thrombolysis on reversible local cerebral ischemic rat model. In the rat model, effect of YROL on parameters concerning anti-oxidation capability, cerebral edema and ultrastructure of brain were observed. RESULTS: YROL could alleviate the cerebral edema after reperfusion, markedly increase the activity of superoxide dismutase in blood plasma, decrease the content of malonyldialdehyde, inhibit the post-reperfusion lipid peroxidation, and significantly reduce the ischemia/reperfusion injury of nerve cells in brain of rat. CONCLUSION: YROL has definite protecting effect on brain.

Mineral elements in root of wild Saposhnikovia divaricata and its rhizosphere soil.

Mineral elements are important components of medicinal herbs, and their concentrations are affected by many factors. In this study, Ca, Mg, Na, K, Fe, Mn, Cu, and Zn concentrations in wild Saposhnikovia divaricata and its rhizosphere soil collected from seven locations at two different times in China were measured, and influences of rhizosphere soil on those minerals in plant were evaluated. The results showed that mean concentrations of eight minerals in plant samples decreased in the order: Ca > Mg > Na > K > Fe > Zn > Mn > Cu, and those in the soil samples followed the following order: Na > Fe > Ca > K > Mg > Mn > Zn > Cu. Mean concentrations of Ca, Na, Mg, and K in plants were higher than those in soils, while higher mean concentrations of the other four minerals were found in soils. It was found that there was a positive correlation of Mg, Na, and Cu concentrations in the plant with those in the soil respectively, but a negative correlation of Mn concentration in plant with that in the soil. Except Ca, K, and Mn, the other five minerals in plant were all directly affected by one or more chemical compositions of soil. The results also indicate that pH value and concentrations of total nitrogen, Mg, Mn, and Cu in soil had significant correlations with multimineral elements in plant. In a word, mineral elements uptake of S. divaricata can be changed by adjusting the soil fertility levels to meet the need of appropriate quality control of S. divaricata.

Determination of aflatoxins in edible oil from markets in Hebei Province of China by liquid chromatography-tandem mass spectrometry.

Analysis of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) in 76 edible oil samples (peanut oil, soybean oil, corn embryo oil and blended oil) was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The oils were sampled from three areas (Shijiazhuang, Baoding and Tangshan) of Hebei Province of China. AFB1 was detected in 22 samples representing 28.9%, followed by AFB2 (7.89%) and AFG1 (3.95%), while no AFG2 contamination was detected in any samples. AFB1 levels in oil samples ranged 0.14-2.72 µg kg(-1) and AFB2 ranged 0.15-0.36 µg kg(-1), while lower levels of 0.01-0.02 µg kg(-1) for AFG1 were recorded. The paper is part of an on-going investigation of aflatoxin contamination in Chinese edible oils.

[Effects of salt stress on mulberry seedlings growth, leaf water status, and ion distribution in various organs].

Taking the seedlings of mulberry (Morus alba) varieties Qiuyu and Tailai from Heilongjiang Province as test materials, this paper studied their growth, leaf water relations, and ion distribution in various organs under the stress of different salt concentration. The resu1ts showed that salt stress decreased the plant height and dry mass of the seedlings obviously, and the dry mass of young leaves was more affected than that of old leaves. With increasing salt stress, leaf water potential, osmotic potential, pressure potential, and relative water content decreased markedly, while the Na+ concentration in root and stem had an obvious increase. When the soil NaCl concentration was 150 mmol L(-1) or higher, the Na+ concentration in leaf, stem, and root reached saturated. Salt stress markedly decreased the K+ and Ca2+ concentrations in leaf, stem, and root as well as the Mg2+ concentration in leaf and stem, but had lesser effects on the Mg2+ concentration in root. The decrease of leaf Ca2+, K+, and Mg2+ concentrations under salt stress caused the ion deficiency in plant and limited plant growth, while the regionalized distribution of Na+ in root, stem, and older leaf could be one of the salt tolerance mechanisms of test varieties.

[Effect of Tongxinluo on nestin and vascular endothehal growth factor mRNA expression in rat brain tissue after cerebral ischemia-reperfusion injury].

OBJECTIVE: To investigate the expressions of nestin and vascular endothehal growth factor (VEGF) mRNAs in rat brain tissue after cerebral ischemia-reperfusion injury and their changes in response to Tongxinluo (a traditional Chinese herbal preparation) treatment. METHODS: Cerebral ischemia was induced in rats by temporary middle cerebral artery occlusion (MCAO) followed by treatment with Tongxinluo at high and low doses. On days 3, 7, 14 and 21 after MCAO, nestin and VEGF mRNA expressions in the ependyma, subventricular zone (SVZ), and hippocampal subdentate gyrus zone (HDG) in the ischemic hemisphere were quantitatively analyzed using immunohistochemistry and RT-PCR. RESULTS: Compared with the sham-operated group, the rats with cerebral ischemia-reperfusion injury showed significantly increased nestin-positive neurons and VEGF mRNA expression in the SVZ and HDG 7, 14 and 21 days after MCAO (P<0.05). Treatment with Tongxinluo, especially at high doses, significantly increased the number of nestin-positive neurons and VEGF mRNA expression in the rats 7, 14 and 21 days after MCAO (P<0.05). CONCLUSION: Focal cerebral ischemia in rats results in rapid response and proliferation of neural stem cells in the SVZ and HDG in the ischemic hemisphere possibly by increasing VEGF mRNA expression in the adjacent tissues around the ischemic focus. Tongxinluo may enhance the differentiation and proliferation capacity of the neural stem cells after MCAO by inducing the expression of VEGF mRNA.

[The effect of PPS on location of rBCG in bladder cancer cell line T24].

AIM: To investigate the effect of polyporus polysaccharide (PPS) on location of green fluorescent recombinant protein BCG in human bladder cancer cell line T24. METHODS: A green fluorescent protein expressed with rBCG was used as reporter gene, and laser scanning confocal microscopy and flow cytometry(FCM) analysis were performed to examine the alteration of T24 cells stimulated with rBCG or rBCG plus PPS. RESULTS: Green fluorescence (86.335+/-5.856) was observed obviously in the cytoplasm of T24 cells after interaction with rBCG for 24 h. By FCM we found scatter dot signal in SS and FS channel from rBCG groups was stronger that a control at 24 h and 48 h respectively. Meanwhile the population of T24 cells deflected to upper right and the percentage of GFP(+) T24 cells detected in FL1 channel was (8.7+/-1.572)%, (13.8+/-2.31)% (P<0.05 compared to the control) respectively. For rBCG plus PPS groups, the fluorescence intensity increased in the nucleus (72.603+/-1.165), while decreased in the cytoplasm (93.06+/-0.958), thus the nucleus/plasm ratio increased (0.78+/-0.005). Compared to rBCG group (62.832+/-2.909, 105.306+/-6.393, 0.597+/-0.012), the difference was statistically significant (P<0.05). CONCLUSION: rBCG is able to enter directly into the cytoplasm of T24 cells.