Controlling Polymer Molecular Weight Distribution through a Latent Mediator Strategy with Temporal Programming.

Polymer molecular weight distribution (MWD) is a key parameter of polymers. Here we present a robust method for controlling polymer MWD in controlled cationic polymerizations. A latent mediator strategy was designed and combined with temporal programming to regenerate mediators at different times during polymerization. Both the breadths and shapes of MWD curves were tuned easily by adjusting an external light source. Bimodal, trimodal, and tetramodal distributions were obtained, and the breadths could be varied from 1.06 to 2.09. Polymers with different MWDs prepared by this method had good chain end fidelity, which was demonstrated with successful chain-extension experiments. In addition, the introduction of temporal programming with a computer-controlled single chip for the light source opened an avenue for the use of artificial intelligence in polymer synthesis.

Role of SIRT2 in regulating the dexamethasone-activated autophagy pathway in skeletal muscle atrophy.

The proteolytic autophagy system is involved in a major regulatory pathway in dexamethasone (Dex)-induced muscle atrophy. Sirtuin 2 (SIRT2) is known to modulate autophagy signaling, exerting effects in skeletal muscle atrophy. We examined the effects of SIRT2 on autophagy in Dex-induced myoatrophy. Tostudy this, mice were randomly distributed among the normal, Dex, and sirtinol groups. C2C12 cells were differentiated into myotubes and transduced with lentivirus carrying Sirt2-green fluorescent protein (GFP) or Sirt2 short hairpin RNA (Sirt2-shRNA)-GFP. To evaluate the mass and function of skeletal muscles, we measured myofiber cross-sectional area, myotube size, gastrocnemius (GA) muscle wet mass:body mass ratio (%), and time to exhaustion. The expression levels of SIRT2, myosin heavy chain, microtubule-associated protein 1 light chain 3 (LC3), and Beclin-1 were measured using Western blotting and quantitative reverse transcription - polymerase chain reaction. Inhibition of SIRT2 markedly attenuated GA muscle mass and endurance capacity. The same phenotype was observed in Sirt2-shRNA-treated myotubes, as evidenced by their decreased size. Conversely, overexpression of SIRT2 alleviated Dex-induced myoatrophy in vitro. Moreover, SIRT2 negatively regulated the expression of LC3b and Beclin-1 in skeletal muscles. These findings suggest that SIRT2 activation protects myotubes against Dex-induced atrophy through inhibition of the autophagy system; this phenomenon may serve as a target for treating glucocorticoid-induced myopathy.

In Vitro Neuroprotection of Rat Hippocampal Neurons by Manninotriose and Astragaloside IV Against Corticosterone-Induced Toxicity.

A chronically elevated glucocorticoid level impairs memory and cognition. Manninotriose is the main oligosaccharide of Prepared Radix Rehmanniae, and Astragaloside IV (AS-IV) is the primary ingredient of Astragali Radix; they have been reported to possess neuroprotective effects. The aim of the present study was to investigate the protective effects of Manninotriose and AS-IV on corticosterone (CORT) induced neurotoxicity and the underlying mechanisms. Primary cultured hippocampal neurons from newborn Sprague Dawley rats were treated with CORT in the absence or presence of Manninotriose and AS-IV. Cell Counting Kit-8 experiments and fluorescein diacetate (FDA)/propidium iodide (PI) double staining were conducted to assess the activity and survival rate of neurons. Quantitative Real-time PCR (qRT-PCR) and western blot analysis were performed to detect the expression of glucocorticoid receptor (GR), zinc finger protein (Zif268) and synapsin 1 (SYN1). DNA methylation of the gene promoter was assessed by bisulfite sequencing (BSP) analysis. The results demonstrated that pre-treatment with Manninotriose and AS-IV significantly improved cell viability and survival rate, and ameliorated the downregulation of GR, Zif268 and SYN1 genes in CORT injured neurons. BSP analysis revealed that CORT was able to improve the CpG island methylation rate of SYN1. AS-IV was observed to decrease the hypermethylation of the SYN1 gene induced by CORT. The results of the present study indicated that Manninotriose and AS-IV may have a protective effect against CORT-induced damage and the downregulation of learning and memory associated genes in hippocampal neurons. Regulation of DNA methylation may be important in the pharmaceutical activities of AS-IV. Thus, Manninotriose and AS-IV may be effective agents against learning and memory impairment.

Beta-Ecdysone Protects Mouse Osteoblasts from Glucocorticoid-Induced Apoptosis In Vitro.

Glucocorticoid-induced osteoporosis is a common form of secondary osteoporosis. Glucocorticoids affect both bone formation and resorption, and prolonged glucocorticoid exposure can suppress osteoblast activities. beta-Ecdysone, found in many plants, is involved in protein synthesis, carbohydrate and lipid metabolism, and immunologic modulation. Here, we evaluated the effects of beta-ecdysone on osteoblast viability by assessing apoptosis following treatment with excess glucocorticoids. Mouse bone marrow stromal cells were induced to differentiate and grow into osteoblasts, and then treated with 10 µM glucocorticoid and 10, 1, or 0.1 µM beta-ecdysone. The expression levels of osteoblast growth and differentiation factors (runt-related transcription factor 2, osteogenic protein-1, and alkaline phosphatase), apoptosis-related genes (transformation-related protein 53, ataxia telangiectasia mutated protein, caspase-3, and caspase-8), and Akt1 and phospho-Akt (Thr308) were then assessed via alkaline phosphatase staining, acridine orange-propidium iodide staining, annexin V/PI apoptosis assay, real-time RT-PCR, and Western blot analyses. Notably, treatment with 10 µM glucocorticoid resulted in reduced osteoblast viability and the specific activity of alkaline phosphatase as well as reduced runt-related transcription factor 2, osteogenic protein-1, and alkaline phosphatase mRNA expression in vitro, indicating that glucocorticoid inhibited osteogenic differentiation. Moreover, glucocorticoid treatment yielded increased transformation-related protein 53, ataxia telangiectasia mutated protein, caspase-3, and caspase-8 expression and decreased Akt1 and phospho-Akt levels, indicating glucocorticoid-induced apoptosis. Meanwhile, beta-ecdysone inhibited glucocorticoid function, preserving the expression of Akt1 and phospho-Akt and reducing the expression of transformation-related protein 53, ataxia telangiectasia mutated protein, caspase-3, and caspase-8. Thus, beta-ecdysone prevented glucocorticoid-induced osteoblast apoptosis in vitro. These data highlight the potential for beta-ecdysone as a treatment for preventing the effects of glucocorticoid on bone growth.

Prevention of glucocorticoid induced bone changes with beta-ecdysone.

Beta-ecdysone (ßEcd) is a phytoecdysteroid found in the dry roots and seeds of the asteraceae and achyranthes plants, and is reported to increase osteogenesis in vitro. Since glucocorticoid (GC) excess is associated with a decrease in bone formation, the purpose of this study was to determine if treatment with ßEcd could prevent GC-induced osteoporosis. Two-month-old male Swiss-Webster mice (n=8-10/group) were randomized to either placebo or slow release prednisolone pellets (3.3mg/kg/day) and treated with vehicle control or ßEcd (0.5mg/kg/day) for 21days. GC treatment inhibited age-dependent trabecular gain and cortical bone expansion and this was accompanied by a 30-50% lower bone formation rate (BFR) at both the endosteal and periosteal surfaces. Mice treated with only ßEcd significantly increased bone formation on the endosteal and periosteal bone surfaces, and increased cortical bone mass were their controls to compare to GC alone. Concurrent treatment of ßEcd and GC completely prevented the GC-induced reduction in BFR, trabecular bone volume and partially prevented cortical bone loss. In vitro studies determined that ßEcd prevented the GC increase in autophagy of the bone marrow stromal cells as well as in whole bone. In summary, ßEcd prevented GC induced changes in bone formation, bone cell viability and bone mass. Additional studies are warranted of ßEcd for the treatment of GC induced bone loss.

Decreased c-rel activation contributes to aberrant interleukin-2 expression in CD4(+)T cells of aged rats.

Studies indicated significantly decreased expression of interleukin-2 (IL-2) with age. This decrease could be a major contributory factor to the increased frequency of morbidity and mortality among the elderly. C-rel is a key coregulator of IL-2 expression. However, it is unknown whether aging inhibits normal c-rel activation, thereby decreasing production of IL-2. We analyzed the dynamics of IL-2 expression in CD4(+)T cells from different aged rats (young group: around 6 months (n=6), aged group: around 24 months (n=6)). The expression of the CD3 receptor and CD28 receptor in the CD4(+)T cells was assessed by flow cytometry. Translocation of c-rel and its protein level in the cytoplasm and nucleus at different time points were detected by confocal microscopy and Western blotting. Chromatin immunoprecipitation (ChIP) was used to analyze the status of c-rel binding to the IL-2 promoter region in the different aged rats. Our results showed the CD4(+)T cells from young rats and aged rats showed different expression kinetics of IL-2 after stimulation. The expression level of IL-2 was higher in young rats compared with aged rats at 24h and 48h. Data showed lower CD3 receptor expression on CD4(+)T cells from aged rats compared with young rats. Although the CD28 receptors declined on the aged CD4(+)T cells, the difference was not significant. After stimulation for 0.5h, more c-rel was translocated into nucleus markedly compared with that in the aged group. ChIP showed that in aged CD4(+)T cells, c-rel DNA binding was inhabited compared with that in young cells. Therefore, reduced IL-2 production in activated CD4(+)T cells from aged rats is associated with concomitant impairments in the activation of c-rel.

Effects of 5-hydroxymethyl furfural extracted from Rehmannia glutinosa Libosch on the expression of signaling molecules relevant to learning and memory among hippocampal neurons exposed to high concentration of corticosterone.

OBJECTIVE: To determine the effects of 5-hydroxymethyl furfural (5-HMF), an extract of Rehmannia glutinosa Libosch, on several down-regulated signaling molecules involved in learning and memory in hippocampal neurons. METHODS: After cultured for 7 days, primary hippocampal neurons were divided into 5 groups: normal, corticosterone model, RU38486, 5-HMF, and donepezil group. Neuron survival rates were calculated 24 h later using SYTO13-PI double-fluorescence staining and an 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ß-galactosidase activity was also assayed. Protein expressed by the glucocorticoid receptor (GCR), brainderived neurotrophic factor (BDNF), and N-methyl-D-aspartate receptor 2B (NR2B), as well as phosphorylationcyclic adenosine monophosphate (cAMP) response element binding protein (p-CREB), phosphorylation-extracellular signal-regulated kinase (p-ERK), and phosphorylation-synapsin (p-synapsin) were quantified with Western blot. RESULTS: Hippocampal neuron survival rates and the above-mentioned proteins were dramatically decreased (P<0.05), ß-galactosidase activity was significantly increased in the model group. but the effect was reversed by 5-HMF, RU38486, and to a lesser extent by donepezil (P<0.05). CONCLUSION: 5-HMF extracts from the Chinese herb Rehmannia glutinosa Libosch could protect hippocampal neurons from glucocorticoid injury and from down-regulated signaling molecules in the GCR-BDNF-NR2B-p-ERK-p-CREB-p-synapsin signal transduction pathway.

Mannotriose regulates learning and memory signal transduction in the hippocampus.

Rehmannia is a commonly used Chinese herb, which improves learning and memory. However, the crucial components of the signal transduction pathway associated with this effect remain elusive. Pri-mary hippocampal neurons were cultured in vitro, insulted with high-concentration (1 × 10(-4) mol/L) cor-ticosterone, and treated with 1 × 10(-4) mol/L mannotriose. Thiazolyl blue tetrazolium bromide assay and western blot analysis showed that hippocampal neuron survival rates and protein levels of glucocorti-coid receptor, serum and glucocorticoid-regulated protein kinase, and brain-derived neurotrophic factor were all dramatically decreased after high-concentration corticosterone-induced injury. This effect was reversed by mannotriose, to a similar level as RU38486 and donepezil. Our findings indicate that mannotriose could protect hippocampal neurons from high-concentration corticosterone-induced injury. The mechanism by which this occurred was associated with levels of glucocorticoid receptor protein, serum and glucocorticoid-regulated protein kinase, and brain-derived neurotrophic factor.

[The crosstalking between aging and autophagy].

Aging is the syndrome caused by the functional decline and physiological disorder during the organism deterioration. Autophagy is the way that cells use to deliver cytoplasmic protein and organelle to lysosomes for degradation. Constitutive autophagy has a housekeeping role and is essential for survival, development and metabolic regulation. Recent studies from model organisms demonstrate that there is close relationship between aging and autophagy. Normal aging and phathological aging are often associated with a reduced autophagic potential. This paper reviewed the cross-talk between aging and autophagy, and possible molecular mechanisms as well.

[Effects of erlong zuoci pills and its effective disassembled prescriptions on gentamycin induced hair cell apoptosis].

OBJECTIVE: To investigate the effects of erlong zuoci (ELZC) pills and its disassembled prescriptions (Shudi-huang-Zexie group and Zexie group) on the enzymatic activity and protein expression changes of the key apoptosis molecules in the gentamycin injured hair cells. METHOD: The model of gentamycin induced ototoxicity in mice cochlear primary cultures was copied. Cochlear organotypic cultures of postnatal day 3-5 (P3-P5) mice were treated with gentamycin alone or in combination with ELZC pills, Shudihuang-Zexie group or Zexie group respectively. The enzymatic activity of Caspase-9 and Caspase-3 was determined by means of fluorescence staining in situ. The protein expression of Bcl-2 and Bax in the hair cell area was examined by immunofluorescence in normal and treated specimens. RESULT: Average optical density analysis indicated that, compared to the normal group, 0.03 mmol x L(-1) gentamycin could significantly activate Caspase-9 and Caspase-3, downregulate the ratio of Bcl-2 and Bax protein expression. Compared to the gentamycin model group, ELZC pills significantly inhibited the enzymatic activity of Caspase-9 and upregulated the ratio of Bcl-2 and Bax protein expression, showing inhibition trend toward the enzymatic activity of Caspase-3. Both Shudihuang-Zexie group and Zexie group could effectively inhibit the enzymatic activity of Caspase-9 and Caspase-3, upregulate the ratio of Bcl-2 and Bax protein expression. CONCLUSION: ELZC pills, Shudihuang-Zexie group and Zexie group can effectively protect hair cells from gentamycin by correcting the abnormal changes of the mitochondrion-dependent signal transduction pathway.

[Analysis of variance of repeated data measured by water maze with SPSS].

OBJECTIVE: To introduce the method of analyzing repeated data measured by water maze with SPSS 11.0, and offer a reference statistical method to clinical and basic medicine researchers who take the design of repeated measures. METHODS: Using repeated measures and multivariate analysis of variance (ANOVA) process of the general linear model in SPSS and giving comparison among different groups and different measure time pairwise. RESULTS: Firstly, Mauchly's test of sphericity should be used to judge whether there were relations among the repeatedly measured data. If any (P

[Effects of recipes for replenishing qi and activating blood on senescence related gene expressions in the liver of aging rats].

OBJECTIVE: To observe the effects of recipes for replenishing qi and activating blood on p16, p21, proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E gene expressions in the liver of aging rats. METHODS: A recipe for replenishing qi and a recipe for activating blood were administered to aging rats respectively, and the effects of the above recipes on the expressions of senescence related genes (p16, p21, PCNA, cyclin D1 and cyclin E) were examined by RT-PCR and Western blotting methods. RESULTS: The expressions of p16, p21 and cyclin D1 mRNAs and proteins in the liver of the untreated aging rats were up-regulated, while the expressions of PCNA and cyclin E mRNAs and proteins decreased. As compared with the untreated aging rats, both recipes could down-regulate the expressions of cyclin D1 mRNA and protein and up-regulate the expressions of cyclin E mRNA and protein, but had no obvious effects on the expressions of mRNAs and proteins of p16, p21 and PCNA. CONCLUSION: Recipes for replenishing qi and activating blood can improve the liver cell proliferation of aging rats via down-regulating the expressions of cyclin D1 mRNA and protein and up-regulating the expressions of cyclin E mRNA and protein.

[Effect of heart benefiting recipe in controlling IL-1beta, IL-6 and APPmRNA expression in brain of beta-amyloid protein induced rat model of dementia].

OBJECTIVE: To investigate the neuro-immune regulatory mechanism of Heart Benefiting recipe (HBR), an effective recipe for treatment of Alzheimer's disease (AD). METHODS: Using immunohistochemical and RT-PCR methods, the neuro-immunological pathological changes in the AD rat model induced by beta-amyloid protein (A beta1-40) via lateral cerebral ventricle injection, including mainly the glial fibrillary acidic protein expression and inflammatory cytokines IL-1beta, IL-6mRNA and beta-amyloid protein precursor (APPmRNA) gene expression were studied. And the effects of HBR on these parameters were observed. RESULTS: Deposition of A beta in cerebral tissue could induce activation of stellate glial cells and abnormal increased levels of inflammatory cytokines (IL-1beta and IL-6mRNA), also the elevation of APPmRNA level. HBR could effectively control the above-mentioned pathological changes. CONCLUSION: HBR could effectively control the inflammation and the A beta immune cascade reaction in brain of AD patients, it is one of the important therapeutic mechanisms of the recipe.

[Establishment of analogous oxidative damaged Alzheimer's disease rat model and effect of tiaoxin recipe on it].

OBJECTIVE: To establish a convenient, economical and practical analogous oxidative damaged Alzheimer's disease rat model (AD model) for exploring the effect of Tiaoxin Recipe (TXR) on the spatial memory capacity and beta-amyloid protein (A beta) deposition in the model. METHODS: The AD model was established by left ventricular injection of DHF-FeCl3-ADP. Spatial memory and learning capacity of the model rat was observed by Morris water maze method, A beta deposition in its cerebral cortex was observed by immunohistochemistry, and the effect of TXR was analysed. RESULTS: Compared with the normal group, the spatial memory capacity in the model group was obviously decreased, with A beta widely deposited in cortex, immunohistochemical examination showed that the number of A beta positive cells and their mean optic density significantly increased. TXR displayed significantly improving effect on the above-mentioned changes. CONCLUSION: The oxidative damaged model could not only express the clinical characteristics (short-term memory impairment), but also partially reflex the pathological changes (A beta deposition) of AD, is an economical and practical analogous AD model. TXR has the effects of improving spatial memory impairment and lowering A beta deposition in the AD model rats.