The effect of protein oxidation on the formation of advanced glycation end products after chicken myofibrillar protein glycation

Investigation that protein oxidation to the formation of advanced glycation end products(AGEs)after chicken myofibrillar protein glycation is limited.Models of protein oxidation induced by different concentrations of hydroxyl radicals(·OH)were developed after the chicken myofibrillar protein mild glycation(MPG).Results exhibited that levels of AGEs and surface hydrophobicity(H_(0))steadily increased with the a ddition of h ydrogen peroxide(H_(2)O_(2))concentration.However,levels of s ulfhydryl group,free amino group,and particle size gradually decreased with the H_(2)O_(2)concentration.The protein carbonyl value increased in H_(2)O_(2)concentration until 10 mmol/L.Pearson's correlation indicated that MPG structure modification(unfolding and degradation)induced by protein oxidation were significantly positively correlated with AGEs concentration(P<0.05).Finally,a mechanism was proposed to hypothesize t he effect of protein oxidation on the formation of AGEs under MPG conditions.

Changes of protein oxidation,lipid oxidation and lipolysis in Chinese dry sausage with different sodium chloride curing salt content

The effect of sodium chloride(NaCl)curing salt content on protein oxidation,lipid oxidation and lipolysis of Chinese dry sausage was investigated.Two groups Chinese dry sausages with 2%and 4%(m/m)salt content were studied.The degree of protein oxidation increased during the processes in two groups sausages,while the content of phospholipids decreased,neutral lipids and free fatty acids increased.The degree of protein oxidation,lipid oxidation and lipolysis in 4%NaCl content group was higher than those in 2%NaCl content group,while 4%NaCl content group has higher lipase activity.In conclusion,4%NaCl may facilitate the protein oxidation,lipid hydrolysis and oxidation in Chinese dry sausage,and the protein oxidation had strong correlation with lipid oxidation and lipolysis.The results could provide a basis for improving the technology of industrial production.

13C Protein Oxidation in Breath: Is It Relevant for the Whole Body Protein Status?

Introduction: The kinetics of protein oxidation, monitored in breath, and its contribution to the whole body protein status is not well established. Objectives: To analyze protein oxidation in various metabolic conditions we developed/validated a 13C-protein oxidation breath test using low enriched milk proteins. Method/Design: 30 g of naturally labeled 13C-milk proteins were consumed by young healthy volunteers. Breath samples were taken every 10 min and 13CO2 was measured by Isotope Ratio Mass Spectrometry. To calculate the amount of oxidized substrate we used: substrate dose, molecular weight and 13C enrichment of the substrate, number of carbon atoms in a substrate molecule, and estimated CO2-production of the subject based on body surface area. Results: We demonstrated that in 255 min 20% ± 3% (mean ± SD) of the milk protein was oxidized compared to 18% ± 1% of 30 g glucose. Postprandial kinetics of oxidation of whey (rapidly digestible protein) and casein (slowly digestible protein) derived from our breath test were comparable to literature data regarding the kinetics of appearance of amino acids in blood. Oxidation of milk proteins was faster than that of milk lipids (peak oxidation 120 and 290 minutes, respectively). After a 3-day protein restricted diet (~10 g of protein/day) a decrease of 31% ± 18% in milk protein oxidation was observed compared to a normal diet. Conclusions: Protein oxidation, which can be easily monitored in breath, is a significant factor in protein metabolism. With our technique we are able to characterize changes in overall protein oxidation under various meta-bolic conditions such as a protein restricted diet, which could be relevant for defining optimal protein intake under various conditions. Measuring protein oxidation in new-born might be relevant to establish its contribution to the protein status and its age-dependent development.

Effects of protein oxidation, cathepsins, and various freezing temperatures on the quality of superchilled sturgeon fillets

Many aquatic products have been stored using superchilling technology, but rarely used for the storage of sturgeon fillets. In this study, we investigated the effects of protein oxidation, cathepsin, and various freezing temperatures on the quality of superchilled sturgeon fillets. Sensory evaluation results showed that the sensory attributes of superchilled (−3 °C) sturgeon fillets were acceptable three times longer (18 days) than samples stored at refrigeration temperatures (4 °C). The sarcoplasmic protein, carbonyl, myofibrillar protein, total sulfhydryl content and the surface hydrophobicity were determined using fluorescence spectrophotometry and SDS-PAGE. Results showed that superchilling might protect myofibrillar proteins from oxidation compared to refrigeration temperatures. The activity of the three cathepsins (B, L, and H) in terms of myofibrillar, mitochondria, lysosomes, and sarcoplasm demonstrated that superchilling can inhibit cathepsins activity in sturgeon and protect its muscle structure. Microscopic observations showed that as the temperature decreased, the gap area of the muscle fibers decreased, and the deformation of cross-sectional slices was gradually reduced. In addition, the freezing rate of ice crystals produced during the freezing process influenced the muscle structure, texture, and sensory attributes. Superchilled sturgeon fillets showed good hardness, chewiness, and water retention. In conclusion, superchilling technology shows promise for its ability to extend the shelf life while maintaining the texture and sensory attributes of fresh sturgeon fillets.

Metformin promotes angiogenesis and functional recovery in aged mice after spinal cord injury by adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury.However,its effect on spinal cord injury in aged mice remains unclear.Considering the essential role of angiogenesis during the regeneration process,we hypothesized that metformin activates the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway in endothelial cells,thereby promoting microvascular regeneration in aged mice after spinal cord injury.In this study,we established young and aged mouse models of contusive spinal cord injury using a modified Allen method.We found that aging hindered the recovery of neurological function and the formation of blood vessels in the spinal cord.Treatment with metformin promoted spinal cord microvascular endothelial cell migration and blood vessel formation in vitro.Furthermore,intraperitoneal injection of metformin in an in vivo model promoted endothelial cell proliferation and increased the density of new blood vessels in the spinal cord,thereby improving neurological function.The role of metformin was reversed by compound C,an adenosine monophosphate-activated protein kinase inhibitor,both in vivo and in vitro,suggesting that the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway likely regulates metformin-mediated angiogenesis after spinal cord injury.These findings suggest that metformin promotes vascular regeneration in the injured spinal cord by activating the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway,thereby improving the neurological function of aged mice after spinal cord injury.

5-Hexylidene-4-propylamino-1,5-dihydroimidazol-2-one formed from Cu-catalyzed oxidation with implication for the structure of a His-Lys cross-link

Cu-catalyzed oxidation of 5-hexyl-1,3-dihydroimidazo-2-one (1) in the presence of propylamine, as surrogates for the oxidized His side chain and Lys side chain, was investigated. 5-Hexylidene-4-propylamino-1,5-dihydroimidazol-2-one (2), a model His-Lys cross-link product, was isolated and structurally characterized by NMR and mass spectrometry.

Influence of dietary canola oil and palm oil blend and refrigerated storage on fatty acids,myofibrillar proteins,chemical composition,antioxidant profile and quality attributes of semimembranosus muscle in goats

Background： Improving the unsaturated fatty acid content of ruminant meat is essential due to the generally saturated nature of fatty acids in ruminant meat and the negative effects this can have on human health. Nonetheless, enhancing the unsaturated fatty acid content of ruminant meat can have adverse effects on the shelf life and quality attributes of the meat. This study assessed the effects of dietary 80 % canola oil and 20 % palm oil blend（CPOB） on fatty acid composition, antioxidants, oxidative spoilage, cholesterol and physicochemical properties of semimembranosus（SM）muscle from goats. Twenty four Boer bucks were randomly assigned to diets containing on dry matter basis 0, 4 and8 % CPOB, fed for 100 d and slaughtered. The carcasses were subjected to a 7 d postmortem refrigerated storage. Al analyses were conducted on the SM muscle.Results： Diet had no effect（P 〉 0.05） on the concentration of free thiol and carbonyl and the band intensity of myosin heavy chain, actin and troponin T. The muscle glycogen, p H, water holding capacity, tenderness, glutathione peroxidase（GPX） activity, total carotenoid, δ-tocopherol, cholesterol and proximate composition did not differ（P 〉 0.05） between diets. The SM muscle from goats fed 4 and 8 % CPOB had lower（P 〈 0.05） concentration of C14：0 and C16：0 and higher（P 〈 0.05） concentration of C18：1 trans-11, C18：1ω-9, C18：3ω-3, C20：5ω-3 and C22：5ω-3 than the SM muscle from the control goats. Dietary CPOB increased（P 〈 0.05） the concentration of α and γ tocopherol and meat redness（a＊） on d1 and 4 postmortem. Regardless of diet, antioxidant vitamins, and shear force decreased（P 〈 0.05） while drip loss, lipid and protein oxidation increased（P 〈 0.05） as postmortem storage progressed.Conclusion： Results evince that dietary CPOB can be used as a management tool to enhance the beneficial fatty acids and antioxidant contents of chevon without deleterious effects on its physicochemical properties and shelf life.

Effect of Triptolide on Expression of Oxidative Carbonyl Protein in Renal Cortex of Rats with Diabetic Nephropathy

The traditional Chinese medicine（Tripterygium wilfordii Hook.f., TWH） has been clinically used to treat primary and secondary renal diseases and proteinuria for nearly 40 years. However, there is a rare literature about the effect of triptolide（the main active ingredient of TWH） on the expression of oxidative carbonyl protein（OCP） in diabetic nephropathy（DN）. This study aimed to provide experimental evidence for triptolide treatment on DN through its effect on the expression of OCP, in order to investigate the effects of triptolide on the expression of OCP in rats with DN. Sixty SD rats were randomly divided into five groups： control group, high-dose triptolide（Th） group, low-dose triptolide（Tl） group, DN model group, and positive control（benazepril） group. The DN model was established using streptozotocin. Urinary protein excretion, fasting blood glucose（FBG）, superoxide dismutase（SOD） in renal homogenate, malondialdehyde（MDA） in renal homogenate and renal nitrotyrosine by immunohistochemistry, and the expression of OCP by oxyblotimmune blotting were detected. In the DN model group, rat urinary protein excretion and renal MDA were significantly increased, while renal SOD significantly decreased and nitrotyrosine expression was obviously upregulated in the kidney. After triptolide treatment, 24-h urinary protein excretion（61.96±19.00 vs. 18.32±4.78 mg/day, P〈0.001）, renal MDA（8.09±0.79 vs. 5.45±0.68 nmol/L, P〈0.001）, and nitrotyrosine expression were decreased. Furthermore, renal OCP significantly decreased, while renal SOD（82.50±19.10 vs. 124.00±20.52 U/L, P〈0.001） was elevated. This study revealed that triptolide can down-regulate the expression of OCP in the renal cortex of DN rats.

The application of Melissa officinalis L.essential oil nanoemulsions protects sea bass(Lateolabrax japonicus)against myofibrillar protein and lipid oxidation during refrigeration

Objectives:The nutrient rich sea bass is prone to oxidation of lipid and protein during refrigeration.Materials and Methods:The research was to investigate the effect of different concentrations of Melissa officinalis L.essential oil(MOEO)nanoemulsions on myofibrillar protein(MP)and lipid oxidation in sea bass(Lateolabrax japonicus)during refrigeration at 4°C.Results:The results of thiobarbituric acid reactive substances and mitochondrial membrane potential showed that carboxymethyl chitosan/locust bean gum active coating solutions incorporating 2%MOEO nanoemulsions(C/L-2M)was the most effective in inhibiting lipid oxidation that occurred in sea bass under attack by reactive oxygen species.Low-field nuclear magnetic resonance results showed that C/L-2M maximally slowed the conversion of bound water to free water during storage.The oxidation of lipids and MP disrupted the secondary and tertiary conformations of MP and accelerated protein aggregation and degradation.Conclusions:C/L-2M slowed the oxidation of lipids and proteins by inhibiting the oxidation of reactive oxygen species.C/L-2M is a very promising preservative emulsion for the preservation of sea bass.

Advanced Oxidation Protein Products Regulate the Pharmacokinetics of Aloe-emodin,Emodin,Rhein,and Chrysophanol in Chronic Kidney Disease Rats

Background:As accelerators and products of the progression of chronic kidney disease(CKD),advanced oxidation protein products(AOPPs)affect the function of the liver.Huang Gan granules(HGGs)are commonly used to prevent the progression of CKD,but the pharmacokinetics of aloe-emodin,emodin,rhein,and chrysophanol in HGGs in CKD remain unknown.Objective:To investigate the influence and its molecular mechanism of AOPPs on the in vivo pharmacokinetics of aloe-emodin,emodin,rhein,and chrysophanol in HGGs.Methods:We constructed 5/6 nephrectomised(5/6 nx),adenine-induced(adenine)and AOPP-treated rat models.After oral administration of HGG,the concentrations of aloe-emodin,emodin,rhein,and chrysophanol in the plasma samples were detected by high-performance liquid chromatography(HPLC),and their pharmacokinetics were analysed with the PKSolver software.The plasma concentrations of IL-6 and TNF-αare detected by enzyme linked immunosorbent assay(ELISA).The RT-PCR was performed in the HepG2 cells to explore the effect of TNF-αand IL-6 on the mRNA expression of CYP1A2 and CYP3A4.Result:The results showed that the method was suitable for the quantification of four anthraquinones in plasma and excreta samples with satisfactory linear(R R^(2)>0.9931),precision(<9.4%)and accuracy(±10%).In 5/6 nx,adenine and AOPPs-treated rats,the concentrations of TNF-αand IL-6 were increased.In 5/6 nx and adenine rats,the pharmacokinetic parameters(t_(1/2),MRT_(0-∞)and AUC_(0-∞))of aloe-emodin,emodin,rhein,and chryso-phanol were,respectively,significantly increased and correlated with the concentration of AOPPs.In AOPPs-treated rats,the concentration of AOPPs was significantly increased and the pharmacokinetic parameters of four anthraquinones were also increased.Conclusion:In summary,inflammatory cytokine production may be one of the important causes in AOPPs’regulat-ing the pharmacokinetic of aloe-emodin,emodin,rhein,and chrysophanol in the CKD rats.Studies of aloe-emodin,emodin,rhein,and chrysophanol in CKD facilitate the appropriate prescription of HGGs in the clinical.

Evaluation of Oxidative Stress in Colorectal Cancer Patients

Objective To evaluate the oxidative stress in patients with colorectal cancer and to investigate the relationship between oxidative stress and colorectal cancer. Methods Seventy-six subjects were divided into two groups （36 colorectal cancer patients as the study group and 40 normal healthy individuals as the control group）. Their protein oxidation, DNA damage, lipid peroxidation and antioxidants, vitamin C, vitamin E, glutathione （GSH）, and antioxidative enzymes in serum were detected. Results The levels of protein carbonyl and advanced oxidation protein products （AOPP） were significantly higher in the study group than in the control group （P〈0.01）. Serum 8-OHdG was significantly increased in the study group compared to the control group （P〈0.01）. However, the mean serum level of MDA and conjugated diene was lower in the study group than in the control group （P〈0.01）. The activity of antioxidative enzymes was significantly decreased in the study group compared to the control group （P〈0.01）. Serum vitamins C and E concentrations were significantly reduced in the study group compared to the control group （P〈0.01）. Conclusion Colorectal cancer is associated with oxidative stress, and assessment of oxidative stress and given antioxidants is important for the treatment and prevention of colorectal cancer.

Application of Natural Antioxidants from Strawberry Tree(Arbutus unedo L.) and Dog Rose(Rosa canina L.) to Frankfurters Subjected to Refrigerated Storage

The effect of the addition of natural antioxidants from strawberry tree （Arbutus unedo L.; AU） and dog rose （Rosa canina L.; RC）, in frankfurters elaborated with or without the addition of antioxidant additives （sodium ascorbate and nitrite） was studied. Six different types of experimental frankfurters were prepared depending on the addition of phenolic-rich extracts from RC and AU and the presence （P） or absence （C） of antioxidant additives. Thiobarbituric acid reactive substances （TBARS）-numbers signiifcantly increased during chilled storage of C-frankfurters while additives and fruit phenolics inhibited lipid oxidation in P-frankfurters. The amount of protein carbonyls signiifcantly increased in all treatments except in P-AU frankfurters. The discoloration process that occurred during the chilled storage was reduced by the addition of substances with proven antioxidant activity （P-frankfurters）. Texture characteristics as hardness, springiness, cohesiveness and gumminess also suffered a signiifcant deterioration in C-frankfurters. The use of phenolic fruit extracts in combination with traditional antioxidant additives is a successful strategy to enhance the oxidative stability of frankfurters without modifying their color and texture properties.

Influence of peanut skin extract on shelf-life of sheep patties

Objective: To evaluate the phenolic profile and antioxidant activity in vitro of peanut skin extract(PSE) and effect of PSE on characteristics of sheep patties during storage.Methods: PSE phenolic profile was evaluated in LC–MS analysis and by total phenolic content, 1,1-diphenyl-2-picrylhydrazyl radical scavenging capacity and ferric reducing/antioxidant power. Patties elaborated with sheep meat were packaged in modified atmosphere and storage at(2 ± 1)C. The analyses were performed every 5 days for 20 days on microbial counts, physico-chemical properties, lipid oxidation, protein stability and sensory characteristics.Results: The major group of phenolic compounds in PSE was the proanthocyanidins followed by other flavonoids, which are related to potential phenolic content and antioxidant activity. The addition of PSE and butyl hydroxytoluene(BHT) reduced the microbial counts during the storage time, caused reduction on the loss of redness and sensory properties over time. The lipid and protein oxidation in sheep patties was effectively inhibited by PSE and BHT.Conclusions: The present results showed the potential application of PSE as a natural alternative to replace synthetic antioxidants(BHT) for increasing the quality and extending the shelf-life of sheep patties.

Comparative oxidation proteomics analyses suggest redox regulation of cytosolic translation in rice leaves upon Magnaporthe oryzae infection

Pathogen attack can increase plant levels of reactive oxygen species(ROS),which act as signaling molecules to activate plant defense mechanisms.Elucidating these processes is crucial for understanding redox signaling pathways in plant defense responses.Using an iodo-tandem mass tag(TMT)-based quantitative proteomics approach,we mapped 3362 oxidized cysteine sites in 2275 proteins in rice leaves.Oxidized proteins were involved in gene expression,peptide biosynthetic processes,stress responses,ROS metabolic processes,and translation pathways.Magnaporthe oryzae infection led to increased oxidative modification levels of 512 cysteine sites in 438 proteins,including many transcriptional regulators and ribosomal proteins.Ribosome profiling(Ribo-seq)analysis revealed that the oxidative modification of ribosomal proteins promoted the translational efficiency of many mRNAs involved in defense response pathways,thereby affecting rice immunity.Our results suggest that increased oxidative modification of ribosomal proteins in rice leaves promotes cytosolic translation,thus revealing a novel function of posttranslational modifications.Furthermore,the oxidation-sensitive proteins identified here provide a valuable resource for research on protein redox regulation and can guide future mechanistic studies.

Advanced oxidation protein products induce monocyte chemoattractant protein-1 expression via p38 mitogen-activated protein kinase activation in rat vascular smooth muscle cells

Background Advanced oxidation protein products （AOPPs） are new uremic toxins reported by Witko-Sarsat in 1996, which are associated with the pathogenesis of atherosclerosis. However, the mechanisms by which AOPPs enhance atherosclerosis have not been fully understood. Monocyte chemoattractant protein-1 （MCP-1） is a chemokine which stimulates migration of monocytes and plays a critical role in the development of atherosclerosis. In this study, we investigated the effect of AOPPs on MCP-1 expression in cultured vascular smooth muscle cells （VSMCs）.

Association between heat stress and oxidative stress in poultry;mitochondrial dysfunction and dietary interventions with phytochemicals

Heat as a stressor of poultry has been studied extensively for many decades; it affects poultry production on a worldwide basis and has significant impact on well-being and production. More recently, the involvement of heat stress in inducing oxidative stress has received much interest. Oxidative stress is defined as the presence of reactive species in excess of the available antioxidant capacity of animal cells. Reactive species can modify several biologically cellular macromolecules and can interfere with cell signaling pathways. Furthermore, during the last decade, there has been an ever-increasing interest in the use of a wide array of natural feed-delivered phytochemicals that have potential antioxidant properties for poultry. In light of this, the current review aims to（1） summarize the mechanisms through which heat stress triggers excessive superoxide radical production in the mitochondrion and progresses into oxidative stress,（2） illustrate that this pathophysiology is dependent on the intensity and duration of heat stress,（3） present different nutritional strategies for mitigation of mitochondrial dysfunction, with particular focus on antioxidant phytochemicals.Oxidative stress that occurs with heat exposure can be manifest in all parts of the body; however, mitochondrial dysfunction underlies oxidative stress. In the initial phase of acute heat stress, mitochondrial substrate oxidation and electron transport chain activity are increased resulting in excessive superoxide production. During the later stage of acute heat stress, down-regulation of avian uncoupling protein worsens the oxidative stress situation causing mitochondrial dysfunction and tissue damage. Typically, antioxidant enzyme activities are upregulated. Chronic heat stress, however, leads to downsizing of mitochondrial metabolic oxidative capacity, up-regulation of avian uncoupling protein, a clear alteration in the pattern of antioxidant enzyme activities, and depletion of antioxidant reserves.Some phytochemicals, such as various types of flavonoids and related compounds, were shown to be beneficial in chronic heat-stressed poultry, but were less or not effective in non-heat-stressed counterparts. This supports the contention that antioxidant phytochemicals have potential under challenging conditions. Though substantial progress has been made in our understanding of the association between heat stress and oxidative stress, the means by which phytochemicals can alleviate oxidative stress have been sparsely explored.

Artificial rearing influences the morphology,permeability and redox state of the gastrointestinal tract of low and normal birth weight piglets

Background： In this study the physiological implications of artificial rearing were investigated. Low（LBW） and normal birth weight（NBW） piglets were compared as they might react differently to stressors caused by artificial rearing. In total, 42 pairs of LBW and NBW piglets from 16 litters suckled the sow until d19 of age or were artificially reared starting at d3 until d19 of age. Blood and tissue samples that were collected after euthanasia at 0, 3, 5, 8 and 19 d of age. Histology, ELISA, and Ussing chamber analysis were used to study proximal and distal small intestine histomorphology, proliferation, apoptosis, tight junction protein expression, and permeability. Furthermore, small intestine,liver and systemic redox parameters（GSH, GSSG, GSH-Px and MDA） were investigated using HPLC.Results： LBW and NBW artificially reared piglets weighed respectively 40 and 33% more than LBW and NBW sowreared piglets at d19（P 〈 0.01）. Transferring piglets to a nursery at d3 resulted in villus atrophy, increased intestinal FD-4 and HRP permeability and elevated GSSG/GSH ratio in the distal small intestine at d5（P 〈 0.05）. GSH concentrations in the proximal small intestine remained stable, while they decreased in the liver（P 〈 0.05）. From d5 until d19, villus width and crypt depth increased, whereas PCNA, caspase-3, occludin and claudin-3 protein expressions were reduced. GSH,GSSG and permeability recovered in artificially reared piglets（P 〈 0.05）.Conclusion： The results suggest that artificial rearing altered the morphology, permeability and redox state without compromising piglet performance. The observed effects were not depending on birth weight.

Stimulatory effect of puerarin on bone formation through co-activation of nitric oxide and bone morphogenetic protein-2/ mitogen-activated protein kinases pathways in mice

Background Estrogen deficiency results in loss of bone mass compounds with estrogen-like activity that bind to estrogen receptors effect of the phytoestrogen puerarin on adult mouse osteoblasts. Methods Osteoblast cells were harvested from 8-month old female Phytoestrogens are plant-derived non-steroidal The main aim of this study was to investigate the mprinting control region （ICR） mice. The effects of puerarin stimulation on the proliferation, differentiation and maturation of osteoblasts were examined. The production of nitric oxide （NO） and the expression of bone morphogenetic protein-2 （BMP-2）, SMAD4, mitogen-activated protein kinases （MAPK）, core binding factor all runt-related transcription factor 2 （Cbfal/Runx2）, osteoprotegerin （OPG）, and receptor activator of NF-KB ligand （RANKL） genes were analyzed. The activation of signal pathways was further confirmed by specific pathway inhibitors. Results The osteoblast viability reached its maximum at 10-8 mol/L puerarin. At this concentration, puerarin increases the proliferation and matrix mineralization of osteoblasts and promotes NO synthesis. With 108 mol/L puerarin treatment, BMP-2, SMAD4, Cbfal/Runx2, and OPG gene expression were up-regulated, while the RANKL gene expression is down-regulated. Concurrent treatment involving the （bone morphogenetic protein） BMP antagonist Noggin or the NOS inhibitor L-NAME diminishes puerarin induced cell proliferation, Alkaline phosphatase （ALP） activity, NO production, as well as the BMP-2, SMAD4, Cbfal/Runx2, OPG, and RANKL gene expression. Conclusions In this in vitro study, we demonstrate that puerarin is a bone anabolic agent that exerts its osteogenic effects through the induction of BMP-2 and NO synthesis, subsequently regulating Cbfal/Runx2, OPG, and RANKL gene expression. This effect may contribute to its induction of osteoblast proliferation and differentiation, resulting in bone formation.

Effects of Berberine on Hepatic Sirtuin 1-uncoupling Protein 2 Pathway in Non-alcoholic Fatty Liver Disease Rats Induced by High-fat Diet

Objective To investigate the involvement of sirtuin 1（SIRT1）-uncoupling protein 2（UCP2） pathway in the development of non-alcoholic fatty liver disease and whether berberine exerts its effects by regulating this pathway. Methods Male SD rats were divided into three groups： normal control group, high-fat diet group, and berberine supplement group. The rats in the normal control group were given normal diet while the rats in the other two groups were fed with high-fat diet. Rats in the berberine supplement group were concurrently given berberine（100 mg/kg body weight） once daily. After 16 weeks, the levels of serum, liver lipids, and serum aminotransferase were measured using an automatic biochemical analyzer. Superoxide dismutase（SOD） activity and malondialdehyde（MDA） content in the liver were measured using commercial kits. Histopathological changes of liver tissues were observed by hematoxylin and eosin（HE） staining and Oil Red O staining. The hepatic m RNA and protein levels of SIRT1 and UCP2 were assayed by reverse transcription polymerase chain reaction（RT-PCR） or Western blotting. Results Berberine supplement could significantly decrease the serum and liver lipid contents in rats fed with high-fat diet. Meanwhile, SOD level was significantly elevated, but MDA level was reduced in the liver. The results of HE and Oil Red O staining showed that the hepatic steatosis was alleviated in berberine supplement group. Furthermore, berberine induced an increase in SIRT1 expression but a decrease in UCP2 expression. Conclusion The regulation of hepatic SIRT1-UCP2 pathway may be an important mechanism by which berberine exerts the beneficial effects in NAFLD rats.

Rapid Inactivation of Chloroplastic Ascorbate Peroxidase is Responsible for Oxidative Modification to Rubisco in Tomato (Lycopersicon esculentum) under Cadmium Stress

To investigate the sensitive site of antioxidant systems in chloroplast under cadmium stress and its consequence on reactive oxygen species production and action, the sub-organellar localization of chloroplast superoxide dismutases （SOD, EC 1.15.1.1） and ascorbic peroxidase （APX, EC 1.11.1.11） isoenzymes and changes of enzymes activities under cadmium stress were investigated in tomato seedlings. Two APX isoforms, one thylakoid-bound and one stromal, were detected. Cd at 50 μM induced a moderate increase of SOD activities but a rapid inactivation of both APX isoenzymes. APX inactivation was mainly related to the decrease of ascorbate concentration, as supported by in vitro treatment of exogenous ascorbate and APX kinetic properties under Cd stress. H2O2 accumulation in chloroplast, as a consequence of APX inactivation, was associated with a 60% loss of Rubisco （EC 4.1.1.39） activity, which could be partially accounted for by a 10% loss of Rubisco content. Protein oxidation assay found that the Rubisco large subunit was the most prominent carbonylated protein; the level of carbonylated Rubisco large subunit increased fivefold after Cd exposure. Thiol groups in the Rubisco large subunit were oxidized, as indicated by non-reducing electrophoresis. Treating crude extract with H2O2 resulted in a similar pattern of protein oxidation and thiols oxidation with that observed in Cd-treated plants. Our study indicates that APXs in the chloroplast is a highly sensitive site of antioxidant systems under Cd stress, and the inactivation of APX could be mainly responsible for oxidative modification to Rubisco and subsequent decrease in its activity.