Two Novel Angiotensin-Converting Enzyme (ACE) Inhibitory and ACE2 Upregulating Peptides from the Hydrolysate of Pumpkin (Cucurbita moschata) Seed Meal.

Pumpkin (Cucurbita moschata) seed meal (PSM), the major byproduct of pumpkin seed oil industry, was used to prepare angiotensin-converting enzyme (ACE) inhibitory and angiotensin-converting enzyme 2 (ACE2) upregulating peptides. These peptides were isolated and purified from the PSM hydrolysate prepared using Neutrase 5.0 BG by ultrafiltration, Sephadex G-15 column chromatography, and reversed-phase high-performance liquid chromatography. Two peptides with significant ACE inhibition activity were identified as SNHANQLDFHP and PVQVLASAYR with IC50 values of 172.07 and 90.69 µM, respectively. The C-terminal tripeptides of the two peptides contained Pro, Phe, and Tyr, respectively, and PVQVLASAYR also had Val in its N-terminal tripeptide, which was a favorable structure for ACE inhibition. Molecular docking results declared that the two peptides could interact with ACE through hydrogen bonds and hydrophobic interactions. Furthermore, the two peptides performed protective function on EA.hy926 cells by decreasing the secretion of endothelin-1, increasing the release of nitric oxide, and regulating the ACE2 activity. In vitro simulated gastrointestinal digestion showed the two peptides exhibited good stability against gastrointestinal enzyme digestion. In conclusion, PSM is a promising material for preparing antihypertensive peptides.

Correction: Marginal zinc deficiency alters the heart proteome of rats.

Correction for 'Marginal zinc deficiency alters the heart proteome of rats' by Yongzhi Sun et al., Food Funct., 2023, https://doi.org/10.1039/d2fo03815c.

Marginal zinc deficiency alters the heart proteome of rats.

Zinc deficiency is closely related to cardiovascular diseases (CVDs), but the effects of marginal zinc deficiency (MZD) after birth on the heart are unknown. In this study, 4-week-old male rats were fed a low zinc diet (10 mg kg-1, 1/3 recommended nutrient intake, RNI) for 8 weeks. Echocardiography and histopathology were performed to assess the functional and morphological alterations of the heart. High-throughput proteomics was used to study the effects of MZD on cardiac protein expression. We found that MZD reduced food intake, body weight, serum zinc, and heart weight; however, the coefficient, zinc concentration, function, and histopathology of the heart were not changed. The heart proteome was altered in the marginal zinc-deficient diet group (MZG), compared with the normal zinc diet group (NZG). A total of 310 differentially expressed proteins (P < 0.05) were significantly changed by MZD, among which 163 proteins were up-regulated and 147 were down-regulated. Of these, 43 proteins are related to CVDs and 18 proteins are zinc-associated proteins. Gene Ontology and Pathway analysis revealed that 74 biological processes (BPs) and 37 pathways were significantly changed by MZD. This included six CVD-related BPs, such as regulation of heart rate, cardiac muscle contraction, regulation of ventricular cardiac muscle cell action potential, and regulation of blood pressure, and eight CVD-related pathways, such as dilated cardiomyopathy, diabetic cardiomyopathy, and hypertrophic cardiomyopathy. Our data show that marginal zinc deficiency after birth significantly alters cardiac protein expression and pathways related to CVDs.

Effects of marginal zinc deficiency on learning and memory ability after birth.

Zinc deficiency during pregnancy and severe zinc deficiency after birth both impaired learning and memory ability, but the effects of marginal zinc deficiency (MZD) after birth on learning and memory are unclear. In the first experiment, 4-week-old male rats were randomly divided into three groups: the marginal zinc-deficient group (MZG, 10 mg kg-1, 1/3 RNI), normal zinc group (NZG, 30 mg kg-1, RNI), and paired zinc group (PZG, 30 mg kg-1). After a 4-week feeding period, the brain weight, brain coefficient, and serum zinc concentration were measured, and hippocampal proteomics analysis was performed. In the second experiment, 4-week-old male rats were fed the same diet for 8 weeks. In addition to the previously mentioned indicators, the Morris water maze results, brain pathology, post-translational modifications (PTMs) of hippocampal proteins, and the concentrations of indicators known to be related to learning and memory were analyzed. In both experiments, compared with those of the NZG, the food intake, body weight and serum zinc of the MZG were significantly decreased, and the brain weight was unchanged, but the brain coefficient was increased. Two hippocampal proteomics analyses and PTM screening showed that MZD did not change the expression and PTM of proteins. The brain pathology, learning, memory and the concentrations of indicators known to be related to learning and memory were not changed by MZD. Our study confirmed that marginal zinc deficiency (10 mg kg-1, 1/3 RNI) had no effect on the learning and memory abilities of rats after birth.

High-Throughput Metabolomics for Discovering Potential Biomarkers and Identifying Metabolic Mechanisms in Aging and Alzheimer's Disease.

Alzheimer's disease (AD) is an aging-related neurodegenerative disease. We aimed to investigate the metabolic mechanisms of aging and AD and to identify potential biomarkers for the early screening of AD in a natural aging population. To analyze the plasma metabolites related to aging, we conducted an untargeted metabolomics analysis using ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry in a two-stage cross-sectional study. Spearman's correlation analysis and random forest were applied to model the relationship between age and each metabolite. Moreover, a systematic review of metabolomics studies of AD in the PubMed, Cochrane and Embase databases were searched to extract the differential metabolites and altered pathways from original studies. Pathway enrichment analysis was conducted using Mummichog. In total, 669 metabolites were significantly altered with aging, and 12 pathways were enriched and correlated with aging. Three pathways (purine metabolism, arginine and proline metabolism, and the TCA cycle) were shared between aging and AD. Arginine and proline metabolism play a key role in the progression from healthy to mild cognitive impairment and to AD in the natural aging population. Three metabolites, 16-a-hydroxypregnenolone, stearic acid and PC[16:0/22:5(4Z,7Z,10Z,13Z,16Z)] were finally proposed as potential markers of AD in the natural aging population. The underlying mechanism shared between aging and AD and the potential biomarkers for AD diagnosis were proposed based on multistep comparative analysis.

Rapid inhibition of atherosclerotic plaque progression by sonodynamic therapy.

Aims: Currently, efficient regimens to reverse atherosclerotic plaques are not available in the clinic. Herein, we present sonodynamic therapy (SDT) as a novel methodology to rapidly inhibit progression of atherosclerotic plaques. Methods and results: In atherosclerotic rabbit and apoE-deficient mouse models, SDT efficiently decreased the atherosclerotic burden within 1 week, revealing a decrease in the size of the atherosclerotic plaque and enlarged lumen. The shrunken atherosclerotic plaques displayed compositional alterations, with a reduction in lesional macrophages and lipids. The rapid efficacy of SDT may be due to its induction of macrophage apoptosis, enhancement of efferocytosis, and amelioration of inflammation in the atherosclerotic plaque. Compared with atorvastatin, the standard of care for atherosclerosis, SDT showed more significant plaque shrinkage and lumen enlargement during 1 week treatment. Furthermore, SDT displayed good safety without obvious side effects. In a pilot clinical trial recruiting the patients suffering atherosclerotic peripheral artery disease, combination therapy of SDT with atorvastatin efficiently reduced progression of atherosclerotic plaque within 4 weeks, and its efficacy was able to last for at least 40 weeks. Conclusion: SDT is a non-invasive and efficacious regimen to inhibit atherosclerotic plaque progression.

[Effects of hydroxyl acetylated curcumin induced sonodynamic therapy on viability, apoptosis and necrosis of THP-1 macrophages].

OBJECTIVE: We aim to investigate the sonodynamic effect induced by hydroxyl acetylated curcumin (HAC) on THP-1 macrophages. METHODS: THP-1 derived macrophages (1 x 10(5) per milliliter) were cultured with HAC at a concentration of 5 µg/mL for 4 h and then exposed to pulse ultrasound treatment (0.5 W/cm2) for 5 min. Six hours later, cell viability analysis was performed with CCK-8 assay, apoptosis and necrosis analysis were detected with Annexin V/PI staining by flow cytometery. RESULTS: The cell viability of THP-1 macrophage decreased significantly in the group treated with the combination of HAC and ultrasound (P < 0.01), and HAC-SDT induced both apoptosis and necrosis in THP-1 macrophages, the apoptotic rate was higher than the necrotic rate with appropriate conditions, the maximum apoptosis/necrosis ratio was detected in sonodynamic therapy (SDT) group (P < 0.01). CONCLUSION: hAC-SDT was effective to induce THP-1 macrophages apoptosis.

Rapid stabilisation of atherosclerotic plaque with 5-aminolevulinic acid-mediated sonodynamic therapy.

5-Aminolevulinic acid-mediated sonodynamic therapy (ALA-SDT) effectively induces the apoptosis of atherogenic macrophages, but whether it can stabilise atherosclerotic plaque in vivo is unclear. Here, we used an animal model to evaluate the effects of ALA-SDT on plaque stabilisation. Sixty rabbits were induced atherosclerotic plaques in the femoral artery with a combination of silastic tube placement with atherogenic diet, and randomly assigned into control (n = 12) and SDT (n = 48) groups. In the SDT group, after intravenous injected with ALA (60 mg/kg) animals underwent the treatment of ultrasound with intensities of 0.75, 1.00, 1.50 and 2.00 W/cm(²) (n = 12 for each intensity). Seven days after the treatment, the plaque disruption assay was performed to test plaque stability. We found that ALA-SDT with ultrasound intensity of 1.5 W/cm(²) showed the strongest efficacy to stabilise plaques. Under this condition, the frequency of plaque disruption decreased by 88% (p<0.01), positive area of macrophages reduced by 94% (p<0.001) and percentage content of lipids dropped by 60% (p < 0.001), while percentage content of collagens increased by 127% (p<0.001). We also found that the plaque stabilisation by ALA-SDT was associated with increased macrophage apoptosis and apoptotic cell clearance. Moreover, ALA-SDT decreased the contents and activities of matrix metalloproteinase-2,9 and increased the levels of tissue inhibitors of matrix metalloproteinase-1,2 in plaques. Our studies demonstrate that ALA-SDT promotes plaque stabilisation by inducing macrophage elimination and inhibiting matrix degradation. This method might be a promising regimen for atherosclerosis therapy.

Hyperbaric Oxygen Preconditioning Attenuates Myocardium Ischemia-Reperfusion Injury Through Upregulation of Heme Oxygenase 1 Expression: PI3K/Akt/Nrf2 Pathway Involved.

BACKGROUND: With the rise of the burden of ischemic heart disease, both clinical and economic evidence show a desperate need to protect the heart against myocardium ischemia-reperfusion injury-related complications following cardiac surgery or percutaneous coronary intervention. However, there is no effective intervention for myocardium ischemia-reperfusion injury as yet. METHODS: We pretreated mice with 4 daily 2.0 absolute atmosphere (ATA) hyperbaric oxygen, then observed its effects on heart function parameters and infarct size following in situ ischemia-reperfusion. Multiple oxidative and inflammation products were measured in the myocardium. Next, we investigated the expression of heme oxygenase 1 (HO-1), phosphatidylinositol 3-kinase (PI3K)/serine/threonine protein kinase (Akt) pathway, and NF-E2-related factor 2 (Nrf2) in the presence of myocardium ischemia-reperfusion injury, hyperbaric oxygen preconditioning, and their inhibitors and their effects on heart function parameters. RESULTS: Hyperbaric oxygen preconditioning ameliorated the cardiac function and histological alterations induced by myocardium ischemia-reperfusion injury, decreased oxidative products and proinflammatory cytokine. Hyperbaric oxygen preconditioning increased expression of HO-1, which was suppressed by PI3K inhibitor LY294002, Nrf2 knockout, and Akt inhibitor triciribine. The expression of Nrf2 was enhanced by hyperbaric oxygen preconditioning, but decreased by LY294002 and triciribine. The Akt was also activated by hyperbaric oxygen preconditioning but suppressed by LY294002. The hemodynamic assays showed that cardiac function was suppressed by LY294002, Nrf2 knockout, and triciribine. CONCLUSION: These data present a novel signaling mechanism by which hyperbaric oxygen preconditioning protects myocardium ischemia-reperfusion injury via PI3K/Akt/Nrf2-dependent antioxidant defensive system.

miR-30a downregulation aggravates pressure overload-induced cardiomyocyte hypertrophy.

miRNAs play an important role in the pathogenesis of cardiac hypertrophy and dysfunction. However, little is known about how miR-30a regulates cardiomyocyte hypertrophy. In the study, Male C57BL/6 mice were subjected to thoracic aortic constriction, and hearts were harvested at 3 weeks. We assayed miR-30a expression level by real-time PCR and defined the molecular mechanisms of miR-30a-mediated cardiomyocyte hypertrophy. We found that myocardial expression of miR-30a was decreased in mouse models of hypertrophy and in H9c2 cells treated with phenylephrine. MiR-30a inhibition markedly increased mRNA expression of cardiac hypertrophy markers such as atrial natriuretic factor and brain natriuretic peptide in H9c2, and cell size was increased after miR-30a inhibitor treatment. Downregulated miR-30a activated autophagy by inhibiting beclin-1 expression in H9c2 cell. More important, autophagy inhibition suppressed miR-30a inhibitor-induced cardiomyocyte hypertrophy. Together, our data demonstrated that downregulated miR-30a aggravates pressure overload-induced cardiomyocyte hypertrophy by activating autophagy, thus offering a new target for the therapy of cardiomyocyte hypertrophy.

An animal model of atherosclerotic plaque disruption and thrombosis in rabbit using pharmacological triggering to plaques induced by perivascular collar placement.

INTRODUCTION: Limited availability of suitable animal model of plaque disruption and thrombosis has hampered the study of mechanism and preclinical evaluation of plaque-stabilizing therapies. This study aims to develop an animal model of atherosclerotic plaque disruption and thrombosis in rabbit femoral artery. METHODS: Silastic collars were placed around the bilateral femoral arteries of rabbits, which had been fed with atherogenic diet for 7 days. After 28 days on the same diet, the rabbits received pharmacological triggering by intraperitoneal injection of Russell's viper venom (RVV, 0.15 mg/kg) followed by intravenous injection of histamine (0.02 mg/kg), and the animals were then processed for imageological and histological examinations. RESULTS: Perivascular collar placement of the femoral artery in high-cholesterol-fed rabbits for 28 days induced marked intimal hyperplasia, which was a lipid- and collagen-rich lesion that contained substantial amount of macrophages and smooth muscle cells. Subsequent histological analysis showed that the pharmacological triggering evoked plaque disruption and platelet- and fibrin-rich thrombi in the collared femoral arteries. CONCLUSION: We demonstrated, for the first time, a rabbit model of plaque disruption and thrombosis induced by the combination of perivascular collar placement, RVV, and histamine injections. This model can be rapidly formed, easily operated, and site controlled.

A novel model of intimal hyperplasia with graded hypoosmotic damage.

BACKGROUND: The purpose was to develop a rabbit model of intimal hyperplasia with controllable lesion. METHODS: Following 1 week of a 2% cholesterol diet, 32 New Zealand White male rabbits underwent right femoral arteries surgical perfusion with distilled water for 1, 3, 5, or 7 min (n=8/group). After a further 4 weeks of the same diet, serum total cholesterol, triglyceride, low-density lipoprotein, and high-density lipoprotein were measured in all rabbits. Intimal hyperplasia in histological sections of arteries were assessed by intimal proliferation ratio. Macrophage numbers and levels of proteins matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 2, and alpha smooth muscle actin in lesions were analyzed by immunohistochemistry. RESULTS: Serum lipids levels showed no statistical difference between experimental groups. Intimal proliferation ratio increased gradually with perfusion time, and a positive linear correlation was calculated between intimal proliferation ratio and duration of distilled water perfusion. Similarly, number of macrophages and levels of matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 2, and alpha smooth muscle actin in lesions increased with perfusion time. CONCLUSIONS: A novel model of intimal hyperplasia was established by intravascular distilled water perfusion in high-cholesterol-fed rabbits. Importantly, this model exhibits time-dependent neointimal proliferation lesions that can be readily controlled in terms of extent, thus providing an avenue for further studies.

Detection and photodynamic therapy of inflamed atherosclerotic plaques in the carotid artery of rabbits.

Photodynamic therapy (PDT) has been applied in the treatment of artery restenosis following balloon injury. This study aimed to detect the accumulation of 5-aminolevulinic acid (ALA)-derived protoporphyrin IX (PpIX) in inflamed atherosclerotic plaque in rabbit model and evaluate the efficacy of PDT. The inflamed atherosclerotic plaque in the common carotid artery was produced by combination of balloon denudation injury and high cholesterol diet. After intravenous administration of ALA, the fluorescence of PpIX in plaque was detected. At the peak time, the correlation between the fluorescence intensity of PpIX and the macrophage infiltration extent in plaque was analyzed. Subsequently, PDT (635nm at 50J/cm(2)) on the atherosclerotic plaques (n=48) was performed and its effect was evaluated by histopathology and immunohistochemistry. The fluorescence intensity of PpIX in the plaque reached the peak 2h after injection and was 12 times stronger than that of adjacent normal vessel segment, and has a positive correlation with the macrophage content (r=0.794, P<0.001). Compared with the control group, the plaque area was reduced by 59% (P<0.001) at 4week after PDT, the plaque macrophage content decreased by 56% at 1week and 64% at 4week respectively, the smooth muscle cells (SMCs) was depleted by 24% at 1week (P<0.05) and collagen content increased by 44% at 4week (P<0.05). It should be pointed out that the SMC content increased by 18% after PDT at 4week compared with that at 1week (P<0.05). Our study demonstrated that the ALA-derived PpIX can be detected to reflect the macrophage content in the plaque. ALA mediated PDT could reduce macrophage content and inhibit plaque progression, indicating a promising approach to treat inflamed atherosclerotic plaques.

Interleukin-18 among atrial fibrillation patients in the absence of structural heart disease.

AIMS: Inflammation plays a role in the genesis and perpetuation of atrial fibrillation (AF). Interleukin (IL)-18 is a pleiotropic proinflammatory cytokine with a central role in the inflammatory cascade. We hypothesize that the circulating IL-18 concentration is elevated in AF patients. METHODS AND RESULTS: In a case-control study design, 56 cases with AF and 26 controls were enrolled. All AF cases were categorized into paroxysmal and persistent AF or lone AF and AF with hypertension. Circulating levels of IL-18, tumour necrosis factor-α, high-sensitivity C-reactive protein, matrix metalloproteinase (MMP)-9, and tissue inhibitor of MMP-1 were measured. In adjusted analyses, only age, MMP-9, and IL-18 were independently associated with AF, in which IL-18 had the most significant association (P = 0.0011, standardized estimate &bgr = 1.76, OR = 1.02, 95% confidence interval: 1.01-1.03). Interleukin-18 levels in persistent AF patients were higher than those in paroxysmal ones (P = 0.0011). Patients who developed AF within 24 h prior to sampling displayed a higher level of IL-18 than those with sinus rhythm (P = 0.0027). Interleukin-18 was positively correlated with left atrial diameter (r = 0.33, P = 0.0117). CONCLUSION: This study documents the elevated IL-18 in AF patients. Interleukin-18 may be superior to other inflammatory markers which are known to be elevated in AF.

Hematoporphyrin monomethyl ether-mediated photodynamic effects on THP-1 cell-derived macrophages.

Photodynamic therapy (PDT) has been shown to attenuate atherosclerotic plaque progression and decrease macrophage-infiltration. The effectiveness of PDT depends strongly on the type of photosensitizers. Hematoporphyrin monomethyl ether (HMME) is a promising second-generation porphyrin-related photosensitizer for PDT. This study is designed to characterize effects of HMME-based PDT on THP-1 cell-derived macrophages and define the cell-death pathway. HMME was identified to accumulate in the macrophages by fluorescence microscopy and confocal scanning laser microscope. Our data demonstrated that the intensity of laser-induced HMME fluorescence in macrophages steadily increased with the increasing incubation concentration of HMME. The survival rate of macrophages determined by MTT assay decreased with the increasing HMME concentration and irradiation time. HMME-based PDT induced macrophage apoptosis via caspase-9 and caspase-3 activation pathway detected by caspase fluorescent assay kit and flow cytometer. The PDT increased the number of apoptotic macrophages by 14-fold at 12 h post irradiation by 9 J/cm(2) 635 nm diode laser. These results imply that photodynamic therapy with HMME may therefore be a useful clinical treatment for unstable atherosclerotic plaques.

Apoptosis of vascular smooth muscle cells induced by photodynamic therapy with protoporphyrin IX.

Photodynamic therapy (PDT) had been shown effective in the treatment of intimal hyperplasia, which contributes to restenosis, by eradicating cells in the vessel wall. This study is designed to evaluate the effects of PDT with protoporphyrin IX (PpIX) on the viability of vascular smooth muscle cells (SMCs) and to define the cell-death pathway. Fluorescence microscopy and laser-induced fluorescence spectroscopic detection showed that SMCs selectively uptake PpIX, and the intracellular PpIX concentration increases with the amount of PpIX in the incubation solution. PDT with PpIX impaired cellular viability from 93+/-3.4% to 36+/-3.9% when the light intensity increases from 2 to 9J/cm(2) and intracellular PpIX concentration increases from 0.5 to 20 microg/ml. Although PDT induced both apoptosis and necrosis, the ratio of apoptotic cells increased with light dosage or intracellular PpIX concentration. The loss of mitochondrial membrane potential coincided with the apoptotic ratio. Our results indicated that the induction of apoptosis of SMCs may be one of the mechanisms by which PDT inhibits restenosis in vivo.

Anti-apoptotic action of hydrogen sulfide is associated with early JNK inhibition.

The mechanism of action of Hydrogen sulfide (H(2)S) as a novel endogenous gaseous messenger and potential cardioprotectant is not fully understood. We therefore investigated the prevention of cardiomyocyte apoptosis by exogenous H(2)S and the signaling pathways leading to cardioprotection. Using a simulated ischemia-reperfusion (I/Re) model with primary cultured rat neonatal cardiomyocytes, I/Re induced a rapid, time-dependent phosphorylation of c-Jun N-terminal kinase (JNK), with significant elevation at 0.25 h and a peak at 0.5h during reperfusion. NaHS (H(2)S donor) significantly inhibited the early phosphorylation of JNK, especially at 0.5h. Both NaHS and SP600125 (specific JNK inhibitor) decreased the number of apoptotic cells, lowered cytochrome C release and enhanced Bcl-2 expression. When NaHS application was delayed 1h after reperfusion, the inhibition of apoptosis by H(2)S was negated. In conclusion, this is novel evidence that early JNK inhibition during reperfusion is associated with H(2)S-mediated protection against cardiomyocyte apoptosis.

Effects of pravastatin on the function of dendritic cells in patients with coronary heart disease.

The aim of the study was to investigate the functional profile of dendritic cells in patients with coronary heart disease and the effects of pravastatin on this. Forty-eight patients with coronary heart disease were divided into three groups: 16 treated with pravastatin 10 mg/day, 16 treated with pravastatin 20 mg/day and 16 not treated with pravastatin. Dendritic cells from 48 patients with coronary heart disease (before and 4 weeks after the treatment) and 16 healthy individuals were derived from peripheral blood. CD86 of dendritic cells was assessed by flow cytometry. Immunostimulatory capacity of dendritic cells was measured by mixed lymphocyte reaction. The levels of cytokines in the medium of mixed lymphocyte reaction were analysed. Blood lipids and high-sensitivity C-reactive protein were measured. Compared to normal group, more CD86+ dendritic cells were expressed in coronary heart disease and greater immunostimulatory capacity of dendritic cells in coronary heart disease was demonstrated. T lymphocytes in coronary heart disease in mixed lymphocyte reaction secreted higher levels of pro-inflammation cytokines and lower levels of anti-inflammation cytokines. CD86 expression significantly correlated with C-reactive protein, but did not correlate with low-density lipoprotein cholesterol. Both dosages of pravastatin markedly inhibited the function of dendritic cells and lowered C-reactive protein, which is independent of plasma cholesterol lowering. The anti-inflammatory effect of pravastatin showed no obvious difference between the two dosage groups. In conclusion, dendritic cells were activated in coronary heart disease and dendritic cell-mediated immune mechanisms may be involved in the pathogenesis of coronary heart disease. Pravastatin can inhibit dendritic cell activation, which is independent of plasma cholesterol lowering. Pravastatin in different doses showed no apparent differences in the inhibition of dendritic cell functions.