Hormonal Regulation and Stimulation Response of Jatropha curcas L. Homolog Overexpression on Tobacco Leaf Growth by Transcriptome Analysis.

The Flowering locus T (FT) gene encodes the florigen protein, which primarily regulates the flowering time in plants. Recent studies have shown that FT genes also significantly affect plant growth and development. The FT gene overexpression in plants promotes flowering and suppresses leaf and stem development. This study aimed to conduct a transcriptome analysis to investigate the multiple effects of Jatropha curcas L. homolog (JcFT) overexpression on leaf growth in tobacco plants. The findings revealed that JcFT overexpression affected various biological processes during leaf development, including plant hormone levels and signal transduction, lipid oxidation metabolism, terpenoid metabolism, and the jasmonic-acid-mediated signaling pathway. These results suggested that the effects of FT overexpression in plants were complex and multifaceted, and the combination of these factors might contribute to a reduction in the leaf size. This study comprehensively analyzed the effects of JcFT on leaf development at the transcriptome level and provided new insights into the function of FT and its homologous genes.

Integrated transcriptome and physiological analysis revealed core transcription factors that promote flavonoid biosynthesis in apricot in response to pathogenic fungal infection.

MAIN CONCLUSION: Integrated transcriptome and physiological analysis of apricot leaves after Fusarium solani treatment. In addition, we identified core transcription factors and flavonoid-related synthase genes which may function in apricot disease resistance. Apricot (Prunus armeniaca) is an important economic fruit species, whose yield and quality of fruit are limited owing to its susceptibility to diseases. However, the molecular mechanisms underlying the response of P. armeniaca to diseases is still unknown. In this study, we used physiology and transcriptome analysis to characterize responses of P. armeniaca subjected to Fusarium solani. The results showed increasing malondialdehyde (MDA) content, enhanced peroxidase (POD) and catalase (CAT) activity during F. solani infestation. A large number of differentially expressed genes (DEGs), which included 4281 upregulated DEGs and 3305 downregulated DEGs, were detected in P. armeniaca leaves exposed to F. solani infestation. Changes in expression of transcription factors (TFs), including bHLH, AP2/ERF, and WRKY indicated their role in triggering pathogen-responsive genes in P. armeniaca. During the P. armeniaca response to F. solani infestation, the content of total flavonoid was changed, and we identified enzyme genes associated with flavonoid biosynthesis. Ectopic overexpression of PabHLH15 and PabHLH102 in Nicotiana benthamiana conferred elevated resistance to Fspa_1. Moreover, PabHLH15 and PabHLH102 positively interact with the promoter of flavonoid biosynthesis-related genes. A regulatory network of TFs regulating enzyme genes related to flavonoid synthesis affecting apricot disease resistance was constructed. These results reveal the potential underlying mechanisms of the F. solani response of P. armeniaca, which would help improve the disease resistance of P. armeniaca and may cultivate high-quality disease-resistant varieties in the future.

Finding a resveratrol analogue as potential anticancer agent with apoptosis and cycle arrest.

Resveratrol has been extensively studied as the anti-cancer agent. A variety of resveratrol analogues have been developed with structural modification to improve its bioactivity. In this work, resveratrol analogues, compound 1-4, were designed and synthesized with the Stille-Heck reaction. These results showed compound 1-4 had better anticancer effect than that of parent resveratrol. Especially compound 1 ((E)-4,4'-(ethene-1,2-diyl)bis(3-methylphenol)) displayed the excellent cytotoxicity and high selectivity. The mechanism research indicated compound 1 inhibited cell proliferation by binary paths of cell cycle arrest in S phase regulated by cyclin A1/A2 and apoptosis induction mediated by Bax/Bcl2 in a prooxidant manner.

Finding an efficient tetramethylated hydroxydiethylene of resveratrol analogue for potential anticancer agent.

With the improvement and advance in cancer diagnosis and treatment, the cancer is still a major cause of morbidity and mortality throughout the world. Obviously, new breakthroughs in therapies remain be urgent needed. In this work, we designed and synthesized the compound 1-4, namely resveratrol analogues with methylation of hydroxy distyrene, to further explore its new anti-cancer potential. Encouragingly, compound 1 ((E)-4,4'-(ethene-1,2-diyl)bis(3,5-dimethylphenol)) exhibited cytotoxicity superior to resveratrol in MCF 7 cells. More importantly, the compound 1 showed greater toxicity to tumor cells than that to normal cells, which proved that it could selectively kill tumor cells. The favorable results encouraged us to explore the inhibitory mechanism of compound 1 on MCF 7 cells. The research finding indicated the compound 1 inhibited tumor cell proliferation by both arresting cell cycle in S phase and apoptosis via a prooxidant manner. In addition, the results further verified compound 1 caused cell cycle arrest in S phase and apoptosis by down-regulation of the cycling A1/cycling A2 expression and the rise of Bax/Bcl-2 ratio in a p21-dependant pathway in MCF 7 cells. Therefore, these results are helpful for the effective design of anticancer reagents and the better understanding of their mechanism of action.

Atorvastatin suppresses vascular hypersensitivity and remodeling induced by transient adventitial administration of lipopolysaccharide in rats.

BACKGROUND: The phenotypic transition of vascular smooth muscle cells (VSMCs) from a contractile to a proliferative state markedly affects the pathophysiology of cardiovascular diseases. The adventitial inflammation can promote neointimal formation and vascular remodeling. We used direct administration of lipopolysaccharide (LPS) into the periphery of the carotid artery to investigate the influence of transient adventitial inflammation on vascular remodeling and its potential mechanism. METHODS: Male 15-week-old Wistar rats were randomly assigned to four groups with six rats in each group. The rats of groups I and II were administered distilled water, and group III and IV were treated with fasudil and atorvastatin respectively. All treatments were given daily for 11 days. On day 8, the adventitia in group I was injected with 5 µL sterile saline, and the group II-IV were injected with 5 µL sterilized LPS. The carotid blood flow and femoral blood pressure were measured in vivo, and the thickness of vascular intima and middle layer was measured in vitro. Serum interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) were determined using enzyme-linked immunosorbent assay (ELISA) assay. And the Rho-associated coiled-coil-containing protein kinase 2 (ROCK2), myosin phosphatase target subunit 1 (MYPT1), myosin light chain (MLC), myocardin, SM-α actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected by western blot. The comparisons were made by one-way analysis of variance with Bonferroni's post hoc test. A value of P<0.05 was considered to represent a statistically significant difference. RESULTS: Transient adventitial inflammation induced by LPS caused no obvious change in basal blood flow, but did lead to vascular hypersensitivity to serotonin. Morphological examinations revealed that the medial layer was the only domain affected, and showed VSMC proliferation and rearrangement. LPS increased serum IL-6 and TNFα contents, ROCK2 expression and activity, and caused changes in the expression levels of some stereotypical VSMC genes. Similar to the Rho-kinase inhibitor fasudil, atorvastatin completely restored the morphological alterations, even increased blood flow. CONCLUSIONS: Our study confirms the beneficial effect of atorvastatin on the vascular system in terms of morphology and function.

Anti-inflammatory constituents from the root of Litsea cubeba in LPS-induced RAW 264.7 macrophages.

Context Litsea cubeba (Lour.) Pers. (Lauraceae) has long been used as a folk remedy in Traditional Chinese Medicine (TCM) for the treatment of rheumatic diseases. Previous studies from our laboratory indicated that L. cubeba extract showed anti-arthritic activity in rats. Objective To study L. cubeba chemically and biologically and to find the potential constituents responsible for its anti-arthritic effect. Materials and methods The compounds were isolated from the root of L. cubeba by column chromatography which eluted with PE:EtOAc gradient system, and the structures were elucidated by detailed spectroscopic data analysis; the anti-inflammatory activity of the isolated compounds was evaluated by lipopolysaccharide (LPS)-induced RAW 264.7 cells and the TNF-α and NO level were measured by ELISA (commercial kit); The iNOS and COX-2 mRNA expression were measured by RT-PCR and the phosphorylation of IκBα, IKKß, P38 and Akt were determined by western blots. Results A novel 9-fluorenone, 1-ethoxy-3,7-dihydroxy-4,6-dimethoxy-9-fluorenone (1), together with 4 known compounds, namely pinoresinol (2), syringaresinol (3), 9,9'-O-di-(E)-feruloyl-meso-5,5'-dimethoxysecoisolariciresinol (4) and lyoniresinol (5) were isolated from the root of L. cubeba for the first time. The IC50 for NO inhibition on compounds 1 and 4 were 56.1 ± 1.2 and 32.8 ± 2.3 µM, respectively. The IC50 for TNF-α inhibition were 28.2 ± 0.9 and 15.0 ± 1.0 µM, respectively. Both 1 and 4 suppress mRNA expression of iNOS, COX-2 and protein phosphorylation of IκBα, IKKß in LPS-induced RAW 264.7 cells. Discussion and conclusion Compounds 1 and 4 isolated from L. cubeba exhibited potent anti-inflammatory activity through the NF-κB signal pathway.

Correlation of in vivo and in vitro methods in measuring choroidal vascularization volumes using a subretinal injection induced choroidal neovascularization model.

BACKGROUND: In vivo quantification of choroidal neovascularization (CNV) based on noninvasive optical coherence tomography (OCT) examination and in vitro choroidal flatmount immunohistochemistry stained of CNV currently were used to evaluate the process and severity of age-related macular degeneration (AMD) both in human and animal studies. This study aimed to investigate the correlation between these two methods in murine CNV models induced by subretinal injection. METHODS: CNV was developed in 20 C57BL6/j mice by subretinal injection of adeno-associated viral delivery of a short hairpin RNA targeting sFLT-1 (AAV.shRNA.sFLT-1), as reported previously. After 4 weeks, CNV was imaged by OCT and fluorescence angiography. The scaling factors for each dimension, x, y, and z (µm/pixel) were recorded, and the corneal curvature standard was adjusted from human (7.7) to mice (1.4). The volume of each OCT image stack was calculated and then normalized by multiplying the number of voxels by the scaling factors for each dimension in Seg3D software (University of Utah Scientific Computing and Imaging Institute, available at http://www.sci.utah.edu/cibc-software/seg3d.html). Eighteen mice were prepared for choroidal flatmounts and stained by CD31. The CNV volumes were calculated using scanning laser confocal microscopy after immunohistochemistry staining. Two mice were stained by Hematoxylin and Eosin for observing the CNV morphology. RESULTS: The CNV volume calculated using OCT was, on average, 2.6 times larger than the volume calculated using the laser confocal microscopy. The correlation statistical analysis showed OCT measuring of CNV correlated significantly with the in vitro method (R 2 =0.448, P = 0.001, n = 18). The correlation coefficient for CNV quantification using OCT and confocal microscopy was 0.693 (n = 18, P = 0.001). CONCLUSIONS: There is a fair linear correlation on CNV volumes between in vivo and in vitro methods in CNV models induced by subretinal injection. The result might provide a useful evaluation of CNV both for the studies using CNV models induced by subretinal injection and human AMD studies.

Transforming growth factor ß suppresses osteoblast differentiation via the vimentin activating transcription factor 4 (ATF4) axis.

ATF4 is an osteoblast-enriched transcription factor of the leucine zipper family. We recently identified that vimentin, a leucine zipper-containing intermediate filament protein, suppresses ATF4-dependent osteocalcin (Ocn) transcription and osteoblast differentiation. Here we show that TGFß inhibits ATF4-dependent activation of Ocn by up-regulation of vimentin expression. Osteoblasts lacking Atf4 (Atf4(-/-)) were less sensitive than wild-type (WT) cells to the inhibition by TGFß on alkaline phosphatase activity, Ocn transcription and mineralization. Importantly, the anabolic effect of a monoclonal antibody neutralizing active TGFß ligands on bone in WT mice was blunted in Atf4(-/-) mice. These data establish that ATF4 is required for TGFß-related suppression of Ocn transcription and osteoblast differentiation in vitro and in vivo. Interestingly, TGFß did not directly regulate the expression of ATF4; instead, it enhanced the expression of vimentin, a negative regulator of ATF4, at the post-transcriptional level. Accordingly, knockdown of endogenous vimentin in 2T3 osteoblasts abolished the inhibition of Ocn transcription by TGFß, confirming an indirect mechanism by which TGFß acts through vimentin to suppress ATF4-dependent Ocn activation. Furthermore, inhibition of PI3K/Akt/mTOR signaling, but not canonical Smad signaling, downstream of TGFß, blocked TGFß-induced synthesis of vimentin, and inhibited ATF4-dependent Ocn transcription in osteoblasts. Thus, our study identifies that TGFß stimulates vimentin production via PI3K-Akt-mTOR signaling, which leads to suppression of ATF4-dependent Ocn transcription and osteoblast differentiation.

Antiplatelet and antithrombotic activities of timosaponin B-II, an extract of Anemarrhena asphodeloides.

1. Antithrombotic agents are effective in the treatment of ischaemic stroke. Timosaponin B-II (TB-II) is a major active component of Anemarrhena asphodeloides Bunge (Liliaceae; rhizome) that has protective effects against cerebral ischaemic damage. The present study examined the antiplatelet and antithrombotic actions of TB-II. 2. In in vitro experiments, TB-II (20, 40 and 80 mg/mL) potently and dose-dependently inhibited ADP-induced platelet aggregation. Furthermore, 1, 3 and 6 mg/kg TB-II prolonged activated partial thromboplastin time by 9.29, 16.86 and 25.50%, respectively, but had no effect on the prothrombin time. Furthermore, 1, 3 and 6 mg/kg TB-II significantly reduced the wet weight, dry weight and length of the thrombi (%inhibition (based on wet weight): 13.6, 19.8 and 24.7%, respectively). 3. In a rabbit arteriovenous shunt model, 1, 3 and 6 mg/kg, i.v., TB-II had no effect on thrombus formation. Plasma euglobulin lysis time and fibrin degradation product were not affected by 1, 3 and 6 mg/kg TB-II, but plasminogen levels were decreased significantly by 14.4, 18.3 and 29.0%, respectively. 4. The results of the present study demonstrate significant antiplatelet and anticoagulation effects of TB-II and suggest that these actions could contribute to its neuroprotective effect against damage following cerebral ischaemia damage.

Timosaponin B-II inhibits pro-inflammatory cytokine induction by lipopolysaccharide in BV2 cells.

It was reported that the total polysaccharides extracts from Anemarrhenae asphodeloides Bge (Liliaceae, rhizome) could inhibit inflammatory responses in various models. In the present study, the effects of Timosaponin B-II, a purified extract from A. asphodeloidesb, on the expressions of IL-1beta, TNF-alpha and IL-6, the activity of NF-kappaB and the activation of signal pathway related to NF-kappaB were explored in vitro. Timosaponin B-II significantly attenuated increase of these cytokines on both mRNA and protein levels from LPS-stimulated BV2 cells in a dose-dependent manner. The reporter gene assay also showed that the activation of NF-kappaB induced by LPS was inhibited by pre-treatment with Timosaponin B-II. Moreover, western blot results showed that the activation of p38, JNK and P65 had been decreased. These results suggest that both NF-kappaB signal pathway and MAPK pathway were involved in the inhibitory effects of Timosaponin B-II on the expression of pro-inflammatory cytokines.

Ameliorative effects of arctiin from Arctium lappa on experimental glomerulonephritis in rats.

Membranous glomerulonephritis (MGN) remains the most common cause of adult-onset nephrotic syndrome in the world and up to 40% of untreated patients will progress to end-stage renal disease. Although the treatment of MGN with immunosuppressants or steroid hormones can attenuate the deterioration of renal function, numerous treatment-related complications have also been established. In this study, the ameliorative effects of arctiin, a natural compound isolated from the fruits of Arctium lappa, on rat glomerulonephritis induced by cationic bovine serum albumin (cBSA) were determined. After oral administration of arctiin (30, 60, 120 mg/kgd) for three weeks, the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) and 24-h urine protein content markedly decreased, while endogenous creatinine clearance rate (ECcr) significantly increased. The parameters of renal lesion, hypercellularity, infiltration of polymorphonuclear leukocyte (PMN), fibrinoid necrosis, focal and segmental proliferation and interstitial infiltration, were reversed. In addition, we observed that arctiin evidently reduced the levels of malondialdehyde (MDA) and pro-inflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor (TNF-alpha), suppressed nuclear factor-kappaB p65 (NF-kappaB) DNA binding activity, and enhanced superoxide dismutase (SOD) activity. These findings suggest that the ameliorative effects of arctiin on glomerulonephritis is carried out mainly by suppression of NF-kappaB activation and nuclear translocation and the decreases in the levels of these pro-inflammatory cytokines, while SOD is involved in the inhibitory pathway of NF-kappaB activation. Arctiin has favorable potency for the development of an inhibitory agent of NF-kappaB and further application to clinical treatment of glomerulonephritis, though clinical studies are required.

Regulative effects of essential oil from Atractylodes lancea on delayed gastric emptying in stress-induced rats.

Gastric motor dysfunction induced by psychological stress results in many symptoms of functional dyspepsia (FD). There are a number of herbal medicines that are reported to improve gastrointestinal motor. However, the mechanisms of considerable herbal medicines are not explicit. In the present study, the effects of an essential oil (EO) extracted from Atractylodes lancea on delayed gastric emptying, gastrointestinal hormone and hypothalamic corticotropin-releasing factor (CRF) abnormalities induced by restraint stress in rats were investigated and the mechanism of the EO is also explored. Oral administration of EO for 7 days did not affect normal gastric emptying, but accelerated delayed gastric emptying induced by restraint stress in rats. The EO significantly increased the levels of motilin (MTL) and gastrin (GAS) and decreased the levels of somatostatin (SS) and CRF. The EO did not modify gastric emptying in vagotomized rats that underwent restraint stress, but antagonized delayed gastric emptying induced by intracisternal injection of CRF. These results suggest that the regulative effects of the EO on delayed gastric emptying are preformed mainly via inhibition of the release of central CRF and activation of vagal pathway, which are also involved in the release of gastrointestinal hormones such as MTL, GAS and SS.

Inhibitory effect of traditional Chinese medicine Zi-Shen Pill on benign prostatic hyperplasia in rats.

In the present study, we investigate the effects of an extract isolated from traditional Chinese medicine Zi-Shen Pill (ZSPE) on benign prostatic hyperplasia (BPH) in rats induced by testosterone after castration. A total of 50 rats were equally divided into five groups: Group 1 served as control (sham-operated group); Group 2 was model group; Group 3 and Group 4 animals were administered with ZSPE at dose levels of 300 mg/kg and 600 mg/kg; Group 5 was served as positive control group and treated with finasteride at a dose of 1 mg/kg. The drugs were administered orally once a day for 28 days consecutively. The prostate weight, prostatic index, and serum dihydrotestosterone (DHT) levels were significantly reduced and the pathological changes in BPH were also by ameliorated ZSPE. Immunohistochemical examination revealed that the expressions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in prostate were inhibited by ZSPE treatment, whereas the levels of transforming growth factor-beta1 (TGF-beta1) were increased. These results suggest that ZSPE has a definite inhibitory effect on BPH and might be an alternative medicine for treatment of human BPH.

Reduction of atherosclerosis in cholesterol-fed rabbits and decrease of expressions of intracellular adhesion molecule-1 and vascular endothelial growth factor in foam cells by a water-soluble fraction of Polygonum multiflorum.

Polygonum multiflorum stilbeneglycoside (PMS) is a water-soluble fraction of Polygonum multiflorum Thunb., one of the most famous tonic traditional Chinese medicines, that has protective effects on the cardiovascular system. The purpose of the present study is to elucidate the effects of PMS on macrophage-derived foam cell functions and the reduction of severity of atherosclerosis in hypercholesterolemic New Zealand White (NZW) rabbits. NZW rabbits were fed for 12 weeks with a normal diet, a high cholesterol diet, or a high cholesterol diet associated with irrigation with different doses of PMS (25, 50, or 100 mg/kg). Treatment of NZW rabbits fed with high cholesterol diet with 100 mg/kg PMS attenuated the increase in plasma cholesterol, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, and plasma triglyceride. Treatment with 50 and 100 mg/kg PMS caused 43% and 60% decrease in atherosclerotic lesioned area ratio to total surface area, respectively. In U937 foam cells, PMS could decrease the high expression of intercellular adhesion molecule (ICAM)-1 protein and the vascular endothelial growth factor (VEGF) protein levels in the medium induced by oxidized lipoprotein when analyzed by flow cytometry. The results proved that PMS is a powerful agent against atherosclerosis and that PMS action could possibly be through the inhibition of the expression of ICAM-1 and VEGF in foam cells.