Docosahexaenoic Acid-Acylated Astaxanthin Monoester Ameliorates Amyloid-ß Pathology and Neuronal Damage by Restoring Autophagy in Alzheimer's Disease Models.

SCOPE: Astaxanthin (AST) is ubiquitous in aquatic foods and microorganisms. The study previously finds that docosahexaenoic acid-acylated AST monoester (AST-DHA) improves cognitive function in Alzheimer's disease (AD), although the underlying mechanism remains unclear. Moreover, autophagy is reportedly involved in amyloid-ß (Aß) clearance and AD pathogenesis. Therefore, this study aims to evaluate the preventive effect of AST-DHA and elucidates the mechanism of autophagy modulation in Aß pathology. METHODS AND RESULTS: In the cellular AD model, AST-DHA significantly reduces toxic Aß1-42 levels and alleviated the accumulation of autophagic markers (LC3II/I and p62) in Aß25-35 -induced SH-SY5Y cells. Notably, AST-DHA restores the autophagic flux in SH-SY5YmRFP-GFP-LC3 cells. In APP/PS1 mice, a 3-month dietary supplementation of AST-DHA exceeded free-astaxanthin (F-AST) capacity to increase hippocampal and cortical autophagy. Mechanistically, AST-DHA restores autophagy by activating the ULK1 signaling pathway and restoring autophagy-lysosome fusion. Moreover, AST-DHA relieves ROS production and mitochondrial stress affecting autophagy in AD. As a favorable outcome of restored autophagy, AST-DHA mitigates cerebral Aß and p-Tau deposition, ultimately improving neuronal function. CONCLUSION: The findings demonstrate that AST-DHA can rectify autophagic impairment in AD, and confer neuroprotection in Aß-related pathology, which supports the future application of AST as an autophagic inducer for maintaining brain health.

Methionine Supplementation Affects Fecal Bacterial Community and Production Performance in Sika Deer (Cervus nippon).

Amino acid balance is central to improving the efficiency of feed protein utilization and for reducing environmental pollution caused by intensive farming. In previous studies, supplementation with limiting amino acids has been shown to be an effective means of improving animal nutrient utilization and performance. In this experiment, the effects of methionine on the apparent digestibility of nutrients, antler nutrient composition, rumen fluid amino acid composition, fecal volatile fatty acids and intestinal bacteria in antler-growing sika deer were investigated by randomly adding different levels of methionine to the diets of three groups of four deer at 0 g/day (CON), 4 g/day (LMet) and 6 g/day (HMet). Methionine supplementation significantly increased the apparent digestibility of organic matter, neutral detergent fiber (NDF) and acid detergent fiber (ADF) in the LMet group (p < 0.05). The crude protein and collagen protein of antlers were significantly higher in the LMet and HMet groups compared to the CON group and also significantly higher in the HMet group compared to the LMet group, while the calcium content of antlers was significantly lower in the HMet group (p < 0.05). Ruminal fluid free amino acid composition was altered in the three groups of sika deer, with significant changes in aspartic acid, citrulline, valine, cysteine, methionine, histidine and proline. At the phylum level, Firmicutes and Bacteroidetes were highest in the rectal microflora. Unidentified bacterial abundance was significantly decreased in the HMet group compared to the CON group. Based on the results of principal coordinate analysis (PCoA) and Adonis analysis, there was a significant difference in the composition of the intestinal flora between the CON and HMet groups (p < 0.05). At the genus level, compared with the CON group, the abundance of Rikenellaceae_RC9_gut_group and Lachnospiraceae_UCG-010 in the LMet group increased significantly (p < 0.05), the abundance of dgA-11_gut_group in the HMet group decreased significantly (p < 0.05) and the abundance of Lachnospiraceae_UCG-010, Saccharofermentans and Lachnospiraceae_NK3A20_group increased significantly. Taken together, the results showed that methionine supplementation was beneficial in increasing the feed utilization efficiency and improving antler quality in sika deer, while affecting the composition of fecal bacteria.

Docosahexaenoic acid-acylated astaxanthin monoester ameliorates chronic high-fat diet-induced autophagy dysfunction via ULK1 pathway in the hypothalamus of mice.

BACKGROUND: Dietary astaxanthin (AST) exhibits the ability to resist lipid accumulation and stimulate hepatic autophagy. Natural AST predominantly exists in stable esterified forms. More importantly, in our previous study, docosahexaenoic acid-acylated AST monoester (AST-DHA) possessed better stability, bioavailability, and neuroprotective ability than AST in free and diester form. However, the AST-DHA mechanisms of action in regulating the obese phenotype and autophagy of the central nervous system remain unclear. RESULTS: High-fat diet (HFD)-fed C57BL/6J mice were orally administered AST-DHA (50 mg/kg body weight/d) for 3 days or 8 weeks. AST-DHA supplementation alleviated HFD-induced abnormal body weight gain, significantly enhanced autophagy with an increased microtubule-associated protein light chain 3 II/I (LC3II/I) ratio, and reduced the accumulation of p62/sequestosome 1 (SQSTM1) in the hypothalamus rather than in the hippocampus. Mechanistically, AST-DHA effectively promoted autophagy and autophagosome formation, and most notably rescued the HFD-impaired autophagosome-lysosome fusion (indicated by the colocalization of LC3 and LAMP1) by regulating mTOR- and AMPK-induced phosphorylation of ULK1. Consequently, AST-DHA enhanced hypothalamic autophagy, leading to pro-opiomelanocortin (POMC) cleavage to produce alpha-melanocyte-stimulating hormone (α-MSH). CONCLUSIONS: This study identified AST-DHA as an enhancer of autophagy that plays a beneficial role in restoring hypothalamic autophagy, and as a new potential therapeutic agent against HFD-induced obesity. © 2023 Society of Chemical Industry.

Hepatoprotective effects of sea cucumber ether-phospholipids against alcohol-induced lipid metabolic dysregulation and oxidative stress in mice.

Sea cucumber is widely consumed as food and folk medicine in Asia, and its phospholipids are rich sources of dietary eicosapentaenoic acid enriched ether-phospholipids (ether-PLs). Emerging evidence suggests that ether-PLs are associated with neurodegenerative disease and steatohepatitis. However, the function and mechanism of ether-PLs in alcoholic liver disease (ALD) are not well understood. To this end, the present study sought to investigate the hepatoprotective effects of sea cucumber ether-PLs, including plasmenyl phosphatidylethanolamine (PlsEtn) and plasmanyl phosphatidylcholine (PlsCho), and their underlying mechanisms. Our results showed that compared with EtOH-induced mice, ether-PL treated mice showed improved liver histology, decreased serum ALT and AST levels, and reduced alcohol metabolic enzyme (ALDH2 and ADH1) expressions. Mechanistic studies showed that ether-PLs attenuated "first-hit" hepatic steatosis and lipid accumulation evoked by alcohol administration. Moreover, PlsEtn more effectively restored endogenous plasmalogen levels than PlsCho, thereby enhancing hepatic antioxidation against "second-hit" reactive oxygen species (ROS) due to the damaged mitochondria and abnormal ethanol metabolism. Taken together, sea cucumber ether-PLs show great potential to become a natural functional food against chronic alcohol-induced hepatic steatosis and lipid metabolic dysregulation.

Effect and neural mechanisms of the transcutaneous vagus nerve stimulation for relapse prevention in patients with remitted major depressive disorder: protocol for a longitudinal study.

INTRODUCTION: After the first episode, patients with remitted major depressive disorder (MDD) have a 60% chance of experiencing a second episode. There are currently no accepted, effective methods to prevent the recurrence of MDD in remission. Transcutaneous vagus nerve stimulation (taVNS) is a non-invasive, safe and economical approach based on the efficacy of VNS in improving clinical depression symptoms. This clinical trial will study the efficacy of taVNS in preventing MDD relapse and investigate the underlying mechanisms of this. METHODS AND ANALYSIS: We will conduct a multicentre, randomised, patient-blinded and evaluators double-blinded trial. We will randomise 90 eligible participants with recurrent MDD in remission in a 1:1 ratio into a real or sham taVNS group. All participants will be given six biopsychosocial assessments: proinflammatory cytokines, serum monoamine neurotransmitters, cognition, affective neuropsychology, multimodal neuroimaging and endocrinology. After the baseline measurements, all participants will be given corresponding interference for 6 months and then complete a 1-year follow-up. The assessments will be performed three times: at baseline, post-treatment and at the end of 1-year follow-up (except for multimodal MRI scanning, which will be conducted at the first two assessments only). Change in 17-item Hamilton Depression Rating Scale scores for MDD is the primary outcome parameter. ETHICS AND DISSEMINATION: The study protocol was approved by the Medical Ethical Committee of Beijing Hospital of Traditional Chinese Medicine on 18 January 2019 (2018BL-076). The trial results will be published in peer-reviewed journals and at conferences. TRIAL REGISTRATION NUMBER: ChiCTR1900022618.

Threshold effects and interactive effects of total zinc and selenium intake on cognitive function in older adults.

BACKGROUND & AIMS: The aim of this study was to assess threshold effects and interactive effects of total zinc and selenium intake on cognitive function in older adults. METHODS: We used data from the National Health and Nutrition Examination Survey (NHANES) 2011-2014. Zinc and selenium intake were obtained through two 24-h dietary recalls. Cognitive performance was evaluated by the Digit Symbol Substitution Test (DSST). Smooth curve fitting, two-piecewise multivariable linear regression models, binary logistic regression model, multiplicative interactions model, and additive interactions model were used to evaluate the association between zinc, selenium intake and their interactive effect on cognitive function. RESULTS: A total of 2450 participants aged 60 years or older were included. Zinc and selenium intake was non-linearly associated with cognitive function. The inflection point for zinc intake was 8.94 mg/d in males and 7.58 mg/d in females. When zinc intake was below inflection point, zinc intake was positively associated with the DSST test in males (ß = 1.02, 95% CI, 0.44 to 1.60) and females (ß = 0.94, 95% CI, 0.26 to 1.62). When zinc intake above inflection point, there is no association between zinc intake and the DSST test in both sexs. The inflection point for selenium intake was 186.33 µg/d in males and 68.40 µg/d in females. Among males, the ß (95% CIs) was 0.03 (0.01,0.06) to the left side of the inflection point and -0.06 (-0.10, -0.02) to the right of the inflection point. Among females, the ß (95% CIs) was 0.13 (0.04,0.22) to the left side of the inflection point and 0.01 (-0.01,0.04) to the right of the inflection point. Besides, zinc and selenium have significant interaction on DSST test only in females (P = 0.028, RERI = 0.418). CONCLUSIONS: The present study demonstrated that zinc and selenium intake was non-linearly associated with cognitive function in different sex. There was an interactive effect between zinc and selenium intake on improving cognitive function, especially in females.

Artesunate attenuates atherosclerosis by inhibiting macrophage M1-like polarization and improving metabolism.

OBJECT: Atherosclerosis (AS) is caused by chronic inflammation. Artesunate (ART), a sesquiterpene lactone endoperoxide isolated from Chinese herbal medicine, displays excellent anti-inflammatory activity. In this study, we investigated the effects of artesunate on atherosclerosis in ApoE knock-out mice, and used untargeted metabolomics to determine metabolite changes in these mice following ART treatment. METHODS: ApoE knock-out mice were fed a western diet and administered ART for eight weeks. Untargeted metabolomics was used to detect differential metabolites following the administration of ART. Oil Red O was used to assess plaque size, western blot and ELISA were used to detect inflammatory factors, and flow cytometry was used to detect the expression of markers on macrophages. RESULTS: Results of the in vivo experiment suggested that ART reduced atherosclerotic plaques in murine aortic root. In addition both in vivo and vitro experiments suggested that ART reduced the expression levels of inflammating cytokines, but enhanced those of the anti-inflammatory cytokines in macrophages. Untargeted metabolomic analysis demonstrated that multiple metabolic pathways, which were blocked in AS mice, showed different degrees of improvement following ART treatment. Furthermore, bioinformatic analyses showed that the HIF-1α pathway was altered in the AS mice and the ART treatment mice. In vitro experiments confirmed that LPS-induced upregulation of HIF-1α expression and activation of the NF-κB signaling pathways was significantly inhibited by ART treatment. CONCLUSION: These results suggest that ART exerts anti-atherosclerosis effects by inhibiting M1 macrophage polarization. One of the molecular mechanisms is that ART inhibits M1-like macrophage polarization via regulating HIF-1α and NF-κB signaling pathways.

Lipidomics Approach in High-Fat-Diet-Induced Atherosclerosis Dyslipidemia Hamsters: Alleviation Using Ether-Phospholipids in Sea Urchin.

Ether-phospholipids (ether-PLs) in sea urchins, especially eicosapentaenoic-acid-enriched plasmenyl phosphatidylethanolamine (PE-P) and plasmanyl phosphatidylcholine (PC-O), exhibit potential lipid-regulating effects. However, their underlying regulatory mechanisms have not yet been elucidated. Herein, we integrated an untargeted lipidomics strategy and biochemical analysis to investigate these mechanisms in high-fat-induced atherosclerotic hamsters. Dietary supplementation with PE-P and PC-O decreased total cholesterol and low-density lipoprotein cholesterol concentrations in serum. The lipid regulatory effects of PE-P were superior to those of PC-O. Additionally, 20 lipid molecular species, including phosphatidylethanolamine, cholesteryl ester, triacylglycerol, and phosphatidylinositol, were identified as potential lipid biomarkers in the serum of hamsters with PC-O and PE-P treatment (95% confidence interval; p < 0.05). The variations of lipids may be attributed to downregulation of adipogenesis genes and upregulation of lipid ß-oxidation genes and bile acid biosynthesis genes. The improved lipid homeostasis by ether-PLs in sea urchins might be a key pathway underlying the antiatherosclerosis effect.

Activating soil microbial community using bacillus and rhamnolipid to remediate TPH contaminated soil.

Soil petroleum contamination has become a global environmental problem. In order to develop a new soil remediation technology, this study established bacteria isolation, surfactant toxicity matching and petroleum contaminated soil remediation practice. The simulated field remediation showed that inoculating the soil with Bacillus methylotrophicus and adding 500 mg kg-1 rhamnolipid (N + RL) to soil can remove 80.24% of aged total petroleum hydrocarbons (TPHs) within 30 days. In particular, although the remediated soil has inoculated sufficient bacterial suspension, the microbial abundance of Bacillus was not a significantly dominant genus after remediation, especially in N + RL (0.73% of the total), but the colonies of indigenous petroleum-degrading bacteria (such as Massilia and Streptomyces) increased significantly. The interaction among genera has been further proved to drive soil non-specific oxidases (such as polyphenol oxidase, laccase and catalase) to remove TPHs. This indicates that the interaction among microorganisms, rather than the degradability of exogenous degrading bacteria, plays more critical role in the degradation of organic pollutants, which enriches the traditional understanding of micro-remediation of contaminated soil. It can be concluded from the obtained results that the remediation of pollutants can be achieved by adjusting the purification capacity of the microbial community and the natural environment.

Yangxue Jiedu Fang Ameliorates Psoriasis by Regulating Vascular Regression via Survivin/PI3K/Akt Pathway.

BACKGROUND: Psoriasis (PA) is a chronic autoimmune disease of the skin that adversely affects patients' quality of life. Yangxue Jiedu Fang (YXJD) has been used for decades to treat psoriasis in China. However, its antipsoriatic mechanisms are still poorly understood. In this study, we explored the effects of YXJD on angiogenesis and apoptosis of microvessels in PA, the underlying mechanisms in HUVEC cells transfected by Survivin overexpression plasmid and in a mouse model of imiquimod-induced psoriasis and the relationship between VEGF (vascular endothelial growth factor) and Survivin. METHODS: A BALB/c mouse model of imiquimod- (IMQ-) induced PA was established, and the mice were treated with YXJD. Cell viability was assessed by CCK8 assay. Apoptosis was detected by annexin V-FITC/PI double-staining and caspase-3 assays. The PI3K/Akt/ß-catenin pathway was analyzed by western blotting, ELISA, and immunochemical analysis. RESULTS: YXJD ameliorated symptoms and psoriasis area and severity index (PASI) scores and also reduced the number of microvessels, as determined by the microvessel density (MVD). The expression of apoptotic protein Survivin in endothelial cells, autophagy-related proteins p62, and angiogenic proteins VEGF was inhibited by YXJD, and the repressed expression of LC3II/I increased by YXJD. The proteins related to the PI3K/Akt pathway and ß-catenin expression and the nuclear entry of ß-catenin were reduced in IMQ-induced PA mice treated with YXJD. In HUVEC cells transfected by Survivin overexpression plasmid, we observed YXJD regulated the expression of Survivin, LC3II/I, and p62, VEGF, and PI3K/Akt pathway-relative proteins and the nuclear entry of ß-catenin. CONCLUSIONS: YXJD inhibited the expression of Survivin via PI3K/Akt pathway to adjust apoptosis, autophagy, and angiogenesis of microvessels and thus improve the vascular sustainability in psoriasis. YXJD may represent a new direction of drug research and development for immunomodulatory therapy for psoriasis.

Effects of Dietary Supplementation with Selenium and Vitamin E on Growth Performance, Nutrient Apparent Digestibility and Blood Parameters in Female Sika Deer (Cervus nippon).

To evaluate the effects of selenium (Se) and vitamin E (Vit E) on female sika deer. This study was conducted using a 3 × 2 + 1 factorial experiment. Depending on treatment design, the deer were fed with the basal diet supplemented with 0.2, 0.3, and 0.4 mg of selenium as well as 100 and 200 IU of vitamin E per kg of dry matter (DM). Accordingly, six groups named G1 to G6 are involved in this study. In addition, group G0 was available in the study, in which the deer were fed with only basal diet. The results show that the final body weight (BW), average daily gain (ADG), and apparent digestibility of crude protein, ether extract, and neutral detergent fiber of the deer in G1 to G6 increased as the selenium level increased from 0.2 to 0.3 mg per kg of DM (P < 0.05). Higher IgG content of the deer was observed with the intake of selenium and vitamin E (P < 0.05). The total content of protein of the deer in G3 was higher than that in G0 (P < 0.05), and the activity of glutathione peroxidase increased following the increase in the supplementation levels of selenium and vitamin E (P < 0.05). Furthermore, selenium had significant effects on the concentration of T4 and T3 (P < 0.05). The optimum levels of selenium and vitamin E for 1-year-old female sika deer were 0.3 mg and 100 IU per kg of dietary DM, respectively.

In situ sodium persulfate/calcium peroxide oxidation in remediation of TPH-contaminated soil in 3D-sand box.

The aim of this article was to obtain the application parameters and conditions of in situ sodium persulfate/calcium peroxide oxidation. For the purposes of remediation, soil from a total petroleum hydrocarbons (TPH)-contaminated site was collected and prepared to reflect the actual stratum condition in a newly developed soil remediation modeling apparatus. Application methods of soil mixture, natural infiltration, direct injection and groundwater circulation were used to simulate in situ sodium persulfate oxidation in TPH-contaminated soil. Results showed that the transfer capability of Na2S2O8 in simulated soil was strong Na2S2O8 migrated to the saturated layer after 3 days of in situ injection, which then continued both horizontal and vertical migration. After 7 days the oxidant was widespread in the saturated layer with a radius of influence of 0.4 m. It was found that mixing CaO2/Fe2+/CA with soil and spraying Na2S2O8 can effectively repair the surface-contaminated soil, and the longitudinal migration of Na2S2O8 in the reaction process can further strengthen the remediation of the upper layer soil. Due to the buffering effect of the soil, the effect of oxidation on the pH and temperature of different soil layers was small, but detectable in comparison to natural environmental factors.

Effects of dietary manganese supplementation on nutrient digestibility and production performance in male sika deer (Cervus Nippon).

This study aimed to investigate the effects of manganese (Mn) supplementation on nutrient digestibility and antler growth in male sika deer. Twenty 3-year-old male sika deer were divided into four groups (Control, T40, T80 and T120). The control diet contained 22.2 mg Mn/kg. The treatment diets were supplemented with 40, 80 and 120 mg of Mn/kg (provided as Mn methionine). The results showed that the digestibility of dry matter and crude protein in the T80 group was greater than other groups. Higher digestibility of neutral detergent fiber and acid detergent fiber was observed in the T80 group than in the control group. Plasma Mn concentrations in the T80 and T120 groups were higher (P < 0.01) than control and T40 group (P < 0.01). The concentration of Mn in feces was increased with increasing Mn amount. Average daily gain of fresh antler and dry antler of T80 was greater than other groups, especially the control group (P < 0.05). In conclusion, the suitable level of Mn supplementation was found to be 80 mg/kg (total Mn content 103.4 mg/kg dry matter), which significantly increased antler daily gain and feed digestibility in 3-year-old sika deer.

[Pulsed electric fields inhibit tumor growth but induce myocardial injury of melanoma-bearing mice].

Objective To investigate the tumor inhibiting effect of pulsed electric fields (PEFs) on melanoma-bearing mice, and understand its influence on myocardial cells and cardial electrical activity. Methods The melanoma models of the BALB/c mice were established by subcutaneously injecting B16 melanoma cells. These mice were randomly divided into four groups. The treated groups received pulsed electric stimulation at pulse width of 1, 3, 5 ms, with field strength of 100 V/cm and frequency of 10 Hz for 10 minutes daily in 15 consecutive days. ECG of mice was recorded. Tumor volume was measured with vernier caliper. Morphological changes of tumors were observed by HE staining. The expression of proliferating cell nuclear antigen (PCNA) mRNA was tested by real-time quantitative PCR, and the expression of PCNA protein was detected by immunofluorescence histochemistry. The ultrastructural changes of the cardiac tissues were observed by transmission electron microscopy (TEM). The serum levels of cardial troponin T (cTnT) and creatine kinase isoenzyme MB (CK-MB) were detected by ELISA. Results Compared with the control group, tumor volumes in all treated groups were reduced 7 days after PEFs treatment; more melanin granules in tumor cells were found in the treated groups; the expressions of PCNA mRNA and protein were down-regulated in all treated groups, and there were greater changes in the groups receiving the bigger pulse width. Myocardial injury was found in 3 ms group and 5 ms group, and the expressions of cTnT and CK-MB were significantly higher than those in the control group. Conclusion PEFs can inhibit tumor growth in melanoma-bearing mice. Increase of pulse width will aggravate myocardial injury.

[Antitumor effect of low-voltage electric pulses on melanoma-bearing mice].

OBJECTIVE: To investigate the antitumor effect of the low-voltage electric pulses (LVEPs) on melanoma-bearing mice. METHODS: Tumor models were established by subcutaneously inoculating BALB/c mice with B16F10 melanoma cells. These mice were randomly divided into control group and treated groups which were exposed to electric pulses of different frequency 10, 20, 30 Hz for consecutive 20 days with 30 minutes per day. In every day, tumor size was measured, and morphological changes of tumors were observed by HE staining. The expressions of caspase-3 and matrix metalloproteinase-9 (MMP-9) mRNAs were tested by real-time quantitative PCR, and the expressions of caspase-3 and MMP-9 proteins were detected by immunohistochemistry. RESULTS: Compared with the control group, seven days after LVEPs treatment, the tumor size in 30Hz treated group was reduced significantly; twenty days after treatment, the tumor size of all treated groups diminished significantly. The tumor growth inhibition rate increased with time. Compared with the control group, nuclear shrinkage and necrosis of more tumor cells were found in the treated groups; the expression of caspase-3 mRNA and protein was significantly enhanced in the treated groups; the expression of MMP-9 mRNA and protein was significantly depressed in the treated groups; and the changes of caspase-3 and MMP-9 expressions became greater with the frequency increasing. CONCLUSION: LVEPs can induce the activation of caspase-3, suppress the expression of MMP-9, and inhibit tumor growth.

Effect of subchronic exposure to arsenic on levels of essential trace elements in mice brain and its gender difference.

The interactions of toxic metals with essential metals may result in disturbances in the homeostasis of essential elements. However, there are few reports about toxic effect of arsenic (As) on the levels of essential trace elements in the central nervous system. To investigate whether subchronic exposure to As disturbs levels of main essential trace elements in the brain of mice and whether the gender difference in the response to As are altered, the concentrations of As, Iron (Fe), copper (Cu), selenium (Se), zinc (Zn) and Chromium (Cr) in the cerebrum and cerebellum of mice exposed to As subchronically were examined by inductively coupled plasma-mass spectrometry (ICP-MS). The gender difference in the changed levels of these essential trace elements was also statistically analyzed. The concentration of As was significantly higher in the cerebrum or cerebellum of mice exposed to As than that in control group (P < 0.05). It indicates that As can accumulate in brain of mice after subchronic exposure. The concentrations of Fe, Se and Cr in the cerebrum or cerebellum were significantly lower in mice exposed to As than those in control group (P < 0.05). On the contrary, the concentration of Cu in the cerebrum or cerebellum was significantly higher in mice exposed to As (P < 0.05). Our results indicate that subchronic exposure to As may decrease the levels of Fe, Se and Cr or increase the level of Cu in the brain of mice. Moreover, the significant gender difference was found relative to the effect of As on concentration of Se in cerebrum and concentrations of Cu and Se in cerebellum of mice. Therefore, more experiments are required to further understand mechanisms whereby As interacts with essential elements in brain and induces the gender difference.

Effects of copper on proliferation and autocrine secretion of insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 (IGFBP-3) in chondrocytes from newborn pigs in vitro.

Chondrocytes from the lateral trochlear ridge of the distal femur taken from 1-day-old piglets were cultured in medium supplemented with 0, 7.8, 15.6, 31.2, and 62.5 µmol/L copper. Insulin-like growth factor-1 (IGF-1) and IGF-binding protein 3 (IGFBP-3) levels in culture medium were determined by radioimmunoassay. DNA synthesis in chondrocytes was measured by tritiated thymidine ((3)H-TdR) incorporation. Proliferation-promoting activity and incorporation of (3)H-TdR in chondrocytes were increased in all culture media supplemented with copper and 15% fetal calf serum (FCS). The contents of IGF-1 and IGFBP-3 were also enhanced significantly in culture media containing 15% FCS and supplemented with copper at 15.6, 31.2, and 62.5 µmol/L. The optimal copper concentration for promoting chondrocyte proliferation and autocrine secretion of IGF-1 and IGFBP-3 was 31.2 µmol/L.

Effect of copper on the expression of TGF-ß in incubated chondrocytes of newborn pigs.

This experiment was conducted to measure the effect of copper supplementation on TGF-ß gene expression in chondrocytes of newborn pigs. Chondrocytes were cultured in media containing 15% fetal calf serum supplemented with 0, 15.6, 31.2, and 62.5 µmol/L copper in 90-mm culture plate. Total RNA was isolated from chondrocytes, and TGF-ß cDNA was synthesized, amplified, and sequenced. The expression level of TGF-ß was examined by reverse transcription polymerase chain reaction. The results showed that the sequence of the cloned TGF-ß gene was 99.4% identical to that in GenBank. The expression of TGF-ß increased in culture media added with final concentration of 15.6, 31.2, and 62.5 µmol/L copper. In this study, the optimal copper concentration and optimal culture time for the highest level of TGF-ß expression were 31.2 µmol/L and 48 h, respectively.

Effect of high dietary copper on somatostatin and growth hormone-releasing hormone levels in the hypothalami of growing pigs.

This experiment was conducted to examine the effect of dietary copper supplementation on somatostatin (SS) and growth hormone-releasing hormone (GHRH) mRNA expression levels in the hypothalami of growing pigs. A total of 45 crossbred pigs were randomly assigned to three groups of 15 pigs each; five replicates of three animals comprised each group. Pigs were allocated to diets that contained 5 mg/kg copper (control), 125 mg/kg copper sulfate, or 125 mg/kg copper methionine. At the end of the experiment, five pigs were selected at random from each group and slaughtered, and hypothalami were collected for determination of SS and GHRH mRNA expression levels. The results showed that the SS expression levels were lower and the GHRH levels were higher in pigs fed the diets with 125 mg/kg copper methionine (P<0.05) and 125 mg/kg copper sulfate (P<0.05), respectively, than in pigs fed the diet with 5 mg/kg copper. Furthermore, the relationship between SS mRNA and GHRH mRNA abundance had a significantly negative correlation (P<0.05). The data indicated that high dietary copper could enhance GHRH mRNA expression levels and suppress SS mRNA expression levels in the hypothalami of pigs. High lever dietary copper (125 mg/kg copper sulfate or 125 mg/kg copper methionine) increased pigs' growth performance and feed efficiency but had no significant effect on daily feed intake; 125 mg/kg copper sulfate or 125 mg/kg copper methionine at the same lever had no difference on growth promoting in pigs.

[Impacts on nutrient export by landscape heterogeneity based on sub-watershed].

Landscape features of a watershed are important factors affecting non-point source (NPS) pollution. Sub-watershed bounds were delineated and landscape heterogeneity was analyzed based on GIS and RS in Xitiaoxi watershed which located the upper reach of Taihu Lake area. Nutrient export intensity of sub-watersheds was estimated by revised export coefficient model. Then the relationships between nutrient export and main landscape types, as well as Shannon diversity index (SHDI) in sub-watershed units were analyzed. Results show, TN and TP export intensity have obvious spatial difference, which changed from 3.01 kg/(hm2 x a) to 15.44 kg/(hm2 x a) and 0.049 kg/(hm2 x a) to 0.355 kg/(hm2 x a) respectively. The dominated landscape types including cultivated land and forest land quantitatively related with nutrient export intensity. TN and TP export intensity will decrease 0.203 1 kg/(hm2 x a) and 0.0152 kg/(hm2 x a) respectively with 10% increased of forest area, and will increase 0.5726 kg/(hm2 x a) and 0.0273 kg/(hm2 x a) with 10% increased of cultivated land area. The relationship between nutrient export intensity and SHDI exhibited second-degree polynomial, export intensity increased by SHDI increasing and to maximum when SHDI equals 1.5, then decreased with SHDI increasing. This research results will provide an important reference value for NPS management.