Target Metabolome and Transcriptome Analysis Reveal Molecular Mechanism Associated with Changes of Tea Quality at Different Development Stages.

This study aimed to explore the molecular mechanisms underlying the differential quality of tea made from leaves at different development stages. Fresh Camellia sinensis (L.) O. Kuntze "Sichuan Colonial" leaves of various development stages, from buds to old leaves, were subjected to transcriptome sequencing and metabolome analysis, and the DESeq package was used for differential expression analysis, followed by functional enrichment analyses and protein interaction analysis. Target metabolome analysis indicated that the contents of most compounds, including theobromine and epicatechin gallate, were lowest in old leaves, and transcriptome analysis revealed that DEGs were significantly involved in extracellular regions and phenylpropanoid biosynthesis, photosynthesis-related pathways, and the oleuropein steroid biosynthesis pathway. Protein-protein interaction analysis identified LOC114256852 as a hub gene. Caffeine, theobromine, L-theanine, and catechins were the main metabolites of the tea leaves, and the contents of all four main metabolites were the lowest in old leaves. Phenylpropanoid biosynthesis, photosynthesis, and brassinosteroid biosynthesis may be important targets for breeding efforts to improve tea quality.

Jianpi Qingchang Decoction Ameliorates Chronic Colitis in Piroxicam-Induced IL-10 Knockout Mice by Inhibiting Endoplasmic Reticulum Stress.

BACKGROUND: Excessive endoplasmic reticulum (ER) stress in intestinal epithelial cells (IEC) may lead to impaired intestinal mucosal barrier function and then participate in the pathogenesis of ulcerative colitis (UC). Jianpi Qingchang decoction (JPQCD) has been shown to have protective effects on UC. However, further studies are needed to determine whether JPQCD regulates PERK/eIF2α/ATF4/CHOP pathways to play a role in treating UC. METHODS: IL-10 -/- mice were randomly assigned into five groups: control, model, low-dose JPQCD (JPQCD L), middle-dose JPQCD (JPQCD M), and high-dose JPQCD (JPQCD H). All groups except for the control group were given model feed containing 200 ppm piroxicam for 10 d to induce colitis. As a comparison, we used wild-type mice that were the progeny of IL-10 +/- matings, bred in the same facility. The control group and wild-type mice were fed with common feed. At the same time, mice in each group were given corresponding drugs by gavage for 14 d. The disease activity index of mice in each group was evaluated daily. Colon tissues of mice were collected, colon length was measured, and pathological changes and ultrastructure of colon epithelial cells were observed. The effects of JPQCD on the PERK/eIF2α/ATF4/CHOP pathways were evaluated by western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The expression of CHOP in colon tissue was detected by tissue immunofluorescence assay. The expression of NF-κB, p-NF-κB p65 protein was analyzed by western blotting; the level of IL-17 in colon tissue was detected by enzyme-linked immunosorbent assay (ELISA) and verified by examining NF-κB and IL-17 mRNA levels by RT-PCR. RESULTS: Compared with the control group, the model group showed significant colitis symptoms and severe colonic tissue damage. The results showed that JPQCD significantly reduced body weight loss, ameliorated disease activity index, and restored colon length in IL-10 -/- mice with piroxicam-induced colitis. Western blotting and RT-PCR showed that the PERK/eIF2α/ATF4/CHOP pathway was activated in colon tissue of model mice, suggesting that the pathway is involved in the pathogenesis of ulcerative colitis (UC) and could become a potential therapeutic target. The JPQCD treatment inhibited the activation of the PERK/eIF2α/ATF4/CHOP pathway, alleviated the ER stress, and played a role in preventing and treating UC. In addition, JPQCD can also downregulate the protein of NF-κB, p-NF-κB p65, downregulate the mRNA expression of NF-κB, and reduce the content of IL-17 and its mRNA expression in colon tissues. CONCLUSION: JPQCD may play a protective role in UC by regulating the PERK/eIF2α/ATF4/CHOP signaling pathway and relieving endoplasmic reticulum stress.

Selenoproteins Protect Against Avian Liver Necrosis by Metabolizing Peroxides and Regulating Receptor Interacting Serine Threonine Kinase 1/Receptor Interacting Serine Threonine Kinase 3/Mixed Lineage Kinase Domain-Like and Mitogen-Activated Protein Kinase Signaling.

Liver necroptosis of chicks is induced by selenium (Se)/vitamin E (VE) deficiencies and may be associated with oxidative cell damage. To reveal the underlying mechanisms of liver necrosis, a pool of the corn-soy basal diet (10 µg Se/kg; no VE added), a basal diet plus all-rac-α-tocopheryl acetate (50 mg/kg), Se (sodium selenite at 0.3 mg/kg), or both of these nutrients were provided to day-old broiler chicks (n = 40/group) for 6 weeks. High incidences of liver necrosis (30%) of chicks were induced by -SE-VE, starting at day 16. The Se concentration in liver and glutathione peroxidase (GPX) activity were decreased (P < 0.05) by dietary Se deficiency. Meanwhile, Se deficiency elevated malondialdehyde content and decreased superoxide dismutase (SOD) activity in the liver at weeks 2 and 4. Chicks fed with the two Se-deficient diets showed lower (P < 0.05) hepatic mRNA expression of Gpx1, Gpx3, Gpx4, Selenof, Selenoh, Selenok, Selenom, Selenon, Selenoo, Selenop, Selenot, Selenou, Selenow, and Dio1 than those fed with the two Se-supplemented diets. Dietary Se deficiency had elevated (P < 0.05) the expression of SELENOP, but decreased the downregulation (P < 0.05) of GPX1, GPX4, SELENON, and SELENOW in the liver of chicks at two time points. Meanwhile, dietary Se deficiency upregulated (P < 0.05) the abundance of hepatic proteins of p38 mitogen-activated protein kinase, phospho-p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, phospho-c-Jun N-terminal kinase, extracellular signal-regulated kinase, phospho-mitogen-activated protein kinase, receptor-interacting serine-threonine kinase 1 (RIPK1), receptor-interacting serine-threonine kinase 3 (RIPK3), and mixed lineage kinase domain-like (MLKL) at two time points. In conclusion, our data confirmed the differential regulation of dietary Se deficiency on several key selenoproteins, the RIPK1/RIPK3/MLKL, and mitogen-activated protein kinase signaling pathway in chicks and identified new molecular clues for understanding the etiology of nutritional liver necrosis.

[Analysis of Water Quality and Exchange Flux of Interstitial Water-Overlying Water in Sediments of Baiyangdian Entrance Area in Summer].

In order to reveal the interaction of overlying water-interstitial water nitrogen and phosphorus nutrient salt in summer at the entrance region of Baiyangdian Lake, this study sampled six main rivers in the region during July 2019. An analysis of the overlying water and interstitial water quality characteristics and the diffusion flux of applied nutrients at the sediment-water interface revealed the effects of nutrient diffusion on sediments and overlying water. The overlying water analysis showed that the water quality was slightly alkaline in the Baiyangdian Lake. The content of dissolved oxygen (DO) was lower, which provided an anaerobic environment for the release of endogenous pollutants from sediments. The ammonia nitrogen (NH4+-N) ranged from 0.35 to 1.76 mg·L-1, and the content of ammonia nitrogen was the highest in the Zhulong River, which was the main source of water supply. The nitrate nitrogen (NO3--N) content ranged from 0.75 to 1.97 mg·L-1. The total dissolved nitrogen (TDN) ranged from 0.99 to 2.70 mg·L-1, and the content of TDN was the highest in Puhe River. The content of total dissolved phosphorus (TDP) was 0.03 to 0.15 mg·L-1, and the content of TDP was the highest was Baigouyin River, which is near the residential area. The results indicated that the content of ammonia nitrogen in the interstitial water was between 5.24 and 10.64 mg·L-1, which was 10 times that of the overlying water, and endogenous pollution in the former was severe. The nitrate nitrogen content ranged from 0.36 to 0.79 mg·L-1. The total dissolved nitrogen content was between 5.36 and 12.02 mg·L-1, which was 5 times higher than that of the overlying water. The total dissolved phosphorus was between 0.03 and 0.3 mg·L-1. According to integrated pollution index, the degree of interstitial water pollution was much higher than that of overlying water, and the sampling points are seriously polluted. The exchange flux analysis of NH4+-N, TDN, and TDP demonstrated that the diffusion flux of NH4+-N was between 1.71 and 7.43 mg·(m2·d)-1, and the diffusion rate of endogenous ammonia nitrogen to the overlying water was fastest in Fu River, the absorbing river in Baoding. The diffusion flux of total dissolved nitrogen was lower in the Baigouyin River, and the other five sample points averaged 9.11 mg·(m2·d)-1. In summer, the dissolved oxygen was lower and the water-sediment had a larger concentration difference, which led to massive nitrogen nutrient of sediment in anaerobic conditions released to the overlying water in great quantities that caused the serious pollution. The diffusion flux of dissolved total phosphorus showed that the sediment of Pinghe River acted as a "sink" of phosphorus nutrients, and the other sampling points ranged from 0.03 to 0.16 mg·(m2·d)-1, showing the state of phosphorus nutrient released upward to the overlying water. Finally, diffusion flux indicated that endogenous pollutants are crucial sources of overlying water pollutants. In order to effectively control the water quality in the entrance area, desilting the nitrogen and phosphorus nutrient salt of sediment is urgently required.

[Progress of antibacterial activity and antibacterial mechanism of isoquinoline alkaloids].

This paper reviewed the antibacterial activity and structure-activity relationship of isoquinoline alkaloids, such as protoberberine, protopine, benzophenanthridine, aporphine, double benzyl isoquinoline, etc. The antibacterial mechanism of alkaloids were illustrated from cell wall and membrane damage, membrane permeability changes, related enzymes and efflux pump inhibition, influence of bacterial DNA and related protein synthesis and so on. In addition, this paper summarized the structure-activity relationship of isoquinoline alkaloids. In order to improve the screening efficiency of drug targets, the complementary effect of biological information science and combinatorial chemistry should be developed abundantly in the development of natural product. This paper will provide a theoretical reference for the research and development of new antibacterial agent.

[Quantitative determination of seven major absorbed volatile constituents in mice brain, liver and blood after intragastric administration of Asari Radix et Rhizoma suspension by headspace-solid phase microextraction-gas chromatography-mass spectrometry].

A headspace-solid phase microextraction-gas chromatography-mass spectrometry method（HS-SPME-GC-MS） was adopted for the quantitative study of 4-allylanisole, methyl eugenol, 2,3,5-trimethoxytoluene, 3,4,5-trimethoxytoluene, sarisan, 3,5-dimethoxytoluene and safrole in mice brain, liver tissues and blood after intragastric administration of Asari Radix et Rhizoma. A VF-WAXms (30 m×0.25 mm, 0.25 µm film thickness) capillary column and SPME fiber coated with 65 µm polydimethylsiloxane/divinylbenzene (PDMS/DVB) were used. The calibration curves of seven volatile constituents were established to validate the method's stability (RSD<15%), repeatability (RSD<9.5%), accuracy (RSD<22%), relative recovery (87.0%-108%) and extraction recovery (74.9%-102%). The validated HS-SPME-GC-MS assay was applied to determine the concentrations of seven constituents in liver, brain and blood. The detected contents were 0.22,0.14 µg•g⁻¹,0.25 mg•L⁻¹ (4-allylanisole), 1.1, 0.39 µg•g⁻¹, 0.69 mg•L⁻¹ (methyl eugenol), 0.45, 0.13 µg•g⁻¹, 0.54 mg•L⁻¹ (2,3,5-trimethoxytoluene), 0.51, 0.15 µg•g⁻¹, 0.45 mg•L⁻¹ (3,4,5-trimethoxytoluene), 0.48, 0.039 µg•g⁻¹, 0.69 mg•L ⁻¹ (sarisan), 2.2, 1.2 µg•g⁻¹, 1.5 mg•L⁻¹ (3,5-dimethoxytoluene) and 1.3, 0.67 µg•g⁻¹, 1.1 mg•L⁻¹ (safrole) respectively. This HS-SPME-GC-MS method is rapid and convenient, with a small sample size, and applicable for the analysis and determination of volatile constituents in traditional Chinese medicines, which provides scientific data for further studies on effective substances and toxic substances in Asari Radix et Rhizoma.

[Qualitative and quantitative analysis of dodecatetraenamides A, B in Asari Radix et Rhizoma].

To develop an analytic method for qualitative and quantitative analysis of dodecatetraenamides A, B in 42 samples of two official species of Asari Radix et Rhizoma( ARR) (37 samples of Asarum heterotropoides var. mandshuricum with different collection time and 5 samples of Asarum sieboldiivar. seoulense). The HPLC-IT-TOF-MS/MS methods for the qualitative and UPLC-PDA methods for the quantitative analysis were established. Dodecatetraenamides A, B were identified by comparing the retention time, UV absorption spectrum and quasi-molecular ion peak [ M + H]+ with the reference compound using HPLC-IT-TOF-MS/MS. The content of dodecatetraenamides A and B in ARR were determined by UPLC-PDA. The separation was successfully carried out on a ACQUITY UPLC BEH C18 (2.1 mm x 100 mm, 1.7 µm) column eluted with mobile phases of water (A) and acetonitrile (B) in gradient program (0-3 min, 35% B; 3-5 min, 35%-36% B; 5-6 min, 36%-43% B; 6 min-11 min 43% B; 11-12 min, 43%-100% B). The column temperature was 45 °C, and the detection wavelength was set at 254 nm. The flow rate was 0.6 mL · min(-1). On one level mass spectrometry scanning, the results showed that the quasi-molecular ion [M + H] + of both dodecatetraenamides A and B were m/z 248.20. The quantitative method with UPLC-PDA has made the baseline separation of the constituents, which were reported as mixtures in the most literatures. The average recovery of dodecatetraenamides A and B were 97.90% and 99.86%, the relative standard deviation were 0.4% and 1.1%, respectively. The contents of dodecatetraenamides A, B in all ARR samples was in the range of 0.11-3.89 and 0.24-6.65 mg · g(-1). Their contents reduced with the extension of storage time. Compared with the samples of 2013, the average content of the two constituents in the samples collected in year 2002-2003 reduced 34% and 36%, respectively (P < 0.05). Compared the A. sieboldii var. seoulense and A. heterotropoides var. mandshuricum with the same collective time and production area, the average contents of the two constituents in latter were up to (1.59 ± 0.75) mg · g(-1) and (2.90 ± 1.17) mg · g(-1), respectively, significantly higher than that in A. sieboldii var. seoulense (dodecatetraenamide A were (0.78 ± 0.52) mg · g(-1), dodecatetraenamide B were (1.69 ± 0.83) mg · g(-1)) (P < 0.05). The content of the dodecatetraenamide A in overground part was in the range of 0.11-0.33 mg · g(-1), dodecatetraenamide B was 0. 24-0.60 mg · g(-1), which were much lower than that of the underground part of ARR (dodecatetraenamide A was in the range of 0.73-3.89 mg · g(-1), dodecatetraenamide B was 2.11-6.24 mg · g(-1)). The method was certified to be simple, accurate and reliable and could be used for qualitative and quantitative analysis of dodecatetraenamide A and B in different species of ARR, also can be used for the comprehensive quality control of traditional Chinese medicine, Asari Radix et Rhizoma.

Possible correlation of selenoprotein W with inflammation factors in chicken skeletal muscles.

The aim of the present study was to investigate the possible correlation of selenoprotein W (SelW) with inflammatory injury induced by dietary selenium (Se) deficiency in chicken. One-day-old male chickens were fed either a commercial diet or a Se-deficient diet for 55 days. Then, the expression levels of SelW messenger RNA (mRNA) and inflammation-related genes (NF-κB, TNF-α, iNOS, COX-2, and PTGES) in chicken skeletal muscles (wing muscle, pectoral muscle, and thigh muscle) were determined at 15, 25, 35, 45, and 55 days old, respectively. In addition, the correlation between SelW mRNA expression and inflammation-related genes were assessed. The results showed that dietary Se deficiency reduced the mRNA expression of SelW in chicken wing, pectorals, and thigh muscles. In contrast, Se deficiency increased the mRNA expression levels of inflammation-related genes in chicken skeletal muscle tissues at different time points. The Pearson's correlation coefficients showed that the mRNA expression levels of inflammation-related genes were significantly negative related to SelW (p < 0.05). These data showed that Se deficiency induced the inflammatory response in chicken skeletal muscle. As one important selenoprotein gene in skeletal muscles, SelW may play a role in the regulation of inflammation reaction in Se-deficiency myopathy.

Selenium deficiency inhibits the conversion of thyroidal thyroxine (T4) to triiodothyronine (T3) in chicken thyroids.

Selenium (Se) influences the metabolism of thyroid hormones in mammals. However, the role of Se deficiency in the regulation of thyroid hormones in chickens is not well known. In the present study, we examined the levels of thyroidal triiodothyronine (T3), thyroidal thyroxine (T4), free triiodothyronine, free thyroxine (FT4), and thyroid-stimulating hormone in the serum and the mRNA expression levels of 25 selenoproteins in chicken thyroids. Then, principal component analysis (PCA) was performed to analyze the relationships between the selenoproteins. The results indicated that Se deficiency influenced the conversion of T4 to T3 and induced the accumulation of T4 and FT4. In addition, the mRNA expression levels of the selenoproteins were generally decreased by Se deficiency. The PCA showed that eight selenoproteins (deiodinase 1 (Dio1), Dio2, Dio3, thioredoxin reductase 2 (Txnrd2), selenoprotein i (Seli), selenoprotein u (Selu), glutathione peroxidase 1 (Gpx1), and Gpx2) have similar trends, which indicated that they may play similar roles in the metabolism of thyroid hormones. The results showed that Se deficiency inhibited the conversion of T4 to T3 and decreased the levels of the crucial metabolic enzymes of the thyroid hormones, Dio1, Dio2, and Dio3, in chickens. In addition, the decreased selenoproteins (Dio1, Dio2, Dio3, Txnrd2, Seli, Selu, Gpx1, and Gpx2) induced by Se deficiency may indirectly limit the conversion of T4 to T3 in chicken thyroids. The information presented in this study is helpful to understand the role of Se in the thyroid function of chickens.

Effect of selenium on selenoprotein expression in the adipose tissue of chickens.

This study describes the effects of selenium (Se) deficiency on the messenger ribonucleic acid (mRNA) expression of 25 selenoproteins (Sels) (including glutathione peroxidases (GPx1-GPx4), thioredoxin reductases (TrxR1-TrxR3), iodothyronine deiodinases (ID1-ID3), selenophosphate synthetase 2 (SPS2), 15-kDa Sel (Sel15), SelH, SelI, SelK, SelM, Sepn1, SelO, Sepx, Selpb, SelS, SelT, SelW, Sepp1, and SelU in the adipose tissues (subcutaneous adipose, visceral adipose, and articular adipose) of chickens. One hundred and fifty 1-day-old chickens were randomly assigned to two groups of 75 each and were fed a low-Se diet (0.032 mg/kg Se) or a control diet (0.282 mg/kg Se). The expression levels of 25 Sel mRNAs were determined on days 35, 45, and 55 from three parts (subcutaneous adipose, visceral adipose, and articular adipose) of the chicken adipose tissues. The results showed that the expression levels of the 25 Sel mRNAs were significantly lower (P < 0.05) in the low-selenium group than in the control group. In addition, the Sel mRNA expression levels in the three adipose tissues were observed to decrease in a time-dependent manner with increasing feeding time.

Protective roles of selenium on nitric oxide-mediated apoptosis of immune organs induced by cadmium in chickens.

Little is known about the influence of subchronic cadmium exposure on apoptosis in the immune organs of birds and the protective effects on apoptosis by selenium against cadmium. The aim of this study was to investigate the effect of subchronic cadmium exposure on nitric oxide and apoptosis in the immune organs of chicken and the protective roles of selenium against cadmium-induced apoptosis. Two hundred ten 30-day-old chickens were randomly assigned to three groups and were fed a basal diet, cadmium+selenium (as 150 mg of CdCl2 per kg of diet+10 mg of Na2SeO3 per kg of diet ) or cadmium (as 150 mg of CdCl2 per kg of diet) in basic diets for 15, 30, 45, and 60 days. Then, the production of nitric oxide, messenger RNA (mRNA level), and the activity of inducible nitric oxide synthase, ultrastructural changes, TUNEL assay, and flow cytometric analysis of apoptosis and Bcl-2 and p53 mRNA levels in the immune organs were examined. The results showed that cadmium exposure caused ultrastructural damage and increased production of nitric oxide, mRNA level, and activity of inducible nitric oxide synthase, the degree, and the number of apoptotic cells in a time-dependent manner. Cadmium exposure decreased Bcl-2 mRNA level and increased p53 mRNA level in a time-dependent manner. Selenium supplementation during dietary cadmium reduced the production of nitric oxide, the mRNA level, and activity of inducible nitric oxide synthase, ultrastructural damage, and apoptosis in the immune organs of chicken. It indicated that cadmium induced nitric oxide-mediated apoptosis of immune organs, and selenium played protective effects against cadmium-induced apoptosis in the immune organs of chickens.

Cadmium supplement triggers endoplasmic reticulum stress response and cytotoxicity in primary chicken hepatocytes.

Cadmium (Cd), a potent hepatotoxin, has been reported to induce endoplasmic reticulum (ER) stress in various cell types. However, whether such effect exists in bird is still unclear. To delineate the effects of Cd exposure on ER stress response, we examined the expression of 78-kDa glucose-regulated protein (GRP78) and alteration in calcium homeostasis in primary chicken hepatocytes treated with 2-22 µM Cd for 24 h. A significant decrease of cell viability was observed in chicken hepatocytes following Cd administration. In cells treated with Cd, GRP78 protein levels increased in a dose-dependent manner. In addition, GRP78 and GRP94mRNA levels were elevated in response to Cd exposure. The increase of the intracellular Ca(2+) concentration in chicken hepatocytes was found during Cd exposure. Cd significantly decreased the CaM mRNA levels in hepatocytes. These results show that Cd regulates the expression of GRP78 and calcium homeostasis in chicken hepatocytes, suggesting that ER stress induced by Cd plays an important role in the mechanisms of Cd cytotoxicity to the bird hepatocytes.

Protective effects of selenium on cadmium-induced brain damage in chickens.

Selenium (Se) is an important dietary micronutrient with antioxidative roles. Cadmium (Cd), a ubiquitous environmental pollutant, is known to cause brain lesion in rats and humans. However, little is reported about the deleterious effects of subchronic Cd exposure on the brain of poultry and the protective roles on the brain by Se against Cd. The aim of this study was to investigate the protective effects of Se on Cd-induced brain damage in chickens. One hundred twenty 100-day-old chickens were randomly assigned to four groups and were fed a basal diet, or Se (as 10 mg Na2SeO3/kg dry weight of feed), Cd (as 150 mg CdCl2/kg dry weight of feed), or Cd + Se in their basic diets for 60 days. Then, concentrations of Cd and Se, production of nitric oxide (NO), messenger RNA (mRNA) level and activity of inducible NO synthase (iNOS), level of oxidative stress, and histological and ultrastructural changes of the cerebrum and cerebellum were examined. The results showed that Cd exposure significantly increased Cd accumulation, NO production, iNOS activities, iNOS mRNA level, and MDA content in the cerebrum and cerebellum. Cd treatment obviously decreased Se content and antioxidase activities and caused histopathological changes in the cerebrum and cerebellum. Se supplementation during dietary Cd obviously reduced Cd accumulation, NO production, mRNA level and activity of iNOS, oxidative stress, and histopathological damage in the cerebrum and cerebellum of chickens. It indicated that Se ameliorates Cd-induced brain damage in chickens by regulating iNOS-NO system changes, and oxidative stress induced by Cd and Se can serve as a potential therapeutic for Cd-induced brain lesion of chickens.

Effects of dietary selenium deficiency or excess on gene expression of selenoprotein N in chicken muscle tissues.

Previous studies have determined the effects of dietary selenium (Se) supplementation on selenoprotein N (SelN, SEPN1), selenophosphate synthetase-1 (SPS1), and selenocysteine-synthase (SecS) mRNA abundance in chicken skeletal and cardiac muscles. To investigate collective responses of these genes to dietary Se concentrations ranging from deficiency to moderately high level in muscle tissues of chicken, 1-day-old chickens were exposed to a diet of deficient Se and supplemented with Se (0.15 mg Se/kg and 1.50 mg Se/kg) as sodium selenite in the feed for 35 days. Muscle tissues (flight, breast, leg, and cardiac muscles) were collected and examined for Se content and mRNA levels of SelN on days 1, 15, 25, and 35 days, respectively. Moreover, SPS1 and SecS mRNA levels were analyzed. The results showed that the expression of SelN gene in cardiac muscle responded to dietary Se concentrations. SelN gene was downregulated in the Se deficiency group (L group), and upregulated in the Se excess group (H group) compared with the moderate Se group (M group) (P < 0.05) in cardiac muscle. Se deficiency mainly unregulated SelN mRNA level in skeletal muscles compared with M group. Excess dietary Se mainly resulted in the upregulation of SelN mRNA level in skeletal muscles compared with the M group. SecS mRNA levels responded to dietary Se concentrations showed a similar change compared with SelN in cardiac muscle. SPS1 mRNA levels responded to dietary Se concentrations showed a downregulation in L group and upregulation in H group. However, SelN mRNA levels displayed a different expression pattern in different skeletal and cardiac muscles. Moreover, Se also regulated the levels of SPS1 and SecS mRNAs. In summary, Se regulated the expression of SelN gene and affected the mRNA levels of SecS and SPS1. The level of Se in the feed may regulate SelN biosynthesis by affecting the levels of SPS1 and SecS mRNA.

The effect of Se-deficient diet on gene expression of inflammatory cytokines in chicken brain.

Selenium (Se) plays an important role in the brain development, function, and degeneration, nutritional encephalomalacia is closely related with dietary Se in avian. However, there is little evidence on the relationship between inflammation and encephalomalacia in avian and the mechanism which Se regulates the inflammatory response in brain tissues remains to be unclear. The present paper describes the effects of Se-deficient granulated diet on one transcription factor-nuclear factor kappaB and four pro-inflammatory cytokines-tumor necrosis factor, cyclooxygenase2, inducible nitric oxide synthase and Prostaglandin E synthase mRNA expression in the chicken brain tissues associated encephalomalacia. One hundred male chickens (1 day old; Weiwei Co. Ltd., Harbin, China) were divided into two groups (50 chickens per group). The expression levels in the brain tissues (cerebral gray matter, cerebral white matter, marrowbrain, cerebellum, thalamus and brain stem) were determined by real-time PCR on days 15, 25, 35, 45, and 55, respectively. The results showed the productions of pro-inflammatory mediators were increased following Se-deficiency. These data indicate the correlations between nutritional encephalomalacia and inflammatory response and the activity of inflammatory response in chicken brain may be induced by Se-deficiency.

[Study on HPLC-fingerprint-based identification of dao-di herb and non dao-di herb of scutellariae radix].

OBJECTIVE: To compare the discrepancies between chemical constituents in Dao-di herb and non Dao-di herb of Huangqin (the root of Scutellaria baicalensis), study the impact of habitat and growth pattern (including cultivated and wild Huangqin) on chemical substances of Huangqin, and then provide evidence for the identification of Dao-di herb and quality evaluation of Huangqin. METHOD: The chemical constituents in Huangqin collected from different habitats and under different growth patterns, were analyzed using HPLC fingerprint. The fingerprints obtained were then evaluated by hierarchical clustering analysis, principal component analysis and components peak area pattern. RESULT: The fingerprints' chemical profiles of Dao-di herb and non Dao-di Huangqin had significant disparity. The fingerprints of modem Dao-di herb Huangqin samples originated from Chengde (Hebei Province) were significantly different from those from other habitats, though the fingerprints of the non Dao-di Huangqin collected from Chifeng (Inner Mongolia) and Chengde had high similarity to each other. The chemical characteristics of Huangqin samples collected from the habitats recorded in ancient herbals, such as Qingyang (Gansu Province), Yan'an (Shaanxi Province), Linyi (Shangdong Province), Changzhi and Jinzhong (Shanxi Province) were similar. The fingerprints of modern non Dao-di samples collected from Dingxi and Longnan (Gansu Province) and Shangluo (Shaanxi Province) had high similarity. In addition, the content of acteoside in wild Huangqin was higher than that in cultivated Huangqin. CONCLUSION: Dao-di herb and non Dao-di herb of Huangqin could be distinguished using the developed HPLC fingerprints. The results obtained may provide evidence for the quality control and pharmcodynamical research of Dao-di herb and non Dao-di Huangqin.

Effects of oxidative stress on apoptosis in manganese-induced testicular toxicity in cocks.

Manganese (Mn) is a trace element known to be essential for maintaining the proper function and regulation of many biochemical and cellular reactions. However, little is known about the reproductive toxicity of Mn in birds. To investigate the toxicity of Mn on male reproduction in birds, 50-day-old cocks were fed either a commercial diet or a Mn-supplemented diet containing 600, 900, and 1800 mg/kg MnCl2. After being treated with Mn for 30, 60, and 90 d, the following were determined: Mn content; histological and ultrastructural changes in the testes, apoptosis; the malondialdehyde (MDA) level; the activities of superoxide dismutase (SOD); the inhibition ability of hydroxyl radicals (OH); the levels of nitric oxide (NO), nitric oxide synthase (NOS), and protein carbonyl in the testes; the DNA-protein crosslinks (DPC); and the activity of the ATP enzyme. Exposure to Mn significantly lowered the activity of SOD and glutathione peroxidase (GPx) and the inhibition ability of OH. Mn exposure also increased the levels of MDA, NO, NOS, DPC, and protein carbonyl; the number of apoptotic cells; and the Mn content and caused obvious histopathological changes in the testes. These findings suggested that Mn exposure resulted in the oxidative damage of cock testicular tissue by altering radical formation, ATP enzyme systems, apoptosis, and DNA damage, which are possible underlying reproductive toxicity mechanisms induced by Mn exposure.

Gene expression of endoplasmic reticulum resident selenoproteins correlates with apoptosis in various muscles of se-deficient chicks.

Dietary selenium (Se) deficiency causes muscular dystrophy in various species, but the molecular mechanism remains unclear. Our objectives were to investigate: 1) if dietary Se deficiency induced different amounts of oxidative stress, lipid peroxidation, and cell apoptosis in 3 skeletal muscles; and 2) if the distribution and expression of 4 endoplasmic reticulum (ER) resident selenoprotein genes (Sepn1, Selk, Sels, and Selt) were related to oxidative damages in these muscles. Two groups of day-old layer chicks (n = 60/group) were fed a corn-soy basal diet (33 µg Se/kg; produced in the Se-deficient area of Heilongjiang, China) or the diet supplemented with Se (as sodium selenite) at 0.15 mg/kg for 55 d. Dietary Se deficiency resulted in accelerated (P < 0.05) cell apoptosis that was associated with decreased glutathione peroxidase activity and elevated lipid peroxidation in these muscles. All these responses were stronger in the pectoral muscle than in the thigh and wing muscles (P < 0.05). Relative distribution of the 4 ER resident selenoprotein gene mRNA amounts and their responses to dietary Se deficiency were consistent with the resultant oxidative stress and cell apoptosis in the 3 muscles. Expression of Sepn1, Sels, and Selt in these muscles was correlated with (r > 0.72; P < 0.05) that of Sepsecs encoding a key enzyme for biosynthesis of selenocysteine (selenocysteinyl-tRNA synthase). In conclusion, the pectoral muscle demonstrated unique expression patterns of the ER resident selenoprotein genes and GPx activity, along with elevated susceptibility to oxidative cell death, compared with the other skeletal muscles. These features might help explain why it is a primary target of Se deficiency diseases in chicks.

Effect of oxygen free radicals and nitric oxide on apoptosis of immune organ induced by selenium deficiency in chickens.

Selenium is an essential element with antioxidant roles in immune regulation, but there is little understanding of how Se acts in apoptosis in the immune organs of birds. The aim of study was to evaluate the influence of Se deficiency on oxygen free radicals, NO and apoptosis in immune organ of chickens. 160 1-day-old chickens were randomly assigned to two groups of 80 each and were fed on a low-Se diet (0.032 mg/kg Se) or a control diet (0.282 mg/kg Se), respectively. OFR production in blood was determined on days 30, 45, 60 and 75, respectively. The iNOS-NO system activity in immune organ (thymus, spleen, bursa of fabricius) was identified by NO content and NOS activity assay on days 30, 45, 60 and 75, respectively. Apoptosis was measured by DNA ladder analysis, ultrastructural observations, TdT-mediated dUTP nick end labeling TUNEL assay and flow cytometric analysis of apoptotic DNA. The transcription of factor-associated suicide, caspase-3 mRNA was tested by fluorescence quantitative PCR. The results showed that OFR production, NO and inducible NO synthases (iNOS) activity in the low-Se group were significantly increased (p < 0.05) than in the control group. In addition, apoptosis was observed in chicken immune organ in the low-Se group. The degree and the number of apoptotic cells rose in a time-dependent manner. The expression of Fas and caspase-3 mRNA increased (p < 0.05) than in the control group. It indicated that the oxidative stress and NO played a causative role in the apoptosis of immune tissues induced by selenium deficiency.

Effects of manganese-toxicity on immune-related organs of cocks.

Manganese (Mn) is a trace element known to be essential for maintaining the proper function and regulation of many biochemical and cellular reactions. However, little is known about the effect of excessive amounts of Mn in immune damage of birds. The aim of this study was to investigate the effect of dietary Mn on immune damage in birds. 50-day-old male Hy-line cocks were fed either a commercial diet or a Mn-supplemented diet containing 600, 900, and 1800 mg kg(-1) MnCl(2). After being treated with Mn for 30, 60 and 90 d, the serum and immune organs (spleen, thymus, and bursa of Fabricius (BF)) were collected respectively and examined for Mn contents, activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), malondialdehyde (MDA) contents, ability to resist OH(·). In addition, DNA damage and apoptosis were observed in cock immune system treated with Mn. The results showed that the contents of Mn and MDA in immune organs and serum were increased, while the activities of SOD, GSH-Px, and ability to resist OH(·) were decreased in the Mn treatment groups. The extensive damage was observed in the immune organs. DNA single strand break and DNA-protein crosslink showed time and dosage effect in lymphocytes of immune organs. It indicated that Mn exposure resulted in oxidative damage of birds immune system by altering antioxidant defense enzyme systems, lipid peroxidation, and apoptosis.