Sequential Growth of High Quality Sub-10 nm Core-Shell Nanocrystals: Understanding the Nucleation and Growth Process Using Dynamic Light Scattering.

Monodisperse sub-10 nm core-shell nanocrystals have been extensively studied owing to their important applications in catalysis, bioimaging, nanomedicine, and so on. In this work, an amorphous shell component crystallization strategy has been proposed to prepare high quality sub-10 nm NaYF4:Yb/Er@NaGdF4 core-shell nanocrystals successfully via a sequential growth process. The dynamic light scattering technique has been used to investigate the secondary nucleation and growth process forming the core-shell nanocrystals. The size and morphology evolution of the core-shell nanocrystals reveals that the secondary nucleation of the shell component is unavoidable after hot-injecting the shell precursor at high temperatures, which was followed by dissolution and recrystallization (an Ostwald ripening process) to partially produce the core-shell nanocrystals. The present study demonstrates that the size of seed nanocrystals and the injection temperature of the shell component precursor play a vital role in the formation of core-shell nanostructures completely. This work will provide an alternative strategy for precisely controlling the fabrication of sub-10 nm core-shell nanostructures for various applications.

Comparison of Ciprofol-Based and Propofol-Based Total Intravenous Anesthesia on Microvascular Decompression of Facial Nerve with Neurophysiological Monitoring: A Randomized Non-Inferiority Trial.

Purpose: Ciprofol is a recently developed short-acting gamma-aminobutyric acid receptor agonist with a higher potency than that of propofol. As a new sedative drug, there are few clinical studies on ciprofol. We sought to examine the safety and efficacy of ciprofol use for general anesthesia in neurosurgical individuals undergoing neurosurgical surgery with intraoperative neurophysiological monitoring (IONM). Patients and Methods: This single-center, non-inferiority, single-blind, randomized controlled trial was conducted from September 13, 2022 to September 22, 2023. 120 patients undergoing elective microvascular decompression surgery (MVD) with IONM were randomly assigned to receive either ciprofol or propofol. The primary outcome of this study was the amplitude of intraoperative compound muscle action potential decline, and the secondary outcome included the indexes related to neurophysiological monitoring and anesthesia outcomes. Results: The mean values of the primary outcome in the ciprofol group and the propofol group were 64.7±44.1 and 53.4±35.4, respectively. Furthermore, the 95% confidence interval of the difference was -25.78 to 3.12, with the upper limit of the difference being lower than the non-inferiority boundary of 6.6. Ciprofol could achieve non-inferior effectiveness in comparison with propofol in IONM of MVD. The result during anesthesia induction showed that the magnitude of the blood pressure drop and the incidence of injection pain in the ciprofol group were significantly lower than those in the propofol group (P<0.05). The sedative drug and norepinephrine consumption in the ciprofol group was significantly lower than that in the propofol group (P<0.05). Conclusion: Ciprofol is not inferior to propofol in the effectiveness and safety of IONM and the surgical outcome. Concurrently, ciprofol is more conducive to reducing injection pain and improving hemodynamic stability, which may be more suitable for IONM-related surgery, and has a broad application prospect.

Spatiotemporal variations of ecosystem security pattern in Horqin sandy dune meadow alternating area, China.

The natural and geographical environment of ecologically fragile areas in northern China is complex. Due to heavy human disturbance and impacts of climate change, the sustainable development of ecosystems is facing serious challenges. Constructing ecological security pattern can provide decision-making basis for ecological environment protection in desertification areas. Based on land use change data of Horqin dune-meadow interphase area from 1985 to 2021, we identified ecological sources with the importance of ecosystem services and ecological sensitivity, and constructed the ecological security pattern using the minimum cumulative resistance model. We further analyzed the ecological security pattern and its development trend in 1985, 1995, 2005, 2015 and 2021, and explored the ecological spatial layout adjustment strategy. The results showed that the proportion of source area in the ecological security pattern of the study area was always small and scattered from 1985 to 2021, the network of ecological corridors was low, and the connectivity between ecological patches was lacking. The ecological security pattern had experienced a trend of deterioration first and then gradually improving. Ecological policies such as returning farmland to forest and grassland and afforestation had significantly improved the environmental security. We optimized the study area by combining the cultivated land suitability evaluation method. The ecological security pattern showed a spatial trend of 'dual-core, scattered and semi-surrounded'. The results could provide references for the construction of county-scale ecological security pattern in ecologically fragile areas and the ecological management of Horqin sands.

Puerarin decreases apoptosis of retinal pigment epithelial cells in diabetic rats by reducing peroxynitrite level and iNOS expression.

The purpose of this study was to investigate the protective effect of puerarin on retina pigment epithelial (RPE) cells of diabetic rats against apoptosis. One hundred and eight Sprague-Dawley (SD) rats were randomly divided into 3 groups: control group, streptozotocin (STZ) group and puerarin group. STZ and puerarin groups received 3 d of STZ injection (45 mg/kg per day, i.p.). Additionally, puerarin groups were treated with puerarin (140 mg/kg, i.p.) from the 4th day to the end of experiment. The rats from different groups were sacrificed on 20, 40 and 60 d after STZ injection for harvesting RPE cells. Western blot analysis, DNA laddering, RT-PCR and immunohistochemistry were used for determining the expression of nitrotyrosine (NT, the foot print of peroxynitrite), cell apoptosis, iNOS mRNA and Fas/Fas ligand (FasL) signal transduction in RPE cells, respectively. The results showed that control group maintained low apoptosis level and little NT, iNOS mRNA, Fas/FasL protein expressions, as well as normal blood glucose and body weight during 60 d of the experiment. Compared with control group, STZ group showed obvious apoptosis and higher NT, iNOS mRNA, Fas/FasL protein expressions from 20 d after STZ injection. Puerarin relieved apoptosis of RPE cells and decreased NT, iNOS mRNA, Fas/FasL protein expressions in puerarin group 20 or 40 d after STZ injection, compared with STZ group. These results suggest puerarin can decrease RPE cells apoptosis in diabetic rats by reducing peroxynitrite level and iNOS expression, thus being a potential therapeutic agent in controlling of diabetic retinopathy.

[Protective effects of puerarin on lens epithelial cells in rat diabetic cataract].

OBJECTIVE: The therapeutic effects of general and local applications of puerarin in the treatment of streptozotocin (STZ)-induced rat diabetic model were compared. METHODS: Experimental research. We equally divided normal Sprague-Dawley (SD) rats into a STZ group, a peritoneal injection group, a peribulbar injection group and a control group. STZ, peritoneal injection and peribulbar injection groups were first treated with STZ. Subsequently, the STZ group was injected with normal saline intraperitoneally, while in the later two groups puerarin was injected through peritoneal and peribular routes, respectively. Control group only received peritoneal injection of saline. The morphology of lens epithelial cells (LEC) and their subcellular structure were examined by bright-field microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) 20, 40 and 60 days after the injection. Nitrogen oxide (NO) and nitric oxide synthase (NOS) were measured by biochemistry methods. Finally, inducible nitric oxide synthase (iNOS) protein and mRNA levels were monitored by Western blot and RT-PCR, respectively. Data was processed with two factorial experiment analysis of variance. RESULTS: Twenty to sixty days after the injection, marked or complete lens opacities appeared in the STZ group, whereas only slight opacities appeared in the lens in peritoneal and peribulbar puerarin groups and the lens in the control group remained clear. At the 20th, 40th and 60th day after the injection, optical microscope detected pathological changes of LEC in the STZ group. The cell volume was decreased with a dense nucleus and many bubbles appeared around the equator area. Under TEM, enlargement of cell gap, vacuoles in the cytoplasm, swelling of mitochondria and unclear structure of rough endoplasmic reticulum appeared in the LEC of the STZ group. Part of the nucleus was in karyopyknosis and peripheral nucleus gap was enlargement. Under SEM, normal fiber conjunction structure of the lens disappeared, fibers were swelling, part of fiber membranes were discontinuous, detached, and accumulated in certain areas. Mild lens opacities detected by bright-field microscope were developed in peritoneal and peribulbar puerarin injection groups. Nucleus and fibers in the lens cells of both groups appeared to be normal, with minor swelling of mitochondria, minor enlargement of endoplasmic reticulum and slight increase of intracellular space. NO, NOS and iNOS protein and mRNA of the lens were increased and up-regulated in STZ group. In the other two groups only minor changes were present and the changes were significantly less than that of the STZ group but greater than that in the control group. CONCLUSION: Peritoneal and peribulbar injection of puerarin have similar therapeutic effects in the treatment of rat diabetic cataract.

[Semenogelin and sperm motility inhibition: an update].

Semen liquefaction and sperm capacitation are the key processes for sperm to acquire forward movement ability. In these processes, semenogelin plays a vital role by directly participating in the formation of semen coagulation, collaborating with other protease and metal ions from the male reproductive tract, and then reacting with the surface of sperm cells, finally involved in the regulation of these processes and ensuring sperm's acquisition of forward movement ability.

[The effect of puerarin on prevention of oxidative damage of cultured lens capsule epithelial cells].

OBJECTIVE: To investigate the peroxynitrite damage to the lens epithelial cells (LEC) and the prevention of this damage by puerarin in vitro. METHODS: This paper was experimental study. Rabbit LEC were isolated and cultured and the third or forth passage LEC were used in this experiment The experiment groups included: (1) CONTROL GROUP: Heat-pathogen free saline (NS) 200 microl was added to the medium; (2) ONOO- group: ONOO- 200 microl was added to obtain the terminal concentration at 0. 5 mmol/L; (3) Puerarin group: 5 microg/ml ONOO- and 10 microg/ml puerarin were added simultaneously. Then, the cells were cultured and collected after 6,12 or 24 hours. The nitrotyrosine (NT), a symbol of the ONOO-, was tested with immunofluorescence technique. The expression of NT protein was examined with Western blot method. The cell morphology was observed with light microscope. Cell apoptosis was examined via DNA ladder, flow cytometry and Fas/FasL immunohistochemical staining. These datas were analyzed by one-way-ANOVA and q test. RESULTS: During the 6 to 24 hours of experiment period, green color could be observed in the cell nucleus and cytoplasm of control group. Staining ranged from yellow to brown-yellow, then to brown color were observed in STZ group. Staining ranged from faint green to yellow green or faint green color were observed in puerarin group. Slight expression of nitrotyrosine (NT) could be seen in the control group. A moderate to strong expression of NT was observed at different stages in the STZ group (A = 77.22 +/- 2.44, 145.00 +/- 3.94, 235. 8 +/- 5.97). At 6 hours, a slight expression of NT could be seen in the control group (A = 72.78 +/- 2.64), this increased at 12 hours (A =89. 94 +/- 3.01) and decreased at 24 hours (A = 74. 44 +/- 3.00). With computer photo-analysis, there were significant differences between the control, STZ and puerarin groups at different period during the experiment (q = 78.12, 82.76, 69.98, P <0. 01). In the control group, cell morphology and gene DNA ladder were normal, minor apoptosis could be observed but no expression of Fas/FasL in the membrane and cytoplasm of the cells. Distinctive cell morphology changes and the typical "ladder bands" as well as the expression of Fas/FasL could be observed in STZ group. All of these aspects were comparatively normal in puerarin group. CONCLUSIONS: The LEC apoptosis induced by ONOO- in vitro could be alleviated by puerarin. Fas/FasL cell signal transduction pathway may affect and strengthen the apoptosis process mediated by ONOO-.

Epitaxial growth of ultrathin layers on the surface of sub-10 nm nanoparticles: the case of ß-NaGdF4:Yb/Er@NaDyF4 nanoparticles.

Upconversion core-shell nanoparticles have attracted a large amount of attention due to their multifunctionality and specific applications. In this work, based on a NaGdF4 sub-10 nm ultrasmall nanocore, a series of core-shell upconversion nanoparticles with uniform size doped with Yb3+, Er3+ and NaDyF4 shells with different thicknesses were synthesized by a facile sequential growth process. NaDyF4 coated upconversion luminescent nanoparticles showed an obvious fluorescence quenching under excitation at 980 nm as a result of energy resonance transfer between Yb3+, Er3+ and Dy3+. NaGdF4:Yb,Er@NaDyF4 core-shell nanoparticles with ultrathin layer shells exhibited a better T 1-weighted MR contrast.

Sequential growth of CaF2:Yb,Er@CaF2:Gd nanoparticles for efficient magnetic resonance angiography and tumor diagnosis.

It is a significant challenge to develop nanoscale magnetic resonance imaging (MRI) contrast agents with high performance of relaxation. In this work, Gd3+-doped CaF2-based core-shell nanoparticles (CaF2:Yb,Er@CaF2:Gd) of sub-10 nm size were controllably synthesized by a facile sequential growth method. The as-prepared hydrophilic CaF2:Yb,Er@CaF2:Gd nanoparticles modified using PEG-PAA di-block copolymer benefited from the presence of Gd only in the outer CaF2 layer of the nanoparticles, which exhibited r1 as high as 21.86 mM-1 s-1 under 3.0 T, seven times as high as that of commercially used gadopentetate dimeglumine (Gd-DTPA). Low cytotoxicity, no hemolysis phenomenon and no potential gadolinium ion leakage phenomenon of the hydrophilic CaF2:Yb,Er@CaF2:Gd nanoparticles have been observed and confirmed. Clear vascular details can be observed in magnetic resonance angiography and obvious MR signal of 4T1 tumor area could be significantly improved by intravenous injection of the hydrophilic CaF2:Yb,Er@CaF2:Gd nanoparticles at a low dosage in mice. A series of in vivo biological safety evaluations confirmed the good biocompatibility of the hydrophilic CaF2:Yb,Er@CaF2:Gd nanoparticles, which might be employed in clinical blood pool imaging and tumor diagnosis as a safe and efficient MRI probe.

The antagonism of cholecystokinin octapeptide-8 to the peroxynitrite oxidation on a diabetic cataractal rat model.

BACKGROUND: Cataracts is considered be formed because of an abnormal glucose metabolic pathway or oxidative stress. We explored the damaging role of ONOO- and antagonism of cholecystokinin octapeptide-8 (CCK-8) in diabetic cataractal rat lenses. METHODS: A diabetic cataractal animal model was established by peritoneal injection of streptozotocine (STZ). Thirty-six normal SD rats were taken as control group; seventy-two were given STZ (45 mg/kg) and then divided into STZ group and CCK-8 group (peritoneal injection CCK-8). STZ induced diabetic rats were treated with CCK-8 for 60 days. Lenses were examined with slit lamp at 20, 40 and 60 days. Immunofluorescent staining and Western blot analysis were used for determining nitrotyrosine (NT, a marker for ONOO-). PT-PCR and gene array analysis were used for determining the expression of inducible nitric oxide synthetase mRNA (iNOS mRNA) in lens epithelium (LEC). RESULTS: STZ group rats developed lens opacity by 20 days that reached a high level by 60 days after STZ injection. CCK-8 group rats delayed the cataract formation. CCK-8 group rats delayed the cataract formation. There was no distinct expression of NT and iNOS mRNA in control group. In STZ group, there were distinct expression of NT and upregulation of iNOS mRNA; however, CCK-8 group showed weak expression of NT and downregulation of iNOS mRNA. CONCLUSIONS: NT, which may be a new form of oxidative stress, was expressed in diabetic rat LEC although CCK-8 could reverse NT damage in LEC. The results suggested that CCK-8 might be a useful therapeutic agent against diabetic cataract. The antagonizing mechanism of CCK-8 may be related to direct antagonism of ONOO- as well as its inhibition of the expression of iNOS mRNA for production of NO and therefore decrease in the formation of ONOO-.

Puerarin decreases lens epithelium cell apoptosis induced partly by peroxynitrite in diabetic rats.

The present study was designed to observe if puerarin decreases lens epithelium cell (LEC) apoptosis induced partly by peroxynitrite (ONOO(-)). One hundred and eight rats were randomly divided into control group (n=36), streptozotocin (STZ) group (n=36) and STZ + puerarin group (n=36). The rats in the control group intraperitoneally (i.p.) received 0.5 ml of saline. The rats in STZ group and STZ + puerarin group received intraperitoneal injection of STZ (45 mg/kg). Three days later, the rats in STZ + puerarin group were given puerarin (140 mg/kg per day, i.p.). On days 20, 40 and 60 of the experiment, morphologic changes of lenses were observed with slit lamp. Then the animals were sacrificed for further analysis. The amount and percentage of apoptotic LECs were determined by flow cytometry. Nitrotyrosine (NT, the foot print of ONOO(-)) was examined by immunohistochemistry. Apoptosis-related genes (iNOS, etc.) were analyzed by gene array. The results showed that in the control group, all the lenses were clear. In STZ group, gradually severe opacity of the lens was observed on days 20, 40 and 60. But in STZ + puerarin group, mild opacity of the lens was observed on day 20 and more severe on day 40, but markedly decreased on day 60. In the control group, mild apoptosis of LECs was observed. In STZ group, time-dependent increase in apoptosis of LECs was observed. In STZ + puerarin group, mild apoptosis of LECs was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. There was no expression of NT in the lens in the control group, but an increased expression of NT in STZ group. In STZ + puerarin group, mild expression of NT was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. There was no expression of iNOS in the lens in the control group, but continuous up-regulation of iNOS expression in STZ group. In STZ + puerarin group, mild expression of iNOS was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. Except the changes of iNOS related to NO production, the other apoptosis-related genes, including BCL-2 and SOD were down-regulated, while NF-kappaB and TNFR1-FADD-caspase signal transduction way were up-regulated in STZ group. The results were opposite in STZ + puerarin group and the control group. These findings show that NT is expressed in diabetic rat lens, which proves that LEC apoptosis in diabetic lens is partly induced by ONOO(-) which may be a new oxidative damage way to form cataract. Puerarin partly decreases LEC apoptosis induced by ONOO(-) and is a potential medicine for therapy of diabetic cataract. The mechanism of puerarin dealing with diabetic cataract may be related to its direct inhibition of LEC apoptosis and antagonism of ONOO(-) in diabetic rats.

[Effects of phosphorus fertilization on biomass accumulation and phosphorus use efficiency of trellis-cultivated melon].

A field experiment applying six rates of P fertilizer (P2O5, 0, 150, 225, 300, 375 and 450 kg . hm-2, respectively) was conducted to investigate the effects of P fertilization on dry matter accumulation (DMA), P uptake and accumulation (PUA) and P use efficiency (PUE) of trellis-cultivated melon. Results showed that, P application increased DMA and PUA, for 150 and 225 kg P2O5 . hm-2 treatments, being 19.9% and 26.3%, 23.0% and 26.3% higher than that in no P fertilizer treatment at fruiting stage. With plant growth, DMA and PUA of different organs and the whole plant gradually increased. DMA and PUA were mainly distributed in the leaves during the early stage of the growth and in the fruit during the latter stage. P application decreased the recovery efficiency of applied P (REP), agronomic efficiency of applied P (AEP) and partial factor productivity of applied P (PFP). At 150 kg . hm-2 P application rate, the maximum REP, AEP and PFP were 11.1%, 152.9 kg . kg-1 and 476.3 kg . kg-1, respectively. Compared with no P fertilizer treatment, melon yields of 150 and 225 kg P2O5 . hm2 treatments increased by 47.3% and 39.7%, respectively. In summary, the vining stage and fruit expanding stage were the key periods for P application in trellis-cultivated melon system. Based on synthesized economic yield and P fertilizer efficiency, the recommendation of P fertilizer for trellis-cultivated melon is 150-225 kg P2O5 . hm-2 under the climatic condition of the experimental area.

[Peroxynitrite-induced formation of diabetic cataract and its prevention by puerarin in rat].

OBJECTIVE: To investigate the effect of peroxynitrite on the formation of diabetic cataract and its reversal by puerarin. in rat. METHODS: Normal Sprague-Dawley (SD) rats were equally divided into control group, streptozotocin group (STZ) and treatment group. STZ and treatment group were treated with STZ to establish animal model of diabetic rat cataract and in treatment group puerarin was given by peritoneal injection. General condition and lens shape of rats were examined at 20th, 40th, 60th day respectively. Lens epithelial cells (LEC) were observed with optical microscope. percentage of apoptotic cells, and fluorescent intensity of positive cells for nitrotyrosine (NT) which is the symbol of peroxynitrite were tested by flow cytometry. The expression of NT in the lens was analyzed by Western blot analysis. RESULTS: On 20th, 40th, 60th day of the experiment, clear, mild and severe lens opacity were found in control group, treatment group, and STZ group respectively and the lens opacity was gradually increased in all of rats included in the study. Under optical microscope, no changes were presented in control group, but mild changes were showed in treatment group and remarkable changes were found in STZ group. On all the specified days, distinguishable differences were found in the amount and percentage of apoptotic cells compared with that of control group in treatment group and STZ groups as well as between treatment and STZ groups (P < 0.05). There was significant increase in the fluorescent intensity and amount of NT in treatment and STZ groups compared with that of control group (P < 0.05), and the similar results were revealed between treatment group and STZ. The amount of expression of NT protein at different time points in different groups as described above was also significant difference (P < 0.001). CONCLUSIONS: Peroxynitrite plays an important role in the formation of diabetic rat cataract and is a participant in the pathogenesis of oxidative stress induced cataract. Puerarin is an effective antioxidative agent in the treatment of early diabetic rat cataract.

Peroxynitrite-induced expression of inducible nitric oxide synthase and activated apoptosis via nuclear factor-kappa B pathway in retinal pigment epithelial cells and antagonism of cholecystokinin octapeptide-8 in vitro.

AIM: To explore that if peroxynitrite induced the expression of inducible nitric oxide synthase (iNOS)via nuclear factor-kappa B (NF-κB) pathway in retinal pigment epithelial (RPE) cells and the antagonism of cholecystokinin octapeptide-8 (Melatonin, CCK-8) in vitro. METHODS: RPE cells were obtained from eyes of C57BL/6 mouse and divided into control, peroxynitrite and CCK-8 groups. Control group was treated with saline, peroxynitrite group was treated with peroxynitrite, and CCK-8 group was treated with CCK-8 after added with peroxynitrite. All changes were observered at 6, 12 and 24 hours after treatment. Gene array analysis, Reverse Transcription Polymerase Chain Reaction (RT-PCR) were used to determine the expression of inducible nitric oxide synthase (iNOS) mRNA in RPE cells. Western blotting was used to test the apoptosis of RPE cells. Immunofluorescent staining was used to determine the NF-κB pathway signal transduction. RESULTS: Compared to the control group, the expression of iNOS mRNA was up-regulated in peroxynitrite group and down-regulated in CCK-8 group with gene array analysis. Apoptosis was increased in peroxynitrite group and decreased in CCK-8 group with western blotting. The NF-κB pathway signal transduction was more and more stronger in the peroxynitrite group. But in CCK-8 group, little stronger could be observed at 12 hours, then weak at 24 hours with immunofluorescent staining (P<0.001). CONCLUSION: This study suggested that apoptosis of RPE cells was partly induced by peroxynitrite, which may be the new way of oxidative damage to the RPE cells. The NF-κB signal transduction may affect and reinforce apoptosis mediated by peroxynitrite. CCK-8 decreased apoptosis of RPE cells induced by peroxynitrite and is a potential agent for therapy of retinopathy. The mechanism of CCK-8 dealing with RPE cells may be related to its direct inhibition of the formation of iNOS to produce peroxynitrite and antagnism of damage of peroxynitrite to the RPE cells.

Control of peroxyntrite-induced production of inducible nitric oxide synthase isoforms and antagonism of cholecystokinin octapeptide -8 in retinal pigment epithelial cells in vivo.

AIM: To explore if peroxyntrite (ONOO(-)) induced iNOS via Fas/Fas/L pathway in diabetic rats and the effection of cholecystokinin octapeptide-8 (CCK-8) as therapeutic agent for decrease diabetic retinopathy. METHODS: Thirty-six rats were taken as control group, seventy two were given (streptozotocin) STZ (45mg/kg) and then divided into ONOO(-) group and CCK-8 group (peritoneal injection CCK-8). STZ-induced diabetic rats were treated with CCK-8 for 60 days. Western blotting analysis, DNA ladder, RT-PCR, immunohistochemistry and flow cytometry were used for determining the expression of nitrotyrosine (NT, the foot print of ONOO(-)); apoptosis and inducible nitric oxide synthase (iNOS) mRNA as well as Fas/Fasl signal transduction in RPE cells. RESULTS: Both RPE cells in ONOO(-) and CCK-8 group developed apoptosis and expressed NT, iNOS mRNA and Fas/Fasl. But latter delayed the all changes in a time-dependent manner compared with control and ONOO(-) group (P<0.001). iNOS and Fas/Fasl were up-regulated and associated with an increase of expression of ONOO(-)in vivo. CONCLUSION: The study suggested that apoptosis of RPE was partly induced by ONOO(-) may be the new way of oxidative damage to the RPE cells. CCK-8 decreased RPE cells apoptosis partly induced by ONOO(-) and is a potential drug for therapy of diabetic retinopathy. The mechanism of CCK-8 dealing with RPE cells may be related to its direct inhibition of the formation of iNOS to produce ONOO(-) and antagnism of damage of ONOO(-) to RPE cells.

Antagonistic effects of ultra-low-molecular-weight heparin on Aß25-35-induced apoptosis in cultured rat cortical neurons.

Low-molecular-weight heparin (LMWH) and ultra-low-molecular-weight heparin (ULMWH) are heparin's derivatives, having various pharmacological effects. The present study aims to investigate the effect of ULMWH on amyloid ß peptide (Aß25-35)-induced neurotoxicity in cultured rat cortical neurons, and LMWH was employed as a positive control agent. The neurons were incubated with Aß25-35 (35µM), Aß25-35 plus ULMWH (2, 10, 50 µg/ml) or LMWH (10 µg/ml) for 24h. The cell viability was assessed by MTT and LDH release. FITC-Annexin V/PI double staining, Hoechst 33258 staining, TUNEL and Western blotting for bcl-2 and caspase-3 were employed to measure the neuron apoptosis. Furthermore, the intracellular Ca(2+) concentration was measured by a fluorescent dye, Fura-2/AM. The results showed that ULMWH significantly increased cell viability and the protein expression levels of bcl-2 and decreased the LDH release, the number of apoptotic cells, the concentration of intracellular Ca(2+) and the protein expression levels of caspase-3 in cortical neurons, suggesting that ULMWH can obviously reduce Aß25-35-induced neurotoxic effects and might act as a potential agent for Alzheimer's disease.

Inducible nitric oxide synthase and Fas/FasL with C3 expression of mouse retinal pigment epithelial cells in response to stimulation by peroxynitrite and antagonism of puerarin.

BACKGROUND: Retinal pigment epithelial (RPE) cell is a monolayer of multifunctional cells between the retina and the choroid. Peroxynitrite (ONOO(-)) is known to induce toxicity on RPE cells. This study aimed to evaluate ONOO(-) induced expression of inducible nitric oxide synthase (iNOS) and complement 3 (C3) via Fas/FasL pathway in RPE cells and the values of puerarin as a therapeutic target for inhibiting the apoptosis of RPE cells. METHODS: RPE cells were obtained from eyes of C57BL/6 mice. RPE cells were divided into control, ONOO(-) and puerarin groups. Control group was treated with saline, ONOO(-) group was treated with ONOO(-), and puerarin group was treated with puerarin after added with ONOO(-). All changes were observered at 6, 12 and 24 hours after treatment. Western blotting analysis was used to determine the expression of nitrotyrosine (NT, the foot print of ONOO(-)) and C3; flow cytometry was used to determine the apoptosis of RPE cells. Immunohistochemistry and Western blotting were used to determine Fas/FasL signal transduction. Gene array analysis, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to determine the expression of iNOS mRNA and iNOS protein in RPE cells. RESULTS: There were minor expression of NT, C3, Fas/FasL and iNOS mRNA in control group, and strong expression of NT and C3 in ONOO(-) group, while in puerarin group weak expressions of NT and C3 were detected as time passed by (P < 0.001). Apoptosis of RPE cells occured and reached a higher level at 6 and 24 hours after addition of ONOO(-) respectively in ONOO(-) group, but delayed apoptosis in puerarin group (P < 0.05). Compared to control group, the expression of Fas/FasL was up-regulated in ONOO(-) group, but was down-regulated in puerarin group (P < 0.001). Similarly, the expressions of iNOS mRNA and iNOS protein in ONOO(-)group were up-regulated in ONOO(-) group, but down-regulated in puerarin group (P < 0.001). CONCLUSIONS: ONOO(-) expresseion in RPE cells may constitute the new way of oxidant stress. Fas/FasL signal transduction pathway and C3 may affect and reinforce apoptosis mediated by ONOO(-). Puerarin could reverse ONOO(-) damage on RPE cells. The antagonizing mechanism of puerarin may be related to its inhibitory to the expression of iNOS mRNA, and therefore decrease ONOO(-) formation as well as directly antagonize the effect of ONOO(-). Furthermore, puerarin may be an useful therapeutic agent against apoptosis of RPE cells.

Toxicity of endogenous peroxynitrite and effects of puerarin on transplanted retinal pigment epithelial sheets in the subretinal space in mice.

AIM: To evaluate the toxicity of endogeneous peroxynitrite on transplanted retinal pigment epithelial (RPE) sheets and the effect of puerarin on their survival in the C57BL/6 mice after RPE sheets have been transplanted into SD rats' subretinal space . METHODS: C57BL/6 mice eyes were used to culture RPE cells. Ninety-six SD rats were involved in the experiment. They were divided into control (block control), streptozotocin (STZ, negative control), untransplanted RPE (positive control) and transplanted RPE groups respectively. Diabetes was induced in SD rats by intra-peritoneal STZ injection in the latter three groups. Saline was injected into the subretinal space of 24 SD rats in the untransplanted RPE group and primary RPE sheets were injected into the subretinal space of 24 SD rats in the transplanted RPE group. Puerarin (45mg/kg) was administrated into both untransplanted RPE and transplanted RPE groups of diabetic rats through intra-peritoneal injection route after RPE sheets transplantation. At 20, 40, 60 days after surgery, Western blotting analysis, DNA ladder and RT-PCR were used for determining the differences in expression of nitrotyrosine (NT, the foot print of peroxynitrite ), apoptosis and iNOS mRNA in the control, STZ, untransplanted RPE and transplanted RPE groups respectively. HE staining was used for determining the RPE survival in the subretinal space of the transplanted RPE group. RESULTS: Apoptosis and expression of NT and iNOS mRNA were observed in STZ, untransplanted RPE and transplanted RPE groups, but were delayed in untransplanted RPE and transplanted RPE groups in a time-dependent manner compared with control and STZ groups (P<0.01). There were no differences between the two groups (P>0.01). NT, DNA ladder, iNOS mRNA were down-regulated, which were associated with the decrease of expression of peroxynitrite. Numerous pigmented cells emerged and increased in number in the subretinal space during the 60-day observation period after transplantation. On day 20, heavily pigmented cells were visible at the transplant site; On day 40, monolayer and multilayered transplant was visible in the subretinal space; On day 60, heavily pigmented monolayer and multilayered transplants with round apical profile were present along Bruch's membrane. CONCLUSION: Puerarin increased the 60-day survival of C57BL/6 mice RPE xenografts in the SD rats' subretinal space, which may be related to its direct inhibition of apoptosis of RPE cells and antagnism of damage of peroxynitrite to RPE cells.

Effect of puerarin on retinal pigment epithelial cells apoptosis induced partly by peroxynitrite via Fas/FasL pathway.

AIM: To evaluate the peroxynitrite (ONOO(-)) of puerarin on retinal pigment epithelial (RPE) cells apoptosis induced partly by peroxynitrite via Fas/FasL. METHODS: RPE cells from C57BL/6 mice eyes were cultured. Diabetes was induced in Sprague-Dawley (SD) rats by streptozotocin (STZ) intraperitoneal injection. Puerarin was administrated to cultured RPE cells and diabetic rats. Western blotting analysis, DNA ladder, RT-PCR, immunohistochemistry were used for determining the expression of nitrotyrosine (NT, the foot print of ONOO(-)), complement 3 (C3); apoptosis and inducible nitric oxide synthase (iNOS) mRNA as well as Fas/FasL signal transduction in RPE cells. RESULTS: Both RPE cells in ONOO(-) and puerarin group developed apoptosis and expressed NT, C3, iNOS mRNA and Fas/FasL. But latter delayed the all changes in a time-dependent manner compared with control and STZ group (P<0.001). iNOS, C3 and Fas/FasL were up-regulated and associated with an increase of expression of ONOO(-)in vivo and in vitro. CONCLUSION: Puerarin decreases RPE cells apoptosis partly induced by ONOO(-) for diabetic retinopathy.