Characterization of individual spores of two biological insecticides, Bacillus thuringiensis and Lysinibacillus sphaericus, in response to glutaraldehyde using single-cell optical approaches.

Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) are the most widely used microbial insecticides. Both encounter unfavorable environmental factors and pesticides in the field. Here, the responses of Bt and Ls spores to glutaraldehyde were characterized using Raman spectroscopy and differential interference contrast imaging at the single-cell level. Bt spores were more sensitive to glutaraldehyde than Ls spores under prolonged exposure: <1.0% of Bt spores were viable after 10 min of 0.5% (v/v) glutaraldehyde treatment, compared to ~ 20% of Ls spores. The Raman spectra of glutaraldehyde-treated Bt and Ls spores were almost identical to those of untreated spores; however, the germination process of individual spores was significantly altered. The time to onset of germination, the period of rapid Ca2+-2,6-pyridinedicarboxylic acid (CaDPA) release, and the period of cortex hydrolysis of treated Bt spores were significantly longer than those of untreated spores, with dodecylamine germination being particularly affected. Similarly, the germination of treated Ls spores was significantly prolonged, although the prolongation was less than that of Bt spores. Although the interiors of Bt and Ls spores were undamaged and CaDPA did not leak, proteins and structures involved in spore germination could be severely damaged, resulting in slower and significantly prolonged germination. This study provides insights into the impact of glutaraldehyde on bacterial spores at the single cell level and the variability in spore response to glutaraldehyde across species and populations.

Theoretical Investigation of HER and OER Electrocatalysts Based on the 2D R-graphyne Completely Composed of Anti-Aromatic Carbon Rings.

Based on the DFT calculations, two-dimensional (2D) R-graphyne has been demonstrated to have high stability and good conductivity, which can be conducive to the relevant electrocatalytic activity of the material. Different from the poor graphene, R-graphyne, which is completely composed of anti-aromatic structural units, can exhibit certain HER catalytic activity. In addition, doping the TM atoms in Group VIIIB can be considered an effective strategy to enhance the HER catalytic activity of R-graphyne. Particularly, Fe@R-graphyne, Os@R-graphyne, Rh@R-graphyne and Ir@R-graphyne can exhibit higher HER catalytic activities due to the formation of more active sites. Usually, the shorter the distance between the TM and C atoms is, the better the HER activity of the C-site is. Furthermore, doping Ni and Rh atoms of Group VIIIB can significantly improve the OER catalytic performance of R-graphyne. It can be found that ΔGO* can be used as a good descriptor for the OER activities of TM@R-graphyne systems. Both Rh@R-graphyne and Ni@R-graphyne systems can exhibit bifunctional electrocatalytic activities for HER/OER. In addition, all the relevant catalytic mechanisms are analyzed in detail. This work not only provides nonprecious and highly efficient HER/OER electrocatalysts, but also provides new ideas for the design of carbon-based electrocatalysts.

Theoretical Study on the High HER/OER Electrocatalytic Activities of 2D GeSi, SnSi, and SnGe Monolayers and Further Improvement by Imposing Biaxial Strain or Doping Heteroatoms.

Under the DFT calculations, two-dimensional (2D) GeSi, SnSi, and SnGe monolayers, considered as the structural analogues of famous graphene, are confirmed to be dynamically, mechanically and thermodynamically stable, and all of them can also possess good conductivity. Furthermore, we systematically investigate their electrocatalytic activities in overall water splitting. The SnSi monolayer can show good HER catalytic activity, while the SnGe monolayer can display remarkable OER catalytic activity. In particular, the GeSi monolayer can even exhibit excellent bifunctional HER/OER electrocatalytic activities. In addition, applying the biaxial strain or doping heteroatoms (especially P atom) can be regarded as the effective strategies to further improve the HER activities of these three 2D monolayers. The doped GeSi and SnSi systems can usually exhibit higher HER activity than the doped SnGe systems. The correlative catalytic mechanisms are also analyzed. This work could open up a new avenue for the development of non-noble-metal-based HER/OER electrocatalysts.

Circadian clock gene BMAL1 controls testosterone production by regulating steroidogenesis-related gene transcription in goat Leydig cells.

Testosterone is produced by Leydig cells (LCs) and undergoes diurnal changes in serum levels in rats, mice, and humans, but little is known in goats. The present study revealed that goat serum testosterone levels displayed diurnal rhythmic changes (peak time at ZT11.2). Immunohistochemical staining showed that BMAL1, a circadian clock protein, is highly expressed in goat LCs. ELISA revealed that both hCG (0-5 IU/ml) and 22R-OH-cholesterol (0-30 µM) addition stimulated testosterone synthesis in primary goat LCs in a dose-dependent manner. Treating goat LCs with hCG (5 IU/ml) significantly increased intracellular cAMP levels. Additionally, real-time quantitative polymerase chain reaction (PCR) analysis revealed that the circadian clock (BMAL1, PER1, PER2, DBP, and NR1D1) and steroidogenesis-related genes (SF1, NUR77, StAR, HSD3B2, CYP17A1, CYP11A1, and HSD17B3) showed rhythmic expression patterns in goat LCs following dexamethasone synchronization. Several Bmal1-Luc circadian oscillations were clearly observed in dexamethasone-treated goat LCs transfected with the pLV6-Bmal1-Luc plasmid. BMAL1 knockdown significantly downregulated mRNA levels of PER2, NR1D1, DBP, StAR, HSD3B2, SF1, NUR77, and GATA4, and dramatically decreased StAR and HSD3B2 protein levels and testosterone production. In contrast, BMAL1 overexpression significantly increased the mRNA and protein expression levels of StAR and HSD17B3 and enhanced testosterone production. Reporter assays revealed that goat BMAL1, or in combination with mouse CLOCK, activated goat HSD17B3 transcription in vitro. These data indicate that BMAL1 contributes to testosterone production by regulating transcription of steroidogenesis-related genes in goat LCs, providing a basis for further exploring the underlying mechanism by which the circadian clock regulates ruminant reproductive capability.

Zearalenone perturbs the circadian clock and inhibits testosterone synthesis in mouse Leydig cells.

Zearalenone (ZEA), a mycotoxin, is known to impair reproductive capability by disrupting the synthesis and secretion of testosterone by Leydig cells (LCs), although the mechanism is unknown. Robust rhythmicity of circadian clock and steroidogenic genes were identified in LCs. The aim of this study was to examine whether ZEA significantly attenuated the transcription of core clock genes (Bmal1, Dbp, Per2, and Nr1d1) as well as steroidogenic genes (StAR, Hsd3b2, and Cyp11a1) in mouse testis Leydig cell line (TM3). Western blotting confirmed declines in BMAL1, NR1D1, and StAR protein levels. ZEA also suppressed secreted testosterone levels. In primary LCs, isolated from PER2::LUCIFERASE reporter gene knock in mice, ZEA diminished the amplitude of PER2::LUC expression, and induced a phase shift and period extension. In primary LCs, ZEA also suppressed the expression levels of core clock and steroidogenic genes, reduced protein levels of BMAL1, and decreased testosterone secretion. In vivo expression of core clock and steroidogenic genes were reduced in testes of mice exposed to ZEA for 1 week leading to decreased serum testosterone levels. In summary, data suggest that ZEA may impair testosterone synthesis through attenuation of the circadian clock in LCs culminating in reproductive dysfunction in male mammals .

Coordination between the circadian clock and androgen signaling is required to sustain rhythmic expression of Elovl3 in mouse liver.

ELOVL3 is a very long-chain fatty acid elongase, and its mRNA levels display diurnal rhythmic changes exclusively in adult male mouse livers. This cyclical expression of hepatic Elovl3 is potentially controlled by the circadian clock, related hormones, and transcriptional factors. It remains unknown, however, whether the circadian clock, in conjunction with androgen signaling, functions in maintaining the rhythmic expression of Elovl3 in a sexually dimorphic manner. Under either zeitgeber or circadian time, WT mouse livers exhibited a robust circadian rhythmicity in the expression of circadian clock genes and Elovl3 In contrast, male Bmal1-/- mice displayed severely weakened expression of hepatic circadian clock genes, resulting in relatively high, but nonrhythmic, Elovl3 expression levels. ChIP assays revealed that NR1D1 binds to the Elovl3 promoter upon circadian change in WT mouse livers in vivo, and a diminished binding was observed in male Bmal1-/- mouse livers. Additionally, female mouse livers exhibited constant low levels of Elovl3 expression. Castration markedly reduced Elovl3 expression levels in male mouse livers but did not disrupt circadian variation of Elovl3 Injection of female mice with 5α-dihydrotestosterone induced Elovl3 rhythmicity in the liver. In AML12 cells, 5α-dihydrotestosterone also elevated Elovl3 expression in a time-dependent manner. In contrast, flutamide efficiently attenuated this induction effect. In conclusion, a lack of either the circadian clock or androgen signaling impairs hepatic Elovl3 expression, highlighting the observation that coordination between the circadian clock and androgen signaling is required to sustain the rhythmic expression of Elovl3 in mouse liver.

Circadian clock and steroidogenic-related gene expression profiles in mouse Leydig cells following dexamethasone stimulation.

Previous studies have shown that circadian clock genes are expressed in mammalian testes; however, it remains unclear if the expression patterns of these genes are cyclic. Furthermore, it is unknown whether Leydig cells, the primary androgen secreting cells in the testis, play a role in the rhythmicity of circadian clock and steroidogenic-related gene transcription. Here, we examine the circadian clock of mouse Leydig cells, and the link to steroidogenic-related gene transcription. We confirm, via sampling over a full circadian time (CT) period, a lack of circadian rhythmicity in mouse testes in comparison with the robust gene expression cycling of circadian clock genes in mouse livers. Immunofluorescence imaging of mouse testes collected at CT0 and CT12 show that the BMAL1 protein is exclusively expressed in mouse Leydig cells, and clearly linked to the circadian oscillation. Furthermore, dexamethasone treatment synchronized the expression of several of these canonical circadian clock and steroidogenic-related genes. Bioinformatic analyses revealed the presence of several circadian clock-related sequence motifs in the promoters of these steroidogenic-related genes. Our results suggest mouse Leydig cells may contain a functional circadian oscillator and the circadian clockwork in mouse Leydig cells regulates steroidogenic-related gene transcription by binding to the E-box, RORE, and D-box motifs in their promoters. However, additional research is required to determine the specific molecular mechanisms involved.

Tetramethylpyrazine Nitrone Improves Neurobehavioral Functions and Confers Neuroprotection on Rats with Traumatic Brain Injury.

Oxidative stress is one of the major secondary injury mechanisms after traumatic brain injury (TBI). 2-[[(1,1-Dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a derivative of the clinically used anti-stroke drug tetramethylpyrazine armed with a powerful free radical-scavenging nitrone moiety, has been demonstrated promising therapeutic efficacy in ischemic stroke and Parkinson's models. The present study aims to investigate the effects of TBN on behavioral function and neuroprotection in rats subjected to TBI. TBN (90 mg/kg) was administered twice daily for 7 days by intravenous injection following TBI. TBN improved neuronal behavior functions after brain injury, including rotarod test and adhesive paper removal test. Compared with the TBI model group, TBN treatment significantly protected NeuN-positive neurons, while decreased glial fibrillary acidic protein (GFAP)-positive cells. The number of 4-hydroxynonenal (4-HNE)-positive and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive cells around the damaged area after TBI were significantly decreased in the TBN treatment group. In addition, TBN effectively reversed the altered expression of Bcl-2, Bax and caspase 3, and the down-regulation of nuclear factor erythroid-derived 2-like 2 (Nrf-2) and hemeoxygenase-1 (HO-1) proteins expression stimulated by TBI. In conclusion, TBN improves neurobehavioral functions and protects neurons against TBI. This protective effect may be achieved by anti-neuronal apoptosis, alleviating oxidative stress damage and up-regulating Nrf-2 and HO-1 expression.

Effect and Significance of High-Quality Nursing on Blood Glucose, Pregnancy Outcome, and Neonatal Complications of Patients with Gestational Diabetes Mellitus.

Objective: To explore the role of high-quality nursing on blood glucose, pregnancy outcome, and neonatal complications in patients with gestational diabetes mellitus. Methods: Altogether, 148 patients with GDM admitted to our hospital were selected as the research participants, 83 of whom received high-quality nursing as the observation group (OG). Another 65 patients received only routine care as the control group (CG). The blood sugar level, blood pressure value, and adverse pregnancy outcomes of the OG and the CG of patients after intervention were compared. The changes of psychological state and nursing satisfaction of the OG and the CG of patients were observed. The blood lipid level and neonatal complications of the OG and the CG were detected. Results: The blood sugar level of the OG was lower than that of the CG (P < 0.05). The systolic pressure and diastolic pressure of patients in the OG were lower than those in the CG after nursing intervention (P < 0.05). The abnormal delivery rate in the CG was evidently higher than that in the OG (P < 0.05). After intervention, SAS and SDS scores in the OG were lower than those in the CG (P < 0.05). The number of people who needed improvement and were dissatisfied in the OG was evidently lower than that in the CG (P < 0.05). TG and LDL-C in the OG were lower than those in the CG (P < 0.05). The total incidence of complications in the CG was evidently higher than that in the OG (P < 0.05). Conclusion: High-quality nursing can reduce blood sugar and blood pressure of GDM patients and has a great protective effect on maternal and infant health, which is worthy of clinical practice.

Controllable growth of Fe-doped NiS2 on NiFe-carbon nanofibers for boosting oxygen evolution reaction.

The construction of high-efficiency and low-cost electrocatalysts toward oxygen evolution reaction (OER) to improve the overall water decomposition performance is a fascinating route to deal with the clean energy application. Herein, Fe-doped NiS2 crystals grown on the surface of carbon nanofibers (CNFs) encapsulated with NiFe alloy nanoparticles ((Ni,Fe)S2/NiFe-CNFs) are fabricated through an electrospinning-calcination-vulcanization process, which has been used as a splendid electrocatalyst for OER. Benefitting from the abundant electrochemical active sites from the incorporation of Fe element in NiS2 and the synergistic effect between NiFe-CNFs and surface sulfides, the obtained (Ni,Fe)S2/NiFe-CNFs catalyst exhibits highly electrochemical activities and satisfactory durability toward OER in an alkaline medium with a low overpotential of only 287 mV at a high current density of 30 mA cm-2, and with a little decline in the current retention after 48 h, suggesting its superior OER performance even compared with some noble metal-based electrocatalysts. Additionally, a two-electrode system conducted by using the (Ni,Fe)S2/NiFe-CNFs and commercial Pt/C as electrodes, only needs a cell voltage of 1.54 V to afford 10 mA cm-2 for overall water splitting, which is even much better than the RuO2||Pt/C electrolyzer. This study offers a promising approach to prepare high-efficiency OER catalysts toward overall water splitting.

Age-related endoplasmic reticulum stress represses testosterone synthesis via attenuation of the circadian clock in Leydig cells.

Senile animals exhibit a high risk of elevated endoplasmic reticulum (ER) stress, attenuated circadian clock, and impaired steroidogenesis in testes. However, how these three processes are intertwined in mouse Leydig cells remains unclear. In this study, a mouse model of aging and hydrogen peroxide (H2O2)-induced senescent TM3 Leydig cells were used to dissect the connections among ER stress, circadian oscillators, and steroidogenesis in Leydig cells. Additionally, thapsigargin (Tg, 60 nM)/tunicamycin (Tm, 60 ng/mL)-induced ER stress were established to investigate the underlying mechanisms by which ER stress regulated testosterone synthesis via circadian clock-related signaling pathways in TM3 cells and primary Leydig cells. Elevated ER stress, attenuated circadian clock, and diminished steroidogenesis were detected in the testes of aged mice (24-month-old) and H2O2-induced (200 µM) senescent TM3 cells in comparison with their control groups. Tg/Tm-induced ER stress reduced the transcription of the circadian clock and steroidogenic genes in TM3 cells and LH-treated (100 ng/mL) primary Leydig cells. Furthermore, 4-phenylbutyric acid (4-PBA, 1 µM), an inhibitor of ER stress, alleviated the inhibitory effect of Tg-mediated ER stress on Per2:Luc oscillations in primary Leydig cells isolated from mPer2Luc knock-in mice, and attenuated the repressive effect of H2O2-induced or Tg-mediated ER stress on the transcription of circadian clock and steroidogenic genes expression and testosterone synthesis in TM3 cells. Collectively, these data indicate that age-related ER stress represses testosterone synthesis via attenuation of the circadian clock in Leydig cells.

circ-ACACA promotes proliferation, invasion, migration and glycolysis of cervical cancer cells by targeting the miR-582-5p/ERO1A signaling axis.

Circular RNAs (circ) have been reported to serve crucial roles in the regulation of cancer occurrence and development. The present study aimed to investigate the role of circ-acetyl-CoA carboxylase α (ACACA) in the progression of cervical cancer (CC). The expression levels of circ-ACACA in several CC cell lines were first determined using reverse transcription-quantitative PCR. circ-ACACA expression was subsequently knocked down to evaluate its effects on the viability, proliferation, apoptosis, invasion and migration of CC cells using MTT, colony formation, TUNEL, transwell and wound healing assays, respectively. 13C-labeling of intracellular metabolites and analysis of glucose consumption and lactate production were performed to determine the levels of glycolysis. In addition, the expression levels of endoplasmic reticulum oxidoreductase 1α (ERO1α; ERO1A) and glycolysis-related proteins were analyzed using western blotting. The binding interactions among circ-ACACA, microRNA (miR)-582-5p and ERO1A were validated using dual-luciferase reporter assays. Subsequently, rescue experiments were performed to determine the potential underlying mechanism by which circ-ACACA affected CC cell functions. The results revealed that circ-ACACA expression was significantly upregulated in CC cells and silencing of circ-ACACA significantly reduced the proliferation, invasion and migration, and promoted the apoptosis of CC cells. Knockdown of circ-ACACA markedly inhibited glycolysis in CC cells. However, the effects of silencing of circ-ACACA on CC cells were reversed following transfection with the miR-582-5p inhibitor or pcDNA3.1-ERO1A overexpression plasmid. In conclusion, to the best of our knowledge, the present study was the first to investigate the role of circ-ACACA in CC progression. The results suggested that circ-ACACA may promote CC tumorigenesis and glycolysis by targeting the miR-582-5p/ERO1A signaling axis. Therefore, circ-ACACA may be a promising biomarker for CC diagnosis and treatment.

Tetrathiafulvalene-based covalent organic frameworks for ultrahigh iodine capture.

To safeguard the development of nuclear energy, practical techniques for capture and storage of radioiodine are of critical importance but remain a significant challenge. Here we report the synergistic effect of physical and chemical adsorption of iodine in tetrathiafulvalene-based covalent organic frameworks (COFs), which can markedly improve both iodine adsorption capacity and adsorption kinetics due to their strong interaction. These functionalized architectures are designed to have high specific surface areas (up to 2359 m2 g-1) for efficient physisorption of iodine, and abundant tetrathiafulvalene functional groups for strong chemisorption of iodine. We demonstrate that these frameworks achieve excellent iodine adsorption capacity (up to 8.19 g g-1), which is much higher than those of other materials reported so far, including silver-doped adsorbents, inorganic porous materials, metal-organic frameworks, porous organic frameworks, and other COFs. Furthermore, a combined theoretical and experimental study, including DFT calculations, electron paramagnetic resonance spectroscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy, reveals the strong chemical interaction between iodine and the frameworks of the materials. Our study thus opens an avenue to construct functional COFs for a critical environment-related application.

Bisphenol A attenuates testosterone production in Leydig cells via the inhibition of NR1D1 signaling.

Bisphenol A (BPA) is an endocrine-disrupting compound that impairs testosterone synthesis in male mammals. A circadian clock gene deficiency leads to diminished fertility and even infertility in male mice. However, whether circadian clock signaling pathways mediate the suppressive effect of BPA on testosterone synthesis in Leydig cells (LCs) remains unknown. The present study aims to detect the effect of BPA on cellular circadian clock and testosterone synthesis in mouse LCs, and examine the mechanisms underlying NR1D1 signaling. BPA treatment significantly attenuated the transcription levels of Nr1d1 and steroidogenic genes (Hsd3b2 and Hsd17b3) in TM3 cells, but increased other circadian clock gene levels (Per2 and Dbp). BPA treatment also significantly downregulated NR1D1 and StAR protein expression, but upregulated BMAL1 protein expression in TM3 cells. Furthermore, there was a marked decline in testosterone production in BPA-treated TM3 cells. Intraperitoneal injection of BPA profoundly reduced NR1D1 and StAR protein levels and steroidogenic gene transcription levels (Cyp11a1, Hsd3b2, and Hsd17b3), while enhancing BMAL1 protein and other circadian clock gene (Per2 and Dbp) levels in mouse testes. Notably, serum testosterone levels were also drastically reduced in BPA-treated mice. Moreover, SR9009, an NR1D1 agonist, augmented testosterone production in TM3 cells via elevated expression of steroidogenic genes (StAR, Cyp11a1 and Hsd17b3). Conversely, Nr1d1 knockdown inhibited testosterone accumulation and attenuated steroidogenic gene expression. Moreover, treatment with SR9009 partially reversed the BPA effect on the circadian clock and testosterone production. Taken together, our study demonstrates that BPA perturbs testosterone production, at least partially, via inhibiting NR1D1 signaling in LCs.

[Situation and assessment of heavy metal pollution in river and mud in one city in Henan Province].

OBJECTIVE: To study the heavy metal contamination status in river water and mud in the suburb of a city in Henan Province. METHODS: Typical sampling method is used to select a farmland irrigation river of the suburb of a city. Use the atomic absorption spectrophotometry, and measure the heavy metal cadmium (Cd), copper (Cu), lead (Pb) in the river water samples and mud samples by graphite furnace method and flame method, respectively. The results of water were compared with GB 3838-2002, Environmental Quality Standards for Surface Water, and GB 5084-2005, Standards for Irrigation Water Quality. The results of mud were compared with national soil background value. The contents of Cu and Cd in the river samples do not exceed the standard, and that of Pb is 3 to 6 times higher than the standard. According to the single factor pollution index method, the single factor pollution indice of Cu, and Cd in the river are less than 0.2 and are of clean level, while that of Pb reaches 6.84, indicating the Pb pollution in river water is severe. Cu in mud is more than 4 times of the soil background value, and that of Cd is more than 69 times of the soil background value, and that of Pb is more than 2 times of the soil background value. The single item pollution index indicates, in mud, the pollution index of Pb is 2.5, medium level pollution. The pollution indice of Cu and Cd in mud are more than 3, is severe pollution, and the Cd pollution is especially heavy, and the single pollution index reaches 67.76. The comprehensive pollution indice of the river and the mud are 5.346 and 84.115, respectively, indicating that both are at heavy pollution level. The main pollution source of the river originates from Pb, and that of the mud is from Cd and it is required to take measure and control as early as possible.

ER stress activation impairs the expression of circadian clock and clock-controlled genes in NIH3T3 cells via an ATF4-dependent mechanism.

Endoplasmic reticulum (ER) stress and circadian clockwork signaling pathways mutually regulate various cellular functions, but the details regarding the cross-talk between these pathways in mammalian cells are unclear. In this study, whether perturbation of ER stress signaling affects the cellular circadian clockwork and transcription of clock-controlled genes was investigated in NIH3T3 mouse fibroblasts. An NIH3T3 cell model stably expressing luciferase (Luc) under the control of the Bmal1 clock gene promoter was established using a lentiviral system. Then, Luc activity was monitored in real-time to detect Bmal1-Luc oscillations. The ER stress activators thapsigargin (Tg) and tunicamycin (Tm) markedly reduced Bmal1-Luc oscillation amplitudes and induced phase delay shifts in NIH3T3 cells. Treatment with Tg/Tm activated ER stress signaling by upregulating GRP78, CHOP, ATF6, and ATF4 and simultaneously significantly decreased BMAL1 protein levels and inhibited the transcription of circadian clock (Bmal1, Per2, Nr1d1, and Dbp) and clock-controlled (Scad1, Fgf7, and Arnt) genes. 4-Phenylbutyric acid, an ER stress inhibitor, alleviated the transcriptional repression of the circadian clock genes and partially restored Bmal1-Luc oscillation amplitudes in Tg- or Tm-treated NIH3T3 cells. More importantly, knock-down of ATF4, but not ATF6, in Tg-treated NIH3T3 cells partially rescued Bmal1-Luc oscillation amplitudes and mRNA expression of the four circadian clock genes. Taken together, our study demonstrates that ER stress activation inhibits the transcription of circadian clock and clock-controlled genes via an ATF4-dependent mechanism.

Neuroprotective Effect and Mechanism of Action of Tetramethylpyrazine Nitrone for Ischemic Stroke Therapy.

Our previous studies demonstrated that the multifunctional agent TBN, a derivative of tetramethylpyrazine armed with a nitrone moiety, displayed high therapeutic efficacy in experimental ischemic stroke models. However, its molecular mechanisms of action underlying the neuroprotective effect need further exploration. In the present study, we found that TBN had significant activities scavenging free radicals such as ·OH, O 2·- and ONOO-, inhibiting Ca2+ overload, maintaining mitochondrial function and preventing neuronal damage in primary cortical cultures. Further, TBN was effective in reducing brain infarction and ameliorating impairment of behavioral functions in the permanent middle cerebral artery occlusion (p-MCAo) rat model. TBN down-regulated the expression of pro-apoptotic factors Bax, while up-regulated the expression of anti-apoptotic factor Bcl-2 and increased the expression of pro-survival factors including p-Akt and p-GSK3ß in the peri-infarct cortex of p-MCAo rats. In addition, LY-294002 (a PI3K inhibitor) and MK2206 (an Akt inhibitor) significantly blocked the protective effect of TBN against OGD-induced death of cortical neurons. Taken together, the multifunctional mechanisms including scavenging free radicals, blocking calcium overload, maintaining mitochondrial function and activating the PI3K/Akt/p-GSK3ß cell survival pathway were possibly involved in the neuroprotective effects of TBN, making it a promising clinical candidate for the treatment of ischemic stroke.

Tetramethylpyrazine nitrone activates the BDNF/Akt/CREB pathway to promote post-ischaemic neuroregeneration and recovery of neurological functions in rats.

BACKGROUND AND PURPOSE: Neuronal regeneration from endogenous precursors is an attractive strategy for the treatment of ischaemic stroke. However, most stroke-generated newborn neurons die over time. Therefore, a drug that is both neuroprotective and pro-neurogenic may be beneficial after stroke. Here, we assessed the neurogenic and oligodendrogenic effects of tetramethylpyrazine nitrone (TBN), a neuroprotective drug candidate for stroke, in a rat model of ischaemic stroke. EXPERIMENTAL APPROACH: We used Sprague Dawley rats with middle cerebral artery occlusion (MCAO). TBN was administered by tail vein injection beginning at 3 h post ischaemia. Therapeutic effect of TBN was evaluated by neurological behaviour and cerebral infarction. Promotion of neurogenesis and oligodendrogenesis was determined by double immunofluorescent staining and Western blotting analyses. Primary cultures of cortical neurons were used to assess the effect of TBN on neuronal differentiation in vitro. KEY RESULTS: TBN reduced cerebral infarction, preserved and/or restored neurological function and promoted neurogenesis and oligodendrogenesis in rats after MCAO. In addition, TBN stimulated neuronal differentiation on primary culture of cortical neurons in vitro. Pro-neurogenic effects of TBN were attributed to its activation of the AKT/cAMP responsive element-binding protein through increasing brain-derived neurotrophic factor (BDNF) expression, as shown by the abolition of the effects of TBN by a specific inhibitor of BDNF receptor ANA-12 and by the PI3K inhibitor LY294002. CONCLUSION AND IMPLICATIONS: As TBN can simultaneously provide neuroprotection and pro-neurogenic effects, it may be a promising treatment for both acute phase neuroprotection and long-term functional recovery after ischaemic stroke.

[Effect of anthocyanin rich fruit extract on PGE2 produced by endothelial cells].

OBJECTIVE: To study the effect of anthocyanin rich fruit extract on PGE2 produced by endothelial cells. METHODS: Normal human endothelial cells, CRL-2606, were cultured in F12K medium (complemented with 10% FBS, 0.1 mg/ml heparin, 0.03 mg/ml ECGS, 50 microg/ml streptomycin and 500U/ml penicillin) in 5% CO2 with 95% air at 37 degrees C. When the cells grow close to confluence, anthocyanin rich fruit extract with/without 100 ng/ml of LPS were added to the medium. After 18 hours of incubation, cells were harvested and the supernatant were collected. Cell viability was assayed. After centrifugation, PGE2 concentration in the supernatant was measured with the STAT-Prostaglandin E2 EIA Kit. RESULTS: 300 microg/ml or higher Chokeberry extract showed cytotoxicity effect on CRL-2606 cells, the viability was lower than 60% and showed a dose-response manner. Under using dosage, Blackcurrant extract (100 - 700 microg/ml) and Blueberry extract (50 - 400 microg/ml) did not show any cytotoxicity. When stimulated by LPS, the production of PGE2 by endothelial cells were increased two fold. Blueberry extract inhibit such action. 100 microg/ml of Blueberry extract keeps the production of PGE2 in normal level. 700 microg/mL of Blackcurrant extract and 500 microg/ml Chokeberry extract also inhibit the releasing of PGE2. CONCLUSION: Anthocyanin rich fruit extract from Blueberry, Blackcurrant, and Chokeberry inhibit PGE2 produced by endothelial cells, there exist antiinflammation and antioxidation.

[Application of modified ABTS+ assay method to monitor antioxidant extraction from fruit].

OBJECTIVE: Modify the ABTS method to measure the antioxidant activity of compound. Apply this method to optimize antioxidant extraction from fruit. METHODS: In microplate, Trolox served as reference. Antioxidant activity was measured according to their ability to scavenge ABTS+ in the reaction mixture. Under different temperatures, solvent solid ratios, solvent concentrations, antioxidants were extracted. RESULTS: The standard error for measuring Trolox antioxidant activity was under 0.2. The antioxidant activities of fourteen standard samples were consistent with that reported in literatures. Using ethanol as solvent, at about 17 degrees C, for 1 gram of blackcurrant 20 ml of 67% ethanol was appropriate. While using sodium metabisulphite as solvent, at about 30 degrees C, for 1 gram of blackcurrant 20 ml of 300 mg/L metabisulphite would be appropriate. CONCLUSION: Modified ABTS+ method for measuring antioxidant activity was simple and feasible. This method could be applied to monitor antioxidant extraction from fruit and high throughput screening for antioxidant.