Quantitative fusion omics reveals that refrigeration drives methionine degradation through perturbing 5-methyltetrahydropteroyltriglutamate-homocysteine activity.

Postharvest senescence and quality deterioration of fresh tea leaves occurred due to the limitation of processing capacity. Refrigerated storage prolongs the shelf life of fresh tea. In this study, quantitative fusion omics delineated the translational landscape of metabolites and proteins in time-series (0-12 days) refrigerated tea by UHPLC-Q-Orbitrap HRMS. Accurate quantification results showed the content of amino acids, especially l-theanine, decreased with the lengthening of the storage duration (15.57 mg g-1 to 7.65 mg g-1) driven by theanine synthetase. Downregulation of enzyme 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase expression led to methionine degradation (6.29 µg g-1 to 1.78 µg g-1). Refrigerated storage inhibited serine carboxypeptidase-like acyltransferases activity (59.49 % reduction in 12 days) and induced the polymerization of epicatechin and epigallocatechin and generation of procyanidin dimer and δ-type dehydrodicatechin, causing the manifestation of color deterioration. A predictive model incorporating zero-order reaction and Arrhenius equation was constructed to forecast the storage time of green tea.

Golgi-localized putative S-adenosyl methionine transporters required for plant cell wall polysaccharide methylation.

Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, so far, no Golgi SAM transporter has been identified in plants. Here we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knockout of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesized polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, NMR revealed the existence of pectin 'egg-box' structures in intact cell walls and showed that their formation is enhanced by reduced methyl esterification. These changes in wall architecture were linked to substantial growth and developmental phenotypes. In particular, anisotropic growth was strongly impaired in the double mutant. The identification of putative transporters involved in import of SAM into the Golgi lumen in plants provides new insights into the paramount importance of polysaccharide methylation for plant cell wall structure and function.

Effects of rumen-protected methionine supplementation on the performance of high production dairy cows in the tropics.

Increasing methionine availability in dairy cow diets during the first third of lactation may enhance their performance and health. The aim of this study was to determine the effect of supplementing rumen-protected methionine (Smartamine® M, SM) in a lactation diet with protein and energy levels calculated according to the literature. Seventy-six multiparous Holstein cows (39.1 ± 6.8 kg of milk/d and 65 ± 28 DIM) were assigned to 1 of 2 dietary treatments (38/treatment) according to a randomized complete block design with a 2-wk (covariate) and 10-wk experimental period. Treatments were a basal diet (CON; 3.77 Lys:1Met); and CON + 23 g SM (2.97 Lys:1 Met). Individual milk samples were taken every 2 weeks to determine milk composition. Blood was collected from 24 cows on d+30 d to measure plasma AA levels. Body weight and body condition score (BCS) were measured at the beginning and the end of the experiment. The SM diet promoted higher milk yield (41.7 vs. 40.1 kg/d; P = 0.03). Energy-corrected milk yield (41.0 vs. 38.0 kg/d), milk protein yield (1.30 vs. 1.18 kg/d), milk protein (3.14% vs. 2.97%) and casein (2.39% vs. 2.28%) were also different (P < 0.01) as well as milk fat yield (1.42 vs. 1.29 kg/d; P = 0.02). A trend (P = 0.06) for higher milk fat % (3.41% vs. 3.21%) was observed. Both diets resulted in similar body weight, but CON-fed cows tended (P = 0.08) to have higher BCS. Higher plasma methionine levels were determined with SM compared with CON (29.6 vs. 18.4 µM; P < 0.01), but lysine and histidine were not different. Dietary supplementation of RPM improved productive performance by increasing milk yield and milk components yields, suggesting better dietary AA utilization when Met levels are adjusted in Lys-adequate lactation diets.

Yellow loosestrife (Lysimachia vulgaris var. davurica) ameliorates liver fibrosis in db/db mice with methionine- and choline-deficient diet-induced nonalcoholic steatohepatitis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH), a liver disease caused by a nonalcoholic fatty liver, is increasing in incidence worldwide. Owing to the complexity of its pathogenic mechanisms, there are no therapeutic agents for this disease yet. The ideal drug for NASH needs to concurrently decrease hepatic lipid accumulation and exert anti-inflammatory, antifibrotic, and antioxidative effects in the liver. Because of their multipurpose therapeutic effects, we considered that medicinal herbs are suitable for treating patients with NASH. METHODS: We determined the efficacy of the alcoholic extract of Lysimachia vulgaris var. davurica (LV), an edible medicinal herb, for NASH treatment. For inducing NASH, C57BLKS/J lar-Leprdb/Leprdb (db/db) male mice were fed with a methionine-choline deficient (MCD) diet ad libitum. After 3 weeks, the LV extract and a positive control (GFT505) were administered to mice by oral gavage for 3 weeks with a continued MCD diet as needed. RESULTS: In mice with diet-induced NASH, the LV extract could relieve the disease symptoms; that is, the extract ameliorated hepatic lipid accumulation and also showed antioxidative and anti-inflammatory effects. The LV extract also activated nuclear factor E2-related factor 2 (Nrf2) expression, leading to the upregulation of antioxidants and detoxification signaling. Moreover, the extract presented remarkable efficacy in alleviating liver fibrosis compared with GFT505. This difference was caused by significant LV extract-mediated reduction in the mRNA expression of fibrotic genes like the alpha-smooth muscle actin and collagen type 3 alpha 1. Reduction of fibrotic genes may thus relate with the downregulation of transforming growth factor beta (TGFß)/Smad signaling by LV extract administration. CONCLUSIONS: Lipid accumulation and inflammatory responses in the liver were alleviated by feeding LV extract to NASH-induced mice. Moreover, the LV extract strongly prevented liver fibrosis by blocking TGFß/Smad signaling. Hence, LV showed sufficient potency for use as a therapeutic agent against NASH.

Use of alternative protein sources for fishmeal replacement in the diet of largemouth bass (Micropterus salmoides). Part II: effects of supplementation with methionine or taurine on growth, feed utilization, and health.

Fishmeal has long been a staple protein feedstuff for fish, but its global shortage and high price have prompted its replacement with alternative sustainable sources. In this experiment involving largemouth bass (a carnivorous fish), a new mixture of feedstuffs (45% poultry byproduct meal, 30% soybean meal, 15% blood meal, and 10% krill shrimp meal) was added to low (14.5%) fishmeal diets along with 0.0%, 0.5% taurine, 0.5% methionine, or 0.5% taurine plus 0.5% methionine (dry matter basis). The positive control diet [65.3% fishmeal (46% crude protein on dry matter basis)] and all low-fishmeal diets contained 40% true protein and 10% lipids. There were 3 tanks per treatment group (20 fish/tank). Fish with the mean initial body weight of 16.6 g were fed to satiety twice daily. Compared with the unsupplemented low-fishmeal group, supplementing either 0.5% methionine or 0.5% methionine plus 0.5% taurine to the low-fishmeal diet improved (P < 0.05) the growth, feed utilization, retention of dietary protein and lipids, and health of largemouth bass, reduced (P < 0.05) the occurrence of black skin syndrome from ~ 40 to ~ 10%. Histological sections of tissues from the fish with black skin syndrome showed retina degeneration, liver damage, and enteritis in the intestine. Compared with methionine supplementation, supplementing 0.5% taurine alone to the low-fishmeal diet did not affect the growth or feed efficiency of fish and had less beneficial effects (P < 0.05) on ameliorating the black skin syndrome. These results indicated that: (a) the basal low-fishmeal diet was inadequate in methionine or taurine; and (b) dietary supplementation with methionine was an effective method to improve the growth performance, feed efficiency, and health of largemouth bass. Further studies are warranted to understand the pathogenesis of the black skin syndrome in largemouth bass.

Citrus reticulata Blanco peel extract ameliorates hepatic steatosis, oxidative stress and inflammation in HF and MCD diet-induced NASH C57BL/6 J mice.

Excessive lipid deposition, oxidative stress and inflammation in liver tissues are regarded as crucial inducers of nonalcoholic steatohepatitis (NASH), which is the most frequent chronic liver disease and closely related to obesity and insulin resistance. In this work, the preventive and therapeutic effects of Citrus reticulata Blanco (Jizigan) peel extract (JZE) on NASH induced by high fat (HF) diet and methionine choline-deficient (MCD) diet in C57BL/6 mice were investigated. We found that daily supplementation of JZE with an HF diet effectively ameliorated glucose tolerance and insulin resistance. In addition, the key indexes of lipid profiles, oxidative stress, hepatic steatosis and inflammatory factors were also ameliorated in both NASH mouse models. Furthermore, JZE treatment activated nuclear factor erythroid-2-related factor 2 (Nrf2) in the livers of diet- induced NASH mice. Our study suggests that JZE might alleviate NASH via the activation of Nrf2 signaling and that citrus Jizigan could be used as a dietary therapy for NASH and related metabolic syndrome.

Maternal supplementation of energy and protein, but not methionine hydroxy analog, enhanced postnatal growth and response to vaccination in Bos indicus-influenced beef offspring.

A 2-yr study evaluated the growth and postvaccination immune response of beef calves born from heifers offered no supplementation or pre- and postpartum supplementation of sugarcane molasses + urea with or without methionine hydroxy analog (MHA). On day 0 of each year (57 ± 5 d prepartum), Brangus crossbred beef heifers (n = 36/yr; 20 to 22 mo of age) were stratified by their initial body weight (BW; 396 ± 24.1 kg) and body condition score (BCS; 5.6 ± 0.43) and randomly allocated into 1 of 12 bahiagrass (Paspalum notatum) pastures (3 heifers/pasture). Treatments were randomly assigned to pastures (4 pastures/treatment/yr) and consisted of no supplementation (NOSUP) and supplementation of sugarcane molasses + urea (7.2 kg of DM/heifer/wk) with (MOL+) or without (MOL-) fortification with 105 g/heifer/wk of MHA. Treatments were provided from 57 ± 5 d prepartum until 17 ± 5 d postpartum (day 0 to 74). On day 74, all heifer-calf pairs were combined and managed as a single group until the end of the breeding season (day 237). Calves were early weaned at 89 ± 5 d of age (day 147), limit-fed at 3.5% of BW (DM basis) in drylot until day 201, and vaccinated against respiratory disease pathogens on days 160 and 188. Prepartum BCS on day 44 did not differ (P = 0.26) between MOL+ and MOL- heifers but both groups had greater (P < 0.0001) BCS than NOSUP heifers. Plasma concentrations of l-methionine on day 44 were the greatest (P ≤ 0.04) for MOL+ heifers and did not differ (P = 0.40) between NOSUP vs. MOL- heifers. Calf birth BW did not differ (P = 0.13) among treatments. Calf average daily gain (ADG) from birth to day 201 did not differ (P ≥ 0.17) between MOL+ vs. MOL- calves, but both groups had greater (P ≤ 0.05) ADG from birth to day 201 than NOSUP calves. Calf postvaccination plasma concentrations of glucose, cortisol, and haptoglobin did not differ among treatments (P ≥ 0.13). However, plasma concentrations of IGF-1 on day 167 and the overall positive vaccine seroconversion did not differ (P ≥ 0.18) between MOL- and MOL+ calves, but both were greater (P ≤ 0.04) compared with NOSUP calves. Hence, maternal supplementation of sugarcane molasses + urea increased BCS at calving and offspring BW gain and response to vaccination against respiratory pathogens compared with no maternal supplementation. MHA inclusion into maternal supplements effectively increased maternal plasma l-methionine concentrations but did not enhance maternal BCS at calving and offspring growth and postvaccination immune response.

The effects of betaine supplementation in diets containing different levels of crude protein and methionine on the growth performance, blood components, total tract nutrient digestibility, excreta noxious gas emission, and meat quality of the broiler chickens.

The effects of betaine supplementation on growth performance, blood components, nutrient digestibility, excreta noxious gas emission, and meat quality of broiler chickens were examined using different dietary crude protein (CP) and methionine (Met) levels. A total of 768 Ross 308 broiler chickens were allotted to four treatments, with 12 replications of each treatment conducted over 6 wk. Treatments were factorially designed, with 2 levels of CP [Starter: CP 21% (low Met) and 23% (high Met); Finisher: CP 18% (low Met) and 20% (high Met)] and 2 levels of betaine supplementation (0 and 0.12%). Body weight gain and feed conversion improved significantly as dietary levels of protein increased (P < 0.05), but the results for betaine supplementation differed. The concentrations of serum total protein, albumin, and glutathione peroxidase (GPx) were elevated by either the supplementary betaine or the CP (P < 0.05). In addition, serum albumin concentration significantly increased in groups fed low CP amounts and betaine 0.12% compared with groups fed low CP only (P < 0.05). Total tract digestibility of nitrogen in broilers fed high CP amounts or 0.12% betaine, was observed to be greater than that in groups fed low CP amounts or no betaine treatment (P < 0.05). Supplemental betaine affected excreta ammonia gas emission, and hydrogen sulfide concentrations decreased significantly in low CP-fed groups (P < 0.05). Breast meat quality and relative organ weights were not influenced by CP levels or dietary betaine supplementation. These results suggest that betaine does not increase productivity, but may affect serum total protein, albumin, GPx, excreta ammonia emission, and nitrogen digestibility in broiler chickens. In addition, betaine supplementation is more effective in increasing serum albumin concentration when it was added in low CP (low Met) diets.

Simultaneous selenium and sulfur speciation analysis in cultivated Pleurotus pulmonarius mushroom.

Selenium (Se) and sulfur (S) speciation analysis in edible and medicinal Se enriched P. pulmonarius extracts was performed. Mycelium, colonized substrate, and fruiting bodies at different harvesting times were analyzed using ion-pairing reversed-phase chromatography coupled to an ICPMS/MS detector. Extraction efficiencies in enzymatically digested and aqueous extracts were between 45.3 and 109% for Se, depending on the sample type. Selenomethionine (Se-Met) was found to be the major Se-compound, together with a number of unknown Se-species. Cystine (Cys2), methionine (Met), and sulfate were also detected and quantified in all samples. Most of the Se-Met (84.0%) and Met (75.8%) were found to be in free form in the fruiting body, in contrast with the mycelium where 53.4% of Se-Met and 80.5% of Met is incorporated into proteins.

Lycium barbarum polysaccharides improve hepatic injury through NFkappa-B and NLRP3/6 pathways in a methionine choline deficient diet steatohepatitis mouse model.

Lycium barbarum polysaccharides (LBP) are major bioactive constituents of wolfberry which possess several pharmacological effects such as antioxidant and immunomodulatory activities. We aimed to evaluate how LBP attenuated the hepatic injury in a non-alcoholic steatohepatitis (NASH) methionine-choline deficient (MCD) mouse model. NASH was induced in C57BL/6N mice by feeding with MCD diet for 6 weeks. During the experiments, 1 mg/kg LBP was intragastrically fed on a daily basis with or without MCD diet lasting from the 4th to 6th week. Control and vehicle-control (LBP + PBS) were fed with a regular animal chow. LBP significantly ameliorated NASH-induced injuries, including the increase of serum ALT and AST levels, hepatic oxidative stress, fibrosis, inflammation, and apoptosis. The hepatoprotective effects of LBP were accompanied by the attenuation of thioredoxin interacting protein, nod-like receptor protein 3/6 (NLRP3/6) and reduced NF-κB (nuclear factor-kappa B) activity. Vehicle LBP fed mice showed no adverse effect on the liver. In conclusion, the suppression of the NLRP3/6 inflammasome pathway and NF-κB activation may partly contribute to the reduction of the hepatic injury during the progression of NASH by therapeutic LBP treatment.

Effect of Phyllanthus emblica L. fruit on methionine and choline-deficiency diet-induced nonalcoholic steatohepatitis.

Phyllanthus emblica L. fruit contains abundant bioactive components and exhibits a variety of biological activities. In this study, the hepatoprotective effect of water extract of P. emblica (WEPE) on nonalcoholic steatohepatitis (NASH) was evaluated. C57BL/6 mice were fed methionine and choline-deficiency diet (MCD diet) for 4 or 8 weeks to induce NASH. Results showed that administration of WEPE could significantly reduce serum AST and ALT as compared to MCD diet-alone group. Administration of WEPE could significantly decrease lipid peroxidation and CYP2E1 mRNA expression, and elevate the antioxidant activities in mice livers. In addition, administration of WEPE after 8 weeks could significantly decrease the mRNA expressions of TNF-α and IL-1ß in mice livers, but have less improving effect of hepatic steatosis and mononuclear cell infiltration. Taken together, MCD diet might cause serious hepatic steatosis and mild inflammation in mice livers, but administration of WEPE could ameliorate the rapid progression of NASH.

Review: The promise and limits for enhancing sulfur-containing amino acid content of soybean seed.

Soybeans are an excellent source of protein in monogastric diets and rations with ∼75% of soybeans produced worldwide used primarily for animal feed. Even though soybeans are protein-rich and have a well-balanced amino acid profile, the nutritive quality of this important crop could be further improved by elevating the concentrations of certain amino acids. The levels of the sulfur-containing amino acids cysteine and methionine in soybean seed proteins are inadequate for optimal growth and development of monogastric animals, which necessitates dietary supplementation. Subsequently, concerted efforts have been made to increase the concentrations of cysteine and methionine in soybean seeds by both classical breeding and genetic engineering; however, these efforts have met with only limited success. In this review, we discuss the strengths and weakness of different approaches in elevating the sulfur amino acid content of soybeans. Manipulation of enzymes involved in the sulfur assimilatory pathway appears to be a viable avenue for improving sulfur amino acid content. This approach requires a through biochemical characterization of sulfur assimilatory enzymes in soybean seeds. We highlight recent studies targeting key sulfur assimilatory enzymes and the manipulation of sulfur metabolism in transgenic soybeans to improve the nutritive value of soybean proteins.

Rapid analysis of traditional Chinese medicine Pinellia ternata by microchip electrophoresis with electrochemical detection.

Traditional Chinese herbal medicine has long enjoyed the reputation of the world's most advanced system of natural medicine. Pinellia ternata is one of the most commonly used herbs in the traditional Chinese medical science. In this study, five representative ingredients of Pinellia ternata guanosine, methionine, glycine, 3,4-dihydroxybenzaldehyde, and homogentisic acid, were assayed using simple derivatization procedures. Under optimized experimental condition, five analytes in Pinellia ternata were rapidly separated and detected using microchip electrophoresis, affording the benefits of speed, minimal sample requirements, and sensitive on-the-chip electrochemical detection, in 5 min with linearity over a concentration of 20-500 µM (R2  = 0.994) with nearly complete recovery (95.6-98.5%).

The effect of antioxidants on quantitative changes of lysine and methionine in linoleic acid emulsions at different pH conditions.

BACKGROUND: Plants are an important source of phenolic compounds. The antioxidant capacities of green tea, thyme and rosemary extracts that contain these compounds have been reported earlier. However, there is a lack of accessible information about their activity against lipid oxidation in emulsions and inhibit the interaction of lipid oxidation products with amino acids. Therefore, the influence of green tea, thyme and rosemary extracts and BHT (butylated hydroxytoluene) on quantitative changes in lysine and methionine in linoleic acid emulsions at a pH of isoelectric point and a pH lower than the isoelectric point of amino acids was investigated. METHODS: Total phenolic contents in plant extracts were determined spectrophotometrically by using Folin-Ciocalteu's reagent, and individual phenols by using HPLC. The level of oxidation of emulsion was determined using the measurement of peroxides and TBARS (thiobarbituric acid reactive substances). Methionine and lysine in the system were reacted with sodium nitroprusside and trinitrobenzenesulphonic acid respectively, and the absorbance of the complexes was measured. RESULTS: Extract of green tea had the highest total polyphenol content. The system containing antioxidants and amino acid protected linoleic acid more efficiently than by the addition of antioxidants only. Lysine and methionine losses in samples without the addition of antioxidants were lower in their isoelectric points than below these points. Antioxidants decrease the loss of amino acids. The protective properties of antioxidants towards methionine were higher in a pH of isoelectric point whereas towards lysine in pH below this point. CONCLUSIONS: Green tea, thyme and rosemary extracts exhibit antioxidant activity in linoleic acid emulsions. Moreover, they can be utilized to inhibit quantitative changes in amino acids in lipid emulsions. However, the antioxidant efficiency of these extracts seems to depend on pH conditions. Further investigations should be carried out to clarify this issue.

Different Effects of Sheep- and Duck-Meat Supplemented Diets on Serum Cytokine Levels of Rats.

UNLABELLED: This study aimed to explore the mechanism underlying the different effects of diet supplemented with sheep- or duck-meat on serum cytokines of rats. METHODS: Male Sprague-Dawley rats were randomly divided into three groups fed on sheep meat, duck meat, or soybean, respectively. The profiles of amino acids and fatty acids of the three diets were examined, and the levels of serum cytokines in rats, including interleukin-1ß (IL-1ß), IL-2, IL-4, IL-6, IL-10 and tumor necrosis factor-α (TNF-α), were detected 30 d after feeding, using radioimmunoassay. The contents of methionine and glycine in the sheep-meat and duck-meat diets were significantly higher than those in the soybean diet. The content of saturated fatty acids in the sheep-meat diet and duck-meat diet was higher than that in soybeans, while the polyunsaturated fatty acids (PUFAs) in the duck-meat diet were highest and those in the sheep-meat diet were lowest. Serum levels of IL-2 and IL-10 in the rats of the sheep-meat and duck-meat groups were significantly higher than those in the rats of the soybean group (p<0.05). IL-10 and TNF-α in the rats of the sheep-meat group were higher than those in the duck-meat group. But the levels of IL-1ß and IL-6 were not significantly different among the three groups. Additionally, there were positive correlations between glycine and IL-1ß as well as glycine and IL-2, while negative correlation existed between C18:2 and TNF-α. Methionine, glycine and PUFAs in a diet supplemented with sheep- or duck-meat might influence the levels of serum cytokines in rats, suggesting the potential regulatory mechanism of amino acids and fatty acids from diet in immune responses.

Effects of castration age, protein level and lysine/methionine ratio in the diet on colour, lipid oxidation and meat acceptability of intensively reared Friesian steers.

A total of 64 intensively reared Friesian steers were used in a 2×2×2 design to study the effects of age of castration (15 days old v. 5 months old), dietary protein level (14.6% v. 16.8%; DM basis) and lysine/methionine (lys/met) ratio (3.0 v. 3.4) on meat quality. The lys/met ratio of 3.0 was reached with supplementation of protected methionine. Animals were slaughtered at a live weight of 443.5 ± 26.2 kg at around 12 months of age. Colour and lipid oxidation were measured in the longissimus thoracis muscle throughout the 14 days of display under modified atmospheric and commercial display conditions. A panel of 17 consumers assessed daily the visual acceptability of the meat on display. A consumer acceptability eating test was also performed with 120 consumers in meat aged for 7 days under vacuum conditions. Lipid oxidation was not influenced by castration age and the protein level in the diet. Castration age did not affect meat colour, but meat from the low protein level diet and the low lys/met ratio showed higher redness (a*) from 3 days of display onwards. Nevertheless, from 6 days onwards, consumer visual acceptability was below the level of acceptance in all treatments, and even from 5 days onwards in those animals that underwent early castration and were fed either a high protein diet or a combination diet low in protein content and high in lys/met ratio. The best accepted treatments throughout the display period were those from late castrated animals fed a low protein diet, probably related to other visual aspects. However, the best accepted meat after consumption was that from late castrated animals fed high protein and high lys/met. The addition of protected methionine to reach lys/met levels of 3.0 did not improve beef acceptability, with the high protein diet being preferred by consumers in terms of palatability in late castrated animals.

[Emodin worsens methionine-choline-deficient diet-induced non-alcoholic fatty liver disease in mice].

OBJECTIVE: To investigate the influence of emodin on methionine-choline-deficient (MCD) diet-induced non-alcoholic fatty liver disease (NAFLD) in mice. METHODS: C57BL/6J mice were randomly divided into three groups: methionine-choline-supplemented (MCS) diet group, MCD diet plus DMSO injection group (MCD), MCD diet plus emodin injection group (MCD-emodin). The mice were fed with MCS or MCD diet for 10 days, and then peritoneally injected with DMSO or emodin for 20 days consecutively. HE staining was performed to observe pathologic changes of livers. The levels of serum alanine aminotransferase (ALT) and aspartate transaminase (AST), triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL), alkaline phosphatase (ALP), glucose (GLU) were measured by automatic biochemical analyzer. The mRNA levels of interleukin 1ß (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α) were determined by real-time quantitative PCR. RESULTS: HE staining showed that there were more lipid accumulation and leukocyte infiltration in the livers of the MCD group compared with the MCS group. The above manifestations were more severe in the MCD-emodin group. Moreover, compared with the MCD group, emodin injection remarkably raised serum AST and ALT levels and greatly increased IL-1ß and IL-6 mRNA levels. CONCLUSION: Emodin worsened MCD diet-induced NAFLD in mice.

Dietary Supplementation of Genistein Alleviates Liver Inflammation and Fibrosis Mediated by a Methionine-Choline-Deficient Diet in db/db Mice.

Nonalcoholic fatty liver disease is a complex disorder which includes simple steatosis, steatohepatitis, fibrosis and ultimately cirrhosis. Previous studies have reported that genistein, a soy phytoestrogen, attenuates steatohepatitis induced in obese and type 2 diabetic models. Here we investigated the effect of dietary genistein supplementation (0.05%) on nonalcoholic steatohepatitis (NASH) development induced by a methionine-choline-deficient (MCD) diet in db/db mice. MCD-diet-fed mice exhibited a significantly lower body weight and a higher degree of steatohepatitis with increased oxidative stress, steatosis, inflammation, stellate cell activation, and mild fibrosis. Although genistein did not inhibit hepatic steatosis, we observed that oxidative stress, endoplasmic reticulum stress, and AMP-dependent kinase inactivation were alleviated by genistein. Genistein also down-regulated the augmented gene expressions associated with hepatic inflammation and fibrosis. Therefore, these results suggest that genistein may protect MCD-diet-mediated NASH development by suppressing lipid peroxidation, inflammation, and even liver fibrosis in db/db mice.

Hepatoprotective and antioxidant activities of extracts from Salvia-Nelumbinis naturalis against nonalcoholic steatohepatitis induced by methionine- and choline-deficient diet in mice.

BACKGROUND: Nonalcoholic steatohepatitis (NASH), the advanced stage of nonalcoholic fatty liver disease that is characterized by both steatosis and severe injury in liver, still lacks efficient treatment. The traditional Chinese formula Salvia-Nelumbinis naturalis (SNN) is effectively applied to improve the symptoms of nonalcoholic simple fatty liver (NAFL) patients. Previous studies have confirmed that SNN could reduce the liver lipid deposition and serum transaminases of NAFL experimental models. This study aims to determine whether SNN is effective for murine NASH model and investigate the underlying pharmacological mechanisms. METHODS: C57BL/6 J mice were fed with methionine- and choline-deficient (MCD) diet for six weeks to induce NASH. Simultaneously, SNN or saline was intragastrically administered daily to the mice in the SNN or model group, respectively. A standard diet was given to the control mice. Serum biochemical indices and tumor necrosis factor-α were measured. Liver histopathology was observed, and the contents of triglycerides and lipid peroxide malondialdehyde (MDA) in liver homogenates were evaluated. The hepatic expression and/or activation of genes associated with inflammation, apoptosis, and oxidative stress were determined by quantitative RT-PCR or Western blot analysis. RESULTS: The prominent liver steatosis displayed in the NASH model was prevented by SNN. The liver injury of NASH mice was obviously manifested by the increased levels of serum transaminases and bilirubin, as well as the lobular inflammation, elevated pro-inflammatory cytokines, and upregulated apoptosis in liver tissues. SNN administration improved the aforementioned pathological changes. The increased hepatic levels of MDA and cytochrome P450 2E1 of the model confirmed the unregulated balance of oxidative stress. The hepatic expression of nuclear factor erythroid 2-related factor 2 and its target genes decreased, whereas c-Jun N-terminal kinase activation in the model mice increased. Treating the mice with SNN significantly improved oxidative stress-related harmful factors. CONCLUSIONS: This study shows that SNN can protect the liver from severe steatosis and damage induced by MCD diet, which suggests the potential use of SNN on the treatment of NASH patient. The results also indicate that improving the hepatic antioxidant capability of the liver may contribute to the underlying hepatoprotective mechanism.

The effects of α-lipoic acid on liver oxidative stress and free fatty acid composition in methionine-choline deficient diet-induced NAFLD.

Development of nonalcoholic fatty liver disease (NAFLD) occurs through initial steatosis and subsequent oxidative stress. The aim of this study was to examine the effects of α-lipoic acid (LA) on methionine-choline deficient (MCD) diet-induced NAFLD in mice. Male C57BL/6 mice (n=21) were divided into three groups (n=7 per group): (1) control fed with standard chow, (2) MCD2 group--fed with MCD diet for 2 weeks, and (3) MCD2+LA group--2 weeks on MCD receiving LA i.p. 100 mg/kg/day. After the treatment, liver samples were taken for pathohistology, oxidative stress parameters, antioxidative enzymes, and liver free fatty acid (FFA) composition. Mild microvesicular hepatic steatosis was found in MCD2 group, while it was reduced to single fat droplets evident in MCD2+LA group. Lipid peroxidation and nitrosative stress were increased by MCD diet, while LA administration induced a decrease in liver malondialdehyde and nitrates+nitrites level. Similary, LA improved liver antioxidative capacity by increasing total superoxide dismutase (tSOD), manganese SOD (MnSOD), and copper/zinc-SOD (Cu/ZnSOD) activity as well as glutathione (GSH) content. Liver FFA profile has shown a significant decrease in saturated acids, arachidonic, and docosahexaenoic acid (DHA), while LA treatment increased their proportions. It can be concluded that LA ameliorates lipid peroxidation and nitrosative stress in MCD diet-induced hepatic steatosis through an increase in SOD activity and GSH level. In addition, LA increases the proportion of palmitic, stearic, arachidonic, and DHA in the fatty liver. An increase in DHA may be a potential mechanism of anti-inflammatory and antioxidant effects of LA in MCD diet-induced NAFLD.