Multi-dimensional deep learning drives efficient discovery of novel neuroprotective peptides from walnut protein isolates.

Neurodegenerative diseases, such as Alzheimer's and Parkinson's, are multi-factor induced neurological disorders that require management from multiple pathologies. The peptides from natural proteins with diverse physiological activity can be candidates as multifunctional neuroprotective agents. However, traditional methods for screening neuroprotective peptides are not only time-consuming and laborious but also poorly accurate, which makes it difficult to effectively obtain the needed peptides. In this case, a multi-dimensional deep learning model called MiCNN-LSTM was proposed to screen for multifunctional neuroprotective peptides. Compared to other multi-dimensional algorithms, MiCNN-LSTM reached a higher accuracy value of 0.850. The MiCNN-LSTM was used to acquire candidate peptides from walnut protein hydrolysis. Following molecular docking, behavioral and biochemical index experimental validation eventually found 4 hexapeptides (EYVTLK, VFPTER, EPEVLR and ELEWER) demonstrating excellent multifunctional neuroprotective properties. Therein, EPEVLR performed the best and can be investigated in depth as a multifunctional neuroprotective agent. This strategy will greatly improve the efficiency of screening multifunctional bioactive peptides, and it will be beneficial for the development of food functional peptides.

Isolation, identification and molecular docking of anti-inflammatory peptides from walnut (Juglans regia L.) meal hydrolysates.

The oil extraction residue of walnuts is rich in proteins and has been employed in the formulation of various functional food products. In this study, alcalase and neutrase were used to hydrolyze defatted walnut meal protein to obtain anti-inflammatory peptides. After separation by ultrafiltration and by using Sephadex G-25, the fraction with the highest anti-inflammatory activity was identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and 579 peptides were obtained. Then, four of the most stable binding tripeptides with the sequences Trp-Pro-Leu (WPL, MW: 414.2 Da), Trp-Ser-Leu (WSL, MW: 404.2 Da), Phe-Pro-Leu (FPL, MW: 375.2 Da) and Phe-Pro-Tyr (FPY, MW: 425.2 Da) were successfully identified by virtual screening. The anti-inflammatory activity determination of the synthetic peptide assay indicated that FPL (200 µM) exhibited excellent anti-inflammatory activity with inhibitory rates of 63.65 ± 2.64%, 68.25 ± 2.19%, 42.52 ± 2.01% and 59.39 ± 2.21% in terms of four inflammatory mediators (NO, TNF-α, IL-6 and IL-1ß), respectively. It was speculated that the anti-inflammatory activity of walnut peptides might be related to hydrophobic amino acids and aromatic amino acids. By molecular docking, further insight into the theoretical interaction mechanism of binding revealed that hydrophobic interactions and hydrogen bonds turned out to be the main interaction forces between the four peptides and iNOS. These results indicated that FPL screened in this study could be expected to be used as a natural anti-inflammatory active substance in the functional food and pharmaceutical industries.

Amelioration of walnut-derived novel peptides against D-galactose-induced cognitive impairment by modulating the gut microbiota composition.

In this work, RLWPF (Arg-Leu-Trp-Pro-Phe) and VLRLF (Val-Leu-Arg-Leu-Phe) were investigated for the effects against D-galactose (D-gal) induced cognitive impairment by modulating the gut microbiota composition. The effects on serum metabolite production were further investigated. The two novel peptides derived from walnut protein alkaline protease hydrolysates were predicted by docking to acetylcholinesterase (AChE) with the highest binding affinities, -10.3 and -9.9 kcal mol-1, considered as the potential neuroprotective peptides. In behavioral experiments, RLWPF and VLRLF treatment significantly restored spatial learning and memory impairment induced by D-gal. The results showed that RLWPF and VLRLF could alleviate cholinergic dysfunction, oxidative stress, and inflammation to varying degrees caused by D-gal-induced aging. Furthermore, 16S rRNA analysis revealed that RLWPF and VLRLF treatment improved cognitive impairment by regulating the composition of the gut microbiota and the abundance of harmful bacteria, including the ratio of Firmicutes to Bacteroidetes, Helicobacter, Allobaculum, Alistipes, Mucispirillum, and Odoribacter. In addition to the same regulation, RLWPF and VLRLF had their marker and regulatory flora. Studies based on the gut microbiota would allow a better understanding of the neuroprotective effects of walnut-derived peptides, supporting that walnut-derived peptides could be potential functional ingredients in foods and nutraceuticals or drug candidates in the treatment of cognitive dysfunction.

Formulated chitosan-sodium tripolyphosphate nanoparticles for co-encapsulation of ellagic acid and anti-inflammatory peptide: characterization, stability and anti-inflammatory activity.

BACKGROUND: Chitosan (CS) and tripolyphosphate (TPP) can be combined in the development of a material with synergistic properties and promising potential for the conservation of food products. In this study, ellagic acid (EA) and anti-inflammatory peptide (FPL)-loaded CS nanoparticles (FPL/EA NPs) were prepared using the ionic gelation method and optimal preparation conditions were obtained through a single factor design. RESULTS: The synthesized nanoparticles (NPs) were characterized using a scanning electron microscope (SEM), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). Nanoparticles were spherical, with an average size of 308.33 ± 4.61 nm, a polydispersity index (PDI) of 0.254, a zeta potential of +31.7 ± 0.08 mV, and a high encapsulation capacity (22.16 ± 0.79%). An in vitro release study showed that EA/FPL had a sustainable release from FPL/EA NPs. The stability of the FPL/EA NPs was evaluated for 90 days at 0, 25, and 37 °C. Significant anti-inflammatory activity of FPL/EA NPs was verified by nitric oxide (NO) and tumor necrosis factor-α (TNF-α) reduction. CONCLUSION: These characteristics support the use of CS nanoparticles to encapsulate EA and FPL and improve their bioactivity in food products. © 2023 Society of Chemical Industry.

Fermentation of rose residue by Lactiplantibacillus plantarum B7 and Bacillus subtilis natto promotes polyphenol content and beneficial bioactivity.

The present study evaluated the effect of fermentation with Lactiplantibacillus plantarum B7 and Bacillus subtilis natto on phenolic compound levels and enzyme activity, as well as antioxidant capacity of the rose residue. Results showed that the polyphenol content of rose residue was significantly increased from 16.37 ± 1.51 mg/100 mL to 41.02 ± 1.68 mg/100 mL by fermentation at 37 °C and 2.0% (v/v) inoculum size for 40 h. The flavone, soluble dietary, and protein contents were also enhanced by almost 1-fold, 3-fold, and 1-fold, respectively. Fifteen phenolic compounds were quantified in the fermented broth, among which the concentration of gallic acid, quercetin, and p-coumaric acid increased by 5-fold, 4-fold, and almost 8-fold, respectively. Chlorogenic acid was a new phenolic compound produced during fermentation. Moreover, the fermented rose residue presented higher superoxide dismutase, α-amylase, and protease activity. ABTS•+, hydroxylradical, and DPPH• scavenging activity increased by 60.93%, 57.70%, and 37.00%, respectively. This provides an effective means of transforming rose residue into a highly bioactive value-added substance.

The Synergistic Anti-inflammatory Activity and Interaction Mechanism of Ellagic Acid and a Bioactive Tripeptide (Phe-Pro-Leu) from Walnut Meal.

The anti-inflammatory effect of the interaction between ellagic acid (EA) and a bioactive tripeptide (FPL) from walnut meal was investigated in this study. We found that lipopolysaccharide (LPS) -induced expression of nitric oxide, tumor necrosis factor-α, interleukin-6, and interleukin-1ß were significantly inhibited by the interaction of EA and FPL in RAW264.7 macrophage cells. Cell viability assays and CompuSyn simulations predicted the highest synergistic effect of the combination at doses of EA-25 µM and FPL-100 µM, with the lowest combination index (CI) values reaching 0.56. Fluorescence spectra revealed the intrinsic fluorescence of phenylalanine in FPL was quenched by interaction with EA. Fourier transform infrared spectroscopy indicated FPL had electrostatic and hydrophobic interactions with EA through N-H, C = O, C-N bonds and the secondary structure of FPL had effectively changed, with a decrease in α-helix when interacting with EA. Our results demonstrated that the synergistic anti-inflammatory effect of EA and FPL as potential inflammatory inhibitors in food industry.

The shelf-life of chestnut rose beverage packaged in PEN/PET bottles under long term storage: A comparison to packaging in ordinary PET bottles.

The shelf life of chestnut rose beverage is largely dependent on packaging method and storage temperature. In this study, we investigated the effects of packaging beverages in bottles made of either polyethylene terephthalate (PET) or PEN (polyethylene naphthalate)/PET and storage temperature (4, 25, 37, and 55 ℃) on the shelf life of chestnut rose beverage. The physicochemical parameters and enzyme activity of beverages were evaluated, and we found that at 4 °C, the vitamin C, superoxide dismutase, and total polyphenol contents of beverages stored in PEN/PET bottles increased by 9.95 ± 0.49%, 2.86 ± 0.13%, and 3.23 ± 0.09% respectively, compared to beverages in ordinary PET bottles. In addition, other characteristic indicators including total soluble solids, browning index, and color value were also significantly improved. A shelf-life model was established based on the Arrhenius equation, and it will help distributors and consumers to determine the storage time and optimal shelf life of chestnut rose beverage.

The impact of chitooligosaccharides and their derivatives on the in vitro and in vivo antitumor activity: A comprehensive review.

Chitooligosaccharides (COS) are the degraded products of chitin or chitosan. COS is water-soluble, non-cytotoxic to organisms, readily absorbed through the intestine, and eliminated primarily through the kidneys. COS possess a wide range of biological activities, including immunomodulation, cholesterol-lowering, and antitumor activity. Although work on COS goes back at least forty years, several aspects remain unclear. This review narrates the recent developments in COS antitumor activities, while paying considerable attention to the impacts of physicochemical properties (such as molecular weight and degrees of deacetylation) and chemical modifications both in vitro and in vivo. COS derivatives not only improve some physicochemical properties, but also expand the range of applications in drug and gene delivery. COS (itself or as a drug carrier) can inhibit tumor cell proliferation and metastasis, which might be attributed to its ability to stimulate the immune response along with its anti-angiogenic activity. Further, an attempt has been made to report limitations and future research. The potential health benefits of COS and its derivatives against cancer may offer a new insight on their applications in food and medical fields.

Separation, identification, and molecular docking of tyrosinase inhibitory peptides from the hydrolysates of defatted walnut (Juglans regia L.) meal.

Defatted walnut meal protein was hydrolyzed using alcalase to yield tyrosinase inhibitory peptides. After separation by ultrafiltration and Sephadex G-25, the fraction with the highest tyrosinase inhibitory activity was identified using liquid chromatography-tandem mass spectrometry and 606 peptides were obtained. Then, molecular docking was used to screen for tyrosinase inhibitory peptides and to clarify the theoretical interaction mechanism between the peptides and tyrosinase. A peptide with the sequence Phe-Pro-Tyr (FPY, MW: 425.2 Da) was identified and the synthesized peptide inhibited tyrosine monophenolase and diphenolase with IC50 values of 1.11 ± 0.05 and 3.22 ± 0.09 mM, respectively. The inhibition of tyrosinase by FPY was competitive and reversible. Good stability of FPY toward digestion was observed in an in vitro gastrointestinal digestion simulation experiment. These results indicated that FPY can be used as a potential tyrosinase inhibitor in the food, medicine, and cosmetics industries.

Effect of whey protein isolate/chitosan/microcrystalline cellulose/PET multilayer bottles on the shelf life of rosebud beverages.

Multilayer bottles consisting of chitosan (CS), microcrystalline cellulose (MCC), whey protein isolate (WPI), and polyethylene terephthalate (PET) were tested as novel materials for packaging and extending shelf life of rosebud beverages. We studied the storage stability at 4 °C, 25 °C, 37 °C, and 55 °C by assessing the physical and biochemical parameters. The results show that multilayer PET bottles had better barrier performance and improved soluble solids content, pH, polyphenol content, color indices, and browning degree in rosebud beverages over the control at all studied temperatures. A shelf life model was established based on the Arrhenius equation, and the number of days when polyphenol contents dropped to <50% of the initial content was defined as the shelf life. Our results highlight the reliability of the prediction model, and we conclude that packaging rosebud beverages in multilayer PET bottles significantly extends the product shelf life, and this benefit was further extended at low temperatures.

In vitro antioxidant activities of Rhodobacter sphaeroides and protective effect on Caco-2 cell line model.

The present study aimed to evaluate the in vitro antioxidant activities and the protective effect of Rhodobacter sphaeroides on H2O2-induced oxidative stress in Caco-2 cells. The results showed that the antioxidant action of R. sphaeroides varied with different cell concentrations and treatments. Also, the intact cells and intracellular cell-free extracts showed better antioxidant activities. Caco-2 cell-based oxidative stress model was developed by optimizing H2O2 concentration and culture time with the half lethal dose and methyl thiazolyl tetrazolium. By increasing the activity of endogenous antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, upregulating the antioxidant ability of the anti-superoxide anion and anti-hydroxyl radical, R. sphaeroides, especially the mutant strain R. sphaeroides (CGMCC No. 8513), exhibited significant protective activity against H2O2-induced oxidative stress in Caco-2 cells. Taken together, R. sphaeroides (CGMCC No. 8513) exhibits strong antioxidant activities and is a candidate to be investigated as a potential probiotic in the future.

Transport of ACE Inhibitory Peptides Ile-Gln-Pro and Val-Glu-Pro Derived from Spirulina platensis Across Caco-2 Monolayers.

This study evaluated transepithelial transport mechanisms of Ile-Gln-Pro (IQP) and Val-Glu-Pro (VEP), two ACE-inhibitory peptides derived from Spirulina platensis, using human intestinal Caco-2 cell monolayers. IQP and VEP were absorbed intact through Caco-2 cell monolayers with Papp values of 7.48 ± 0.58 × 10-6 and 5.05 ± 0.74 × 10-6 cm/s, respectively. The transport of IQP and VEP were affected neither by Gly-Pro nor by wortmannin, indicating that they were not PepT1-mediated and did not involve endocytosis. However, transport of IQP and VEP were increased significantly by sodium deoxycholate, suggesting that the major transport mechanism was paracellular. In addition, the increased transport of VEP and IQP were followed with the addition of sodium azide, suggesting influence of energy to the process. The transport of VEP was also increased by verapamil, indicating an apical-to-basolateral flux mediated by P-gp. PRACTICAL APPLICATION: Bioactive peptides derived from food proteins have been considered as potentially ideal products to reduce hypertension because of their safety and positive impacts on health. IQP and VEP are the 2 ACE inhibitory peptides derived from Spirulina platensis, a kind of edible cyanobacteria with rich nutrition and multiple physiological functions, and were demonstrated to inhibit ACE and lower blood pressure in spontaneously hypertensive rats. However, it is prerequisite that such bioactive peptides must be absorbed intact across the intestinal epithelium, so as to exert antihypertensive effects in vivo. This study evaluated transepithelial transport mechanisms of IQP and VEP. It contributes to the study of Spirulina in lowering blood pressure and supports the development of bioactive peptide products.

Mixed fermentation of Spirulina platensis with Lactobacillus plantarum and Bacillus subtilis by random-centroid optimization.

Spirulina platensis is a high-nutrient blue-green alga with a long history as a food supplement. In this study, the mixed fermentation of Spirulina with Lactobacillus plantarum and Bacillus subtilis was investigated using random-centroid optimization to improve deodorization of off-flavor and hydrolysis of protein. Fermented Spirulina with the maximum total viable counts of both organisms achieved best sensory characteristics and degree of proteolysis among those with different maximum biomass. The mixed fermentation noticeably reduced the volatile compounds of Spirulina, and yielded the highest relative contents (>85%) of acetoin and other odorants collectively producing a creamy aroma. Approximately one-third of the Spirulina proteins were hydrolyzed, yielding over 16% polypeptides and increasing the ratio of essential amino acids to total free amino acids to 1.5-fold compared with unfermented Spirulina. Fermentation with B. subtilis and L. plantarum effectively improved the odor and protein availability of Spirulina.

Protective effect of Letinous edodes foot peptides against ethanol­induced liver injury in L02 cells.

The aim of the present study was to evaluate the protective effect and mechanism of Letinous edodes foot peptides on ethanol­induced L02 cells. A cell model of ethanol­induced damage was established in vitro to study the effects of the Letinous edodes foot peptides on human L02 hepatocytes. The expression and activity of superoxide dismutase (SOD), malondialdehyde (MDA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), following treatment were examined to determine the anti­alcoholism and hepatoprotective functions of Letinous edodes foot peptides. Taking Letinous edodes foot peptides prior to ethanol exposure was more beneficial, which significantly increased SOD activity and the mRNA expression of ADH and ALDH suppressed by ethanol. In addition, the intracellular MDA content, and AST and ALT activity decreased in ethanol­induced L02 cells pretreated with the peptides, when compared with the control. Furthermore, Letinous edodes foot peptides inhibited the ethanol­induced activation of the proinflammatory cytokines, interleukin­6 and tumor necrosis factor­α, and promoted the metabolic regulation factors, AMP­activated protein kinase­α2 and peroxisome proliferator­activated receptor­α.

Antioxidant and hypoglycemic effects of Diospyros lotus fruit fermented with Microbacterium flavum and Lactobacillus plantarum.

Diospyros lotus, a member of the Ebenaceae family, has long been used as a traditional sedative in China. In this study, the antioxidant and hypoglycemic effects of non-fermented and microorganism-fermented D. lotus were explored. The total phenolic and vitamin C contents of microorganism-fermented D. lotus for 24-72 h were less than those of non-fermented. High-performance liquid chromatography showed that the tannic, catechinic, and ellagic acid contents increased significantly upon fermentation for 24 h. D. lotus fermented with Microbacterium flavum for 24 h exhibited the highest DPPH radical scavenging activity (IC50 = 4.18 µg mL-1), and the highest ABTS radical scavenging activity was exhibited at 72 h of fermentation (IC50 = 29.18 µg mL-1). The anti-α-glucosidase activity of fermented D. lotus was higher (2.06-4.73-fold) than that of non-fermented one. Thus, fermented D. lotus is a useful source of natural antioxidants, and a valuable food, exhibiting antioxidant and hypoglycemic properties.

Effects of IQP, VEP and Spirulina platensis hydrolysates on the local kidney renin angiotensin system in spontaneously hypertensive rats.

The aim of the present study was to investigate the antihypertensive effects of the bioactive Spirulina platensis peptides Ile­Gln­Pro (IQP), Val­Glu­Pro (VEP), as well as Spirulina platensis hydrolysates (SH), and assessed whether the synthesis of components of the myocardial and renal local renin angiotensin system (RAS) are regulated differentially in spontaneously hypertensive rats (SHR). The SHR were administrated with IQP, VEP and SH respectively (10 mg/kg/day) for 6 weeks and received continuous monitoring of blood pressure (BP) for two more weeks. During the trial, the rats' kidney tissues were removed from these rats and collected at weeks 3, 6 and 8. The expression of the main components of local kidney RAS was measured at the mRNA levels by reverse transcription­quantitative polymerase chain reaction, and at the protein levels by ELISA or western blotting. Oral administration of IQP, VEP and SH into SHR resulted in marked antihypertensive effects. IQP, VEP and SH decreased rats' BP by affecting the expression of local kidney RAS components via downregulating the angiotensin­converting enzyme (ACE), Ang II and angiotensin II (Ang II) and angiotensin type­1 receptor (AT 1), while upregulating ACE2, Ang (1­7), Mas and AT 2. The comparisons of SH effects on local tissue RAS demonstrated that local kidney RAS regulated BP via the ACE­Ang II­AT 1/AT 2 axis and the ACE2­Ang (1­7)­Mas axis primarily at the mRNA level, while the local myocardium RAS mainly at the protein level. This preliminary study suggests that the main components of local RAS presented different expression levels in myocardium and kidney, which is important to the development of bioactive peptides targeting for lowering BP by changing the levels of some components in local RAS in specific tissues.

Role of the ACE2­Ang­(1­7)­Mas axis in blood pressure regulation and its potential as an antihypertensive in functional foods (Review).

The renin­angiotensin system (RAS) serves a critical role in blood pressure regulation and prevention of cardiovascular diseases. Efforts to develop functional foods that enhance the RAS have focused on inhibition of angiotensin­converting enzyme (ACE) activity in the ACE­angiotensin II (Ang II)­Ang II type 1 receptor axis. ACE2 and the Mas receptor are important components of this axis. ACE2 catalyzes Ang II into Ang­(1­7), which then binds to the G­protein­coupled receptor Mas. In addition, it induces nitric oxide release from endothelial cells and exerts antiproliferative, vasodilatory and antihypertensive effects. The present review examined recent findings regarding the physiological and biological roles of the ACE2­Ang­(1­7)­Mas axis in the cardiovascular system, discussed potential food­derived ACE2­activating agents, and highlighted initiatives, based on this axis, that aim to develop functional foods for the treatment of hypertension.

Ultra-high-pressure processing improves proteolysis and release of bioactive peptides with activation activities on alcohol metabolic enzymes in vitro from mushroom foot protein.

This study was to find an effective process to extract bioactive peptides from mushroom foot and determine their effects on activation of alcohol metabolic enzymes in vitro. The optimum extraction assisted by ultra-high-pressure processing of mushroom foot peptides was obtained with a pressure of 400MPa and a processing time of 10min. After ultrafiltration, peptides with molecular weight of 0-3kDa had the highest activity to activate alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) by 70.79% and 71.35%, respectively. Following dextran gel chromatography, two peaks (p-I and p-II) appeared and the activation activities on ADH and ALDH of p-I were 72.00% and 73.43%, both higher than p-II. Nine peptides were found in p-I as determined by LC-MS/MS, and two of them (IPLH and IPIVLL) were synthesized. IPLH activated ADH and ALDH by 42.7% and 29.2% respectively, which were higher than IPIVLL.

Preparation, Identification and Antioxidant Properties of Black-Bone Silky Fowl (Gallus gallus domesticus Brisson) Iron(II)-Oligopeptide Chelate.

Black-bone silky fowl iron(II)-oligopeptide chelate was synthesized from iron(II) solution and the black-bone silky fowl oligopeptide, which was extracted from the muscle protein of black-bone silky fowl (Gallus gallus domesticus Brisson). Orthogonal array analysis was used to determine the optimal conditions for the iron(II)-oligopeptide chelate preparation. Ultraviolet-visible (UV-Vis) spectroscopy, electron microscopy, and Fourier transform infrared (FTIR) spectroscopy were used to identify the structure of iron(II)-oligopeptide chelate. 2-Diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical scavenging assays were performed to compare the antioxidant abilities of the black-bone silky fowl oligopeptide and iron(II)-oligopeptide chelate. The optimal conditions for iron(II)-oligopeptide chelate preparation were 4% of the black-bone silky fowl oligopeptide and a ratio of the black- -bone silky fowl oligopeptide to FeCl2·4H2O of 5:1 at pH=4. Under these conditions, the chelation rate was (84.9±0.2) % (p<0.05), and the chelation yield was (40.3±0.1) % (p<0.05). The structures detected with UV-Vis spectroscopy, electron microscopy and FTIR spectra changed significantly after chelation, suggesting that Fe(II) ions formed coordinate bonds with carboxylate (-RCOOZ) and amino (-NH2) groups in the oligopeptides, confirming that this is a new oligopeptide-iron chelate. The iron(II)-oligopeptide chelate had stronger scavenging activity towards DPPH and superoxide radicals than did the black-bone silky fowl oligopeptide.

Long-Term Regulation of the Local Renin-Angiotensin System in the Myocardium of Spontaneously Hypertensive Rats by Feeding Bioactive Peptides Derived from Spirulina platensis.

This study investigated the long-term (8 weeks) anti-hypertensive effects of 10 mg/kg tripeptides isolated from Spirulina platensis, Ile-Gln-Pro (IQP) and Val-Glu-Pro (VEP), and S. platensis hydrolysates (SH) on spontaneously hypertensive rats. The treatment period was 6 weeks, and observation continued for another 2 weeks. After treatment, weighted systolic blood pressure, weighted diastolic blood pressure, left ventricular mass index, and right ventricular mass index of groups treated with IQP, VEP, and SH were significantly lower than those of the group treated with distilled water, even when the treatments had been withdrawn for 2 weeks. Quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting showed the mRNA expression levels and protein/peptide concentrations of the main components of the renin angiotensin system in myocardium were significantly affected by treatment: angiotensin converting enzyme, angiotensin II, and angiotensin type 1 receptor were down-regulated, whereas angiotensin type 2 receptor, angiotensin converting enzyme 2, angiotensin-(1-7), and Mas receptor were up-regulated.